EA040257B1 - LIPIDS AND LIPID COMPOSITIONS FOR DELIVERY OF ACTIVE AGENTS - Google Patents

Description

The field of technology to which the invention belongs

This invention relates to cationic lipid compounds and compositions containing such compounds. This invention also relates to methods for the preparation of such compounds and compositions and to methods and uses of such compounds and compositions, for example, for the delivery of biologically active agents such as RNAi agents to cells and tissues.

State of the art

Delivery of biologically active agents (including therapeutically useful compounds) to subjects is often complicated by difficulties in the compounds reaching the target cell or tissue. In particular, the delivery of many biologically active agents into living cells is severely limited by the complex membrane system of cells. These limitations may lead to the need to use higher concentrations of biologically active agents, which is desirable to achieve the result, which increases the risk of toxic effects and side effects. One solution to this problem is the use of specific carrier molecules that provide selective entry into the cell. Lipid carriers, biodegradable polymers, and various conjugated systems can be used to improve the delivery of biologically active agents to cells.

One class of biologically active agents that is most difficult to deliver to cells is the biotherapeutic agents (including nucleosides, nucleotides, polynucleotides, nucleic acids, and derivatives such as RNAi agents). Typically, nucleic acids are stable for only a limited period in cells or plasma. The development of RNA interference, RNAi therapy, RNA drugs, antisense therapy and gene therapy, among other things, has increased the need for effective means of introducing active agents based on nucleic acids into cells. For these reasons, compositions that can stabilize and deliver nucleic acid agents to cells are of particular interest.

The most well studied approaches to improve the transport of foreign nucleic acids into cells involve the use of viral vectors or cationic lipids. Viral vectors can be used to transfer genes efficiently into some cell types, but they generally cannot be used to introduce chemically synthesized molecules into cells.

An alternative approach is to use delivery compositions containing cationic lipids that interact with the biologically active agent in one part and interact with the membrane system in another part (for a review, see Feigner, 1990, Advanced Drug Delivery Reviews, 5, 162-187 and Feigner , 1993, J. Liposome Res., 3, 3-16). Such compositions are reported to contain liposomes.

Since the first description of liposomes in 1965 by Bangham (J. Mol. Biol. 13, 238-252), there has been a steady interest and effort in the development of lipid-based carrier systems for the delivery of biologically active agents. A method for introducing functional nucleic acids into cultured cells using positively charged liposomes was first described by Philip Feigner et al. Proc. Natl. Acad. Sci., USA, 84, 7413-7417 (1987). The method was later demonstrated in vivo by K. L. Brigham et al., Am. J. Med. Sci., 298, 278-281 (1989).

Liposomes are attractive carriers because they protect biological molecules from degradation while improving their cellular uptake. Of the various classes of liposomes, liposomes that contain cationic lipids are commonly used to deliver polyanions (eg, nucleic acids). Such liposomes can be prepared using cationic lipids per se, and optionally including other lipids and amphiphiles such as phosphatidylethanolamine. It is well known in the art that both the composition of the lipid formulation, as well as the method of its preparation, affect the structure and size of the resulting aggregate.

The use of cationic lipids for cellular delivery of biologically active agents has several advantages. Encapsulation of anionic compounds using cationic lipids is essentially quantitative due to electrostatic interaction. In addition, cationic lipids are believed to interact with negatively charged cell membranes to initiate cell membrane transport (Akhtar et al., 1992, Trends Cell Bio., 2, 139; Xu et al., 1996, Biochemistry 35, 5616).

There is a need for additional cationic lipids that facilitate systemic and local delivery of biologically active agents such as RNAi agents into cells. There is also a need for cationic lipids that, when compared to those cationic lipids known in the art, improve systemic and local delivery of biologically active agents to cells. There is an additional need for lipid formulations that have optimized physical characteristics for improved systemic and local delivery of biologically active agents to certain organs and tumors, in particular tumors outside the liver.

The essence of the invention

In one aspect, this invention provides a compound of formula (I)

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or its pharmaceutically acceptable salt, in which R1-R ⁴ , L and X are defined in the present description. The compounds of formula (I) and their pharmaceutically acceptable salts are cationic lipids useful in the delivery of biologically active agents to cells and tissues.

In a second aspect, this invention provides a lipid composition containing a compound according to formula (I) (ie, a lipid composition of the invention) or a pharmaceutically acceptable salt thereof. In another embodiment, at least one other lipid component is present. In another embodiment, the lipid composition also contains a biologically active agent, optionally in combination with one or more other lipid components. In another embodiment, the lipid composition is in the form of a liposome. In another embodiment, the lipid composition is in the form of a lipid nanoparticle (LNP). In another embodiment, the lipid composition is suitable for delivery to the liver. In another embodiment, the lipid composition is suitable for delivery to a tumor. In another embodiment, the lipid composition is suitable for immunization purposes.

In a third aspect, this invention provides a pharmaceutical composition (i.e. formulation) comprising a lipid composition of the invention and a pharmaceutically acceptable carrier or excipient. In another embodiment, at least one other lipid component is present in the lipid composition. In another embodiment, the lipid composition is in the form of a liposome. In another embodiment, the lipid composition is in the form of a lipid nanoparticle. In another embodiment, the lipid composition is suitable for delivery to the liver. In another embodiment, the lipid composition is suitable for delivery to a tumor. In another embodiment, the biologically active agent is DNA or RNA. In another embodiment, the biologically active agent is a siRNA. In another embodiment, the biologically active agent is an mRNA. In another embodiment, the lipid composition is suitable for immunization purposes and the biologically active agent is an RNA that encodes an immunogen.

In a fourth aspect, this invention provides a method of treating a disease or condition, comprising the step of administering a therapeutically effective amount of a lipid composition of the invention to a patient in need of such treatment. In one embodiment, the disease or condition is treatable by administering an siRNA agent. In another embodiment, the disease or condition is treatable by administering an mRNA agent.

In a fifth aspect, this invention provides the use of a lipid composition of the invention for the treatment of a disease or condition in a patient. In one embodiment, the disease or condition is treatable by administering an RNAi agent. In another embodiment, the disease or condition is treatable by administering an mRNA agent.

In a sixth aspect, the invention provides a method of immunizing a subject against an immunogen of interest, comprising the step of administering an immunologically effective amount of a lipid composition of the invention to the subject in combination with an RNA that encodes the immunogen.

In a seventh aspect, this invention provides the use of a lipid composition of the invention for immunizing a subject against an immunogen of interest. The lipid is used in combination with the RNA that codes for the immunogen.

Detailed description of the invention

The present invention provides a compound of formula (I)

wherein

L is _C1-6 alkylene, *-C ₁ . ₄ alkylene-L2-, *-C ₁ . ₄ αkylene-L2-С ₁ . ₄ alkylene-,

or

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in which * denotes the accession of the fragment to the NR1R ² group;

L2 attached in either direction is -C(O)O-, -OC(O)O- or -CONH;

each s is independently 0, 1, or 2;

each t is independently 0, 1, 2, 3, or 4;

u is 0, 1, 2, 3, 4, 5 or 6;

R ¹ and R ² are each independently optionally substituted C _1-6 alkyl; wherein said C _1-6 alkyl is optionally substituted with one or two substituents, each independently selected from the group consisting of OH, COOH and COO-C _1-4 alkyl, or R ¹ and R ² are joined together to form an optionally substituted 4-7- a membered heterocyclic ring containing 1-2 heteroatoms selected from N and O, said heterocyclic ring being optionally substituted with one to three OH substituents;

R ³ and R ⁴ are each independently -R ^c , where the R ^c chain is

(s14), (s15)

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or

R is C _5-22 alkyl, C _5-22 alkenyl, or C _5-22 alkynyl;

n is 0-12;

m, p and q are each independently 0, 1, 2, 3 or 4;

provided that the chain R ^c has 12-70 carbon atoms;

X is CR ⁶ or N; and R ⁶ is H, halo, C _1-6 alkyl, or R ⁴ .

Embodiments

In one embodiment, R ¹ and R ² are each independently optionally substituted C _1-6 alkyl. In another embodiment, R ¹ and R ² are each independently optionally substituted methyl or optionally substituted ethyl. In another embodiment, R ¹ is methyl and R ² is optionally substituted ethyl. In another embodiment, R ¹ and R ² are both methyl.

In another embodiment, R ¹ and R ² are joined together to form an optionally substituted 47-membered heterocyclic ring. In another embodiment, a 4-7 membered heterocyclic

- 4 040257 the ring is an optionally substituted azetidinyl, an optionally substituted pyrrolyl or an optionally substituted piperidinyl. In another embodiment, the 4-7 membered heterocyclic ring is azetidinyl, pyrrolyl, or piperidinyl, each optionally substituted with one OH group. In another embodiment, the 4-7 membered heterocyclic ring is azetidinyl, pyrrolyl, or piperidinyl.

In another embodiment, L is C _1-6 alkylene, *-C _1-4 alkylene-L2-, *-C _1-4 alkylene-L2-C _1-4 alkylene-,

In another embodiment, *-C1 _-4 alkylene-L2-C _{1 -2} alkylene

L is *-C _1-3 alkylene-L2-,

where s is 0 and u is 1, or

where s is 0, t is 1 and u is 1.

In another embodiment, L is methylene, ethylene, or propylene. In another embodiment, L is methylene.

In another embodiment, L2 attached in either direction is -C(O)O-, -OCOO- or -CONH-.

In another embodiment, L is *-C _1-4 alkylene-L2-C _1-2 alkylene, where L2 attached in either direction is -C(O)O- or -OC(O)O-.

In another embodiment, L is:

In one embodiment, L-NR ¹ R ² -rpynny formula (I) is selected from the list in table. 1.

Table 1

L-NR ¹ R ² groups of formula (I), where the dotted line indicates the point of attachment to formula (I).

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- 6 040257

In another embodiment, R ^c is (c1) or (c3). In another embodiment, R ^c is (c1) or (c3), where n is 1 or 2; m is 0 or 1; and p is 1.

In another embodiment, X is CR ⁶ where R ⁶ is H, chlorine, bromine, or C _1-3 alkyl. Preferably X is CH.

In another embodiment, X is N.

One embodiment of the present invention is a compound selected from the group consisting of

1-(3,5-bis((Z)-octadec-9-en-1-yloxy)phenyl)-N,N-dimethylmethyl anamine,

1-(3,5-bis ((9Z,12Z)-octadeca-9,12-dien-1-yloxy)phenyl)N,N-dimethylmethanamine,

2,2'- ((3,5-bis((9Z,12Z)-octadeca-9,12-dien-1yloxy)benzyl)azanediyl)diethanol,

1-(3,5-bis((9Z,12Z)-octadeca-9,12-dien-1yloxy)benzyl)pyrrolidine,

1-(3,5-bis ((9Z,12Z)-octadeca-9,12-dien-1yloxy)benzyl)azetidin-3-ol, ethyl-2-((3,5-bis((Z)-octadec -9-en-1yloxy)benzyl)(methyl)amino)acetate,

1-(3,5-bis ((Z)-octadec-9-en-1-yloxy)benzyl)pyrrolidine, 2-((3,5-bis((Z)-octadec-9-en-1yloxy)benzyl )(methyl)amino)acetic acid,

1-((2-(3,5-bis((Z)-octadec-9-en-1-yloxy)phenyl)-1,3-7040257 dioxolan-4-yl)methyl)pyrrolidine,

1-(2-(3,5-bis((9Z,12Z)-octadeca-9,12-dien-1-yloxy)phenyl) 1,3-dioxolan-4-yl)-N,N-dimethylmethanamine,

1- ((2-(3,5-bis((9Z,12Z)-octadeca-9,12-dien-1-yloxy)phenyl)-1,3-dioxolan-4-yl)methyl)piperidine, (2 -(3,5-bis((Z)-octadec-9-en-1-yloxy)phenyl)-1,3dioxolan-4-yl)methyl-2-(dimethylamino)acetate,

3,5-bis ((9Z,12Z)-octadeca-9,12-dien-1-yloxy)benzyl-4(dimethylamino)butanoate,

3,5-bis ((9Z,12Z)-octadeca-9,12-dien-1-yloxy)benzyl-3(dimethylamino)propanoate,

3,5-bis ((9Z,12Z)-octadeca-9,12-dien-1-yloxy)benzyl-2(dimethylamino)acetate,

4-methyl-3,5-bis ((9Z,12Z)-octadeca-9,12-dien-1yloxy)benzyl-3-(dimethylamino)propanoate,

3,5-bis ((9Z,12Z)-octadeca-9,12-dien-1-yloxy)benzyl-3(dimethylamino)-2-methylpropanoate,

2-(dimethylamino)ethyl-2,6-bis ((9Z,12Z)-octadeca-9,12-dien1-yloxy)isonicotinate,

3-(dimethylamino)propyl-2, 6-bis ( (9Z,12Z)-octadeca-9,12dien-1-yloxy)isonicotinate, (9Z,9'Z,12Z,12'Z)-5- ( ( ( 3(dimethylamino)propanoyl)oxy)methyl)-1,3-phenylene-bis(octadeca9, 12-dienoate), (9Z,9'Z,12Z,12'Z)-5- ( ((4(dimethylamino)butanoyl )oxy)methyl)-1,3-phenylene-bis(octadeca9,12-dienoate), (2,6-bis((9Z,12Z)-octadeca-9,12-dien-1-yloxy)pyridin-4yl) methyl 3-(diethylamino)propanoate, (9Z, 9' Z, 12Z, 12' Z) - (5- ( ( ((3(dimethylamino)propanoyl)oxy)methyl)-1,3phenylene)bis(methylene)bis( octadeca-9,12-dienoate), (2,6-bis ((9Z,12Z)-octadeca-9,12-dien-1-yloxy)pyridin-4yl)methyl-4-(dimethylamino)butanoate, (2, 6-bis ((9Z,12Z)-octadeca-9,12-dien-1-yloxy)pyridin-4yl)methyl-3-(dimethylamino)propanoate,

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1-(2,6-bis ((9Z,12Z)-octadeca-9,12-dien-1-yloxy)pyridin-4 yl)-N,N-dimethylmethanamine, (9Z,9'Z,12Z,12' Z)- (5- (((dimethylamino)methyl)-1,3phenylene)bis(methylene)bis(octadeca-9,12-dienoate), (5-(((dimethylamino)methyl)-1,3phenylene)bis(methylene) ditridecanoate, (5-((dimethylamino)methyl)-1,3-phenylene)bis(methylene)bis(3octylundecanoate), (9Z, 12Z)-3-(((dimethylamino)methyl)-5-(((3octylundecanoyl)oxy )methyl)benzyloctadeca-9,12-dienoate, di ( ( ( 9Z,12Z) -octadeca-9,12-dien-1-yl) -5- ( ( (3 (dimethylamino) propanoyl) oxy) methyl) isophthalate, ( 9Z,9'Z,12Z,12'Z)- ( ( ( (5-( ((dimethylamino)methyl)-1,3phenylene)bis(oxy))bis(ethane-2,1-diyl))bis(oxy) )bis(ethane-2,1diyl)bis(octadeca-9,12-dienoate), (9Z,9'Z,12Z,12'Z) — ( ( ( (5-((diethylamino)methyl)-1,3phenylene ) bis(oxy) )bis(ethane-2,1-diyl) )bis(oxy) )bis(ethane-2,1diyl)bis(octadeca-9,12-dienoate) ,

3,5-bis ((9Z,12Z)-octadeca-9,12-dien-1-ylcarbamoyl)benzyl 3-(dimethylamino)propanoate,

3,5-bis ((9Z,12Z)-octadeca-9,12-dien-1-ylcarbamoyl)benzyl4-(dimethylamino)butanoate,

3,5-bis ((9Z,12Z)-octadeca-9,12-dien-1-yloxy)phenethyl-3(dimethylamino)propanoate,

3,5-bis ((9Z,12Z)-octadeca-9,12-dien-1-yloxy)benzyl(3(dimethylamino)propyl)carbonate, (9Z,9'Z,12Z,12'Z)-(5 - ( ( ( ( 3(diethylamino)propoxy)carbonyl)oxy)methyl)-1,3phenylene)bis(methylene)bis(octadeca-9,12-dienoate), (9Z,9'Z,12Z,12'Z) - (5-((((2(dimethylamino)ethoxy)carbonyl)oxy)methyl)-1,3phenylene)bis(methylene)bis(octadeca-9,12-dienoate), (9Z,9'Z,12Z,12 'Z)- (5-((((3(dimethylamino)propoxy)carbonyl)oxy)methyl)-1,3phenylene)bis(methylene)bis(octadeca-9,12-dienoate), (9Z,9'Z, 12Z,12'Z)-5-((((2- 9 040257 (dimethylamino)ethoxy)carbonyl)oxy)methyl)-1,3-phenylenebis(octadeca-9,12-dienoate),

3-(dimethylamino)propyl-4-isopropyl-3,5-bis((9Z,12Z)octadeca-9,12-dien-1-yloxy)benzyl carbonate,

4-bromo-3,5-bis ((9Z,12Z)-octadeca-9,12-dien-1yloxy)benzyl (3-(dimethylamino)propyl)carbonate,

4-chloro-3,5-bis ((9Z,12Z)-octadeca-9,12-dien-1yloxy)benzyl(3-(dimethylamino)propyl)carbonate,

N-(3,5-bis ((9Z,12Z)-octadeca-9,12-dien-1-yloxy)benzyl)-2(dimethylamino)acetamide,

O,O' - ((5-(((dimethylamino)methyl)-1,3phenylene)bis(methylene))di((92.122)-octadeca-9,12-dien-1yl)disuccinate,

O,O' -((5-((dimethylamino)methyl)-1,3phenylene)bis(methylene))bis(10-(octanoyloxy)decyl)disuccinate,

O ' \ O ¹ - ((5- ( (dimethylamino) methyl) -1,3-phenylene) bis (methylene) 8-dinonyldioctanedioate,

O ' \ O ¹ - ((5- ( (dimethylamino) methyl) -1,3-phenylene) bis (methylene)) 9-dioctyldinonanedioate,

3-(3,5-bis ((9Z,12Z)-octadeca-9,12-dien-1yloxy)phenyl)propyl-3-(dimethylamino)propanoate, (9Z,9'Z,12Z,12'Z)- ( ( (5- (((dimethylamino)methyl)-1,3phenylene)bis(methylene))bis(oxy))bis(4-oxobutane-4,1diyl)bis(octadeca-9,12-dienoate) , ((( 5-((dimethylamino)methyl)-1,3phenylene) bis(methylene))bis(oxy))bis(8-oxooctane-8,1diyl)bis(decanoate), (((5-((dimethylamino)methyl)- 1,3phenylene) bis(methylene))bis(oxy))bis(4-oxobutan-4,1diyl)dioctanoate, (((5-(((dimethylamino)methyl)-1,3phenylene)bis(methylene))bis(oxy ))bis(6-oxohexane-6,1diyl)dioctanoate, (5-((dimethylamino)methyl)-1,3-phenylene)bis(methylene)bis(10-(octanoyloxy)decanoate), (5-((dimethylamino )methyl)-1,3-phenylene)bis(methylene)bis(8-10 040257 (octanoyloxy)octanoate), (((5-((dimethylamino)methyl)-1,3phenylene)bis(methylene))bis(oxy ))bis(6-oxohexane-6,1diyl)bis(decanoate),

4,4'— ((5—(((dimethylamino)methyl)-1,3phenylene)bis(oxy))bis(butane-1,2-diyl)tetraoctanoate, (R)-4-(3-((S) -3,4-bis(octanoyloxy)butoxy)-5((dimethylamino)methyl)phenoxy)butane-1,2-diyldioctanoate, (S)-4-(3-((S)-3,4-bis(octanoyloxy )butoxy)-5((dimethylamino)methyl)phenoxy)butane-1,2-diyldioctanoate, (R)-4-(3-((R)-3,4-bis(octanoyloxy)butoxy)-5((dimethylamino )methyl)phenoxy)butane-1,2-diyldioctanoate,

N,N-dimethyl-1-(3,4,5-tris ((9Z,12Z)-octadeca-9,12-dien-1yloxy)phenyl)methanamine, ((((5-((dimethylamino)methyl)- 1,3phenylene)bis(oxy) )bis(propan-3, 1-diyl) )bis(oxy) )bis(4oxobutan-4,1-diyl)bis(decanoate),

O ' \ O ¹ - ( ( ( (5- ( (dimethylamino) methyl) -1,3 phenylene) bis (oxy) ) bis (propane-3, 1-diyl) ) -9-dioctyldinonanedioate,

3-((dimethylamino)methyl)-5-(((8(octanoyloxy)octanoyl)oxy)methyl)benzyl-3-octylundecanoate,

2-(3,5-bis ((9Z,12Z)-octadeca-9,12-dien-1-yloxy)phenyl)N,N-dimethylethanamine,

3-(3,5-bis((9Z,12Z)-octadeca-9,12-dien-1-yloxy)phenyl)N,N-dimethylpropan-1-amine,

- (3- (4 - (5- ( (dimethylamino) methyl) -2-methyl-3- ( ( 9Z,12Z) octadeca-9,12-dien-1-yloxy) phenoxy) butoxy) -3 oxopropyl) propane- 1,3-diyldihexanoate, (5-((dimethylamino)methyl)-1,3-phenylene)bis(methylene)bis(8decanamidooctanoate), (5-((dimethylamino)methyl)-1,3-phenylene)bis(methylene )bis(6(((nonyloxy)carbonyl)oxy)hexanoate), bis(1,3-bis(octanoyloxy)propan-2-yl)-0.0^-((5((dimethylamino)methyl)-1, 3-phenylene)bis(methylene))disuccinate, (((5-((dimethylamino)methyl)-1,3phenylene)bis(oxy))bis(butane-4, 1-diyl))bis(propane-3,2 ,1triyl)tetraoctanoate, ( ( ( 5- (((dimethylamino)methyl)-1,3phenylene)bis(oxy))bis(methylene))bis(propane-3,2,1triyl)tetraoctanoate, (5-((dimethylamino) methyl)-1,3-phenylene)bis(methylene)bis(5heptyldodecanoate), (5-((dimethylamino)methyl)-1,3-phenylene)bis(methylene)bis(7hexyltridecanoate), (5-((dimethylamino) methyl)-1,3-phenylene)bis(methylene)bis(9pentyltetradecanoate), ((((5-((dimethylamino)methyl)-1,3phenylene)bis(oxy))bis(butane-4,1- diyl))bis(oxy))bis(propane-

3, 2,1-triyl)tetraoctanoate,

1-(3,5-bis(4,4-bis(octyloxy)butoxy)phenyl)-Ν,Νdimethylmethyl an amine, or a pharmaceutically acceptable salt thereof.

In the context of the present description, the term alkyl refers to a fully saturated branched or unbranched hydrocarbon chain containing a certain number of carbon atoms. For example, _C1-6 alkyl refers to an alkyl group containing 1 to 6 carbon atoms. Alkyl

- 11 040257 groups may be optionally substituted with one or more substituents as defined in formula (I). Typical examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, t-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3methylhexyl, 2, 2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecanyl, n-dodecanyl, n-tridecanyl, 9-methylheptadecanyl and the like.

In the context of the present description, the term alkylene refers to a divalent alkyl group, as defined above in the present description. Representative examples of alkylene include, but are not limited to, methylene, ethylene, n-propylene, isopropylene, n-butylene, sec-butylene, isobutylene, t-butylene, n-pentylene, isopentylene, neopentylene, n-hexylene, 3-methylhexylene, 2, 2-dimethylpentylene, 2,3dimethylpentylene, n-heptylene, n-octylene, n-nonylene, n-decylene and the like.

In the context of the present description, the term alkenyl refers to an unsaturated branched or unbranched hydrocarbon chain containing a certain number of carbon atoms and one or more carbon-carbon double bonds in the chain. For example, _C2-6 alkenyl refers to an alkenyl group containing 2 to 6 carbon atoms with one or more carbon-carbon double bonds in the chain. In some embodiments, the implementation of alkenyl groups contain one carbon-carbon double bond in the chain. In other embodiments, the implementation of alkenyl groups contain more than one carbon-carbon double bond in the chain. Alkenyl groups may be optionally substituted with one or more substituents as defined in formula (I). Representative examples of alkenyl include, but are not limited to, ethyleneyl, propenyl, butenyl, pentenyl, hexenyl, and the like. Other examples of alkenyl include, but are not limited to: Z-octadec-9-enyl, Z-undec-7enyl, Z-heptadeca-8-enyl, (9Z,12Z)-octadec-9,12-dienyl, (8Z,11Z )-heptadeca-8,11-dienyl; and (8Z,11Z,14Z)-heptadeca-8,11,14-trienyl.

In the context of the present description, the term alkynyl refers to an unsaturated branched or unbranched hydrocarbon chain containing a certain number of carbon atoms and one or more carbon-carbon triple bonds. For example, C ₂ . ₆ alkynyl refers to an alkynyl group containing 2 to 6 carbon atoms with one or more carbon-carbon triple bonds in the chain. In some embodiments, alkynyl groups contain one carbon-carbon triple bond in the chain. In other embodiments, alkynyl groups contain more than one carbon-carbon triple bond in the chain. Alkynyl groups may be optionally substituted with one or more substituents as defined in formula (I). Representative examples of alkynyl include, but are not limited to, ethynyl, 1-propynyl, propargyl, butynyl, pentynyl, hexynyl, and the like.

In the context of the present description, the term halogen refers to fluorine, chlorine, bromine and iodine.

In the context of the present description, the term heterocyclic refers to a 4-7 membered saturated or unsaturated monocyclic or bicyclic ring containing from 1 to 2 heteroatoms. Heterocyclic ring systems are not aromatic. Heterocyclic groups containing more than one heteroatom may contain different heteroatoms. The heterocyclic groups may be optionally substituted with one or more substituents as defined in formula (I). Heterocyclic groups are monocyclic, spiro or fused or bridged bicyclic ring systems. Examples of monocyclic heterocyclic groups include tetrahydrofuranyl, dihydrofuranyl, 1,4-dioxanyl, morpholinyl, 1,4dithianyl, azetidinyl, piperazinyl, piperidinyl, 1,3-dioxolanyl, imidazolidinyl, imidazolinyl, pyrrolinyl, pyrrolidinyl, tetrahydropyranyl, dihydropyranyl, 1,2,3 ,6-tetrahydropyridinyl, oxathiolanyl, dithiolanyl, 1,3-dioxanyl, 1,3-dithianyl, oxathianyl, thiomorpholinyl, 1,4,7-trioxa-10azacyclododecanyl, azapanil, and the like.

Examples of spiroheterocyclic rings include, but are not limited to, 1,5-dioxa-9azaspiro[5.5]undecanyl, 1,4-dioxa-8-azaspiro[4.5]decanyl, 2-oxa-7-azaspiro[3.5]nonanyl, and the like. Fused heterocyclic ring systems have 8 to 11 ring atoms and include groups in which the heterocyclic ring is fused to a phenyl ring. Examples of fused heterocyclic rings include, but are not limited to, decahydroquinilinyl, (4aS,8aR)-decahydroisoquinolinyl, (4aS,8aS)-decahydroisoquinolinyl, octahydrocyclopenta[c]pyrrolyl, isoinolinyl, (3aR,7aS)-hexahydro-[1,3] dioxolo[4,5-c]pyridinyl, octahydro-1Hpyrrolo[3,4-b]pyridinyl, tetrahydroisoquinolinyl and the like.

In the context of the present description, the term optical isomer or stereoisomer refers to any of the various stereoisomeric configurations that may exist for a given compound of the present invention, and includes geometric isomers. It should be understood that the substituent may be attached to the chiral center of the carbon atom. The term chiral refers to molecules that have the property of non-superimposable mirror images of the molecule, while the term achiral refers to molecules whose mirror images can be superimposed on each other. Therefore, the invention includes the enantiomers, diastereomers or racemates of the compound. Enantiomers are a pair of stereoisomers that are non-mirror images that can be superimposed on each other. A 1:1 mixture of enantiomer pairs is a racemic mixture. The term is used to refer to a racemic mixture when appropriate.

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Diastereoisomers are stereoisomers that contain at least two asymmetric atoms, but which are not mirror images of each other. Absolute stereochemistry is given according to the Cahn-Ingold-Prelog R-S system. When a compound is the pure enantiomer, the stereochemistry at each chiral carbon atom can be defined as either R or S. Separated compounds whose absolute configuration is unknown can be designated (+) or (-) depending on the direction (right or left handed) , in which they rotate the plane of polarized light at the wavelength of the sodium D-line. Some compounds described in this application contain one or more asymmetric centers or axes and may thus form enantiomers, diastereomers and other stereoisomeric forms, which can be defined, based on absolute stereochemistry, as (R)- or (S) -.

Depending on the choice of starting materials and techniques, the compounds may be present in the form of one of the possible isomers or as mixtures of them, for example, as pure optical isomers or as mixtures of isomers, such as racemates and diastereomeric mixtures, depending on the number of asymmetric carbon atoms . The present invention is intended to include all such possible isomers, including racemic mixtures, diastereomeric mixtures, and optically pure forms. Optically active (R)- and (S)-isomers can be obtained using chiral synthons or chiral reagents or separated using standard techniques. If the compound contains a double bond, the substituent may be in the E or Z configuration. If the compound contains a disubstituted cycloalkyl, the cycloalkyl substituent may be in the cis or trans configuration. All tautomeric forms must also be included.

Any asymmetric atom (eg, carbon or the like) of the compound(s) of the present invention may be present in a racemic or enantiomerically enriched, eg (R)-, (S)-, or (R,S)-configuration. In some embodiments, each asymmetric atom has at least 50% enantiomeric excess, at least 60% enantiomeric excess, at least 70% enantiomeric excess, at least 80% enantiomeric excess, at least 90% enantiomeric excess, at least at least 95% enantiomeric excess or at least 99% enantiomeric excess in the (R)- or ^-configuration. Substituents on atoms with unsaturated double bonds may, if possible, be in the cis-(Z)- or trans-(E)-form.

Accordingly, as used herein, the compound of the present invention may be in the form of one of the possible isomers, rotamers, atropisomers, tautomers, or mixtures thereof, such as substantially pure geometric (cis or trans) isomers, diastereomers, optical isomers. (antipodes), racemates or mixtures thereof.

Any mixtures of isomers obtained can be separated on the basis of the physicochemical differences of the constituents into pure or essentially pure geometric or optical isomers, diastereomers, racemates, for example by chromatography and/or fractional crystallization.

Any resulting racemates of end products or intermediates may be resolved into optical antipodes by known methods, for example by separating their diastereomeric salts prepared with an optically active acid or base and releasing the optically active acidic or basic compound. In particular, the basic fragment can be used in this way to separate the compounds of the present invention into their optical antipodes, for example, by fractional crystallization of a salt formed with an optically active acid, for example, tartaric acid, dibenzoyltartaric acid, diacetyltartaric acid, di-O, O'-ptoluoyltartaric acid, mandelic acid, malic acid or camphor-10-sulfonic acid. Racemic products can also be separated using chiral chromatography, such as high performance liquid chromatography (HPLC) using a chiral adsorbent.

As used herein, the terms salt or salts refer to an acid addition or base addition salt of a compound of the invention. Salts include, in particular, pharmaceutically acceptable salts. The term pharmaceutically acceptable salts refers to salts which retain the biological efficacy and properties of the compounds of this invention and which are generally not biologically or otherwise undesirable. In many cases, the compounds of the present invention are capable of forming acidic and/or basic salts due to the presence of amino and/or carboxyl groups or the like.

Pharmaceutically acceptable acid addition salts can be formed with inorganic acids and organic acids, e.g. acetate, aspartate, benzoate, besylate, bromide/hydrobromide, bicarbonate/carbonate, bisulfate/sulfate, camphorsulfonate, chloride/hydrochloride, chlorotheophyllonate, citrate, ethanedisulfonate, fumarate , gluceptate, gluconate, glucuronate, hippurate, hydroiodide/iodide, isethionate, lactate, lactobionate, lauryl sulfate, malate, maleate, malonate, mandelate, mesylate, methyl sulfate, naphthoate, napsylate, nicotinate, nitrate, octadecanoate, oleate, oxalate, palmitate, pamoate , phosphate/hydrophosphate/dihydrogen phosphate, polygalacturonate, propionate, stearate, succinate, sulfosalicylate, tartrate, tosylate, and trifluoroacetate.

Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid.

- 13 040257 slot and the like.

Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, sulfosalicylic acid and the like. Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases.

Inorganic bases from which salts may be derived include, for example, ammonium and metal salts of Groups I-XII of the Periodic Table. In some embodiments, the salts are derived from sodium, potassium, ammonium, calcium, magnesium, iron, silver, zinc, and copper; most preferred are the ammonium, potassium, sodium, calcium and magnesium salts.

Organic bases from which salts may be derived include, for example, primary, secondary, and tertiary amines, substituted amines, including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like. Some organic amines include isopropylamine, benzathine, choline, diethanolamine, diethylamine, lysine, meglumine, piperazine, and tromethamine.

Pharmaceutically acceptable salts of the present invention can be synthesized from a basic or acid moiety using standard chemical methods. Typically, such salts can be prepared by reacting the free acid forms of these compounds with a stoichiometric amount of the appropriate base (such as Na, Ca, Mg or K hydroxide, carbonate, bicarbonate or the like) or by reacting the free base forms of these compounds with a stoichiometric amount of the corresponding acid. Such reactions are usually carried out in water, or in an organic solvent, or in a mixture of the two. Generally, the use of non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile is desirable when possible. Lists of additional useful salts can be found in, for example, Remington's Pharmaceutical Sciences, 20th ed., Mack Publishing Company, Easton, Pa., (1985); and in Handbook of Pharmaceutical Salts: Properties, Selection, and Use by Stahl and Wermuth (Wiley-VCH, Weinheim, Germany, 2002).

General methods for the synthesis of cationic lipids

The present invention also includes methods for preparing compounds of formula (I). In the reactions described, it may be necessary to protect reactive functional groups, for example hydroxyl, amino, imino, thio, or carboxy groups, where they are desired in the final product, to avoid their undesirable participation in the reactions.

The compounds and methods of the present invention will be better understood using the following synthetic schemes, which are only intended to illustrate the methods by which compounds can typically be prepared and are not intended to limit the scope of the invention as defined in the claims.

The final compounds of formula (I) can be obtained as described in Scheme I.

Scheme I

A compound of formula (I) can be prepared by reacting a compound of formula 2 with a compound of formula 3 using an appropriate reducing agent (eg sodium acetoxyborohydride and the like) and optionally a Lewis acid (eg titanium tetraisopropoxide and the like) in an appropriate solvent such as ethanol . The reaction can be carried out at room temperature to 80° C. and may take up to 24 hours to complete.

The compounds of formula (I) can also be obtained using the procedure as described in Scheme II.

Scheme II

A compound of formula (I) can be prepared by reacting a compound of formula 4 where Y is chlorine, bromine, iodine, mesyl, tosyl or another leaving group with a compound of formula 5 in DMF or other appropriate solvent at a temperature of 20 to 180° WITH.

The final compounds of formula (Ia) can be obtained as described in Scheme III.

- 14 040257

Scheme III

The compound of formula (Ia) can be prepared by reacting an alcohol of formula 6 with an acid of formula 7 in dichloromethane or other appropriate solvent using EDC or other appropriate coupling agent with an optional base or catalyst such as DMAP at a temperature of 20 to 150°C.

The compounds of formula 4 and 6 can be obtained from the corresponding precursor of formula 7 using methods known to the person skilled in the art, for example, as described in Scheme IV.

Scheme IV

A compound of formula 6 can be prepared by reacting a compound of formula 2 with sodium borohydride or other suitable reducing agent (eg, diisobutylaluminum hydride, lithium borohydride, etc.) in ethanol or other appropriate solvent at -20 to 150°C. The compound of formula 4 can be prepared from the compound of formula 6 by reaction with mesylanhydride or other appropriate activating agent (eg tosyl chloride, phosphorus oxychloride, etc.) in dichloromethane or other suitable solvent at -20 to 80°C.

Compounds of formula 2 can be prepared as described in Scheme V.

Scheme V

A compound of formula 2 can be prepared from a compound of formula 8 by reaction with a compound of formula 9 in the presence of DIAD or another appropriate diazo compound (e.g. DEAD etc.) with triphenylphosphine or another appropriate phosphine (e.g. trimethylphosphine) in dichloromethane or other appropriate solvent at a temperature of -20 to 50°C.

Alternatively, the compounds of formula 2 can be obtained in accordance with Scheme VI.

Scheme VI

A compound of formula 2 can be prepared from a compound of formula 8 by reacting with a compound of formula 10 where Y is a halogen, mesylate, or other suitable leaving group, in the presence of potassium carbonate or another appropriate base (e.g., cesium carbonate, potassium orthophosphate, etc.). e) in DMF or other suitable solvent at a temperature of 20 to 180°C. A compound of formula 10 can be prepared from a compound of formula 9 by reaction with mesyl chloride or another appropriate activating agent (eg tosyl chloride, phosphorus oxychloride, etc.) in the presence of pyridine or another appropriate base in dichloromethane or another appropriate solvent at a temperature of - 20 to 180°C.

Alternatively, the compounds of formula 2 can be obtained in accordance with Scheme VII.

Scheme VII

A compound of formula 2 can be prepared from a compound of formula 11 by reaction with a compound of formula 12 and an EDC or other appropriate coupling agent (e.g.,

DIC, HATU, etc.) in the presence of DMAP or other appropriate catalyst and DIEA or other appropriate base in dichloromethane or other appropriate solvent (eg DMF, DCE, etc.) at a temperature of 0 to 180°C.

Alternatively, the compounds of formula 2 can be obtained in accordance with Scheme VIII.

Scheme VIII

A compound of formula 2 can be prepared from a compound of formula 13 by reaction with a compound of formula 14 and EDC or other appropriate coupling agent (e.g. DIC, HATU, etc.) in the presence of DMAP or other appropriate catalyst and DIEA or other appropriate base in dichloromethane or other suitable solvent (eg DMF, DCE, etc.) at a temperature of from 0 to 180°C.

pKa for cationic lipids

The compounds of formula (I) are cationic lipids used to deliver biologically active agents to cells and tissues. It has been found that lipid formulations for the delivery of biologically active agents can be tailored to preferentially target one cell or organ type over another by changing only the cationic lipid in the formulation. For example, a cationic lipid with a pKa of about 5.1 to about 7.4 is generally effective when used in a liver delivery formulation. In one embodiment, the pKa of the cationic lipid is from about 5.1 to about 7.4 for liver delivery. In another embodiment, the pKa of the cationic lipid is from about 5.3 to about 7.0 for liver delivery. In another embodiment, the pKa of the cationic lipid is from about 5.3 to about 6.6 for liver delivery. For tumor delivery, a cationic lipid with a pKa of about 5.3 to about 6.4 is most effective when used in a formulation to deliver a biologically active agent to a tumor. Thus, in one embodiment, the pKa of the cationic lipid is from about 5.3 to about 6.4 for tumor delivery. In another embodiment, the pKa of the cationic lipid is from about 5.4 to about 6.2 for tumor delivery. In another embodiment, the pKa of the cationic lipid is from about 5.8 to about 6.1 for tumor delivery. For immunization purposes, a pKa of a cationic lipid is effective from 5.0 to 7.6, for example 5.7 to 5.9 (see WO 2012/006378).

Lipid compositions

The present invention provides a lipid composition containing at least one compound of formula (I), i. lipid composition of the invention. In one embodiment, at least one other lipid component is present. Such compositions may also contain a biologically active agent, optionally in combination with one or more other lipid components.

One embodiment of the present invention provides a lipid composition containing a compound of formula (I) and another lipid component. Such other lipid components include, but are not limited to, cationic lipids, neutral lipids, anionic lipids, helper lipids, and hidden lipids.

Cationic lipids suitable for use in the lipid composition of the invention include, but are not limited to, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearylN,N-dimethylammonium bromide (DDAB), N- (1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), N-(1-(2,3- dioleyloxy)propyl)-N,N,Ntrimethylammonium chloride (DOTMA), 1,2-dioleoylcarbamyl-3-dimethylammonium-propane (DOCDAP), 1,2dilineoyl-3-dimethylammonium-propane (DLINDAP), dilauryl (C _12:0 ) trimethylammonium propane (DLTAP), dioctadecylamidoglycylspermine (DOGS), DC-Choi, dioleoyloxy-N-(2-(sperminecarboxamido)ethyl)N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), 1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide (DMRIE), 3-dimethylamino-2-(cholest-5-en-3-beta-hydroxybutane-4-oxy)-1(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), N,N- dimethyl-2,3-dioleyloxy)propylamine (DODMA), 2-[5'-(cholest-5-en-3[beta]-oxy)-3'-oxapentoxy)-3-dimethyl-1-(cis,cis -9',12'-octadec dienoxy)propane (CpLinDMA), and N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), and 1,2-N,N'dioleycarbamyl-3-dimethylaminopropane (DOcarbDAP). In one embodiment, the cationic lipid is DOTAP or DLTAP.

Neutral lipids suitable for use in the lipid composition of the invention include, for example, various neutral, uncharged, or zwitterionic lipids. Examples of neutral phospholipids suitable for use in the present invention include, but are not limited to, 5-heptadecylbenzene-1,3-diol (resorcinol), cholesterol hemisuccinate (CHEMS), di

- 16 040257 palmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), phosphocholine (DOPC), dimyristoylphosphatidylcholine (DMPC), phosphatidylcholine (PLPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DAPC), phosphatidylethanolamine (PE), egg phosphatidylcholine (nPC), dilauriloylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), 1-myristoyl-2-palmitoylphosphatidylcholine (MPPC), 1-palmitoyl-2-myristoylphosphatidylcholine (PMPC), 1-palmitoyl-2-stearoylphosphatidylcholine (PSPC), 1,2-diaracidoyl-sn -glycero-3-phosphocholine (DBPC), 1-stearoyl-2-palmitoylphosphatidylcholine (SPPC), 1,2-diacosenoyl-sn-glycero-3-phosphocholine (DEPC), palmitoyloleoylphosphatidylcholine (POPC), lysophosphatidylcholine, dioleoylphosphatidylethanolamine (DOPE), dilinoleoylphosphatidylcholine, distearoylphosphatidylethanolamine (DSPE), dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPFE), palmitoyloleoylphosphatidylethanolamine (POFE), lysophosphatidylethanolamine and combinations thereof. In one embodiment, the neutral phospholipid is selected from the group consisting of distearoylphosphatidylcholine (DSPC) and dimyristoylphosphatidylethanolamine (DMPE).

Anionic lipids suitable for use in the present invention include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, and lysylphosphatidylglycerol.

Suitable neutral and anionic lipids also include those described in US 2009/0048197.

Helper lipids are lipids that enhance transfection to some extent (eg, transfection of a nanoparticle comprising a biologically active agent). The mechanism by which the helper lipid enhances transfection may include, for example, increased particle stability and/or increased membrane fusogenicity. Helper lipids include steroids and alkylresorcinols. Helper lipids suitable for use in the present invention include, but are not limited to, cholesterol, 5-heptadecylresorcinol, and cholesterol hemisuccinate.

Hidden lipids are lipids that increase the period of time that the nanoparticles can exist in vivo (eg, in blood). Hidden lipids suitable for use in the lipid composition of the invention include, but are not limited to, hidden lipids containing a hydrophilic head associated with a lipid moiety. Examples of such latent lipids include compounds of formula (XI) as described in WO 2011/076807,

or their salt or pharmaceutically acceptable derivative, where

Z is a hydrophilic head component selected from PEG and polymers based on poly(oxazoline), poly(ethylene oxide), poly(vinyl alcohol), poly(glycerol), poly(N-vinylpyrrolidone), poly[N-(2-hydroxypronyl)methacrylamide ], polysaccharides and poly(amino acids), in which the polymer may be linear or branched and in which the polymer may be optionally substituted;

in which Z is polymerized with n subunits;

n is a number average degree of polymerization from 10 to 200 Z units, where n is optimized for different types of polymers;

L1 is optionally substituted with a C ₁ _ ₁₀ alkylene or C _1-10 heteroalkylene linker including zero, one, two or more of an ether (e.g. -O-), an ester (e.g. C(O)O-), a succinate ( e.g. -O(O) C-CH ₂ -CH ₂ -C(O)O-)), carbamate (e.g. -OC(O)-NR'-), carbonate (e.g. -OC(O)O- ), ketone (e.g. -C-C(O)-C-), carbonyl (e.g. -C(O)-), urea (e.g. -NRC(O)NR'-), amine (e.g. -NR '-), amide (e.g. -C(O)NR'-), imine (e.g. -C(NR')-), thioether (e.g. -S-), xanthate (e.g. -OC(S)S -) and a phosphodiester (eg -OP(O) ₂ O-); any of which may be substituted with zero, one or more Z groups;

where R' is independently selected from -H, -NH-, -NH ₂ , -O-, -S-, phosphate or optionally substituted C ₁ _ ₁₀ alkylene;

X1 and _X2 are independently selected from carbon or a heteroatom selected from -NH-, -O-, -S- or phosphate;

A1 and A ₂ are independently selected from C ₆ - ₃₀ alkyl, C ₆ - ₃₀ alkenyl and C ₆ - ₃₀ alkynyl, where A1 and A ₂ may be the same or different, or where A1 and A ₂ together with the carbon atom to which they attached, form an optionally substituted steroid.

Certain hidden lipids include, but are not limited to, the lipids listed in Table. 3.

- 17 040257

Table 3 Hidden lipids

- 18 040257

- 19 040257

S018 Η- -Lo - n γγ S019 Η- Tw ⁰ '^'' -HoY— n S020 -ay- S021 -N'-- θ n / < > < > < '> <' \ 2 S022 n ⁰ \ / > > S023 ϋ O HE To S024 ]45O^ 0 ^-LoX- n S025 Ίο Lo^_ Ί23- at O Yc4? ''O J— '3^0 n S026 G ⁰ ' 14b S027 nH O S028 nH But- -ΝγΟ^-ο^, 0

- 20 040257

Other hidden lipids suitable for use in the lipid composition of the present invention and information on the biochemistry of such lipids can be found in Romberg et al., Pharmaceutical Research, Vol. 25, no. 1, 2008, p. 55-71 and Hoekstra et al., Biochimica et Biophysica Acta 1660 (2004) 41-52.

In one embodiment, a suitable hidden lipid contains a group selected from PEG (sometimes referred to as poly(ethylene oxide), and polymers based on poly(oxazoline), poly(vinyl alcohol), poly(glycerol), poly(P-vinylpyrrolidone), polyamino acids, and poly[K-(2hydroxypropyl)methacrylamide] Additional suitable PEG lipids are disclosed, for example, in WO 2006/007712.

Certain suitable hidden lipids include polyethylene glycol-diacylglycerol or polyethylene glycol-diacylglycamide (PEG-DAG) conjugates, including those containing a dialkylglycerol or dialkylglycamide group having an alkyl chain length independently containing from about C ₄ to about C ₄₀ saturated or unsaturated carbon atoms. The dialkylglycerol or dialkylglycamide group may additionally contain one or more substituted alkyl groups. In any of the embodiments described herein, the PEG conjugate may be selected from PEG-dilaurylglycerol, PEG-dimyristylglycerol (PEG-DMG) (catalog # GM-020 from NOF, Tokyo, Japan), PEG-dipalmitoylglycerol, PEG-disterylglycerol , PEG-dilaurylglycamide, PEG-dimyristylglycamide, PEG-dipalmitoylglycamide and PEG-disterylglycamide, PEG-cholesterol (1-[8'-(cholest-5-en-3[beta]oxy)carboxamido-3',6'-dioxaoctanyl] carbamoyl-[omega]-methyl-poly(ethylene glycol), PEG-DMB (3,4-ditradecoxybenzyl-[omega]-methylpoly(ethylene glycol) ether), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol)-2000] (catalog # 880150P from Avanti Polar Lipids, Alabaster, Alabama, USA).

In one embodiment, the latent lipid is S010, SO11, or S024.

In another embodiment, the hidden lipid is PEG-dimyristylglycerol (PEG-DMG).

Unless otherwise indicated, in the context of this description, the term PEG means any polymer of polyethylene glycol or other polyalkylene ether. In one embodiment, the PEG is an optionally substituted linear or branched polymer of ethylene glycol or ethylene oxide. In one embodiment, the PEG is unsubstituted. In one embodiment, the PEG is substituted, for example, with one or more alkyl, alkoxy, acyl, or aryl groups. In one embodiment, the term includes PEG copolymers such as PEG-polyurethane or PEG-polypropylene (see, for example, J. Milton Harris, Poly(ethylene glycol) chemistry: biotechnical and biomedical applications (1992)); in another embodiment, the term does not include PEG copolymers. In one embodiment, the PEG has a molecular weight of from about 130 to about 50,000, in a sub-embodiment from about 150 to about 30,000, in a sub-embodiment from about 150 to about 20,000, in a sub-embodiment from about 150 to about 15,000, in a sub-embodiment from at

- 21 040257 about 150 to about 10000, in a sub-embodiment from about 150 to about 6000, in a sub-embodiment from about 150 to about 5000, in a sub-embodiment from about 150 to about 4000, in a sub-embodiment from about 150 to about 3000, in about 300 to about 3000 in a sub-embodiment, about 1000 to about 3000 in a sub-embodiment, and about 1500 to about 2500 in a sub-embodiment.

In some embodiments, the PEG is PEG-2K, also referred to as PEG 2000, which has an average molecular weight of about 2000 daltons. PEG-2K is represented herein by the following formula (XIIa), in which n is 45, implying that the number average degree of polymerization is approximately 45 subunits. However, other PEG embodiments known in the art may be used, including, for example, those in which the number average degree of polymerization is approximately 23 subunits (n=23) and/or 68 subunits (n=68).

(XIIa)

The lipid compositions of the invention may also contain one or more biologically active agents, including, but not limited to, antibodies (for example, monoclonal, chimeric, humanized, nanobodies and their fragments, etc.), cholesterol, hormones, peptides, proteins, chemotherapeutic and other types of anticancer agents, small molecule drugs, vitamins, cofactors, nucleosides, nucleotides, oligonucleotides, enzymatic nucleic acids, antisense nucleic acids, triplex-forming oligonucleotides, antisense DNA or RNA compositions, chimeric DNA:RNA compositions, allozymes, aptamers, ribozymes, traps and their analogs, plasmids and other types of expression vectors, and small nucleic acid molecules, RNAi agents, short interfering nucleic acid (siNA) molecules, DSPC (siRNA), double-stranded RNA (dsRNA), micro-RNA (siRNA) ) and short hairpin RNA (shRNA), peptido nucleic acid (PNA), locked nucleic acid ribonucleotide ( LNA), morpholinonucleotide, threosonucleic acid (TNA), glycol nucleic acid (GNA), sisiRNA (short internally segmented interfering RNA), siRNA (asymmetric interfering RNA), and siRNA with 1, 2 or more mismatches between sense and antisense strands in the respective cells, and /or tissues, such as in a cell culture, object or organism. The lipid compositions of the invention may also include mRNA (messenger RNA) as a biologically active agent. Such compounds may be purified or partially purified, and may be naturally occurring or synthetic, and may be chemically modified. In one embodiment, the biologically active agent is an RNAi agent, a short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), miRNA (siRNA), or short hairpin RNA (shRNA) molecule. In one embodiment, the biologically active agent is an RNAi agent useful for mediating RNA interference (RNAi). In another embodiment, the biologically active agent is an siRNA agent. In another embodiment, the biologically active agent is an mRNA.

Various methods of loading biologically active agents into lipid compositions such as liposomes and lipid nanoparticles are available in the art, including both passive and active loading methods. The exact method used can be selected based on numerous factors, which include, but are not limited to, for example, the biologically active agent to be loaded, the method of storage to be used after loading, the size of the resulting particle, and the intended dosing regimen. Methods include, for example, mechanical mixing of the drug and lipids during formation or reconstitution of liposomes, dissolving all components in an organic solvent and concentrating them in a dry film, forming a pH or ion gradient to introduce the active agent into the interior of the liposome, creating a transmembrane potential, and ionophore-mediated loading. See, for example, PCT Publication WO 95/08986, US Patent No. 5837282, US Patent No. 5837282 and US Patent No. 7811602.

By lipid nanoparticle is meant a particle that contains a plurality (ie, more than one) of lipid molecules physically linked to each other by intermolecular forces. The lipid nanoparticles can be, for example, microspheres (including single-layer and multilayer vesicles, eg liposomes), dispersed phase in emulsion, micelles or dispersed phase in suspension.

The lipid nanoparticles have a size of from about 1 to about 2500 nm, from about 1 to about 1500 nm, from about 1 to about 1000 nm, in a sub-embodiment from about 50 to about 600 nm, in a sub-embodiment from about 50 to 400 nm, in a sub-embodiment, from about 50 to about 250 nm; and in a sub-embodiment, from about 50 to about 150 nm. If not specified

- 22 040257 otherwise, all dimensions mentioned in the present description are the average dimensions (diameters) of a fully formed nanoparticle, as measured using the dynamic light scattering method on the Malvern Zetasizer. A nanoparticle sample is diluted in phosphate buffered saline (PBS) such that the intensity is approximately 200-400 kimp. Data are presented as a weighted average of intensity measurements.

One embodiment of the present invention provides a lipid composition containing a compound of formula (I) and another lipid component. Another embodiment provides a lipid composition comprising a compound of formula (I) and a helper lipid such as cholesterol. Another embodiment provides a lipid composition comprising a compound of formula (I), a helper lipid such as cholesterol and a neutral lipid such as DSPC. Another embodiment of the present invention provides a lipid composition comprising a compound of formula (I), a helper lipid, eg cholesterol, a neutral lipid, eg DSPC, and a hidden lipid, eg PEG-DMG, S010, S011 or S024. Another embodiment of the present invention provides a lipid composition comprising a compound of formula (I), a helper lipid such as cholesterol, a neutral lipid such as DSPC, a hidden lipid such as PEG-DMG, S010, S011 or S024, and a biologically active agent, such as siRNA. Another embodiment of the present invention provides a lipid composition comprising a compound of formula (I), a helper lipid, such as cholesterol, a neutral lipid, such as DSPC, a hidden lipid, such as PEG-DMG, S010, S011, or S024, and a biologically active agent, such as , mRNA. Another embodiment of the present invention provides a lipid nanoparticle comprising a compound of formula (I), a helper lipid such as cholesterol, a neutral lipid such as DSPC, and a hidden lipid such as PEG-DMG, S010, S011 or S024, and a biologically active agent such as siRNA. Another embodiment of the present invention provides a lipid nanoparticle comprising a compound of formula (I), a helper lipid, such as cholesterol, a neutral lipid, such as DSPC, and a hidden lipid, such as PEG-DMG, S010, S011, or S024, and a biologically active agent, such as mRNA .

Embodiments of the present invention also provide lipid compositions described according to the respective molar ratios of the components of the lipids in the composition, in which the slash (/) denotes the respective components, as provided in the present description.

Another embodiment of the present invention is a lipid composition comprising a compound of formula (I) and a helper lipid, eg cholesterol, in a lipid molar ratio of 55-40 of the compound of formula (I)/55-40 of the helper lipid. Another embodiment provides a lipid composition comprising a compound of formula (I), a helper lipid such as cholesterol and a neutral lipid such as DSPC in a lipid molar ratio of 55-40 compound of formula (I)/55-40 helper lipid/15-5 neutral lipid. Another embodiment provides a lipid composition comprising a compound of formula (I), a helper lipid such as cholesterol, a neutral lipid such as DSPC and a hidden lipid such as PEG-DMG, S010, S011 or S024 in a lipid molar ratio of 55-40 of the compound formula (I)/55-40 helper lipid/15-5 neutral lipid/10-1 latent lipid.

Another embodiment of the present invention is a lipid composition comprising a compound of formula (I) and a helper lipid, eg cholesterol in a lipid molar ratio of 50-40 of the compound of formula (I)/50-40 of the helper lipid. Another embodiment provides a lipid composition comprising a compound of formula (I), a helper lipid such as cholesterol and a neutral lipid such as DSPC in a lipid molar ratio of 50-40 compound of formula (I)/50-40 helper lipid/15-5 neutral lipid. Another embodiment provides a lipid composition comprising a compound of formula (I), a helper lipid such as cholesterol, a neutral lipid such as DSPC, a hidden lipid such as PEG-DMG, S010, S011 or S024 in a lipid molar ratio of 50-40 of the compound of formula (I )/50-40 helper lipid/15-5 neutral lipid/5-1 latent lipid.

Another embodiment of the present invention is a lipid composition comprising a compound of formula (I) and a helper lipid, eg cholesterol, in a lipid molar ratio of 47-43 of the compound of formula (I)/47-43 of the helper lipid. Another embodiment provides a lipid composition comprising a compound of formula (I), a helper lipid such as cholesterol and a neutral lipid such as DSPC in a lipid molar ratio of 47-43 compound of formula (I)/47-43 helper lipid/12-7 neutral lipid. Another embodiment provides a lipid composition comprising a compound of formula (I), a helper lipid such as cholesterol, a neutral lipid such as DSPC, a hidden lipid such as PEG-DMG, S010, S011 or S024 in a lipid molar ratio of 47-43 of the compound of formula (I )/47-43 helper lipid/12-7 neutral lipid/4-1 hidden lipid.

Another embodiment of the present invention is a lipid composition comprising a compound of formula (I) and a helper lipid, such as cholesterol in a lipid molar ratio of about 45 of the compound of formula (x/about 44 of the helper lipid. Another embodiment provides a lipid composition containing a compound of formula (I), a helper lipid, e.g., cholesterol, and a neutral lipid, e.g., DSPC in a lipid molar ratio of about 45 compound of formula II/about 44 helper lipid/about 9 neutral lipid. I), a helper lipid, such as cholesterol, a neutral lipid, such as DSPC, an occult lipid, such as PEG-DMG, S010, S011, or S024 in a lipid molar ratio of approximately 45 compounds of the formula II/approximately 44 helper lipid/approximately 9 neutral lipid/ approximately 2 hidden lipid, for example PEG-DMG, S010, S011 or S024.

Preferred compounds of formula (I) for use in the above lipid compositions are shown in examples 38, 40, 41, 42, 43, 44, 47, 52, 62, 63, 92, 93, 94 and 112. Most preferred compounds are shown in examples 38 and 52. Preferred biologically active agents for use in the above lipid compositions are siRNAs. Another preferred biologically active agent for use in the above lipid compositions is mRNA.

The lipid compositions of the present invention can be further optimized by one of skill in the art by combining cationic lipids with a desired pKa range, hidden lipids, helper lipids, and neutral lipids in formulations including, for example, liposome formulations, lipid nanoparticle (LNP) formulations, and the like. , for delivery to specific cells and tissues in vivo. In one embodiment, further optimization is obtained by adjusting the lipid molar ratio between these different types of lipids. In one embodiment, further optimization is obtained by adjusting one or more of the desired particle size, N/P ratio, preparation methods, and/or dosing regimen (e.g., number of doses administered over time, actual dose in mg/kg, adjusting dosing time, combination with other therapeutic agents, etc.). Various optimization techniques known to those skilled in the art relating to the above embodiments are considered part of the present invention.

General methods for obtaining lipid nanoparticles

The following methods can be used to create lipid nanoparticles of the invention. To achieve size reduction and/or to improve particle size uniformity, one skilled in the art can use the process steps outlined below, experimenting with various combinations. In addition, a person skilled in the art can apply sonication, filtration, or other sizing techniques that are used in liposomal formulations.

The process for preparing a composition of the invention typically includes providing an aqueous solution, such as a citrate buffer, containing the biologically active agent in a first reservoir, providing a second reservoir containing an organic solution, such as an organic alcohol, such as ethanol, of the lipid(s), and then mixing the aqueous solution with organic lipid solution. The first reservoir is not necessarily in fluid communication with the second reservoir. The mixing step is optionally followed by an incubation step, a filtration step, and a dilution and/or concentration step. The incubation step includes keeping the solution from the mixing step in a vessel for about 0 to about 100 hours (preferably about 0 to about 24 hours) at about room temperature and optionally protected from light. In one embodiment, a dilution step follows an incubation step. The dilution step may include dilution with an aqueous buffer (eg citrate buffer), eg using a pump (eg a peristaltic pump). The filtration step is ultrafiltration. Ultrafiltration involves concentrating a dilute solution followed by diafiltration, for example, using a suitable pumping system (eg, a pump such as a peristaltic pump or equivalent) in combination with a suitable ultrafiltration membrane (eg, GE hollow fiber cartridges or equivalent).

In one embodiment, the mixing step provides a clear single phase.

In one embodiment, after the mixing step, the organic solvent is removed to obtain a suspension of particles in which the biologically active agent is encapsulated in a lipid(s), such as a lipid bilayer.

The choice of an organic solvent will typically include consideration of the polarity of the solvent and the ease with which the solvent can be removed in subsequent particle formation steps. The organic solvent, which is also used as a solubilizing agent, is preferably in an amount sufficient to provide a clear single phase mixture of biologically active agents and lipids. The organic solvent may be selected from one or

- 24 040257 more (for example, two) from chloroform, dichloromethane, diethyl ether, cyclohexane, cyclopentane, benzene, toluene, methanol and other aliphatic alcohols (for example, C ₁ -C ₈ ), such as ethanol, propanol, isopropanol, butanol, tert-butanol, isobutanol, pentanol and hexanol.

The mixing step may be carried out by any means, for example by mechanical means such as a vortex mixer.

The methods used to remove the organic solvent will typically include diafiltration, or evaporation under reduced pressure, or purging a stream of inert gas (eg, nitrogen or argon) through the mixture.

In other embodiments, the method further comprises adding non-lipid polycations that are suitable for effecting cell transformation using the present compositions. Examples of suitable non-lipid polycations include, but are limited to, hexadimethrin bromide (sold under the tradename POLYBREN® from Aldrich Chemical Co., Milwaukee, WI, USA) or other hexadimethrin salts. Other suitable polycations include, for example, salts of polyA-ornithine, poly-L-arginine, poly-L-lysine, poly-D-lysine, polyallylamine and polyethyleneimine. In some embodiments, the formation of lipid nanoparticles can be carried out either in a single phase system (eg single phase Bligh and Dyer or a similar mixture of aqueous and organic solvents) or a two phase system with suitable agitation.

The lipid nanoparticle can be formed in a one- or two-phase system. In a one-phase system, the cationic lipid(s) and biologically active agent are each dissolved in the volume of the one-phase mixture. Combining the two solutions provides a single mixture in which the complexes form. In a two-phase system, cationic lipids bind to the biologically active agent (which is present in the aqueous phase) and draw it into the organic phase. In one embodiment, lipid nanoparticles are produced by a method that includes: (a) reacting a biologically active agent with a solution containing non-cationic lipids and a detergent to form a compound-lipid mixture; (b) reacting the cationic lipids with the compound-lipid mixture to neutralize a portion of the negative charge of the biologically active agent and form a charge-neutralized mixture of biologically active agent and lipids; and (c) removing detergent from the charge-neutralized mixture.

In one group of embodiments, the solution of neutral lipids and detergent is an aqueous solution. The interaction of the biologically active agent with a solution of neutral lipids and detergent is usually carried out by mixing together the first solution of the biologically active agent and the second solution of lipids and detergent. Preferably, the biologically active agent solution is also a detergent solution. The amount of neutral lipid that is used in the present method is usually determined based on the amount of cationic lipid used, and is usually from about 0.2 to 5 times the amount of cationic lipid, preferably from about 0.5 to about 2 times the amount of cationic lipid used. .

The thus obtained mixture of biologically active agent-lipid interacts with cationic lipids to neutralize part of the negative charge that is associated with the molecule of interest (or other polyanionic materials). The amount of cationic lipid used is usually an amount sufficient to neutralize at least 50% of the negative charge of the biologically active agent. Preferably the negative charge will be at least 70% neutralized, more preferably at least 90% neutralized.

The methods used to remove the detergent typically include dialysis. When organic solvents are present, removal is usually accomplished by diafiltration or evaporation under reduced pressure, or by purging a stream of inert gas (eg, nitrogen or argon) through the mixture.

In the present description, a device for obtaining the composition of the present invention is disclosed. The device typically includes a first reservoir for storing an aqueous solution containing a biologically active agent and a second reservoir for storing an organic lipid solution. The device also typically includes a pump mechanism capable of pumping the aqueous and organic lipid solutions into the mixing area or mixing chamber at substantially the same flow rate. In one embodiment, the mixing area or mixing chamber includes a Tee or equivalent that allows aqueous and organic fluid streams to combine as input materials into the T-piece and the resulting combined aqueous and organic solutions exit the T-piece into a reservoir. to collect or its equivalent.

Methods for delivering biologically active agents and treating disease

The cationic lipids of formula (I) and their lipid compositions are useful in pharmaceutical compositions or formulations used to deliver biologically active agents. Formulations containing cationic lipids of formula (I) or lipid compositions thereof may be in various forms, including, but not limited to, particle delivery agents, including microparticles, nanoparticles, and transfection agents that are suitable for delivery of various molecules to cells. Certain formulations are effective in transfecting or delivering biologically active agents such as antibodies (e.g., monoclonal, chimeric, humanized, nanobodies and fragments thereof, etc.), cholesterol, hormones, peptides, proteins, chemotherapeutic and other types of anticancer agents, small molecule drugs, vitamins, cofactors, nucleosides, nucleotides, oligonucleotides, enzymatic nucleic acids, antisense nucleic acids, triplex-forming oligonucleotides, antisense DNA or RNA compositions, chimeric DNA:RNA compositions, allozymes, aptamers, ribozymes, traps and their analogues, plasmids and other types of expression vectors, and small nucleic acid molecules, RNAi agents, short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (siRNA) and short hairpin RNA (shRNA) molecules ), molecules of peptidonucleic acid (PNA), ribonucleotide of a locked nucleic acid acids (LNA), morpholinonucleotide, threosonucleic acid (TNA), glycol nucleic acid (GNA), sisiRNA (short internally segmented interfering RNA), siRNA (asymmetric interfering RNA), and siRNA with 1, 2 or more mismatches between sense and antisense strand in appropriate cells and/or tissues, such as in a cell culture, object or organism. The foregoing list of biologically active agents is exemplary only and is not intended to be limiting. For example, lipid formulations are also effective in transfecting or delivering mRNA agents into a cell. Such compounds may be purified or partially purified, and may be naturally occurring or synthetic, and may be chemically modified.

Such formulations containing biologically active agents are useful, for example, in providing compositions for the prevention, inhibition, or treatment of diseases, conditions, or traits in a cell, object, or organism. Diseases, conditions, or features include, but are not limited to, proliferative diseases including cancer, inflammatory disease, graft and/or tissue rejection, autoimmune disease or condition, age-related disease, neurological or neurodegenerative disease, respiratory disease, cardiovascular disease , eye disease, metabolic disease, dermatological disease, hearing disease, liver disease, kidney disease, etc.

The amount of active agent administered in a single dose is an amount above the minimum therapeutic dose but below the toxic dose. The actual amount per dose may be determined by the attending physician depending on a number of factors such as the patient's medical history, the use of other therapies, the biologically active agent being provided, and the nature of the disease. The amount of biologically active agent administered may be adjusted during treatment depending on the patient's response to treatment and the presence or severity of any treatment-related side effects. Approximate dosages and treatments for compounds that have been approved by the appropriate regulatory authority are known and available to those skilled in the art. See, for example, Physician's Desk Reference, 64th ed., Physician's Desk Reference Inc. (2010), Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa. (1985) and Remington The Science and Practice of Pharmacy, 21st ed., Lippincott Williams & Williams Publishers (2005).

In one embodiment, a single dose of a biologically active agent is administered to a patient in need of such treatment. In one embodiment, multiple doses are administered, wherein the multiple doses may be administered simultaneously, sequentially, or alternately. In one embodiment, the same formulation is administered in multiple doses. In one embodiment, the compositions differ by several doses. In various embodiments, the implementation of the dose can be administered once a day or for one, two, three, four or more consecutive days. In one embodiment, the doses are administered once a week. In one embodiment, doses are administered once every two weeks. In one embodiment, patients receive at least two treatments and potentially more depending on the patient's response to treatment. In single agent regimens, general courses of treatment are determined by the patient and the treating physician based on observed responses and toxicity. The above dosing regimens should be considered as non-limiting examples. Other dosing regimens are considered to be within the scope of the invention and depend on the desired therapeutic effect.

The invention also provides a method of treating a disease or condition, comprising the step of administering a therapeutically effective amount of a lipid composition of the invention to a patient in need of such treatment. In one embodiment, the disease or condition is treatable by administering a siRNA agent. In another embodiment, the disease or condition is treatable by administering an mRNA agent.

The invention also provides the use of the lipid composition of the invention for the treatment of a disease or condition in a patient. In one embodiment, the disease or condition is treatable by administering a siRNA agent. In another embodiment, the disease or condition is treatable by administering an mRNA agent.

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The total amount of lipids in the composition that is administered is in one embodiment from about 5 to about 30 mg of lipid per mg of biologically active agent (e.g., siRNA), in another embodiment from about 5 to about 25 mg of lipid per mg of biologically active agent (e.g., siRNA), in another embodiment, from about 7 to about 25 mg lipid per mg of biologically active agent (e.g., siRNA), and in one embodiment from about 7 to about 15 mg lipid per mg of biologically active agent (e.g., kiRNA).

In another embodiment, the total amount of lipids in the composition that is administered is from about 5 to about 30 mg lipid per mg of biologically active agent (e.g., mRNA), in another embodiment, from about 5 to about 25 mg lipid per mg of biologically active agent (e.g., mRNA), in another embodiment, from about 7 to about 25 mg of lipid per mg of biologically active agent (e.g., mRNA), and in one embodiment, from about 7 to about 15 mg of lipid per mg of biologically active agent (e.g., mRNA).

In the context of the present description, treatment includes ameliorative, radical and prophylactic treatment. In the context of the present description, the patient means an animal, preferably a mammal, preferably a human, in need of treatment.

The term "therapeutically effective amount" refers to the amount of a compound of the invention and a biologically active agent (eg, therapeutic compound) required to treat or alleviate the disease or condition of interest.

The term immunologically effective amount refers to the amount of a compound of the invention and RNA that encodes an immunogen necessary to elicit an immune response that recognizes the immunogen (eg, in relation to a pathogen). The term immunogen refers to any substance or organism that elicits an immune response when introduced into the body. An RNA phrase that encodes an immunogen refers to a polynucleotide, such as messenger RNA or a replicon, that a cell or organism is able to translate into a polypeptide according to the codon sequence of such RNA.

By proliferative disease as used herein is meant any disease, condition, trait, genotype, or phenotype characterized by unregulated cell growth or replication as is known in the art. In one embodiment, the proliferative disease is cancer. In one embodiment, the proliferative disease is a tumor. In one embodiment, the proliferative disease includes, but is not limited to, for example, liquid tumors such as, for example, leukemias, such as acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), acute lymphocytic leukemia (ALL), multiple myeloma, and chronic lymphocytic leukemia; and solid tumors, for example, AIDS-associated cancer such as Kaposi's sarcoma; mammary cancer; bone cancer; brain cancer; head and neck cancer, non-Hodgkin's lymphoma, adenoma, squamous cell carcinoma, laryngeal cancer, gallbladder and bile duct cancer, retinal cancer, esophageal cancer, gastrointestinal cancer, ovarian cancer, uterine cancer, thyroid cancer, testicular cancer, endometrial cancer, melanoma , colon and rectal cancer, lung cancer, bladder cancer, prostate cancer, lung cancer (including non-small cell lung carcinoma), pancreatic cancer, sarcomas, Wilms tumor, cervical cancer, head and neck cancer, skin cancer, nasopharyngeal carcinoma , liposarcoma, epithelial carcinoma, renal cell carcinoma, gallbladder adenocarcinoma, endometrial sarcoma, multidrug-resistant cancer. In one embodiment, the proliferative disease includes neovascularization associated with tumor angiogenesis, macular degeneration (eg, wet/dry age-related macular degeneration), corneal neovascularization, diabetic retinopathy, neovascular glaucoma, myopic degeneration. In one embodiment, the proliferative disease includes restenosis and polycystic kidney disease.

By autoimmune disease as used herein is meant any disease, condition, trait, genotype, or phenotype characterized by autoimmunity as is known in the art. Autoimmune diseases include, but are not limited to, for example, multiple sclerosis, diabetes mellitus, lupus, scleroderma, fibromyalgia, transplant rejection (eg, preventive effect against allograft rejection), pernicious anemia, rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, severe myasthenia gravis, lupus erythematosus, multiple sclerosis, and Graves' disease.

By infectious disease is meant any disease, disorder, or condition associated with an infectious agent, such as a virus, bacterium, fungus, prion, or parasite.

The invention can be used for immunization against pathogens that cause an infectious disease. Examples of such pathogens are given below.

By neurological disease is meant any disease, disorder or condition affecting the central or peripheral nervous system. Neurological diseases include, but are not limited to, diseases or disorders of either the peripheral or central nervous system, including, for example, Alzheimer's disease, aneurysm, brain injury, carpal tunnel syndrome, cerebral aneurysm, chronic pain, Creutzfeldt-Jakob disease , epilepsy, Huntington's chorea, meningitis, epilepsy and other neurological diseases, disorders and syndromes.

Respiratory disease refers to any disease or condition that affects the respiratory tract. Respiratory diseases include, but are not limited to, for example, asthma, chronic obstructive pulmonary disease (COPD), allergic rhinitis, sinusitis, allergic reactions, breathing difficulties, respiratory distress syndrome, cystic fibrosis, pulmonary hypertension or vasoconstriction, and emphysema.

By cardiovascular disease is meant a disease or condition that affects the heart and vascular system. Cardiovascular diseases include, but are not limited to, for example, coronary artery disease (CHD), cerebrovascular disease (CVD), aortic stenosis, peripheral vascular disease, myocardial infarction (heart attack), arrhythmias, and congestive heart failure.

By ocular disease as used herein is meant any disease, condition, trait, genotype or phenotype of the eye and associated structures. Eye diseases include, but are not limited to, for example, cystic macular edema, diabetic retinopathy, lattice degeneration, retinal vein occlusion, retinal artery occlusion, macular degeneration (for example, age-related macular degeneration such as wet AMD or dry AMD), toxoplasmosis, pigmentary retinitis, conjunctival rupture, corneal rupture, glaucoma and the like.

By metabolic disease is meant any disease or condition that affects metabolic pathways. A metabolic disease can result in an abnormal metabolic process, either congenital due to an inherited enzyme disorder (congenital metabolic disorder) or acquired due to endocrine organ disease or failure of a metabolically important organ such as the liver. In one embodiment, the metabolic disease includes obesity, insulin resistance, and diabetes (eg, type 1 and/or type 11 diabetes).

By dermatological disease is meant any disease or condition of the skin, dermis or any substructure therein, such as hair, follicle, etc. Dermatological diseases, disorders, conditions, and signs may include psoriasis, ectopic dermatitis, skin cancers such as melanoma and basal cell carcinoma, hair loss, hair removal, and changes in pigmentation.

By hearing disease is meant any disease or condition of the auditory system, including the ear, such as the inner ear, middle ear, outer ear, auditory nerve, and any substructures therein. Hearing disorders, disorders, conditions, and signs may include hearing loss, deafness, tinnitus, dizziness, balance, and incoordination.

As used herein, the term short interfering nucleic acid (snNA) refers to any nucleic acid molecule capable of inhibiting or repressing gene expression or viral replication by mediating RNA interference (RNAi) or gene silencing in a sequence-specific manner. It includes molecules of short interfering RNA (siRNA), miRNA (miRNA), short interfering oligonucleotides, and chemically modified short interfering nucleic acid. siRNAs are responsible for RNA interference, the process of sequence-specific post-transcriptional gene silencing in animals and plants. siRNAs are generated by ribonuclease III cleavage from a longer double-stranded RNA (dsRNA) that is homologous or specific to the target suppressed gene.

The term RNA interference (RNAi) is a post-transcriptionally targeted gene silencing technique that uses an RNAi agent to cleave messenger RNA (mRNA) containing a sequence that is the same as or similar to an RNAi agent. See: Zamore and Haley, 2005, Science, 309, 1519-1524; Zamore et al., 2000, Cell, 101, 25-33; Elbashir et al., 2001, Nature, 411, 494-498; and Kreutzer et al., PCT Publication WO 00/44895; Fire, PCT Publication WO 99/32619; Mello and Fire, PCT Publication WO 01/29058 and the like.

In the context of the present description, RNAi corresponds to other terms used to describe sequence-specific RNA interference, such as post-transcriptional gene silencing, translation inhibition, transcriptional inhibition, or epigenetics. For example, compositions containing lipids of the invention can be used in combination with kink molecules to epigenetically silence genes, either at the post-transcriptional level and/or pre-transcriptional level. In a non-limiting example, modulation of gene expression by kink molecules can result from kink-mediated RNA (either coding or non-coding RNA) cleavage by RISC, or alternatively translation inhibition, as is known in the art. In another embodiment, modulation of gene expression by kink may result from transcriptional inhibition, such as reported, for example, in Janowski et al., 2005, Nature Chemical Biology, 1, 216-222.

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The term RNAi inhibitor is any molecule that can suppress (eg, reduce or inhibit) the function or activity of RNA interference in a cell or patient. An RNAi inhibitor can suppress, reduce, or inhibit RNAi (e.g., RNAi-mediated cleavage of a target polynucleotide, inhibition of translation, or suppression of transcription) by interacting with or interfering with the function of any component of the RNAi pathway, including protein components such as RISCs or nucleic acid components such as like miRNA or siRNA. An RNAi inhibitor can be a siNA molecule, an antisense molecule, an aptamer, or a small molecule that interacts with or interferes with the function of a RISC, siRNA, or siRNA, or any other component of the RNAi pathway in a cell or patient. When inhibiting RNAi (eg, RNAi-mediated cleavage of a target polynucleotide, inhibition of translation or suppression of transcription), an RNAi inhibitor can be used to modulate (eg, activate or suppress) the expression of a target gene. In one embodiment, an RNA inhibitor is used to activate gene expression by interfering with (eg, reducing or preventing) endogenous suppression or inhibition of gene expression via translational inhibition, transcriptional inhibition, or RISC-mediated polynucleotide (eg, mRNA) cleavage. By interfering with the mechanisms of endogenous repression, silencing or inhibition of gene expression, the RNAi inhibitors of the invention can therefore be used to activate gene expression to treat diseases or conditions resulting from loss of function. The term RNAi inhibitor is used interchangeably with the term siRNA in various embodiments herein.

The term messenger ribonucleic acid (messenger RNA, mRNA) refers to the ribonucleic acid (RNA) molecule that mediates the transfer of genetic information to ribosomes in the cytoplasm, where it serves as the basis for protein synthesis. It is synthesized from template DNA during the transcription process. See The American Heritage® Dictionary of the English Language, Fourth Edition (Updated in 2009). Houghton Mifflin Company.

Ribonucleic acid (RNA) is a polymer of nucleotides linked by a phosphodiester bond, in which each nucleotide contains ribose or its modification as a sugar component. Each nucleotide contains adenine (A), guanine (G), cytosine (C), uracil (U) or their modifications as a base. The genetic information in an mRNA molecule is encoded in the sequence of nucleotide bases of the mRNA molecule, which are arranged in codons of three nucleotide bases each. Each codon codes for a specific amino acid of the polypeptide, with the exception of stop codons, which stop translation (protein synthesis). In a living cell, mRNA is transported to the ribosome, the site of protein synthesis, where it provides the genetic information for protein synthesis (translation). For a detailed description, see Alberts B et al. (2007) Molecular Biology of the Cell, Fifth Edition, Garland Science.

In eukaryotes, mRNA is transcribed in vivo in chromosomes by the cellular enzyme RNA polymerase. During or after in vivo transcription, a 5' cap (also called RNA cap, RNA-7methylguanosine cap, or m7G RNA cap) is added in vivo to the 5' end of the mRNA. The 5'-cap is a terminal 7-methylguanosine residue that is linked via a 5'-5'-triphosphate bond to the first nucleotide to be transcribed. In addition, most eukaryotic mRNA molecules have a polyadenylyl fragment (poly(A) tail) at the 3' end of the mRNA molecule. In vivo, the eukaryotic cell adds a poly(A) tail after transcription, often about 250 adenosine residues in length. Thus, a typical mature eukaryotic mRNA has a structure that starts at the 5' end of the mRNA with a nucleotide cap followed by a 5' untranslated region (5'UTR) of nucleotides, then an open reading frame that starts with an initiation codon that is the AUG triplet nucleotide bases, i.e. the coding sequence for the protein, and which ends with a stop codon, which can be a triplet of UAA, UAG or UGA nucleotide bases, then the 3' untranslated region (3'UTR) of nucleotides, and ending with a polyadenosine tail. Whereas typical mature eukaryotic mRNA features are naturally produced in the eukaryotic cell in vivo, the same or structurally and functionally equivalent features can be generated in vitro using molecular biology techniques. Accordingly, any RNA having a structure similar to a typical mature eukaryotic mRNA can function as mRNA and is within the scope of the term messenger ribonucleic acid.

The mRNA molecule is typically of a size at which it can be encapsulated in a lipid nanoparticle of the invention. While the size of an mRNA molecule varies in nature depending on the identification of the types of mRNA that codes for a particular protein, the average size of an mRNA molecule is 500-10,000 bases.

As used herein, the term enzymatic nucleic acid refers to a nucleic acid molecule that has complementarity in the substrate-binding region to a particular target gene and also has an enzymatic activity that acts to specifically cleave the target RNA while inactivating the target RNA molecule. Complementary

- 29 040257 plots provide sufficient hybridization of the enzymatic nucleic acid molecule to the target RNA and thus allow cleavage. Complementarity of 100% is preferred, but complementarity of only 50-75% may also be useful in the present invention (see, for example, Werner and Uhlenbeck, 1995, Nucleic Acids Research, 23, 2092-2096; Hammann et al., 1999, Antisense and Nucleic Acid Drug Dev., 9, 25-31). Nucleic acids may be modified in base, sugar and/or phosphate groups. The term enzymatic nucleic acid is used interchangeably with phrases such as ribozymes, catalytic RNA, enzymatic RNA, catalytic DNA, aptazyme or aptamer-binding ribozyme capable of regulating ribozyme, catalytic oligonucleotides, nucleozyme, DNAzyme, RNA enzyme, endoribonuclease, endonuclease, minizyme , lydzyme, oligozyme, or DNA enzyme. All of these terminologies describe nucleic acid molecules with enzymatic activity. The main features of an enzymatic nucleic acid molecule are that it has a specific substrate-binding site that is complementary to one or more target nucleic acid regions, and that it has nucleotide sequences within or surrounding that substrate-binding site that confer cleavage activity and /or ligation of a nucleic acid molecule (see, for example, Cech et al., US patent 4987071; Cech et al., 1988, 260 JAMA 3030). The ribozymes and enzymatic nucleic acid molecules of the invention may be chemically modified, for example, as described in the art and hereinafter.

As used herein, the term antisense nucleic acid refers to a non-enzymatic nucleic acid molecule that binds to a target RNA via RNARNA or RNA-DNA or RNA-PNA (peptide nucleic acid; Egholm et al., 1993 Nature 365, 566) interactions and alters the activity of the target RNA (for a review, see Stein and Cheng, 1993 Science 261, 1004 and Woolf et al., US Pat. No. 5,849,902). Antisense DNA can be chemically synthesized or expressed using a single stranded DNA expression vector or equivalent. The antisense molecules of the invention may be chemically modified, for example, as described in the art.

As used herein, the term RNase H activating region refers to a region (usually greater than or equal to 4-25 nucleotides in length, preferably 5-11 nucleotides in length) of a nucleic acid molecule capable of binding to a target RNA to form a non-covalent complex that is recognized the cellular enzyme RNase H (see, for example, Arrow et al. US Pat. No. 5,849,902; Arrow et al. US Pat. No. 5,989,912). The RNase H enzyme binds to the nucleic acid-target RNA complex and cleaves the target RNA sequence.

As used herein, the term 2-5A antisense chimera refers to an antisense oligonucleotide containing a 5'-phosphorylated 2'-5'-linked adenylate residue. These chimeras bind to the target RNA in a sequence-specific manner and activate the cellular 2-5A dependent ribonuclease, which in turn cleaves the target RNA (Torrence et al., 1993 Proc. Natl. Acad. Sci. USA 90, 1300; Silverman et al., 2000, Methods Enzymol., 313, 522-533; Player and Torrence, 1998, Pharmacol. Ther., 78, 55-113). Molecules 2-5A antisense chimera can be chemically modified, for example, as described in the art.

As used herein, the term triplex-forming oligonucleotides means an oligonucleotide that can bind to double-stranded DNA in a sequence-specific manner to form a three-stranded helix. Formation of such a triple helix structure has been shown to inhibit transcription of the target gene (Duval-Valentin et al., 1992 Proc. Natl. Acad. Sci. USA 89, 504; Fox, 2000, Curr. Med. Chem., 7, 17- 37; Praseuth et al., 2000, Biochim Biophys Acta 1489, 181-206). The triplex-forming oligonucleotide molecules of the invention may be chemically modified, for example, as described in the art.

As used herein, the term RNA decoy refers to an RNA molecule or aptamer that is designed to preferentially bind to a predetermined ligand. Such binding may result in inhibition or activation of the target molecule. An RNA decoy or aptamer can compete with a naturally occurring binding target for binding a specific ligand. Similarly, an RNA decoy may be designed to bind to a receptor and block binding of an effector molecule, or may be designed to bind to a receptor of interest and prevent interaction with the receptor. The trap molecules of the invention may be chemically modified, for example as described in the art.

In the context of the present description, the term single stranded DNA (ssDNA) refers to a naturally occurring or synthetic deoxyribonucleic acid molecule containing a linear single strand, for example, ssDNA can be the sense and antisense gene sequence or EST (expressed marker sequence).

In the context of the present description, the term allozyme refers to an allosteric enzymatic nucleic acid molecule, including, for example, US patent No. 5834186, 5741679, 5589332, 5871914 and PCT publication No. 29842.

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In the context of the present description, the term aptamer means a polynucleotide composition that specifically binds to a target molecule, in which the polynucleotide has a sequence that differs from the sequence normally recognized by the target molecule in the cell. On the other hand, an aptamer may be a nucleic acid molecule that binds to a target molecule, wherein the target molecule does not naturally bind to the nucleic acid. The target molecule can be any molecule of interest. The aptamer molecules of the invention may be chemically modified, for example as described in the art.

Composition of lipid compositions

For pharmaceutical use, the lipid compositions of the invention may be administered by enteral or parenteral routes, including intravenous, intramuscular, subcutaneous, transdermal, respiratory (aerosol), oral, intranasal, rectal, vaginal, buccal, nasopharyngeal, gastrointestinal, or sublingual administration. The introduction can be systemic or local. Topical administration may include, for example, catheterization, implantation, osmotic pump, direct injection, dermal/percutaneous application, stenting, ear/eye drops, or portal vein injection. The compounds of formula (I) must be evaluated with respect to their biopharmaceutical properties such as solubility and solution stability (via pH), permeability, etc., in order to select the most appropriate dosage form and route of administration for the treatment of the intended indication.

Compositions of the invention will usually, but not necessarily, be formulated in combination with one or more pharmaceutically acceptable excipients. The term excipient includes any ingredient other than the compound(s) of the invention, other lipid component(s), and the biologically active agent. The excipient can impart either functional (eg, control of drug release rate) and/or non-functional (eg, excipient or diluent) characteristics to the formulations. The choice of excipient will largely depend on factors such as the particular route of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form.

Typical pharmaceutically acceptable excipients include: diluents such as lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine; lubricants, for example, silicon dioxide, talc, stearic acid, its magnesium or calcium salt and/or polyethylene glycol; binders, for example magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone; disintegrants, for example starches, agar, alginic acid or its sodium salt, or effervescent mixtures; and/or absorbents, colorants, flavors and/or sweeteners.

The excipient may be an aqueous solution carrier which may optionally contain a buffer (eg PBS buffer) and/or sugar.

A comprehensive discussion of pharmaceutically acceptable excipients is available in Gennaro, Remington: The Science and Practice of Pharmacy 2000, 20th edition (ISBN: 0683306472).

Compositions of the invention may be administered orally. Oral administration may include swallowing, so that the compound enters the gastrointestinal tract and/or buccal, lingual or sublingual administration, by which the compound enters the bloodstream directly from the oral cavity.

Compositions of the invention may be administered parenterally. The compounds and compositions of the invention may be administered directly into the bloodstream, into subcutaneous tissue, into muscle, or into an internal organ. Suitable routes of administration include intravenous, intraarterial, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular, intraarticular and subcutaneous. Suitable devices for administration include needle (including microneedle) injectors, needleless injectors and infusion techniques.

Parenteral formulations are usually aqueous or oily solutions. When the solution is aqueous, excipients are such as sugars (including, but not limited to, glucose, mannitol, sorbitol, etc.), salts, carbohydrates, and buffering agents (preferably pH 3 to 9), but for some applications, they may more suitably be formulated as a sterile non-aqueous solution or as a dry form to be used in combination with a suitable vehicle such as sterile pyrogen-free water (WFI).

Parenteral formulations may include implants made from degradable polymers such as polyesters (ie, polylactic acid, polylactide, polylactide-co-glycolide, polycaprolactone, polyhydroxybutyrate), polyorthoesters, and polyanhydrides. These formulations may be administered through a surgical incision into subcutaneous tissue, muscle tissue, or directly into specific organs.

The preparation of parenteral formulations under sterile conditions, for example by lyophilization, can be readily accomplished using standard pharmaceutical techniques well known to those skilled in the art.

The solubility of compounds and compositions used in the preparation of parenteral solutions can be increased by the use of appropriate preparation techniques such as the inclusion of co-solvents and/or solubility enhancing agents such as surfactants, micellar structures and cyclodextrins.

The compositions of the invention can be administered intranasally or by inhalation, usually in the form of a dry powder (or alone, as a mixture, for example dry-mixed with lactose, or as mixed components, for example, mixed with phospholipids such as phosphatidylcholine) from a dry powder inhaler, in the form of an aerosol spray from a sealed container, pump, nebulizer, nebulizer (preferably an electrohydrodynamic nebulizer to produce a fine mist) or nebulizer with or without the use of a suitable propellant such as 1,1,1,2-tetrafluoroethane or 1,1,1,2,3,3,3-heptafluoropropane or in the form of nasal drops. For intranasal use, the powder may contain a bioadhesive agent such as chitosan or cyclodextrin.

The sealed container, pump, dispenser, nebulizer or nebulizer contains a solution or suspension of the compound(s) of the invention containing, for example, ethanol, aqueous ethanol or a suitable alternative agent for dispersing, solubilizing or prolonging the release of the compositions of the invention, the propellant(s) as solvent and an optional surfactant such as sorbitan trioleate, oleic acid, or oligolactic acid.

Prior to use in a dry powder or suspension formulation, the composition is finely ground to a size suitable for inhalation delivery (typically less than 5 microns). This can be achieved by any suitable grinding method such as spiral jet milling, fluidized bed jet milling, supercritical fluid treatment to form nanoparticles, high pressure homogenization, or spray drying.

Capsules (made, for example, from gelatin or hydroxypropyl methylcellulose), blisters and cartridges for use in an inhaler or insufflator may contain a powdered mixture of a compound or composition of the invention, a suitable powdered base such as lactose or starch and a performance modifier such as β-leucine, mannitol or magnesium stearate. The lactose may be anhydrous or in the form of a monohydrate, preferably the latter. Other suitable excipients include dextran, glucose, maltose, sorbitol, xylitol, fructose, sucrose and trehalose.

Formulations for inhalation/nasal administration can be prepared for immediate and/or modified release using, for example, PGLA. Modified release formulations include delayed, sustained, intermittent, controlled, targeted, and programmed release.

Suitable formulations for transdermal use include a therapeutically effective amount of a compound or composition of the invention with a carrier. Preferred carriers include easily absorbed, pharmacologically acceptable solvents to aid penetration through the skin of the host. Typically, transdermal devices are in the form of a dressing comprising a support member, a reservoir containing the compound, optionally admixed with carriers, an optional rate-controlling barrier to deliver the compound to the host's skin at a controlled and predetermined rate over an extended period of time, and means for delivery. devices into the skin.

The lipid compositions of the invention are administered by any of a number of routes, including parenteral, intravenous, systemic, topical, oral, intratumoral, intramuscular, subcutaneous, intraperitoneal, inhalation, or any such mode of delivery. In one embodiment, the compositions are administered parenterally, i. intraarticular, intravenous, intraperitoneal, subcutaneous or intramuscular. In a specific embodiment, the liposomal compositions are administered by intravenous infusion or intraperitoneally by bolus injection.

The lipid compositions of the invention may be formulated into pharmaceutical compositions suitable for delivery to a subject. Pharmaceutical compositions of the invention will often additionally contain one or more buffer solutions (for example, neutral buffered solution or phosphate buffered saline), carbohydrates (for example, glucose, mannose, sucrose, dextrose or dextrans), mannitol, proteins, polypeptides or amino acids, such as glycine, antioxidants, bacteriostatic agents, chelating agents such as EDTA or glutathione, adjuvants (eg aluminum hydroxide), solutes which render the formulation isotonic, hypotonic or mildly hypertonic with respect to the blood of the recipient, suspending agents, thickening agents and /or preservatives. Alternatively, the compositions of the present invention can be obtained in the form of a lyophilisate.

Suitable formulations for use in the present invention can be found, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17.sup.th Ed. (1985). Often, intravenous compositions will contain a solution of liposomes suspended in a suitable vehicle, such as an aqueous vehicle.

In one embodiment, this invention provides a pharmaceutical composition (i.e. formulation) comprising a lipid composition of the invention and a pharmaceutically acceptable

- 32 040257 carrier or excipient. In another embodiment, at least one other lipid component is present in the lipid composition. In another embodiment, the lipid composition is in the form of a liposome. In another embodiment, the lipid composition is in the form of a lipid nanoparticle. In another embodiment, the lipid composition is suitable for delivery to the liver. In another embodiment, the lipid composition is suitable for delivery to a tumor. In another embodiment, the biologically active agent is DNA or RNA. In another embodiment, the biologically active agent is a siRNA. In another embodiment, the biologically active agent is an mRNA.

For immunization purposes, the composition will typically be prepared as an injectable and will be administered by injection (eg, intramuscular injection).

The invention also provides a delivery device (eg, syringe, nebulizer, nebulizer, inhaler, skin patch, etc.) containing the composition of the invention. This device can be used to administer a pharmaceutical composition to an object, such as a human for immunization.

Cells and organs to which the invention is directed

The compounds, compositions, methods, and uses of the invention may be used to deliver a biologically active agent to one or more of the following in a patient: liver or liver cells (eg, hepatocytes); kidney or kidney cells; tumor or tumor cells; CNS or CNS (central nervous system, eg brain and/or spinal cord) cells; PNS or PNS cells (peripheral nervous system); lung or lung cells; the vascular system or cells of the vascular system; skin or skin cells (eg, dermal cells and/or follicular cells); eye or eye cells (eg, macula, fovea, cornea, retina); and ear or ear cells (eg, cells of the inner ear, middle ear, and/or outer ear).

The compounds, compositions, methods, and uses of the invention can also be used to deliver a biologically active agent (eg, RNA that encodes an immunogen) to cells of the immune system.

In one embodiment, the compounds, compositions, methods, and uses of the invention serve to deliver a biologically active agent to liver cells (eg, hepatocytes). In one embodiment, the compounds, compositions, methods, and uses of the invention serve to deliver a biologically active agent to a tumor or tumor cells (eg, primary tumor or metastatic cancer cells).

To deliver a biologically active agent to the liver or liver cells, in one embodiment, the composition of the invention interacts with the liver or liver cells of the patient, as is well known in the art, for example, through parenteral administration (for example, intravenous, intramuscular, subcutaneous administration) or topical administration (eg, direct injection, portal vein injection, catheterization, stenting) to facilitate delivery.

To deliver a biologically active agent to the kidney or cells of the kidney, in one embodiment, the composition of the invention interacts with the kidney or cells of the kidney of the patient, as is well known in the art, for example, through parenteral administration (for example, intravenous, intramuscular, subcutaneous administration) or topical administration (eg, direct injection, catheterization, stenting) to facilitate delivery.

To deliver a biologically active agent to a tumor or tumor cells, in one embodiment, the composition of the invention interacts with the tumor or tumor cells of the patient, as is generally known in the art, for example, through parenteral administration (for example, intravenous, intramuscular, subcutaneous administration) or topical administration (eg, direct injection, catheterization, stenting) to facilitate delivery.

To deliver a biologically active agent to the CNS or CNS cells (e.g., brain cells and/or spinal cord cells), in one embodiment, the composition of the invention interacts with the CNS or CNS cells (e.g., brain cells and/or spinal cord cells) of a patient , as is generally known in the art, for example, via parenteral administration (eg, intravenous, intramuscular, subcutaneous administration) or topical administration (eg, direct injection, catheterization, stenting, osmotic pump insertion (eg, intrathecal or ventricular)) to facilitate delivery.

To deliver a biologically active agent to the PNS or PNS cells, in one embodiment, the composition of the invention interacts with the PNS or PNS cells of a patient, as is generally known in the art, for example, via parenteral administration (eg, intravenous, intramuscular, subcutaneous administration) or topical administration. (eg direct injection) to facilitate delivery.

To deliver the biologically active agent to the lung or lung cells, in one embodiment, the composition of the invention interacts with the lung or cells of the lung of the patient, as is well known in the art, for example, through parenteral administration (for example, intravenous, intramuscular, subcutaneous administration) or topical administration (for example, inhalation

- 33 040257 directly into lung tissue and cells) to facilitate delivery.

To deliver a biologically active agent to the vascular system or cells of the vascular system, in one embodiment, the composition of the invention interacts with the vascular system or cells of the vascular system of the patient, as is well known in the art, for example, through parenteral administration (for example, intravenous, intramuscular, subcutaneous administration ) or topical (eg, clamp, cath, stent) to facilitate delivery.

To deliver the biologically active agent to the skin or skin cells (e.g., dermis and/or follicular cells), in one embodiment, the composition of the invention interacts with the skin or skin cells (e.g., dermis and/or follicular cells) of a patient, as is commonly known in art, for example, through parenteral administration (eg, intravenous, intramuscular, subcutaneous administration) or topical administration (eg, direct application to the skin, iontophoresis) to facilitate delivery.

To deliver a biologically active agent to the eye or eye cells (eg, macula, fovea, cornea, retina), in one embodiment, the composition of the invention interacts with the eye or eye cells (eg, macula, fovea, cornea, retina) of a patient, as commonly known in the art, for example, via parenteral administration (eg, intravenous, intramuscular, subcutaneous administration) or topical administration (eg, direct injection, intraocular injection, periocular injection, subretinal administration, iontophoresis, eye drops, implants) to facilitate delivery.

To deliver the biologically active agent to the ear or ear cells (e.g., cells of the inner ear, middle ear, and/or outer ear), in one embodiment, the composition of the invention interacts with the ear or ear cells (e.g., cells of the inner ear, middle ear, and/or external ear) of the patient, as is generally known in the art, for example, via parenteral administration (eg, intravenous, intramuscular, subcutaneous administration) or topical administration (eg, direct injection) to facilitate delivery.

To deliver a biologically active agent (e.g., an RNA encoding an immunogen) to cells of the immune system (e.g., antigen-presenting cells, including professional antigen-presenting cells), in one embodiment, the composition of the invention is delivered intramuscularly, after which immune cells can enter the delivery site and process the delivered RNA. Such immune cells may include macrophages (eg, bone marrow-derived macrophages), dendritic cells (eg, bone marrow-derived plasmacytoid dendritic cells and/or bone marrow-derived myeloid dendritic cells), monocytes (eg, human peripheral blood monocytes), and etc. (for example, see WO 2012/006372).

Immunization according to the invention

For immunization purposes, the invention includes the delivery of an RNA that encodes an immunogen. The immunogen elicits an immune response that recognizes the immunogen and can therefore be used to confer immunity against a pathogen, or against an allergen, or against a tumor antigen. Immunization against disease and/or infection caused by a pathogen is preferred.

The RNA is delivered with a lipid composition of the invention (eg, with a liposome or LNP), and typically the invention uses liposomes or LNP into which the immunogen-coding RNA is encapsulated. Encapsulation in liposomes can protect RNA from RNase cleavage.

In one embodiment, the invention provides a liposome having a lipid bilayer encapsulating an aqueous core, wherein: (i) the lipid bilayer comprises a lipid of the invention; and (ii) the water core includes RNA that encodes the immunogen. If the composition contains a population of liposomes with different diameters, it may be useful for immunization purposes if (i) at least 80% of the number of liposomes have diameters in the range from 60 to 180 nm and preferably in the range from 80 to 160 nm and/or (ii) the average diameter (by intensity, eg Z-mean) of the population is in the range of 60 to 180 nm and preferably in the range of 80 to 160 nm. The diameters within the set should ideally have a polydispersity factor of <0.2.

Following in vivo administration of the immunization composition, the delivered RNA is released and translated in the cell to provide the immunogen in situ. RNA is plus (+) stranded so that it can be translated by cells without the need for any intermediate steps of replication such as reverse transcription. It can also bind to TLR7 receptors expressed by immune cells, thus initiating an adjuvant effect.

Preferred plus (+)-stranded RNAs are self-replicating. A self-replicating RNA molecule (replicon), when delivered into a vertebrate cell, even without any proteins, can lead to the production of multiple daughter RNAs by transcription from itself (through the antisense copy it generates from itself). A self-replicating RNA molecule is thus usually a ± stranded molecule that can be directly translated

- 34 040257 after delivery to the cell, and this translation produces an RNA-dependent RNA polymerase, which then produces both antisense and sense transcripts from the delivered RNA. Thus, the delivered RNA leads to the formation of many daughter RNAs. These progeny RNAs, as well as collinear subgenomic transcripts, can be translated on their own to allow for in situ expression of the encoded immunogen, or can be transcribed to provide additional transcripts with the same meaning as the delivered RNA, which is translated to allow for in situ expression. immunogen. The overall result of this transcription sequence is a huge amplification in the number of RNA replicons introduced, and thus the encoded immunogen becomes the major polypeptide product of the cells.

One suitable system for achieving self-replication is the use of an alpha virus-based RNA replicon. These ± thread replicons are translated after delivery into the cell to produce a replicase (or replicase-transcriptase). The replicase is translated as a polyprotein that self-cleaves to provide a replication complex that creates genomic strand copies ± strand delivered RNA. These .beta.-stranded transcripts can themselves be transcribed to make additional copies of the +/- stranded parental RNA and also to produce a subgenomic transcript that encodes the immunogen. Translation of the subgenomic transcript thus results in in situ expression of the immunogen by the infected cell. Suitable alphavirus replicons can use replicase from Sindbis virus, Semliki forest virus, Eastern equine encephalitis virus, Venezuelan equine encephalitis virus, etc. Mutant or wild type virus sequences can be used, for example an attenuated TC83 VEEV mutant has been used in replicons.

The preferred self-replicating RNA molecule thus encodes (i) an RNA-dependent RNA polymerase that can transcribe RNA from the self-replicating RNA molecule and (ii) an immunogen. The polymerase may be an alpha virus replicase, for example, comprising one or more of the alpha virus proteins nsP1, nsP2, nsP3 and nsP4.

While natural alpha virus genomes encode structural virion proteins in addition to the non-structural replicase polyprotein, it is preferred that the self-replicating RNA molecule of the invention does not encode alpha virus structural proteins. Thus, a preferred self-replicating RNA can result in the production of genomic RNA copies of itself in the cell, but not in the production of RNA-containing virions. The inability to obtain these virions means that, unlike the wild-type alpha virus, a self-replicating RNA molecule cannot maintain itself in an infectious form. Structural alpha virus proteins that are essential for persistence in wild-type viruses are absent from the self-replicating RNAs of the invention and their position is taken from the gene(s) encoding the immunogen of interest, such that the subgenomic transcript encodes the immunogen and not the structural virion alpha virus proteins.

Thus, a suitable self-replicating RNA molecule of the invention may have two open reading frames. The first (5') open reading frame encodes a replicase; the second (3') open reading frame encodes the immunogen. In some embodiments, the RNA may have additional (eg, downstream) open reading frames, eg, to encode additional immunogens (see below) or to encode additional polypeptides.

The self-replicating RNA molecule may have a 5' sequence that is compatible with the encoded replicase.

Self-replicating RNA molecules can be of various lengths, but are typically 5000-25000 nucleotides in length, eg 8000-15000 nucleotides or 9000-12000 nucleotides. Thus, RNAs are longer than those observed during siRNA delivery.

The RNA molecule may have a 5' cap (eg 7-methylguanosine). This cap can enhance RNA translation in vivo.

Suitable 5'-nucleotide of the RNA molecule according to the invention may have a 5'-triphosphate group. In capped RNAs, it can be linked to 7-methylguanosine via a 5'-5' bridge. 5'-triphosphate can enhance RIG-I binding and thus activate adjuvant effects.

An RNA molecule may have a 3' poly(A) tail. It may also include a poly(A) polymerase recognition sequence (eg, AAUAAA) near its 3' end.

A suitable RNA molecule of the invention for immunization purposes will generally be single stranded. Single-stranded RNAs can usually initiate an adjuvant effect by binding to TLR7, TLR8, RNA helicases and/or PKRs. The delivered double-stranded RNA (dsRNA) can bind to TLR3, and this receptor can also be activated by dsRNA, which is produced either during the replication of the single-stranded RNA or within the secondary structure of the single-stranded RNA.

RNA molecules for immunization purposes can be easily obtained using in vitro transcription (IVT). IVT may use engineered template (cDNA) and disseminated in the form of plasmids in bacteria, or synthetically produced (for example, through gene synthesis and/or engineering).

- 35 040257 methods of polymerase chain reaction (PCR)). For example, a DNA dependent RNA polymerase (such as bacteriophage T7, T3 or SP6 RNA polymerase) can be used to transcribe RNA from messenger DNA. Appropriate capping and poly-A addition reactions can be used as needed (although the replicon poly-A is usually encoded in template DNA). These RNA polymerases may have stringent requirements for the 5' nucleotide(s) to be transcribed, and in some embodiments, these requirements must match those of the encoded replicase to ensure that the IVT-transcribed RNA can effectively function as a substrate for its self-encoding replicase. .

As discussed in WO 2011/005799, a self-replicating RNA may include (in addition to any 5' cap structure) one or more nucleotides having a modified nucleobase. For example, the self-replicating RNA may include one or more modified pyrimidine nucleobases such as pseudouridine and/or 5-methylcytosine residues. In some embodiments, however, the RNA contains unmodified nucleobases and may include unmodified nucleotides, ie. all nucleotides in RNA are standard A, C, G, and U ribonucleotides (with the exception of any 5'-cap structure, which may include 7'-methylguanosine). In other embodiments, the RNA may include any 5' cap containing 7'-methylguanosine and the first 1, 2, or 3 5' ribonucleotides may be methylated at the 2' position of the ribose.

The RNA used in the invention for immunization purposes ideally includes only phosphodiester linkages between nucleosides, but in some embodiments it may contain phosphoramidate, phosphorothioate and/or methylphosphonate linkages.

The amount of RNA in a liposome can vary. The number of individual self-replicating RNA molecules in a liposome is typically <50, such as <20, <10, <5 or 1-4 per liposome.

The RNA molecules used in the invention for immunization purposes encode a polypeptide immunogen. Once administered, the RNA is translated in vivo and the immunogen can elicit an immune response in the recipient. An immunogen may elicit an immune response against a pathogen (eg, a bacterium, virus, fungus, or parasite), but in some embodiments, it elicits an immune response against an allergen or tumor antigen. The immune response may include a humoral immune response (usually including IgG) and/or a cell-mediated immune response. The polypeptide immunogen will typically elicit an immune response that recognizes the corresponding pathogen (or allergen, or tumor) polypeptide, but in some embodiments, the polypeptide may act as a mimotope to elicit an immune response that recognizes the saccharide. The immunogen will typically be a surface polypeptide, such as adhesion, hemagglutinin, envelope glycoprotein, spike glycoprotein, etc.

An RNA molecule may encode a single polypeptide immunogen or multiple polypeptides. Numerous immunogens can be presented as a single polypeptide immunogen (combined polypeptide) or as individual polypeptides. If the immunogens are expressed as individual polypeptides from the replicon, then one or more of them may have a 3'5' direction of the IRES or an additional viral promoter element. In addition, multiple immunogens can be expressed from a polyprotein that encodes individual immunogens coupled to a short autocatalytic protease (eg FMDV protein 2A) or as inteins.

In some embodiments, the immunogen elicits an immune response against one of these bacteria:

Neisseria meningitidis: Suitable immunogens include, but are not limited to, membrane proteins such as adhesins, autotransporters, toxins, iron binding proteins, and factor H binding protein. A combination of three suitable polypeptides is disclosed in Giuliani et al. (2006) Proc Natl Acad Sci USA 103(29): 10834-9.

Streptococcus pneumoniae: Useful polypeptide immunogens are disclosed in WO 2009/016515. These include, but are not limited to, RrgB pilus subunit, beta-M-acetylhexosaminidase precursor (spr0057), spr0096, total stress protein GSP-781 (spr2021, SP2216), serine/threonine kinase StkP (SP1732), and pneumococcal surface adhesion PsaA.

Streptococcus pyogenes: Suitable immunogens include, but are not limited to, the polypeptides disclosed in WO 02/34771 and WO 2005/032582.

Moraxella catarrhalis

Bordetella pertussis: Suitable pertussis immunogens include, but are not limited to, pertussis toxin or toxoid (PT), filamentous haemagglutinin (FHA), pertactin, and agglutinogens 2 and 3.

Staphylococcus aureus: Suitable immunogens include, but are not limited to, polypeptides disclosed in WO 2010/119343 such as hemolysin, esxA, esxB, ferrichrome binding protein (sta006) and/or sta011 lipoprotein.

Clostridium tetani: A typical immunogen is tetanus toxoid.

- 36 040257

Cornynebacterium diphtheriae: A typical immunogen is a diphtheria toxoid.

Haemophilus influenzae: Suitable immunogens include, but are not limited to, the polypeptides disclosed in WO 2006/110413 and WO 2005/111066.

Pseudomonas aeruginosa

Streptococcus agalactiae: Suitable immunogens include, but are not limited to, the polypeptides disclosed in WO02/34771.

Chlamydia trachomatis: Suitable immunogens include, but are not limited to, PepA, LcrE, ArtJ, DnaK, CT398, OmpH-like, L7/L12, OmcA, AtoS, CT547, Eno, HtrA, and MurG (for example, as disclosed in WO 2005/ 002619). LcrE (WO2006/138004) and HtrA (WO 2009/109860) are two preferred immunogens.

Chlamydia pneumoniae: Suitable immunogens include, but are not limited to, the polypeptides disclosed in WO 02/02606.

Helicobacter pylori: Suitable immunogens include, but are not limited to, CagA, VacA, NAP and/or urease (WO 03/018054).

Escherichia coli: Suitable immunogens include, but are not limited to, those derived from enterotoxigenic E. coli (ETEC), enteroaggregating E. coli (EAggEC), diffusely adherent E. coli (DAEC), enteropathogenic E. coli (EPEC), extraintestinal pathogenic E. coli (ExPEC) and/or enterohemorrhagic E. coli (EHEC). ExPEC strains include uropathogenic E. coli (UPEC) and meningitis/sepsis associated E. coli (MNEC). Suitable UPEC immunogens are disclosed in WO 2006/091517 and WO 2008/020330. Suitable MNEC immunogens are disclosed in WO 2006/089264. A suitable immunogen for some types of E. coli is AcfD (WO 2009/104092).

Bacillus anthracis

Yersinia pestis: Suitable immunogens include, but are not limited to, those disclosed in WO 2007/049155 and WO 2009/031043.

Staphylococcus epidermis

Clostridium perfringens or Clostridium botulinums

Legionella pneumophila

Coxiella burnetii

Brucella such as B. abortus, B. canis, B. melitensis, B. neotomae, B. ovis, B. suis, B. pinnipediae.

Francisella such as F. novicida, F. philomiragia, F. tularensis.

Neisseria gonorrhoeae

Treponema pallidum

Haemophilus ducreyi

Enterococcus faecalis or Enterococcus faecium

Staphylococcus saprophyticus

Yersinia enterocolitica

Mycobacterium tuberculosis

Rickettsia

Listeria monocytogenes

Vibrio cholerae

Salmonella Typhi

Borrelia burgdorferi

Porphyromonas gingivalis

Klebsiella

In some embodiments, the immunogen elicits an immune response against one of these viruses:

Orthomyxovirus: Suitable immunogens can be derived from influenza A, B or C, including hemagglutinin, neuraminidase, or M2 matrix proteins. Where the immunogen is influenza A hemagglutinin, it may be from any subtype, for example, H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, or H16.

Paramyxoviruses: Immunogens include, but are not limited to, those derived from pneumoviruses (eg, respiratory syncytial virus, RSV), rubulaviruses (eg, mumps virus), paramyxoviruses (eg, parainfluenza virus), metapneumoviruses, and morbilliviruses (eg, measles virus) .

Poxviruses: Immunogens include, but are not limited to, those derived from orthopoxvirus such as variola, including but not limited to Variola major and Variola minor.

Picornavirus: Immunogens include, but are not limited to, those derived from picornaviruses such as enteroviruses, rhinoviruses, heparnaviruses, cardioviruses, and aphthoviruses. In one embodiment, the enterovirus is a poliovirus, eg, type 1, type 2, and/or type 3 poliovirus. In another embodiment, the enterovirus is an EV71 enterovirus. In another embodiment, the enterovirus is coxsackievirus A or B.

Bunyavirus: Immunogens include, but are not limited to, those derived from orthobunyavirus,

- 37 040257 such as California encephalitis virus, phlebovirus such as Rift Valley fever virus or nairovirus such as Crimean Congo hemorrhagic fever virus.

Heparnavirus: Immunogens include, but are not limited to, those derived from a heparnavirus such as hepatitis A virus (HAV).

Filovirus: Immunogens include, but are not limited to, derived from filovirus such as Ebola virus (including Zaire, Ivory Coast, Reston or Sudan ebolavirus) or Marburg virus.

Togavirus: Immunogens include, but are not limited to, those derived from togavirus such as rubivirus, alphavirus, or arterivirus. It includes the rubella virus.

Flavivirus: Immunogens include, but are not limited to, those derived from a flavivirus such as tick-borne encephalitis virus (TBEV), Dengue virus (type 1, 2, 3, or 4), yellow fever virus, Japanese encephalitis virus, Kyasanur forest disease virus, West Nile virus, Saint Louis encephalitis virus, Russian spring-summer encephalitis virus, Powassan encephalitis virus.

Pestivirus: Immunogens include, but are not limited to, those derived from a pestivirus such as bovine diarrhea virus (BVDV), classical swine fever (CSF), or border disease of sheep (PBO).

Hepadnavirus: Immunogens include, but are not limited to, derived from hepadnavirus, such as hepatitis B virus. The composition may include hepatitis B surface antigen (HBsAg).

Other Hepatitis Viruses: The composition may include an immunogen from hepatitis C virus, hepatitis delta virus, hepatitis E virus, or hepatitis G virus.

Rhabdovirus: Immunogens include, but are not limited to, those derived from rhabdovirus such as lyssavirus (eg, rabies virus) and vesiculovirus (VSV).

Calicivirus: Immunogens include, but are not limited to, those derived from calicivirus such as Norwalk virus (norovirus) and Norwalk-like viruses such as Hawaii virus and Snow Mountain virus.

Coronavirus: Immunogens include, but are not limited to, those derived from SARS coronavirus, avian infectious bronchitis (AIB), murine hepatitis virus (MHV), and porcine transmissible gastroenteritis virus (TGV). The coronavirus immunogen may be a spike polypeptide.

Retrovirus: Immunogens include, but are not limited to, those derived from an oncovirus, a lentivirus (eg, HIV-1 or HIV-2), or a spumavirus.

Reovirus: Immunogens include, but are not limited to, those derived from orthoreovirus, rotavirus, orbivirus, or coltivirus.

Parvovirus: Immunogens include, but are not limited to, those derived from parvovirus B19.

Herpesvirus: Immunogens include, but are not limited to, those derived from human herpesvirus, such as, by way of example only, herpes simplex virus (HSV) (e.g., HSV types 1 and 2), varicella-zoster virus (VZV), Epstein-Barr virus (EBV ), cytomegalovirus (CMV), human herpesvirus type 6 (HHV-6), human herpesvirus type 7 (HHV-7), and human herpesvirus type 8 (HHV-8).

Papovaviruses: Immunogens include, but are not limited to, those derived from papillomaviruses and polyomaviruses. The papillomavirus (human) virus can be serotype 1, 2, 4, 5, 6, 8, 11, 13, 16, 18, 31, 33, 35, 39, 41, 42, 47, 51, 57, 58, 63, or 65, such as one or more serotypes 6, 11, 16 and/or 18.

Adenovirus: Immunogens include those derived from serotype 36 (Ad-36).

In some embodiments, the immunogen elicits an immune response against a virus that infects fish, such as infectious salmon anemia virus (ISAV), salmon pancreatic disease virus (SPDV), infectious pancreatic necrosis virus (IPNV), channel catfish virus (CCV ), fish lymphocystosis virus (FLDV), salmon infectious hematopoietic tissue necrosis virus (IGTL), koi herpes, salmon picorna-like virus (also known as Atlantic salmon picorna-like virus), freshwater salmon virus (LSV), Atlantic salmon rotavirus ( ASR), rainbow trout scale disease virus (TSD), oncogenic coho salmon virus (CSTV), or viral hemorrhagic septicemia (HCV).

Fungal immunogens can be derived from dermatophytes, including Epidermophyton floccusum, Microsporum audouini, Microsporum canis, Microsporum distortum, Microsporum equinum, Microsporum gypsum, Microsporum nanum, Trichophyton concentricum, Trichophyton equinum, Trichophyton gallinae, Trichophyton gypseum, Trichophyton megnini, Triteschophyton, chophyton mentagro , Trichophyton rubrum, Trichophyton schoenleini, Trichophyton tonsurans, Trichophyton verrucosum, T. verrucosum var. album, var. discoides, var. ochraceum, Trichophyton violaceum and/or Trichophyton faviforme; or from Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus nidulans, Aspergillus terreus, Aspergillus sydowi, Aspergillus flavatus, Aspergillus glaucus, Blastoschizomyces capitatus, Candida albicans, Candida enolase, Candida tropicalis, Candida glabrata, Candida krusei, Candida parapsilosis Can- 38 040257 dida kusei, Candida parakwsei, Candida lusitaniae, Candida pseudotropicalis, Candida guilliermondi, Cladosporium carrionii, Coccidioides immitis, Blastomyces dermatidis, Cryptococcus neoformans, Geotrichum clavatum, Histoplasma capsulatum, Klebsiella pneumoniae, Microsporiata, Encephalitoziodia, and Encephalitozoidia beneusi; наименее распространенными являются Brachiola spp, Microsporidium spp., Nosema spp., Pleistophora spp., Trachipleistophora spp., Vittaforma spp Paracoccidioides brasiliensis, Pneumocystis carinii, Pythiumn insidiosum, Pityrosporum ovale, Sacharomyces cerevisae, Saccharomyces boulardii, Saccharomyces pombe, Scedosporium apiosperum, Sporothrix schenckii , Trichosporon beigelii, Toxoplasma gondii, Penicillium marneffei, Malassezia spp., Fonsecaea spp., Wangiella spp., Sporothrix spp., Basidiobolus spp., Conidiobolus spp., Rhizopus spp, Mucor spp, Absidia spp, Mortierella spp, Cunninghamena spp, Saksea spp., Alternaria spp, Curvularia spp, Helminthosporium spp, Fusarium spp, Aspergillus spp, Penicillium spp, Monolinia spp, Rhizoctonia spp, Paecilomyces spp, Pithomyces spp and Cladosporium spp.

In some embodiments, the immunogen elicits an immune response against a parasite of the genus Plasmodium, such as P. falciparum, P. vivax, P. malariae, or P. ovale. Thus, the invention can be used for immunization against malaria. In some embodiments, the immunogen elicits an immune response against a parasite from the Caligidae family, in particular from the genera Lepeophtheirus and Caligus, for example sea lice such as Lepeophtheirus salmonis or Caligus rogercresseyi.

In some embodiments, the immunogen elicits an immune response against pollen allergens (tree pollen, aromatic grass, plants, and grass allergens); insect or arachnid allergens (inhaled, saliva and venom allergens, eg mite allergens, cockroach and midge allergens, hymenoptera venom allergens); allergens from animal hair and dander (for example, from dogs, cats, horses, rats, mice, etc.); and food allergens (eg, gliadin). Important tree, grass, and plant pollen allergens are those derived from the taxonomic orders Fagales, Oleales Pinales, and platanaceae, including, but not limited to, birch (Betula), alder (Alnus), hazel (Corylus), hornbeam (Carpinus) and olive (Olea), cedar (Cryptomeria and Juniperus), sycamore (Platanus), order Poales, including grasses of the genera Lolium, Phleum, Poa, Cynodon, Dactylis, Holcus, Phalaris, Secale and Sorghum, orders Asterales and Urticales, including plants of the genera Ambrosia , Artemisia and Parietaria. Other important inhaled allergens are those derived from house dust mites of the genera Dermatophagoides and Euroglyphus, barn mites such as Lepidoglyphys, Glycyphagus and Tyrophagus which are derived from cockroaches, midges and fleas, such as Blatella, Periplaneta, Chironomus and Ctenocepphalides and which are derived from animals, such as cat, dog, and horse, allergens to poisons, including those derived from stinging or biting insects, such as those derived from the taxonomic order Hymenoptera, including bees (Apidae), wasps (Vespidea), and ants (Formicoidae) .

In some embodiments, the immunogen is a tumor antigen selected from: (a) testicular cancer antigens, such as NY-ESO-1, SSX2, SCP1, as well as RAGE, BAGE, GAGE, and MAGE family polypeptides, e.g., GAGE-1, GAGE-2, MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-5, MAGE-6 and MAGE-12 (which can be used, for example, to target melanoma, lung, head and neck tumors , NSCLC, breast, gastrointestinal, and bladder tumors), (b) mutated antigens, e.g. p53 (associated with various solid tumors, e.g. , with melanoma, pancreatic and rectal cancer), CDK4 (associated with e.g. melanoma), MUM1 (associated with e.g. melanoma), caspase-8 (associated with e.g. head and neck cancer), CIA 0205 (associated with e.g. bladder cancer), HLA-A2-R1701, beta-catenin (associated with e.g. melanoma), TCR (associated with e.g. T-cell non-Hodgkin's lymphoma), BCR-abl (associated with e.g. chronic myelogenous leukemia), triose phosphate isomerase, KIA 0205, CDC-27 and LDLR-FUT, (c) overexpressed antigens e.g. Galectin 4 (associated with e.g. rectal cancer), Galectin 9 (associated, for example, with Hodgkin's disease), proteinase 3 (associated, for example, with chronic myelogenous leukemia), WT 1 (associated, for example, with various leukemias), carbonic anhydrase (associated, for example, with cancer kidney), aldolase A (associated with e.g. lung cancer), PRAME (associated with e.g. melanoma), HER-2/neu (associated with e.g. breast, colon, lung and ovarian cancer), mammaglobin, alphafetoprotein (associated, for example, with hepatoma), KSA (associated, for example, with colorectal cancer), gastrin (associated, for example, with pancreatic and gastric cancer), telomerase catalytic protein, MUC-1 (associated, for example, with cancer breast and ovaries), G-250 (associated th, for example, with renal cell carcinoma), p53 (associated, for example, with cancer of the breast and colon), and carcinoembryonic antigen (associated, for example, with breast cancer, lung cancer, and cancer of the gastrointestinal tract, such as cancer of the rectum gut), (d) being common antigens, e.g. melanoma-melanocyte differentiation antigens such as MART-1/Melan A, gp100, MC1R, melanocyte-stimulating hormone receptor, tyrosinase, tyrosinase-related protein-1/TRPI and tyrosinase- associated protein-2/TRP2 (associated, for example, with melanoma), (e) prostate-associated antigens such as PAP, PSA, PSMA, PSH-P1, PSM-P1, PSM-P2, associated

- 39 040257 ny, for example, with prostate cancer, (f) immunoglobulin idiotypes (associated with myeloma and B-cell lymphoma, for example). In some embodiments, tumor immunogens include, but are not limited to, p15, Hom/Mel-40, H-Ras, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein-Barr virus antigens, EBNA, antigens human papillomavirus (HPV), including E6 and E7, hepatitis B and C virus antigens, human T-lymphotropic virus antigens, TSP-180, p185erbB2, p180erbB-3, c-met, mn-23H1, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, p16, TAGE, PSCA, CT7, 43-9F, 5T4, 791 Tgp72, beta-HCG, BCA225, BTAA, CA 125, CA 15- 3 (CA 27.29\BCAA), CA 195, CA 242, CA-50, CAM43, CD68\KP1, CO-029, FGF-5, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag , MOV18, NB/70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2 binding protein/cyclophilin associated protein), TAAL6, TAG72, TLP, TPS and the like.

The pharmaceutical composition of the invention, in particular one suitable for immunization, may include one or more small molecular weight immunostimulants. For example, the composition may include a TLR2 agonist (e.g., Pam3CSK4), a TLR4 agonist (e.g., aminoalkyl glucosamine phosphate, such as E6020), a TLR7 agonist (e.g., imiquimod), a TLR8 agonist (e.g., resiquimod), and/or a TLR9 agonist (e.g., IC31) . Any such agonist ideally has a molecular weight of <2000 Da. Such agonist(s), in some embodiments, may be encapsulated with RNA in liposomes, but in other embodiments, they are unencapsulated.

Examples

Cationic lipids of Formula (I)

The following examples are intended to illustrate the invention and should not be construed as limiting it. Temperatures are presented in degrees Celsius. Unless otherwise indicated, all evaporative concentrations are carried out under reduced pressure, preferably from about 15 mm Hg. up to 100 mm Hg (=20-133 mbar). The structure of the final products, intermediates and starting materials is confirmed using standard analytical methods, such as microanalysis or spectral characterization, such as MS, IR or NMR. The abbreviations used are standard in the art, some of which are described below.

Flash column purification is preferably carried out on silica gel using an appropriate isocratic or gradient composition eluent.

HPLC analysis was performed on a Waters Atlantis dC18 column (4.6 x 150 mm, 3 mm) with gradient elution (0 to 95% acetonitrile in water modified with 0.1% v/v trifluoroacetic acid, over 20 min and a flow rate of 1.4 ml/min) unless otherwise noted.

1H-NMR spectra were recorded on a Bruker Avance II 400 MHz spectrometer. All chemical shifts are given in parts per million (δ) relative to tetramethylsilane. The following abbreviations are used to designate the signal model: s=singlet, d=doublet, t=triplet, k=quartet, q=quintet, m=multiplet, br=broadened. ER-MS data were recorded using a Waters LTC Premier mass spectrometer with a dual electrospray ionization source on an Agilent 1100 liquid chromatograph. ™ every third scan.

Abbreviations

C Celsius

DCM dichloromethane degrees degrees

DIEA N,N-diisopropylethylamine

DIPEA N,N-diisopropylethylamine

DMAP 4-dimethylaminopyridine

DMF N,N-dimethylformamide

DMSO dimethyl sulfoxide

EDC 1 -ethyl-3-(3-dimethylaminopropyl)carbodiimide

ER-MS electrospray mass spectrometry

EtOAc ethyl acetate

EtOH ethanol g grams h hour(s)

HATU 2-(1 H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate

HOBt hydroxybenzotriazole

HPLC HPLC kg kilogram l liter

LAG lithium aluminum hydride

LC liquid chromatography

LC-MS liquid chromatography and mass spectrometry

- 40 040257

MeOH methanol

MS Mass Spectrometry mbar millibar min minutes ml milliliter(s) mm millimeter µM micromolar m/z mass-to-charge ratio nm nanometer nM nanomolar

N normal

NaOEt sodium ethylate

NMP N-methylpyrrolidone

NMR nuclear magnetic resonance

Pd/C palladium on carbon

PdCl ₂ (dppf).CH ₂ Cl ₂ 1,1'-bis(diphenylphosphino)ferrocene-palladium(II) dichloride dichloromethane complex psi psi

ppm pTsOH p-toluenesulfonic acid quintuplet rac racemic

Rt retention time

TBAF tetrabutylammonium fluoride

TBDPS tert-butyldiphenylsilyl ether

TBTU O-(Benzotriazol-1 -yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate

TCEP tris(2-carboxyethyl)phosphine

TEA triethylamine

TFA trifluoroacetic acid

THF tetrahydrofuran

THP tetrahydropyran

TLC thin layer chromatography

TMS-CN trimethylsilyl cyanide

TsOH toluenesulfonic acid

Synthesis of example 1:

Intermediate 1а: 3,5-bis((2)-octadec-9-en-1-yloxy)benzaldehyde

To a flask containing DMF (40 ml) were added 3,5-dihydroxybenzaldehyde (2 g, 14.2 mmol), potassium carbonate (5.88 g, 42.6 mmol) and oleyl mesylate (11.3 g, 32.6 mmol ). The resulting mixture was heated to 80° C. overnight with stirring. The reaction mixture was cooled and water and EtOAc were added. The organic layer was collected, washed with brine and dried over magnesium sulfate. The mixture was filtered and the filtrate was concentrated under reduced pressure to give a crude product which was purified on silica gel using heptane/EtOAc as eluent to provide 8.53 g of desired product.

TLC (silica gel, 10% EtOAc in heptane): Rf=0.56.

Example 1 compound: 1-(3,5-bis((Z)-octαdec-9-en-1-yloxy)phenyl)-N,N-dimethylmethaneαmin

Intermediate 1a (4 g, 6.26 mmol) was stirred in EtOH (25 ml) and dimethylamine hydrochloride (1.02 g, 12.5 mmol) was added followed by TEA (1.21 ml, 8.76 mmol) and tetraisopropoxide titanium (1.8 ml, 6.3 mmol). The resulting mixture was stirred for 3 hours at room temperature and sodium borohydride (355 mg, 9.39 mmol) was added in one portion. The mixture was stirred at room temperature overnight. The reaction mixture was quenched with 7 N ammonia in MeOH (8.94 ml, 62.6 mmol) and the resulting white suspension was filtered through celite, washing with DCM. The filtrate was concentrated under reduced pressure to give a crude product which was purified on silica gel with 0 to 50% EtOAc in heptane to provide 2.66 g of the desired product.

- 41 040257

1 H NMR (400 MHz, CDCl ₃ ) δ: 6.46 (d, J=2.0 Hz, 2H), 6.36 (t, J=2.5 Hz, 1H), 5.41-5.31 (m, 4H), 3.93 (t,

J=6.9 Hz 4H), 3.34(s, 2H), 2.24(s, 6H), 2.09-1.98(m, 8H), 1.81-1.69(m, 4H), 1.50-1.21 (m, 44H), 0.89 (t, J=7.0

Hz, 6H) ppm

ER-MS m/z=668.8 (MH+).

Examples 2-7 can be obtained using methods similar to those used to obtain example 1.

Example 2: 1-(3,5-6uc((9Z, 12Z)-octadeca-9,12-dien-1-yloxy)phenyl)-N,N-dimethylmethanamine

1H NMR (400 MHz, CDCl ₃ ) δ: 6.46 (d, J=2.5 Hz, 2H), 6.35 (t, J=2.3 Hz, 1H), 5.44-5.30 (m, 8H), 3.93 (t, J=6.5 Hz, 4H), 3.34 (s, 2H), 2.78 (t, J=6.2 Hz, 4H), 2.24 (s, 6H), 2.13-1.99 (m, 8H), 1.81-1.71 (m, 4H), 1.49-1.23 (m, 32H), 0.90 (t , J=6.8 Hz, 6H) ppm

ER-MS m/z=664.9 (MH+).

Example 3: 2,2'-((3,5-6uc((9Z, 12Z)-octadeca-9,12-dien-1-yloxy)benzyl)azanediyl)diethanol

1H NMR (400 MHz, CDCl ₃ ) δ: 6.46-6.44 (m, 2H), 6.38-6.34 (m, 1H), 5.44-5.29 (m, 8H), 3.92 (t, J=6.5 Hz, 4H), 3.69-3.63 (m, 6H), 2.82-2.71 (m, 8H), 2.12-2.00 ( m, 8H), 1.82-1.70 (m, 4H), 1.51-1.22 (m, 32H), 0.90 (t, J=6.9 Hz, 6H) ppm.

ER-MS m/z=724.6 (MH+).

Example 4: 1-(3,5-bis((9Z,12Z)-octadeca-9,12-dien-1-yloxy)benzyl)pyrrolidine

1H NMR (400 MHz, CDCl ₃ ) δ: 6.52-6.47 (m, 2H), 6.36-6.32 (m, 1H), 5.47-5.26 (m, 8H), 3.93 (t, J=6.6 Hz, 4H), 3.55 (s, 2H), 2.83-2.74 (m, 4H), 2.57-2.47 (m, 4H) , 2.14-2.00 (m, 8H), 1.86-1.70 (m, 8H), 1.52-1.21 (m, 32H), 0.90 (t, J=6, 8 Hz, 6H) ppm

ER-MS m/z=690.5 (MH+).

Example 5: 1-(3,5-bis((9Z,12Z)-octadecα-9,12-dien-1-yloxy)benzyl)azetidin-3-ol

1H NMR (400 MHz, CDCl ₃ ) δ: 6.49 (d, J=2.0 Hz, 2H), 6.42 (t, J=2.1 Hz, 1H), 5.33-5.47 (m, 7H), 4.46 (tt, J=6.7, 3.6 Hz, 1H), 3.94 (t, J=6.7 Hz, 4H), 3.76-3.85 ( m, 4H), 3.59 (d, J=10.3 Hz, 2H), 2.80 (t, J=6.5 Hz, 4H), 2.00-2.12 (m, 8H), 1.78 (dtd, J=7.8, 6.8, 5.8 Hz, 4H), 1.41-1.51 (m, 4H), 1.25-1.41 (m, 27H), 0.83-0.95 (m, J=6.8, 6.8 Hz, 5H) ppm

ER-MS m/z=692.5 (MH+).

Example 6 Ethyl 2-((3,5-bis((Z)-octadec-9-en-1-yloxy)benzyl)(methyl)amino)acetate

1H NMR (400 MHz, CDCl3) δ 6.50 (s, 2H), 6.36 (s, 1H), 5.39-5.32 (m, 4H), 4.18 (q, J=7, 0 Hz 2H), 3.92 (t, J=6.5 Hz, 4H), 3.60 (s, 2H), 3.24 (s, 2H), 2.40 (s, ZN), 2, 10-1.95 (m, 8H), 1.80-1.60 (m, 4H), 1.50-1.15 (m, 47H), 0.88 (t, J=7.0 Hz, 6H) ppm

ER-MS m/z=740.9 (MH+).

Example 7: 1-(3,5-bis((Z)-octadec-9-en-1-yloxy)benzyl)pyrrolidine

1H NMR (400 MHz, CDCl3) δ 6.53 (s, 2H), 6.35 (s, 1H), 5.38-5.30 (m, 4H), 3.92 (t, J=6, 5 Hz, 4H), 3.62 (br. s, 2H), 2.62 (m, 4H), 2.10-1.95 (m, 8H), 1.90-1.80 (m, 4H ), 1.80-1.65 (m, 4H), 1.50-1.20 (m, 43H), 0.87 (t, J=7.0 Hz, 6H) ppm

ER-MS m/z=694.9 (MH+).

Example 8: 2-((3,5-bis((2)-octadec-9-en-1-yloxy)benzyl)(methyl)amino)acetic acid

To a solution of the compound from example 6 (160 mg, 0.22 mmol) in dioxane (7 ml) was added 50% HCl in

- 42 040257 water (6.57 ml). The mixture was heated to reflux overnight and then cooled to room temperature. The volatiles were removed under reduced pressure, and the resulting product was purified using a strong cation exchange resin followed by silica gel column chromatography using DCM/MeOH as the eluent to provide 123 mg of the desired product.

1H NMR (400 MHz, CDCl3) δ 10.34 (br. s 1H), 6.57 (s, 2H), 6.42 (s, 1H), 5.38-5.30 (m, 4H), 4.17 (s, 2H), 3.87 (t, J=6.5 Hz, 4H), 3.51 (s, 2H), 2.77 (s, 3H), 2.10-1.95 (m, 8H), 1.80-1.60 (m, 4H), 1.50-1.15 (m, 44H), 0.87 (t, J=7.0 Hz, 6H) ppm .

¹³ C NMR (100 MHz, CDCl3) δ 168.6, 160.6, 132.3, 129.9, 129.7, 109.0, 101.8, 68.1, 60.0, 57.6, 41.1, 31.8, 29.7, 29.5, 29.5, 29.4, 29.4, 29.2, 29.2, 27.2, 26.0, 22.6, 14, 1 ppm

Synthesis of example 9:

Intermediate 9а: ((2-(3,5-bis((Z)-octadec-9-en-1-yloxy)phenyl)-1,3-dioxolan-4-yl)methoxy)(tert-butyl)diphenylsilane

To Intermediate 1a (1 g, 1.56 mmol) in MePh (30 mL) was added TBDPS-protected glycerol (0.52 g, 1.56 mmol) and TsOH monohydrate (0.03 g, 0.16 mmol). The mixture was heated at reflux overnight and then cooled to room temperature. The volatiles were removed under reduced pressure and the resulting product was purified on silica gel using heptane/EtOAc as eluent to provide 1.28 g of a mixture containing the desired product.

TLC (silica gel, 10% EtOAc in heptane): Rf=0.45.

Intermediate 9b: (2-(3,5-bis(^)-octadec-9-en-1-yloxy)phenyl)-1,3-dioxolan-4-yl)methanol

To a solution of Intermediate 9a (1.28 g, 1.34 mmol) in THF (10 mL) was added TBAF (9.9 mL, 1.0 M in THF, 9.93 mmol). The resulting solution was stirred overnight at room temperature. Volatiles were removed under reduced pressure and the resulting product was purified on silica gel using heptane/EtOAc as eluent, providing 638 mg (67%) of the desired product.

TLC (silica gel, 50% EtOAc in heptane): Rf=0.65.

Intermediate 9c: (2-(3,5-bis((Z)-octadec-9-en-1-yloxy)phenyl)-1,3-dioxolan-4-yl)methylmethanesulfonate

To a solution of Intermediate 9b (638 mg, 0.895 mmol) in DCM (20 ml) was added DIEA (0.78 ml, 4.5 mmol) and MsCl (0.35 ml, 4.5 mmol). The resulting solution was stirred for 30 minutes. The volatiles were removed under reduced pressure and the resulting product was used without further purification.

ER-MS m/z=791.4 (MH+).

Compound of Example 9: 1-((2-(3,5-bis(^)-octadec-9-en-1-yloxy)phenyl)-1,3-dioxolan-4-yl)methyl)pyrrolidine

A solution of Intermediate 9c (708 mg, 0.895 mmol) was dissolved in pyrrolidine (3.0 ml, 36.2 mmol) and heated to 140° C. in a microwave reactor. The volatiles were removed under reduced pressure and the resulting product was purified on silica gel using heptane/EtOAc as eluent to provide 292 mg of desired product.

1H NMR (400 MHz, CDCl3) δ 6.62 (dd, J=6.0 Hz, 2.5 Hz, 2H), 6.44-6.42 (m, 1H), 5.89 (s, 0 .50H), 5.75 (s, 0.50H), 5.37-5.35 (m, 4H), 4.42-4.33 (m, 1H), 4.26-4.23 (m , 0.50H), 4.12 (dd, J=7.8 Hz, 6.8 Hz, 0.50H), 3.93 (t, J=6.3 Hz, 4H), 3.80 (dd , J=7.8 Hz, 6.8 Hz, 0.50N), 3.71-3.67 (m, 0.50N), 2.81-2.73 (m, 2H), 2.65- 2.57 (m, 4H), 2.05-2.00 (m, 8H), 1.81-1.73 (m, 8H), 1.48-1.27 (m, 44H), 0, 89 (m, 6H) ppm

ER-MS m/z=766.6 (MH+).

Examples 10 and 11 can be obtained using methods similar to those used to obtain example 9.

Example 10: 1-(2-(3,5-6uc((9Z, 12Z)-octadeca-9,12-dien-1-yloxy)phenyl)-1,3-dioxolan-4-yl)-N,N - 43 040257 dimethylmethanamine

1H NMR (400 MHz, CDCl ₃ ) δ 6.59-6.65 (m, 2H), 6.44 (t, J=2.3 Hz, 1H), 5.75 (s, 1H), 5, 32-5.44 (m, 6H), 4.32-4.42 (m, 1H), 4.08-4.18 (m, 1H), 3.94 (t, J=6.5 Hz, 4H), 3.78 (dd, J=8.0, 6.5 Hz, 1H), 2.74-2.83 (m, 4H), 2.51-2.66 (m, 2H), 2 .31-2.37 (m, 6H), 2.00-2.11 (m, 8H), 1.68-1.85 (m, 6H), 1.20-1.50 (m, 32H) , 0.84-0.93 (m, 6H) ppm

ER-MS m/z=736.5 (MH+).

Example 11: 1-((2-(3,5-bis((92,122)-octadeca-9,12-dien-1-yloxy)phenyl)-1,3-dioxolan-4-yl)methyl)piperidine

1H NMR (400 MHz, CDC13) δ 6.63 (d, J=2.0 Hz, 2H), 6.45 (t, J=2.3 Hz, 1H), 5.74 (s, 1H), 5.29-5.48 (m, 8H), 4.38 (t, J=6.1 Hz, 1H), 4.07-4.15 (m, 1H), 3.96 (t, J= 6.6 Hz, 4H), 3.78 (dd, J=8.1, 6.6 Hz, 1H), 2.80 (t, J=6.3 Hz, 4H), 2.62-2, 72 (m, 1H), 2.50-2.62 (m, 4H), 2.40-2.50 (m, 2H), 2.08 (d, J=7.1 Hz, 4H), 1 .70-1.83 (m, 4H), 1.53-1.70 (m, 7H), 1.23-1.53 (m, 34H), 0.91 (t, J=7.1 Hz , 6H) ppm

ER-MS m/z 776.7 (MH+).

Example 12: (2-(3,5-bis((2)-octadec-9-en-1-yloxy)phenyl)-1,3-dioxolan-4-yl)methyl-2-(dimethylamino)acetate

To a solution of Intermediate 9b (2.42 g, 3.39 mmol) in DCM (30 mL) was added K,K-dimethylglycine (0.38 g, 3.73 mmol) followed by HATU (1.55 g, 4. 07 mmol) and pyridine (1.1 ml, 13 mmol). The mixture was stirred at room temperature overnight. The volatiles were removed under reduced pressure and the resulting product was purified on silica gel using heptane/EtOAc as eluent to provide 1.14 g of desired product.

1H NMR (400 MHz, CDCl3) δ 6.60 (dt, J=8.8 Hz, 2.0 Hz, 2H), 6.44-6.41 (m, 1H), 5.87 (s, 0 .33H), 5.73 (s, 0.33H), 5.45 (s, 0.33H), 5.36-5.30 (m, 4H), 4.74 (m, 0.33H), 3.91(m, 0.33H), 3.76(m, 0.33H), 4.50-4.06(m, 4H), 3.91(t, J=6.5Hz, 4H) , 3.31 (s, 0.66H), 3.22 (s, 0.66H), 3.19 (s, 0.66H), 2.38 (s, 2H), 2.36 (s, 2H ), 2.34 (s, 2H), 2.04-1.99 (m, 8H), 1.78-1.71 (m, 4H), 1.50-1.26 (m, 44H), 0.87 (t, J=7.0 Hz, 6H) ppm

ER-MS m/z=798.5 (MH+).

Synthesis of example 13:

Intermediate 13а: (3,5-bis((92,122)-octadeca-9,12-dien-1-yloxy)phenyl)methanol o _x

To Intermediate 1a (1.5 g, 2.4 mmol) in THF (10 ml) and MeOH (5 ml) was added sodium borohydride (0.116 g, 3.07 mmol). The resulting mixture was stirred overnight and then quenched with MeOH and water. The resulting product was extracted with EtOAc and the organic layers were dried over sodium sulfate. The product was decanted and the volatiles were removed under reduced pressure. The product was used in the next step without further purification.

Compound of Example 13: 3,5-bis((92,122)-octadeca-9,12-dien-1-yloxy)benzyl-4-(dimethylamino)butanoate

To Intermediate 13a (486 mg, 0.762 mmol) in DCM (5 mL) was added 4-dimethylaminobutanoic acid (100 mg, 0.762 mmol) followed by DIEA (0.32 mL, 1.83 mmol), DMAP (50 mg, 0 .41 mmol) and EDC (175 mg, 0.92 mmol). The resulting mixture was stirred overnight at room temperature and purified directly on silica gel using heptane/EtOAc as eluent, providing 319 mg (56%) of the desired product.

1H NMR (400 MHz, CDCl ₃ ) δ 6.48-6.45 (m, 2H), 6.41-6.38 (m, 1H), 5.44-5.29 (m, 8H), 5 .04 (s, 2H), 3.92 (t, J=6.5 Hz, 4H), 2.81-2.75 (m, 4H), 2.41 (t, J=7.5 Hz, 2H), 2.29 (t, J=7.3 Hz, 2H), 2.21 (s, 6H), 2.10-2.00 (m, 8H), 1.87-1.71 (m , 6H), 1.51-1.23 (m, 32H), 0.90 (t, J=6.8 Hz, 6H) ppm.

ER-MS m/z=750.7 (MH+).

- 44 040257

Examples 14-17 can be obtained using methods similar to those used to obtain example 13.

Example 14 3,5-6uc((9Z,12Z)-octadeca-9,12-dien-1-yloxy)benzyl-3-(dimethylamino)propanoate

1H NMR (400 MHz, CDCl ₃ ) δ 6.49-6.46 (m, 2H), 6.42-6.39 (m, 1H), 5.44-5.29 (m, 8H), 5 .05 (s, 2H), 3.93 (t, J=6.8 Hz, 4H), 2.82-2.75 (m, 4H), 2.68-2.61 (m, 2H), 2.59-2.52 (m, 2H), 2.25 (s, 6H), 2.12-2.01 (m, 8H), 1.821.71 (m, 4H), 1.52-1, 23 (m, 32H), 0.90 (t, J=6.8 Hz, 6H) ppm

ER-MS m/z=736.8 (MH+).

Example 15 3,5-6uc((9Z,12Z)-octadeca-9,12-dien-1-yloxy)benzyl-2-(dimethylamino)acetate

1H NMR (400 MHz, CDCl3) δ 6.51-6.46 (m, 2H), 6.42-6.39 (m, 1H), 5.46-5.29 (m, 8H), 5, 09 (s, 2H), 3.92 (t, J=6.5 Hz, 4H), 3.23 (s, 2H), 2.83-2.75 (m, 4H), 2.37 (s , 6H), 2.12-2.00 (m, 8H), 1.83-1.70 (m, 4H), 1.50-1.23 (m, 32H), 0.89 (t, J =6.8 Hz, 6H) ppm

ER-MS m/z=722.4 (MH+).

Example 16 4-methyl-3,5-6uc((9Z, 12Z)-octadeca-9,12-dien-1-yloxy)benzyl-3-(dimethylamino)propanoate

1H NMR (400 MHz, CDCl ₃ ) δ 6.51 (s, 2H), 5.31-5.45 (m, 8H), 5.07 (s, 2H), 3.97 (t, J=6 .6 Hz, 4H), 2.80 (dd, J=6.3, 6.3 Hz, 4H), 2.68 (t, J=7.1 Hz, 2H), 2.54 (t, J = 7.3 Hz, 2H), 2.27 (s, 6H), 2.04-2.13 (m, 11H), 1.761.85 (m, 4H), 1.46-1.53 (m, 4H), 1.26-1.45 (m, 32H), 0.92 (t, J=6.8 Hz, 5H), 0.93 (br.s, 1H) ppm.

ER-MS m/z=750.5 (MH+).

Example 17: 3,5-6uc((9Z, 12Z)-octadeca-9,12-dien-1-yloxy)benzyl-3-(dimethylamino)-2-methylpropanoate

¹ H NMR (400 MHz, CDCl ₃ ) δ 6.40 (d, J=2.0 Hz, 2H), 6.34-6.29 (m, 1H), 5.36-5.21 (m, 8H), 4.98 (s, 2H), 3.85 (t, J=6.6 Hz, 4H), 2.70 (t, J=6.3 Hz, 4H), 2.66-2, 54 (m, 2H), 2.14 (s, 6H), 1.98 (q, J=6.6 Hz, 8H), 1.72-1.64 (m, 4H), 1.42-1 .34 (m, 4H), 1.33-1.16 (m, 36H), 1.10 (d, J=6.6 Hz, 3H), 0.85-0.79 (m, 6H) m .d.

ER-MS m/z=750.3 (MH+).

Synthesis of Example 18:

Intermediate 18а: (9Z,12Z)-2-hydroxyethyl-octadeca-9,12-dienoate

Linoleic acid (5.0 g, 17.83 mmol) was dissolved in 39.9 ml of ethylene glycol with stirring. EDC (5.136 g, 26.7 mmol) and HOBt (4.10 g, 26.7 mmol) were added to the mixture, followed by triethylamine (7.45 ml, 53.5 mmol). The reaction mixture was stirred at room temperature for 48 h and the completion of the reaction was monitored by TLC. The crude material was diluted with 100 ml dichloromethane and washed with 50 ml water and 50 ml brine. The organic layer was separated and dried over anhydrous sodium sulfate. The crude product was loaded dry on celite and purified by silica gel chromatography with a gradient of 10% to 40% EtOAc in heptane. The product was recovered as a clear oil (3.884 g, 67.1%). TLC (silica gel, 20% EtOAc in heptane): Rf=0.22.

Intermediate 18b: (9Z,9'Z,12Z,12'Z)-((5-formyl-1,3-phenylene)bis(oxy))bis(ethane-2,1-diyl)bis(octadeca-9, 12-dienoate)

Intermediate 18a (1.5 g, 4.62 mmol), 3,5-dihydroxybenzaldehyde (0.319 g, 2.311 mmol) and triphenylphosphine (1.273 g, 4.85 mmol) were dissolved in 19 ml of anhydrous THF. DIAD (0.944 mL, 4.85 mmol) was added and the reaction mixture was allowed to stir for 48 hours at room temperature. reactionary

- 45 040257 the mixture was directly concentrated on celite and purified by silica gel chromatography with a gradient of 10 to 20% EtOAc in heptane. The product was isolated as a colorless oil (1.077 g,

62.1%).

1H NMR (400 MHz, CDCl3) δ 9.92 (s, 1H), 7.06 (d, J=2.3 Hz, 2H), 6.76 (t, J=2.3 Hz, 1H), 5.31-5.43 (m, 8H), 4.43-4.49 (m, 4H), 4.20-4.26 (m, 4H), 2.78 (t, J=6.4 Hz, 4H), 2.37 (t, J=7.7 Hz, 4H), 2.01-2.11 (m, 8H), 1.58-1.71 (m, 5H), 1.24 -1.42 (m, 30H), 0.90 (t, J=6.8 Hz, 6H) ppm

Intermediate 18c: (9Z,9'Z, 12Z, 12'Z)-((5-(hydroxymethyl)-1,3-phenylene)bis(oxy))bis(ethane-2,1diyl)bis(octadeca-9 ,12-dienoate) o

Intermediate 18b (465.2 mg, 0.619 mmol) was dissolved in 4.1 ml of dry ethanol under nitrogen atmosphere. Sodium borohydride (46.9 mg, 1.239 mmol) was added in one portion and stirred at room temperature for 30 minutes. The completion of the reaction was monitored by TLC. The reaction mixture was quenched with acetic acid, diluted with 10 ml of water and extracted into 30 ml of DCM. The resulting organic layers were combined, dried over sodium sulfate, filtered and concentrated. The product was reconstituted as 429 mg of a clear oil.

TLC (silica gel, 30% EtOAc in heptane): Rf=0.55

Compound of Example 18: (9Z,9'Z,12Z,12'Z)-((5-(((3-(dimethylamino)propanoyl)oxy)methyl)-1,3phenylene)bis(oxy))bis(ethane- 2,1-diyl)bis(octadeca-9,12-dienoate) o o

Intermediate 18c (50 mg, 0.066 mmol) and X,X-dimethylaminopropionic acid (10.20 mg, 0.066 mmol) were dissolved in 4 ml DCM. HATU (37.9 mg, 0.100 mmol) was added followed by triethylamine (9.25 µl, 0.066 mmol). The reaction mixture was stirred for 18 hours at room temperature and monitored by LCMS. The reaction mixture was diluted with 100 ml DCM and 50 ml water. The organic layers were separated, then washed with brine, dried over sodium sulfate, filtered and concentrated.

The crude product was purified on silica gel with methanol and dichloromethane as eluent. The product was further purified by HPLC (Waters Sunfire C8 column with 5 to 100% 1:1 acetonitrile:isopropanol in water modified with 0.1% TFA). Fractions containing product were partitioned between dichloromethane and saturated aqueous sodium bicarbonate over one hour. The dichloromethane layer was dried over sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to give the title compound.

1H NMR (400 MHz, CDCl3) δ 6.54 (d, J=2.26 Hz, 2H), 6.45 (t, J=2.26 Hz, 1H), 5.28-5.49 (m , 8H), 5.07 (s, 2H), 4.35-4.48 (m, 4H), 4.08-4.22 (m, 4H), 2.79 (t, J=6.53 Hz, 4H), 2.60-2.72 (m, 2H), 2.49-2.60 (m, 2H), 2.37 (t, J=7.65 Hz, 4H), 2.27 (s, 6H), 1.97-2.14 (m, 8H), 1.56-1.74 (m, 4H), 1.23-1.44 (m, 28H), 0.85-0 .97 (m, 6H)

ppm

ER-MS m/z=852.7 (MH+).

Examples 19-23 can be obtained using methods similar to those used to obtain example 18.

Example 19: (9Z,9'Z,12Z,12'Z)-((5-(((3-(dimethylamino)propanoyl)oxy)methyl)-1,3-phenylene)bis(oxy))bis(propane -3,1-diyl)bis(octadeca-9,12-dienoate)

1H NMR (400 MHz, CDCl3) δ 6.50 (d, J=2.3 Hz, 2H), 6.41 (t, J=2.3 Hz, 1H), 5.33-5.44 (m , 8H), 5.07 (s, 2H), 4.27 (t, J=6.4 Hz, 4H), 4.03 (t, J=6.1 Hz, 4H), 2.73-2 .78 (m, 4H), 2.64 (d, J=7.3 Hz, 2H), 2.29-2.37 (m, 10H), 2.12 (dd, J=6.3, 6 .3 Hz, 4H), 2.02-2.10 (m, 8H), 1.63 (dd, J=7.3, 7.3 Hz, 5H), 1.25-1.41 (m, 32H), 0.91 (t, J=6.8 Hz, 6H) ppm

ER-MS m/z=880.4 (MH+).

Example 20: (9Z,9'Z, 12Z, 12'Z)-((5-((2-(dimethylamino)acetoxy)methyl)-1,3-phenylene)bis(oxy))bis(propane-3, 1-diyl)bis(octadeca-9,12-dienoate)

1H NMR (400 MHz, CDCl ₃ ) δ 6.51 (d, J=2.0 Hz, 2H), 6.42 (t, J=2.0 Hz, 1H), 5.27-5.47 ( m, 8H), 5.11 (s,

- 46 040257

2H), 4.27 (dd, J=6.4, 6.4 Hz, 4H), 4.03 (dd, J=6.1, 6.1 Hz, 4H), 3.30 (s, 2H ), 2.79 (dd, J=6.4, 6.4 Hz, 4H), 2.44 (s, 6H), 2.32 (dd, J=7.5, 7.5 Hz, 4H) , 2.03-2.15 (m, 12H), 1.53-1.77 (m, 14H), 1.25-1.43 (m, 32H), 0.91 (t, J=6, 8

Hz, 6H) ppm

ER-MS m/z=866.4 (MH+).

Example 21: (9Z,9'Z,12Z,12'Z)-((5-((2-(dimethylamino)acetoxy)methyl)-1,3-phenylene)bis(oxy))bis(butane-4, 1-diyl)bis(octadeca-9,12-dienoate)

1H NMR (400 MHz, CDCl3) δ 6.48 (d, J=2.3 Hz, 2H), 6.40 (t, J=2.1 Hz, 1H), 5.43-5.32 (m , 8H), 5.11 (s, 2H), 4.17-4.10 (m, 4H), 3.96 (dd, J=5.6, 5.6 Hz, 4H), 3.54 ( s, 2H), 3.20-3.27 (m, J=7.3 Hz, 3H), 2.81 (s, 22H), 2.77 (dd, J=6.7, 6.7 Hz , 4H), 2.63 (s, 6H), 2.30 (dd, J=7.7, 7.7 Hz, 4H), 2.01-2.08 (m, 8H), 1.86- 1.78 (m, 8H), 1.66-1.58 (m, 5H), 1.42-1.37 (m, 7H), 1.37-1.23 (m, 36H), 0. 91-0.87 (m, 6H) ppm

ER-MS m/z=894.5 (MH+).

Example 22: (9Z,9'Z,12Z,12'Z)-((5-(((3-(dimethylamino)propanoyl)oxy)methyl)-1,3-phenylene)bis(oxy))bis(butane -4,1-diyl)bis(octadeca-9,12-dienoate)

1H NMR (400 MHz, CDCl3) δ: 6.48 (d, J=2.3 Hz, 2H), 6.40 (br. s, 1H), 5.32-5.43 (m, 8H), 5.11 (s, 2H), 4.10-4.17 (m, 4H), 3.96 (t, J=5.6 Hz, 4H), 3.54 (s, 2H), 3.23 (k, J=7.3 Hz, 2H), 2.77 (dd, J=6.7, 6.7 Hz, 4H), 2.63 (s, 6H), 2.30 (dd, J= 7.7, 7.7 Hz, 4H), 2.02-2.07 (m, 8H), 1.79-1.88 (m, 8H), 1.58-1.68 (m, 5H) , 1.23-1.39 (m, 38H), 0.89 (t, J=6.5 Hz, 7H) ppm

ER-MS m/z=908.7 (MH+).

Example 23: (9Z,9'Z, 12Z, 12'Z)-((5-(((4-(dimethylamino)butanoyl)oxy)methyl)-1,3-phenylene)bis(oxy))bis(ethane -2,1-diyl)bis(octadeca-9,12-dienoate)

1H NMR (400 MHz, CDCl3) δ 6.84 (s, 1H), 6.74 (d, J=1.5 Hz, 2H), 5.21-5.35 (m, 8H), 5.07 (s, 2H), 4.31 (t, J=4.8 Hz, 4H), 4.03-4.08 (m, 4H), 2.69 (t, J=6.1 Hz, 4H) , 2.47-2.58 (m, 8H), 2.35-2.41 (m, 7H), 2.23-2.28 (m, 5H), 1.95-2.00 (m, 8H), 1.79-1.86 (m, 2H), 1.52-1.59 (m, 5H), 1.17-1.32 (m, 37H), 0.82 (t, J= 6.8 Hz, 6H) ppm

ER-MS m/z=866.4 (MH+).

Synthesis of example 24:

Intermediate 24а: (9Z, 12Z)-octadeca-9,12-dien-1-yl-2,6-bis((9Z, 12Z)-octadeca-9,12-dien-1-yloxy)isonicotinate

A solution of citrazic acid (1.8 g, 11.6 mmol) in DMF (60 ml) was stirred at room temperature and linoleyl mesylate (16.0 g, 46.4 mmol) and potassium carbonate (8.02 g, 58.0 mmol). The mixture was heated to 80°C overnight and then cooled to room temperature, and added water (50 ml) and EtOAc (100 ml). The organic phase was collected and dried over sodium sulfate and then the volatiles were removed under reduced pressure. The resulting crude product was purified on silica gel using heptane/EtOAc as eluent to provide 5.2 g of desired product.

¹³ C NMR (100 MHz, CDCl3) δ 165.3, 163.5, 142.9, 130.2, 130.1, 130.1, 120.8, 127.9, 101.2, 66.6, 65.7, 31.6, 29.7, 29.7, 29.5, 29.4, 29.4, 29.3, 29.3, 29.1, 28.6, 27.2, 26, 1, 26.0, 25.6, 22.6, 14.1 ppm

Intermediate 24b: 2,6-bis((9Z,12Z)-octadeca-9,12-dien-1-yloxy)isonicotinic acid

ABOUT

ABOUT.

Intermediate 24a (3.06 g, 3.40 mmol) was stirred in EtOH (15 mL) and potassium hydroxide (329 mg, 5.10 mmol) was added. The cloudy solution turned clear and water (10 ml) and THF (8 ml) were added. The resulting mixture was stirred overnight at room temperature and then the volatiles were removed under reduced pressure. The resulting residue was purified on silica gel using heptane/EtOAc as eluent to provide 1.6 g of desired product.

ER-MS m/z=652.4 (MH+).

Compound of Example 24: 2-(dimethylamino)ethyl-2,6-bis((9Z,12Z)-octadeca-9,12-dien-1-yloxy)isonicotinate

Intermediate 24b (311 mg, 0.477 mmol) was stirred in DMF (15 ml) and HBTU (651 mg, 1.717 mmol), HOBt (120 mg, 0.444 mmol) and DIEA (0.582 ml, 3.34 mmol) were added. The reaction mixture was stirred at room temperature overnight, the reaction mixture was poured into water (50 ml) and the resulting mixture was extracted with EtOAc. The organic phases were collected, dried over sodium sulfate and then concentrated under reduced pressure. The resulting crude product was purified on silica gel using heptane/EtOAc as eluent to provide 207 mg of desired product.

¹ H NMR (400 MHz, CDCl ₃ ) δ 6.85 (s, 2H), 5.32-5.44 (m, 8H), 4.42 (t, J=8 Hz, 2H), 4.27 (t, J=8 Hz, 2H), 2.79 (t, J=8 Hz, 2H), 2.71 (t, J=8 Hz, 2H), 2.34 (s, 6H), 2, 07 (dd, J=8 Hz, 8H), 1.78 (k, J=8 Hz, 4H), 1.27-1.48 (m, 32H), 0.91 (t, J=8 Hz, 6H) ppm

ER-MS m/z=723.4 (MH+).

Example 25 3-(dimethylamino)propyl-2,6-bis((9Z,12Z)-octadeca-9,12-dien-1-yloxy)isonicotinate

Example 25 can be prepared using methods similar to those used to prepare Example 24.

1H NMR (400 MHz, CDCl3) δ 6.84 (s, 2H), 5.32-5.44 (m, 8H), 4.37 (t, J=8 Hz, 2H), 4.28 (t , J=8 Hz, 4H), 2.79 (t, J=8 Hz, 4H), 2.45 (t, J=8 Hz, 2H), 2.29 (s, 6H), 2.07 ( dd, J=8 Hz, 8H), 1.97 (k, J=5 Hz, 2H), 1.78 (k, J=8 Hz, 6H), 1.27-1.48 (m, 32H) , 0.90 (t, J=8 Hz, 6H) ppm

ER-MS m/z=737.5 (MH+).

Synthesis of example 26:

Intermediate 26а: (9Z,9'Z, 12Z, 12'Z)-5-formyl-1,3-phenylene-bis(octadeca-9,12-dienoate)

A solution of 3,5-dihydroxybenzaldehyde (500 mg, 3.62 mmol) in DCE (9 ml) was placed in a microwave vial and linoleic acid (2.03 g, 7.24 mmol), DIEA (1.26 ml, 7. 24 mmol), DMAP (442 mg, 3.62 mmol) and EDC (1.74 g, 9.05 mmol). The reaction mixture was heated to 80°C in a microwave reactor for 20 min and then stored at 4°C for 2 days. The volatiles were removed under reduced pressure and the resulting product was purified on silica gel using heptane/EtOAc as eluent to provide 1.44 g of desired product.

¹³ C NMR (100 MHz, CDCl ₃ ) δ 190.2, 171.6, 151.7, 138, 130.3, 130.0, 128.1, 127.9, 121.5, 119.9, 34 .3, 31.5, 29.6, 29.4, 29.2, 29.1, 29.1, 27.2, 27.2, 25.6, 24.8, 22.6, 14.1 ppm

Intermediate 26b: (9Z,9'Z, 12Z, 12'Z)-5-(hydroxymethyl)-1,3-phenylene-bis(octadeca-9,12-dienoate)

Intermediate 26a (1.44 g, 2.17 mmol) was stirred in THF (18 ml) and EtOH (18 ml) and the resulting solution was cooled in an ice bath. Sodium borohydride (25 mg, 0.65 mmol) was added and the reaction mixture was stirred at 0° C. for 1 hour. The reaction mixture was diluted with EtOAc and washed twice with water. The resulting organic layer was dried over sodium sulfate, and then the volatiles were removed under reduced pressure. The crude product was purified on silica gel using heptane/EtOAc followed by DCM/MeOH as eluent to provide 850 mg (59%) of the desired product.

1H NMR (400 MHz, CDCl ₃ ) δ 7.01-6.97 (m, 2H), 6.82-6.80 (m, 1H), 5.45-5.30 (m, 8H), 4 .71 (s, 2H), 2.82.75 (m, 4H), 2.58-2.51 (m, 4H), 2.12-2.01 (m, 8H), 1.80-1.70 (m, 4H), 1.45-1.23 (m, 28H), 0.93-0.86 (m, 6H)

ppm

Compound of Example 26: (9Z,9'Z,12Z,12'Z)-5-(((3-(dimethylamino)propanoyl)oxy)methyl)-1,3phenylene-bis(octadeca-9,12-dienoate)

- 48 040257

To Intermediate 26b (330 mg, 0.496 mmol) in DCM (30 ml) was added 3-dimethylaminopropionic acid hydrochloride (114 mg, 0.744 mmol), EDC (143 mg, 0.744 mmol), DMAP (6 mg, 0.05 mmol) and TEA (0.277 ml, 1.98 mmol). The resulting mixture was stirred overnight at room temperature, then purified directly on formic acid modified silica gel using heptane/EtOAc followed by DCM/MeOH as eluent to provide 428 mg of title product as the formic acid salt and 697 mg as free base. Characterization of salt of formic acid.

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.41 (d, J=1.26 Hz, 0.5H, formate), 6.99 (d, J=2.01 Hz, 2H), 6.89 (t, J=2.13 Hz, 1H), 5.26-5.48 (m, 8H), 5.13 (s, 2H), 2.93 (k, J=7.03 Hz, 2H) , 2.80 (t, J=6.80 Hz, 4H), 2.67-2.77 (m, 2H), 2.56 (t, J=7.53 Hz, 4H), 2.45 ( d, J=5.52 Hz, 6H), 1.97-2.17 (m, 8H), 1.75 (kv, J=7.47 Hz, 4H), 1.23-1.50 (m , 28H), 0.81-0.98 (m, 6H) ppm ER-MS m/z=7 64.6 (MH+).

Example 27: (9Z,9'Z, 12Z, 12'Z)-5-(((4-(dimethylamino)butanoyl)oxy)methyl)-1,3-phenylene-bis(octadeca-9,12-dienoate)

Example 27 can be obtained using methods similar to those used to obtain example 26.

¹ H NMR (400 MHz, CDCl ₃ ) δ 6.97 (d, J=2.0 Hz, 2H), 6.87 (t, J=2.1 Hz, 1H), 5.27-5.48 (m, 8H), 5.10 (s, 2H), 2.78 (t, J=6.5 Hz, 4H), 2.54 (t, J=7.5 Hz, 4H), 2.41 (t, J=7.4 Hz, 2H), 2.27-2.35 (m, 2H), 2.22 (s, 6H), 2.002.12 (m, 8H), 1.82 (q, J=7.3Hz, 2H), 1.74(q, J=7.5Hz, 4H), 1.23-1.46(m, 28H), 0.89(t, J=7.0 Hz, 6H) ppm

ER-MS m/z=778.5 (MH+).

Synthesis of example 28.

Intermediate 28а: (2,6-6uc((9Z, 12Z)-octadeca-9,12-dien-1-yloxy)pyridin-4-yl)methanol

Intermediate 24b (3.5 g, 3.89 mmol) was stirred in THF (50 ml) and the solution was cooled in an ice bath. Lithium aluminum hydride (570 mg, 15 mmol) was slowly added to this cold solution. After the addition, the reaction mixture was allowed to warm to room temperature and stirred overnight. Ice was added carefully and the resulting mixture was extracted with EtOAc. The organic layers were collected and dried over sodium sulfate and then concentrated under reduced pressure. The resulting crude product was purified on silica gel using heptane/EtOAc as eluent to provide 1.3 g of desired product.

¹ H NMR (400 MHz, CDCl ₃ ) δ 6.27 (s, 2H), 5.44-5.27 (m, 8H), 4.63 (s, 2H), 4.23 (t, J= 5.7 Hz, 4H), 2.82.75 (m, 4H), 2.10-2.02 (m, 8H), 1.80-1.72 (m, 4H), 1.47-1.23 (m, 32H), 0.93-0.86 (m, 6H) ppm

Example 28: (2,6-bis((9Z,12Z)-octadeca-9,12-dien-1-yloxy)pyridin-4-yl)methyl-3(diethylamino)propanoate

Intermediate 28a (334 mg, 0.523 mmol) was stirred in DCM (20 ml) with 3-diethylaminopropionic acid hydrochloride (143 mg, 0.785 mmol). HATU (397 mg, 1.05 mmol) was added and the reaction mixture was stirred overnight at room temperature. Volatiles were removed under reduced pressure and the crude product was purified on silica gel using heptane/EtOAc as eluent to provide 171 mg of desired product.

¹ H NMR (400 MHz, CDCl3) δ 6.25 (s, 2H), 5.32-5.44 (m, 8H), 5.04 (s, 2H), 4.24 (t, J=8 Hz, 4H), 2.78-2.87 (m, 6H), 2.55 (dd, J=8 Hz, 6H), 2.07 (dd, J=8 Hz, 8H), 1.78 ( k, J=8 Hz, 4H), 1.27-1.48 (m, 32H), 1.05 (t, J=8 Hz, 6H), 0.91 (t, J=8 Hz, 6H) ppm

ER-MS m/z=765.7 (MH+).

Example 29 (9Z,9'Z,12Z,12'Z)-(5-(((3-(dimethylamino)propanoyl)oxy)methyl)-1,3-phenylene)bis(methylene)bis(octadeca-9 ,12-dienoate)

Example 29 can be prepared using methods similar to those used

- 49 040257 to obtain Intermediate 33a and example 18.

1H NMR (400 MHz, CDCl3) δ 7.30 (s, 3H), 5.28-5.45 (m, 8H), 5.14 (s, 2H), 5.11 (s, 4H), 2 .77 (t, J=6.5 Hz, 4H), 2.61-2.70 (m, 2H), 2.51-2.59 (m, 2H), 2.37 (t, J=7 .5 Hz, 4H), 2.25 (s, 6H), 2.05 (k, J=6.9 Hz, 8H), 1.59-1.73 (m, 6H), 1.22-1 .43 (m, 26H), 0.89 (t, J=7.0 Hz, 6H) ppm

ER-MS m/z=792.4 (MH+).

Examples 30-31 can be obtained using methods similar to those used to obtain example 28.

Example 30 (2,6-6uc((9Z, 12Z)-Octadeca-9,12-dien-1-yloxy)pyridin-4-yl)methyl-4-(dimethylamino)butanoate

1H NMR (400 MHz, CDCl ₃ ) δ 6.24 (s, 2H), 5.46-5.33 (m, 8H), 5.05 (s, 2H), 4.24 (t, J=6 .7 Hz, 4H), 2.812.72 (m, 7H), 2.66-2.61 (m, 2H), 2.34 (s, 6H), 2.02-2.11 (m, 8H) , 1.80-1.72 (m, 4H), 1.49-1.40 (m, J=6.0, 13.8 Hz, 5H), 1.40-1.25 (m, 32H) , 0.90 (t, J=6.9 Hz, 6H) ppm

ER-MS m/z=751.7 (MH+).

Example 31 (2,6-6uc((9Z, 12Z)-Octadeca-9,12-dien-1-yloxy)pyridin-4-yl)methyl-3-(dimethylamino)propanoate

1H NMR (400 MHz, CDCl3) δ 6.51 (d, J=2.0 Hz, 2H), 6.42 (t, J=2.0 Hz, 1H), 5.44-5.30 (m , 8H), 5.11 (s, 2H), 4.27 (t, J=6.4 Hz, 4H), 4.03 (t, J=6.1 Hz, 4H), 3.30 (s , 2H), 2.79 (t, J=6.4 Hz, 4H), 2.44 (s, 6H), 2.32 (t, J=7.5 Hz, 4H), 2.17-2 .10 (m, 4H), 2.09-2.03 (m, 9H), 1.68-1.57 (m, 10H), 1.42-1.26 (m, 34H), 0.91 (t, J=6.8 Hz, 6H).

ER-MS m/z=737.5 (MH+).

Synthesis of Example 32:

Intermediate 32а: (2,6-6uc((9Z, 12Z)-Octadeca-9,12-dien-1-yloxy)pyridin-4-yl)methylmethane-

Intermediate 28a (330 mg, 0.517 mmol) was stirred in DCM (30 ml) with TEA (0.290 ml, 2.07 mmol) and the resulting solution was cooled in an ice bath. MsCl (0.08 ml, 1.0 mmol) was added and the resulting mixture was allowed to warm to room temperature with stirring for 4 h. The reaction mixture was treated with HCl (30 ml, 1M in water) and DCM (50 ml) and the organic layer was collected . The product was dried over sodium sulfate and the volatiles removed under reduced pressure to give 360 mg of product which was used without further purification.

1H NMR (400 MHz, CDCl3) δ 6.27-6.36 (s, 2H), 5.32-5.44 (m, 10H), 5.14 (s, 1H), 4.39-4, 50 (m, 1H), 4.174.30 (m, 5H), 3.41 (k, J=7.03 Hz, 1H), 3.01-3.06 (m, 2H), 2.74-2 .84 (m, 5H), 2.07 (k, J=6.86 Hz, 10H), 1.70-1.87 (m, 5H), 1.66 (s, 2H), 1.24- 1.55 (m, 34H), 0.91 (t, J=6.78 Hz, 6H) ppm

Example 32 1-(2,6-bis((9Z,12Z)-Octadeca-9,12-dien-1-yloxy)pyridin-4-yl)-N,Ndimethylmethanamine

Intermediate 32a (360 mg, 0.503 mmol) was stirred in DMF (3 mL) with dimethylamine (3 mL, 2M, 11.9 mmol) and the mixture was heated in a microwave to 140°C for 30 min. This heating was repeated until all of the starting material had reacted as determined by TLC. Volatiles were removed under reduced pressure and the resulting crude product was purified on silica gel using heptane/EtOAc as eluent to provide 123 mg of desired product.

1H NMR (400 MHz, CDCl ₃ ) δ 6.27 (s, 2H), 5.32-5.44 (m, 8H), 4.26 (t, J=8 Hz, 4H), 3.33 ( s, 2H) 2.80 (t, J=8 Hz, 4H), 2.26 (s, 6H), 2.07 (dd, J=8 Hz, 8H), 1.78 (c, J=5 Hz, 4H), 1.27-1.48 (m, 32H), 0.92 (t, J=8 Hz, 6H) ppm

ER-MS m/z=665.5 (MH+).

Synthesis of example 33.

Intermediate 33а: (9Z,9'Z,12Z, 12'Z)-(5-(hydroxymethyl)-1,3-phenylene)bis(methylene)bis(octadeca9,12-dienoate)

- 50 040257 about

Linoleic acid (3.42 g, 12.19 mmol) was stirred with EDC (2.33 g, 12.2 mmol) in DCM (30 ml). After dissolution, DIEA (2.60 ml, 14.9 mmol) and DMAP (145 mg, 1.19 mmol) were added. After 10 minutes of stirring, benzene-1,3,5-triyl trimethanol (1.0 g, 6.0 mmol) was added and the resulting mixture was stirred at room temperature for 3 days. The volatiles were removed under reduced pressure and the resulting crude product was purified on silica gel using heptane/EtOAc as eluent to provide 1.36 g of desired product.

TLC (silica gel, 20% EtOAc in heptane): Rf=0.12.

Intermediate 33b: (9Z,9'Z, 12Z, 12'Z)-(5-formyl-1,3-phenylene)bis(methylene)bis(octadeca-9,12-dienoate)

Intermediate 33a (214 mg, 0.309 mmol) was stirred in DCM (30 ml) and PDC (244 mg, 0.648 mmol) was added. The reaction mixture was stirred at room temperature overnight. The volatiles were removed under reduced pressure and the resulting crude product was purified on silica gel using heptane/EtOAc as eluent to provide 210 mg of desired product.

TLC (silica gel, 20% EtOAc in heptane): Rf=0.44.

Compound of Example 33: (9Z,9'Z,12Z,12'Z)-(5-((dimethylamino)methyl)-1,3-phenylene)bis(methylene)bis(octadeca-9,12-dienoate)o

Intermediate 33b (210 mg, 0.30 mmol) was stirred in DCE (10 ml) and dimethylamine (0.53 ml, 2.0 M in THF, 1.06 mmol) was added. Acetic acid (0.017 ml, 0.304 mmol) and sodium triacetoxyborohydride (129 mg, 0.608 mmol) were added and the product was stirred at room temperature overnight. Volatiles were removed under reduced pressure and the resulting crude product was purified on silica gel using heptane/EtOAc followed by DCM/MeOH as eluent to provide 186 mg of desired product.

1H NMR (400 MHz, CDCl ₃ ) δ 7.29-7.25 (m, 2H), 7.25-7.22 (m, 1H), 5.44-5.27 (m, 8H), 5 .10 (s, 4H), 3.44 (s, 2H), 2.81-2.74 (m, 4H), 2.36 (t, J=7.5 Hz, 4H), 2.25 ( s, 6H), 2.10-2.01 (m, 8H), 1.70-1.58 (m, 4H), 1.42-1.21 (m, 28H), 0.89 (t, J=6.9 Hz, 6H) ppm

ER-MS m/z=721.1 (MH+).

Examples 34-36 can be prepared using methods similar to those used to prepare example 33.

Example 34 (5-((Dimethylamino)methyl)-1,3-phenylene)bis(methylene)ditridecanoate

1H NMR (400 MHz, CDCl ₃ ) δ 7.21 (s, 2H); 7.18 (s, 1H), 5.03 (s, 4H); 3.42 (br. s, 2H); 2.28 (t, J=7.5 Hz, 4H); 2.22 (s, 6H); 1.61-1.53 (m, 4H); 1.27-1.15 (m, 36H); 0.81 (t, J=6.8 Hz, 6H) ppm

ER-MS m/z=588.5 (MH+).

Example 35 (5-((Dimethylamino)methyl)-1,3-phenylene)bis(methylene)bis(3-octylundecanoate)

1H NMR (400 MHz, CDCls) δ 7.26 (s, 2H), 7.24 (s, 1H), 5.10 (s, 4H), 3.43 (s, 2H), 2.29 (d , J=7.4 Hz, 2H), 2.25 (s, 6H), 1.87 (m, 2H), 1.31-1.25 (m, 58H), 0.91-0.87 ( m, 12H) ppm

ER-MS m/z=756.6 (MH+).

Example 36 (9Z,12Z)-3-((Dimethylamino)methyl)-5-(((3-octylundecanoyl)oxy)methyl)benzyloctadeca-9,12-dienoate

- 51 040257

1H NMR (400 MHz, CDCl ₃ ) δ 7.28-7.25 (m, 2H), 7.25-7.22 (m, 1H), 5.44-5.29 (m, 4H), 5 .10 (s, 4H), 3.43 (s, 2H), 2.83-2.73 (m, 2H), 2.36 (t, J=7.5 Hz, 2H), 2.29 ( d, J=6.8 Hz, 2H), 2.25 (s, 6H), 2.10-2.00 (m, 4H), 1.921.80 (m, 1H), 1.71-1.57 (m, 2H), 1.43-1.17 (m, 42H), 0.94-0.83 (m, 9H) ppm.

ER-MS m/z=736.6 (MH+).

Synthesis of example 37.

Intermediate 37a: 5-formylisophthalic acid

To a solution of 3-formyl-5-(methoxycarbonyl)benzoic acid (1 g, 4.80 mmol) in THF (25 ml) was added LiOH (12.01 ml, 24.02 mmol) and stirred at room temperature for 16 h Partial hydrolysis was observed. The reaction mixture was heated to 50° C. for an additional 16 hours. The reaction mixture was diluted with EtOAc (50 ml) and water (50 ml) and the pH was adjusted to neutral with 1 N HCl. The organic layer was collected, washed with water (2x50 ml) and dried over sodium sulfate. The volatiles were removed under reduced pressure and the resulting product was used without further purification.

Intermediate 3 7b: di((92,122)-octadeca-9, 12-dien-1 -yl)-5-formyl isophthalate

Intermediate 37a (700 mg, 0.36 mmol) was stirred in DCM (15 ml) and oxalyl chloride (3.16 ml, 36 mmol) was added along with a drop of DMF. The resulting mixture was stirred overnight at room temperature. The volatiles were removed under reduced pressure and the resulting residue was redissolved in THF (10 ml). Linoleyl alcohol (2.02 g, 7.57 mmol) was added followed by TEA (2.51 ml, 18.0 mmol) and the resulting mixture was stirred in an ice bath for 3 h. The reaction mixture was diluted with EtOAc (50 ml) and water (50 ml). The organic layer was collected, washed with water (2x50 ml) and dried over sodium sulfate. Volatiles were removed under reduced pressure and the resulting crude product was purified on silica gel using heptane/EtOAc as eluent to provide 350 mg of desired product.

1H NMR (400 MHz, CDCl3) δ 8.66 (s, 1H), 8.26 (s, 2H), 5.25-5.53 (m, 8H), 4.16-4.43 (m, 4H), 3.47-3.67 (m, 1H), 2.77 (t, J=5.68 Hz, 4H), 2.05 (d, J=6.32 Hz, 7H), 1, 91 (br.s, 2H), 1.65-1.85 (m, 4H), 1.37-1.53 (m, 5H), 1.09-1.37 (m, 28H), 0. 88 (t, J=6.19 Hz, 6H) ppm

Intermediate 37c: di((92,122)-octadeca-9,12-dien-1-yl)-5-(hydroxymethyl)isophthalate o

Sodium borohydride (17.8 mg, 0.47 mmol) was added to a solution of Intermediate 37b (250 mg, 0.36 mmol) in THF (30 ml) and EtOH (15 ml). The reaction mixture was stirred for 30 min at room temperature and then diluted with EtOAc (50 ml) and water (50 ml). The organic layer was collected, washed with water (2x50 ml) and dried over sodium sulfate. The volatiles were removed under reduced pressure and the resulting crude product was purified on silica gel using heptane/EtOAc as eluent to provide 260 mg of desired product.

1H NMR (400 MHz, CDCl ₃ ) δ 8.60 (t, J=1.51 Hz, 1H), 8.13-8.32 (m, 2H), 5.22-5.46 (m, 8H ), 4.82 (d, J=5.77 Hz, 2H), 4.34 (t, J=6.78 Hz, 4H), 2.77 (t, J=6.53 Hz, 4H), 1.93-2.13 (m, 9H), 1.70-1.86 (m, 4H), 1.201.49 (m, 32H), 0.76-1.00 (m, 6H) ppm .

Example 37: di((92,122)-octadeca-9,12-dien-1-yl)-5-(((3-(dimethylamino)propanoyl)oxy)methyl)isophthalate

To a solution of 3-dimethylaminopropionic acid (25 mg, 0.16 mmol) in DCM (3 mL) was added EDC (31 mg, 0.16 mmol) and DMAP (1.32 mg, 0.011 mmol) followed by TEA (0. 06 ml, 0.43 mmol). The resulting solution was stirred for 30 min at room temperature and added

- 52 040257

Intermediate 37c (75 mg, 0.11 mmol). The reaction mixture was stirred for 16 h and then diluted with DCM (20 ml) and water (20 ml). The organic layer was collected, washed with water (2x20 ml) and then dried over magnesium sulfate. The volatiles were removed under reduced pressure and the resulting crude product was purified on silica gel using heptane/EtOAc as eluent to provide 41 mg of desired product.

1H NMR (400 MHz, CDCl ₃ ) δ 8.63 (t, J=1.6 Hz, 1H), 8.20 (d, J=1.8 Hz, 2H), 5.47-5.26 ( m, 8H), 5.22 (s, 2H), 4.35 (t, J=6.8 Hz, 4H), 2.89-2.74 (m, 6H), 2.74-2.65 (m, J=7.0 Hz, 2H), 2.39 (s, 6H), 2.12-1.95 (m, 8H), 1.87-1.71 (m, 4H), 1, 49-1.19 (m, 33H), 0.89 (t, J=7.0 Hz, 6H) ppm

ER-MS m/z=792.4 (MH+).

Synthesis of example 38.

Intermediate 38a: 4-(vinyloxy)butylmethanesulfonate

A solution of 4-(vinyloxy)butan-1-ol (50 g, 430 mmol) in DCM (430 ml) was cooled in an ice bath. To this solution was added TEA (90 ml, 646 mmol) followed by dropwise addition of methanesulfonyl chloride (36.9 ml, 473 mmol). During the second half of the addition, a white precipitate formed, and as the addition was complete, the reaction mixture turned pale orange. The reaction mixture was stirred overnight, allowing the ice to melt and the reaction mixture to reach ambient temperature. The reaction mixture was poured into a separatory funnel and diluted with 400 ml saturated sodium bicarbonate solution (aq) and 400 ml EtOAc. The layers were separated and the aqueous layer was extracted with 300 ml EtOAc two more times. The EtOAc layers were washed with 200 ml of saturated sodium bicarbonate solution, then with water, and the EtOAc layer was then dried over sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to give 84 g (100%) of the desired product.

Ή NMR (400 MHz, CDCl3) δ 6.47 (dd, J=6.8, 14.3 Hz, 1H), 4.29 (t, J=6.3 Hz, 2H), 4.19 (dd , J=2.0, 14.3 Hz, 1H), 4.02 (dd, J=2.0, 6.8 Hz, 1H), 3.74 (t, J=5.9 Hz, 2H) , 3.03 (s, 3H), 1.96-1.85 (m, 2H), 1.86-1.73 (m, 2H)

ppm

Intermediate 38b: 3,5-bis(4-(vinyloxy)butoxy)benzaldehyde

Intermediate 38a (84 g, 435 mmol) was stirred in DMF (400 ml) and 3,5 dihydroxybenzaldehyde (27.3 g, 198 mmol) was added followed by potassium carbonate (109 mg, 791 mmol). The reaction mixture was heated to 80° C. overnight. After cooling to room temperature, the reaction mixture was diluted with EtOAc (600 ml) and water (700 ml). The organic layer was collected and the aqueous layer was back extracted with EtOAc (300 ml). The organic layers were combined and washed with water (4x300 ml), dried over sodium sulfate and then the volatiles were removed under reduced pressure. The resulting crude product was purified on silica gel using heptane/EtOAc as eluent to provide 56 g of desired product.

ER-MS m/z=335.1 (MH+).

Intermediate 38с: 1-(3,5-bis(4-(vinyloxy)butoxy)phenyl)-N,N-dimethylmethanamine

Intermediate 38b (37 g, 111 mmol) was stirred with dimethylamine (116 ml, 2M in THF, 332 mmol) and acetic acid (6.33 ml, 111 mmol) in DCM (400 ml). To this mixture was added sodium triacetoxyborohydride (58.6 g, 277 mmol) and the reaction mixture was stirred at room temperature overnight. Saturated sodium bicarbonate solution (800 ml) and EtOAc (1000 ml) were added to the reaction mixture. The organic phase was collected and the aqueous phase was extracted with EtOAc (500 ml). The combined organic phases were dried over sodium sulfate and the volatiles were removed under reduced pressure. The resulting crude product was purified on silica gel using heptane/EtOAc as eluent to provide 34 g of desired product.

ER-MS m/z=364.9 (MH+).

Intermediate 38d: 4,4'-((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(butan-1-ol)

To a solution of Intermediate 38с (42 g, 116 mmol) in EtOAc (200 mL) was added HCl (87 mL, 4M in dioxane, 348 mmol). After completion of the reaction under TLC monitoring, saturated aqueous sodium bicarbonate (500 ml) was added and the pH was adjusted to 10 by adding solid potassium carbonate. EtOAc (600 ml) was added and the organic layer was collected. The aqueous layer was extracted with EtOAc (3x500 ml) and the combined organic layers were dried over sodium sulfate. The volatiles were removed under reduced pressure to give 35 g of product which was used without further purification.

1H NMR (400 MHz, CDCl ₃ ) δ 6.50 (d, J=2.2, Hz, 2H), 6.37 (t, J=2.3 Hz, 1H), 4.01 (t, J = 6.1 Hz, 4H), 3.793.68 (m, 6H), 3.36 (s, 2H), 2.26 (s, 6H), 1.93-1.82 (m, 4H), 1 .82-1.67 (m, 4H) ppm

Compound of Example 38: (9Z,9'Z,12Z,12'Z)-((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(butane-4,1-diyl) bis(octadeca-9,12-dienoate)

To a solution of Intermediate 36 (10 g, 32 mmol) in DCM (161 ml) were added DMAP (392 mg, 3.21 mmol), DIEA (16.8 ml, 96 mmol) and linoleic acid (18.9 g, 67 4 mmol). Added EDC (14.8 g, 77 mmol) and the substance was left to mix at room temperature overnight. Saturated aqueous sodium bicarbonate solution (500 ml) was added and the resulting mixture was extracted with EtOAc (3x600 ml). The combined organic layers were dried over sodium sulfate and then the volatiles were removed under reduced pressure. The resulting crude product was purified on silica gel using heptane/EtOAc as eluent to give 18.6 g of the expected product.

1H NMR (400 MHz, CDCl3) δ 6.50 (br.s, 2H), 6.36 (br.s, 1H), 5.28-5.45 (m, 8H), 4.14 (t, J=6.05 Hz, 4H), 3.98 (t, J=5.70 Hz, 4H), 3.39 (br.s, 2H), 2.78 (t, J=6.55 Hz, 4H), 2.16-2.41 (m, 10H), 1.98-2.12 (m, 8H), 1.77-1.90 (m, 8H), 1.52-1.71 ( m, 4H), 1.20-1.43 (m, 28H), 0.90 (t, J=6.85 Hz, 6H) ppm.

ER-MS m/z=836.7 (MH+).

Synthesis of example 39:

Intermediate 3 9a: (9Z,9'Z, 12Z, 12'Z)-((5-formyl-1,3-phenylene)bis(oxy))bis(propane-3,1-diyl)bis(octadeca-9 ,12-dienoate)

Intermediate 39a can be prepared in a manner analogous to that of Intermediate 18b using linoleic acid and 1,3-propanediol as starting materials.

TLC (silica gel, 40% EtOAc in heptane): R _f = 0.74.

Compound of Example 39: (9Z,9'Z,12Z,12'Z)-((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(propane-3,1-diyl) bis(octadeca-9,12-dienoate)

To a solution of Intermediate 39a (2.42 g, 3.11 mmol) in DCE (20 ml) was added dimethylamine (3.25 ml, 2.0 M in THF, 6.5 mmol) and acetic acid (0.18 ml, 3.1 mmol) followed by the addition of sodium triacetoxyborohydride (1.32 g, 6.21 mmol). The reaction mixture was stirred at room temperature overnight, and then a saturated aqueous sodium bicarbonate solution was added, followed by DCM. The organic layer was collected and then washed with additional saturated aqueous sodium bicarbonate. The resulting aqueous layer was again extracted with DCM and the organic layers were combined and dried over sodium sulfate. Volatiles were removed under reduced pressure. The resulting crude product was purified on silica gel using heptane/EtOAc followed by DCM/MeOH as eluent to give 1.4 g of the desired product.

1H NMR (400 MHz, CDCl3) δ 0.89 (t, J=8.00 Hz, 6H) 1.21-1.43 (m, 28H) 1.55-1.71 (m, 4H) 1, 98-2.14 (m, 12H) 2.25 (s, 6H) 2.30 (t, J=7.65 Hz, 4H) 2.77 (t, J=8.00 Hz, 4H) 3, 35 (s, 2H), 4.02 (t, J=6.15 Hz, 4H) 4.25 (t, J=6.40 Hz, 4H) 5.24-5.52 (m, 8H) 6 .35 (t, J=2.26 Hz, 1H) 6.48 (d, J=2.26 Hz, 2H) ppm

ER-MS m/z=809.2 (MH+).

Examples 40-64 can be prepared using methods similar to those used to prepare example 38, example 39 and example 52.

Example 40 (9Z,9'Z, 12Z, 12'Z)-((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(ethane-2,1diyl)bis( octadeca-9,12-dienoate)

- 54 040257

1H NMR (400 MHz, CDCl3) δ 0.88 (t, J=8.00 Hz, 6H) 1.22-1.42 (m, 28H) 1.56-1.71 (m, 4H) 1, 98-2.10 (m,

8H) 2.23 (s, 6H) 2.34 (t, J=7.53 Hz, 4H) 2.76 (t, J=6.53 Hz, 4H) 3.34 (s, 2H), 4 .08-4.20 (m, 4H) 4.35-4.46 (m,

4H) 5.26-5.44 (m, 8H), 6.38 (t, J=2.26 Hz, 1H) 6.51 (d, J=2.26 Hz, 2H) ppm.

ER-MS m/z=781.3 (MH+).

Example 41 ((5-((Dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(octane-8,1-diyl)dihexanoate

1H NMR (400 MHz, CDCl ₃ ) δ 0.90 (t, J=8.00 Hz, 6H) 1.29-1.52 (m, 24H) 1.63 (q, J=7.45 Hz, 8H) 1.711.82 (m, 4H) 2.26 (s, 6H) 2.30 (t, J=7.58 Hz, 4H) 3.37 (s, 2H), 3.93 (t, J= 6.57 Hz, 4H) 4.06 (t, J=6.69 Hz, 4H) 6.35 (t, J=2.27 Hz, 1H) 6.47 (d, J=2.27 Hz, 3H) ppm

ER-MS m/z=620.2 (MH+).

Example 42 ((5-((Dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(octane-8,1-diyl)dioctanoate

1H NMR (400 MHz, CDCI3) δ 0.88 (t, J=8.00 Hz, 6H) 1.19-1.50 (m, 32H) 1.55-1.68 (m, 8H) 1, 71-1.85 (m, 4H) 2.21-2.39 (m, 10H) 3.40 (br.s, 2H) 3.94 (t, J=6.44 Hz, 4H) 4.07 (t, J=6, 69 Hz, 4H) 6.36 (t, J=2.27 Hz, 1H) 6.49 (d, J=2.02 Hz, 2H) ppm

ER-MS m/z=676.6 (MH+).

Example 43 ((5-((Dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(decane-10,1-diyl)dioctanoate

1H NMR (400 MHz, CDCl ₃ ) δ 0.88 (t, J=8.00 Hz, 6H) 1.21-1.50 (m, 40H) 1.62 (q, J=6.95 Hz, 8H) 1.701.82 (m, 4H) 2.25 (s, 6H) 2.29 (t, J=7.58 Hz, 4H) 3.35 (s, 2H), 3.93 (t, J= 6.57 Hz, 4H) 4.06 (t, J=6.82 Hz, 4H) 6.35 (t, J=2.27 Hz, 1H) 6.46 (d, J=2.27 Hz, 2H) ppm

ER-MS m/z=732.1 (MH+).

Example 44 ((5-((Dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(decane-10,1-diyl)dihexanoate

1H NMR (400 MHz, CDCl3) δ 0.89 (t, J=8.00 Hz, 6H) 1.23-1.51 (m, 32H) 1.57-1.69 (m, 8H) 1, 70-1.82 (m, 4H) 2.24-2.39 (m, 10H) 3.43 (br.s, 2H) 3.94 (t, J=6.57 Hz, 4H) 4.06 (t, J=6.69 Hz, 4H) 6.37 (t, J=2.15 Hz, 1H) 6.50 (d, J=1.52 Hz, 2H) ppm

ER-MS m/z=676.6 (MH+).

Example 45 (9Z,9'Z, 12Z, 12'Z)-((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(hexane-6,1diyl)bis( octadeca-9,12-dienoate)

1H NMR (400 MHz, CDCl ₃ ) δ 0.90 (t, J=8.00 Hz, 6H) 1.23-1.52 (m, 36H) 1.65 (dt, J=14.59.7 .48 Hz, 8H) 1.73-1.83 (m, 4H) 1.99-2.11 (m, 8H) 2.24 (s, 6H) 2.30 (t, J=7.58 Hz , 4H) 2.78 (t, J=6.57 Hz, 4H) 3.39 (br.s, 2H) 3.94 (t, J=6.44 Hz, 4H) 4.08 (t, J =6.69 Hz, 4H) 5.25-5.49 (m, 8H) 6.35 (t, J=2.27 Hz, 1H) 6.46 (d, J=2.27 Hz, 2H) ppm

ER-MS m/z=892.8 (MH+).

Example 46 (9Z,9'Z, 12Z, 12'Z)-((5-((Dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(octane-8,1diyl)bis( octadeca-9,12-dienoate)

1H NMR (400 MHz, CDCl ₃ ) δ 0.89 (t, J=8.00 Hz, 6H) 1.23-1.51 (m, 44H) 1.55-1.69 (m, 8H) 1 .71-1.82 (m, 4H) 1.99-2.11 (m, 8H) 2.24 (s, 6H) 2.30 (t, J=7.58 Hz, 4H) 2.78 ( t, J=6.57 Hz, 4H) 3.35 (s, 2H), 3.93 (t, J=6.57 Hz, 4H) 4.07 (t, J=6.69 Hz, 4H) 5.28-5.45 (m, 8H) 6.35 (t, J=2.27 Hz, 1H) 6.46 (d, J=2.27 Hz, 2H) ppm

- 55 040257

ER-MS m/z=948.8 (MH+).

Example 47 ((5-((Dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(hexane-6,1-diyl)bis(decanoate)

1H NMR (400 MHz, CDCl ₃ ) δ 6.49-6.43 (m, 2H), 6.37-6.32 (m, 1H), 4.07 (t, J=6.6 Hz, 4H ), 3.93 (t, J=6.4 Hz, 4H), 3.35 (s, 2H), 2.29 (t, J=7.6 Hz, 4H), 2.25 (s, 6H ), 1.84-1.72 (m, 4H), 1.72-1.55 (m, 8H), 1.55-1.36 (m, 8H), 1.36-1.16 (m , 24H), 0.88 (t, J=6.8 Hz, 6H) ppm

ER-MS m/z=676.4 (MH+).

Example 48 ((5-((Dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(hexane-6,1-diyl)dioctanoate

1H NMR (400 MHz, CDCl ₃ ) δ: 6.65-6.58 (m, 2H), 6.44-6.39 (m, 1H), 4.07 (t, J=6.6 Hz, 4H), 3.96 (t, J=6.3 Hz, 4H), 3.72 (s, 2H), 2.52 (s, 6H), 2.29 (t, J=7.5 Hz, 4H), 1.84-1.73 (m, 4H), 1.72-1.56 (m, 8H), 1.55-1.36 (m, 8H), 1.36-1.18 ( m, 16H), 0.91-0.84 (m, 6H) ppm

ER-MS m/z=620.4 (MH+).

Example

49.

(9Z,9'Z, 12Z, 12'Z)-((5-((3-hydroxyazetidin-1-yl)methyl)-1,3-phenylene)bis(oxy))bis(butane-4,1 -diyl)bis(octadeca-9,12-dienoate)

1H NMR (400 MHz, CDCl ₃ ) δ 6.45 (d, J=2.01 Hz, 2H) 6.35 (t, J=1.00 Hz 1H), 5.31-5.44 (m, 8H), 4.11-4.19 (m, 5H), 3.97 (t, J=1.00 Hz, 4H), 3.65-3.76 (m, 2H), 3.60 (s , 2H), 2.79 (t, J=1.00 Hz, 4H), 2.32 (t, J=7.53 Hz, 4H), 2.07 (k, J=6.78 Hz, 8H ), 1.80-1.89 (m, 8H), 1.64 (d, J=16.81 Hz, 6H), 1.24-43 (m, 31H), 0.91 (t, J= 1.00 Hz, 6H) ppm

ER-MS m/z=8 64.6 (MH+).

Example 50 (8Z,8'Z)-((5-((Dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(hexane-6,1-diyl)bis(dodec-8-enoate )

1H NMR (400 MHz, CDCl ₃ ) δ 6.48 (d, J=2.0 Hz, 2H), 6.35 (t, J=2.2 Hz, 1H), 5.43-5.25 ( m, 4H), 4.08 (t, J=6.8 Hz, 4H), 3.94 (t, J=6.4 Hz, 4H), 3.38 (s, 2H), 2.30 ( t, J=7.6 Hz, 4H), 2.27 (s, 6H), 2.07-1.90 (m, 8H), 1.851.70 (m, 4H), 1.71-1.55 (m, 8H), 1.55-1.29 (m, 24H), 0.90 (t, J=7.4 Hz, 6H) ppm ER-MS m/z=728.5 (MH+).

Example 51 (9Z,9'Z,12Z,12'Z)-((5-((Dimethylamino)methyl)-2-methyl-1,3-phenylene)bis(oxy))bis(butane4,1-diyl )bis(octadeca-9,12-dienoate)

1H NMR (400 MHz, CDCl ₃ ) δ 6.48 (s, 2H), 5.32-5.44 (m, 8H), 4.17 (t, J=8 Hz, 4H), 4.01 ( t, J=8 Hz, 4H), 3.37 (s, 2H), 2.79 (t, J=8 Hz, 4H), 2.32 (t, J=8 Hz, 4H), 2.25 (s, 6H), 2.10 (s, 3H), 2.08 (dd, J=8 Hz, 8H), 1.87 (k, J=5 Hz, 8H), 1.64 (m, 4H ), 1.27-1.48 (m, 28H), 0.90 (t, J=8 Hz, 6H) ppm

ER-MS m/z=850.6 (MH+).

Synthesis of example 52.

Intermediate 52а: 8-hydroxyoctyldecanoate

Octane-1,8-diol (21.90 g, 150 mmol), ethyl decanoate (10 g, 49.9 mmol) and p-toluenesulfonic acid trihydrate (0.4 g, 2.103 mmol) were mixed in a round bottom flask. The flask was placed on a rotary evaporator under reduced pressure (100 mbar) and slowly rotated at 100° C. in an oil bath to form a clear melt. After 6 hours the pressure was reduced to 10 mbar and the reaction mixture was held for 15 minutes. The reaction mixture was then diluted with 150 ml of heptane and stirred until the octane diol began to solidify. The reaction mixture was placed in an ice bath and stirred for 10 min, then filtered. The residue was washed with additional 150 ml of heptane and the combined filtrates were transferred to a separating funnel. The heptane layer was washed with 3x150 ml of water, then

- 56 040257 the combined washings were again extracted with 100 ml of heptane. The combined heptane extracts were dried over magnesium sulfate, filtered and concentrated under reduced pressure to give the desired product (with an admixture of the bis-acylated product) which was used without further purification.

In addition, Intermediate 52a can be synthesized as follows. In a round bottom flask, octane-1,8-diol (58.5 g, 400 mmol) was dissolved in pyridine (100 ml). Decanoyl chloride (41.5 ml, 200 mmol) was added dropwise to the resulting solution. The reaction mixture was stirred overnight at ambient temperature. The reaction mixture was concentrated under reduced pressure to give a solid residue. Heptane (300 ml) was added and the resulting suspension was vigorously stirred for 30 minutes. The reaction mixture was filtered and the residue was washed with 500 ml of additional heptane. The filtrate was washed with 1 N aqueous HCl (3x200 ml) and the combined aqueous layers were again extracted with 150 ml of heptane. The combined heptane layers were dried over magnesium sulfate and filtered. The filtrate was concentrated under reduced pressure to give the desired product (contaminated with bis-acylated product), which was used without further purification.

ER-MS m/z=301.6 (MH+).

Intermediate 52b: 8-((methylsulfonyl)oxy)octyldecanoate o

In a round bottom flask, Intermediate 52a (62.7 g, 210 mmol Intermediate 52a) and triethylamine (32.2 ml, 231 mmol) were taken up in dichloromethane (anhydrous, 400 ml). The solution was cooled in an ice bath and methanesulfonyl chloride (16.38 mL, 210 mmol) was added dropwise over 5 minutes. The reaction mixture was stirred in an ice bath for 2 hours, after which time the cooling bath was removed and the reaction mixture was warmed to ambient temperature. The reaction mixture was transferred to a separating funnel and washed with 1N aqueous HCl solution (3x200 ml). The combined aqueous washings were back-extracted with 100 ml dichloromethane. The combined DCM extracts were dried over magnesium sulfate and filtered. The filtrate was concentrated under reduced pressure to give the title compound (68% by weight purity) which was used without further purification.

ER-MS m/z=378.7 (MH+).

Intermediate 52с: ((5-formyl-1,3-phenylene)bis(oxy))bis(octane-8,1-diyl)bis(decanoate)

In a round bottom flask, Intermediate 52b (78.0 g, 206 mmol Intermediate 52b), 3,5 dihydroxybenzaldehyde (12.5 g, 91 mmol) and cesium carbonate (88 g, 272 mmol) were placed in DMF (500 ml). The reaction mixture was heated in an oil bath from ambient temperature to 80°C. After 16 hours at 80° C., the reaction mixture was cooled to ambient temperature, transferred to a separating funnel and diluted with 500 ml of ethyl acetate, 500 ml of heptane and 1500 ml of water. The total volume of the reaction mixture was filtered through a pad of celite, which was washed separately with 500 ml of ethyl acetate and 500 ml of heptane. The filtrate was separated into layers, and the aqueous layer was extracted with a washing solution of celite after filtration, followed by 1000 ml of heptane. The combined organic layers were washed with 500 ml of water, dried over magnesium sulfate and filtered, the dark brown filtrate was concentrated under reduced pressure. The product was purified on silica gel with ethyl acetate and heptane as eluent to give the title compound.

ER-MS m/z=703.4 (MH+).

Compound of Example 52 ((5-((Dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(octane-8,1-diyl)bis(decanoate)

In a round bottom flask, Intermediat 52c (47.5 g, 67.6 mmol) was taken up in dichloromethane (300 ml) followed by the addition of dimethylamine (2M in THF, 203 ml, 406 mmol) and acetic acid (3.87 ml, 67.6 mmol). Sodium triacetoxyborohydride (35.8 g, 169 mmol) was added to the resulting solution. After 16 hours at ambient temperature, the reaction mixture was quenched with 300 ml of saturated aqueous sodium bicarbonate and 300 ml of water, and the resulting layers were separated. The organic layer was washed with 2x200 ml saturated aqueous sodium bicarbonate. The combined aqueous layers were extracted with 2x300 ml dichloromethane. The combined dichloromethane extracts were dried over magnesium sulfate and filtered. The filtrate was concentrated under reduced pressure. The residue was purified on silica gel with acetone and heptane as eluent to give the title compound.

- 57 040257

Ή NMR (400 MHz, CDCl ₃ ) δ 0.88 (t, J=8.00 Hz, 6H) 1.18-1.50 (m, 40H) 1.56-1.70 (m, 8H) 1 .70-1.81 (m,

4H) 2.24 (s, 6H) 2.30 (t, J=7.53 Hz, 4H) 3.34 (s, 2H), 3.93 (t, J=6.53 Hz, 4H) 4 .06 (t, J=6.78 Hz, 4H) 6.35 (t,

J=2.26 Hz, 1H) 6.46 (d, J=2.26 Hz, 2H) ppm

ER-MS m/z=732.5 (MH+).

Example 53 ((5-((Dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(octane-8,1-diyl)didodecanoate

1H NMR (400 MHz, CDCl3) δ 0.83-0.97 (m, 6H) 1.19-1.51 (m, 48H) 1.56-1.68 (m, 8H) 1.71-1 .83 (m, 4H) 2.24 (s, 6H) 2.30 (t, J=7.53 Hz, 4H) 3.34 (s, 2H) 3.93 (t, J=6.53 Hz , 4H) 4.06 (t, J=6.78 Hz, 4H) 6.35 (t, J=2.26 Hz, 1H) 6.46 (d, J=2.01 Hz, 2H) m. d.

ER-MS m/z=788.6 (MH+).

Example 54 (9Z,9'Z,12Z,12'Z)-((((5-((Dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(ethane-2,1diyl)) bis(oxy))bis(ethane-2,1-diyl)bis(octadeca-9,12-dienoate)

Ή NMR (400 MHz, CDCl ₃ ) δ 6.59 (br.s, 2H), 6.44 (br.s, 1H), 5.27-5.44 (m, 8H), 4.26 (t , J=4.9 Hz, 4H), 4.13 (t, J=4.65 Hz, 4H), 3.85 (t, J=4.65 Hz, 4H), 3.77 (t, J =4.9 Hz, 4H), 3.50 (br. s, 2H), 2.77 (t, J=6.65 Hz, 4H), 2.21-2.52 (m, 10H), 1 .96-2.12 (m, 8H), 1.55-1.70 (m, 4H), 1.19-1.45 (m, 28H), 0.89 (t, J=6.90 Hz , 6H) ppm

ER-MS m/z=868.9 (MH+).

Example 55 (9Z,9'Z, 12Z,12'Z)-((5-((Diethylamino)methyl-2-methyl-1,3-phenylene)bis(oxy))bis(butane4,1-diyl) bis(octadeca-9,12-dienoate)

1H NMR (400 MHz, CDCl3) δ 6.52 (s, 2H), 5.32-5.44 (m, 8H), 4.16 (t, J=8 Hz, 4H), 4.00 (t , J=8 Hz, 4H), 3.52 (s, 2H), 2.79 (t, J=8 Hz, 4H), 2.54 (dd, J=8 Hz, 4H), 2.32 ( t, J=8 Hz, 4H), 2.09 (s, 3H), 2.08 (dd, J=8 Hz, 8H), 1.87 (c, J=5 Hz, 8H), 1.64 (m, 4H), 1.27-1.48 (m, 28H), 1.06 (t, J=8 Hz, 6H), 0.91 (t, J=8 Hz, 6H) ppm.

ER-MS m/z=878.6 (MH+).

Example 56

Ή NMR (400 MHz, CDCl3) δ 6.48 (d, J=2.02 Hz, 2H) 6.35 (t, J=2.27 Hz, 1H) 4.25 (t, J=6.32 Hz, 4H) 4.03 (t, J=6.19 Hz, 4H) 3.35 (s, 2H), 2.20-2.30 (m, 10H) 2.10 (q, J=6, 25 Hz, 4H) 1.84 (br.s, 2H) 1.19-1.36 (m, 56H) 0.81-0.97 (m, 12H) ppm

ER-MS m/z=844.7 (MH+).

Example 57 Didecyl-8,8'-((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))dioctanoate

1H NMR (400 MHz, CDCl ₃ ) δ 6.51-6.45 (m, 2H), 6.38-6.33 (m, 1H), 4.06 (t, J=6.8 Hz, 4H ), 3.93 (t, J=6.5 Hz, 4H), 3.40 (s, 2H), 2.30 (t, J=7.5 Hz, 4H), 2.28 (s, 6H ), 1.82-1.71 (m, 4H), 1.70-1.56 (m, 8H), 1.54-1.20 (m, 40H), 0.93-0.84 (m , 6H) ppm

ER-MS m/z=732.4 (MH+).

Example 5 8. (9Z,9'Z, 12Z, 12'Z)-((((5-((Diethylamino)methyl)-1,3-phenylene)bis(oxy))bis(ethane-2,1 diyl ))bis(oxy))bis(ethane-2,1-diyl)bis(octadeca-9,12-dienoate)

1H NMR (400 MHz, CDCl3) δ 6.54 (d, J=2.3 Hz, 2H), 6.37 (t, J=2.25 Hz, 1H), 5.25-5.46 (m , 8H), 4.26 (t, J=4.8 Hz, 4H), 4.11 (t, J=4.75 Hz, 4H), 3.85 (t, J=4.75 Hz, 4H ), 3.77 (t, J=4.9 Hz, 4H), 3.48 (s, 2H), 2.77 (t, J=6.5 Hz, 4H), 2.50 (k, J = 7.1 Hz, 4H), 2.34 (t, J=7.7 Hz, 4H), 1.98-2.13 (m, 8H), 1.55-1.70 (m, 4H) , 1.22-1.42

- 58 040257 (m, 28H), 1.03 (t, J=7.2 Hz, 6H), 0.89 (t, J=6.9 Hz, 6H) ppm

ER-MS m/z=896.8 (MH+).

Example 59 ((5-((Dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(hexane-6,1-diyl)didodecanoate

1H NMR (400 MHz, CDCl ₃ ) δ 6.48 (d, J=2.26 Hz, 2H) 6.36 (t, J=2.51 Hz, 1H) 4.08 (t, J=6, 65 Hz, 4H) 3.94 (t, J=6.40 Hz, 4H) 3.43 (s, 2H) 2.23-2.35 (m, 10H) 2.03 (s, 1H) 1, 78 (ddt, J=14.05, 13.30, 7.53, 7.53 Hz, 4H) 1.57-1.70 (m, 8H) 1.38-1.53 (m, 8H) 1 .22-1.34 (m, SP) 0.88 (t, J=6.02 Hz, 6H) ppm

ER-MS m/z=732.6 (MH+).

Example 60 (Z)-((5-((Dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(butane-4,1-diyl)dioleate

¹ H NMR (400 MHz, CDCl ₃ ) δ 6.49 (br. s, 2H), 6.36 (t, J=2.0 Hz, 1H), 5.44-5.28 (m, 4H) , 4.15 (t, J=5.8 Hz, 4H), 3.98 (t, J=5.8 Hz, 4H), 3.36 (br.s, 2H), 2.38-2, 18 (m, 10H), 2.10-1.95 (m, 8H), 1.92-1.76 (m, 8H), 1.711.56 (m, J=7.3, 7.3 Hz, 4H), 1.30 (d, J=15.6 Hz, 40H), 0.90 (t, J=6.5 Hz, 6H) ppm.

ER-MS m/z=840.7 (MH+).

Example 61 ((5-((Dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(butane-4,1-diyl)ditradecanoate

1H NMR (400 MHz, CDCl ₃ ) δ 6.54 (br.s, 2H), 6.37 (br.s, 1H) 4.14 (t, J=6.30 Hz, 4H) 3.98 ( t, J=5, 65 Hz, 4H) 3.48 (br.s, 2H), 2.22-2.51 (m, 10H) 1.73-1.93 (m, 8H) 1.62 ( m, 4H) 1.19-1.49 (m, 40H), 0.88 (t, J=6.9 Hz, 6H) ppm

ER-MS m/z=732.7 (MH+).

Example 62 (9Z,9'Z, 12Z, 12'Z, 15Z, 15'Z)-((5-((Dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(butane-4 ,1-diyl)bis(octadeca-9,12,15-trienoate)

1H NMR (400 MHz, CDCl ₃ ) δ 6.51 (d, J=1.8 Hz, 2H), 6.36 (t, J=2.3 Hz, 1H), 5.55-5.18 ( m, 12H), 4.15 (t, J=5.5 Hz, 4H), 3.98 (t, J=5.6 Hz, 4H), 3.41 (br.s, 2H), 2, 82 (t, J=6.0 Hz, 8H), 2.39-2.22 (m, 10H), 2.17-2.00 (m, 8H), 1.92-1.74 (m, 9H), 1.71-1.55 (m, 4H), 1.45-1.22 (m, 16H), 0.99 (t, J=7.5 Hz, 6H) ppm.

ER-MS m/z=833.0 (MH+).

Example 63 (9Z,12Z)-4-(3-((Dimethylamino)methyl)-5-(4-(oleoyloxy)butoxy)phenoxy)butyloctadeca-9,12-dienoate

1H NMR (400 MHz, CDCl ₃ ) δ 6.51 (s, 2H), 6.37 (t, J=2.0 Hz, 1H), 5.49-5.23 (m, 6H), 4, 15 (t, J=6.5 Hz, 4H), 3.99 (t, J=5.6 Hz, 4H), 3.42 (br.s, 2H), 2.79 (t, J=6 .7 Hz, 2H), 2.39-2.21 (m, 10H), 2.18-1.95 (m, 8H), 1.94-1.73 (m, 8H), 1.73- 1.55 (m, 4H), 1.44-1.19 (m, 34H), 0.95-0.86 (m, J=4.0, 6.8, 6.8 Hz, 6H) m .d.

ER-MS m/z=838.7 (MH+).

Example 64: (9Z, 12Z, 15Z)-4-(3-((dimethylamino)methyl)-5-(4-((9Z, 12Z)-octadeca-9,12-dienoyloxy)butoxy)phenoxy)butyloctadeca-9 ,12,15-trienoate

1H NMR (400 MHz, CDCl ₃ ) δ 6.48 (d, J=2.0 Hz, 2H), 6.35 (t, J=2.3 Hz, 1H), 5.48-5.27 ( m, 10H), 4.15 (t, J=5.8 Hz, 4H), 3.98 (t, J=5.8 Hz, 4H), 3.36 (s, 2H), 2.90- 2.73 (m, 6H), 2.32 (t, J=7.7 Hz, 4H), 2.26 (s, 6H), 2.162.00 (m, 8H), 1.93-1.75 (m, 8H), 1.64 (t, J=7.3 Hz, 4H), 1.44-1.22 (m, 22H), 0.99 (t, J=7.5 Hz, 3H) , 0.91 (t, J=7.0 Hz, 3H) ppm

ER-MS m/z=835.0 (MH+).

- 59 040257

Synthesis of example 65.

Intermediate 65 a: 5-(hydroxymethyl)isophthalic acid o no^o

To a solution of diethyl 5-(hydroxymethyl)isophthalate (509 mg, 2.02 mmol) in THF (5 ml) was added NaOH (5.04 ml, 1.0 M in water, 5.04 mmol). The reaction mixture was stirred for 3 days at room temperature. The volatiles were removed under reduced pressure and the resulting product was used without further purification.

Intermediate 65b: 5-(hydroxymethyl)-N ¹ , N ³ -di((9Z, 12Z)-octadeca-9,12-dien-1-yl)isophthalamide o i ⁿ n

Intermediate 65a (168 mg, 0.694 mmol) was stirred in DCM (25 mL) and EDC (399 mg, 2.08 mmol) and HOBt (319 mg, 2.08 mmol) were added followed by TEA (0.481 mL, 3.47 mmol). The reaction mixture was stirred at room temperature for 5 minutes and linoleylamine hydrochloride (419 mg, 1.39 mmol) was added. The reaction mixture was stirred overnight at room temperature and then diluted with DCM (100 ml) and water (100 ml). The organic layer was collected and washed with water (2x50 ml) and dried over magnesium sulfate. Volatiles were removed under reduced pressure. The resulting crude product was purified on silica gel using heptane/EtOAc as eluent to provide 150 mg of desired product.

ER-MS m/z=691.4 (MH+).

Compound of Example 65: 3,5-bis((9Z,12Z)-octadeca-9,12-dien-1-ylcarbamoyl)benzyl-3-(dimethylamino)propanoate o o ¹ u ⁿ n

Example 65 can be prepared from Intermediate 65b using conditions similar to those used to prepare Example 37.

1H NMR (400 MHz, CDCl3) δ 8.11 (t, J=1.5 Hz, 1H), 7.89 (d, J=1.5 Hz, 2H), 6.43 (t, J=5 .6 Hz, 2H), 5.255.48 (m, 8H), 5.19 (s, 2H), 3.33-3.53 (m, 4H), 2.77 (t, J=6.5 Hz , 4H), 2.60-2.70 (m, 2H), 2.49-2.60 (m, 2H), 2.26 (s, 6H), 2.05 (k, J=6.9 Hz, 8H), 1.55-1.71 (m, 4H), 1.18-1.47 (m, 32H), 0.89 (t, J=7.0 Hz, 6H) ppm

ER-MS m/z=790.4 (MH+).

Example 66 3,5-bis((9Z,12Z)-Octadeca-9,12-dien-1-ylcarbamoyl)benzyl-4-(dimethylamino)butanoate

Example 66 can be obtained using methods similar to those used to obtain Example 65.

1H NMR (400 MHz, CDCl ₃ ) δ 8.11 (t, J=1.6 Hz, 1H), 7.88 (d, J=1.5 Hz, 2H), 6.50 (t, J= 5.6 Hz, 2H), 5.205.48 (m, 8H), 5.15 (s, 2H), 3.31-3.55 (m, 4H), 2.66-2.85 (m, 4H ), 2.41 (t, J=7.4 Hz, 2H), 2.28 (t, J=7.3 Hz, 2H), 2.20 (s, 6H), 2.04 (k, J \u003d 6.9 Hz, 8H), 1.82 (k, J \u003d 7.4 Hz, 2H), 1.54-1.67 (m, 4H), 1.17-1.45 (m, 32H) , 0.88 (t, J=7.0 Hz, 6H) ppm

ER-MS m/z=804.5 (MH+).

Synthesis of example 67.

Intermediate 67а: methyl 2-(3,5-6uc((9Z, 12Z)-octadeca-9,12-dien-1-yloxy)phenyl)acetate

To a solution of methyl 3-(3,5-dihydroxyphenyl)acetate (1.0 g, 5.4 mmol) in DMF (25 ml) was added linoleyl mesylate (4.16 g, 12.1 mmol) and potassium carbonate (3. 0 g, 21.6 mmol). The reaction mixture was heated to 100°C for 4 h, after which the reaction mixture was cooled to room temperature and water (100 ml) was added. The resulting mixture was extracted with EtOAc (3x50 ml). The organic layers were combined, washed with brine, dried over sodium sulfate and the volatiles were removed under reduced pressure. The resulting crude product was purified on silica gel using n-hexane/EtOAc as eluent to give 3.3 g of the expected product.

- 60 040257

ER-MS m/z=680 (MH+).

Intermediate 67b: 2-(3,5-6uc((9Z, 12Z)-octadeca-9,12-dien-1-yloxy)phenyl)ethanol HO^

Intermediate 67a (3.3 g, 4.8 mmol) was stirred in THF (50 ml) and cooled in an ice bath. A solution of lithium aluminum hydride (370 mg, 97 mmol) in THF (3 ml) was slowly added to the stirred reaction mixture. After the addition, the reaction mixture was stirred at room temperature for 3 hours. The product was then cooled again in an ice bath and water (5 ml) and EtOAc (5 ml) were added. After 10 minutes, the resulting suspension was filtered through Celite and the filtrate was concentrated under reduced pressure to give 2.8 g of product, which was used without further purification.

Compound of Example 67: 3,5-bis((9Z,12Z)-octadeca-9,12-dien-1-yloxy)phenethyl-3-(dimethylamino)propanoate

To Intermediate 67b (75 mg, 0.12 mmol) in DCM (3 mL) was added 3-dimethylaminopropionic acid hydrochloride (26 mg, 0.17 mmol) and HATU (88 mg, 0.23 mmol) followed by TEA (0.016 ml, 0.12 mmol). The reaction mixture was stirred overnight at room temperature and then water (2 ml) was added. The mixture was extracted with DCM (3x5 ml) and the combined organic layers were dried over sodium sulfate. The volatiles were removed under reduced pressure and the resulting crude product was purified on silica gel using a DCM/MeOH mixture as eluent to give 72 mg of the expected product.

¹ H NMR (400 MHz, CDCl ₃ ) δ 6.32-6.37 (m, 3H), 5.28-5.44 (m, 8H), 4.37 (t, J=7.2 Hz, 2H), 3.92 (t, J=6.6 Hz, 4H), 3.36 (t, J=6.4 Hz, 2H), 2.86-2.92 (m, 8H), 2, 75-2.81 (m, 6H), 2.06 (ddd, J=6.7, 6.7, 6.7 Hz, 8H), 1.711.80 (m, 4H), 1.41-1, 49 (m, 4H), 1.24-1.41 (m, 32H), 0.90 (t, J=7.1 Hz, 6H) ppm.

ER-MS m/z=750.5 (MH+).

Example 68

O.

Intermediate 13a (82.5 mg, 0.130 mmol) was dissolved in dry CDCl ₃ (2 ml) 4-nitrophenyl chloroformate (28.7 mg, 0.142 mmol) was added followed by pyridine (0.011 ml, 0.129 mmol). The resulting mixture was stirred at 50° C. for 30 minutes. After this time, the heating was turned off and the reaction mixture was allowed to stir overnight at room temperature. The reaction was monitored by TLC, which showed the total consumption of IV (starting material). The intermediate was concentrated and redissolved in dichloromethane (3 mL) 3-(dimethylamino)propan-1-ol (66.8 mg, 0.648 mmol) was added followed by DMAP (3.16 mg, 0.026 mmol). The reaction mixture was stirred for 72 h at room temperature. The reaction mixture was quenched with water (2 ml) and extracted with additional DCM (3x5 ml). The organic layers were concentrated. The crude product was purified on silica gel using 0 to 6% MeOH in DCM as eluent to give 47 mg of the desired product.

¹ H NMR (400 MHz, CDCl3) δ 6.51 (d, J=2.3 Hz, 2H), 6.42 (t, J=2.3 Hz, 1H), 5.32-5.48 ( m, 8H), 5.09 (s, 2H), 4.23 (t, J=6.7 Hz, 2H), 3.94 (t, J=6.5 Hz, 4H), 2.80 ( dd, J=6.4, 6.4 Hz, 4H), 2.37 (t, J=7.0 Hz, 2H), 2.24 (s, 6H), 1.99-2.16 (m , 8H), 1.82-1.91 (m, 2H), 1.72-1.82 (m, 4H), 1.41-1.50 (m, 5H), 1.27-1.41 (m, 29H), 0.83-0.97 (m, J=6.8, 6.8 Hz, 5H) ppm

ER-MS m/z=766.5 (MH+).

Example 69 (9Z,9'Z, 12Z, 12'Z)-(5-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)-1,3-phenylene)bis(methylene)bis( octadeca-9,12-dienoate)

Example 69 can be prepared using methods similar to those described for Intermediate 33a and Example 68.

1H NMR (400 MHz, CDCl ₃ ) δ 7.33 (s, 2H), 7.31 (s, 1H), 5.25-5.45 (m, 8H), 5.16 (s, 2H), 5.11 (s, 4H), 4.22 (t, J=6.5 Hz, 2H), 2.73-2.82 (m, 4H), 2.43-2.55 (m, 6H) , 2.37 (t, J=7.5 Hz, 4H), 2.01-2.09 (m, 8H), 1.83 (q, J=6.5 Hz, 2H), 1.57- 1.71 (m, 5H), 1.21-1.42 (m, 28H), 1.01 (t, J=7.2 Hz, 6H), 0.89 (t, J=6.8 Hz , 6H) ppm

- 61 040257

ER-MS m/z=850.6 (MH+).

Examples 70 and 71 can be prepared using methods similar to those used to prepare example 69.

Example 70 (9Z,9'Z, 12Z, 12'Z)-(5-((((2-(dimethylamino)ethoxy)carbonyl)oxy)methyl)-1,3-phenylene)bis(methylene)bis( octadeca-9,12-dienoate)

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.33 (s, 2H), 7.30 (s, 1H), 5.29-5.43 (m, 8H), 5.16 (s, 2H) , 5.11 (s, 4H), 4.25 (t, J=5.8 Hz, 2H), 2.77 (t, J=6.5 Hz, 4H), 2.60 (t, J= 5.8 Hz, 2H), 2.37 (t, J=7.7 Hz, 4H), 2.29 (s, 6H), 2.05 (k, J=6.8 Hz, 8H), 1 .52-1.75 (m, 4H), 1.24-1.40 (m, 29H), 0.88 (t, J=6.8 Hz, 5H) ppm

ER-MS m/z=808.5 (MH+).

Example 71 (9Z,9'Z, 12Z, 12'Z)-(5-((((3-(dimethylamino)propoxy)carbonyl)oxy)methyl)-1,3-phenylene)bis(methylene)bis( octadeca-9,12-dienoate)

1H NMR (400 MHz, CDCl ₃ ) δ 7.34 (s, 2H), 7.32 (s, 1H), 5.29-5.45 (m, 8H), 5.17 (s, 2H), 5.12 (s, 4H), 4.24 (t, J=6.5 Hz, 2H), 2.79 (t, J=6.5 Hz, 4H), 2.34-2.41 (m , 6H), 2.24 (s, 6H), 2.06 (q, J=6.7 Hz, 8H), 1.87 (q, J=6.5 Hz, 2H), 1.61-1 .72 (m, 4H), 1.25-1.42 (m, 29H), 0.91 (t, J=7.0 Hz, 6H) ppm

ER-MS m/z=822.6 (MH+).

Example 72 (9Z,9'Z, 12Z, 12'Z)-5-((((2-(dimethylamino)ethoxy)carbonyl)oxy)methyl)-1,3-phenylenebis(octadeca-9,12-dienoate )

Example 72 can be prepared using methods similar to those used in the synthesis of Intermediate 26b and Example 68.

J NMR (400 MHz, CDCl ₃ ) δ 7.01 (d, J=2.0 Hz, 2H), 6.89 (t, J=2.1 Hz, 1H), 5.30-5.46 ( m, 8H), 5.14 (s, 2H), 4.26 (t, J=5.8 Hz, 2H), 2.79 (t, J=6.4 Hz, 4H), 2.61 ( t, J=5.8 Hz, 2H), 2.54 (t, J=7.5 Hz, 4H), 2.29 (s, 6H), 2.01-2.11 (m, 8H), 1.74 (dt, J=14.7, 7.5 Hz, 4H), 1.24-1.46 (m, 31H), 0.90 (t, J=7.0 Hz, 6H) m. d.

ER-MS m/z=780.4 (MH+).

Synthesis of example 73.

Intermediate 73 a: methyl 4-bromo-3,5-dimethoxybenzoate

To a solution of 4-bromo-3,5-dihydroxybenzoic acid (28 g, 120.7 mmol) in acetone (300 ml) was added dimethyl sulfate (53 g, 422.4 mmol) and potassium carbonate (58 g, 422.4 mmol) . The reaction mixture was refluxed for 4 h, then cooled to ambient temperature. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure. The residue was recrystallized from methanol to give the title compound.

TLC (silica gel, 10% ethyl acetate in hexane): Rf=0.82 Intermediate 73b: methyl 3,5-dimethoxy-4-(prop-1-en-2-yl)benzoate

To a solution of Intermediate 73а (6.0 g, 21.9 mmol) in DMF (100 ml) was added isopropenyltributylstannan (7.98 g, 24.1 mmol), cesium fluoride (6.66 g, 43.8 mmol) and Hb [(t-butyl) ₃ P] ₄ (225 mg, 0.44 mmol). The reaction mixture was heated in a bath at 100°C for 3 h, then cooled to ambient temperature. The reaction mixture was diluted with water and extracted with ethyl acetate (2x100 ml). The combined organic extracts were washed with brine, dried over sodium sulfate and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified on silica gel using ethyl acetate and hexane as eluent to give the title compound.

TLC (silica gel, 10% ethyl acetate in hexane): Rf=0.45

- 62 040257

Intermediate 73с: methyl 4-isopropyl-3,5-dimethoxybenzoate

To a solution of Intermediate 73b (4.0 g, 16.94 mmol) in methanol (200 ml) was added 10% Pd/C (4.0 g) and ammonium formate (21.35 g, 339.0 mmol). The reaction mixture was heated at 90° C. in a heating bath for 48 hours. The reaction mixture was cooled to ambient temperature and filtered through celite. The filtrate was concentrated under reduced pressure and the residue was diluted with water and extracted with dichloromethane (2x100 ml), the combined DCM extracts were washed with brine, dried over sodium sulfate and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified on silica gel using ethyl acetate and hexane as eluent to give the title compound.

TLC (silica gel, 10% ethyl acetate in hexane): Rf=0.50

Intermediate 73 d: methyl 3,5-dihydroxy-4-isopropyl benzoate

To a solution of Intermediate 73c (2.0 g, 8.40 mmol) in dichloromethane (200 ml) cooled in an ice bath was added boron tribromide (42 ml, 1M in DCM, 42 mmol) and tetrabutylammonium iodide (15.53 g, 42.01 mmol). After 3 hours the reaction mixture was poured into a saturated aqueous ammonium chloride solution. The reaction mixture was extracted with dichloromethane (2x100 ml) and the combined DCM extracts were washed with brine, dried over sodium sulfate and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified on silica gel using ethyl acetate and hexane as eluent to give the title compound.

TLC (silica gel, 10% ethyl acetate in hexane): Rf=0.05

Intermediate 73е: methyl 4-isopropyl-3,5-bis((92, 122)-octadeca-9,12-dien-1-yloxy)benzoate

To a solution of Intermediate 73 d (1.4 g, 6.66 mmol) in DMF (60 ml) was added linoleyl mesylate (5.05 g, 14.66 mmol) and potassium carbonate (3.68 g, 26.66 mmol). The reaction mixture was heated in an oil bath at 100°C for 2 h, then cooled to ambient temperature. The reaction mixture was diluted with water and extracted with ethyl acetate (2x200 ml). The combined organic extracts were washed with brine, dried over sodium sulfate and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified on silica gel using ethyl acetate and hexane as eluent to give the title compound.

TLC (silica gel, 10% ethyl acetate in hexane): Rf=0.82

Intermediate 73f: (4-isopropyl-3,5-bis((9Z, 122)-octadeca-9,12-dien-1-yloxy)phenyl)methanol

To a solution of Intermediate 73e (3.8 g, 5.38 mmol) in THF (80 ml) cooled in an ice bath was added lithium aluminum hydride (410 mg, 10.76 mmol). The reaction mixture was stirred for 30 minutes then quenched with water. The reaction mixture was filtered through celite and the filtrate was extracted with ethyl acetate (2x150 ml). The combined organic extracts were washed with brine, dried over sodium sulfate and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified on silica gel using ethyl acetate and hexane as eluent to give the title compound.

TLC (silica gel, 10% ethyl acetate in hexane): Rf=0.21

Example 73 3-(Dimethylamino)propyl-4-isopropyl-3,5-bis((92,122)-octadeca-9,12dien-1-yloxy)benzyl carbonate

Example 73 can be prepared from Intermediate 73f using methods similar to those used in the synthesis of example 68.

1H NMR (400 MHz, CDCl3) δ 6.52 (s, 2H), 5.50-5.25 (m, 8H), 5.07 (s, 2H), 4.21 (t, J=6, 5 Hz, 2H), 3.93 (t,

- 63 040257 j=6.4 Hz, 4H), 3.63 (kv, J=7.1 Hz, 1H), 2.79 (t, J=6.4 Hz, 4H), 2.36 (t , J=7.5 Hz, 2H), 2.22 (s, 6H), 2.06 (k, J=6,

Hz, 8H), 1.92-1.71 (m, 6H), 1.56-1.41 (m, 4H), 1.41-1.20 (m, 34H), 0.90 (t, J=6.8 Hz, 3H) ppm

ER-MS m/z=808.8 (MH+).

Synthesis of Example 74.

Intermediate 74a: methyl 4-bromo-3,5-dihydroxybenzoate

To a solution of 4-bromo-3,5-dihydroxybenzoic acid (7.0 g, 30.2 mmol) in methanol was added chlorotrimethylsilane (8.11 g, 75.4 mmol). The reaction mixture was heated at reflux for 3 h, then cooled to ambient temperature. The reaction mixture was concentrated under reduced pressure, the residue was diluted with water and extracted with ethyl acetate (2x50 ml). The combined organic extracts were washed with saturated aq. sodium bicarbonate solution and brine, dried over sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to give the title compound, which was used without further purification.

TLC (silica gel, 30% ethyl acetate in hexane): Rf=0.33

Intermediate 74b: methyl 4-bromo-3,5-bis((92,122)-octadeca-9,12-dien-1-yloxy)benzoate

Intermediate 74b can be prepared from Intermediate 74a using methods similar to those used in the synthesis of Intermediate 73e.

TLC (silica gel, 10% ethyl acetate in hexane): Rf=0.70

Intermediate 74с: (4-bromo-3,5-bis((92,122)-octadeca-9,12-dien-1-yloxy)phenyl)methanol

To a solution of Intermediate 74b (500 mg, 0.67 mmol) in dichloromethane (20 ml) cooled in a dry ice/acetone bath was added DIBAL-H (25% in toluene, 0.96 ml). The reaction mixture was stirred for 15 min, then quenched with saturated aqueous ammonium chloride. The reaction mixture was filtered through celite and the filtrate was extracted with dichloromethane (2x50 ml). The combined DCM extracts were washed with brine, dried over sodium sulfate and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified on silica gel using ethyl acetate and hexane as eluent to give the title compound.

TLC (silica gel, 20% ethyl acetate in hexane): Rf=0.49

Example 74: 4-bromo-3,5-bis((92,122)-octadeca-9,12-dien-1-yloxy)benzyl(3-(dimethylamino)propyl)carbonate

Example 74 can be prepared from Intermediate 74c using methods similar to those used in the synthesis of example 68.

1H NMR (400 MHz, CDCI3) δ 6.55 (s, 2H), 5.44-5.30 (m, 8H), 5.08 (s, 2H), 4.22 (t, J=6, 7 Hz, 2H), 4.02 (t, J=6.5 Hz, 4H), 2.78 (t, J=6.3 Hz, 4H), 2.35 (t, J=7.4 Hz , 2H), 2.22 (s, 6H), 2.06 (k, J=6.7 Hz, 8H), 1.90-1.77 (m, 6H), 1.55-1.46 ( m, 4H), 1.42-1.22 (m, 28H), 0.89 (t, J=6.8 Hz, 6H) ppm.

ER-MS m/z=844.4 (MH+) observed the isotopic distribution of bromine.

Synthesis of example 75:

Intermediate 75а: 4-chloro-3,5-dihydroxybenzoic acid

To a solution of 3,5-dihydroxybenzoic acid (4 g, 26.0 mmol) in methanol (25 ml) was added a solution of N-chlorosuccinimide (3.64 g, 27.3 mmol) in methanol (10 ml) over 1 h. 16 h the reaction mixture was poured into cold water and was extracted with ethyl acetate (2x50 ml). The combined organic extracts were washed with brine, dried over sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to give the title compound, which was used without further purification.

TLC (silica gel, 10% ethyl acetate in hexane): Rf=0.15

Example 75: 4-chloro-3,5-bis((9Z,12Z)-octadeca-9,12-dien-1-yloxy)benzyl(3-(dimethylamino)propyl)carbonate

Example 75 can be prepared from Intermediate 75a using methods similar to those used in the synthesis of example 74.

¹ H NMR (400 MHz, CDCl ₃ ) δ 6.59 (d, J=2.8 Hz, 1H), 6.48 (d, J=2.8 Hz, 1H), 5.51-5.29 (m, 8H), 5.26 (s, 2H), 4.25 (t, J=6.5 Hz, 2H), 4.00 (t, J=6.5 Hz, 2H), 3.94 (t, J=6.5 Hz, 2H), 2.80 (t, J=6.4 Hz, 4H), 2.38 (t, J=7.0 Hz, 2H), 2.24 (s , 6H), 2.07 (k, J=6.7 Hz, 8H), 1.91-1.75 (m, 6H), 1.55-1.44 (m, 4H), 1.44- 1.24 (m, 30H), 0.91 (t, J=6.8 Hz, 6H) ppm

ER-MS m/z=800.6 (MH+) observed the isotopic distribution of chlorine.

Synthesis of example 76:

Intermediate 7 6a: 2-(3,5-6uc((9Z, 12Z)-octadeca-9,12-dien-1-yloxy)benzyl)isoindoline-1,3-dione

Intermediate 13a (120 mg, 0.188 mmol), isoindoline-1,3-dione (34.6 mg, 0.235 mmol) and triphenylphosphine (64.2 mg, 0.245 mmol) were dissolved in THF (1.5 ml). DIAD (0.044 ml, 0.226 mmol) was then added dropwise. The reaction mixture was stirred at room temperature for 16 hours. Completion of the reaction was monitored by LCMS. The reaction mixture was concentrated, then washed with water, then brine, and dried over sodium sulfate, and re-concentrated. The title product was obtained as a mixture with triphenylphosphine oxide to give 144.0 mg of product.

Intermediate 76b: (3,5-6uc((9Z, 12Z)-octadeca-9,12-dien-1-yloxy)phenyl)methanamine

Intermediate 76a (144 mg, 0.188 mmol, mixture) was dissolved in EtOH (3.7 mL). Hydrazine (0.030 ml, 0.940 mmol) was added and the reaction mixture was heated for 4 hours at 50°C. The completion of the reaction was monitored by LCMS. The reaction mixture was concentrated and suspended in DCM (10 ml). The reaction mixture was filtered. The filtrate was then loaded onto a DCM pre-equilibrated BondElute SCX column. The column was washed with 3CV DCM and then the product was eluted with DCM + 5% 7N ammonia in methanol to recover 57 mg of the desired product.

ER-MS m/z=636.5 (MH+).

Example 76: amino)acetamide

N-(3,5-6uc((9Z, 12Z)-octadeca-9,12-dien-1-yloxy)benzyl)-2-(dimethyl-

Intermediate 76b (36.1 mg, 0.057 mmol), 2-(dimethylamino)acetic acid hydrochloride (23.77 mg, 0.170 mmol) and HATU (43.2 mg, 0.114 mmol) was dissolved in DCM (4 mL). Then triethylamine (0.032 ml, 0.227 mmol) was added and the reaction mixture was stirred for 18 hours at room temperature and checked by LCMS. The reaction mixture was quenched with water (2 ml) and extracted with additional DCM (3x5 ml). The reaction mixture was purified by silica gel chromatography with 0 to 5% MeOH in DCM to give 20 mg of the desired product.

1H NMR (400 MHz, CDCl ₃ ) δ 7.34 (br. s, 1H), 6.43 (d, J=2.0 Hz, 2H), 6.40-6.33 (m, 1H), 5.54-5.30 (m, 8H), 4.40 (d, J=6.1 Hz, 2H), 3.94 (t, J=6.6 Hz, 4H), 3.01 (s , 2H), 2.80 (t, J=6.3 Hz, 4H), 2.30 (s, 6H), 2.13-1.98 (m, 8H), 1.70-1.82 ( m, J=6.6 Hz, 4H), 1.53-1.23 (m, 41H), 0.91 (t, J=7.1 Hz, 6H) ppm.

ER-MS m/z=721.5 (MH+).

Synthesis of Example 77:

Intermediate 77а: diethyl 5-((dimethylamino)methyl)isophthalate

In a round bottom flask, diethyl 5-(hydroxymethyl)isophthalate (15.53 g, 47.6 mmol) and DIPEA (10.39

- 65 040257 ml, 59.5 mmol) were placed in chloroform (40 ml). The resulting suspension was stirred for 1 hour. Toluenesulfonic acid anhydride (10 g, 39.6 mmol) was added to the resulting suspension, and the reaction mixture was stirred at ambient temperature. After 20 h, the reaction solution was added dropwise to dimethylamine (60 ml, 120 mmol) over ~30 min to control the exothermic reaction below the reflux temperature. The reaction mixture was stirred at ambient temperature for 3 hours. The reaction mixture was diluted with dichloromethane (200 ml), saturated aqueous sodium bicarbonate solution (200 ml) and water (50 ml). The layers were separated and the aqueous layer was extracted with dichloromethane (3x100 ml). The combined organic layers were washed with saturated aqueous sodium bicarbonate (3x100 ml). The organic layers were dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, 0-10% methanol in dichloromethane) followed by flash chromatography (silica gel, 0-100% ethyl acetate in dichloromethane, 0-10% methanol in ethyl acetate) to give 7.3 g of the desired product.

1H NMR (400 MHz, CDCl3) δ 1.44 (t, J=7.2 Hz, 6H); 2.29 (s, 6H); 3.55 (s, 2H); 4.43 (k, J=7.1 Hz, 4H); 8.20 (d, J=1.5 Hz, 2H); 8.61 (t, J=1.5 Hz, 1H) ppm

Intermediate 77b: (5-((dimethylamino)methyl)-1,3-phenylene)dimethanol

In a round bottom flask, LAH (2.480 g, 65.3 mmol) was loaded into tetrahydrofuran (50 ml) and the reaction mixture was placed in a water bath at ambient temperature. Intermediate 77a (7.3 g, 26.1 mmol) was dissolved in THF (10 mL) and added dropwise over 10 min to the LAG suspension to maintain the exothermic reaction below reflux. The resulting green suspension was stirred overnight at ambient temperature, after which time the color changed to dark gray. The reaction mixture was diluted to 150 ml with additional THF and quenched with water (2.5 ml) via dropwise addition to keep the temperature below reflux. After stirring at ambient temperature for 15 minutes, the reaction mixture was further quenched with 2.5M aqueous NaOH (5 ml) by dropwise addition over 5 minutes. The reaction mixture was stirred at ambient temperature for 5 minutes and water (7.5 ml) was added dropwise over 1 minute resulting in a white suspension. The reaction mixture was stirred at ambient temperature for 2 hours, after which the salts were filtered through celite, washing with ethyl acetate. The filtrate was collected and concentrated under reduced pressure to give a viscous colorless oil. The material was dissolved in dichloromethane and purified by flash column chromatography (silica gel 050% methanol in dichloromethane). The product fractions were collected and the solvents were removed under reduced pressure to give a colorless oil. The material was dissolved in dichloromethane (100 ml) and filtered. Washing the residue with ethyl acetate resulted in additional precipitation. The solvents were removed under reduced pressure and the material was redissolved in ethyl acetate (100 ml), filtered and the solvents were removed under reduced pressure to give 4 g of the expected product.

L NMR (400 MHz, CDCl3) δ 2.21 (s, 6H); 3.37 (s, 2H); 3.77 (br. s, 2H); 4.56 (s, 4H); 7.16 (s, 2H); 7.21 (s, 1H) ppm

Intermediate 77с: 4-((9Z,12Z)-octadeca-9,12-dien-1-yloxy)-4-oxobutanoic acid

In a borosilicate glass vial, linoleyl alcohol (2 g, 7.51 mmol) and DMAP (0.046 g, 0.375 mmol) were stirred in chloroform (7 ml). Succinic anhydride (1.127 g, 11.26 mmol) was added and the reaction mixture was stirred at ambient temperature. After 3 days, the reaction mixture was directly purified by flash column chromatography (silica gel, 0-10% methanol in dichloromethane) which provided 2.73 g of the desired product.

¹ H NMR (400 MHz, CDCl3) δ 0.91 (t, J=7.0 Hz, 3H); 1.27-1.41 (m, 17H); 1.64 (m, 2H); 2.07 (dd, J=7.0, 13.8 Hz, 4H); 2.62-2.73 (m, 4H); 2.79 (t, J=6.7 Hz, 2H); 4.11 (t, J=6.8 Hz, 2H); 5.32-5.44 (m, 4H) ppm

Compound of Example 77: O,O'-((5-((dimethylamino)methyl)-1,3-phenylene)bis(methylene))di((9Z,12Z)octadeca-9,12-dien-1-yl) disuccinate

In a borosilicate glass vial, Intermediate 77b (250 mg, 1.280 mmol) was stirred in di

- 66 040257 chloromethane (10 ml). DIPEA (0.671 ml, 3.84 mmol), DMAP (15.64 mg, 0.128 mmol), EDC (736 mg, 3.84 mmol) and substance from Intermediate 77c (1032 mg, 2.82 mmol) were added successively. The reaction mixture was sealed and stirred at ambient temperature. After 2 days, the reaction mixture was directly purified by flash column chromatography (silica gel equilibrated with 1% formic acid in dichloromethane, purification with 0-10% methanol in dichloromethane) to give 916 mg of the desired product.

1H NMR (400 MHz, CDCl3) δ 8.24 (s, 0.5H, formate); 7.55 (s, 2H); 7.41 (s, 1H); 5.43-5.31 (m, 8H); 5.20 (s, 4H); 4.16 (s, 2H); 4.08 (t, J=6.8 Hz, 4H); 2.79 (t, J=6.8 Hz, 4H); 2.75 (s, 6H); 2.75-2.66 (m, 8H); 2.06 (k, J=6.6 Hz, 8H); 1.66-1.59 (m, 4H); 1.40-1.27 (m, 32H); 0.90 (t, J=6.5 Hz, 6H) ppm

ER-MS m/z=892.7 (MH+).

Example 78: hydroxy)decyl)disuccinate

O,O'-((5-((dimethylamino)methyl)-1,3-phenylene)bis(methylene))bis(10-(octanoyl-

Example 78 can be prepared using methods similar to those used in the synthesis of Intermediate 18a and Example 77.

1H NMR (400 MHz, CDCl ₃ ) δ 8.37 (s, 1.6H, formate); 7.42 (s, 2H); 7.38 (s, 1H); 5.18 (s, 4H); 4.10-4.05 (m, 10H); 2.74-2.65 (m, 14H); 2.31 (t, J=7.5 Hz, 4H); 1.66-1.59 (m, 12H); 1.40-1.25 (m, 40H); 0.90 (t, J=7.0 Hz, 6H) ppm

ER-MS m/z=960.9 (MH+)

Synthesis of example 79:

Intermediate 79а: 8-(nonyloxy)-8-oxooctanoic acid

In a 250 ml round bottom flask equipped with a magnetic stirrer, suberic acid (5 g, 28.7 mmol) and EDC (6.60 g, 34.4 mmol) were dissolved in DCM (150 ml). DIPEA (15.04 ml, 86 mmol) was added followed by DMAP (1.403 g, 11.48 mmol) and the mixture was stirred at room temperature for 1 hour before adding 1-nonanol (5.01 ml, 28.7 mmol) . The mixture was stirred at room temperature overnight. The volatiles were removed under reduced pressure and the resulting product was purified by silica gel chromatography using heptane/EtOAc as eluent to give 1 g of the expected product.

Example 79: O' ¹ ,O ¹ -((5-((dimethylamino)methyl)-1,3-phenylene)bis(methylene)-8-dinonyldioctanedioate

Example 79 can be obtained using methods similar to those used for example 77.

1H NMR (400 MHz, CDCl3) δ 7.26 (s, 2H), 7.23 (s, 1H), 5.10 (s, 4H), 4.05 (t, J=6.8 Hz, 4H ), 3.44 (s, 2H), 2.36 (t, J=7.7 Hz, 4H), 2.31-2.25 (m, 10H), 1.69-1.58 (m, 12H), 1.36-1.27 (m, 32H), 0.88 (t, J=7.0 Hz, 6H) ppm.

ER-MS m/z=760.4 (MH+).

Example 80: O' ¹ ,O ¹ -((5-((dimethylamino)methyl)-1,3-phenylene)bis(methylene))-9-dioctyldinone andioate

Example 80 can be obtained using methods similar to those used to obtain example 79.

1H NMR (400 MHz, CDCl3) δ 7.27 (s, 2H), 7.24 (s, 1H), 5.11 (s, 4H), 4.07 (t, J=6.8 Hz, 4H ), 3.44 (s, 2H), 2.37 (t, J=7.7 Hz, 4H), 2.30 (t, J=7.5 Hz, 4H), 2.26 (s, 6H ), 1.67-1.59 (m, 12H), 1.33-1.29 (m, 32H), 0.90 (t, J=8.0 Hz, 6H) ppm.

ER-MS m/z=760.4 (MH+).

Synthesis of Example 81:

Intermediate 81а: methyl 3-(3,5-bis((9Z, 122)-octadeca-9,12-dien-1-yloxy)phenyl)acrylate

- 67 040257

Trimethylphosphonoacetate (357 mg, 1.96 mmol) was added to a suspension of NaH (78 mg, 1.96 mmol) in THF (10 ml) which was stirred in an ice bath. After 10 minutes, the starting aldehyde (1 g, 1.63 mmol) obtained using a procedure similar to that described in Example 1a was dissolved in THF (5 ml) and added slowly. The reaction mixture was stirred for 1 h and then ice-cold water (5 ml) was added and the resulting mixture was extracted with EtOAc (3x30 ml). The combined organic layers were dried over sodium sulfate and concentrated under reduced pressure. The resulting product was purified by silica gel chromatography using heptane/EtOAc as eluent to give 1.2 g of expected product.

TLC (silica gel, 10% ethyl acetate in hexanes): Rf=0.77.

Intermediate 81b: 3-(3,5-bis((9Z,12Z)-octadeca-9,12-dien-1-yloxy)phenyl)propan-1-ol

Intermediate 81a (2.6 g, 3.7 mmol) in THF (75 ml) was cooled in an ice bath and lithium aluminum hydride (300 mg, 7.9 mmol) was added in portions. The reaction mixture was stirred for 45 min in a cooling bath and then quenched with ice-cold water. The resulting product was filtered through Celite, and the filtrate was concentrated under reduced pressure. The resulting product was purified by silica gel chromatography using heptane/EtOAc as eluent to give 2.5 g of expected product.

TLC (silica gel, 10% EtOAc in hexanes): Rf=0.21

Example 81: 3-(3,5-bis((9Z,12Z)-octadeca-9,12-dien-1-yloxy)phenyl)propyl-3-(dimethylamino)propanoate

Example 81 can be prepared from Intermediate 81b using methods similar to those used for the synthesis of example 13.

¹ H NMR (400 MHz, CDCl ₃ ) δ 6.34 (d, J=2.0 Hz, 2H), 6.32 (d, J=2.3 Hz, 1H), 5.27-5.47 (m, 8H), 4.16 (t, J=6.5 Hz, 2H), 3.93 (t, J=6.5 Hz, 4H), 2.92-3.03 (m, 2H) , 2.80 (dd, J=6.4, 6.4 Hz, 4H), 2.61-2.69 (m, 4H), 2.58 (s, 6H), 2.07 (k, J =6.9 Hz, 8H), 1.91-2.03 (m, 2H), 1.71-1.85 (m, J=7.8 Hz, 4H), 1.42-1.52 ( m, 4H), 1.23-1.41 (m, 29H), 0.91 (t, J=6.8 Hz, 6H) ppm.

ER-MS m/z=764.6 (MH+).

Synthesis of example 82:

Intermediate 82a: 4-(( 9Z, 12Z)-octadeca-9,12-dienoyloxy)butanoic acid

The starting alcohol, (9Z, 12Z)-4-hydroxybutyloctadeca-9,12-dienoate (1.0 g, 2.8 mmol), obtained using the procedure as for Intermediate 18a, was stirred in acetone (25 ml) and cooled in ice-cold bath. Jones reagent (2.27 ml, 2.5 M, 5.67 mmol) was added dropwise and the ice bath was removed. After 1 hour stirring, MeOH (5 ml) was added followed by EtOAc (220 ml). The resulting mixture was washed with 1:1 water:brine and then with brine. The resulting organic layer was dried over sodium sulfate, and then the volatiles were removed under reduced pressure. The crude product was purified by silica gel chromatography using heptane/EtOAc as eluent to give 672 mg of desired product.

¹³ C NMR (100 MHz, CDCl ₃ ) δ 6. 178.3, 173.8, 130.2, 130.0, 128.0, 127.9, 63.0, 34.2, 31.5, 30 .4, 29.6, 29.3, 29.2, 29.1, 27.2, 25.6, 24.9, 23.7, 22.5, 14.1 ppm

Example 82: (9Z,9'Z,12Z,12'Z)-(((5-((dimethylamino)methyl)-1,3-phenylene)bis(methylene))bis(oxy))bis(4- oxobutane-4,1-diyl)bis(octadeca-9,12-dienoate)

Intermediate 82a (672 mg, 1.83 mmol) was stirred in DCE (40 ml). EDC (501 mg, 2.6 mmol) was added followed by the addition of the substance from Intermediate 77b (170 mg, 0.87 mmol) as a solution in DCE (10 mL). TEA (0.485 ml, 3.48 mmol) and DMAP (21 mg, 0.17 mmol) were added and the reaction mixture was stirred for 3 days at room temperature. The reaction mixture was concentrated at

- 68 040257 under reduced pressure and the crude product was purified by chromatography on silica gel using heptane/EtOAc as eluent to obtain 359 mg of the desired product.

1H NMR (400 MHz, CDCl3) δ 7.31-7.28 (m, 2H), 7.25-7.23 (m, 1H), 5.44-5.28 (m, 8H), 5, 12 (s, 4H), 4.11 (t, J=6.4 Hz, 4H), 2.81-2.73 (m, 4H), 2.46 (t, J=7.4 Hz, 4H ), 2.32-2.24 (m, 9H), 2.09-1.94 (m, 12H), 1.69-1.51 (m, 5H), 1.42-1.22 (m , 28H), 0.89 (t, J=6.9 Hz, 6H) ppm

ER-MS m/z=892.7 (MH+).

Examples 83-88 can be prepared using methods similar to those used to prepare example 82.

Example 83

1H NMR (400 MHz, CDCI3) δ 7.28-7.25 (m, 2H), 7.24-7.21 (m, 1H), 5.10 (s, 4H), 4.05 (t, J=6.8Hz, 4H), 3.43(s, 2H), 2.36(t, J=7.6Hz, 4H), 2.29(t, J=7.6Hz, 4H) , 2.25 (s, 6H), 1.72-1.52 (m, 12H), 1.42-1.14 (m, 36H), 0.95-0.75 (m, 6H) m. d.

ER-MS m/z=788.4 (MH+).

Example 84

Example 84 was characterized as a salt of formic acid.

1H NMR (400 MHz, CDCl ₃ ) δ 8.33 (s, 1H, formate); 7.54 (s, 2H); 7.39 (s, 1H); 5.17 (s, 4H); 4.15 (s, 2H), 4.12 (t, J=6.4 Hz, 4H); 2.74 (s, 6H); 2.50 (t, J=7.4 Hz, 4H); 2.30 (t, J=7.5 Hz, 4H); 2.04-1.97 (m, 4H); 1.65-1.58 (m, 4H); 1.34-1.23 (m, 16H); 0.88 (t, J=6.7 Hz, 6H) ppm

LCMS m/z=620.2 (MH+).

Example 85

1H NMR (400 MHz, CDCl ₃ ) δ 8.30 (s, 0.6H, formate); 7.54 (s, 2H); 7.40 (s, 1H); 5.15 (s, 4H); 4.15 (s, 2H); 4.06 (t, J=6.5 Hz, 4H); 2.74 (s, 6H); 2.42 (t, J=7.5 Hz, 4H); 2.29 (t, J=7.5 Hz, 4H); 1.73-1.58 (m, 12H); 1.44-1.36 (m, 4H); 1.34-1.23 (m, 16H); 0.88 (t, J=6.3 Hz, 6H) ppm

LCMS m/z=676.2 (MH+).

Example 86 (5-((Dimethylamino)methyl)-1,3-phenylene)bis(methylene)bis(10-(octanoyloxy)decanoate)

1H NMR (400 MHz, CDCl3) δ 7.50 (s, 2H); 7.38 (s, 1H); 5.14 (s, 4H); 4.06 (t, J=6.8 Hz, 4H); 3.97 (s, 2H); 2.63 (s, 6H); 2.39 (t, J=7.7 Hz, 4H); 2.30 (t, J=7.5 Hz, 4H); 1.69-1.59 (m, 12H); 1.38-1.25 (m, 36H); 0.89 (t, J=7.0, 6H) ppm

LCMS m/z=788.8 (MH+).

Example 87 (5-((Dimethylamino)methyl)-1,3-phenylene)bis(methylene)bis(8-(octanoyloxy)octanoate)

1H NMR (400 MHz, CDCl3) δ 7.52 (s, 2H); 7.38 (s, 1H); 5.15 (s, 4H); 4.06 (t, J=6.7 Hz, 4H); 3.99 (s, 2H); 2.65 (s, 6H); 2.40 (t, J=7.5 Hz, 4H); 2.30 (t, J=7.5 Hz, 4H); 1.70-1.59 (m, 12H); 1.38-1.25 (m, 28H); 0.89 (t, J=6.8 Hz, 6H) ppm

LCMS m/z=732.8 (MH+).

Example 88 (((5-((Dimethylamino)methyl)-1,3-phenylene)bis(methylene))bis(oxy))bis(6-oxohexane-6,1diyl)bis(decanoate)

- 69 040257 'H NMR (400 MHz, CDCl ₃ ) δ 7.54 (s, 2H); 7.38 (s, 1H); 5.15 (s, 4H); 4.07 (t, J=6.7 Hz, 4H); 4.02 (s, 2H);

2.66 (s, 6H); 2.42 (t, J=7.5 Hz, 4H); 2.30 (t, J=7.7 Hz, 4H); 1.74-1.58 (m, 12H); 1.45-1.37 (m, 4H); 1.35-1.22 (m,

24H); 0.89 (t, J=7.0 Hz, 6H) ppm LCMS m/z=732.8 (MH+).

Synthesis of example 89:

Intermediate 89а: dimethyl 5,5'-((5-formyl-1,3-phenylene)bis(oxy))dipentanoate

To a solution of 3,5-dihydroxybenzaldehyde (1.0 g, 7.24 mmol) in acetone (35 ml) was added methyl 5-bromopentanoate (3.53 g, 18.1 mmol). Potassium carbonate (3.0 g, 22 mmol) was added and the reaction mixture was heated to reflux in an oil bath. After heating overnight, the reaction mixture was cooled to room temperature and stirred for 4 days. The volatiles were removed under reduced pressure and the product was resuspended in DCM. The resulting mixture was filtered and the filtrate was concentrated under reduced pressure to give a crude product, which was purified by silica gel chromatography using heptane/EtOAc as eluent to give 824 mg of the desired product.

1H NMR (400 MHz, CDCl ₃ ) δ 9.89 (s, 1H), 6.99 (s, 2H), 6.68 (s, 1H), 4.01 (m, 4H), 3.68 ( s, 6H), 2.41 (m, 4H), 1.84 (m, 8H) ppm.

Intermediate 89b: 5,5'-((5-formyl-1,3-phenylene)bis(oxy))dipentanoic acid

Intermediate 89a (824 mg, 2.25 mmol) was stirred in EtOH (15 ml). Potassium hydroxide (505 mg, 9.0 mmol) and water (5 ml) were added and the reaction mixture was stirred at room temperature for 3 hours. The product was then diluted with EtOAc (100 ml) and washed with 1M HCl (2x50 ml). The resulting organic phase was dried over sodium sulfate and concentrated under reduced pressure to give 710 mg of the expected product.

1H NMR (400 MHz, DMSO-d6) δ 9.90 (s, 1H), 7.03 (s, 2H), 6.81 (s, 1H), 4.02 (m, 4H), 2.29 (m, 4H), 1.61.8 (m, 8H) ppm

Intermediate si)) dipentanoate

89c: di((92,122)-octadeca-9,12-dien-1 -yl)-5,5'-((5-formyl-1,3-phenylene)bis(oc-

Intermediate 89b (710 mg, 2.1 mmol) was stirred in DCM (20 ml). Linoleyl alcohol (1.40 g, 5.25 mmol) was added along with DMAP (64 mg, 0.52 mmol) and p-toluenesulfonic acid monohydrate (100 mg, 0.52 mmol). EDC (1.0 g, 5.25 mmol) was then added and the reaction mixture was stirred at room temperature for 48 h. The product was purified directly by silica gel chromatography using heptane/EtOAc as eluent to give 1.32 g of product containing the target product and approximately 30% linoleyl alcohol. The product was used without further purification.

Example 89: di((92,122)-octadeca-9,12-dien-1 -yl)-5,5'-((5-((dimethylamino)methyl)-1,3phenylene)bis(oxy))dipentanoate

Example 89 can be prepared from Intermediate 89c using methods similar to those described in Example 33.

1H NMR (400 MHz, CDCl ₃ ) δ 6.46 (d, J=2.20 Hz, 2H), 6.33 (t, J=2.30 Hz, 1H), 5.27-5.45 ( m, 8H), 4.07 (t, J=6.80 Hz, 4H), 3.88-4.01 (m, 4H), 3.34 (s, 2H), 2.78 (t, J = 6.65 Hz, 4H), 2.33-2.44 (m, 4H), 2.24 (s, 6H), 1.982.13 (m, 8H), 1.75-1.88 (m, 8H), 1.55-1.70 (m, 4H), 1.22-1.43 (m, 32H), 0.90 (t, J=6.90 Hz, 6H) ppm.

- 70 040257

ER-MS m/z=864.5 (MH+).

Example 90 Didodecyl 6,6'-((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))dihexanoate

Example 90 can be obtained using methods similar to those used to obtain example 89.

¹ H NMR (400 MHz, CDCl ₃ ) δ 6.48 (br.s, 2H) 6.35 (br.s, 1H) 4.00-4.11 (m, 4H) 3.94 (t, J =6.44 Hz, 4H) 2.34 (t, J=7.45 Hz, 5H) 2.27 (br.s, 3H) 1.57-1.85 (m, 12H) 1.40-1 .57 (m, 4H) 1.20-1.39 (m, 40H) 0.80-0.98 (m, 6H) ppm

ER-MS m/z=732.1 (MH+).

Synthesis of example 91:

Intermediate 91а: 2-(2,2-dimethyl-1,3-dioxolan-4-yl)ethyl-4-methylbenzenesulfonate

Pyridine (25 ml) was added to a suspension of 2-(2,2-dimethyl-1,3-dioxolan-4-yl)ethanol (10 g, 68.4 mmol) in DCM (100 ml). Toluenesulfonic acid anhydride (26.8 g, 82 mmol) was added and the reaction mixture was stirred overnight at room temperature. The volatiles were removed under reduced pressure and the resulting product was purified by silica gel chromatography using heptane/EtOAc as eluent to give 8.92 g of the expected product.

'and NMR (400 MHz, CDCl ₃ ) δ 7.82-7.77 (m, 2H), 7.38-7.32 (m, 2H), 4.21-4.07 (m, 3H), 4.05-3.99 (m, 1H), 3.55-3.49 (m, 1H), 2.45 (s, 3H), 1.97-1.83 (m, 2H), 1, 34 (s, 3H), 1.29 (s, 3H) ppm.

Intermediate 91b: 3,5-bis(2-(2,2-dimethyl-1,3-dioxolan-4-yl)ethoxy)benzaldehyde

To a flask containing Intermediate 91a (8.92 g, 29.7 mmol) was added 3,5-dihydroxybenzaldehyde (1.9 g, 13.8 mmol) and DMF (50 ml). Potassium carbonate (5.7 g, 41.3 mmol) was added and the reaction mixture was heated to 80° C. overnight. The reaction mixture was cooled and water was added. The resulting product was extracted with EtOAc and the combined organic layers were dried over sodium sulfate. The volatiles were removed under reduced pressure and the resulting residue was purified by silica gel chromatography using heptane/EtOAc as eluent to give 1.9 g of the expected product.

'and NMR (400 MHz, CDCl ₃ ) δ 9.89 (s, 1H), 7.02-6.99 (m, 2H), 6.72-6.68 (m, 1H), 4.36- 4.26 (m, 2H), 4.184.07 (m, 6H), 3.68-3.62 (m, 2H), 2.10-2.02 (m, 4H), 1.43 (s, 3H), 1.37 (s 3H) ppm.

Intermediate 91с: 1-(3,5-bis(2-(2,2-dimethyl-1,3-dioxolan-4-yl)ethoxy)phenyl)-N,N-dimethylmethanamine

Intermediate 91b (1.4 g, 3.55 mmol) was stirred in DCE (35 ml). Dimethylamine (7.10 ml, 2M in THF, 14.2 mmol) was added followed by acetic acid (0.20 ml, 3.6 mmol) and then sodium triacetoxyborohydride (1.88 g, 8.87 mmol). The reaction mixture was closed and left to stir at room temperature overnight. The reaction mixture was quenched with saturated aqueous sodium bicarbonate and the resulting mixture was extracted with EtOAc. The combined organic layers were dried over sodium sulfate and concentrated under reduced pressure to 1.34 g of the crude product, which was used without further purification.

Intermediate 91d: 4,4'-((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(butane-1,2-diol)

Intermediate 91c (1.34 g, 3.16 mmol) was stirred in MeOH (20 ml). A concentrated aqueous HCl solution (0.19 ml, 6.33 mmol) was added and the reaction mixture was stirred at room temperature overnight. The volatiles were removed under reduced pressure and the product was used without further purification.

- 71 040257

ER-MS m/z=344.2 (MH+).

Example 91: diyl)tetraoctanoate

4,4'-((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(butane-1,2-

Intermediate 91d (1.0 g, 2.63 mmol) was stirred in DMF (6 ml) until complete dissolution. DCM (6 ml) was added followed by pyridine (1.7 ml, 21 mmol) and DMAP (0.096 mg, 0.79 mmol). Octanoyl chloride (2.14 g, 13.16 mmol) was slowly added to the stirred reaction mixture and the resulting mixture was stirred for 3 days at room temperature. The reaction mixture was diluted with water and saturated aqueous sodium bicarbonate and the resulting mixture was extracted with DCM and EtOAc. The combined organic layers were dried over sodium sulfate and the volatiles were removed under reduced pressure. The product was purified using silica gel that had been pre-washed with 1% acetic acid (v/v) in DCM. The compound was eluted with EtOAc/heptane and the product containing fractions were washed with saturated aqueous sodium bicarbonate. The resulting organic layers were dried over sodium sulfate and then concentrated under reduced pressure. The resulting product was purified a second time on silica gel using heptane/EtOAc as eluent to give 1.65 g of the desired product.

1H NMR (400 MHz, CDCl ₃ ) δ 6.59 (br.s, 2H) 6.36 (br.s, 1H) 5.25-5.37 (m, 2H) 4.35 (dd, J= 12.05, 3.26 Hz, 2H) 4.12 (dd, J=11.92, 6.15 Hz, 2H) 3.91-4.09 (m, 4H), 3.58 (br.s , 2H), 2.25-2.55 (m, 14H) 2.01-2.13 (m, 4H) 1.49-1.70 (m, 8H) 1.13-1.39 (m, 32H) 0.77-0.99 (m, 12H) ppm

ER-MS m/z=848.6 (MH+).

Examples 92-94 can be obtained using methods similar to those used to obtain example 91.

Example 92 (R)-4-(3-((S)-3,4-bis(Octanoyloxy)butoxy)-5-((dimethylamino)methyl)phenoxy)butane1,2-diyldioctanoate

1H NMR (400 MHz, CDCl3) δ 0.74-0.97 (m, 12H) 1.14-1.40 (m, 32H) 1.51-1.71 (m, 8H) 2.07 (q , J=5, 94 Hz, 4H) 2.21-2.46 (m, 14H) 3.44 (s, 2H) 3.89-4.07 (m, 4H) 4.12 (dd, J= 11.92, 6.15 Hz, 2H) 4.34 (dd, J=12.05, 3.26 Hz, 2H), 5.20-5.40 (m, 2H) 6.32 (t, J =2.26 Hz, 1H) 6.49 (d, J=2.01 Hz, 2H) ppm

ER-MS m/z=848.3 (MH+).

Example 93 (S)-4-(3-((S)-3,4-bis(Octanoyloxy)butoxy)-5-((dimethylamino)methyl)phenoxy)butane1,2-diyldioctanoate

1H NMR (400 MHz, CDCl ₃ ) δ 0.87 (m, 12H) 1.14-1.38 (m, 32H) 1.48-1.72 (m, 8H) 2.07 (q, J= 6.19 Hz, 4H) 2.22-2.41 (m, 14H) 3.44 (s, 2H), 3.87-4.07 (m, 4H) 4.12 (dd, J=11, 92, 6.15 Hz, 2H) 4.34 (dd, J=12.05, 3.26 Hz, 2H) 5.20-5.40 (m, 2H) 6.32 (t, J=2, 26 Hz, 1H) 6.49 (d, J=2.01 Hz, 2H) ppm

ER-MS m/z=848.3 (MH+).

Example 94 (R)-4-(3-((R)-3,4-bis(Octanoyloxy)butoxy)-5-((dimethylamino)methyl)phenoxy)butane1,2-diyldioctanoate

¹ H NMR (400 MHz, CDCl ₃ ) δ 0.87 (m, 12H) 1.12-1.40 (m, 32H) 1.47-1.71 (m, 8H) 2.07 (q, J =6.44Hz, 4H)

- 72 040257

2.18-2.38 (m, 14H) 3.47 (s, 2H), 3.85-4.06 (m, 4H) 4.12 (dd, J=11.92, 6.15 Hz, 2H) 4.34 (dd, J=11.92, 3.39 Hz,

2H) 5.20-5.40 (m, 2H) 6.33 (t, J=2.26 Hz, 1H) 6.49 (d, J=2.01 Hz, 2H) ppm

ER-MS m/z=848.3 (MH+).

Synthesis of Example 95:

Intermediate 95 a: 3,4,5-tris((9Z,12Z)-octadeca-9,12-dien-1-yloxy)benzaldehyde

3,4,5-trihydroxybenzaldehyde (600 mg, 3.89 mmol), LinOMs (4427 mg, 12.85 mmol) and potassium carbonate (2690 mg, 19.47 mmol) were stirred in DMF (30 ml) and heated to 80 °C during the night. The reaction mixture was poured into ice water (100 ml) and extracted with diethyl ether (100 ml x 2). The organic phase was collected and dried over sodium sulfate and the volatiles were removed under reduced pressure. The residue was purified by silica gel chromatography using heptane/EtOAc as eluent to give 2.76 g of expected product.

1H NMR (400 MHz, CDCl ₃ ) δ 9.84 (s, 1H), 7.72 (dd, J=5.77, 3.26 Hz, 1H), 7.50-7.58 (m, 1H ), 7.09 (s, 2H), 5.22-5.47 (m, 13H), 4.23 (t, J=6.02 Hz, 1H), 3.94-4.12 (m, 7H), 2.78 (t, J=6.40 Hz, 7H), 2.06 (k, J=6.69 Hz, 13H), 1.78-1.91 (m, 5H), 1, 73-1.78 (m, 2H), 1.70 (dd, J=11.42, 5.40 Hz, 1H), 1.58 (s, 1H), 1.42-1.55 (m, 8H) 1.22-1.42 (m, 51H) 0.82-0.98 (m, 13H) ppm

Compound of Example 95: N,N-dimethyl-1-(3,4,5-tris((9Z,12Z)-octadeca-9,12-dien-1-yloxy)phenyl)methanamine

Example 95 can be prepared from Intermediate 95a using reaction conditions similar to those used to prepare Example 33.

1H NMR (400 MHz, CDCl ₃ ) δ 6.50 (s, 2H), 5.44-5.29 (m, 12H), 4.00-3.91 (m, 6H), 3.32 (s , 2H), 2.81-2.75 (m, 6H), 2.24 (s, 6H), 2.10-2.01 (m, 12H), 1.85-1.65 (m, 8H ), 1.52-1.24 (m, 50H), 0.92-0.86 (m, 9H) ppm.

¹³ C NMR (100 MHz, CDCl ₃ ) δ 152.9, 137.5, 134.1, 130.2, 130.1, 130.1, 128.0, 127.9, 107.3, 73.3 , 69.0, 64.7, 45.4, 31.3, 30.3, 29.7, 29.7, 29.6, 29.6, 29.5, 29.4, 29.4, 29 .3, 29.3, 27.3, 27.2, 27.2, 26.1, 25.6, 22.6, 14.1

ppm

Synthesis of example 96:

Intermediate 96a: 4-hydroxybutyldecanoate o

To a solution of butanediol (3.54 g, 39.3 mmol) in DCM (80 ml) was added pyridine (1.65 ml, 20.4 mmol) and DMAP (0.38 g, 3.2 mmol). Decanoyl chloride (3.0 g, 15.7 mmol) was added and the reaction mixture was stirred at room temperature for 1 hour. The volatiles were removed under reduced pressure and the resulting product was purified by silica gel chromatography using heptane/EtOAc as eluent with obtaining 3.3 g of the target product.

¹ H NMR (400 MHz, CDCl ₃ ) δ 4.1 (m, 2H), 3.7 (m, 2H), 2.3 (m, 2H), 1.7 (m, 2H), 1.6 (m, 4H), 1.3 (m, 12H), 0.9 (m, 3H) ppm

Intermediate 96b: 4-(decanoyloxy)butanoic acid o no.^.^ o

Intermediate 96b can be prepared from Intermediate 96a using conditions similar to those used in the synthesis of Intermediate 84a.

1H NMR (400 MHz, CDCl ₃ ) δ 4.1 (m, 2H), 2.5 (m, 2H), 2.3 (m, 2H), 2.0 (m, 2H), 1.6 ( m, 2H), 1.3 (m, 12H), 0.9 (m, 3H) ppm.

Intermediate 96с: 4-(3-hydroxypropoxy)-4-oxobutyldecanoate

Intermediate 96c can be prepared from Intermediate 96b and 1,3-propanediol using conditions similar to those used to prepare Intermediate 18a.

¹ H NMR (400 MHz, CDCl3) δ 4.3 (m, 2H), 4.1 (m, 2H), 3.7 (m, 2H), 2.4 (m, 2H), 2.3 ( m, 2H), 2.0 (m, 2H), 1.9 (m, 3H), 1.6 (m, 2H), 1.3 (m, 12H), 0.9 (m, 3H) m .d.

Intermediate 96d: ((((5-formyl-1,3-phenylene)bis(oxy))bis(propane-3,1-diyl))bis(oxy))bis(4-oxobutan-4,1-diyl) bis(decanoate)

- 73 040257

Intermediate 96d can be prepared from Intermediate 96c using conditions similar to those used in the synthesis of Intermediate 18b.

'H NMR (400 MHz, CDCl ₃ ) δ 9.9 (s, 1H), 7.0 (s, 2H), 6.7 (s, 1H), 4.3 (m, 4H), 4.1 (m, 8H), 2.4 (m, 4H),

2.3 (m, 4H), 2.2 (m, 4H), 2.0 (m, 4H), 1.6 (m, 4H), 1.3 (m, 24H), 0.9 (m , 6H) ppm

Example 96 compound: ((((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(propane-3,1diyl))bis(oxy))bis(4-oxobutane-4, 1-diyl)bis(decanoate)

Example 96 can be prepared from Intermediate 96d using reaction conditions similar to those used in the synthesis of example 33.

'H NMR (400 MHz, CDCl ₃ ) δ 0.81-0.92 (m, 6H) 1.18-1.37 (m, 24H) 1.53-1.67 (m, 4H) 1.96 (kv, J=6.90Hz, 4H) 2.09 (kv, J=6.27Hz, 4H) 2.23 (s, 6H) 2.28 (t, J=7.53Hz, 4H) 2.40 (t, J=7.53 Hz, 4H) 3.33 (s, 2H), 4.02 (t, J=6.02 Hz, 4H) 4.09 (t, J=6.00 Hz, 4H) 4.26 (t, J=6.27 Hz, 4H) 6.33 (t, J=2.26 Hz, 1H) 6.47 (d, J=2.26 J Hz, 2H) ppm

ER-MS m/z=764.5 (MH+).

Synthesis of example 97:

Intermediate 97a: ethyl 3-octylundec-2-enoate

A solution of 9-heptadecanone (15 g, 59 mmol) and triethylphosphonoacetate (13.2 g, 59 mmol) was stirred in THF (100 ml). To this reaction mixture was added NaOEt (26.4 ml, 21% in EtOH, 70.7 mmol) and the resulting solution was heated at reflux for 48 hours. The reaction mixture was acidified with 1M HCl and then diluted with EtOAc. The organic layer was collected and washed with saturated aqueous sodium bicarbonate. The resulting organic product was dried over sodium sulfate and the volatiles removed under reduced pressure to give a crude product which was purified by silica gel chromatography using heptane/EtOAc as eluent to provide 11.7 g of desired product.

Ή NMR (400 MHz, CDCl ₃ ) δ 5.62 (s, 1H), 4.01-4.26 (m, 2H) 2.49-2.68 (m, 2H) 2.13 (m, 2H ) 1.44 (dd, J=7.33, 4.80 Hz, 4H) 1.17-1.35 (m, 23H) 0.83-0.98 (m, 6H) ppm

Intermediate 97b: ethyl 3-octylundecanoate

Intermediate 97a (11.75 g, 36.2 mmol) was stirred in DCM (16.5 ml) and MeOH (165 ml). Pd/C (3.85 g, 10% Pd) was added and the reaction flask was fitted with a balloon filled with hydrogen. The reaction mixture was stirred at room temperature for 24 hours. The reaction mixture was degassed with nitrogen and filtered through celite, washing with DCM and MeOH. The filtrate was collected and the volatiles were removed under reduced pressure to give 10.6 g of product which was used without further purification.

1H NMR (400 MHz, CDCl ₃ ) δ 4.13 (q, J=7.16 Hz, 2H) 2.39 (t, J=7.45 Hz, 2H) 2.22 (d, J=6, 82 Hz, 2H) 1.84 (br.s, 1H) 1.56 (t, J=7.20 Hz, 2H) 1.19-1.36 (m, 27H) 0.81-0.95 ( m, 6H) ppm

Intermediate 97с: 3-octylundecanoic acid no^.o

Intermediate 97b (10.6 g, 32.5 mmol) was stirred with NaOH (9.74 ml, 10 M, 97.4 mmol) in MeOH (100 ml) and DCM (10 ml). The reaction mixture was heated under reflux overnight. Aqueous HCl was added to neutralize the solution, the volatiles were removed under reduced pressure, and the resulting product was returned to DCM. The organic layers were washed with an aqueous saturated sodium bicarbonate solution, and the resulting aqueous layer was extracted with DCM. The combined organic layers were dried over magnesium sulfate, filtered and concentrated under reduced pressure. The product was purified by silica gel chromatography using heptane/EtOAc

- 74 040257 as eluent. The resulting product was taken up in DCM and loaded onto an NH2 functionalized column. The column was washed with DCM and then with DCM/MeOH. The product was eluted with acidified methanol and the eluent was concentrated under reduced pressure. The residue was taken up in DCM and washed with saturated aqueous sodium bicarbonate, dried over magnesium sulfate, filtered and concentrated under reduced pressure to give 6.5 g of the expected product.

1H NMR (400 MHz, CDClj) δ 2.28 (d, J=7.07 Hz, 2H) 1.86 (br. s, 1H) 1.15-1.44 (m, 28H) 0.82- 0.97 (m, 6H) ppm

Intermediate 97d: 3,5-bis(3-((tetrahydro-2H-pyran-2-yl)oxy)propoxy)benzaldehyde

A solution of 3,5-dihydroxybenzaldehyde (3 g, 22 mmol) and 2-(3-bromopropoxy)tetrahydro-2H-pyran (8.11 ml, 47.8 mmol) was stirred in DMF (100 ml). Was added potassium carbonate (15 g, 109 mmol) and the reaction mixture was heated to 80°C during the night. The reaction mixture was diluted with brine and EtOAc followed by the addition of saturated aqueous sodium bicarbonate. The resulting mixture was filtered, the organic layer was collected, dried over sodium sulfate and the volatiles were removed under reduced pressure. The resulting product was purified by silica gel chromatography using heptane/EtOAc as eluent to give 3.8 g of expected product.

¹ H NMR (400 MHz, CDCl ₃ ) δ 9.88 (s, 1H) 6.84-7.12 (m, 2H) 6.68 (t, J=2.40 Hz, 1H) 4.52- 4.76 (m, 1H) 4.13 (t, J=6.32 Hz, 2H) 3.78-4.05 (m, 2H) 3.44-3.73 (m, 2H) 1.97 -2.27 (m, 2H) 1.79-1.92 (m, 1H) 1.67-1.79 (m, 1H) 1.41-1.67 (m, 4H) ppm

Intermediate 97е: 3,5-bis(3-hydroxypropoxy)benzaldehyde

Intermediate 97d (2.6 g, 6.15 mmol) was stirred in MeOH (40 ml), THF (40 ml) and HCl (24.6 ml, 1 N in water, 24.6 mmol). The reaction mixture was stirred at room temperature for 4 hours. Saturated aqueous sodium bicarbonate solution was added and the reaction mixture was concentrated under reduced pressure. The resulting mixture was extracted with dichloromethane and the combined organic layers were dried over sodium sulfate. The volatiles were removed under reduced pressure and the resulting product was purified by silica gel chromatography using heptane/EtOAc as eluent to give 1.4 g of the expected product.

1H NMR (400 MHz, CDClj) δ 9.90 (s, 1H) 7.03 (d, J=2.27 Hz, 2H) 6.73 (t, J=2.27 Hz, 1H) 4.17 (t, J=6.06 Hz, 4H) 3.88 (t, J=5.81 Hz, 4H) 1.98-2.20 (m, 4H) ppm

Intermediate 97f: 3-(3-formyl-5-(3-hydroxypropoxy)phenoxy)propyl-3-octyldodecanoate

Intermediate 97e (1.0 g, 3.93 mmol), acid from Intermediate 97c (1.41 g, 4.72 mmol), EDC (0.90 g, 4.7 mmol), DIEA (2.06 ml, 11.8 mmol) and DMAP (0.48 g, 3.93 mmol) were dissolved in DCE (20 ml) and the resulting solution was divided into two portions. Each part was heated in a microwave reactor for 20 min at 70°C. The resulting mixtures were combined and purified directly by silica gel chromatography using heptane/EtOAc as eluent to give 680 mg of the desired product.

1H NMR (400 MHz, CDCl ₃ ) δ 9.90 (s, 1H) 7.02 (ddd, J=7.83, 2.27, 1.26 Hz, 1H) 6.72 (t, J=2 .27 Hz, 1H) 4.22-4.35 (m, 2H) 4.13-4.22 (m, 2H) 4.09 (t, J=6.06 Hz, 1H) 3.88 (k , J=5.81 Hz, 2H) 2.25 (d, J=6.82 Hz, 2H) 1.99-2.19 (m, 4H) 1.84 (br.s, 1H) 1.14 -1.38 (m, 30H) 0.89 (td, J=6.95, 3.54 Hz, 6H) ppm

Intermediate 97g: (9Z, 12Z)-3-(3-formyl-5-(3-((3-octyldodecanoyl)oxy)propoxy)phenoxy)propyloctadeca-9, 12-dienoate

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Intermediate 97g can be prepared from Intermediate 97f using the conditions used to prepare Intermediate 18a.

1H NMR (400 MHz, CDCl3) δ 9.90 (s, 1H) 7.01 (d, J=2.27 Hz, 2H) 6.70 (t, J=2.27 Hz, 1H) 5.28 -5.44 (m, 4H) 4.27 (t, J=6.32 Hz, 4H) 4.02-4.17 (m, 4H) 2.78 (t, J=6.44 Hz, 2H ) 2.32 (t, J=7.45 Hz, 2H) 2.25 (d, J=6, 82 Hz, 2H) 2.14 (q, J=6.19 Hz, 4H) 1.98- 2.09 (m, 4H) 1.84 (br.s, 1H) 1.58-1.70 (m, 2H) 1.18-1.41 (m, 42H) 0.770.99 (m, 9H) ppm

Example 97 compound: (9/,122)-3-(3-((dimethylamino)methyl)-5-(3-((3-octylundecanoyl)oxy)propoxy)phenoxy)propyloctadeca-9,12-dienoate

Example 97 can be prepared from Intermediate 97g using conditions similar to those used to prepare Example 39.

¹ H NMR (400 MHz, CDCl3) δ 6.49 (br. s, 2H) 6.27-6.43 (m, 1H) 5.29-5.46 (m, 4H) 4.26 (t, J=6.32 Hz, 4H) 4.03 (t, J=6.19 Hz, 4H) 3.36 (br.s, 2H) 2.78 (t, J=6.57 Hz, 2H) 2 .18-2.39 (m, 10H) 1.96-2.18 (m, 8H) 1.84 (br.s, 1H) 1.48-1.73 (m, 4H) 1.17-1 .47 (m, 40H) 0.74-1.03 (m, 9H) ppm

ER-MS m/z=826.3 (MH+).

Synthesis of example 98:

Intermediate 98a: 1-(3-hydroxypropyl)-8-nonyl octanedioate o o

Intermediate 98a can be prepared from Intermediate 79a using conditions similar to those used to prepare Intermediate 18a.

TLC (silica gel, 5% MeOH in DCM): Rf=0.51.

Example 98: O' ¹ ,O ¹ -(((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(propane-3,1diyl))-9-dioctyldinonanedioate

Example 98 can be prepared from Intermediate 98a using conditions similar to those described for the preparation of Example 33.

1H NMR (400 MHz, CDCl ₃ ) δ 6.47 (s, 2H), 6.34 (s, 1H), 4.25 (t, J=6.3 Hz, 4H), 4.07-4, 01 (m, 8H), 3.34 (s, 2H), 2.29 (k, J=7.2 Hz, 8H), 2.24 (s, 6H), 2.09 (q, J=6 .2 Hz, 4H), 1.65-1.58 (m, 12H), 1.31-1.28 (m, 32H), 0.88 (t, J=6.8 Hz, 6H) m. d.

ER-MS m/z=848.5 (MH+).

Synthesis of example 99:

Intermediate 99a: 10-hydroxydecyloctanoate

Prepared from 1,10-decanediol and octanoyl chloride using conditions similar to those used to prepare Intermediate 96a.

1H NMR (400 MHz, CDCl ₃ ) δ 4.06 (t, J=6, 65 Hz, 2H), 3.64 (t, J=6.65 Hz, 2H), 2.29 (t, J= 7.65 Hz, 2H), 1.50-1.70 (m, 5H), 1.18-1.41 (m, 19H), 0.88 (t, J=7.28 Hz, 3H) m .d.

Intermediate 99b: 10-((methylsulfonyl)oxy)decyloctanoate

O

A mixture of Intermediate 99a (3.34 g, 11.1 mmol) and triethylamine (6.2 ml, 44 mmol) in DCM (40 ml) was stirred in an ice bath and MsCl (1.04 ml, 13.3 mmol) was added. The reaction mixture was allowed to warm to room temperature and stirred overnight. The reaction mixture was then poured into ice water. The resulting organic phase was collected, dried over sodium sulfate and concentrated under reduced pressure to give 4.2 g of product, which was used without further purification.

1H NMR (400 MHz, CDCl3) δ 4.23 (t, J=6.57 Hz, 1H), 4.06 (t, J=6.82 Hz, 2H), 3.15 (s, 1H), 3.01 (s, 1H), 2.30 (t, J=7.58 Hz, 2H), 1.75 (dd, J=8.08, 6.82 Hz, 1H), 1.62 (t , J=6, 95 Hz, 5H), 1.40 (t, J=7.33 Hz, 4H), 1.181.34 (m, 17H), 0.89 (t, J=7.10 Hz, 3H ) ppm

Example 99: ((5-((dimethylamino)methyl)benzene-1,2,3-triyl)tris(oxy))tris(decane-10,1diyl)trioctanoate

- 76 040257

The final compound was then prepared from Intermediate 99b following procedures similar to those used for the preparation of Intermediate 97d and Example 33.

'H NMR (400 MHz, CDCl3) δ 6.51 (s, 2H), 4.03-4.12 (m, 6H), 3.89-4.03 (m, 6H), 3.33 (s , 2H), 2.31 (t, J=7.53 Hz, 6H), 2.25 (s, 6H), 1.69-1.87 (m, 6H), 1.55-1.69 ( m, 12H), 1.42-1.55 (m, 6H), 1.21-1.42 (m, 54H), 0.82-0.96 (m, 9H) ppm.

ER-MS m/z=1030.7 (MH+).

Example 100

Example 100 can be obtained using methods similar to those used to obtain example 99.

1H NMR (400 MHz, CDCl ₃ ) δ 6.55 (s, 2H), 4.02-4.12 (m, 6H), 3.90-4.02 (m, 6H), 3.48 (s , 2H), 2.53 (k, J=7.11 Hz, 4H), 2.31 (t, J=7.53 Hz, 6H), 1.69-1.87 (m, 6H), 1 .57-1.69 (m, 12H), 1.41-1.54 (m, 6H), 1.23-1.41 (m, 54H), 1.05 (t, J=7.03 Hz , 6H), 0.84-0.96 (m, 9H) ppm.

ER-MS m/z=1058.7 (MH+).

Example 101 3-((Dimethylamino)methyl)-5-(((8-(octanoyloxy)octanoyl)oxy)methyl)benzyl-3-octylundecanoate

Example 101 can be prepared from Intermediate 77b, an acid prepared similarly to Intermediate 79a, and Intermediate 97c using methods similar to those used to prepare Example 77.

1H NMR (400 MHz, CDCl ₃ ) δ 7.27 (s, 2H), 7.24 (s, 1H), 5.11 (s, 4H), 4.06 (t, J=6.8 Hz, 2H), 3.44 (s, 2H), 2.37 (t, J=7.5 Hz, 2H), 2.32-2.29 (m, 4H), 2.26 (s, 6H), 1.88 (m, 1H), 1.70-1.59 (m, 6H), 1.35-1.26 (m, 42H), 0.89 (t, J=6.8 Hz, 9H) ppm

ER-MS m/z=744.4 (MH+).

Synthesis of example 102:

Intermediate 102а: methyl 2-(3,5-bis((9Z, 12Z)-octadeca-9,12-dien-1-yloxy)phenyl)acetate

To a suspension of potassium carbonate (12.1 g, 88 mmol) in dimethylformamide (100 ml) was added methyl 2(3,5-dihydroxyphenyl)acetate (4.0 g, 22 mmol) and linoleyl mesylate (16.6 g, 48 mmol ). The reaction mixture was heated in a bath at 100°C for 4 h, then left to cool to ambient temperature. Water (100 ml) was added and the reaction mixture was extracted with ethyl acetate (3x100 ml). The combined organic layers were washed with brine, dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel chromatography using ethyl acetate and n-hexane as eluent to give the title compound.

ER-MS m/z=680 (MH+).

Intermediate 102b: 2-(3,5-6uc((9Z, 12Z)-octadeca-9,12-dien-1-yloxy)phenyl)ethanol

To a suspension of lithium aluminum hydride (671 mg, 17.6 mmol) in tetrahydrofuran (50 ml) was slowly added a solution of Intermediate 104a (6 g, 8.8 mmol) in THF (38 ml). The reaction mixture was then stirred at ambient temperature for 3 h, after which time the reaction mixture was cooled in a bath at 0° C. and quenched with water (5 ml) and ethyl acetate (5 ml). The reaction mixture was stirred for 10 min, then filtered through celite. The filtrate was concentrated under reduced pressure to give the title compound.

ER-MS m/z=652 (MH+).

Intermediate 102с: 2-(3,5-6uc((9Z, 12Z)-octadeca-9,12-dien-1-yloxy)phenyl)acetaldehyde

- 77 040257

To a solution of Intermediate 102b (5.3 g, 8.1 mmol) in dichloromethane (86 ml) was added Dess-Martin periodinan (10.3 g, 24 mmol). The reaction mixture was stirred at ambient temperature for 2 hours, after which time the reaction mixture was quenched by adding aqueous sodium bicarbonate solution. The reaction mixture was extracted with dichloromethane and the combined organic layers were dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel chromatography using ethyl acetate and n-hexane as eluent to give the title compound.

ER-MS m/z=649 (MH+).

Example 102: 2-(3,5-bis((9Z,12Z)-octadeca-9,12-dien-1-yloxy)phenyl)-N,N-dimethylethanamine

To a solution of Intermediate 102c (2.5 g, 3.8 mmol) in ethanol (10 ml) and tetrahydrofuran (10 ml) was added dimethylamine hydrochloride (627 mg, 5.7 mmol) followed by the addition of triethylamine (1.1 ml, 7 .7 mmol) and titanium (IV) isopropoxide (2.1 g, 7.7 mmol). The reaction mixture was stirred at ambient temperature after which sodium borohydride (216 mg, 5.7 mmol) was added. The reaction mixture was stirred at ambient temperature for an additional 10 hours.

The reaction mixture was quenched by slow addition of water (2 ml) and the resulting mixture was filtered through celite. The residue was washed with tetrahydrofuran and the combined filtrates were concentrated under reduced pressure. The residue was purified by silica gel chromatography using methanol in dichloromethane with 0.1% ammonium hydroxide as eluent to give the title compound.

1H NMR (400 MHz, CDCl3) δ 6.36 (d, J=2.3 Hz, 2H); 6.32 (t, J=2.0 Hz, 1H); 5.44-5.32 (m, 8H), 3.93 (t, J=6.5 Hz, 4H); 2.81-2.74 (m, 6H); 2.62-2.58 (m, 2H); 2.36 (s, 6H); 2.08 (t, J=6.9 Hz, 4H); 2.06 (t, J=6.9 Hz, 4H); 1.78 (kv, J=7.0 Hz, 4H); 1.48-1.27 (m, 32H), 0.90 (t, J=7.0 Hz, 6H).

ER-MS m/z=678.7 (MH+).

Synthesis of example 103:

Intermediate 103 a: methyl 3-(3,5-6uc((9Z, 12Z)-octadeca-9,12-dien-1-yloxy)phenyl)acrylate

Trimethylphosphonoacetate (1.0 g, 18 mmol) was added to a suspension of sodium hydride (60% dispersion, 18 mmol) in tetrahydrofuran (50 ml) cooled in a bath at 0°C. The reaction mixture was stirred for 10 minutes, then a solution of aldehyde prepared using conditions analogous to those in Example 1a (7.5 g, 12 mmol) in tetrahydrofuran (25 ml) was slowly added. The reaction mixture was stirred for an additional 1 hour, then ice water (5 ml) was added.

The reaction mixture was extracted with ethyl acetate (3x100 ml), the combined organic phases were dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel chromatography using ethyl acetate and n-hexane as eluent to give the title compound.

TLC (silica gel, 10% EtOAc in hexanes): R _f = 0.77.

Intermediate 103b: 3-(3,5-6uc((9Z, 12Z)-octadeca-9,12-dien-1-yloxy)phenyl)propan-1-ol

Lithium aluminum hydride (927 mg, 24 mmol) was added to a solution of Intermediate 103a (7.7 g, 11.1 mmol) in THF (100 ml) cooled in a bath at 0°C. The reaction mixture was stirred for 45 minutes, then ice water was added slowly. The resulting mixture was filtered through Celite, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography using ethyl acetate and n-hexane as eluent to give the title compound.

TLC (silica gel, 10% EtOAc in hexanes): Rf=0.21.

Intermediate 103c: 3-(3,5-6uc((9Z, 12Z)-octadeca-9,12-dien-1-yloxy)phenyl)propanal

- 78 040257

A solution of Intermediate 105b (2.5 g, 3.8 mmol) in tetrahydrofuran (40 ml) was added to a second solution of 2-iodoxybenzoic acid (2.3 g, 8.3 mmol) in DMSO (8 ml). The reaction mixture was stirred at ambient temperature for 2 hours. The reaction mixture was diluted with ether and filtered through celite. Water was added and the reaction mixture was extracted with dichloromethane. The combined organic layers were dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel chromatography using ethyl acetate and nhexane as eluent to give the title compound.

TLC (silica gel, 10% EtOAc in hexanes): Rf=0.75.

Example 103: 3-(3,5-bis((9Z,12Z)-octadeca-9,12-dien-1-yloxy)phenyl)-N,N-dimethylpropan-1-amine

Dimethylamine hydrochloride (742 mg, 9.0 mmol), triethylamine (913 mg, 9.0 mmol), and titanium (IV) isopropoxide were added to a solution of Intermediate 103c (2.0 g, 3.0 mmol) in ethanol (40 ml). (2.5 g, 9.0 mmol). The reaction mixture was stirred at amine temperature for 10 h after which sodium borohydride (172 mg, 4.5 mmol) was added. The reaction mixture was stirred at ambient temperature for an additional 10 h, after which water (2 ml) was added. The reaction mixture was filtered through celite and the residue was washed with tetrahydrofuran. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography using methanol and dichloromethane modified with 0.1% ammonium hydroxide as eluent to give the title compound.

1H NMR (400 MHz, CDCl3) δ 6.26 (d, J=2.0 Hz, 2H), 6.21 (t, J=2.3 Hz, 1H), 5.22-5.35 (m , 8H), 3.84 (t, J=6.7 Hz, 4H), 2.71 (t, J=6.3 Hz, 4H), 2.49 (t, J=7.8 Hz, 2H ), 2.22 (t, J=7.3 Hz, 2H), 2.15 (s, 6H), 1.98 (k, J=6.9 Hz, 8H), 1.65-1.74 (m, J=7.0 Hz, 6H), 1.33-1.42 (m, 5H), 1.15-1.33 (m, 33H), 0.82 (t, J=6.8 Hz, 6H) ppm

ER-MS m/z=692.5 (MH+).

Synthesis of example 104:

Intermediate 104a: diethyl 2-(3-(benzyloxy)propyl)malonate

To a suspension of sodium hydride (1.05 g, 26 mmol) in tetrahydrofuran (40 ml) was added diethyl malonate (7 g, 44 mmol). After gas evolution ceased, ((3-bromopropoxy)methyl)benzene (5 g, 22 mmol) was added. The reaction mixture was heated in a bath at 90°C for 6 h, then cooled to ambient temperature. The reaction mixture was diluted with diethyl ether (100 ml) and washed with water (100 ml). The aqueous layer was separated and extracted with diethyl ether (2x100 ml). The combined organic layers were dried over magnesium sulfate and concentrated under reduced pressure. The residue was purified by silica gel chromatography using ethyl acetate and heptane as eluent to give the title compound.

1H NMR (400 MHz, CDCl ₃ ) δ 7.31-7.38 (m, 4H), 7.25-7.31 (m, 1H), 4.50 (s, 2H), 4.10-4 .27 (m, 7H), 3.50 (t, J=6.3 Hz, 2H), 3.32-3.41 (m, 2H), 1.93-2.08 (m, 2H), 1.60-1.73 (m, 2H), 1.20-1.38 (m, 10H) ppm

Intermediate 104b: 2-(3-(benzyloxy)propyl)propane-1,3-diol

Lithium aluminum hydride (2.58 g, 68 mmol) was added to a solution of Intermediate 104a (7 g, 23 mmol) in tetrahydrofuran (100 ml) cooled in a bath at 0°C. The cooling bath was removed and the mixture was stirred at ambient temperature overnight. Ice was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic phase was dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel chromatography using ethyl acetate and heptane as eluent to give the title compound.

1H NMR (400 MHz, CDCl ₃ ) δ 7.27-7.39 (m, 5H), 4.51 (s, 2H), 3.75-3.88 (m, 2H), 3.60-3 .73 (m, 2H), 3.413.55 (m, 2H), 2.39 (t, J=5.1 Hz, 2H), 1.76 (dt, J=7.2, 3.5 Hz, 1H), 1.59-1.71 (m, 3H), 1.32-1.45 (m, 2H)

Intermediate 104c: 2-(3-(benzyloxy)propyl)propane-1,3-diyldihexanoate

Hexanoyl chloride (792 mg, 5.9 mmol).

- 79 040257

The reaction mixture was stirred at ambient temperature for 1 h, then poured into

M aqueous HCl (20 ml). The mixture was extracted with diethyl ether (2x40 ml).

The combined organic phases were dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel chromatography using ethyl acetate and heptane as eluent to give the title compound.

1H NMR (400 MHz, CDCl ₃ ) δ 7.27-7.39 (m, 5H), 4.43 (s, 2H), 3.96-4.06 (m, 4H), 3.39-3 .42 (t, 2H), 2.202.24 (t, 4H), 1.94 (m, 1H), 1.52-1.60 (m, 8H), 1.39 (m, 2H), 1, 17-1.26 (m, 10H), 0.80-0.85 (t, 6H) ppm

Intermediate 104d: 2-(3-hydroxypropyl)propane-1,3-diyldihexanoate

Intermediate 104c (1 g, 2.4 mmol) and palladium (10 wt% on charcoal, 20 mg) were taken up in methanol (10 ml). The reaction mixture was pressurized with hydrogen to 54 psi and stirred at ambient temperature overnight. The pressure was reduced and the reaction mixture was filtered. The filtrate was concentrated under reduced pressure to give the title compound.

¹ H NMR (400 MHz, CDCl ₃ ) δ 4.06-4.14 (m, 4H), 3.66-3.70 (m, 2H), 2.03-2.06 (m, 1H), 1.59-1.69 (m, 6H), 1.46-1.48 (m, 2H), 1.29-1.36 (m, 10H), 0.89-0.93 (m, 6H ) ppm

Intermediate 104е: 5-(hexanoyloxy)-4-((hexanoyloxy)methyl)pentanoic acid

Jones reagent (2 M, 1.8 mmol) was added to a solution of Intermediate 104d (760 mg, 2.3 mmol) in acetone (10 ml) cooled in an ice bath. The reaction mixture was stirred at ambient temperature for 2 hours. Methanol (1 ml) was added and the reaction mixture was stirred for 5 minutes, then concentrated under reduced pressure. The residue was taken up in ethyl acetate (50 ml) and water (540 ml) and the organic phase was collected, dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel chromatography using ethyl acetate and heptane as eluent to give the expected product.

ER-MS m/z=343 (M-H).

Intermediate 104f: 4-((tetrahydro-2H-pyran-2-yl)oxy)butylmethanesulfonate

Triethylamine (16.0 g 158 mmol). The cooling bath was removed and the reaction mixture was stirred at ambient temperature for 30 minutes. The reaction mixture was poured into ice water. The organic phase was separated, dried over sodium sulfate, and concentrated under reduced pressure to give the title compound.

1H NMR (400 MHz, CDCl ₃ ) δ 4.49-4.51 (m, 1H), 4.22 (t, 2H), 3.69-3.80 (m, 2H), 3.33-3 .47 (m, 2H), 2.95 (s, 3H), 1.44-1.83 (m, 11H) ppm.

Intermediate 104g: 4-((tetrahydro-2H-pyran-2-yl)oxy)butyl-3-hydroxy-4-methyl-5-(4-((tetrahydro-2H-pyran-2-yl)oxy)butoxy) benzoate

Mixture of Intermediate 104f (20.4 g, 81 mmol), 3,5-dihydroxy-4-methylbenzoic acid (4.12 g, 25 mmol) and potassium carbonate (13.55 g, 98 mmol) in dimethylformamide (100 ml) heated in a bath at 80°C during the night. The reaction mixture was cooled to ambient temperature and poured into ice water (150 ml). The mixture was extracted with diethyl ether (2x150 ml) and the combined organic phases were dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel chromatography using ethyl acetate and heptane as eluent to give the title compound.

ER-MS m/z=479 (M-H).

Intermediate 104h: 4-((tetrahydro-2Н-pyran-2-yl)oxy)butyl-4-methyl-3 -((9Z, 12Z)-octadeca-9,12dien-1-yloxy)-5 -(4- ((tetrahydro-2H-pyran-2-yl)oxy)butoxy)benzoate

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A mixture of Intermediate 104g (2.07 g, 4.3 mmol), linoleyl mesylate (1.78 g, 5.2 mmol) and calcium carbonate

lium (2.38 g, 17.2 mmol) in dimethylformamide (20 ml) was heated in a bath at 80° C. overnight. The reaction mixture was cooled to ambient temperature and poured into ice water (150 ml). The mixture was extracted with diethyl ether (2x100 ml). The organic phases were combined, dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel chromatography using ethyl acetate and heptane as eluent to give the desired product.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.20 (s, 2H), 5.34-5.42 (m, 4H), 4.60-4.63 (m, 2H), 4.36 ( t, 2H), 4.00-4.08 (m, 4H), 3.83-3.86 (m, 4H), 3.47-3.51 (m, 4H), 2.80 (t, 2H), 2.16 (s, 3H), 2.06-2.08 (m, 5H), 1.73-1.84 (m, 14H), 1.51-1.60 (m, 20H) , 1.29-1.37 (m, 18H), 0.90 (m, 4H) ppm.

Intermediate 104i: (4-methyl-3 -((9Z, 12Z)-octadeca-9,12-dien-1-yloxy)-5 -(4-((tetrahydro-2Н-pyran2-yl)oxy)butoxy)phenyl )methanol

Lithium aluminum hydride (89 mg, 2.3 mmol) was added to a solution of Intermediate 104h (1.55 g, 2.1 mmol) in tetrahydrofuran (25 ml) cooled in an ice bath. The cooling bath was removed and the reaction mixture was stirred overnight. Ice was added and the mixture was extracted with ethyl acetate. The organic phase was separated, dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel chromatography using ethyl acetate and heptane as eluent to give the title compound.

¹ H NMR (400 MHz, CDCl ₃ ) δ 6.54 (s, 2H), 5.34-5.44 (m, 4H), 3.97-4.03 (m, 4H), 3.85- 3.87 (m, 2H), 3.493.52 (m, 2H), 2.80 (t, 2H), 2.11 (s, 3H), 2.07-2.08 (m, 4H), 1 .72-1.90 (m, 18H), 1.26-1.35 (m, 16H), 0.91 (t, 4H)

ppm

Intermediate 104j: 4-methyl-3-((9Z, 12Z)-octadeca-9,12-dien-1-yloxy)-5-(4-((tetrahydro-2H-pyran2-yl)oxy)butoxy)benzylmethanesulfonate

O.

Triethylamine (420 mg, 4.2 mmol) was added to a mixture of Intermediate 104i (580 mg, 1.0 mmol) and methanesulfonyl chloride (143 mg, 1.2 mmol) in dichloromethane (20 mL) cooled in an ice bath. The cooling bath was removed and the reaction mixture was stirred at ambient temperature for 30 minutes, after which the reaction mixture was poured into ice water. The organic phase was collected, dried over sodium sulfate and concentrated under reduced pressure to give the title compound, which was used without further purification.

Intermediate 104k: N,K-dimethyl-1 -(4-methyl-3-((9Z, 12Z)-octadeca-9,12-dien-1-yloxy)-5-(4-((tetrahydro-2H-pyran -2-yl)oxy)butoxy)phenyl)methanamine

O.

Intermediate 104j (660 mg, 1.0 mmol), sodium iodide (600 mg, 4.0 mmol) and dimethylamine (2M in tetrahydrofuran, 2 ml) were taken up in dimethylformamide (5 ml). The reaction mixture was sealed and heated to 120° C. with microwave radiation for 40 min. The reaction mixture was cooled to ambient temperature and concentrated under reduced pressure. The residue was dissolved in ethyl acetate (50 ml) and washed with water (50 ml). The organic phase was collected, dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel chromatography using dichloromethane and methanol as eluent to give the title compound.

ER-MS m/z=586.3 (MH+).

Intermediate 1041: si)phenoxy)butan-1-ol

4-(5-((dimethylamino)methyl)-2-methyl-3 -((9Z, 12Z)-octadeca-9,12-dien-1-ylok-

To a solution of Intermediate 104k (520 mg, 0.89 mmol) in methanol (10 ml) was added aqueous HCl

- 81 040257 ml). The reaction mixture was stirred at ambient temperature for 30 minutes, then concentrated under reduced pressure. The residue was dissolved in toluene (3 ml) and concentrated under reduced pressure to give the title compound as the hydrochloride salt.

ER-MS m/z=502.3 (MH+).

Example 104: 2-(3-(4-(5-((dimethylamino)methyl)-2-methyl-3-((92.122)-octadeca-9.12 dien-1-yloxy)phenoxy)butoxy)-3 -oxopropyl)propane-1,3-diyldihexanoate

To a solution of Intermediate 104e (250 mg, 0.73 mmol) were added EDCI (167 mg, 0.871 mmol), diisopropylethylamine (0.190 ml, 1.1 mmol) and 4-(dimethylamino)pyridine (1.8 mg, 0.015 mmol). The reaction mixture was stirred for 1 h at ambient temperature, then Experiment 1041 alcohol (480 mg, 0.892 mmol) was added. The reaction mixture was stirred at ambient temperature overnight and then concentrated under reduced pressure. The residue was purified by silica gel chromatography using dichloromethane and methanol with a modifier of 1% acetic acid. Fractions containing product were washed with sodium bicarbonate solution and concentrated under reduced pressure. The residue was repurified by silica gel chromatography using ethyl acetate and heptane as eluent to give the title compound.

1H NMR (400 MHz, CDCl3) δ 6.48 (s, 1H), 6.47 (s, 1H), 5.32-5.44 (m, 4H), 3.95-4.19 (m, 10H), 3.37 (s, 2H), 2.80 (t, J=8 Hz, 2H), 2.42 (t, J=8 Hz, 2H), 2.32 (t, J=8 Hz , 4H), 2.25 (s, 6H), 2.10 (s, 3H), 2.08 (dd, J=8 Hz, 4H), 1.86 (k, J=5 Hz, 4H), 1.64 (m, 4H), 1.27-1.48 (m, 24H), 0.91 (t, J=8 Hz, 9H) ppm

ER-MS m/z=828.4 (MH+).

Synthesis of example 105:

Intermediate 105a: 8-decanamidooctanoic acid

To a solution of decanoyl chloride (3.73 g, 20 mmol) and pyridine (3.10 g, 39 mmol) in dichloromethane (50 ml) was added 8-aminocaprylic acid (3.27 g, 21 mmol). The mixture was stirred at ambient temperature for 2 hours. The mixture was diluted with water and dichloromethane and the aqueous phase was adjusted to pH 4 to 6 with solutions of 1 N aqueous HCl and sodium bicarbonate. The organic phase was separated and washed with water. The organic phase was dried over magnesium sulfate and concentrated under reduced pressure. The residue was purified by silica gel chromatography using ethyl acetate and heptane as eluent to give the title compound.

TLC (silica gel, 5% MeOH in DCM): Rf=0.25.

Example 105: (5-((dimethylamino)methyl)-1,3-phenylene)bis(methylene)bis(8-decanamidooctanoate)

Example 105 can be prepared from Intermediate 105a using conditions similar to those described for the synthesis of example 77.

1H NMR (400 MHz, CDCl3) δ 7.21 (s, 2H), 7.18 (s, 1H), 5.90 (t, J=5.4 Hz, 2H), 5.05 (s, 4H ), 3.38 (s, 2H), 3.17 (k, J=6.9 Hz, 4H), 2.31 (t, J=7.5 Hz, 4H), 2.20 (s, 6H ), 2.11 (t, J=7.7 Hz, 4H), 1.62-1.53 (m, 8H), 1.46-1.39 (m, 4H), 1.27-1, 21 (m, 36H), 0.82 (t, J=6.9 Hz, 6H) ppm

ER-MS m/z=786.5 (MH+).

Synthesis of example 106:

Intermediate 106а: tert-butyl-6-(((nonyloxy)carbonyl)oxy)hexanoate

To a solution of 4-nitrophenyl chloroformate (3.75 g, 19 mmol), 4-(dimethylamino)pyridine (0.65 g, 5.3 mmol) and pyridine (3.15 g, 40 mmol) in dichloromethane (30 ml) was added tert-butyl-6-hydroxyhexanoate (2.5 g, 13 mmol). The reaction mixture was stirred at ambient temperature overnight. Nonanol (5.75 g, 40 mmol) was added and the mixture was stirred at ambient temperature overnight. The mixture was concentrated under reduced pressure, and the residue was purified by silica gel chromatography using ethyl acetate and heptane as eluent to give the title compound.

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TLC (silica gel, 20% EtOAc in heptane): Rf=0.58.

Intermediate 106b: 6-(((nonyloxy)carbonyl)oxy)hexanoic acid

A solution of Intermediate 106a (2.42 g, 6.7 mmol) in trifluoroacetic acid (3.0 ml) was stirred with rotation for 1 min, then concentrated under reduced pressure. The residue was dissolved in dichloromethane (10 ml) and concentrated under reduced pressure to give the title compound.

'H NMR (400 MHz, CDCl ₃ ) δ 10.20 (br. s, 1H), 4.18-4.13 (m, 4H), 2.45 (t, J=7.3 Hz, 2H) , 1.76-1.64 (m, 6H), 1.50-1.27 (m, 14H), 0.89 (m, 3H) ppm.

Example 106: (5-((dimethylamino)methyl)-1,3-phenylene)bis(methylene)bis(6-(((nonyloxy)carbonyl)oxy)hexanoate)

Example 106 can be prepared from Intermediate 106b using conditions similar to those used for the synthesis of example 77.

1H NMR (400 MHz, CDCl ₃ ) δ 7.26 (s, 2H), 7.23 (s, 1H), 5.10 (s, 4H), 4.12 (t, J=6.7 Hz, 8H), 3.43 (s, 2H), 2.38 (t, J=7.5 Hz, 4H), 2.25 (s, 6H), 1.73-1.62 (m, 12H), 1.46-1.27 (m, 28H), 0.88 (m, 6H) ppm

ER-MS m/z=764.3 (MH+).

Synthesis of example 107:

Intermediate 107а: 4-((1,3-bis(octanoyloxy)propan-2-yl)oxy)-4-oxobutanoic acid

To a solution of 1,3-dicaprylin (1.0 g, 2.9 mmol) in toluene (12 ml) was added succinic anhydride (0.320 g, 3.2 mmol). The reaction mixture was sealed and heated under microwave irradiation at 140°C for 40 min. The reaction mixture was cooled to ambient temperature and concentrated under reduced pressure. The residue was suspended in DCM and filtered through celite. The filtrate was concentrated under reduced pressure to give the title compound.

'H NMR (400 MHz, CDCl ₃ ) δ 13.26 (br. s, 1H), 5.32-5.26 (m, 1H), 4.35-4.28 (m, 2H), 4, 23-4.15 (m, 2H), 2.70-2.65 (m, 4H), 2.38-2.31 (m, 4H), 1.70-1.55 (m, 4H), 1.40-1.20 (m, 16H), 0.91-0.88 (m, 6H) ppm

Example 107: bis(1,3-bis(octanoyloxy)propan-2-yl)-O,O'-((5-((dimethylamino)methyl)1,3-phenylene)bis(methylene))disuccinate

Example 107 can be prepared from Intermediate 107a using conditions similar to those described for the synthesis of example 77.

1H NMR (400 MHz, CDCl ₃ ) δ 7.27 (s, 2H), 7.23 (s, 1H), 5.30-5.25 (m, 2H), 5.13 (s, 4H), 4.34-4.28 (m, 4H), 4.21-4.13 (m, 4H), 3.43 (s, 2H), 2.70-2.67 (m, 8H), 2, 35-2.30 (m, 8H), 2.25 (s, 6H), 1.65-1.58 (m, 8H), 1.321.27 (m, 32H), 0.88 (m, 12H) ppm

ER-MS m/z=1048.5 (MH+).

Synthesis of example 108:

Intermediate 108a: (9Z, 12Z)-4-(3-formyl-5-(4-hydroxybutoxy)phenoxy)butyloctadeca-9,12dienoate

To a solution of linoleic acid (2.33 g, 8.3 mmol) in dichloroethane (21 ml) were added EDCI (2.41 g, 12.6 mmol), DIPEA (1.63 g, 12.6 mmol) and DMAP ( 0.10 g, 0.84 mmol). A diol prepared using conditions similar to those described in Example 99e was added and the reaction mixture was sealed and heated under microwave irradiation at 80° C. for 20 minutes. The reaction mixture was cooled to ambient temperature and concentrated under reduced pressure. The residue was purified by silica gel chromatography using ethyl acetate and heptane as eluent, followed by methanol and dichloromethane as eluent to give the title compound.

1H NMR (400 MHz, CDCl3) δ 9.90 (s, 1H), 7.00 (d, J=2.3 Hz, 1H), 6.69 (d, J=2.3 Hz, 1H), 5.22-5.47 (m, 4H), 4.15 (t, J=6.1 Hz, 2H), 3.97-4.10 (m, 2H), 2.77 (t, J= 6.5 Hz, 2H), 2.31 (t, J=7.7 Hz, 2H), 1.97-2.14 (m, 4H), 1.72-1.97 (m, 5H), 1.46-1.70 (m, 4H), 1.23-1.45 (m, 15H), 0.89 (t, J=6.8 Hz, 3H) ppm

Intermediate 108b: (9Z,12Z)-4-(3-(4-((Z)-dodec-8-enoyloxy)butoxy)-5-formylphenoxy)butyloctadeca-9,12-dienoate

To a solution of dodec-8-enoic acid (60.1 mg 0.30 mmol) in dichloroethane (690 µl) were added EDCI (79 mg 0.41 mmol), diisopropylethylamine (53 mg, 0.41 mmol) and 4-(dimethylamino )pyridine (3.4 mg, 0.03 mmol). Intermediate 110a (150 mg, 0.28 mmol) was added and the reaction mixture was sealed and heated under microwave irradiation at 80°C for 20 min. The reaction mixture was concentrated under reduced pressure. The residue was purified by silica gel chromatography using ethyl acetate and heptane as eluent, followed by methanol and dichloromethane as eluent to give the title compound.

¹ H NMR (400 MHz, CDCl3) δ 9.90 (s, 1H), 7.00 (d, J=2.3 Hz, 2H), 6.69 (t, J=2.3 Hz, 1H) , 5.23-5.45 (m, 6H), 4.15 (t, J=6.1 Hz, 4H), 4.03 (t, J=5.9 Hz, 4H), 2.77 ( t, J=6.5 Hz, 2H), 2.31 (t, J=7.7 Hz, 4H), 1.94-2.12 (m, 8H), 1.75-1.94 (m , 8H), 1.51-1.72 (m, 6H), 1.21-1.45 (m, 23H), 0.79-1.02 (m, 6H) ppm.

Example 108 compound: (9Z,12Z)-4-(3-((dimethylamino)methyl)-5-(4-((Z)-dodec-8-enoyloxy)butoxy)phenoxy)butyloctadeca-9,12-dienoate

Example 108 can be prepared from Intermediate 108b using conditions similar to those used for the synthesis of example 33.

1H NMR (400 MHz, CDCl3) δ 6.48 (d, J=2.26 Hz, 2H) 6.35 (t, J=1.00 Hz, 1H) 5.37 (d, J=4.77 Hz, 5H) 4.15 (s, 4H) 3.98 (s, 4H) 3.36 (s, 2H), 2.74-2.83 (m, 2H) 2.32 (t, J=7 .53 Hz, 4H) 2.25 (s, 6H) 1.96-2.11 (m, 8H) 1.771.91 (m, 8H) 1.60 (s, 8H) 1.27-1.43 ( m, 23H) 0.88-0.95 (m, 6H) ppm

ER-MS m/z=754.5 (MH+).

Example 109 (9Z, 12Z)-4-(3-((Dimethylamino)methyl-5-(4-((3-octylundecanoyl)oxy)butoxy)phenoxy)butyloctadeca-9,12-dienoate

Example 109 can be prepared using methods similar to those used to prepare Intermediate 97c and Example 108.

1H NMR (400 MHz, CDCl ₃ ) δ 6.49 (d, J=2.01 Hz, 2H) 6.36 (t, J=4.30 Hz, 1H) 5.37 (d, J=5, 77 Hz, 4H) 4.11-4.19 (m, 4H) 3.98 (t, J=5.65 Hz, 4H) 3.40 (s, 2H) 2.79 (t, J=6, 65 Hz, 2H) 2.23-2.34 (m, 10H) 2.07 (d, J=7.78 Hz, 5H) 1.79-1.90 (m, 9H) 1.23-1, 40 (m, 44H) 0.86-0.94 (m, 9H) ppm

ER-MS m/z=854.5 (MH+).

Synthesis of example 110:

Intermediate 110а: 2-(4-hydroxybutyl)propane-1,3-diyldioctanoate

Intermediate 110a can be prepared using conditions similar to those used in the synthesis of Intermediate 104d. This intermediate was used directly in the next step without purification.

Intermediate 110b: (((5-formyl-1,3-phenylene)bis(oxy))bis(butane-4,1-diyl))bis(propan-3,2,1-triyl)tetraoctanoate

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Intermediate 110b can be prepared from Intermediate 110a using conditions similar to those used in the synthesis of Intermediate 104f and Intermediate 104g.

1H NMR (400 MHz, CDCl ₃ ) δ 9.87 (s, 1H), 6.97 (s, 2H), 6.67 (s, 1H), 3.96-4.11 (m, 12H), 2.29 (t, 8H), 2.00 (m, 2H), 1.76-1.82 (m, 4H), 1.58-1.61 (m, 12H), 1.41-1, 45 (m, 4H), 1.25-1.27 (m, 34H), 0.87 (t, 12H) ppm.

Example 110: (((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(butane-4,1diyl))bis(propane-3,2,1-triyl)tetraoctanoate

Example 110 can be prepared from Intermediate 110c using conditions similar to those described for the preparation of Example 33.

1H NMR (400 MHz, CDCl ₃ ) δ 6.49-6.44 (m, 2H), 6.35-6.31 (m, 1H), 4.14-4.01 (m, 8H), 3 .97-3.90 (m, 4H), 3.35 (s, 2H), 2.34-2.27 (m, 8H), 2.25 (s, 6H), 2.08-1.98 (m, 2H), 1.82-1.71 (m, 4H), 1.68-1.38 (m, 16H), 1.361.19 (m, 32H), 0.93-0.84 (m , 12H) ppm

ER-MS m/z=932.6 (MH+).

Synthesis of example 111:

Intermediate 111а: 2-(hydroxymethyl)propane-1,3-diyldioctanoate

To a solution of octanoyl chloride (12.3 g, 75 mmol) in dichloromethane (50 ml) were added DMAP (1.84 g, 15 mmol) and pyridine (11.9 g, 150 mmol). The mixture was stirred at ambient temperature for 5 minutes, then 2-(hydroxymethyl)-1,3-propanediol (4.0 g, 38 mmol) was added. The reaction mixture was stirred at ambient temperature overnight, then concentrated under reduced pressure. The residue was purified by silica gel chromatography using ethyl acetate and heptane to give the desired product.

TLC (silica gel, 20% EtOAc in heptane): Rf=0.21.

Intermediate 111b: (((5-formyl-1,3-phenylene)bis(oxy))bis(methylene))bis(propane-3,2,1-triyl)tetraoctanoate

Intermediate 111b can be prepared from Intermediate 111a using conditions similar to those used to prepare Intermediate 18b.

TLC (silica gel, 20% EtOAc in heptane): Rf=0.44.

Example 111: (((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(methylene))bis(propane-3,2,1-triyl)tetraoctanoate

Example 111 can be prepared from Intermediate 111b using conditions similar to those used to prepare Example 33.

¹ H NMR (400 MHz, CDCl ₃ ) δ 6.51 (s, 2H), 6.37 (s, 1H), 4.25-4.22 (m, 8H), 4.00-3.98 ( m, 4H), 3.52 (br. s,

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2H), 2.53-2.50 (m, 2H), 2.34-2.30 (m, 14H), 1.65-1.58 (m, 8H), 1.31-1.25 ( m, 32H), 0.87 (m, 12H) ppm

ER-MS m/z=848.4 (MH+).

Example 112 (92.9'2.122.12'2)-((5-(pyrrolidin-1-ylmethyl)-1,3-phenylene)bis(oxy))bis(butane-4,1diyl)bis(octadeca- 9,12-dienoate)

Example 112 can be prepared from Intermediate 38b using conditions similar to those used to prepare Example 38.

¹ H NMR (400 MHz, CDCl ₃ ) δ 6.52 (s, 2H), 6.35 (s, 1H), 5.44-5.32 (m, 8H), 4.15 (t, J= 5.6 Hz, 4H), 3.98 (t, J=5.1 Hz, 4H), 3.57 (s, 2H), 2.79 (t, J=6.4 Hz, 4H), 2 .54 (br.s, 4H), 2.32 (t, J=7.5 Hz, 4H), 2.07 (c, J=6.7 Hz, 8H), 1.79-1.90 ( m, 12H), 1.68-1.59 (m, 5H), 1.42-1.27 (m, 27H), 0.91 (t, J=6.8 Hz, 6H) ppm.

ER-MS m/z=862.8 (MH+).

Synthesis of example 113:

Intermediate 113а: methyl 3-hexylnon-2-enoate

To a suspension of sodium hydride (60% in paraffin oil, 14.16 g, 335 mmol) in THF (500 ml) cooled in an ice bath was added trimethylphosphonoacetate (50.74 g, 278.8 mmol) slowly. After 2 h, tridecan-7-one (6.5 g, 32.8 mmol) was added slowly and the reaction mixture was warmed to ambient temperature. After 1 h the reaction mixture was heated to reflux. After 4 days the reaction mixture was cooled and 1 N HCl (aq.) was added to quench the reaction mixture. The reaction mixture was extracted with ethyl acetate (3x200 ml). The combined organic extracts were dried over sodium sulfate, filtered and concentrated under reduced pressure. The concentrate was purified on silica gel using ethyl acetate/hexane as eluent to give 8.0 g of the expected product.

TLC (silica gel, 10% ethyl acetate in hexanes): Rf=0.72.

Intermediate 113b: 3-hexylnon-2-en-1-ol

To a solution of Intermediate 113a (8.1 g, 31.9 mmol) in THF (100 ml) cooled in an ice bath was added diisobutylaluminum hydride (25% in toluene, 54.4 ml, 95.6 mmol). After 30 minutes the reaction mixture was brought to ambient temperature. After 6 h the reaction mixture was cooled in an ice bath and quenched with ice water (50 ml) and 1N HCl (aq., 15 ml). The reaction mixture was extracted with ethyl acetate (2x50 ml). The combined organic extracts were washed with water (2x60 ml) and brine (60 ml). The organic extract was dried over sodium sulfate, filtered and concentrated under reduced pressure. The concentrate was purified on silica gel using ethyl acetate/hexane as eluent to give 6.8 g of the expected product.

TLC (silica gel, 20% ethyl acetate in hexanes): R _f = 0.29.

Intermediate 113c: 3-hexylnon-2-enal n

To a stirred suspension of IBX (21.0 g, 75.12 mmol) in DMSO (30 ml) heated to 30°C was added Intermediate 113b in THF (100 ml). The reaction mixture was kept at 25-30° C. for 2 hours. The reaction mixture was diluted with diethyl ether and filtered through celite, washing with diethyl ether. The filtrate was washed with water (2x200 ml) and brine (200 ml). The organic extract was dried over sodium sulfate, filtered and concentrated under reduced pressure to give 6.0 g of the expected product, which was used without further purification.

TLC (silica gel, 10% ethyl acetate in hexanes): R _f = 0.50.

Intermediate 113d: 7-hexyl trideca-4,6-dienoic acid

ABOUT

NaHMDS (1.0 M in THF, 111 ml , 111 mmol). Intermediate 113c (5.0 g, 22.3 mmol) in THF (20 ml) was slowly added and the reaction mixture was heated to 30°C. After 16 hours the reaction mixture was diluted with 200 ml of water and acidified with 2N HCl (aq.). The reaction mixture was extracted with ethyl acetate (3x100 ml). The combined organic extracts were dried over sodium sulfate, filtered and concentrated under reduced pressure. The concentrate was purified on silica gel using ethyl acetate/hexane as eluent to give 4.0 g of the expected product.

TLC (silica gel, 30% EtOAc in n-hexane): Rf=0.21.

Intermediate 113e: 7-hexyl tridecanoic acid

To a suspension of Intermediate 113d (4.0 g, 13.6 mmol) in methanol (100 ml) in a Raag shaker vessel was added Pd/C (10% Pd/C, 2.0 g). The reaction mixture was placed on a shaker device and pressurized to 60 psi with hydrogen. After 2 h, the reaction mixture was filtered through celite, washing with methanol (2x50 ml). The filtrate was concentrated under reduced pressure and the concentrate was purified on silica gel using ethyl acetate/n-hexane as eluent to give 3.9 g of the expected product.

TLC (silica gel, 30% EtOAc in n-hexane): Rf=0.21.

Example 113: ((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(butane-4,1diyl)bis(7-hexyl tridecanoate)

Example 113 can be prepared from Intermediate 38d and Intermediate 113e using methods similar to those used to prepare example 38.

¹ H NMR (400 MHz, CDCl ₃ ) δ 6.58 (br.s, 2H), 6.38 (br.s, 1H), 4.08-4.19 (m, 4H), 3.93- 4.08 (m, 4H), 3.56 (br.s, 2H), 2.41 (br.s, 6H), 2.23-2.35 (m, 4H), 1.75-1, 93 (m, 8H), 1.51-1.73 (m, 6H), 1.13-1.41 (m, 52H), 0.751.02 (m, 12H) ppm.

¹³ C NMR (101 MHz, CDCl ₃ ) δ 174.03 (2C), 160.29 (2C), 107.85 (3C), 101.26, 67.46 (2C), 63.89 (3C), 44.26(2C), 37.37(2C), 34.40(2C), 33.63(4C), 33.51(2C), 31.98(4C), 29.85(4C), 29 .71 (2C), 26.66 (4C), 26.38 (2C), 25.84, 25.80, 25.48 (2C), 25.07 (2C), 22.74 (4C), 14 .18 (4C) ppm

Example 114 Pentyltetradecanoate) ((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(butane-4,1-diyl)bis(9-

Example 114 can be obtained from Intermediate 38d using methods similar to those used to obtain example 113.

1H NMR (400 MHz, CDCl ₃ ) δ 6.49 (br. s, 2H), 6.35 (s, 1H), 4.09-4.23 (m, 4H), 3.98 (t, J \u003d 5.56 Hz, 4H), 3.38 (br. s, 2H), 2.19-2.37 (m, 10H), 1.75-1.94 (m, 8H), 1.48- 1.75 (m, 10H), 1.14-1.39 (m, 48H), 0.89 (t, J=7.07 Hz, 12H) ppm

ER-MS m/z=872.9 (MH+).

Synthesis of example 115:

Intermediate 115a: 3-heptyldec-2-enal

Intermediate 115a can be prepared using methods similar to those used to prepare Intermediate 113c in the synthesis of Example 113.

TLC (silica gel, 10% EtOAc in hexanes): R _f = 0.63.

Intermediate 115b: methyl 5-heptyldodeca-2,4-dienoate

To a suspension of sodium hydride (55% in paraffin oil, 3.5 g, 74.3 mmol) in THF (70 ml) cooled in an ice bath was added trimethylphosphonoacetate (9.6 ml, 59.5 mmol). After 10 min, Intermediate 115a (7.5 g, 29.7 mmol) in THF (10 ml) was added and the reaction mixture was allowed to warm to ambient temperature. After 2 hours, the reaction mixture was quenched with the slow addition of ice water (20 ml). The reaction mixture was extracted with ethyl acetate (2x100 ml). The organic extracts were washed with water and brine. The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure to give 8.0 g of the expected product.

TLC (silica gel, 10% EtOAc in hexanes): R _f = 0.75.

Intermediate 115с: methyl 5-heptyl dodecanoate

- 87 040257

To a solution of Intermediate 115b (8.0 g, 25.95 mmol) in methanol (350 ml) was added palladium on carbon (10% Pd/C, 1.0 g). The reaction was carried out under a pressure of 1 atm of hydrogen supplied by a balloon. After 14 h, the reaction mixture was filtered through celite, rinsing with methanol. The filtrate was concentrated under reduced pressure to give 7.7 g of the expected product.

TLC (silica gel, 5% methanol in dichloromethane): R _f = 0.63.

Intermediate 115d: 5-heptyldodecanoic acid

To a mixture of 5N sodium hydroxide (aq., 125 ml) and methanol (350 ml) was added Intermediate 115c (7.7 g, 24.7 mmol) and the reaction mixture was heated to reflux. After 16 h, the reaction mixture was cooled in an ice bath and quenched with the addition of concentrated aqueous HCl until acidic. The mixture was extracted with ethyl acetate (2x250 ml). The organic extracts were dried over sodium sulfate, filtered and concentrated under reduced pressure. The concentrate was purified on silica gel using ethyl acetate/hexane as eluent to give 7.0 g of the expected product.

TLC (silica gel, 50% EtOAc in heptane): Rf=0.82.

Example compound diyl)bis(5-heptyldodecanoate)

115: ((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(butane-4,1-

Example 115 can be prepared from Intermediate 38d and Intermediate 115d using methods similar to those used to prepare example 38.

1H NMR (400 MHz, CDCI3) δ 6.48 (d, J=1.76 Hz, 2H), 6.35 (t, J=2.13 Hz, 1H), 4.14 (t, J=5 .90 Hz, 4H), 3.97 (t, J=5.52 Hz, 4H), 3.38 (br.s, 2H), 2.19-2.36 (m, 10H), 1.76 -1.93 (m, 8H), 1.60 (dt, J=15.31, 7.65 Hz, 4H), 1.15-1.40 (m, 54H), 0.79-0.95 (m, 12H) ppm

ER-MS m/z=873.0 (MH+).

Example 116: ((5-((Dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(butan-4,1-diyl)bis(3-octylundecanoate)

Example 116 can be prepared from Intermediate 3 8d and Intermediate 97c using methods similar to those used to prepare example 38.

1H NMR (400 MHz, CDCl ₃ ) δ 6.63-6.82 (m, 2H), 6.38-6.52 (m, 1H), 4.09-4.23 (m, 4H), 4 .02 (t, J=5.56 Hz, 4H), 2.49-2.84 (m, 6H), 2.24 (d, J=7.07 Hz, 4H), 1.75-1, 98 (m, 10H), 1.49-1.69 (m, 2H), 1.16-1.39 (m, 56H), 0.79-0.94 (m, 12H) ppm

ER-MS m/z=872.3 (MH+).

Example 117 (5-((Dimethylamino)methyl)-1,3-phenylene)bis(methylene)bis(5-heptyldodecanoate)

Example 117 can be prepared from Intermediate 77b and Intermediate 115d using methods similar to those used to prepare example 77.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.35 (br.s, 2H), 7.29 (br.s, 1H), 5.12 (s, 4H), 3.62 (br.s, 2H), 2.35 (t, J=7.58 Hz, 10H), 1.62 (dt, J=15.16, 7.58 Hz, 4H), 1.14-1.43 (m, 54H ), 0.80-0.99 (m, 12H) ppm

ER-MS m/z=756.7 (MH+).

Example 118 (5-((Dimethylamino)methyl)-1,3-phenylene)bis(methylene)bis(7-hexyl tridecanoate)

Example 118 can be prepared from Intermediate 77b and Intermediate 113e using methods similar to those used to prepare example 77.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.30 (s, 2H), 7.22-7.29 (m, 1H), 5.11 (s, 4H), 3.55 (br. s, 2H), 2.23-2.46 (m, 10H), 1.65 (q, J=7.40 Hz, 4H), 1.18-1.41 (m, 54H), 0.79-0 .94 (m, 12H) ppm

- 88 040257 ¹³ C NMR (101 MHz, CDCl ₃ ) δ 173.70 (2C), 136.83 (2C), 128.86 (3C), 127.01, 65.67 (2C), 63.60, 45.01(2C), 37.38(2C), 34.34(2C), 33.63(4C), 33.53(2C), 31.97(4C), 29.85(4C), 29 .70 (2C), 26.66 (4C), 26.40 (2C), 25.02 (2C), 22.74 (4C), 14.17 (4C) ppm

Example 119 (5-((Dimethylamino)methyl)-1,3-phenylene)bis(methylene)bis(9-pentyltetradecanoate)

Example 119 can be prepared from Intermediate 77b using methods similar to those used to prepare example 118.

1H NMR (400 MHz, CDCl ₃ ) δ 7.24-7.34 (m, 3H), 5.11 (s, 4H), 3.55 (br. s, 2H), 2.23-2.43 (m, 10H), 1.64 (kv, J=7.28Hz, 4H), 1.11-1.41 (m, 54H), 0.88 (t, J=7.03Hz, 12H) ppm

ER-MS m/z=756.5 (MH+).

Synthesis of example 120.

Intermediate 120a: 4-((tert-butyldimethylsilyl)oxy)butylmethanesulfonate

To a solution of 4-((t-butyldimethylsilyl)oxy)butan-1-ol (10.0 g, 49.0 mmol) in dichloromethane (100 ml) was added triethylamine (20.4 ml, 147 mmol) and methanesulfonyl chloride (4. 93 ml, 63.7 mmol). After 5 hours the reaction mixture was quenched with water (150 ml) and extracted with dichloromethane (2x150 ml). The combined DCM layers were washed with brine (100 ml), dried over sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to give the title compound. TLC (silica gel, 20% ethyl acetate in hexanes): Rf=0.42

Intermediate 120b: 3,5-bis(4-((tert-butyldimethylsilyl)oxy)butoxy)benzaldehyde

To a solution of 3,5-dihydroxybenzaldehyde (2.5 g, 18.1 mmol) in DMF (50 mL) was added potassium carbonate (12.50 g, 90.5 mmol) followed by Intermediate 120a (12.76 g, 45 .3 mmol). The reaction mixture was heated to 80° C. and stirred for 24 hours. The reaction mixture was cooled, quenched with water (200 ml) and extracted with ethyl acetate (2x200 ml). The combined organic layers were washed with brine (150 ml), dried over sodium sulfate and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified on silica gel using ethyl acetate and hexane as eluent to give the title compound.

TLC (silica gel, 30% ethyl acetate in hexane): Rf=0.71

Intermediate 120с: methyl 3-(3,5-bis(4-((tert-butyldimethylsilyl)oxy)butoxy)phenyl)acrylate

To a solution of trimethylphosphonoacetate (3.0 ml, 20.6 mmol) in THF (70 ml) cooled in an ice bath was added sodium hydride (898 mg, 55% dispersion, 20.6 mmol). The reaction mixture was stirred for 30 min, then a solution of Intermediate 120b (7.0 g, 13.7 mmol) in THF (30 ml) was added. The cooling bath was removed and the reaction continued for an additional 90 minutes. The reaction mixture was quenched with water (100 ml) and extracted with ethyl acetate (2x200 ml). The combined organic extracts were washed with brine (200 ml), dried over sodium sulfate and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified on silica gel using ethyl acetate and hexane as eluent to give the title compound.

ER-MS m/z=567.5 (MH+).

Intermediate 120d: 3-(3,5-bis(4-((tert-butyldimethylsilyl)oxy)butoxy)phenyl)propan-1-ol

To a solution of Intermediate 120c (7.0 g, 12.4 mmol) in THF (100 ml) cooled in an ice bath was added lithium aluminum hydride (1.88 g, 49.4 mmol) and the reaction mixture was stirred for 3 h. the mixture was quenched with cold water and filtered through celite. The filtrate was extracted with ethyl acetate (2x200 ml) and the combined organic extracts washed with brine (200

- 89 040257 ml), dried over sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to give the desired product, which was used without further purification.

ER-MS m/z=541.4 (MH+).

Intermediate 120e: 1-(3-(3,5-bis(4-((tert-butyldimethylsilyl)oxy)butoxy)phenyl)propyl)piperidine

To a solution of tosyl anhydride (Ts ₂ O, 1.81 g, 5.55 mmol) in dichloromethane (25 ml) was added triethylamine (1.03 ml, 7.40 mmol) followed by the addition of Intermediate 120d (2.0 g, 3, 70 mmol) in DCM (15 ml). After 1 h, piperidine (3.15 g, 18.50 mmol) was added and the reaction mixture was sealed. After an additional 15 h the reaction mixture was diluted with water (100 ml) and was extracted with dichloromethane (2x100 ml). The combined dichloromethane extracts were washed with brine (200 ml), dried over sodium sulfate and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified on silica gel using methanol:dichloromethane as eluent to give the title compound.

ER-MS m/z=608.3 (MH+).

Intermediate 120f: 4,4'-((5-(3-(piperidin-1-yl)propyl)-1,3-phenylene)bis(oxy))bis(butan-1-ol)

To a solution of Intermediate 120e (1.3 g, 2.14 mmol) in diethyl ether (10 ml) was added HCl in dioxane (20 ml). After 2 hours the reaction mixture was concentrated under reduced pressure and washed with diethyl ether. The residue was diluted with saturated aqueous sodium bicarbonate and extracted with ethyl acetate (2x50 ml). The combined organic extracts were dried over sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to give the title compound, which was used without further purification.

ER-MS m/z=380.1 (MH+).

Example 120 compound: (9Z,9'Z,12Z,12'Z)-((5-(3-(piperidin-1-yl)propyl)-1,3-phenylene)bis(oxy))bis(butane- 4,1-diyl)bis(octadeca-9,12-dienoate)

Example 120 can be prepared from Intermediate 120f using methods similar to those used to prepare Example 38.

¹ H NMR (400 MHz, CDCl ₃ ) δ 6.33 (d, J=2.01 Hz, 2H), 6.21-6.31 (m, 1H), 5.22-5.51 (m, 8H), 4.07-4.22 (m, 4H), 3.95 (t, J=5.52 Hz, 4H), 2.78 (t, J=6, 65 Hz, 4H), 2, 56 (t, J=7.53 Hz, 2H), 2.35-2.54 (m, 4H), 2.26-2.35 (m, 4H), 2.05 (k, J=6, 61 Hz, 8H), 1.77-1.98 (m, 10H), 1.55-1.76 (m, 8H), 1.42-1.55 (m, 2H), 1.20-1 .42 (m, 30H), 0.85-0.99 (m, 6H) ppm

¹³ C NMR (101 MHz, CDCl3) δ 173.98 (2C), 160.07 (2C), 130.24 (2C), 130.07 (2C), 128.05 (2C), 127.91 (2C ), 106.92 (3C), 98.74 (1C), 67.21 (2C), 63.91 (2C), 58.48, 54.34 (2C), 34.36 (2C), 34, 02, 31.55 (2C), 29.63 (2C), 29.37 (2C), 29.21 (2C), 29.17 (2C), 29.15 (2C), 27.22 (4C) , 25.90 (2C), 25.64 (2C), 25.47 (2C), 25.00 (2C), 24.21 (2C), 22.61 (2C), 14.12 (2C) m .d. ER-MS m/z=904.9 (MH+).

Example 121: (9Z,9'Z, 12Z, 12'Z)-((5-(3-(dimethylamino)propyl)-1,3-phenylene)bis(oxy))bis(butane-4,1diyl) bis(octadeca-9,12-dienoate)

Example 121 can be obtained from Intermediate 120d using methods similar to those used to obtain example 120.

1H NMR (400 MHz, CDCl ₃ ) δ 6.33 (d, J=2.02 Hz, 2H) 6.25-6.31 (m, 1H) 5.26-5.47 (m, 8H) 4 .07-4.22 (m, 4H), 3.96 (t, J=5.56 Hz, 4H) 2.78 (t, J=6.57 Hz, 4H) 2.62 (t, J= 7.33 Hz, 4H) 2.52 (br.s, 6H) 2.30 (t, J=7.58 Hz, 4H) 2.05 (c, J=6, 65 Hz, 10H) 1.76 -1.94 (m, 8H) 1.55-1.70 (m, 4H) 1.21-1.46 (m, 28H) 0.81-0.98 (m, 6H) ppm

ER-MS m/z=864.6 (MH+).

Example 122 (9Z,9'Z, 12Z, 12'Z)-((5-(3-morpholinopropyl)-1,3-phenylene)bis(oxy))bis(butane-4,1diyl)bis(octadeca -9,12-dienoate)

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Example 122 can be prepared from Intermediate 120d using methods similar to those used to prepare example 120.

¹ H NMR (400 MHz, CDCl ₃ ) δ 6.33 (d, J=2.26 Hz, 2H), 6.23-6.30 (m, 1H), 5.23-5.48 (m, 8H), 4.07-4.22 (m, 4H), 3.95 (t, J=5.52 Hz, 4H), 3.75 (br.s, 4H), 2.78 (t, J \u003d 6.53 Hz, 4H), 2.56-2.64 (m, 2H), 2.35-2.51 (m, 4H), 2.30 (t, J \u003d 7.65 Hz, 4H) , 2.05 (d, J=6.78 Hz, 8H), 1.75-1.97 (m, 10H), 1.53-1.72 (m, 6H), 1.16-1.44 (m, 28H), 0.79-1.01 (m, 6H) ppm

ER-MS m/z=906.9 (MH+).

Example 123

Example 123 can be prepared using methods similar to those used to prepare Example 18 and Example 52.

¹ H NMR (400 MHz, CDCl ₃ ) δ 6.47 (d, J=2.2 Hz, 2H), 6.40 (t, J=2.2 Hz, 1H), 5.07 (s, 2H ), 4.05 (t, J=6.7 Hz, 4H), 3.92 (t, J=6.5 Hz, 4H), 3.00 (t, J=7.0 Hz, 2H), 2.71 (t, J=7.1 Hz, 2H), 2.56 (s, 6H), 2.29 (t, J=7.6 Hz, 4H), 2.07 (s, 2H), 1.76 (q, J=6.7 Hz, 4H), 1.61 (td, J=7.4, 7.0, 3.9 Hz, 8H), 1.43 (d, J=7, 9 Hz, 4H), 1.39-1.11 (m, 34H), 0.91-0.83 (m, 6H).

ER-MS m/z=805.0 (MH+)

Example 124 ((5-(((4-(Dimethylamino)butanoyl)oxy)methyl)-1,3-phenylene)bis(oxy))bis(octane-8,1 diyl)bis(decanoate)

Example 124 can be obtained using methods similar to those used to obtain example 18 and example 52.

1H NMR (400 MHz, CDCl ₃ ) δ 6.46 (d, J=2.2 Hz, 2H), 6.39 (t, J=2.2 Hz, 1H), 5.04 (s, 2H) , 4.05 (t, J=6.7 Hz, 4H), 3.92 (t, J=6.5 Hz, 4H), 2.62 (s, 6H), 2.51 (t, J= 6.9 Hz, 2H), 2.29 (t, J=7.5 Hz, 4H), 1.95 (k, J=7.0 Hz, 2H), 1.80-1.71 (m, 4H), 1.61 (dc, J=7.2, 3.9 Hz, 8H), 1.44 (t, J=7.7 Hz, 4H), 1.40-1.20 (m, 38H ), 0.87 (t, J=6.7 Hz, 6H).

ER-MS m/z=819.0 (MH+)

Example 125.

Intermediate 125 a: 2,2,3,3,9,9,10,10-octamethyl-4,8-dioxa-3,9-disiloundecan-6-ol

A suspension of glycerol (5 g, 54.3 mmol), imidazole (8.1 g, 119 mmol) and tert-butylchlorodimethylsilane (16.37 g, 109 mmol) in THF (40 ml) was stirred for 15 h. The reaction mixture was quenched with water ( 300 ml) and extracted with ethyl acetate. The combined organic extracts were washed with brine, dried over magnesium sulfate and filtered.

The filtrate was concentrated under reduced pressure, and the residue was purified on silica gel using ethyl acetate and heptane as eluent to give the title compound.

1H NMR (400 MHz, CDCl ₃ ) δ 3.55-3.76 (m, 4H), 2.47 (br.s, 1H), 1.61 (br.s, 1H), 0.79-1 .06 (m, 18H), 0.06-0.21 (m, 12H) ppm

Intermediate 125b: 6-(((tert-butyldimethylsilyl)oxy)methyl)-2,2,3,3-tetramethyl-4,7,12-trioxa-3silatetradec-13-ene

Sodium hydride (0.618 g, 60% dispersion, 15.44 mmol). After 1 hour the cooling bath was removed and the reaction mixture was allowed to warm to ambient temperature for 4 hours followed by reflux for 1 hour. The mixture was cooled to ambient temperature and quenched with ice water. The reaction mixture was extracted with ethyl acetate (100 ml). The organic extract was dried over magnesium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified on silica gel using ethyl acetate and heptane as eluent to give the title compound.

1H NMR (400 MHz, CDCl3) δ 6.47 (dd, J=14.31, 6.78 Hz, 1H), 4.11-4.22 (m, 1H), 3.98 (dt, J= 7.03, 2.01 Hz, 1H), 3.56-3.76 (m, 6H), 3.42-3.56 (m, 2H), 3.30-3.42 (m, 1H) , 1.60-1.81 (m, 4H), 0.80-1.01 (m, 18H), 0.000.12 (m, 12H) ppm.

Intermediate 125с: 2-(4-(vinyloxy)butoxy)propane-1,3-diol

To a solution of Intermediate 125b (1.4 g, 3.34 mmol) in THF (10 ml) was added tetrabutylammonium fluoride (10 ml, 1M in THF, 10 mmol). After 2 h the reaction mixture was concentrated under reduced pressure. The residue was diluted with ethyl acetate and washed with water and brine, dried over magnesium sulfate and filtered. The filtrate was concentrated under reduced pressure and the residue was purified on silica gel using ethyl acetate and hexane as eluent to give the expected product.

TLC (silica gel, 50% ethyl acetate in hexanes): Rf=0.10.

Intermediate 125d: 2-(4-(vinyloxy)butoxy)propane-1,3-diyldioctanoate

To a solution of octanoic acid (584 mg, 4.05 mmol), Intermediate 125c (350 mg, 1.84 mmol), DMAP (22.5 mg, 0.184 mmol) and diisopropylethylamine (713 mg, 5.52 mmol) in dichloromethane ( 10 ml) cooled in an ice bath was added EDCI (846 mg, 4.42 mmol). The reaction mixture was stirred at ambient temperature overnight, after which the reaction mixture was concentrated under reduced pressure. The residue was purified on silica gel using ethyl acetate and heptane as eluent to give the title compound. TLC (silica gel, 50% ethyl acetate in heptane): Rf=0.85

Intermediate 125e: 2-(4-hydroxybutoxy)propane-1,3-diyldioctanoate

To a solution of Intermediate 125d (520 mg, 1.18 mmol) in dichloromethane (10 ml) was added trifluoroacetic acid (0.181 ml, 2.35 mmol). After 1 hour, the reaction mixture was concentrated under reduced pressure, and the residue was purified on silica gel using ethyl acetate and heptane as eluent to give the title compound.

¹ H NMR (400 MHz, CDCl ₃ ) δ 4.23 (dd, J=11.54, 5.02 Hz, 2H), 4.12 (dd, J=11.54, 5.52 Hz, 2H) , 3.71 (kv, J=5.14 Hz, 1H), 3.64 (dt, J=15.43, 5.83 Hz, 4H), 2.34 (t, J=7.00 Hz, 4H), 1.56-1.73 (m, 8H), 1.19-1.41 (m, 16H), 0.81-1.01 (m, 6H) ppm.

Compound of Example 125: ((((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(butane-4,1diyl))bis(oxy))bis(propane-3,2, 1-triyl)tetraoctanoate

Example 125 can be prepared from Intermediate 125e using methods similar to those used to prepare Example 39 or Example 52.

1H NMR (400 MHz, CDCl ₃ ) δ 6.49 (s, 2H), 6.34 (t, J=2.01 Hz, 1H) 4.17-4.26 (m, 4H) 4.08- 4.17 (m, 4H) 3.95 (t, J=6.15 Hz, 4H) 3.71 (kv, J=5.14 Hz, 2H) 3.63 (t, J=6.27 Hz , 4H) 3.42 (br.s, 2H) 2.24-2.40 (m, 14H) 1.79-1.90 (m, 4H) 1.69-1.79 (m, 4H) 1 .62 (kv, J=7.34 Hz, 8H) 1.21-1.38 (m, 32H) 0.83-0.94 (m, 12H) ppm

ER-MS m/z=964.7 (MH+).

Synthesis of example 126:

Intermediate 126а: 4,4-bis(octyloxy)butanenitrile

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To a mixture of 4,4-diethoxybutanenitrile (15 g, 95 mmol) and octanol (37.3 g, 286 mmol) was added pyridinium ptoluenesulfonate (1.2 g, 4.77 mmol) and the mixture was heated to 105°C. After 72 hours, the reaction mixture was cooled and purified on silica gel using ethyl acetate/heptane as eluent to give 9.34 g of expected product.

1H NMR (400 MHz, CDCl ₃ ): δ 4.56 (t, J=5.40 Hz, 1H), 3.61 (dt, J=9.16, 6.59 Hz, 2H), 3.44 (dt, J=9.22, 6.68 Hz, 2H), 2.43 (t, J=7.28 Hz, 2H), 1.95 (td, J=7.34, 5.40 Hz, 2H), 1.50-1.66 (m, 4H), 1.17-1.44 (m, 20H), 0.80-0.95 (m, 6H) ppm.

Intermediate 126b: 4,4-bis(octyloxy)butan-1-ol

To a solution of Intermediate 126a (5 g, 15.4 mmol) in dichloromethane (60 ml) cooled in a dry ice/acetone bath was added DIBAL-H (1.0 M in toluene, 15.4 ml, 15.4 mmol) . After 1.5 h, the cooling bath was removed and the reaction mixture was warmed to ambient temperature. The reaction mixture was quenched with saturated aqueous ammonium chloride (20 ml) and water (10 ml) and extracted with dichloromethane. The DCM extracts were washed with brine, dried over magnesium sulfate and filtered. The filtrate was concentrated and the residue was redissolved in methanol (20 ml). Sodium borohydride (0581 g, 15.4 mmol) was added. After 1 h, the reaction mixture was quenched with water and the reaction mixture was concentrated under reduced pressure to remove methanol. The resulting mixture was extracted with ethyl acetate and the ethyl acetate extracts were washed with saturated aqueous sodium bicarbonate, dried over sodium sulfate and filtered. The filtrate was concentrated under reduced pressure and the residue was purified on silica gel using ethyl acetate and heptane as eluent.

¹ H NMR (400 MHz, DMSO-d6) δ 4.34-4.46 (m, 2H), 3.47 (dt, J=9.03, 6.53 Hz, 2H), 3.28-3 .41 (m, 6H), 1.35-1.58 (m, 8H), 1.13-1.35 (m, 18H), 0.79-0.91 (m, 6H) ppm

Example 126: 1-(3,5-bis(4,4-bis(octyloxy)butoxy)phenyl)-N,N-dimethylmethanamine

Example 126 can be prepared from Intermediate 126b using methods similar to those used to prepare Example 39 or Example 52.

¹ H NMR (400 MHz, CDCl ₃ ) δ 6.48 (d, J=2.01 Hz, 2H), 6.35 (t, J=2.13 Hz, 1H), 4.48-4.58 (m, 2H), 3.96 (t, J=5, 90 Hz, 4H), 3.58 (dt, J=9.29, 6.65 Hz, 4H), 3.33-3.49 ( m, 6H), 2.28 (s, 6H) 1.73-1.92 (m, 8H), 1.57 (q, J=6, 96 Hz, 8H), 1.18-1.42 ( m, 40H), 0.79-0.97 (m, 12H) ppm

ER-MS m/z=792.8 (MH+).

Lipid compositions

Lipid nanoparticles (LNPs) were prepared by mixing equal volumes of lipids dissolved in alcohol with siRNA dissolved in citrate buffer using the flow jet method. The lipid solution contains a cationic lipid compound of the invention, a helper lipid (cholesterol), an optional neutral lipid (DSPC), and a PEG (PEG) lipid at a concentration of 8-16 mg/mL for a target of 12 mg/mL in alcohol. The relative molar ratios of each lipid component in the compositions of this invention are presented in table. 4 and 5. The ratio of siRNA to total lipid is approximately 0.05 (w/w). Where the LNP formulation contains four lipid components, the molar ratios correspond to the lipid type as seen in the first four columns of the table, in the order in which they appear. The lipid ratio is in the range of 20 to 70 mol% for cationic lipids with a target of 40-60, the mole percentage of helper lipids is in the range of 20 to 70 with a target of 30 to 50, the mole % of neutral lipids is in the range of 0 to 30 , the lipid molar percentage of PEG is in the range of 1 to 6 with a target of 2 to 5. The concentration of the siRNA solution is in the range of 0.7 to 1.0 mg/mL with a target of 0.8 to 0.9 mg/mL in sodium citrate: sodium chloride buffer pH 4-6 to 4.5-5.5. LNPs are prepared by mixing equal volumes of a lipid solution in ethanol with siRNA dissolved in citrate buffer using the outflow method using a mixer with an ID ranging from 0.25 to 2.0 mm at a flow rate of 10 to 640 ml/min. The LNP mixed solution is kept at room temperature for 0-24 hours before the dilution step is carried out. The solution is then concentrated and diafiltered with a suitable buffer by an ultrafiltration method using membranes with a 30 to 500 kDa MW diaphragm. The final product is filtered under sterile conditions and stored at 4°C.

siRNA The siRNA used in the lipid nanoparticles described above was prepared from double-stranded siRNA sequences specific to the target mRNA sequence.

1. FVII siRNA double-stranded sequence 5' UUu AAU UGA AAC cAA GAc Auu 3' (SEQ ID NO: 1) 5' uGu cuu GGu uuc AAu uAA Auu 3' (SEQ ID NO: 2)

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2. PLK1-424 siRNA double-stranded sequence 5' UAU UUA AgG AGG GUG AuC Uuu 3' (SEQ ID NO: 3)

5' AGA Uca cCC Ucc uuA AAU auu 3' (SEQ ID NO:4)

The following abbreviations are used in these sequences:

A=adenosine

U=uridine

G=guanosine

C=cytosine a=2'-O-methyladenosine u=2'-O-methyluridine g=2'-O-methylguanosine c=2'-O-methylcytosine

pKa measurements

Unless otherwise noted, all pKa mentioned in the present description were measured at standard temperature and pressure. In addition, unless otherwise indicated, all pKa references are pKa references measured using the following methodology.

mm solution of lipids in ethanol was obtained by weighing the lipids and then dissolving them in ethanol. A 0.3 mM solution of the TNS fluorescent sample in 9:1 ethanol:methanol was prepared by first making a 3 mM TNS solution in methanol and then diluting to 0.3 mM with ethanol.

Received an aqueous buffer containing 200 mm dibasic sodium phosphate and 100 mm citric acid. The buffer was divided into twelve parts and the pH was adjusted with either 12N HCl or 6N NaOH to 4.21-4.33, 4.86-4.99, 5.23-5.37, 5.46-5.54, 5.65-5.74, 5.82-5.89, 6.09-6.18, 6.21-6.32, 6.45-6.52, 6.66-6.72, 6, 83-6.87, 7.19-7.28. 400 μl of 2mM lipid solution and 800 μl of 0.3mM TNS solution were mixed.

Using the Hamilton Microlab Star high throughput fluid device and Hamilton control software, 7.5 µl of the sample/lipid mixture was added to 242.5 µl of buffer in a 1 ml 96-well plate (NUNC model 260252, Nalgae Nunc International). This was done with all twelve buffers.

After mixing in 1 ml of a 96-well plate, 100 μl of each sample/lipid/buffer mixture was transferred to a 250 μl black clear-bottomed 96-well plate (model COSTAR 3904, Corning).

Fluorescence measurements are carried out on a SpectraMax M5 spectrophotometer using SoftMax pro 5.2 software and the following parameters:

Reading mode:

Fluorescence, top reading

Wavelengths: Ex 322 nm, Em 431 nm, Auto Cutoff On 420 nm

Sensitivity: Reading 6, PMT: Auto

Automatic mixing: First: Off position

Automatic calibration: On position

Type of test plate: 96-well standard clrbtm

Read wells: Read the entire plate

Settling time: Off position

Wav column. priority: column priority

Carriage Speed: Normal

Auto-read: Off position

After measurement, the background fluorescence value of the empty well of the 96-well plate was subtracted from each value for the sample/lipid/buffer mixture. The fluorescence intensity values were then normalized to the value at the lowest pH. The reported fluorescence intensities versus pH values were then plotted in Microsoft Excel software. Twelve points were connected by a line with uniformly distributed constants.

A point was found on the line at which the reduced fluorescence intensity was 0.5. A pH value corresponding to a reduced fluorescence intensity of 0.5 was found and the pKa of the lipid was calculated.

The pKa determined by this method is accurate to about 0.1 pKa units.

Polydispersity Index (PDI) measurements

Unless otherwise indicated, all PDIs mentioned herein are the PDI of a fully formed nanoparticle as measured by the dynamic light scattering method on a Malvern Zetasizer. The nanoparticle sample was diluted in phosphate buffered saline (PBS) so that the intensity was approximately 200-400 kimp. The data are presented in tables 4 and 5 as a weighted average of the intensity measurements.

Particle size of lipid nanoparticles

Unless otherwise indicated, all particle size measurements listed in Table. 4 and 5 are

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Z is the average particle size of a fully formed nanoparticle as measured by the dynamic light scattering method on the Malvern Zetasizer. A nanoparticle sample was diluted in phosphate buffered saline (PBS) such that the intensity is approximately 200-400 kimp.

Biological analyzes

Dosing Factor VII to Mice

Female CD-I mice were obtained from Harlan Labs and maintained under standard laboratory conditions without food or water restriction. Animals weighed approximately 25 g at the time of dosing. Formulated Factor VII siRNA was administered as a single dose intravenously via the lateral tail vein. Approximately 48 hours after injection, mice were sacrificed by CO ₂ inhalation followed by vena cava haemorrhage. Blood was collected in tubes containing the anticoagulant 0.105M sodium citrate for analysis of Factor VII plasma activity.

Factor VII activity assay

Collected plasma from injected mice was analyzed for factor VII enzymatic activity using the Biophen FVII kit from Hyphen Biomedical (catalog number 221304). A test calibration curve was generated using pooled aliquots of plasma from vehicle-fed control animals. All samples were diluted to be within the linear range of the calibration curve and Factor VII activity was reported compared to control plasma.

Lipid nanoparticles containing the lipid compounds of formula (I) and the FVII siRNA double strand sequence listed above were tested in a Factor VII activity assay. The results of this analysis are given in table. 4 below as percentage lowest plasma Factor VII enzymatic activity at 0.3 and 0.03 mg/kg.

Table 4. Results of analysis of Factor VII activity using FVII siRNA lipid nanoparticles

Cationic lipid of formula (I) Helper lipid Neutral lipid Hidden lipid ¹ Molar ratio of lipids Size (nm) PDI ² rKa KD FVII 0.3 mg/kg KD FVII 0.03 mg/kg Etc. 1 Hol DSPC S010 45/44/9/2 87.16 0.124 5.59 — 30, 8 Etc. 1 Hol DSPC PEG-DMG 45/44/9/2 98.2 0.05 5.59 75.1 — Etc. 2 Hol DSPC S010 45/44/9/2 92.85 0.067 5.57 — 55 Etc. 2 Hol DSPC PEG-DMG 45/44/9/2 97.4 0.04 5.46 95.5 — Etc. 14 Hol DSPC S010 45/44/9/2 81.74 0.059 6, 11 — — Etc. 26 Hol DSPC S010 45/44/9/2 121.6 0.046 6.45 — — Etc. 26 Hol DSPC PEG-DMG 45/44/9/2 104.3 0.04 6.45 58.1 — Etc. 31 Hol DSPC S010 45/44/9/2 81.16 0.049 6, 01 — — Etc. 33 Hol DSPC S010 45/44/9/2 79, 15 0.042 5.85 — — Etc. 33 Hol DSPC PEG-DMG 45/44/9/2 107.8 0.06 5.75 90.4 — Etc. 34 Hol DSPC S010 45/44/9/2 113.8 0.036 5.95 — — Etc. 34 Hol DSPC PEG-DMG 45/44/9/2 126.0 0.06 5, 61 0 — Etc. 3 8 Hol DSPC S010 45/44/9/2 120 0.04 5, 93 — 43.6 Etc. 3 8 Hol DSPC PEG-DMG 45/44/9/2 108.0 0.02 5, 9 99 — Etc. 3 9 Hol DSPC S010 45/44/9/2 122.9 0.044 5, 91 — —

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Etc. 4 0 Hol DSPC S010 45/44/9/2 94.2θ 0.062 6, 17 — 8.2 Etc. 4 0 Hol DSPC PEG-DMG 45/44/9/2 BUT, 9 0.02 6, 17 89, 8 — Etc. 41 Hol DSPC S010 45/44/9/2 170.7 0.119 5, 83 — — Etc. 42 Hol DSPC 3010 45/44/9/2 120 0.084 5, 93 — 92.8 Etc. 43 Hol DSPC 3010 45/44/9/2 121.9 0.066 5.85 — — Etc. 4 3 Hol DSPC PEG-DMG 45/44/9/2 157.8 0.08 5.85 97.4 — Etc. 4 4 Hol DSPC S010 45/44/9/2 172.9 0.077 5, 66 — — Etc. 4 5 Hol DSPC S010 45/44/9/2 102.8 0.072 5.57 — — Etc. 4 6 Hol DSPC S010 45/44/9/2 102.4 0.135 6, 18 — — Etc. 4 7 Hol DSPC S010 45/44/9/2 125.9 0.035 6, 07 — — Etc. 4 7 Hol DSPC PEG-DMG 45/44/9/2 109.3 0.06 6, 07 99 — Etc. 4 8 Hol DSPC S010 45/44/9/2 152.3 0.105 5, 93 — — Etc. 4 9 Hol DSPC 3010 45/44/9/2 111.1 0.046 5.99 — — Etc. 50 Hol DSPC S010 45/44/9/2 126 0.056 5, 92 — — Etc. 51 Hol DSPC S010 45/44/9/2 103.2 0.052 5, 91 — — Etc. 52 Hol DSPC S010 45/44/9/2 145.4 0.052 5.96 — 73.5 Etc. 52 Hol DSPC PEG-DMG 45/44/9/2 109.4 0, 1 5.96 — 72.5 Etc. 53 Hol DSPC S010 45/44/9/2 113.3 0.079 6, 03 — — Etc. 53 Hol DSPC PEG-DMG 45/44/9/2 129, 1 0, 1 5, 84 96.6 — Etc. 55 Hol DSPC 3010 45/44/9/2 106.9 0.043 6, 37 — — Etc. 55 Hol DSPC PEG-DMG 45/44/9/2 101.9 0.10 6, 03 98.0 — Etc. 56 Hol DSPC SO 10 45/44/9/2 99, 47 0.114 5.93 --- Etc. 56 Hol DSPC PEG-DMG 45/44/9/2 110.1 0.01 5.71 71.0 — Etc. 57 Hol DSPC S010 45/44/9/2 123/8 0.059 5.94 — .--- Etc. 57 Hol DSPC PEG-DMG 45/44/9/2 1'08.4 0.03 5.94 9 a, 7 — Etc. 58 Hol DSPC S010 45/44/9/2 138.9 0.022 5, 9 — 15.25 Etc. 58 Hol DSPC PEG-DMG 45/44/9/2 120.6 0.06 6.02 78.1 — Pr- 59 H0L DSPC S.D10 45/44/9/2 92.56 0.064 6.09 — 48.24 Etc. 60 Hol. DSPC before u 45/44/9/2 95.55 Oh 098 6, 21 — 4 9.75 Etc. 61 Hol DSPC SO 10 45/44/9/2 81.79 0.076 5.82 — 0 Pr, 62 Hol .DSPH S010 45/44/9/2 102.5 0.066 5, 9 --- . 85.8 Etc. 63 Hol DSPC ZOYU 45/44/9/2 91.66 0.057 5.92 — 75 Etc. 69 Hol DSPC S010 45/44/9/2 98, 13 0g 053 5, 3 — -- Etc. 77 Hol DSPC ZOYU 45/44/9/2 154.1 0.014 6.09 — — Etc. 78 Hol DSPC ZOYU 45/44/9/2 178.2 0.043 6.02 — ___ Etc. 79 Hol DSPC noah 45/44/9/2 6, 48 — _— Etc. 82 Hol DSPC z.oyu 45/4.4/9/2 119.2 Oh 064 6, 03 --- — Etc. 83 Hol DSPC before u 45/44/9/2 126.5 0, 00.5. .6.15 — ^: -- Etc. 85 Hol DSPC SO 10 45/44/9/2 5.72 — — ' Etc. 8 6 Hol DSPC ZOYU 45/44/9/2 120.3. Oh 058 5.84 — 0 Etc. 8 6 Hol. DSPC PEG-DMG 45/44/9/2 128.7 0.05 5.81 55.3 — Etc. 87 Hol DSPC ZOYU 45/44/9/2 159.2 0.089 6.07 ___ —

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Etc. 80 Hol DSPC SOLO 4 5/44/9/2 152.4 0.0'46 5.99 — — Etc. 89 Hol DSPC S010 .45/ 44 /9/2 101.5 0.076 6, 02 ---- — Etc. 90 Hol DSPC solo 45/44/9/2 87.54 0.073 5, 81 — — Etc. 91 Hol DSPC S310 45/44/9/2 111.9 0.053 6, 01 — -- Etc. 91 Hol DSPC PEG-DMG 45/44/9/2 124.3 0.06 6, 01 99.0 -.—. Etc. 92 Hol DSPC SO 10 45/44/9/2 117.3 0.065 5, 92 — 89.4 Etc. 93 Hol DSPC sow 45/44/9/2 121.5 0.071 6.00 .--- 89.71 Etc. 94 Hol DSPC sow 45/44/9/2 116.4 0.073 6.16 — 90 Etc. .96 HOL DSPC SOLO 45/44/9/2 197, 3 0-,14. ' 5, 71 ---- Etc. 97 Hol DSPC sow. 45/44/9/2 98, 01 0.106 5, 82 - — Etc. .98 Hol DSPC SO 10 45/44/9/2 164.6 C-096 5, 94 - — _ — Etc. 99 Hol DSPC SO I.Q. 45/44/9/2 96, 61 0.061 5.75 — — Etc. 100 Hol DSPC sow 45/44/9/2 105.9 0.121 6, 17 — 26, 9 Etc. 100 Hol ..DSPH PEG-DMG 45/44/9/2 106.4 0.07 5, 69 94, Q ------ rip. 101 Hol DSPC SOLO 45/44/9/2 — -- '6,.32 — — Etc. 104 Hol DSPC SOLO 45/44/9/2 — — 6, 02 — — Etc. 106 Hol DSPC SOW 45/44/9/2 149.3 ^: 0.083 6, 04 — — project 107 Hol DSPC SOLO 45/44/9/2 191.7- 0.071 6, 11 — — Etc. 109 Hol DSPC St 10 45/44/9/2 91.99' 0.09 5.98 — 68, 97 Etc. 110 Hol DSPC solo . 45/44/9/2 183.3 0.071 5, 66 . --- --- . Etc. 111 Hol dsph SOLO 45/44/9/2 161.9 0.048 6, 27 ---- — Etc. 112 Hol DSPC PEG-DMG 45/44/9/2 96.1 o, 1 6, 08 — 86 Etc. 113 Hol DSPC PEG-DMG 45/44/9/2 87.5 0.132 6, 03 — 57.6 Etc. 114 Hol DSPC PEG-DMG 45/44/9/2 94.9 0, 1 5.97 — 44 Etc. 115 Hol DSPC PEG-DMG 45/44/9/2 89.0 0, 1 5, 87 — 78 Etc. 116 Hol DSPC PEG-DMG 45/44/9/2 87.5 0.094 5, 67 — 29 Etc. 117 — — — — — — 5.46 — — Etc. 118 Hol DSPC PEG-DMG 45/44/9/2 84.8 0.058 5, 67 — 46, 3 Etc. 119 Hol DSPC PEG-DMG 45/44/9/2 85.3 0.073 5, 84 — 7.8 Etc. 120 Hol DSPC PEG-DMG 45/44/9/2 96.5 0.056 6, 31 — 83.7 Etc. 121 — — — — — — — — Etc. 122 — — — — — — 5, 06 — — Etc. 123 — — — — — — 6, 08 — — Etc. 124 — — — — — — 7, 11 — — Etc. 125 Hol DSPC PEG-DMG 45/44/9/2 194.7 0.086 5.75 — — Etc. 126 Hol DSPC PEG-DMG 45/44/9/2 95.2 0.075 5, 54 — 31.7

1 The order of lipid types as they appear in molar ratio corresponds to the order in which the lipids appear in the first four columns of the table.

² pKa refers to the pKa of the cationic lipid of formula (I)

LS411N xenograft analysis

Female Nu/Nu mice (6-8 weeks old) were implanted subcutaneously with 5x106 LS411N cells. Tumor growth was monitored by caliper measurement prior to treatment. Mice having 150-250 mm ³ subcutaneous tumors were randomized and included in the study. Basic siRNA formulations were diluted to 0.3 m/ml PBS for dosing. Animals included in different groups received a single daily IV bolus injection of 3 mg/kg siRNA for three days. Tumors were sampled 24 hours after the last injection to evaluate target gene regulation by real-time PCR (qRT-PCR).

Lipid nanoparticles containing the lipid compounds of formula (I) and the PLK1-424 siRNA double strand sequence listed above were examined in the LS411N xenograft assay. The results of this analysis are shown in table. 5 lower as percentage lowest PLK-1 mRNA compared to control when administered as a single daily dose for three days at a dose of 3 mg/kg.

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Table 5. Lipid nanoparticles containing PLK1-424 siRNA and results of analysis of xenograft LS411N

Cationic lipid of formula (I) Neutral lipid Helper lipid Hidden lipid ³ Molar ratio of lipids Size (nm) PDI ⁴ rKa LS411 KD 3x3 mg/kg Example 1 Hol DSPC S010 45/44/9/2 74.56 0.152 5.59 — Example 2 Hol DSPC son 45/44/9/2 88, 19 0, 1 5.57 — Example 3 8 Hol DSPC S010 45/44/9/2 116.8 0.036 5, 93 60 Example 3 8 Hol DSPC PEG-DSG 45/44/9/2 116.8 0.036 5, 93 58 Example 3 9 Hol DSPC S010 45/44/9/2 114.3 0.039 5, 91 55 Example 42 Hol DSPC S010 45/44/9/2 181.1 0.06 5, 93 50 Example 4 3 Hol DSPC S010 45/44/9/2 150.7 0.046 5.85 40 Example 52 Hol DSPC S010 45/44/9/2 132.2 0.06 5.96 50 Example 52 Hol DSPC PEG-DSG 45/44/9/2 132.2 0.06 5.96 69 Example 57 Hol DSPC S010 45/44/9/2 116.3 0.038 5, 94 60 Example 63 Hol DSPC n3r_DSG 45/44/9/2 108.6 0.047 5, 9 66 Example 91 Hol DSPC S010 45/44/9/2 136.1 0.032 6, 01 45

³ The order of lipid types as they appear in molar ratio corresponds to the order in which the lipids appear in the first four columns of the table.

⁴ pKa refers to the pKa of the cationic lipid of formula (I)

Immunization research

A population of BALB/c mice were immunized with liposomes containing DSPC, cholesterol and various lipids of the invention (or for comparison, the cationic lipid 1,2-dilinoleyloxy-K,K-dimethyl3-aminopropane or DLinDMA). The liposomes were encapsulated with 0.1 μg of the vA317 self-replicating replicon RNA that encodes the respiratory syncytial virus F protein (see WO 2012/031043). As a negative control, mice received 1 μg of naked replicon. Doses were given either on days 0 & 21 or days 0 & 42. Immune responses were assessed 2 weeks after each dose (2wp1, 2wp2) and in some cases also 4 weeks after the second dose (4wp2). Conducted four series of experiments, each with its own naked control. In all experiments, sera were evaluated in two pools, and anti-PCB-F titers are shown in table. 6 below.

Table 6. Immunization titers against RSV F-protein

Cationic lipid Formula (I) o δ 2wpl pool 1 2wpl pool 2 2wp2 pool 1 2wp2 pool 2 4wp2 pool 1 4wp2 pool 2 Naked A 115 264 692 5371 - - DLinDMA A 819 954 18318 21731 - - Example 2 A 217 259 12785 8232 - - Example 13 A 495 620 7157 12316 - - Example 14 A 1234 2089 44059 45246 - - Example 9 A 1832 1352 29707 18788 - - Naked IN 155 48 3264 1440 4747 1954 DLinDMA IN 13 4 18290 5538 13936 7020 Example 12 IN 88 128 4550 4828 4787 3972 Naked A 219 255 855 1052 1157 1726 DLinDMA A 4336 6460 19155 19141 26504 28008 Example 2 6 A 4 4 10 10 10 10 Example 14 A 3098 3539 12785 14582 13687 18851 Example 25 A 1217 1883 1611 1156 2850 1767 Naked A 2699 905 5253 5491 4323 5008

DLinDMA A 4585 3390 60395 56229 50799 35992 Example 14 A 1192 1206 34906 28736 21375 21347 Example 66 A 75 75 75 75 75 75 Example 68 A 1031 494 11315 7800 12963 17734 Example 81 A 1779 1553 24648 15304 41921 34684 Example 2 9 A 2101 3584 12529 18669 23930 37278 Example 7 0 A 1416 3110 31550 20124 18779 18358 Example 69 A 2853 4481 23851 20793 19075 21504

Course: A=days 0 &21;

B=days 0 & 42.

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mRNA transcription protocol

A circular plasmid DNA template was constructed that contained an mRNA transcription cassette consisting of the following features: bacteriophage T7 DNA dependent RNA polymerase consensus promoter, 5' untranslated region (UTR), Kozak sequence and open reading frame, and 3' UTR, and 120 nucleotides long polyadenosine (polyA120) tail. The plasmid DNA template was replicated in E. coli, isolated and linearized by restriction enzyme digestion directly at the 3' poly120 tail. Plasmid DNA was combined with T7 RNA polymerase, ribonucleotide triphosphates, RNase inhibitor, pyrophosphatase enzyme, dithiothreitol, spermidine and enzyme reaction buffer and incubated for 1 hour at 37°C. The enzyme DNase I was added to cleave the plasmid DNA template and incubated for 0.5 h at 37°C. mRNA was isolated by successive lithium chloride precipitation, washing the precipitate in 70% ethanol, resuspending the mRNA precipitate in water, reprecipitating with isopropanol and sodium acetate, and washing the precipitate again in 70% ethanol. The final mRNA pellet was resuspended in water.

Reagent Concentration Notes

Water free of nucleases Remaining volume

Tris-HC1 pH 8.0 (mM)40

MgS1 ₂ (mM) 20

ATP, CTP, GTP, UTP (mM)4

Pseudouridine (mM) 4

To create 100%

PsU mRNA does not include

UTP in the reaction. To create 100% unmodified mRNA, do not include PsU in the reaction

DTT (mM) 10

Spermidine (mM) 2

Linearized

0.05 plasmid DNA (μg/μl)

Pyrophosphatase (u/µl) 0.004

RNase inhibitor (u/µl) 1

T7 RNA polymerase (u/µl)

DNase I (u/µl) 0.04

TEV-h leptin-GAopt-2xhBG-120A (SEQ ID NO: 5)

Sequence features:

Tobacco gravure virus (TGV) 5' UTR: 14-154

Optimal Kozak sequence: 155-163

Human leptin encoding amino acids 1-167 of access protein # NP_000221, codon sequence optimized by GeneArt: 164-664 2 stop codons: 665-670 2 copies of human beta globin 3'UTR: 689-954 120 bp of polyA tail: 961- 1080

GGGAGACGCGUGUUAAAUAACAAAUCUCAACACAACAUAUACAAAACAAACGAAUCUC

AAGCAAUCAAGCAUUCUACUUCUAUUGCAGCAAUUUAAAUCAUUUCUUUUAAAGCAAAAGCAA

UUUUCUGAAAAUUUUCACCAUUUACGAACGAUAGCCGCCACCAUGCACUGGGGAACCCUGUGC

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GGAUUCCUGUGGCUGUGGCCCUACCUGUUCUAUGUGCAAGCCGUGCCCAUCCAGAAGGUGCAG GAG GACAC CAAGAC C CUGAUCAAGAC CAUC GUGAC С C GGAUCAAC GACAUCAGC CACAC C GAG AGCGUGUCCAGCAAGCAGAAAGUGACCGGCCUGGACUUCAUCCCCGGCCUGCACCCUAUCCUG ACCCUGUCCAAGAUGGACCAGACCCUGGCCGUGUACCAGCAGAUCCUGACCAGCAUGCCCAGC CGGAACGUGAUCCAGAUCAGCAACGACCUGGAAAACCUGCGGGACCUGCUGCACGUGCUGGCC UUCAGCAAGAGCUGCCAUCUGCCUUGGGCCAGCGGCCUGGAAACCCUGGAUUCUCUGGGCGGA GUGCUGGAAGCCAGCGGCUACUCUACAGAGGUGGUGGCCCUGAGCAGACUGCAGGGCAGCCUG CAGGAUAUGCUGUGGCAGCUGGAUCUGAGCCCCGGCUGCUAAUAGCGGACCGGCGAUAGAUGA AGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUA AACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUU UCAUUGCAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCA ACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACA UUUAUUUUCAUUGCGGCCGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAA (SEQ ID NO: 5)

mRNA packaging

All disposable equipment and supplies were certified RNase-free by the manufacturer or cleared of RNase using the RNaseZap reagent (LifeTechnologies). The mRNA was encapsulated in a cationic lipid amine to mRNA phosphate (N:P) molar ratio of 4:1. Lipids (cationic lipid, DSPC, cholesterol and lipidated PEG or hidden lipid) were dissolved in ethanol. The molar ratios were 40:10:38:2, respectively. The mixture was briefly sonicated, then gently stirred for 5 min and then kept at 37°C until use. mRNA was replaced in citrate buffer pH 5.8-6.0 using centrifuge concentrators Amicon Ultra-15, the final concentration was adjusted to 0.5 mg/ml and kept at 37°C until use. Equal volumes of lipids in ethanol, mRNA in citrate buffer, and citrate buffer alone were drawn into disposable syringes. The inlet tubes of syringes containing lipids and mRNA were attached to the T-arm and the inlet tube of the syringe containing citrate buffer alone was paired with the tube exiting the T-arm through a collection vessel containing a magnetic stirrer on an active mixing plate. The syringes were placed in a syringe pump set to expel the contents at a flow rate of 1 ml per minute.

The pump was activated and the collected lipid nanoparticle mRNAs were transferred to a SnakeSkin dialysis tube (10,000 MWCO, Thermo Scientific). The material was dialyzed against RNase and pyrogen-free 1x phosphate-buffered saline overnight at 4°C.

Measurement of mRNA encapsulation

The percentage of mRNA encapsulation in lipid nanoparticles was determined using the Quant-iT Ribogreen RNA Assay (Life Technologies). The LNP-mRNA suspension was analyzed in buffer (mRNA outside the particle) and buffer plus detergent Triton X-100 (total mRNA). The calculated difference was the mRNA within the particle. 1000 ng/ml of base material from the resulting RNA in the kit was prepared and used to generate a calibration curve (0 ng/ml, 15.63-1000 ng/ml) in TE and TE + 0.75% Triton X-100. LNP-mRNA samples in TE buffer and TE buffer + 0.75% Triton X-100 were prepared by appropriate dilution so that the reading was in the range of the calibration curve (400-2000-fold). In a 384-well plate (Costar raw #3573), 0.04 ml of standard (in duplicate) or sample (in triplicate) was added to each well. Ribogreen reagent was diluted 240-fold with TE buffer and 0.06 ml per well was added. The contents of the wells were mixed and the fluorescence was measured (excitation=480 nm, emission=520 nm). Background values (not RNA) were subtracted from the values of standard and test samples, and RNA concentrations were determined in the samples using calibration curves. Percent sample encapsulation was determined by dividing the difference in concentration between sample + Triton and sample in buffer separately by the concentration of Sample + Triton.

h Leptin

Intravenous tail vein injection of a modified synthetic leptin mRNA

Prior to tail vein injection, body weights of mice were recorded and diets were weighed with mice grouped according to their body weights. Mice were prepared by heating them under a heat lamp for ~2 minutes, with the mice approximately 12 inches from the heat lamp.

For the tail vein injection method, mice were placed in a fixator and tails cleaned

- 100 040257 or 70% alcohol. A size 27 needle (Becton Dickinson, catalog # 305109) connected to a 1 ml syringe (Becton Dickinson, catalog # 309659) was inserted into the tail vein with the bevel surface up, and the syringe plunger was pulled back to allow blood to enter the syringe. The desired volume of modified synthetic leptin mRNA was manually injected with moderate pressure and speed. The needle was then removed and bleeding was stopped by applying pressure to the gauze injection site.

Single 8-9 week old male C57BL/6 mice were used for the in vivo study. HPLC-purified modified synthetic leptin mRNA (SEQ ID NO: 5) in which uridines were substituted with pseudouridines was packaged in a cationic lipid (N:P molar ratio=8:1) and then diluted in injection saline to a dose of 10 μg per average group body weight.

On day 0, animals were weighed and distributed according to average body weight. Mice were dosed and food intake (FI) was recorded for each of days 1-7 and days 9, 11 and 16.

Subcutaneous injection of modified synthetic leptin mRNA into mice

Prior to subcutaneous injection, the body weights of mice were recorded and the diet was weighed with mice grouped according to their body weights. Mice were clamped by hand and placed on a work surface. They were pulled by the scruff of the neck and the subjacent muscle was lifted, a space into which a size 25 needle connected to a 1 ml syringe was inserted. The plunger of the syringe was pulled back in such a way as to ensure that no liquid entered the syringe, and then the desired volume of leptin mRNA was injected manually at moderate pressure and speed. The needle was then removed and the mice returned to their cages.

8-9 week old male C57BL/6 mice were used for the in vivo study. HPLC-purified modified synthetic leptin mRNA (SEQ ID NO: 5) in which uridines were substituted with pseudouridines (N:P molar ratio=8:1) packaged in several cationic lipids was diluted in injection saline to a dose of 10 μg per average group body weight.

On day 0, animals were weighed and distributed according to average body weight. Mice were dosed at 9 am and bled at 9 am on day 0. Blood was also taken at 9 am on each of days 1 and 2 and leptin protein levels were assessed. Body weight and food intake were also recorded.

Human plasma leptin in mice was measured by ELISA. Antibodies purchased from R&D Systems duoset (Cat# DY398E, part# 840279 for capture antibody and part# 840280 for detection antibody) were reconstituted with PBS and titrated again with PBS. The immobilized antibody was applied at 4 μg/ml in 30 μl/well to a white 384-well Nunc® Maxisorp plate (Cat. # 460372). After incubation overnight at room temperature, the immobilized antibody was aspirated and the plate was blocked for 2 hours at room temperature with 90 μl/well KPL milk blocker (Cat. # 50-82-00). After incubation was complete, the plate was aspirated and recombinant standards and samples were added to the plate at 30 μl/well for 2 hours at 37° C. with shaking at 600 rpm. Sample/standard dilutions were made using casein-based sample diluent. Wash/aspirate 3 times at 100 µl/well was performed using Teknova Plate Wash Solution (Cat. # P1192). Next, the detection antibody was diluted with 12.5 ng/ml casein-based detection antibody diluent and added at 30 µl/well over 2 hours at room temperature. After this incubation, the plate was washed again and a solution of polystreptavidin-HRP (Cat. # 21140) at a 1:1250 dilution in HRP dilution buffer was added to each well (30 µl/well) and incubated for 30 min at room temperature. A final wash/aspirate removed the HRP solution and 30 µl/well chemiluminescent substrate was added (Cat. # 1859678 & 1859679). The plate was quickly read using a Spectramax M5 plate reader with an integration time of 50 ms. The dynamic range of the ELISA is 100 to 2000 pg/ml (6.25-125 pM) of human leptin. The assay is applicable to plasma from mice, rats and macaque crabs.

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Example number % Encapsulation Size (nm) PDI Leptin expression IV-C57B1/6, 24 h (ng/ml) Leptin expression SC-С57В1/6, 24 h (ng/ml) 38 90.9 142.3 0.083 17.21 0.99 39 73, 1 156.0 0.090 10.47 0.79 43 71.1 176.0 0.042 13.56 0.66 52 95.1 143.9 0.109 21.18 0.85 57 96.0 127.8 0.102 9, 17 59 87.0 134.7 0.106 8, 08 1.16 78 71.1 164.0 0.150 0.96 79 91.6 140.5 0.116 1.36 (6h) 85 93.1 134.8 0.125 1.78 90 80, 9 164.7 0.090 0.74 92 72.5 134.2 0.073 1.34

Numbered Embodiments

Embodiment 1.

The present invention provides a compound of formula (I) f (I) where

L is C _1-6 alkylene, *-C _1-4 alkylene-L2-, *-C _1-4 alkylene-L2-C _1-4 alkylene w ^s or

Г°\ h г Г / Н s 'НГ L2^HO \ where * denotes the addition of a fragment to the NR1R ² group;

L2 attached in either direction is -C(O)O-, -OC(O)O- or -CONH-; each s is independently 0, 1, or 2;

t is independently 0, 1, 2, 3 or 4;

each u is 0, 1, 2, 3, 4, 5, or 6;

R ¹ and R ² are each independently unsubstituted or substituted C _1-6 alkyl; where the specified C _1-6 alkyl is optionally substituted with one or two substituents, each independently selected from the group consisting of OH, COOH and COO-C _1-4 alkyl, or R ¹ and R ² are connected together, forming an unsubstituted or substituted 4-7 -membered heterocyclic ring containing 1-2 heteroatoms selected from N and O, wherein said heterocyclic ring is optionally substituted with one to three OH substituents;

R ³ and R ⁴ are each independently a -R ^c chain, where R ^c is

- 102 040257

- 103 040257

- 104 040257

or

R is C _5-22 alkyl, C _5-22 alkenyl, or C _5-22 alkynyl;

n is 0-12;

m, p and q are each independently 0, 1, 2, 3 or 4;

provided that the chain R ^c has 12-70 carbon atoms;

X is CR ⁶ or N and

R ⁶ is H, halo, C _1-6 alkyl or R ⁴ ;

or a pharmaceutically acceptable salt thereof.

Embodiment 2.

The compound of Embodiment 1 wherein X is CR ⁶ ;

or its pharmaceutically acceptable salt.

Embodiment 3.

The compound of Embodiment 2 wherein R ⁶ is H, chlorine, bromine, or

C _1-3 alkyl, or a pharmaceutically acceptable salt thereof.

Embodiment 4.

The compound of embodiment 3 wherein R ⁶ is H; or a pharmaceutically acceptable salt thereof.

Embodiment 5.

The compound of Embodiment 4 wherein L is methylene, ethylene, or propylene, or L is *-C _1-3 alkylene-O-C(O)-, or L is *-C _1-4 alkylene- L2C _1-2 alkylene, where L2 attached in any direction is C(O)O or OC(O)O; or a pharmaceutically acceptable salt thereof.

Embodiment 6.

The compound of Embodiment 5, wherein R ¹ and R ² are each independently unsubstituted or substituted methyl, or unsubstituted or substituted ethyl; or a pharmaceutically acceptable salt thereof.

Embodiment 7.

The compound of Embodiment 4 wherein the L-NR ¹ R ² group of formula (I) is selected from the group consisting of

- 106 040257

- 107 040257

where the dotted line indicates the point of attachment to the formula (I); or a pharmaceutically acceptable salt thereof.

Embodiment 8. The compound of Embodiment 7 wherein R ^c is c1 or c3; n is 1 or 2; m is 0 or 1 and p is 1; or a pharmaceutically acceptable salt thereof.

Embodiment 9 The compound of Embodiment 8 wherein R ⁴ = R ³ ; or a pharmaceutically acceptable salt thereof.

Embodiment 10. A compound selected from the group consisting of

- 108 040257

1-(3,5-bis((Z)-octadec-9-en-1-yloxy)phenyl)-Ν,Νdimethylmethyl anamine,

1-(3,5-bis((92.12g)-octadeca-9,12-dien-1-yloxy)phenyl)N,N-dimethylmethanamine,

2,2'- ((3,5-bis((9Ζ,12Ζ)-octadeca-9,12-dien-1yloxy)benzyl)azanediyl)diethanol,

1-(3,5-bis((9Ζ,12Ζ)-octadeca-9,12-dien-1yloxy)benzyl)pyrrolidine,

1-(3,5-bis((9Ζ,12Ζ)-octadeca-9,12-dien-1-yloxy)benzyl)azetidin-3-ol, ethyl-2-((3,5-bis((Ζ) -octadec-9-en-1yloxy)benzyl)(methyl)amino)acetate,

1-(3,5-bis((Ζ)-octadec-9-en-1-yloxy)benzyl)pyrrolidine,

2-((3,5-bis((Ζ)-octadec-9-en-1yloxy)benzyl)(methyl)amino)acetic acid,

1- ( (2 - (3,5-bis ( (Ζ) -octadec-9-en-1-yloxy)-phenyl)-1,3-dioxolan-4-yl)-methyl)-pyrrolidine,

1-(2-(3,5-bis((9Ζ,12Ζ)-octadeca-9,12-dien-1-yloxy)phenyl)-

1,3-dioxolan-4-yl)-N,N-dimethylmethanamine,

1- ((2-(3,5-bis((9Ζ,12Ζ)-octadeca-9,12-dien-1-yloxy)phenyl)-1,3-dioxolan-4-yl)methyl)piperidine, (2 -(3,5-bis((Ζ)-octadec-9-en-1-yloxy)phenyl)-1,3dioxolan-4-yl)methyl-2-(dimethylamino)acetate,

3,5-bis ((9Ζ,12Ζ)-octadeca-9,12-dien-1-yloxy)benzyl-4(dimethylamino)butanoate,

3,5-bis ((9Ζ,12Ζ)-octadeca-9,12-dien-1-yloxy)benzyl-3(dimethylamino)propanoate,

3,5-bis ((9Ζ,12Ζ)-octadeca-9,12-dien-1-yloxy)benzyl-2(dimethylamino)acetate,

4-methyl-3,5-bis ((9Ζ,12Ζ)-octadeca-9,12-dien-1yloxy)benzyl-3-(dimethylamino)propanoate,

3,5-bis ((9Ζ,12Ζ)-octadeca-9,12-dien-1-yloxy)benzyl-3(dimethylamino)-2-methylpropanoate,

2-(dimethylamino)ethyl-2,6-bis((9Ζ,12Ζ)-octadeca-9,12-dien1-yloxy)isonicotinate,

3-(dimethylamino)propyl-2, 6-bis ((9Ζ,12Ζ)-octadeca-9,12dien-1-yloxy)isonicotinate, (9Ζ,9'Ζ,12Ζ,12'Ζ)-5- ( ( ( 3(dimethylamino)propanoyl)oxy)methyl)-1,3-phenylene-bis(octadeca-

9, 12-dienoate), (9Ζ,9'Ζ,12Ζ,12'Ζ)-5-(((4(dimethylamino)butanoyl)oxy)methyl)-1,3-phenylene-bis(octadeca-

9, 12-dienoate), (2,6-bis ((9Ζ,12Ζ)-octadeca-9,12-dien-1-yloxy)pyridin-4yl)methyl-3-(diethylamino)propanoate, (9Ζ, 9' Ζ, 12Ζ, 12' Ζ) - (5- ( ( ( (3 (dimethylamino) propanoyl) oxy) methyl) -1,3 phenylene) bis (methylene) bis (octadeca-9,12-dienoate), (2,6- bis ((9Ζ,12Ζ)-octadeca-9,12-dien-1-yloxy)pyridin-4yl)methyl-4-(dimethylamino)butanoate, (2,6-bis((9Ζ,12Ζ)-octadeca-9, 12-dien-1-yloxy)pyridin-4yl)methyl-3-(dimethylamino)propanoate,

- 109 040257

1-(2,6-bis ((9Z,12Z)-octadeca-9,12-dien-1-yloxy)pyridin-4 yl)-N,N-dimethylmethanamine, (9Z,9'Z,12Z,12' Z)- (5- (((dimethylamino)methyl)-1,3phenylene)bis(methylene)bis(octadeca-9,12-dienoate), (5-((dimethylamino)methyl)-1,3phenylene)bis(methylene) ditridecanoate, (5-((dimethylamino)methyl)-1,3-phenylene)bis(methylene)bis(3octylundecanoate), (9Z,12Z)-3- ((dimethylamino)methyl)-5-(((3octylundecanoyl)oxy )methyl)benzyloctadeca-9,12-dienoate, di ((9Z,12Z)-octadeca-9,12-dien-1-yl)-5-((((3(dimethylamino)propanoyl)oxy)methyl)isophthalate, ( 9Z,9'Z,12Z,12'Z)- ( ( ( (5- ( ((dimethylamino)methyl)-1,3phenylene)bis(oxy))bis(ethane-2,1-diyl))bis(oxy) )bis(ethane-2,1diyl)bis(octadeca-9,12-dienoate), (9Z,9'Z,12Z,12'Z)-( ( ( (5 -((diethylamino)methyl)-1,3phenylene ) bis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1diyl)bis(octadeca-9,12-dienoate),

3,5-bis ((9Z,12Z)-octadeca-9,12-dien-1-ylcarbamoyl)benzyl 3-(dimethylamino)propanoate,

3,5-bis ((9Z,12Z)-octadeca-9,12-dien-1-ylcarbamoyl)benzyl4-(dimethylamino)butanoate,

3,5-bis ((9Z,12Z)-octadeca-9,12-dien-1-yloxy)phenethyl-3(dimethylamino)propanoate,

3,5-bis ((9Z,12Z)-octadeca-9,12-dien-1-yloxy)benzyl(3(dimethylamino)propyl)carbonate, (9Z,9'Z,12Z,12'Z)- (5 -((((3(diethylamino)propoxy)carbonyl)oxy)methyl)-1,3phenylene)bis(methylene)bis(octadeca-9,12-dienoate) , (9Z,9'Z,12Z,12'Z) - (5-((((2(dimethylamino)ethoxy)carbonyl)oxy)methyl)-1,3phenylene)bis(methylene)bis(octadeca-9,12-dienoate), (9Z, 9' Z, 12Z, 12 'Z)- (5- ( ( ( (3(dimethylamino)propoxy)carbonyl)oxy)methyl)-1,3phenylene)bis(methylene)bis(octadeca-9,12-dienoate), (9Z,9'Z, 12Z,12'Z)-5-((((2- 110 040257 (dimethylamino)ethoxy)carbonyl)oxy)methyl)-1,3-phenylenebis(octadeca-9,12-dienoate),

3-(dimethylamino)propyl-4-isopropyl-3,5-bis((9Z,12Z)octadeca-9,12-dien-1-yloxy)benzyl carbonate,

4-bromo-3,5-bis ((9Z,12Z)-octadeca-9,12-dien-1yloxy)benzyl (3-(dimethylamino)propyl)carbonate,

4-chloro-3,5-bis ((9Z,12Z)-octadeca-9,12-dien-1yloxy)benzyl(3-(dimethylamino)propyl)carbonate,

N-(3,5-bis ((9Z,12Z)-octadeca-9,12-dien-1-yloxy)benzyl)-2(dimethylamino)acetamide,

O,O' -((5-(((dimethylamino)methyl)-1,3phenylene)bis(methylene))di((9Z,12Z)-octadeca-9,12-dien-1yl)disuccinate,

O,O' -((5-(((dimethylamino)methyl)-1,3phenylene)bis(methylene))bis(10-(octanoyloxy)decyl)disuccinate,

O' ¹ , O ¹ - ((5- ((dimethylamino) methyl) -1,3-phenylene) bis (methylene) 8-dinonyldioctanedioate,

O' ¹ , O ¹ - ((5- ((dimethylamino) methyl) -1,3-phenylene) bis (methylene)) 9-dioctyldinonanedioate,

3-(3,5-bis ((9Z,12Z)-octadeca-9,12-dien-1yloxy)phenyl)propyl-3-(dimethylamino)propanoate, (9Z, 9' Z, 12Z, 12' Z) - ( ( (5- ( ((dimethylamino) methyl) -1,3phenylene)bis(methylene))bis(oxy))bis(4-oxobutane-4,1diyl)bis(octadeca-9,12-dienoate), ( ( ( 5-(((dimethylamino)methyl)-1,3phenylene)bis(methylene))bis(oxy))bis(8-oxooctane-8,1diyl)bis(decanoate), (((5-((dimethylamino)methyl)- 1,3phenylene)bis(methylene))bis(oxy))bis(4-oxobutan-4,1diyl)dioctanoate, (((5-((dimethylamino)methyl)-1,3phenylene)bis(methylene))bis(oxy ))bis(6-oxohexane-6,1diyl)dioctanoate, (5-(((dimethylamino)methyl)-1,3-phenylene)bis(methylene)bis(10-(octanoyloxy)decanoate), (5-((dimethylamino )methyl)-1,3-phenylene)bis(methylene)bis(8-111 040257 (octanoyloxy)octanoate), (((5-((dimethylamino)methyl)-1,3phenylene)bis(methylene))bis(oxy ))bis(6-oxohexane-6, 1diyl)bis(decanoate),

4,4'-((5-((dimethylamino)methyl)-1,3phenylene)bis(oxy))bis(butane-1,2-diyl)tetraoctanoate, (R)-4-(3-((S) -3,4-bis(octanoyloxy)butoxy)-5((dimethylamino)methyl)phenoxy)butane-1,2-diyldioctanoate, (S)-4-(3-((S)-3,4-bis(octanoyloxy )butoxy)-5((dimethylamino)methyl)phenoxy)butane-1,2-diyldioctanoate, (R)-4-(3-((R)-3,4-bis(octanoyloxy)butoxy)-5((dimethylamino )methyl)phenoxy)butane-1,2-diyldioctanoate,

N,N-dimethyl-1-(3,4,5-tris ((9Z,12Z)-octadeca-9,12-dien-1yloxy)phenyl)methanamine, ((((5-((dimethylamino)methyl)- 1,3phenylene)bis(oxy) )bis(propan-3, 1-diyl) )bis(oxy) )bis(4oxobutan-4,1-diyl)bis(decanoate),

O ' \ O ¹ - ( ( (5 - ( (dimethylamino) methyl) -1,3 phenylene) bis (oxy) ) bis (propane-3, 1-diyl) ) -9-dioctyldinonanedioate,

3-((dimethylamino)methyl)-5-(((8(octanoyloxy)octanoyl)oxy)methyl)benzyl-3-octylundecanoate,

2-(3,5-bis ((9Z,12Z)-octadeca-9,12-dien-1-yloxy)phenyl)N,N-dimethylethanamine,

3-(3,5-bis((9Z,12Z)-octadeca-9,12-dien-1-yloxy)phenyl)N,N-dimethylpropan-1-amine,

2-(3-(4-(5-((dimethylamino)methyl)-2-methyl-3-((9Z,12Z)octadeca-9,12-dien-1-yloxy)phenoxy)butoxy)-3oxopropyl)propane -1,3-diyldihexanoate, (5-((dimethylamino)methyl)-1,3-phenylene)bis(methylene)bis(8decanamidooctanoate), (5-((dimethylamino)methyl)-1,3-phenylene)bis( methylene) bis (6 (((nonyloxy) carbonyl) oxy) hexanoate), bis (1,3-bis (octanoyloxy) propan-2-yl) -O, O ' - ( (5 ((dimethylamino) methyl) -1 ,3-phenylene)bis(methylene))disuccinate, (((5-(((dimethylamino)methyl)-1,3phenylene)bis(oxy))bis(butane-4,1-diyl))bis(propane-3, 2,1triyl)tetraoctanoate, (((5-((dimethylamino)methyl)-1,3phenylene)bis(oxy))bis(methylene))bis(propane-3,2,1triyl)tetraoctanoate, (5-((dimethylamino )methyl)-1,3-phenylene)bis(methylene)bis(5heptyldodecanoate), (5-((dimethylamino)methyl)-1,3-phenylene)bis(methylene)bis(7hexyltridecanoate), (5-((dimethylamino )methyl)-1,3-phenylene)bis(methylene)bis(9pentyltetradecanoate), ( ( ( ( 5- ((dimethylamino)methyl)-1,3phenylene)bis(oxy) )bis(butane-4,1-diyl ) )bis (hydroxy)) bis (propane-

3, 2,1-triyl)tetraoctanoate,

1-(3,5-bis(4,4-bis(octyloxy)butoxy)phenyl)-N,N-dimethylmethyl anamine, or a pharmaceutically acceptable salt thereof.

Embodiment 11 A lipid composition comprising a compound of any one of Embodiments 1-10, or a pharmaceutically acceptable salt thereof, and a biologically active agent.

Embodiment 12 The lipid composition of Embodiment 11 wherein the biologically active agent is a short interfering RNA (siRNA).

Embodiment 13. The lipid composition of Embodiment 13 further comprising a helper lipid.

Embodiment 14. The lipid composition of Embodiment 13 further comprising a neutral lipid.

Embodiment 15. The lipid composition of Embodiment 14 further comprising an occult lipid.

Embodiment 16 The lipid composition of Embodiment 15 wherein the helper lipid is cholesterol, the neutral lipid is distearoylphosphatidylcholine (DSPC), and the hidden lipid is PEG-DMG, o ^{[ J} 45 ^H (S 010 ) or o

(soil).

Embodiment 17. The lipid composition of Embodiment 15 is in the form of a lipid nanoparticle.

Embodiment 18 The lipid composition of Embodiment 16 having a compound of formula (I)/cholesterol/DSPC/S010 or S011 molar ratio of 45/44/9/2, respectively.

Embodiment 19. The lipid composition of Embodiment 16 having a molar ratio of compound of formula (I)/cholesterol/DSPC/S010 or S011 of 55-40/55-40/15-5/10-1, respectively.

Embodiment 20. The lipid composition of Embodiment 16 having a molar ratio of compound of formula (I)/cholesterol/DSPC/S010 or S011 of 50-40/50-40/15-5/5-1, respectively.

Embodiment 21. The lipid composition of Embodiment 16 having a compound of formula (I)/cholesterol/DSPC/S010 or S011 molar ratio of 47-43/47-43/12-7/4-1, respectively.

Embodiment 22. A pharmaceutical composition comprising the lipid composition of any of Embodiments 11-21 and a pharmaceutically acceptable carrier or excipient.

Embodiment 23. A method of treating a disease or condition, comprising the step of administering a therapeutically effective amount of a lipid composition according to any one of Embodiments 11-21 to a patient in need of such treatment, wherein the disease or condition is selected from the group consisting of proliferative disease, cancer, inflammatory disease , graft and/or tissue rejection, autoimmune disease or age-related disease, neurological or neurodegenerative disease, respiratory disease, cardiovascular disease, eye disease, metabolic disease, dermatological disease, hearing disease, liver disease, kidney disease.

Embodiment 24. A method of treating a disease or condition, comprising the step of administering a therapeutically effective amount of the pharmaceutical composition of Embodiment 22 to a patient in need of such treatment, wherein the disease or condition is selected from the group consisting of proliferative disease, cancer, inflammatory disease, transplant rejection, and /or tissue, autoimmune disease or age-related disease, neurological or neurodegenerative disease, respiratory disease, cardiovascular disease, eye disease, metabolic disease, dermatological disease, hearing disease, liver disease, kidney disease.

Embodiment 25 The lipid composition of Embodiment 11 wherein the biologically active agent is messenger RNA (mRNA).

Embodiment 26. The lipid composition of Embodiment 25 further comprising a helper lipid.

Embodiment 27. The lipid composition of Embodiment 26 further comprising a neutral lipid.

Embodiment 28. The lipid composition of Embodiment 27 further comprising an occult lipid.

Embodiment 29 The lipid composition of Embodiment 26 wherein the helper lipid is cholesterol, the neutral lipid is DSPC, and the hidden lipid is PEG-DMG, S010, S011, or

- 113 -

Claims (34)

Embodiment 30. The lipid composition of Embodiment 29 is in the form of a lipid nanoparticle. Embodiment 31. The lipid composition of Embodiment 29 having a compound of formula (I)/cholesterol/DSPC/S010, S011 or S024 molar ratio of 40/38/10/2, respectively. Embodiment 32. A pharmaceutical composition comprising the lipid composition of any of Embodiments 25-31 and a pharmaceutically acceptable carrier or excipient. Embodiment 33. A method of treating a disease or condition comprising the step of administering a therapeutically effective amount of a lipid composition according to any one of Embodiments 25-31 to a patient in need of such treatment, wherein the disease or condition is selected from the group consisting of proliferative disease, cancer, inflammatory disease , graft and/or tissue rejection, autoimmune disease or age-related disease, neurological or neurodegenerative disease, respiratory disease, cardiovascular disease, eye disease, metabolic disease, dermatological disease, hearing disease, liver disease, kidney disease. Embodiment 34. A method of treating a disease or condition comprising the step of administering a therapeutically effective amount of the pharmaceutical composition of Embodiment 32 to a patient in need of such treatment, wherein the disease or condition is selected from the group consisting of proliferative disease, cancer, inflammatory disease, transplant rejection, and /or tissue, autoimmune disease or age-related disease, neurological or neurodegenerative disease, respiratory disease, cardiovascular disease, eye disease, metabolic disease, dermatological disease, hearing disease, liver disease, kidney disease. CLAIM 1. Compound of formula (I) (I) where L is C _1-6 alkylene, *-C _1-4 alkylene-L2-, *-C _1-4 alkylene-L2-C _1-4 alkylene-, or where * denotes the attachment of the fragment to the NR1R ² group; L2 attached in either direction is -C(O)O-, -OC(O)O- or -CONH-; each s is independently 0.1 or 2; t is independently 0, 1, 2, 3 or 4; each u is 0, 1, 2, 3, 4, 5, or 6; R ¹ and R ² are each independently unsubstituted or substituted C _1-6 alkyl; where said C _1-6 alkyl is optionally substituted with one or two substituents, each independently selected from the group consisting of: OH, COOH and COO-C _1-4 alkyl, or R ¹ and R ² are joined together to form an unsubstituted or substituted 4-7 membered heterocyclic ring containing 1-2 heteroatoms selected from N and O, said heterocyclic ring being optionally substituted with one to three OH substituents; R ³ and R ⁴ are each independently a -R ^c chain, where R ^c is - 114 040257 - 115 040257 or - 116 040257 R is C _5-22 alkyl, C _5-22 alkenyl, or C _5-22 alkynyl; n is 0-12; m, p and q are each independently 0, 1, 2, 3 or 4; provided that the chain R ^c has 12-70 carbon atoms; X is CR ⁶ or N and R ⁶ is H, halo, C _1-6 alkyl or R ⁴ ; or a pharmaceutically acceptable salt thereof. 2. The compound according to claim 1, in which X represents CR ⁶ ; or a pharmaceutically acceptable salt thereof. 3. A compound according to claim 2, wherein R ⁶ is H, chlorine, bromine, or C _1-3 alkyl, or a pharmaceutically acceptable salt thereof. 4. The compound according to claim 3, in which R ⁶ represents H; or a pharmaceutically acceptable salt thereof. 5. The connection according to claim 4, in which L is methylene, ethylene, or propylene, or L is *-C _1-3 alkylene-O-C(O)-, or L is *-C ₁ . ₄ alkylene-L2-C ₁ . ₂ alkylene, where L2 attached in any direction is C(O)O or OC(O)O; or a pharmaceutically acceptable salt thereof. 6. The compound according to claim 5, in which R ¹ and R ² represent each independently unsubstituted or substituted methyl, or unsubstituted or substituted ethyl; or a pharmaceutically acceptable salt thereof. 7. The compound according to claim 4, in which the L-NR ¹ R ² group in the compound of formula (I) is selected from the group consisting of - 117 040257 - 118 040257 where the dotted line indicates the point of attachment to the formula (I); or a pharmaceutically acceptable salt thereof. 8. The compound according to claim 7, in which R ^c represents c1 or c3; n is 1 or 2; m is 0 or 1 and p is 1; or a pharmaceutically acceptable salt thereof. 9. Connection according to any one of paragraphs.1-8, in which R ⁴ = R ³ ; or a pharmaceutically acceptable salt thereof. 10. A compound selected from the group consisting of 1-(3,5-bis((Z)octadec-9-en-1-yloxy)phenyl)-N,N-dimethylmethanamine, 1 -(3,5-6uc((9Z, 12Z)octadeca-9,12-dien-1-yloxy)phenyl)-N,N-dimethylmethanamine, 2,2'-((3,5-6uc((9Z, 12Z)octadeca-9,12-dien-1-yloxy)benzyl)azanediyl)diethanol, 1 - (3,5-6uc ((9Z, 12Z) octadeca-9,12-dien-1 -yloxy) benzyl) pyrrolidine, 1 -(3,5-6uc((9Z, 12Z)octadeca-9,12-dien-1-yloxy)benzyl)azetidin-3-ol, ethyl-2-((3,5-bis((Z)octadec -9-en-1-yloxy)benzyl)(methyl)amino)acetate, 1-(3,5-bis((Z)octadec-9-en-1-yloxy)benzyl)pyrrolidine, 2-((3, 5-bis((Z)octadec-9-en-1-yloxy)benzyl)(methyl)amino)acetic acid, 1-((2-(3,5-bis((Z)octadec-9-en-1 -yloxy)phenyl)-1,3-dioxolan-4-yl)methyl)pyrrolidine, 1 -(2-(3,5-6uc((9Z, 12Z)octadeca-9,12-dien-1-yloxy)phenyl)-1,3-dioxolan-4-yl)-N,N-dimethylmethanamine, 1 -((2-(3,5-6uc((9Z, 12Z)-octadeca-9,12-dien-1-yloxy)phenyl)-1,3-dioxolan-4-yl)methyl)piperidine, (2 -(3,5-bis((Z)octadec-9-en-1-yloxy)phenyl)-1,3-dioxolan-4-yl)methyl-2-(dimethylamino)acetate, 3,5-6uc((9Z, 12Z)octadeca-9,12-dien-1-yloxy)benzyl-4-(dimethylamino)butanoate, 3,5-bis((9Z,12Z)octadeca-9,12-dien-1-yloxy)benzyl-3-(dimethylamino)propanoate, - 119 040257 3,5-6uc((9Z, 12Z)oKTageKa-9,12-dien-1-yloxy)benzyl-2-(dimethylamino)acetate, 4-methyl-3,5-bis ((9Z, 12Z) oKTageKa-9,12-dien-1 -yloxy) benzyl-3 - (dimethylamino) propanoate, 3,5-6uc((9Z, 12Z)oKTageKa-9,12-dien-1-yloxy)benzyl-3-(dimethylamino)-2-methylpropanoate, 2-(dimethylamino)ethyl-2,6-bis((9Z, 12Z)oKTageKa-9,12-dien-1-yloxy)isonicotinate, 3-(dimethylamino)propyl-2,6-bis((9Z,12Z)octadeca-9,12-dien-1-yloxy)isonicotinate, (9Z,9'Z, 12Z, 12'Z)-5-(( (3-(dimethylamino)propanoyl)oxy)methyl)-1,3-phenylene-bis(octadeca-9,12dienoate), (9Z,9'Z, 12Z, 12'Z)-5-(((4-( dimethylamino)butanoyl)oxy)methyl)-1,3-phenylene-bis(octadeca-9,12dienoate), (2,6-6uc((9Z, 12Z)oKTageKa-9,12-dien-1-yloxy)pyridine- 4-yl)methyl-3-(diethylamino)propanoate, (9Z,9'Z, 12Z, 12'Z)-(5-(((3-(dimethylamino)propanoyl)oxy)methyl)-1,3-phenylene )bis(methylene)bis(octadeca-9,12-dienoate), (2,6-bis((9Z, 12Z)octadeca-9,12-dien-1-yloxy)pyridin-4-yl)methyl-4- (dimethylamino)butanoate, 2,6-bis((9Z, 12Z)octadeca-9,12-dien-1-yloxy)pyridin-4-yl)methyl-3-(dimethylamino)propanoate, 1 -(2,6-bis((9Z, 12Z)octadeca-9,12-dien-1-yloxy)pyridin-4-yl)-N,N-dimethylmethanamine, (9Z,9'Z, 12Z, 12' Z)-(5-((dimethylamino)methyl)-1,3-phenylene)bis(methylene)bis(octadeca-9,12-dienoate), (5-((dimethylamino)methyl)-1,3-phenylene) bis(methylene)ditridecanoate, (5-((dimethylamino)methyl)-1,3-phenylene)bis(methylene)bis(3-octylundecanoate), (9Z,12Z)-3-((dimethylamino)methyl)-5- (((3-octylundecanoyl)oxy)methyl)benzyloctadeca-9,12-dienoate, di((9Z, 12Z)octadeca-9,12-dien-1-yl)-5-(((3-(dimethylamino)propanoyl )oxy)methyl)isophthalate, (9Z,9'Z, 12Z, 12'Z)-((((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(ethane-2 ,1-diyl))bis(oxy))bis(ethane-2,1-diyl)bis(octadeca-9,12-dienoate), (9Z,9'Z, 12Z, 12'Z)-(((( 5-((diethylamino)methyl)-1,3-phenylene)bis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl)bis(octadeca- 9,12-dienoate), 3,5-6uc((9Z,12Z)octadeca-9,12-dien-1-ylcarbamoyl)benzyl-3-(dimethylamino)propanoate, 3,5-bis((9Z,12Z)octadeca-9,12-diene -1-ylcarbamoyl)benzyl-4-(dimethylamino)butanoate, 3,5-6uc((9Z, 12Z)octadeca-9,12-dien-1-yloxy)phenethyl-3-(dimethylamino)propanoate, 3,5-6uc((9Z, 12Z)octadeca-9,12-dien-1-yloxy)benzyl(3-(dimethylamino)propyl)carbonate, (9Z,9'Z, 12Z, 12'Z)-(5 -((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)-1,3-phenylene)bis(methylene)bis(octadeca-9,12-dienoate), (9Z,9'Z,12Z,12 'Z)-(5-((((2-(dimethylamino)ethoxy)carbonyl)oxy)methyl)-1,3-phenylene)bis(methylene)bis(octadeca-9,12-dienoate), (9Z,9 'Z, 12Z, 12'Z)-(5-((((3-(dimethylamino)propoxy)carbonyl)oxy)methyl)-1,3-phenylene)bis(methylene)bis(octadeca-9,12-dienoate ), (9Z,9'Z,12Z,12'Z)-5-((((2-(dimethylamino)ethoxy)carbonyl)oxy)methyl)-1,3-phenylene-bis(octadeca9,12-dienoate) , 3-(dimethylamino)propyl-4-isopropyl-3,5-bis((9Z,12Z)octadeca-9,12-dien-1-yloxy)benzyl carbonate, 4-6pom-3,5-6uc((9Z, 12Z)octadeca-9,12-dien-1-yloxy)benzyl(3-(dimethylamino)propyl)carbonate, 4-chloro-3,5-bis((9Z , 12Z) octadeca-9,12-dien-1 -yloxy) benzyl (3 - (dimethylamino) propyl) carbonate, N- (3,5-6uc ((9Z, 12Z) octadeca-9,12-diene-1 - yloxy)benzyl)-2-(dimethylamino)acetamide, O,O'-((5-((dimethylamino)methyl)-1,3-phenylene)bis(methylene))di((9Z, 12Z)octadeca-9, 12-dien-1 yl) disuccinate, O,O'-((5-((dimethylamino)methyl)-1,3-phenylene)bis(methylene))bis(10-(octanoyloxy)decyl)disuccinate, O' ¹ ,O ¹ -((5-((dimethylamino)methyl)-1,3-phenylene)bis(methylene)-8-dinonyldioctanedioate, O' ¹ ,O ¹ -((5-((dimethylamino)methyl) -1,3-phenylene)bis(methylene))-9-dioctyldinonanedioate, 3 -(3,5-6uc((9Z, 12Z)-octadeca-9,12-dien-1-yloxy)phenyl)propyl-3-(dimethylamino)propanoate, (9Z,9'Z, 12Z, 12'Z )-(((5-((dimethylamino)methyl)-1,3-phenylene)bis(methylene))bis(oxy))bis(4-oxobutan-4,1-diyl)bis(octadeca-9,12- dienoate), (((5-((dimethylamino)methyl)-1,3-phenylene)bis(methylene))bis(oxy))bis(8-oxooctane-8,1-diyl)bis(decanoate), (( (5-((dimethylamino)methyl)-1,3-phenylene)bis(methylene))bis(oxy))bis(4-oxobutan-4,1-diyl)dioctanoate, (((5-((dimethylamino)methyl )-1,3-phenylene)bis(methylene))bis(oxy))bis(6-oxohexane-6,1-diyl)dioctanoate, (5-((dimethylamino)methyl)-1,3-phenylene)bis( methylene)bis(10-(octanoyloxy)decanoate), (5-((dimethylamino)methyl)-1,3-phenylene)bis(methylene)bis(8-(octanoyloxy)octanoate), (((5-((dimethylamino )methyl)-1,3-phenylene)bis(methylene))bis(oxy))bis(6-oxohexane-6,1-diyl)bis(decanoate), 4,4'-((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(butane-1,2-diyl)tetraoctanoate, - 120 040257 (R)-4-(3-((S)-3,4-bis(octanoyloxy)butoxy)-5-((dimethylamino)methyl)phenoxy)butane-1,2-diyldioctanoate, (S)- 4-(3-((S)-3,4-bis(octanoyloxy)butoxy)-5-((dimethylamino)methyl)phenoxy)butane-1,2-diyldioctanoate, (R)-4-(3-(( R)-3,4-bis(octanoyloxy)butoxy)-5-((dimethylamino)methyl)phenoxy)butane-1,2-diyldioctanoate, Ν,Ν-dimethyl-1 -(3,4,5-tris((92,12Z)octadeca-9,12-dien-1-yloxy)phenyl)methanamine, ((((5-((dimethylamino)methyl) -1,3-phenylene)bis(oxy))bis(propane-3,1-diyl))bis(oxy))bis(4oxobutan-4,1-diyl)bis(decanoate), O' ¹ ,O ¹ -(((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(propane-3,1-diyl))-9-dioctyldinonanedioate, 3-((dimethylamino)methyl)-5-(((8-(octanoyloxy)octanoyl)oxy)methyl)benzyl-3-octylundecanoate, 2-(3,5-6uc((9Z, 12Z)octadeca-9,12-dien-1-yloxy)phenyl)-N,N-dimethylethanamine, 3 -(3,5-6uc((9Z, 12Z)octadeca-9,12-dien-1-yloxy)phenyl)-N,N-dimethylpropan-1-amine, 2-(3-(4-(5-((dimethylamino)methyl)-2-methyl-3-((9Z,12Z)-octadeca-9,12-dien-1-yloxy)phenoxy)butoxy)-3- oxopropyl)propane-1,3-diyldihexanoate, (5-((dimethylamino)methyl)-1,3-phenylene)bis(methylene)bis(8-decanamidooctanoate), (5-((dimethylamino)methyl)-1,3 -phenylene)bis(methylene)bis(6-(((nonyloxy)carbonyl)oxy)hexanoate), bis(1,3-bis(octanoyloxy)propan-2-yl)-O,O'-((5-( (dimethylamino)methyl)-1,3-phenylene)bis(methylene))disuccinate, (((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(butane-4,1 - diyl))bis(propane-3,2,1 triyl)tetraoctanoate, (((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(methylene))bis(propane-3, 2,1-triyl)tetraoctanoate, (5-((dimethylamino)methyl)-1,3-phenylene)bis(methylene)bis(5-heptyldodecanoate), (5-((dimethylamino)methyl)-1,3-phenylene )bis(methylene)bis(7-hexyltridecanoate), (5-((dimethylamino)methyl)-1,3-phenylene)bis(methylene)bis(9-pentyltetradecanoate), ((((5-((dimethylamino)methyl )-1,3-phenylene)bis(oxy))bis(butane-4,1-diyl))bis(oxy))bis(propane3,2,1-triyl)tetraoctanoate, 1-(3,5-bis(4,4-bis(octyloxy)butoxy)phenyl)-N,N-dimethylmethanamine, or a pharmaceutically acceptable salt thereof. 11. A lipid composition containing a compound according to any one of claims 1 to 10, or a pharmaceutically acceptable salt thereof, and a biologically active agent. 12. The lipid composition of claim 11 wherein the biologically active agent is a short interfering RNA (siRNA). 13. The lipid composition of claim 12 further comprising a helper lipid. 14. A lipid composition according to claim 13 further comprising a neutral lipid. 15. The lipid composition of claim 14, further comprising an occult lipid. 16. The lipid composition of claim 15 wherein the helper lipid is cholesterol, the neutral lipid is distearoylphosphatidylcholine (DSPC), and the hidden lipid is PEG-DMG, o or about (soil). 17. The lipid composition according to claim 15 in the form of a lipid nanoparticle. 18. A lipid composition according to claim 16, having a molar ratio of compound of formula (I)/cholesterol/DSPC/S010 or S011, 45/44/9/2, respectively. 19. A lipid composition according to claim 16 having a molar ratio of a compound of formula (I)/cholesterol/DSPC/S010 or S011 of 55-40/55-40/15-5/10-1, respectively. 20. A lipid composition according to claim 16 having a molar ratio of a compound of formula (I)/cholesterol/DSPC/S010 or S011 of 50-40/50-40/15-5/5-1, respectively. 21. A lipid composition according to claim 16 having a molar ratio of a compound of formula (I)/cholesterol/DSPC/S010 or S011 of 47-43/47-43/12-7/4-1, respectively. 22. A pharmaceutical composition comprising a lipid composition according to any one of claims 11 to 21 and a pharmaceutically acceptable carrier or excipient. 23. A method of treating a disease or condition, comprising the step of administering a therapeutically effective amount of a lipid composition according to any one of paragraphs. 11-21 to a patient in need of such - 121 040257 treatment, where the disease or condition is selected from the group consisting of a proliferative disease, cancer, inflammatory disease, graft and/or tissue rejection, autoimmune disease or age-related disease condition, neurological or neurodegenerative disease, respiratory disease, cardiovascular vascular disease, eye disease, metabolic disease, dermatological disease, auditory disease, liver disease, kidney disease. 24. A method of treating a disease or condition, comprising the step of administering a therapeutically effective amount of a pharmaceutical composition according to claim 22 to a patient in need of such treatment, wherein the disease or condition is selected from the group consisting of a proliferative disease, cancer, inflammatory disease, transplant rejection, and/or tissue, autoimmune disease or age-related disease, neurological or neurodegenerative disease, respiratory disease, cardiovascular disease, eye disease, metabolic disease, dermatological disease, hearing disease, liver disease, kidney disease. 25. Lipid composition according to π. 11, wherein the biologically active agent is messenger RNA (mRNA). 26. The lipid composition of claim 25 further comprising a helper lipid. 27. The lipid composition of claim 26 further comprising a neutral lipid. 28. The lipid composition of claim 27, further comprising an occult lipid. 29. A lipid composition according to any one of claims 26-28, wherein the helper lipid is cholesterol, the neutral lipid is DSPC, and the hidden lipid is PEGDMG, S010, S011, or 30. The lipid composition according to claim 29 in the form of a lipid nanoparticle. 31. A lipid composition according to claim 29, having a molar ratio of compound of formula (1)/cholesterol/DSPC/8010, S011 or S024, 40/38/10/2, respectively. 32. A pharmaceutical composition comprising a lipid composition according to any one of claims 25-31 and a pharmaceutically acceptable carrier or excipient. 33. A method of treating a disease or condition, comprising the step of administering a therapeutically effective amount of a lipid composition according to any one of paragraphs. 25-31 to a patient in need of such treatment, wherein the disease or condition is selected from the group consisting of a proliferative disease, cancer, inflammatory disease, graft and/or tissue rejection, an autoimmune disease or an age-related disease condition, a neurological or neurodegenerative disease, respiratory disease, cardiovascular disease, eye disease, metabolic disease, dermatological disease, hearing disease, liver disease, kidney disease. 34. A method of treating a disease or condition, comprising the step of administering a therapeutically effective amount of a pharmaceutical composition according to claim 32 to a patient in need of such treatment, wherein the disease or condition is selected from the group consisting of a proliferative disease, cancer, inflammatory disease, transplant rejection, and/or tissue, autoimmune disease or age-related disease, neurological or neurodegenerative disease, respiratory disease, cardiovascular disease, eye disease, metabolic disease, dermatological disease, hearing disease, liver disease, kidney disease. Eurasian Patent Organization, EAPO Russia, 109012, Moscow, Maly Cherkassky per., 2 - 122 -