WO2008144728A1 - Conjugates which bind a blood protein such as human serum albumin and methods of using the same in diagnostic and therapeutic applications - Google Patents

Conjugates which bind a blood protein such as human serum albumin and methods of using the same in diagnostic and therapeutic applications Download PDF

Info

Publication number
WO2008144728A1
WO2008144728A1 PCT/US2008/064359 US2008064359W WO2008144728A1 WO 2008144728 A1 WO2008144728 A1 WO 2008144728A1 US 2008064359 W US2008064359 W US 2008064359W WO 2008144728 A1 WO2008144728 A1 WO 2008144728A1
Authority
WO
WIPO (PCT)
Prior art keywords
meg
do3a
composition
imaging
compound
Prior art date
Application number
PCT/US2008/064359
Other languages
French (fr)
Inventor
Edmund R. Marinelli
Natarajan Raju
Radhakrishna K. Pillai
Michael F. Tweedle
Kondareddiar Ramalingam
Original Assignee
Bracco Imaging S.P.A.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Bracco Imaging S.P.A. filed Critical Bracco Imaging S.P.A.
Publication of WO2008144728A1 publication Critical patent/WO2008144728A1/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/0474—Organic compounds complexes or complex-forming compounds, i.e. wherein a radioactive metal (e.g. 111In3+) is complexed or chelated by, e.g. a N2S2, N3S, NS3, N4 chelating group
    • A61K51/0482—Organic compounds complexes or complex-forming compounds, i.e. wherein a radioactive metal (e.g. 111In3+) is complexed or chelated by, e.g. a N2S2, N3S, NS3, N4 chelating group chelates from cyclic ligands, e.g. DOTA
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/74—Synthetic polymeric materials
    • A61K31/785—Polymers containing nitrogen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
    • A61K49/085—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier conjugated systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
    • A61K49/10—Organic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/0402—Organic compounds carboxylic acid carriers, fatty acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/0474—Organic compounds complexes or complex-forming compounds, i.e. wherein a radioactive metal (e.g. 111In3+) is complexed or chelated by, e.g. a N2S2, N3S, NS3, N4 chelating group
    • A61K51/0478—Organic compounds complexes or complex-forming compounds, i.e. wherein a radioactive metal (e.g. 111In3+) is complexed or chelated by, e.g. a N2S2, N3S, NS3, N4 chelating group complexes from non-cyclic ligands, e.g. EDTA, MAG3
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/0497—Organic compounds conjugates with a carrier being an organic compounds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D257/00—Heterocyclic compounds containing rings having four nitrogen atoms as the only ring hetero atoms
    • C07D257/02—Heterocyclic compounds containing rings having four nitrogen atoms as the only ring hetero atoms not condensed with other rings

Definitions

  • the invention relates to conjugates that bind a blood protein such as human serum albumin (HSA), and are useful in diagnostic and/or therapeutic applications.
  • HSA human serum albumin
  • the invention also includes methods for making and using such conjugates in therapeutic and diagnostic applications.
  • IV-DSA Intravenous digital subtraction angiography
  • the heart is always in motion; the patient may not be able to endure breath-holding for more than 15-20 seconds; the hardware is not yet powerful and fast enough (non contrast techniques such as spiral K space sampling or techniques employing single pass CVMRA); and finally, spatial resolution must be at least 1.0 mm x 1.0 mm x 1.0 mm in order to achieve meaningful results. This forces a scan time of at least 7 minutes with hardware that is considered possible to construct.
  • known contrast agents such as MS- 325 (gadofosveset trisodium) a member of a class of blood pool agents for magnetic resonance angiography (MRA) have been used, for example, to diagnose vascular disease under the above constraints.
  • an agent that provides Tl shortening ( ⁇ 50 ms) of principally the intravascular space for an extended period of time is highly desirable, and can greatly facilitate CVMRA.
  • the agent preferably should be restricted from diffusing into the extravascular space, and preferably should undergo clearance from the blood with a half time less than that of extracellular space agents.
  • the present invention is directed, in one embodiment, to a compound for the imaging or treatment of a disease in a mammal, having the structure: Ch- Ll - R - L2 - Q wherein Ch is a chelating moiety, optionally complexed with M, a metal or a radionuclide; Ll is a first linker;
  • R is a hydrophobic amino acid or a hydrophobic non-amino acid compound which renders the entire construct capable of binding a blood protein, particularly of binding human serum albumin (HSA); and
  • L2 is an optional second linker that is further linked to an optional targeting vector Q, which can direct the entire construct to a tissue bearing the target.
  • Ch is a chelating moiety, which is optionally complexed with a metal or a radionuclide.
  • Ch comprises DTPA, DOTA, EDTA, TETA, EHPG, HBED, NOTA, DOTMA, TETMA, PDTA, TTHA, LICAM, MECAM or derivatives thereof.
  • Ch comprises DOTA or a derivative thereof.
  • Ch comprises DO3 A or derivatives thereof.
  • Ch comprises DO3A10CM, an abbreviation of 10-carbonylmethyl- 1,4, 7,10-tetraazacyclododecane- 1,4,7- triacetic acid.
  • This designation indicates that the compound has 3 carboxylate moieties chelating the gadolinium or other metal, and a fourth chelating group one component of which is a carbonyl group which is not the carbonyl group of a carboxylic acid, but rather the carbonyl group of another functionality such as a carboxamide or a hydroxamate, or an acylhydrazine, or of a semicarbazide, or a semicarbazone for example, or related chelators.
  • Ch is a chelating moiety having high relaxivity.
  • Ch may be selected from the following table:
  • Ch is selected from CE-DTPA; CM-DOTMA; CM-DOTA; 7MP-
  • Ch is complexed with a metal M or a radionuclide.
  • M may be a metal ion such as, for example, a paramagnetic or radioactive metal ion.
  • M is a paramagnetic metal ion.
  • M comprises Mn 2+ , Cu 2+ ,
  • M is Gd 3+ .
  • M is a radioactive metal ion or a radionuclide.
  • M is selected from 18 F, 124 1, 125 1, 131 I, 123 1, 77 Br, 76 Br, 99m Tc,
  • M is 99m Tc, 67 Ga, 68 Ga, 90 Y, 88 Y, 111 In or 177 Lu.
  • Ll the first linker, links Ch, the chelating moiety to one or more other components of the conjugate.
  • Ll comprises functional groups sufficient to allow such linking.
  • Ll links the chelator to the hydrophobic molecule R.
  • Ll comprises a substituted or unsubstituted alkyl chain, a polyethylene glycol derivative, one or more amino acids, a sugar, an aliphatic or aromatic spacer, a lipid molecule, or combinations of any of the foregoing.
  • Ll comprises an amino acid.
  • Ll may comprise GIy or Lys.
  • Ll comprises trans-4-methylaminocyclohexanecarboxylic acid (referred to as a).
  • Ll comprises two amino acids.
  • Preferably Ll is Gly-Lys (referred to as GK); Gly-Gly (referred to as GG); Pro- trans-4- aminomethylcyclohexanecarbonyl (referred to as Pa); Asp- trans-4- aminomethylcyclohexanecarbonyl (referred to as Da); isonipecotic acid- trans-4- aminomethylcyclohexanecarbonyl (referred to as ia); GIy- Asp (referred to as GD); GIy- trans-4-aminomethylcyclohexanecarbonyl (referred to as Ga); GIu- trans-4- aminomethylcyclohexanecarbonyl (referred to as Ea); trans-4- aminomethylcyclohexanecarbonyl-Gly (referred to as aG); Asp-Asp (referred to as GK); Gly-Gly (referred
  • R is a hydrophobic moiety which renders the entire conjugate able to bind to a blood protein such as HSA.
  • R is a hydrophobic amino acid, a hydrophobic moiety derived from a hydrophobic amino acid or a hydrophobic organic group.
  • R comprises a hydrophobic amino acid.
  • R may be tryptophan or a derivative thereof; /-biphenylalanine (referred to as B) or /-3,3- diphenylalanine (referred to as e).
  • both R and Ll comprise amino acids (or are derived from amino acids).
  • Ll comprises two amino acids and R comprises a hydrophobic amino acid which binds HSA.
  • Ll-R comprises glycyl-trans-4-aminomethylcyclohexanecarbonyl-/-4-biphenyl (refereed to herein as GaB) or trans-4-aminomethylcyclohexanecarbonyl-/-4-biphenyl (referred to herein as aB) .
  • R is a hydrophobic organic group.
  • R is a hydrophobic organic group of similar size to a hydrophobic amino acid. More preferably, R is a hydrophobic aliphatic or aromatic acyl group In a preferred embodiment R is selected from:
  • the compounds of the present invention optionally include L2, a second linker.
  • L2 is a linker which includes sufficient functional groups to link the targeting vector Q to the remainder of the conjugate.
  • L2 comprises a substituted or unsubstituted alkyl chain, a polyethylene glycol derivative, one or more amino acids, a sugar, an aliphatic or aromatic spacer, a lipid molecule, or combinations of any of the foregoing.
  • L2 may be the same or different as Ll .
  • Preferably L2 comprises one or more amino acids.
  • Q is an optional targeting vector, which directs the entire conjugate to tissue bearing the target.
  • the target may be a site of disease or a pathological process, such as, for example, an angiogenic site in a tumor.
  • Q may be a targeting protein, antibody, peptide or equivalent, derivative or analog thereof; Q may also be, e.g., an enzyme or a molecule that binds to an enzyme or it may be an antibiotic.
  • Q may be a protein such as an antibody, antibody fragment, diabody, triabody, a small molecule which functions as a targeting vector a carbohydrate or an aptamer.
  • Q and Ll both comprise amino acids.
  • Particularly preferred conjugates of the invention include DO3A10CM-a-B (where Ch is DO3A10CM, Ll is a and R is B); DO3A10CM- G-a-B (where Ch is DO3A10CM, Ll is G-a and R is B); DO3A10CM -D-a-B (where Ch is DO3A10CM, Ll is Da and R is B); DO3A10CM -G-L-B (where Ch is DO3A10CM, Ll is Gl and R is B); CE-DTP A-a-B (where Ch is CE-DTPA, Ll is a and R is B); CM-DOTMA-a-B (where Ch is CM-DOTMA, Ll is a and R is B); CM-DOTA-a-B (where Ch is CM-DOTA, Ll is a and R is B); and 7MP- DO3A-G-a-B (where Ch is 7MP-DO3A, Ll is G-A and R
  • Each of these conjugates may optionally further include a second linking group L2 and/or a targeting vector Q. Any of these conjugates may be optionally complexed with a metal M. In a preferred embodiment these conjugates are complexed with a paramagnetic metal, particularly Gd. In another preferred embodiment, these conjugates are complexed with a radioactive metal or radionuclide, particularly with a radioactive lanthanide.
  • Conjugates of the present invention are capable of binding a blood protein such as HSA, which increases the half time of the conjugate in the blood compared to a comparable agent unable to bind a blood protein.
  • the increased blood half time results in conjugates better able to accumulate at target tissue.
  • the binding to a blood protein such as HSA results in an increase of the relaxivity of the chelator which is commonly referred to as enhanced relaxivity.
  • Such conjugates when bound to the blood proteins diffuse into the extravascular space much more slowly than those which cannot bind to proteins in the blood. Therefore their effective residence time in the blood is much longer and because they are bound to blood proteins such as HSA they exhibit enhanced relaxivity and enhanced signal vs chelators which are not protein bound, permitting improved imaging of the intravascular space and facilitating CVMRI.
  • such conjugates also include a chelating moiety Ch whose relaxivity changes to a greater degree then that of a standard chelator when a molecule to which it is conjugated to binds proteins in the blood.
  • a chelating moiety Ch whose relaxivity changes to a greater degree then that of a standard chelator when a molecule to which it is conjugated to binds proteins in the blood.
  • Such agents provide a Tl shortening ( ⁇ 50 ms) of only the intravascular space for an extended period of time, further facilitating CVMRI.
  • Such chelating moieties include for example, : CM-DOTMA, CM-DOTA, CEMP-D03A, 7MP-DO3A, 7MP-CM- DO3 A, TE-DOTA and 4-CEMP-DTTA whose structures are shown in Chart 4 .
  • the present invention is also directed to a method of preparing a conjugate capable of binding a blood protein such as human serum albumin (HSA), wherein said method comprises the steps of: attaching a chelating moiety to the first linker, attaching said first linker to the hydrophobic group R via a protecting group having a carboxylic group thereto, attaching an optional second linker to said hydrophobic group, and attaching an optional targeting vector Q to said optional second linker.
  • the conjugate may then be optionally complexed with M, a metal/radionuclide.
  • the present invention is also directed to a method of preparing a conjugate capable of binding a blood protein such as human serum albumin wherein said method comprises attaching a hydrophobic group R which is capable of binding HSA to a first linker, then attaching the first linker to the chelating moiety Ch, then attaching another conjugatable group in the first linker to a second linker followed by attachment of the second linker to an optional molecule which can serve as a targeting vector.
  • the conjugate may then be optionally complexed with M, a metal/radionuclide.
  • the present invention is also directed to a method of preparing a conjugate capable of binding a blood protein such as HSA, wherein said method comprises preparing the components of the entire construct as modules such as Ch-Ll, R-P (where P is an optional protecting group for a reactive function other than that to be employed for assembling the construct) and L2-Q and then attaching the modules in any desired order to obtain constructs such as Ch - Ll - R - L2 - Q, Ch-L1(L2-Q)-R or Ch(L2-Q)-Ll-R.
  • the conjugate may then be optionally complexed with M, a metal/radionuclide.
  • the present invention is also directed to assembly on solid phase of the entire conjugate or a subset of the entire conjugate described above using orthogonal or quasiorthogonal series of protecting groups which allow creation of any of the conjugate Ch - Ll - R - L2 - Q, Ch-L1(L2-Q)-R, Ch(L2-Q)-Ll-R or any part of the conjugate on solid phase in fully or partially protected form followed by simultaneous cleavage from the solid phase and deprotection of the desired construct.
  • the conjugate may then be optionally complexed with M, a metal/radionuclide.
  • the present invention is further directed to compounds which provide a high fraction bound to serum.
  • compounds of the invention may have a fraction bound to serum greater than about 0.87 (87%, expressed as a percentage).
  • the present invention is also directed to compounds which have high measured relaxivity in SeronormTM.
  • compounds of the invention may have a measured relaxivity in SeronormTM of greater than about 36.57.
  • the present invention is also directed to compounds which have both high fraction bound to serum and high measured relaxivity.
  • compounds of the invention may have fraction bound to serum greater than about 87% and a measured relaxivity in SeronormTM of greater than about 36.57.
  • conjugates of the invention may be used in methods of treating a disease in a mammal comprising the step of administering a composition comprising the conjugates of the present invention.
  • conjugates of the invention may be used in methods of imaging a site in a mammal, comprising the steps of administering a composition comprising the conjugates of the present invention and imaging the mammal.
  • the term "therapeutic" includes at least partial alleviation of symptoms of a given condition.
  • treatment of an individual can result in a decrease in the size of a tumor or diseased area, or prevention of an increase in size of the tumor or diseased area or partial alleviation of other symptoms.
  • Treatment can result in reduction in the number of blood vessels in an area of interest or can prevent an increase in the number of blood vessels in an area of interest.
  • Treatment can also prevent or lessen the number or size of metastic outgrowths of the main tumor(s).
  • peptide is used interchangeably herein with “polypeptide” but is generally used to refer to polypeptides having fewer than 40, and preferably fewer than 25 amino acids.
  • the term "relaxivity” means the ability of a compound to shorten the Tl (longitudinal relaxation time) of water protons or other atoms which can be detected by Tl- weighted MRI imaging.
  • the symbol for relaxivity is r and where the Tl relaxivity is quoted the symbol is rl .
  • This value is quoted as being measured at a given field strength, such as 20MHz, for example, and the value is given in units of mM ⁇ sec "1 .
  • the aggregate relaxivity may be quoted and often the relaxivity per gadolinium atom is also quoted in order to clarify the interpretation of the data.
  • the term "high relaxivity" refers to values of rl provided by MRI agents of a given class, particularly MRI agents based on gadolinium chelates, which are significantly greater than those provided by typical gadolinium chelate-based MRI agents under the same conditions. By significantly greater is meant at least 10% greater than the relaxivity of the comparison gadolinium chelate. By 'same conditions' is meant that the chelators in question are employed under the same conditions of measurement and if conjugated to another molecular species such as a targeting vector or a protein binding moiety, they are conjugated to the same molecular species.
  • the term 'enhanced relaxivity' refers to an increase in relaxivity, rl, which is obtained when a MRI agent, typically based on a gadolinium chelator, binds to a macromolecular species such as a blood protein.
  • the comparison is made between the relaxivity of the MRI agent measured in water or optionally in aqueous solutions of a buffer such a pH 7 phosphate buffer or phosphate buffered saline solution and the relaxivity of the MRI agent in the presence of a defined concentration of a macromolecular target.
  • the macromolecular target is human serum albumin (HSA).
  • binding refers to the determination by standard assays, including those described herein, that a binding moiety such as a hydrophobic group, a small molecule, a peptide, or a polypeptide binds reversibly to a given target.
  • standard assays include, but are not limited to, equilibrium dialysis, gel filtration, surface plasmon resonance and the monitoring of spectroscopic changes, such as increased or decreased absorption, a bathochromic or hypsochromic shift, an increase or decrease or an elimination of or an induction of fluorescence, for example, that result in the bound state.
  • binding species including hydrophobic groups, small molecules, peptides or polypeptides
  • homologues will be understood to fall within the scope of the present invention and the definition of "binding species” so long as the substitution, addition, or deletion of functionality, or in the case of peptidic species, amino acids or other such modification does not eliminate its ability to bind to the target.
  • homologous as used herein, with regard to peptides and polypeptides refers to the degree of sequence similarity between two polymers (i.e. polypeptide molecules or nucleic acid molecules).
  • polymers are homologous at that position. For example, if the amino acid residues at 60 of 100 amino acid positions in two polypeptide sequences match or are homologous, then the two sequences are 60% homologous.
  • the homology percentage figures referred to herein reflect the maximal homology possible between the two polymers, i.e., the percent homology when the two polymers are so aligned as to have the greatest number of matched (homologous) positions.
  • Polypeptide homologues within the scope of the present invention will be at least 70% and preferably greater than 80% homologous to at least one of the binding sequences disclosed herein.
  • binding specificity refers to a binding polypeptide having a higher binding affinity for one target over another. Binding specificity may be characterized by a ratio of the dissociation equilibrium constants (K D ) or association equilibrium constants (K a ) for the two tested target materials.
  • the term "patient” as used herein refers to any mammal, especially humans.
  • pharmaceutically acceptable carrier or excipient refers to a non-toxic carrier or excipient that may be administered to a patient, together with a compound of this invention, and which does not destroy the pharmacological activity thereof.
  • target or “target molecule” refers to any substance that a binding moiety or binding polypeptide (including for example a targeting vector or hydrophobic compound) can bind to, such as a protein or polypeptide, a cell, a receptor, a carbohydrate, a lipid, an oligonucleotide, etc.
  • target also includes a family of receptors, such as, for example, protein-tyrosine kinase receptors.
  • therapeutic agent refers to a compound or an agent having a beneficial, therapeutic or cytotoxic effect in vivo.
  • Therapeutic agents include those compositions referred to as, for example, bioactive agents, cytotoxic agents, drugs, chemotherapy agents, radiotherapeutic agents, genetic material, etc.
  • the present invention is directed, in one embodiment, to novel conjugates that exhibit superior ability to bind a blood protein such as Human Serum Albumin (HSA) and which may be used in diagnostic and/or therapeutic applications.
  • HSA Human Serum Albumin
  • the present invention is further directed, in another embodiment, to novel conjugates that exhibit the ability to bind human serum at greater than about 87% and which may be used in diagnostic and/or therapeutic applications.
  • the present invention is further directed, in still another embodiment, to novel conjugates that exhibit a measured relaxivity in SeronormTM of greater than about 36.57 and which may be used in diagnostic and/or therapeutic applications.
  • the present invention is further directed, in another embodiment, to novel conjugates that exhibit the ability to bind human serum at greater than about 87% and a measured relaxivity in SeronormTM (a solution of human serum albumin commercially available from
  • Ny corned- Amersham of greater than about 36.57 which may be used in diagnostic and/or therapeutic applications.
  • the present invention is further directed, in yet another embodiment, to compounds that comprise one or more moieties that bind a blood protein such as HSA, to which are conjugated to a metal chelator.
  • the compounds comprise one or more agents that bind the protein HSA, as well as a metal chelator linked to the HSA-binding portion by one or more linkers.
  • conjugates have the structure:
  • Ch is a chelating moiety, optionally complexed with M, a metal or a radionuclide;
  • Ll is a first linker;
  • R is a hydrophobic amino acid or a hydrophobic non-amino acid compound which renders the entire construct capable of binding a blood protein, particularly of binding human serum albumin (HSA); and
  • L2 is an optional second linker that is further linked to an optional targeting vector Q, which can direct the entire construct to a tissue bearing the target.
  • CH, Ll, R, L2 and Q need not be attached in the order shown but may also be attached according to, for example: Ch-L1(L2-Q)-R ; and Ch(L2-Q)-Ll-R.
  • the conjugates comprise : Ch-Ll-R, wherein Ch, Ll and R have the above meanings.
  • Ch is a chelating moiety, which is optionally complexed with a metal or a radionuclide.
  • Ch is a paramagnetic metal chelate, which, when complexed with paramagnetic metal may serve as a contrast agent for use in MRI.
  • Suitable chelators are known in the art and include, e.g. , acids with methylene phosphonic acid groups, methylene carbohydroxamine acid groups, carboxyethylidene groups, carboxymethylene groups or methylene carboxamide groups.
  • chelators include, but are not limited to, diethylenetriamine pentaacetic acid (DTPA), l,4,7,10-tetraazacyclotetradecane-l,4,7,10-tetraacetic acid (DOTA), 1 -substituted 1,4,7,- tricarboxymethyl 1,4,7,10 teraazacyclododecane triacetic acid (DO3A), ethylenediaminetetraacetic acid (EDTA), and 1,4,8,11-tetraazacyclotetradecane-l, 4, 8,11- tetraacetic acid (TETA).
  • DTPA diethylenetriamine pentaacetic acid
  • DO3A 1 -substituted 1,4,7,- tricarboxymethyl 1,4,7,10 teraazacyclododecane triacetic acid
  • EDTA ethylenediaminetetraacetic acid
  • TETA 1,4,8,11-tetraazacyclotetradecane-l
  • Additional useful chelating ligands are ethylenebis-(2-hydroxy-phenylglycine) (EHPG), and derivatives thereof, including 5-Cl-EHPG, 5Br-EHPG, 5-Me-EHPG, 5- t-Bu-EHPG, and 5-sec-Bu-EHPG; benzodiethylenetriamine pentaacetic acid (benzo-DTPA) and derivatives thereof, including dibenzo-DTPA, phenyl-DTPA, diphenyl-DTPA, benzyl-DTPA, and dibenzyl DTPA; bis-2 (hydroxybenzyl)-ethylene-diaminediacetic acid (HBED) and derivatives thereof; the class of macrocyclic compounds which contain at least 3 carbon atoms, more preferably at least 6, and at least two heteroatoms (O and/or N), which macrocyclic compounds can consist of one ring, or two or three rings joined together at the hetero ring elements, e.g., benzo-DOTA, dibenz
  • Ch comprises DTPA, DOTA, EDTA, TETA, EHPG, HBED, NOTA, DOTMA, TETMA, PDTA, TTHA, LICAM, MECAM or derivatives thereof.
  • Ch comprises DOTA or a derivative thereof.
  • Ch comprises D03A or DO3A10CM.
  • Ch is a chelator for a radioactive metal or radionuclide.
  • chelators include, for example, linear, macrocyclic, terpyridine, and N3S, N 2 S 2 , or N 4 chelants (see also U.S. 5,367,080, U.S. 5,364,613, U.S. 5,021,556, U.S. 5,075,099, U.S. 5,886,142), and other chelators known in the art including, but not limited to, HYNIC, DTPA, EDTA, DOTA, TETA, and bisamino bisthiol (BAT) chelators (see also U.S. 5,720,934).
  • BAT bisamino bisthiol
  • N 4 chelators are described in U.S. Patent Nos. 6,143,274; 6,093,382; 5,608,110; 5,665,329; 5,656,254; and 5,688,487.
  • Certain N 3 S chelators are described in PCT/CA94/00395, PCT/CA94/00479, PCT/CA95/00249 and in U.S. Patent Nos. 5,662,885; 5,976,495; and 5,780,006.
  • the chelator may also include derivatives of the chelating ligand mercapto-acetyl-acetyl-glycyl-glycine (MAG3), which contains an N 3 S, and N 2 S 2 systems such as MAMA (monoamidemonoaminedithiols), DADS (N 2 S diaminedithiols), CODADS and the like.
  • MAG3 chelating ligand mercapto-acetyl-acetyl-glycyl-glycine
  • MAMA monoamidemonoaminedithiols
  • DADS N 2 S diaminedithiols
  • CODADS CODADS
  • the chelator may also include complexes containing ligand atoms that are not donated to the metal in a tetradentate array.
  • complexes containing ligand atoms that are not donated to the metal in a tetradentate array include the boronic acid adducts of technetium and rhenium dioximes, such as are described in U.S. Patent Nos. 5,183,653; 5,387,409; and 5,118,797, the disclosures of which are incorporated by reference herein, in their entirety.
  • disulfide bonds of a binding polypeptide of the invention are used as two ligands for chelation of a radionuclide such as 99m Tc.
  • a radionuclide such as 99m Tc.
  • Tc peptide-S-S-peptide changed to peptide-S-Tc-S- peptide.
  • the other chelating groups for Tc can be supplied by amide nitrogens of the backbone, another cystine amino acid or other modifications of amino acids.
  • Particularly preferred metal chelators include those of Formula 20, 21, and 22 (for 111 In and lanthanides such as paramagnetic gadolinium (Gd 3+ ) and radioactive lanthanides, such as, for example 177 Lu, 90 Y, 153 Sm, and 166 Ho) and those of Formula 23, 24, and 25 (for radioactive 99m Tc, 186 Re, and 188 Re) set forth below.
  • 111 In and lanthanides such as paramagnetic gadolinium (Gd 3+ ) and radioactive lanthanides, such as, for example 177 Lu, 90 Y, 153 Sm, and 166 Ho
  • Formula 23, 24, and 25 for radioactive 99m Tc, 186 Re, and 188 Re
  • R is alkyl, preferably methyl.
  • X is either CH 2 or O
  • Y is either C 1 -C 10 branched or unbranched alkyl
  • Y is aryl, aryloxy, arylamino, arylaminoacyl
  • Y is arylkyl - where the alkyl group or groups attached to the aryl group are C 1 -C 10 branched or unbranched alkyl groups, C 1 -C 10 branched or unbranched hydroxy or polyhydroxyalkyl groups or polyalkoxyalkyl or polyhydroxy-polyalkoxyalkyl groups
  • the chelators may be covalently linked to the HSA-binding moiety via Ll, or directly to the HSA binding moiety which is optionally further linked to L2 and/or Q, the targeting vector, as described previously, and then directly labeled with the radioactive metal of choice (see, e.g., WO 98/52618, U.S. 5,879,658, and U.S. 5,849,261).
  • a preferred chelator used in conjugates of the present invention is 1 -substituted 4,7,10-tricarboxymethyl 1,4,7,10 tetraazacyclododecane triacetic acid (DO3 A).
  • Another preferred chelating moiety is l,4,7,10-tetraazacyclotetradecane-l,4,7,10-tetraacetic acid (DOTA).
  • the present invention also contemplates chelators comprising DOTA derivatives; e.g., substituted derivatives of DOTA having 4 carboxyl groups chelating gadolinium or another metal and wherein the linkage of the macrocyclic system to the linker Ll is via either a group emanating from one of the alpha-carbon atoms of one of the chelating sidearms or from the macrocyclic carbon backbone.
  • DOTA derivatives e.g., substituted derivatives of DOTA having 4 carboxyl groups chelating gadolinium or another metal and wherein the linkage of the macrocyclic system to the linker Ll is via either a group emanating from one of the alpha-carbon atoms of one of the chelating sidearms or from the macrocyclic carbon backbone.
  • Yet another chelator moiety is DO3A10CM, an abbreviation of 10-carbonylmethyl- l,4,7,10-tetraazacyclododecane-l,4,7-triacetic acid.
  • This designation indicates that the compound has 3 carboxylate moieties chelating the gadolinium or other metal, and a fourth chelating group one component of which is a carbonyl group which is not the carbonyl group of a carboxylic acid, but rather the carbonyl group of another functionality such as a carboxamide or a hydroxamate, or an acylhydrazine, or of a semicarbazide, or a semicarbazone for example.
  • Ch is a chelating moiety having high or enhanced relaxivity.
  • high or enhanced relaxivity chelators that are useful for the present invention are described in, e.g., WO 99/45967, WO 99/45968 (manganese chelates), WO 98/05625 and WO 98/05626, each of which is hereby incorporated by reference in its entirety.
  • Ch may be selected from the following table: Chart 4. Enhanced Relaxivity (ER) Chelate Systems
  • Ch is a chelator with enhanced relaxivity selected from CE-DTPA; CM-DOTMA; CM-DOTA; 7MP-DO3A and TE-DOTA.
  • a most preferred high relaxivity chelator for use in contrast agents for MRI is an aza ligand that is able to complex metal ions, in particular paramagnetic ions.
  • Such an aza ligand should be able to form chelates with the transition metals or metals of the Lanthanide series, exhibiting a high level of affinity and selectivity for the relevant paramagnetic ions, as well as suitable pharmacokinetic properties such as excretion, binding to plasma proteins, metabolical inertia, and the like.
  • Aza ligands that are particularly useful for the present invention are described in WO 03/008390, which is hereby incorporated by reference in its entirety.
  • DO3A10CM was the standard chelating function that was employed for screening the candidate conjugates.
  • Other chelators were employed in an attempt to optimize the conjugates for use in MRI. These chelates were chosen because they were found to display a greater increase in relaxivity than standard chelators in an assay that measures the relaxivity of the Gd-chelate when its motion is restricted artificially.
  • the artificial restriction of the motion of the chelate is designed to mimic the binding of a chelator containing molecule to a blood protein, such as HSA (Human Serum Albumin).
  • the increase in relaxivity is often referred to as enhanced relaxivity - ER for short - by comparison with the unbound state.
  • pairing up of one of the ER chelating molecules with a strong binder of HSA is expected to provide a greater increase in relaxivity upon binding the HSA than would be afforded by the compound comprising the same HSA binding molecule with the standard chelating function DO3A10CM.
  • the chelating moiety Ch is complexed with a metal M or a radionuclide.
  • M may be a metal ion such as, for example, a paramagnetic or radioactive metal ion.
  • M is a paramagnetic metal ion.
  • Preferred paramagnetic metal ions have atomic numbers 21-29, 42, 44, or 57-83, including ions of the transition metal or lanthanide series which have one, and more preferably five or more, unpaired electrons and a magnetic moment of at least 1.7 Bohr magneton.
  • Preferred paramagnetic metals include, but are not limited to, chromium (III), manganese (II), manganese (III), iron (II), iron (III), cobalt (II), nickel (II), copper (II), praseodymium (III), neodymium (III), samarium (III), gadolinium (III), terbium (III), dysprosium (III), holmium (III), erbium (III), europium (III) and ytterbium (III).
  • compounds of the present invention may also be conjugated with one or more superparamagnetic particles.
  • M comprises
  • One skilled in the art will be able to select a metal according to the dose required to detect target containing tissue, as well as considering other factors such as, e.g., toxicity of the metal to the subject. See, Tweedle et al., Magnetic Resonance Imaging (2nd ed.), vol. 1, Partain et al., eds. (W.B. Saunders Co. 1988), pp. 796-97.
  • the desired dose for an individual metal will be proportional to its relaxivity, modified by factors such as the biodistribution, pharmacokinetics and metabolism of the metal.
  • Gd(III) or Gd 3+ The trivalent cation of gadolinium, referred to as Gd(III) or Gd 3+ , is particularly preferred for MRI contrast agents, due to its high relaxivity and low toxicity, with the further advantage that it exists in only one biologically accessible oxidation state. This minimizes undesired metabolization of the metal by a patient.
  • Gd(III) chelates have been used for clinical and radiologic MR applications since 1988, and approximately 30% of all MR examinations currently employ a gadolinium-based contrast agent.
  • Another preferred metal is Cr + , which is advantageous and relatively inexpensive.
  • M is a radioactive metal ion or a radionuclide.
  • M is selected from 18 F, 124 1, 125 1, 131 1, 123 1, 77 Br, 76 Br, 99m Tc, 51 Cr, 67 Ga, 68 Ga, 47 Sc, 51 Cr, 167 Tm, 141 Ce, 111 In, 168 Yb, 175 Yb, 140 La, 90 Y, 88 Y, 153 Sm, 166 Ho, 165 Dy, 166 Dy, 62 Cu, 64 Cu, 67 Cu, 97 Ru, 103 Ru, 186 Re, 188 Re, 203 Pb, 211 Bi, 212 Bi, 213 Bi, 214 Bi, 105 Rh, 109 Pd, 117m Sn, 149 Pm, 161 Tb, 177 Lu, 198 Au or 199 Au and the like.
  • M is 99m Tc, 67 Ga, 68 Ga, 90 Y, 88 Y, 111 In Or
  • one or more linkers may be present between the chelating agent and the hydrophobic compound, and optionally, one or more additional linkers (L2) may be present between the targeting vector and the remainder of the conjugate.
  • linkers/spacers may improve the relevant properties of the conjugates (e.g., by increasing serum stability, etc.).
  • Linkers that are useful for the present invention may include, but are not restricted to, substituted or unsubstituted alkyl chains, polyethylene glycol derivatives, amino acid spacers, sugars, aliphatic or aromatic spacers, lipid molecules, or combinations of any of the foregoing.
  • the conjugates of the invention include Ll and optionally L2.
  • Ll (and L2 if present) comprise at least one amino acid.
  • Ll may comprise GIy or Lys.
  • Ll comprises trans-4-methylaminocyclohexanecarboxylic acid (referred to as a).
  • Ll comprises two amino acids.
  • Ll can be Gly-Lys (referred to as GK)
  • GK Gly-Lys
  • Ll can be Lys(Gly) which indicates that the GIy is appended to the epsilon nitrogen of the Lys component of Ll instead of the alpha nitrogen of the Lys.
  • the linker comprises Lys attachment can be at the alpha and epsilon amines, at the alpha amine and carboxylic acid or at the carboxylic acid and epsilon amine.
  • Gly-Gly referred to as GG
  • Pro- trans-4 aminomethylcyclohexanecarbonyl referred to as Pa
  • Asp- trans-4- aminomethylcyclohexanecarbonyl referred to as Da
  • isonipecotic acid- trans-4- aminomethylcyclohexanecarbonyl referred to as ia
  • GIy- Asp referred to as GD
  • GIy- trans-4-aminomethylcyclohexanecarbonyl referred to as Ga
  • GIu- trans-4- aminomethylcyclohexanecarbonyl referred to as Ea
  • trans-4- aminomethylcyclohexanecarbonyl-Gly referred to as aG
  • Asp-Asp DD
  • L2 is present and includes sufficient functional groups to link the targeting vector Q to the remainder of the conjugate.
  • L2 comprises one or more amino acids.
  • R is a hydrophobic moiety which renders the entire conjugate able to bind to a blood protein such as HSA.
  • R is a hydrophobic amino acid, a hydrophobic moiety derived from a hydrophobic amino acid or a hydrophobic organic group.
  • R comprises a hydrophobic amino acid.
  • the hydrophobic amino acid is comprised of an entity containing an indole ring substituted at the indole nitrogen with a hydrophobic substituent such as a substituted benzene sulfonamide or substituted benzamide moiety.
  • R is tryptophan or a derivative thereof; /-biphenylalanine (referred to as B); or /- 3,3-diphenylalanine (referred to as e).
  • both R and Ll comprise amino acids (or are derived from amino acids).
  • Ll comprises two amino acids and R comprises a hydrophobic amino acid which binds HSA.
  • Ll-R comprises glycyl-trans-4- aminomethylcyclohexanecarbonyl-/-4-biphenylalanyl (referred to herein as GaB) or trans-4- aminomethylcyclohexanecarbonyl-/-4-biphenylalanyl (referred to herein as aB) .
  • R is a hydrophobic organic group.
  • R is a hydrophobic organic group of similar size to a hydrophobic amino acid.
  • R is a hydrophobic molecule that contains one or more benzene rings or 6- membered carbon aryl groups.
  • R is a hydrophobic aliphatic or aromatic carboxylic acid.
  • R is selected from:
  • the purpose of the hydrophobic, blood protein binding portion is to prolong circulation of the conjugate in the bloodstream, in contrast with non-protein binding molecules of similar molecular weight.
  • these non-protein binding molecules would be expected to be rapidly distributed from the blood stream to the extravascular space and then excreted from the body, e.g. , through the urine or the feces.
  • binding of the molecule to a protein in the blood such as HSA, increases the blood residence time of the compound, because the molecular size of the bound blood protein is such that it is only very slowly excreted from the bloodstream, as compared to smaller molecules.
  • the compounds of the present invention are bound to the blood protein, they are unavailable to the pathways for excretion of small molecules such as, for example, passive diffusion across the endothelial cell junctions into the extravascular space, or uptake by the kidneys or the liver via a first pass effect.
  • the conjugates of the present invention may comprise an optional targeting vector Q, which directs the entire conjugate to tissue bearing the target.
  • Q directs the conjugate to the appropriate site, or involves the compounds in a reaction, where the desired diagnostic or therapeutic activity will occur.
  • Q may be a targeting protein, antibody, peptide or equivalent, derivative or analog thereof; Q may also be, e.g., an enzyme or a molecule that binds to an enzyme or it may be an antibiotic.
  • Q may be a protein such as an antibody, antibody fragment, diabody, or triabody, a small molecule which functions as a targeting vector a carbohydrate or an aptamer.
  • the targeting vector Q may be a peptide, equivalent, derivative or analog thereof which functions as a ligand that binds to a particular site, or which has binding affinity for a particular site or a specific metabolic function.
  • the targeting vector may be a peptide that binds to a receptor or enzyme of interest.
  • the targeting vector may be an enzyme, or a molecule that binds to an enzyme.
  • the targeting moiety may be an antibiotic.
  • the targeting moiety is a peptide.
  • the targeting moiety may be a peptide hormone such as, for example, luetinising hormone releasing hormone (LHRH) such as that described in the literature [e.g.
  • Peptides Chemistry, Structure and Biology Pravin T. P. Kaumaya, and Roberts S. Hodges (Eds)., Mayflower Scientific LTD., 1996, pp. 174-175]; gastrin releasing peptide (GRP); bombesin and other known hormone peptides, as well as analogues and derivatives thereof.
  • GFP gastrin releasing peptide
  • Other useful targeting peptides include analogs of somatostatin which, for example, are Lanreotide (Nal-Cys-Thr-DTrp-Lys-Val-Cys-Thr-N ⁇ ), Octreotide (Nal-Cys-Thr-DTrp- Lys-Val-Cys-Thr-ol), and Phe-Cys-Thr-DTrp-Lys-Val-Cys-Thr-ol.
  • somatostatin which, for example, are Lanreotide (Nal-Cys-Thr-DTrp-Lys-Val-Cys-Thr-N ⁇ ), Octreotide (Nal-Cys-Thr-DTrp- Lys-Val-Cys-Thr-ol), and Phe-Cys-Thr-DTrp-Lys-Val-Cys-Thr-ol.
  • Still other useful targeting peptides include Substance P agonists [e.g., G. Bitan, G. Byk, Y. Mahriki, M. Hanani, D. Halle, Z. Selinger, C. Gilon, Peptides: Chemistry, Structure and Biology, Pravin T. P. Kaumaya, and Roberts S. Hodges (Eds), Mayflower Scientific LTD., 1996, pp. 697-698; G Protein Antagonists A novel hydrophobic peptide competes with receptor for G protein binding, Hidehito Mukai, Eisuke Munekata, Tsutomu Higashijima, J. Biol. Chem.
  • NPY(Yl) e.g., Novel Analogues of Neuropeptide Y with a Preference for the Yl -receptor, Richard M. Soil, Michaela, C. Dinger, Ingrid Lundell, Dan Larhammer, Annette G. Beck-Sickinger, Eur. J. Biochem. 2001, 268, 2828-2837; 99mTc-Labeled Neuropeptide Y Analogues as Potential Tumor Imaging Agents, Michael Langer, Roberto La Bella, Elisa Garcia-Garayoa, Annette G. Beck-Sickinger, Bioconjugate Chem.
  • analogs of a targeting peptide can be used. These analogs include molecules that target a desired site receptor with avidity that is greater than or equal to the targeting peptide itself, as well as muteins, retropeptides and retro-inverso-peptides of the targeting peptide.
  • modifications which include substitutions, and/or deletions and/or additions of one or several amino acids, insofar that these modifications do not negatively alter the biological activity of the peptides described therein. These substitutions may be carried out by replacing one or more amino acids by their synonymous amino acids.
  • Synonymous amino acids within a group are defined as amino acids that have sufficiently similar physicochemical properties to allow substitution between members of a group in order to preserve the biological function of the molecule.
  • Synonymous amino acids as used herein include synthetic derivatives of these amino acids (such as for example the D-forms of amino acids and other synthetic derivatives), and may include, for example, those listed in the following Table.
  • amino acids are abbreviated interchangeably either by their three letter or single letter abbreviations, which are well known to the skilled artisan.
  • T or Thr stands for threonine
  • K or Lys stands for lysine
  • P or Pro stands for proline
  • R or Arg stands for arginine.
  • Deletions or insertions of amino acids may also be introduced into the defined sequences provided they do not alter the biological functions of said sequences. Preferentially such insertions or deletions should be limited to 1, 2, 3, 4 or 5 amino acids and should not remove or physically disturb or displace amino acids which are critical to the functional conformation.
  • Muteins of the peptides or polypeptides described herein may have a sequence homologous to the sequence disclosed in the present specification in which amino acid substitutions, deletions, or insertions are present at one or more amino acid positions. Muteins may have a biological activity that is at least 40%, preferably at least 50%, more preferably 60-70%, most preferably 80-90% of the peptides described herein.
  • Analogues of targeting peptides also include peptidomimetics or pseudopeptides incorporating changes to the amide bonds of the peptide backbone, including thioamides, methylene amines, and E-olefins. Also peptides based on the structure of a targeting peptide or its peptide analogues with amino acids replaced by N-substituted hydrazine carbonyl compounds (also known as aza amino acids) are included in the term analogues as used herein.
  • the targeting peptide may be attached to the linker via the N or C terminus or via attachment to the epsilon nitrogen of lysine, the gamma nitrogen or ornithine or the second carboxyl group of aspartic or glutamic acid.
  • the targeting peptide may be LHRH, insulin, oxytosin, somatostatin, NK-I, VIP, GRP, bombesin or any other hormone peptides known in the art, as well as analogs and derivatives thereof.
  • LHRH insulin, oxytosin, somatostatin, NK-I, VIP, GRP, bombesin or any other hormone peptides known in the art, as well as analogs and derivatives thereof.
  • Table 1 Table 2 and Table 2a list conjugates of the present invention complexed with Gd. The complexes are divided into two "classes” based on the structure of the HSA binding portion of the conjugates that were evaluated.
  • the first class includes conjugates of the formula: chelator-linker Ll- hydrophobic AA, complexed with Gd and the tested compounds in this class are listed in Table 1.
  • “Class 2” includes compounds having the formula: chelator-linkerLl-hydrophobic non-AA, complexed with Gd and the tested compounds in this class are listed in Tables 2 and 2a.
  • the distinction between the linkerLl and the hydrophobic AA or hydrophobic non-AA is somewhat formalistic as it was understood that all of these might contribute to binding of the constructs to HSA.
  • the tables also include and evaluation of the enhanced relaxivity of the constructs. The Examples provide details regarding the synthesis, screening and evaluation of representative compounds of the two classes.
  • Chelate class refers to the type of chelating function that is present in the compound:
  • Tl relaxivity by comparison with the standard chelating function. Not all cases provided enhanced Tl relaxivity, by comparison with their standard chelator analogs..
  • the parameter gi refers to the relaxation gain, which is the ratio of the Tl relaxivity of the substance in a solution of HSA called SeronormTM (commercially available from
  • Ny corned- Amersham to the relaxivity of the same compound in water at a specified concentration. Values close to 1 indicate that no binding to protein is occurring and that the compound is unsuitable. On the other hand, higher values (generally, about 3 and above) are more desirable because they highlight, in an MRI image, the bloodstream (intravascular compartment) due to the much higher Tl relaxivity (which is the source of the MRI signal) when bound to the HSA in the blood.
  • the parameter rl ⁇ is the Tl relaxivity of the substance in a solution of human serum albumin called SeronormTM (commercially available from Ny corned- Amersham).
  • the parameter rl w is the Tl relaxivity of the compound in water, where no protein is present.
  • the parameter Fb is the fraction of the compound bound to human serum albumin
  • HSA hypoxia-sensitive styrene-maleic anhydride
  • Fb fraction bound
  • Compounds in Class 1 and Class 2 were evaluated, and the results are as follows. The abbreviations used in these tables and throughout the application are known to those skilled in the art and/or set forth herein, particularly in the sections regarding chealators, linking groups and hydrophobic groups.
  • Table 2b shows the primary screening results for the Gd complexes of conjugates of the invention in Class 2 with the structure chelator-linker-hydrophobic carboxylic acid.
  • the listed conjugates of the general structure DO3A10CM-G-K-hydrophobic carboxylic acid were prepared and evaluated.
  • the "Group Number” refers to the groups as labeled in the chart above.
  • Conjugates DO3A10CM-D-a-B; DO3A10CM-G-a-B(Meg); DO3 Al OCM-G-L-B; CM-DOTMA-a-B; CE-DTP A-a-B; 7MP-DO3A-G-a-B and CM-DOTA-a-B, optionally conjugated to a second linker L2 and/or a targeting vector Q are preferred.
  • CM-DOTMA-a-B; CE-DTP A-a-B; 7MP-DO3A-G-a-B and CM-DOTA-a-B, optionally conjugated to a second linker L2 and/or a targeting vector Q are particularly preferred.
  • conjugates may be complexed with a metal, or a paramagnetic metal such as for example, Gd, or others described above or with a radionuclide from amongst those described above.
  • a metal or a paramagnetic metal such as for example, Gd, or others described above or with a radionuclide from amongst those described above.
  • Compounds of the invention with fractions bound to serum of at least 0.87 are particularly preferred. A higher fraction bound to serum will result in a longer lifetime in blood, thus providing a more persistant signal.
  • compound so f the invention with higher relaxivity values are also particularly preferred. Higher relaxivity value will result in more signal for a given compound after administration.
  • Table 6 in Example 8 shows fraction bound to serum and relaxivity data collected for representative compounds of the present invention compared to a reference compound, MS-325, whose structure is presented below.
  • the following compounds each provide higher bound fractions than the MS325 comparator: Gd-( 10CM-DO3 A)-G-G- W-(Nin-2,4,6- trimethylbenzenesulfonyl)-O " Meg; Gd-(10CM-DO3A)-aaB a; Gd-(10CM-DO3A)-aak a,b; Gd-(10CM-DO3 A)-DDk a; Gd-(10CM-DO3 A)-EEB a,b; Gd-(10CM-DO3A)-Pak a,c; Gd- (10CM-DO3A)-Pak a,b,c; Gd-(10CM-DO3A)-Dak a,b meg; Gd-(10CM-DO3 A)-DDj a,c; Gd-(10CM-DO3 A)-DDX c; Gd-(10CM-DO3 A)-PaJ
  • the conjugates of the present invention are useful for to detect and treat various medical conditions.
  • the conjugates of the present invention can be employed for diagnostic imaging.
  • the conjugates bind a blood protein such as HSA they are useful for prolonged imaging of the intravascular space, or simply the vasculature, because of their ability to be confined to that region for longer periods of time.
  • compounds of the present invention are useful for imaging the vascular space by, for example, MRI or nuclear medicine imaging, depending on the type of metal complexed with the chelator.
  • Methods of the invention include the steps of administering to a patient a pharmaceutical preparation that includes a conjugate of the invention complexed with a metal or a radionuclide and imaging the compound after administration to the patient.
  • the imaging step includes magnetic resonance imaging, or nuclear imaging.
  • the administering step may include, e.g., inhalation, transdermal absorption, intramuscular injection, subcutaneous injection, intravenous injection, intraperitoneal injection, intraarterial injection or parenteral administration.
  • conjugates of the present invention which are useful for MRI (e.g. those that are complexed with a paramagnetic metal ion) are useful as CVMRA agents in therapeutic and diagnostic applications.
  • the invention further includes novel methods for preparation of such agents.
  • the conjugates of the present invention useful for MRI exhibit superior relaxivity.
  • the term rls is defined as the relaxivity of the compound measured in SeronormTM (a commercially available preparation of human serum albumin) at 20 MHz in mM ' V 1 .
  • the term rl w is defined as the relaxivity of the compound measured in water at 20 MHz in mM ' V 1 .
  • Agents of the invention were shown to have superior properties using the methods of parallel synthesis and mass primary screening of candidate compounds, as described in the Examples.
  • Fb is the fraction of the construct bound to protein in blood
  • rls is the Tl relaxivity of the test compound measured in Seronorm solution at a Standard concentration of the test compound
  • gi is the Standard relaxation gain or the relaxation enhancement defined as the ratio rls/rlw under a defined standard of concentration and temperature.
  • the first term of the addition equation, (l-Fb)*rl w *Cbiood, gives the contribution of the unbound compound to the total signal in blood.
  • the second term, gi*rl w *F b *Cbiood, which contains the multiplier gi, gives the contribution of the HSA-bound compound to the total signal in blood. It can be seen that when F b is high, most of the signal in the blood is due to the bound fraction of the compound. This signal is magnified by the factor of gi (the relaxation gain). This parameter is also referred to as the enhanced relaxivity or relaxation enhancement which is realized upon binding of the contrast agent to the macromolecule.
  • This ratio is time dependent and, except in the period immediately after administration, is a likely underestimate (the concentration of the non-protein binding compounds in blood is reduced versus their protein binding congeners with time).
  • the compound is first administered, and clearance of unbound compound from blood is then allowed to occur before the vascular space is imaged.
  • this process is not always strictly necessary.
  • the administrator In the case where imaging is conducted after a waiting period, the administrator must account for the fraction of compound that may migrate to the extravascular space, which also contains HSA.
  • the concentration of the compound in the extravascular space were to be equal to that in the intravascular space (an unlikely circumstance), both areas would provide signal in the MRI, and the ability to distinguish the extravascular space from the intravascular space might thus be diminished. However, even in such a case, signal intensity in the intravascular space would still be greater in most instances. This is because the concentration of HSA in the extravascular spaces (which has been measured) is generally between 10-16 g/L, except in the gut, where it is 25-30 g/L. These values should be compared with the value of ⁇ 25g/L in the vascular space (which includes both the volume of plasma and the other blood components, e.g., erythrocytes and white cells). Therefore, independent of the fractional distribution of the compound in the vascular space versus the extravascular space, the present invention provides an advantage for signal strength in the blood due to the higher concentration of HSA in blood, except in the case of the gut.
  • Imaging according to the present invention should preferably be conducted at time point early enough to allow binding of the constructs of the present invention to intravascular HSA (on the order of one circulation time, i.e. 1-2 min) and clearance of unbound compound to the extravascular space (Ty 2 ⁇ 16 min). Both of these rates of clearance are much faster than the rate of clearance of intravascular HSA (with or without the molecule bound) to the extravascular space (4%/h).
  • diagnostic imaging methods can be employed to provide morphological imaging of the vasculature for detection of a variety of conditions, including, for example, vascular abnormalities, stenosis or even sites of angiogenesis that can be characterized.
  • the morphology and permeability of tumor vasculature can be similarly assessed in accordance with the present invention.
  • the constructs of the present invention are applicable to MRI; they are also applicable to other forms of imaging such, as e.g., ultrasound imaging, wherein metal chelators facilitate the process, and/or light imaging; and additionally to other diagnostic and therapeutic applications that may be contemplated by those skilled in the art.
  • the conjugates of the present invention are applicable to other modes of diagnostic imaging as well as to therapeutic applications.
  • the conjugates of the invention are complexed with metals having different properties than, for example, paramagnetic metals
  • other applications can be contemplated by those of ordinary skill in the art.
  • the HSA- binding portion of the molecule may be used to prolong the residence time in blood of the constructs such as, e.g., targeted pharmaceuticals, including therapeutics and diagnostics.
  • radiometals may be chelated by the chelating function and a construct that utilizes the HSA binding sequence and the chelator bound to a targeting function which directs the construct to tumor sites or to other diseased tissues. The radiometal may also be useful for diagnosis or radiotherapy.
  • the conjugates of the invention bind to a blood protein such as HAS, the blood residence time is prolonged, allowing more opportunity for binding of the radiopharmaceutical to the target site.
  • the present invention also contemplates the delivery of other compounds such as, e.g., chemotherapeutics.
  • a conjugate of the invention which includes a targeting vector that targets a specific type of tumor can be linked to a chemotherapeutic.
  • the conjugate by virtue of the fact that it binds to the intravascular HSA, can prolong the blood residence time of the construct.
  • the constructs of the present invention may also be used to target genetic material to specific cells.
  • the conjugates of the invention may further include genetic material and may be used to localize genetic material to cells or tissue containing the desired target.
  • the genetic material may include nucleic acids, such as RNA or DNA, of either natural or synthetic origin, including recombinant RNA and DNA and antisense RNA and DNA.
  • Types of genetic material that may be used include, for example, genes carried on expression vectors such as plasmids, phagemids, cosmids, yeast artificial chromosomes (YACs) and defective or "helper" viruses, antigene nucleic acids, both single and double stranded RNA and DNA and analogs thereof, such as phosphorothioate and phosphorodithioate oligodeoxynucleotides. Additionally, the genetic material may be combined, for example, with lipids, proteins or other polymers.
  • Delivery vehicles for genetic material may include, for example, a virus particle, a retroviral or other gene therapy vector, a liposome, a complex of lipids (especially cationic lipids) and genetic material, a complex of dextran derivatives and genetic material, etc.
  • MRI Magnetic Resonance Imaging
  • MRI contrast agents including conjugates of the invention may be used in the same manner as conventional MRI contrast reagents.
  • target-containing tissue such as, for example, a site of angiogenesis
  • certain MR techniques and pulse sequences may be preferred to enhance the contrast of the site to the background blood and tissues.
  • These techniques include (but are not limited to), for example, black blood angiography sequences that seek to make blood dark, such as fast spin echo sequences (see, e.g., Alexander et al., Magnetic Resonance in Medicine, 40(2): 298-310 (1998)) and flow-spoiled gradient echo sequences (see, e.g., Edelman et al., Radiology, 177(1): 45-50 (1990)).
  • the labeled reagent may be administered to the patient in the form of an injectable composition.
  • the method of administering the MRI contrast agent is preferably parenterally, meaning intravenously, intraarterially, intrathecally, interstitially, or intracavitarilly.
  • intravenous or intraarterial administration is preferred.
  • the subject will receive a dosage of contrast agent sufficient to enhance the MR signal at the target (e.g. , a site of angiogenesis) at least 10%.
  • the target e.g. , a site of angiogenesis
  • the patient is scanned with a MRI machine to determine the location of any sites containing the target.
  • a cytotoxic or therapeutic agent can be immediately administered, if necessary, and the patient can be subsequently scanned to determine the location of any targeted sites.
  • the present invention may also be useful in the field of nuclear medicine.
  • Conjugates of the present invention may be complexed with a radioactive metal or other radionuclide appropriate for scintigraphy, SPECT or PET imaging or with a radionuclide appropriate for radiotherapy.
  • a conjugate of the present invention may be complexed with one of the various positron emitting metal ions, such as 51 Mn, 52 Fe, 60 Cu, 68 Ga, 72 As, 94m Tc, or 110 In.
  • the constructs of the present invention can also be labeled by halogenation using radionuclides, such as 18 F, 124 I, 125 I, 131 I, 123 I, 77 Br, and 76 Br.
  • Preferred metal radionuclides for scintigraphy or radiotherapy include 99m Tc, 51 Cr, 67 Ga, 68 Ga, 47 Sc, 51 Cr, 167 Tm, 141 Ce, 111 In, 168 Yb, 175 Yb, 140 La, 90 Y, 88 Y, 153 Sm, 166 Ho, 165 Dy, 166 Dy, 62 Cu, 64 Cu, 67 Cu, 97 Ru, 103 Ru, 186 Re, 188 Re, 203 Pb, 211 Bi, 212 Bi, 213 Bi, 214 Bi, 105 Rh, 109 Pd, 117m Sn, 149 Pm, 161 Tb, 177 Lu, 198 Au and 199 Au.
  • the preferred radionuclides include 64 Cu, 67 Ga, 68 Ga, 99m Tc, and 111 In.
  • the preferred radionuclides include 64 Cu, 90 Y, 105 Rh, 111 In, 117m Sn, 149 Pm, 153 Sm, 161 Tb, 166 Dy, 166 Ho, 175 Yb, 177 Lu, 1867188 Re, and 199 Au.
  • a radioactive lanthanide such as, for example, 177 Lu, 90 Y, 153 Sm,
  • 111 In, or 166 Ho is used with DOTA or D03A in radiopharmaceutical conjugates of the invention.
  • 99m Tc is preferred for diagnostic applications because of its low cost, availability, imaging properties, and high specific activity.
  • the nuclear and radioactive properties of 99m Tc make this isotope an ideal scintigraphic imaging agent.
  • This isotope has a single photon energy of 140 keV and a radioactive half- life of about 6 hours, and is readily available from a 99 Mo- 99m Tc generator.
  • Other appropriate radionuclides for preferred embodiments of the present invention will be easily determined by one of ordinary skill in the art.
  • the metal radionuclides may be chelated by any of the chelating moieties discussed herein, supra.
  • One skilled in the art can select an appropriate chelator for a desired radionuclide.
  • Radioactive technetium are particularly useful for diagnostic imaging and complexes of radioactive rhenium are particularly useful for radiotherapy.
  • the technetium complex preferably a salt of 99m Tc pertechnetate
  • the technetium complex is reacted with the reagent in the presence of a reducing agent.
  • Preferred reducing agents are dithionite, stannous and ferrous ions; the most preferred reducing agent is stannous chloride.
  • Means for preparing such complexes are conveniently provided in a kit form comprising a sealed vial containing a predetermined quantity of a reagent of the invention to be labeled and a sufficient amount of reducing agent to label the reagent with 99m Tc.
  • the complex may be formed by reacting a conjugate of the invention with a pre-formed labile complex of technetium and another compound known as a transfer ligand. This process is known as ligand exchange and is well known to those skilled in the art.
  • the labile complex may be formed using such transfer ligands as tartrate, citrate, gluconate or mannitol, for example.
  • the 99m Tc pertechnetate salts useful with the present invention are included the alkali metal salts such as the sodium salt, or ammonium salts or lower alkyl ammonium salts.
  • Preparation of the complexes of the present invention where the metal is radioactive rhenium may be accomplished using as starting materials rhenium compounds wherein the metal is in the +5 or +7 oxidation state.
  • rhenium compounds wherein the metal is in the +5 or +7 oxidation state.
  • Examples of compounds in which rhenium is in the Re(VII) state are NH 4 ReO 4 or KReO 4 .
  • Re(V) is available as, for example,
  • Bu is n-butyl
  • Radioactively-labeled scintigraphic imaging agents provided by the present invention must contain a suitable amount of radioactivity. In forming 99m Tc complexes, it is generally preferred to form radioactive complexes in solutions containing radioactivity at concentrations of from about 0.01 millicurie (mCi) to 100 mCi per mL.
  • the unit dose to be administered has a radioactivity of about 0.01 mCi to about 100 mCi, preferably 1 mCi to 20 mCi.
  • the solution to be injected at unit dosage is from about 0.01 mL to about 10 mL.
  • a PET camera or a gamma camera calibrated for the gamma ray energy of the nuclide incorporated in the imaging agent is used to image areas of uptake of the agent and quantify the amount of radioactivity present in the site.
  • Imaging of the site in vivo can take place in a matter of a few minutes. However, imaging can take place, if desired, in hours or even longer, after the radiolabeled peptide is injected into a patient. In most instances, a sufficient amount of the administered dose will accumulate in the area to be imaged within about 0.1 of an hour to permit the taking of scintiphotos.
  • the radiotherapeutic and radiodiagnostic compositions of the invention can include physiologically acceptable buffers, and can require radiation stabilizers to prevent radiolytic damage to the compound prior to injection.
  • Radiation stabilizers are known to those skilled in the art, and may include, for example, para-aminobenzoic acid, ascorbic acid, gentistic acid and the like.
  • the present invention also contemplates a single, or multi-vial kit that contains all of the components needed to prepare the radiopharmaceuticals of this invention, other than the radionuclide.
  • a single-vial kit preferably contains a conjugate of the invention, a source of a stannous salt (if reduction is required, e.g., when using technetium), or other pharmaceutically acceptable reducing agent, and is appropriately buffered with pharmaceutically acceptable acid or base to adjust the pH to a value of about 3 to about 9.
  • the quantity and type of reducing agent used would depend highly on the nature of the exchange complex to be formed. The proper conditions are well known to those who are skilled in the art. It is preferred that the kit contents be in lyophilized form.
  • Such a single vial kit may optionally contain labile or exchange ligands such as glucoheptonate, gluconate, mannitol, malate, citric or tartaric acid and can also contain reaction modifiers such as diethylenetriamine-pentaacetic acid (DPTA), ethylenediamine tetraacetic acid (EDTA), or ⁇ -, ⁇ -, or ⁇ -cyclodextrin that serve to improve the radiochemical purity and stability of the final product.
  • the kit may also contain stabilizers, bulking agents such as mannitol, that are designed to aid in the freeze-drying process, and other additives known to those skilled in the art.
  • a multi-vial kit preferably contains the same general components but employs more than one vial in reconstituting the radiopharmaceutical.
  • one vial may contain all of the ingredients that are required to form a labile Tc(V) complex on addition of pertechnetate (e.g., the stannous source or other reducing agent).
  • pertechnetate e.g., the stannous source or other reducing agent.
  • Pertechnetate is added to this vial, and after waiting an appropriate period of time, the contents of this vial are added to a second vial that contains the conjugate of the invention, as well as buffers appropriate to adjust the pH to its optimal value. After a reaction time of about 5 to 60 minutes, the complexes of the present invention are formed. It is advantageous that the contents of both vials of this multi-vial kit be lyophilized.
  • reaction modifiers, exchange ligands, stabilizers, bulking agents, etc. may be present in either or both vials.
  • Conjugates of the invention may be administered by any convenient route customary for diagnostic or therapeutic agents; e.g. , parenterally, enterally or intranasaly, and preferably by infusion or bolus injection, or by depot or slow release formulation.
  • the composition may be formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings.
  • compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
  • compositions include, but are not limited to, sterile water, saline solution, buffered saline (including buffers like phosphate or acetate), alcohol, vegetable oils, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, paraffin, etc.
  • the composition may also include a solubilizing agent and a local anesthetic such as, e.g., lidocaine to ease pain at the site of the injection; and further may include preservatives, stabilizers, wetting agents, emulsif ⁇ ers, salts, lubricants, etc. as long as they do not react deleteriously with the active compounds.
  • the composition may comprise conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for parenteral, enteral or intranasal application which do not deleteriously react with the active compounds.
  • the ingredients will be supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as, e.g. , an ampoule or sachette indicating the quantity of active agent in activity units.
  • the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade "water for injection" or saline.
  • an ampoule of sterile water for injection or saline may be provided so that the ingredients may be mixed prior to administration.
  • reaction mixture was then evaporated on a rotary evaporator to remove dichloromethane and the residue was taken-up in ethyl acetate (200 mL).
  • the organic layer was washed sequentially with dilute HCl (0.05 N, 2 x 200 mL), NaHCO 3 (5%, 2 x 200 mL) and water (2 x 200 mL).
  • the organic layer was washed sequentially with dilute HCl (0.05 N, 2 x 150 mL), aqueous sodium carbonate (5%, 2 x 150 mL) and water (150 mL). The organic layer was then separated, dried (Na 2 SO 4 ), filtered and evaporated on a rotary evaporator to provide DO3A10CM-(l,4,7-tris- t-butyl ester)-glycyl-tr ⁇ /?5-4-aminomethylcyclohexanecarbonyl-L-biphenyl-alanine benzyl ester (9) (19.80 g, 90.40 % yield) as an off-white foam.
  • TLC Rf 0.42 (silica gel, 5% methanol in dichloromethane)
  • HPLC t R 13.44 min; Column: YMC-C18, 4.6 mm x 250 mm, S-IOP ⁇ m, 12 ⁇ A; Eluents: A: H 2 O (0.1% TFA), B: CH 3 CN (0.1% TFA); Elution: Initial condition: 45% B, then linear gradient to 95% B over 25 min; Flow rate: 1.0 mL/min, Detection: UV @ 230 nm.
  • Pd-C catalyst 5 g, Degussa type, 10%
  • DOTA(tri-t-butyl ester)-Gly-a-Bip-OH (10) (17.0 g, 17.15 mmol) was treated with trifluoroacetic acid:water:phenol:triisopropylsilane (88:5:5:2) (Reagent B) (300 rnL) with stirring for 4 h under a nitrogen atmosphere. After removal of the solvent the paste thus obtained was triturated with ether (50 mL), and the mixture was centrifuged and the ether layer was decanted. This process was repeated twice to completely remove phenol. The resulting solid was dried to provide 13.5 g (95.5% yield) of DO3A10CM-Gly-a-Bip-OH as an off-white solid.
  • the material was purified by anion exchange (Bio-Rad, AG1-X2 resin, 100-200 mesh, formate form) by elution of the column with 4 L each of 0.1M, 0.2M and 0.3M aqueous formic acid followed by elution with 0.4M formic acid ( ⁇ 12 L) whereupon the desired compound was eluted (eluant monitored by UV detector at 230 nm).
  • anion exchange Bio-Rad, AG1-X2 resin, 100-200 mesh, formate form
  • the product was purified in three portions by reverse phase HPLC as follows: A reversed phase C18 preparative column (Rainin, 41.4 x 250 mm, 8 ⁇ , 6 ⁇ A), was equilibrated with water (no TFA). The sample was applied to the column, and the column was eluted with water-acetonitrile (8:2 v/v), and then a linear gradient elution from 20% acetonitrile to 50% acetonitrile in 60 min was performed. The fractions were analyzed on an analytical reversed phase Cl 8 column, and the fractions containing the product in >99.5% purity were pooled and freeze-dried to provide the Gd- DO3A10CM-G-a-B-OH as a colorless fluffy solid.
  • the gadolinium chelate was converted into its meglumine salt by passing it through a cation exchange column (Bio-Rad, AG 50W- X8 resin, sulfonic acid, meglumine form).
  • the fractions containing the product (detected by UV at 230 nm) were collected and lyophilized to provide Gd-DO3 Al OCM-GIy cyl-trans -4- aminomethylcyclohexanecarboxyl-L-Biphenylalanine (Gd-DO3A10CM-Gly-a-Bip- OH » Meg) (12) as a colorless fluffy solid.
  • the entire sample purified in this manner provided 5.49 g (82% yield) of compound 12.
  • the product (12) was then screened using an IBM PC20-minispec relaxometer for relaxivity in water and SeronormTM, and for HSA binding by the centrifree micro partition method.
  • the SeronormTM/Gd-chelate-peptide and water-peptide mixtures were warmed for not longer than 20 min at 38 0 C prior to the relaxivity measurement, so that the sample was at the same temperature as the PC20 relaxometer (also referred to as the NMR).
  • the NMR signal Prior to measurement of sample Tl 's (in triplicate), the NMR signal was optimized (SOP 830-210 IBM PC/20 Multispec Relaxometer 12/18/97). Tl is also measured for a pure water sample and a fresh aqueous SeronormTM sample. The average Tl for each SeronormTM/Gd-conjugate sample was used in the equation below to calculate relaxivity/mM in SeronormTM rls.
  • SeronormTM/Gd-conjugate mixture was removed from the NMR tubes and 1 mL was placed in a Centrifree Micropartition Device (Millipore, Beverly, MA). The device was centrifuged at 500xg for 45 min in a fixed angle rotor (Beckman Model J2-21M, JA-20 rotor) and 0.5 mL was removed from below the filter for Gd measurement by ICP. The [Gd] in 0.5 mL of the uncentrifuged SeronormTM/peptide mixture was measured as a control. The fraction of Gd- chelate -peptide bound was calculated using the equation below:
  • Fraction Bound (F b ) [Gd] control - [Gd]SeronormTM/Gd-conjugate) / [Gd] control
  • F b 0 05 mmol/Kg dose, , e - 0 15 mM, g - 0 12 mM F b s -F b r -F b m refer to the fraction bound in human, rabbit and monkey serum albumin respectively
  • the compound was optimized using ligand diversity, i.e., the d and / isomers of the compound were prepared as their meglumine salts.
  • the d,l mixture was constituted from the individual compounds.
  • HPLC purification gave the pure ligand which was converted to the chelate and purified by prep HPLC.
  • Table 4 The results of primary screening for the new compounds prepared using the strategy of ligand diversity are shown in Table 4.
  • Gd-DO3A10CM-G-a-(d,/)-Bip displayed g l5 rls and rl w values essentially identical to those found for either the d- or 1-isomer alone. This indicates that the binding of this compound does not appear to be chiroselective. Also indicated is that methods for synthesis of racemic amino acids can be employed to prepare the ⁇ i,/-Bip, thus reducing the cost of a Bip-based compound.
  • benzyl bromide (3.602 g, 21.06 mmol, Aldrich) was added via syringe dropwise over a 1 minute period. The mixture was stirred rapidly for 18h at ambient temperature using a vent needle in the septum cap to relieve pressure from evolved carbon dioxide. TLC analysis was performed using both dichloromethane/ethylacetate 9/1 v/v and dichloromethane/methanol 9/1 v/v; this showed that the reaction was essentially complete.
  • the organic layer was separated and the aqueous layer was extracted with 50 mL of dichloromethane.
  • the dichloromethane layers were combined, volatiles were removed and the residue was redissolved in 200 mL of ethyl acetate.
  • the organic phase was washed with water (4 x 200 mL) to remove tetrabutylammonium salts (visible by development in the iodine chamber) and then dried (sat. brine, then MgSO 4 ).
  • the volatiles were removed to give a residue which was applied (as a solution in 60 mL of dichloromethane) to a 35 cm high x 54 mm wide column of Merck 9385 silica gel packed in dichloromethane.
  • the column was eluted with dichloromethane (1300 mL), dichloromethane-ethyl acetate 95/5 v/v (1000 mL), dichloromethane-ethyl acetate 90/10 v/v (1000 mL) and then dichloromethane-ethyl acetate 85/15 v/v for the remainder of the elution.
  • the fraction size was 50 mL and the desired compound was obtained in 48-84.
  • Fractions 56-74 were taken as the heart cut. The volatiles were removed to provide a foam which was triturated with ether-hexanes to initiate precipitation.
  • N ⁇ -(9-Fluorenylmethoxycarbonyl)-N ⁇ -(Boc)-lysine benzyl ester (10.5 g, 18.79 mmol) was dissolved in CF ⁇ Cb/anisole 45/5 v/v (50 mL) with stirring. To this was added trifluoroacetic acid (50 mL) and the mixture was stirred 1.5 h at ambient temperature. The volatiles were removed on the rotary evaporator and then by pumping at 0.1 mm Hg for 0.5 h. Then anhydrous diethyl ether (250 mL) was added, the mixture was agitated and the side of the flask was scratched with the oil using a glass rod.
  • the HCl salt (which we have prepared on a small scale previously) is a known compound: (Matsoukas, John M.; Hondrelis, John; Agelis, George; Barlos, Kleomenis; Gatos, Dimitrios; Ganter, Renee; Moore, Diana; Moore, Graham J. Novel Synthesis of Cyclic Amide-Linked Analogs of Angiotensins II and III. J. Med. Chem. (1994), 37(18), 2958-69.)
  • the deep yellow mother liquor was examined by TLC employing E. Merck silica gel plates and dichloromethane-ethyl acetate 9/1 v/v as the developing solvent. This showed the presence of the major product (same R/ as the collected solid) and a higher R/ impurity later identified as 9-fluorenone; the starting acid was consumed.
  • the mother liquor was processed by washing with 0.5M NaHSO 4 (200 mL), saturated NaHCO 3 (2 x 200 mL), 5% Na 2 CO 3 (2 x 200 mL), 0.5M NaHSO 4 (200 mL), saturated brine (200 mL) and dried (MgSO 4 ). Removal of the drying agent and evaporation gave 1.11 g of a yellow solid.
  • Mass Spectrum ESI+ 783.2 (M+Cs) + (Cs from residual CsI used by the vendor for machine calibration), 689.3 (M+K) + , 674.2 (M+1+Na) + , 673.3 (M+Na) + , 652.3 (M+l+H) + , 651.3 (M+H) + , 567.2 (unassigned), 489.2 (unassigned), 456.2, 455.2 (M - 9-fluorene-methyleneoxy radical) + , 441 (estimated, not delineated by the operator, unassigned), 429.3 (M-Fmoc+2H) + .
  • the column was then eluted with water-acetonitrile solvent mixture (both containing 0.1 % TFA), starting a linear gradient from 10% acetonitrile to 80% acetonitrile in 80 min.
  • the fractions were analyzed on an analytical reversed phase Cl 8 column, and those containing the pure product were pooled and freeze-dried to provide DO3A10CM-glycinyl-N ⁇ -(9-fluorenecarbonyl)-lysine (7), (4.7 g, yield, 58 %) as a colorless fluffy solid.
  • the solution containing the product was loaded onto a reversed phase Cl 8 preparative column (Rainin, 41.4 x 250 mm, 8 ⁇ , 60 A) which was equilibrated with water.
  • the column was then eluted with 10% aqueous acetonitrile and then a linear gradient from 10% acetonitrile to 80% acetonitrile in 80 min was run.
  • the fractions were analyzed on an analytical reversed phase Cl 8 column and the fractions containing the product in > 99.5% purity were pooled and freeze-dried to provide (Gd-DOTA)-Glycinyl-N ⁇ -(9-fluorenecarbonyl)-lysine, meglumine salt (8) as a colorless fluffy solid.
  • the compound was prepared as follows: a suspension of L-malic acid (1) (Aldrich Chemical Company, 120.0 g, 0.895 mol) in 2,2-dimethoxyethane (1200 mL) was heated under reflux with stirring until all of the material went into solution (2 h). The solution was concentrated under reduced pressure using a rotary evaporator and the resulting oil was dissolved in about 1000 mL of 7:3 hot hexane/isopropyl ether and filtered. The solid that separated from the cooled filtrate was collected and washed with 7:3 hexane/isopropyl ether (2 x 50 mL). The combined filtrates were again concentrated to about 250 mL, yielding more of the product L-malic acid acetonide (2) as colorless crystalline solid. Yield: 131.O g (84%).
  • the reaction mixture was poured into crushed ice (250 g) and the organic layer was washed with ice-cold 2N HCl (2 x 100 niL), ice-cold water (2 x 100 rnL) and dried with anhydrous calcium sulfate in a freezer before its use.
  • the t-butyl-3- benzyloxycarbonyl-2-O-[trifluoromethanesulfonyl) propionate (8) was kept as a solution in dichloromethane.
  • the triflate 8 was found to be very unstable at room temperature and therefore was used immediately as a solution in dichloromethane as soon as it was prepared.
  • TACD 1,4,7, 10-tetraazacyclododecane
  • the reaction mixture was diluted with 200 mL of ethyl acetate and filtered through a bed of celite and washed with 1 : 1 dichloromethane/ethyl acetate (2 x 50 mL).
  • the combined organic layers were washed with water (3 x 200 mL) and dried (Na 2 SO 4 ). Removal of the solvent yielded the crude monoalkylated TCAD (10) as a pale brown paste. This was used directly in the next step in order to avoid decomposition by adventitious moisture. Yield: 28.57 g (87.8%, crude).
  • trans-4-Aminomethylcyclohexanecarboxylic acid (17) (31.0 g, 0.197 mol) was added to a solution of sodium carbonate (23.32 g, 0.22 mol) in water (350 mL) with stirring. Upon dissolution of the solid the solution was cooled in an ice bath and a solution of di-t- butyldicarbonate (48.0 g, 0.22 mol in 350 mL of THF). was added dropwise with rapid stirring. After the addition, the ice bath was removed and the solution was allowed to stir at RT for 4 h.
  • Boc-biphenylalanine (16a) (13.93 g, 0.03 mol) was added to a stirred solution of phenol (2.5 g) in TFA (50 mL). The mixture was stirred for 30 min at RT and then the TFA was removed under reduced pressure. The residue was triturated with 100 mL of anhydrous ether and the solid that separated was filtered and washed with anhydrous ether (2 x 25 niL). The solid, L-biphenylalanine benzyl ester » TFA (16b) was dried under vacuum ( ⁇
  • HPLC t R 8.72 min; Column: YMC C18-ODS 4.6 mm x 250 mm; S-IOP ⁇ particle; 12 ⁇ A pore; Eluents: A: H 2 O (0.1% TFA), B: CH 3 CN (0.1% TFA); Elution: Initial condition: 45% B, then linear gradient 45-95% B over 25 min; Flow rate: 1 mL/min;
  • CM-DOTMA acid (14) (19.45 g, 0.0216 mol) was dissolved in anhydrous dichloromethane (100 mL) and cooled to -10° C under a nitrogen atmosphere.
  • HATU (9.12 g, 0.024 mol) was added followed by diisopropylethylamine (3.1 g, 0.024 mol) and the mixture was stirred for 10 min at -10° C.
  • a hydrogenation bottle was charged with a solution of fully protected CM- DOTMA-a-B (21) (19.0 g, 0.016 mol) in CH 3 OH:Water 9:lv/v , (100 mL) and Pd/C (10% Pd content, 50% water by weight, 3.8 g).
  • the vessel was evacuated and refilled with hydrogen several times. Then the hydrogenation was conducted for 4 h at 50 psi at ambient temperature. .
  • the catalyst was filtered off through a bed of CeliteTM and washed with CH 3 OHiH 2 O 9:1 v/v (2 x 20 mL).
  • the column was eluted with 5% CH3CN/H2O (both containing 0.05% TFA) for 5 min., 5-20% in 10 min., and finally 20-50% in 60 min.
  • the desired fractions were collected and lyophilized. This gave 25 mg of the product with purity of 89% (HPLC).
  • the material was then purified by DEAE-sephadex resin.
  • the compound in 5 mM triethylammonium bicarbonate solution was loaded onto the reconditioned resin and it was eluted with a gradient of aqueous triethylammonium bicarbonate solution (5 mM-800 mM, ) total volume of 360 mL).
  • the desired fractions were combined and lyophilized to give 25 mg of (5) (97% pure as determined by analytical HPLC), as a white fluffy solid.
  • N-methyl-D-glucamine 35 g in 100 mL of H 2 O was passed through the column; then the column was washed with a large excess of H 2 O until the conductivity was ⁇ 0.9 ⁇ S/cm
  • the Gd-chelate (5) 25 mg in 20 mL of H 2 O was applied to the column and the eluate was monitored by UV at 230 nm, employing a Knauer Variable Wavelength UV Detection using a 0.4 mm flow cell. The fractions containing the compound were combined and lyophilized. A total of 25 mg of the compound 6 was obtained as a white fluffy solid.
  • the compound was prepared as follows: To a solution of 1 (7.0 g, 11.75 mmol) in CH 2 Cl 2 (120 mL) was added 2 (11.6 g, 15.3 mmol,) (L-Glutamic acid and L-lysine as useful building blocks for the preparation of bifunctional DTP A- like ligands.
  • Anelli Pier Lucio; Fedeli, Franco; Gazzotti, Ornella; Lattuada, Luciano; Lux, Giovanna; Rebasti, Fabrizio.
  • the column was eluted at 50 mL/min and a gradient elution (0% CH 3 CN:H 2 O v/v over 10 min., then ramp from 0-20 % over 10 min., then ramp to 75 % over 30 min) was employed. Pure product-containing fractions were lyophilized to give compound 6 as a white fluffy solid (4.8 g, 81.3% yield).
  • a glass column was packed with 100 mL of BioRad AG50W-X8 ion exchange resin and the outlet was connected to a conductivity meter.
  • the column was washed with 400 mL of 3N HCl, rinsed with H 2 O to pH 7, washed with 400 mL of 2N NaOH, rinsed with H 2 O to pH 7, then washed with 400 mL of 3N HCl and then rinsed with H 2 O until pH 7.
  • the column was washed with a solution of 70 g of N-methyl-D-glucamine in 300 mL of H 2 O followed by distilled deionized water until the conductivity was ⁇ 0.9 ⁇ S/cm.
  • the Gd- chelate 6 (3.6 g, 3.7 mmol) in 400 mL of H 2 O/CH 3 CN 80/20 (v/v) was applied to the column and the eluate was monitored by UV at 230 nm. The product-containing fractions were combined and lyophilized. A total of 5.3 g (88% yield) of the desired meglumine salt 7 was obtained as a white fluffy solid.
  • the volatiles were removed on a rotary evaporator and the residue was treated with water (150 mL) and the entirety of the contents of the flask was extracted with ethyl acetate (250 mL). The ethyl acetate layer was separated and washed sequentially with aqueous NaHCO 3 solution (200 mL), water (200 mL) and dried (Na 2 SO 4 ). The volatiles were removed and the oil obtained was chromatographed over silica gel.
  • Lysine 3.2 (s, 2H, CH 2 -N(FmOc)) 4.17-4.82 (m, 4H, CH 2 -O-CO) CH-CH 2 , and CH-CO).
  • Boc-Lys(Fmoc)-benzyl ester (1) (14.5 g, 26.0 mmol) was dissolved in CH 2 Cl 2 (80 mL) and anisole (6 mL). The mixture was stirred for 5 min. TFA (80 mL) was added and the mixture was stirred for 1.5 h at ambient temperature. HPLC analysis indicated complete reaction. The volatiles were removed, and ether (200 mL) was added. A solid formed after 15 min of stirring and was collected after 3h. This was dried overnight to give pure Lys(Fmoc)-OBn»TFA (14.4 g, 97% yield).
  • a cation exchange column (Bio-Rad AG50W-X8 resin in the meglumine form) was prepared. A dilute aqueous solution of the free acid 8a was applied to the column and the column was washed with water and fractions were collected. The fractions containing the desired product, compound 8b, were collected, combined and lyophilized to give the pure product (3.95 g) as a colorless fluffy solid. The yield from compound 7 was 91%. HPLC analysis indicated no free gadolinium ion in the final product.
  • a higher fraction bound to serum will result in a longer lifetime in blood, thus providing a more persistant signal.
  • Higher relaxivity value will result in more signal for a given compound after administration.
  • Chelator type refers to the chelator used in the compound.
  • Relaxation gain determined refers to the relaxation gain, which is the ratio of the Tl relaxivity of the substance in a solution of HSA called SeronormTM (commercially available from Nycomed-Amersham) to the relaxivity of the same compound in water at a specified concentration. Values close to 1 indicate that no binding to protein is occurring and that the compound is unsuitable. On the other hand, higher values are more desirable because they highlight, in an MRI image, the bloodstream (intravascular compartment) due to the much higher Tl relaxivity (which is the source of the MRI signal) when bound to the HSA in the blood.
  • the parameter rls is the Tl relaxivity of the substance in a solution of human serum albumin called SeronormTM (commercially available from Nycomed- Amersham)
  • the parameter rl w is the Tl relaxivity of the compound in water, where no protein is present.
  • the parameter Fb is the fraction of the compound bound to human serum albumin (HSA) under standard conditions of defined concentration and temperature. A value of 0.90 indicates that 90% of the molecules of the test compound are bound to HSA. A higher fraction bound (Fb) means that more of the compound should provide the higher signal characteristic of the bound molecule due to immobilization of the chelating function, or significant slowing of the chelating function upon binding of the chelate-HSA binding ligand to the HSA.
  • Gd-(TE-DOTA)-aB Meg provides a relaxivity (ri) in
  • SeronormTM HSA
  • n Seronorm 36.57
  • the following compounds each provide higher bound fractions than the MS325 comparator: Gd-(10CM-DO3 A)-G-G- W-(Nin-2,4,6- trimethylbenzenesulfonyl)-Cr Meg; Gd-(10CM-DO3A)-aaB Na;Gd-(10CM-DO3A)-aak Meg; Gd-(10CM-DO3 A)-DDk Meg; Gd-(10CM-DO3 A)-EEB Meg; Gd-(10CM-DO3A)-Pak Na; Gd-(10CM-DO3A)-Pak Meg; Gd-(10CM-DO3A)-Dak Meg; Gd-(10CM-DO3 A)-DDj Na; Gd-(10CM-DO3 A)-DDX Na; Gd-(10CM-DO3 A)-PaJ Na; Gd-(10CM-DO3A)-aaX Na;
  • m L- (N in -2,4,6-trimethybenzensulphonyl)-tryptophan
  • n L-(N in -2,4,6- triisopropybenzensulphonyl)-tryptophan
  • o L-(N in -2,4,6-trimethylbenzoyl)-tryptophan
  • p O-benzyl-L-threonine
  • X L-2-naphthylalanine.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Organic Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Radiology & Medical Imaging (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

Novel constructs and agents useful for diagnostic and therapeutic applications have been developed. The constructs comprise a chelator, a first linker and either a hydrophobic amino acid or a 'hydrophobe' (hydrophobic non-amino acid), optionally attached to a second linker and a targeting vector. The structure of such compounds enables prolonged residence time in the blood, and therefore increases their usefulness in imaging and treating disease. The present invention is further directed to compounds which provide a high fraction bound to serumand/or to compounds which have high measured relaxivity in Seronorm™.

Description

CONJUGATES WHICH BIND A BLOOD PROTEIN SUCH AS HUMAN
SERUM ALBUMIN AND METHODS OF USING THE SAME IN DIAGNOSTIC
AND THERAPEUTIC APPLICATIONS
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to and benefit of U.S. Provisional Application No. 60/931,090, filed May 21, 2007, the contents of which are hereby incorporated by reference in their entirety.
FIELD OF THE INVENTION
[0002] The invention relates to conjugates that bind a blood protein such as human serum albumin (HSA), and are useful in diagnostic and/or therapeutic applications. The invention also includes methods for making and using such conjugates in therapeutic and diagnostic applications.
BACKGROUND OF THE INVENTION
[0003] New and better compounds are needed for the diagnosis and treatment of disease. For example, the diagnosis and prognosis of coronary and peripheral vascular disease is important in medicine. X-ray angiography with contrast agents has been done in coronary artery imaging. Due to the dilution of these contrast agents in the blood and their rapid transit into the extravascular space, imaging must be performed rapidly during dosing. Intravenous digital subtraction angiography (IV-DSA) was a technique widely studied in the 1980s in an attempt to address the need for better imaging; however, this technique was largely unsuccessful. See Saccani S. et al, "Digital angiography and surgery of the carotid: our experience," Acta Biomed Ateneo Parmense 1987; 58:99-103; Fiedler V., "Image exactness in digital subtraction angiography," ROFO Fortschr Geb Rontgenstr Nuklearmed 1986; 145:708-713; Crocker EF Jr. et al., "The role of intravenous digital subtraction angiography in the evaluation of extra cranial carotid artery disease. Can the decision for carotid artery surgery be made solely on the basis of its findings?" J Vase Surg 1986; 4:157-163; Ball JB Jr. et al., "Complications of intravenous digital subtraction angiography," Arch Neurol 1985; 42:969-972, all of which are hereby incorporated by reference in their entirety. [0004] The applicability of magnetic resonance imaging (MRI) to the diagnosis of cardiovascular and cerebrovascular abnormalities has steadily increased in recent years, due in no small part to the development of sophisticated hardware and special pulse sequences that allow the vasculature to be imaged selectively in the presence of other tissues. Standard extracellular MRI contrast agents (in combination with fast, gated pulse sequences and breath-holding on the part of the patient) have been employed in magnetic resonance angiography (MRA). Indeed, it appears that specialized agents for MRA are unnecessary for certain peripheral vascular examinations. See, e.g., Botnar RM et al., "A fast 3D approach for coronary MRA," JMagn Reson Imaging 1999;10:821-825; van Geuns RJ et al., "Magnetic resonance imaging of the coronary arteries: clinical results from three dimensional evaluation of a respiratory gated technique," Heart 1999; 82:515-519; Earls JP et al., "MR angiography of the thoracic, abdominal, and extremity venous system," Magn Reson Imaging Clin NAm 1998; 6:417-435; Talagala SL et al., "Fast three-dimensional time-of-fiight MR angiography of the intra-cranial vasculature," J Magn Reson Imaging 1995; 5:317-323; Siebert JE et al., "Physical principles and application of magnetic resonance angiography," Semin Ultrasound CTMR 1992;.13:227-245; Leiner T. et al., "Contrast-enhanced magnetic resonance angiography," Ned Tijdschr Geneeskd 1999; 143:1087-1093; Shetty AN et al., "3D Breath-hold contrast-enhanced MRA: a preliminary experience in aorta and iliac vascular disease," J Comput Assist Tomogr 1998; 22:179-185; all of which are hereby incorporated by reference in their entirety.
[0005] Blood pool agents for MRI diagnostics and other applications have been investigated in recent years. See, e.g., WO 00/38738, directed to metal ion complexes of conjugates of bile acids with molecules having chelating activity as contrast agents in MRI, in particular as blood pool agents, which is hereby incorporated by reference in its entirety. [0006] An additional attractive feature of MRA is the fact that employment of a lower dose of contrast agent is a positive attribute for any new technique. However, for cardiovascular magnetic resonance angiography (CVMRA), the situation is more complex for several reasons. For example, the heart is always in motion; the patient may not be able to endure breath-holding for more than 15-20 seconds; the hardware is not yet powerful and fast enough (non contrast techniques such as spiral K space sampling or techniques employing single pass CVMRA); and finally, spatial resolution must be at least 1.0 mm x 1.0 mm x 1.0 mm in order to achieve meaningful results. This forces a scan time of at least 7 minutes with hardware that is considered possible to construct. Thus, known contrast agents such as MS- 325 (gadofosveset trisodium) a member of a class of blood pool agents for magnetic resonance angiography (MRA) have been used, for example, to diagnose vascular disease under the above constraints.
[0007] In view of these factors, an agent that provides Tl shortening (<50 ms) of principally the intravascular space for an extended period of time is highly desirable, and can greatly facilitate CVMRA. In order for this to be possible, the agent preferably should be restricted from diffusing into the extravascular space, and preferably should undergo clearance from the blood with a half time less than that of extracellular space agents.
[0008] The major shortcoming of extracellular MRI agents, their short blood half time, is also faced by targeted imaging agents generally. For example in the case of a targeted diagnostic or therapeutic radiopharmaceutical, even if the radiometal is tightly chelated and the metal chelate is conjugated to a targeting vector, the concentration of the targeted radiopharmaceutical is decreased by distribution to the extravascular space, active and passive excretion via the kidney and possibly hepatobiliary excretion. These processes may be so rapid that residence time of the targeted radiopharmaceutical in the blood may be insufficient to permit its accumulation at an intravascular target or even an extravascular target site. It should be remembered that even if the radiopharmaceutical is distributed rapidly to the extravascular space, if clearance from the extravascular space via the kidney, for example, is rapid, targeting of an extravascular site will be ineffective. Therefore diagnostic and therapeutic compounds bearing a moiety capable of extending the blood half time and even the half time in the extravascular space may have an advantage in targeting sites of disease vs those that cannot. There is therefore a need for diagnostic and therapeutic compounds having superiority over existing compounds with respect to the fraction of the compound bound to serum and with respect to measured relaxivity in Seronorm™ (a solution of human serum albumin ("HSA") commercially available from Nycomed-Amersham).
SUMMARY OF THE INVENTION
[0009] The present invention is directed, in one embodiment, to a compound for the imaging or treatment of a disease in a mammal, having the structure: Ch- Ll - R - L2 - Q wherein Ch is a chelating moiety, optionally complexed with M, a metal or a radionuclide; Ll is a first linker;
R is a hydrophobic amino acid or a hydrophobic non-amino acid compound which renders the entire construct capable of binding a blood protein, particularly of binding human serum albumin (HSA); and
L2 is an optional second linker that is further linked to an optional targeting vector Q, which can direct the entire construct to a tissue bearing the target.
[0010] Note that the components CH, Ll, R, L2 and Q need not be attached in the order shown but may also be attached according to, for example: Ch-L1(L2-Q)-R ; and Ch(L2-Q)-Ll-R. [0011] Ch is a chelating moiety, which is optionally complexed with a metal or a radionuclide. In one embodiment, Ch comprises DTPA, DOTA, EDTA, TETA, EHPG, HBED, NOTA, DOTMA, TETMA, PDTA, TTHA, LICAM, MECAM or derivatives thereof. In a preferred embodiment Ch comprises DOTA or a derivative thereof. In a more preferred embodiment Ch comprises DO3 A or derivatives thereof. Most preferably, Ch comprises DO3A10CM, an abbreviation of 10-carbonylmethyl- 1,4, 7,10-tetraazacyclododecane- 1,4,7- triacetic acid. This designation indicates that the compound has 3 carboxylate moieties chelating the gadolinium or other metal, and a fourth chelating group one component of which is a carbonyl group which is not the carbonyl group of a carboxylic acid, but rather the carbonyl group of another functionality such as a carboxamide or a hydroxamate, or an acylhydrazine, or of a semicarbazide, or a semicarbazone for example, or related chelators. In another preferred embodiment Ch is a chelating moiety having high relaxivity. For example, Ch may be selected from the following table:
Chart 4. Enhanced Relaxivity (ER) Chelate Systems
Figure imgf000005_0001
7MP-DO3A
Figure imgf000005_0002
Figure imgf000005_0003
7MP-CM-DO3A 4-CE-DTPA TE-DOTA 4-CEMP-DTTA
In a preferred embodiment Ch is selected from CE-DTPA; CM-DOTMA; CM-DOTA; 7MP-
D03A and TE-DOTA.
[0012] Optionally Ch is complexed with a metal M or a radionuclide. M may be a metal ion such as, for example, a paramagnetic or radioactive metal ion. In one embodiment
M is a paramagnetic metal ion. Preferably in this embodiment M comprises Mn2+, Cu2+,
Fe2+, Co2+, Ni2+, Gd3+, Eu3+, Dy3+, Pr3+, Cr3+, Co3+, Fe3+, Ti3+, Tb3+, Nd3+, Sm3+, Ho3+, Er3+,
Pa4+ or Eu2+. Most preferably in this embodiment M is Gd3+.
[0013] In another embodiment M is a radioactive metal ion or a radionuclide.
Preferably, in this embodiment M is selected from 18F, 1241, 1251, 131I, 1231, 77Br, 76Br, 99mTc,
51Cr, 67Ga, 68Ga, 47Sc, 51Cr, 167Tm, 141Ce, 111In, 168Yb, 175Yb, 140La, 90Y, 88Y, 153Sm, 166Ho,
165Dy, 166Dy, 62Cu, 64Cu, 67Cu, 97Ru, 103Ru, 186Re, 188Re, 203Pb, 211Bi, 212Bi, 213Bi, 214Bi, 105Rh, 109Pd, 117mSn, 149Pm, 161Tb, 177Lu, 198Au or 199Au and the like. Preferably M is 99mTc, 67Ga, 68Ga, 90Y, 88Y, 111In or 177Lu.
[0014] Ll, the first linker, links Ch, the chelating moiety to one or more other components of the conjugate. Thus, Ll comprises functional groups sufficient to allow such linking. In a preferred embodiment Ll links the chelator to the hydrophobic molecule R. Preferably Ll comprises a substituted or unsubstituted alkyl chain, a polyethylene glycol derivative, one or more amino acids, a sugar, an aliphatic or aromatic spacer, a lipid molecule, or combinations of any of the foregoing. In a particularly preferred embodiment, Ll comprises an amino acid. For example, Ll may comprise GIy or Lys. Similarly, in a preferred embodiment Ll comprises trans-4-methylaminocyclohexanecarboxylic acid (referred to as a). In a more preferred embodiment Ll comprises two amino acids. Preferably Ll is Gly-Lys (referred to as GK); Gly-Gly (referred to as GG); Pro- trans-4- aminomethylcyclohexanecarbonyl (referred to as Pa); Asp- trans-4- aminomethylcyclohexanecarbonyl (referred to as Da); isonipecotic acid- trans-4- aminomethylcyclohexanecarbonyl (referred to as ia); GIy- Asp (referred to as GD); GIy- trans-4-aminomethylcyclohexanecarbonyl (referred to as Ga); GIu- trans-4- aminomethylcyclohexanecarbonyl (referred to as Ea); trans-4- aminomethylcyclohexanecarbonyl-Gly (referred to as aG); Asp-Asp (referred to as DD); Glu-Leu (referred to as EL); Asp-Leu (referred to as DL) or Leu-Leu (referred to as LL) . Of these, the most preferred are GK, a, Ga and aa.
[0015] R is a hydrophobic moiety which renders the entire conjugate able to bind to a blood protein such as HSA. Preferably R is a hydrophobic amino acid, a hydrophobic moiety derived from a hydrophobic amino acid or a hydrophobic organic group. In a preferred embodiment of the present invention, R comprises a hydrophobic amino acid. For example, R may be tryptophan or a derivative thereof; /-biphenylalanine (referred to as B) or /-3,3- diphenylalanine (referred to as e). In a particularly preferred embodiment both R and Ll comprise amino acids (or are derived from amino acids). In an even more preferred embodiment, Ll comprises two amino acids and R comprises a hydrophobic amino acid which binds HSA. For example, in preferred compounds of the invention Ll-R comprises glycyl-trans-4-aminomethylcyclohexanecarbonyl-/-4-biphenyl (refereed to herein as GaB) or trans-4-aminomethylcyclohexanecarbonyl-/-4-biphenyl (referred to herein as aB) . [0016] In another embodiment R is a hydrophobic organic group. Preferably in this embodiment R is a hydrophobic organic group of similar size to a hydrophobic amino acid. More preferably, R is a hydrophobic aliphatic or aromatic acyl group In a preferred embodiment R is selected from:
Figure imgf000007_0001
Figure imgf000007_0002
Figure imgf000007_0003
Figure imgf000007_0004
* Indicates point of attachment of G in the general structure.
[0017] The compounds of the present invention optionally include L2, a second linker. In one embodiment L2 is a linker which includes sufficient functional groups to link the targeting vector Q to the remainder of the conjugate. Preferably L2 comprises a substituted or unsubstituted alkyl chain, a polyethylene glycol derivative, one or more amino acids, a sugar, an aliphatic or aromatic spacer, a lipid molecule, or combinations of any of the foregoing. L2 may be the same or different as Ll . Preferably L2 comprises one or more amino acids. [0018] Q is an optional targeting vector, which directs the entire conjugate to tissue bearing the target. For example, the target may be a site of disease or a pathological process, such as, for example, an angiogenic site in a tumor. Q may be a targeting protein, antibody, peptide or equivalent, derivative or analog thereof; Q may also be, e.g., an enzyme or a molecule that binds to an enzyme or it may be an antibiotic. Q may be a protein such as an antibody, antibody fragment, diabody, triabody, a small molecule which functions as a targeting vector a carbohydrate or an aptamer. In a preferred embodiment Q and Ll both comprise amino acids.
[0019] Particularly preferred conjugates of the invention include DO3A10CM-a-B (where Ch is DO3A10CM, Ll is a and R is B); DO3A10CM- G-a-B (where Ch is DO3A10CM, Ll is G-a and R is B); DO3A10CM -D-a-B (where Ch is DO3A10CM, Ll is Da and R is B); DO3A10CM -G-L-B (where Ch is DO3A10CM, Ll is Gl and R is B); CE-DTP A-a-B (where Ch is CE-DTPA, Ll is a and R is B); CM-DOTMA-a-B (where Ch is CM-DOTMA, Ll is a and R is B); CM-DOTA-a-B (where Ch is CM-DOTA, Ll is a and R is B); and 7MP- DO3A-G-a-B (where Ch is 7MP-DO3A, Ll is G-A and R is B). Each of these conjugates may optionally further include a second linking group L2 and/or a targeting vector Q. Any of these conjugates may be optionally complexed with a metal M. In a preferred embodiment these conjugates are complexed with a paramagnetic metal, particularly Gd. In another preferred embodiment, these conjugates are complexed with a radioactive metal or radionuclide, particularly with a radioactive lanthanide.
[0020] Conjugates of the present invention are capable of binding a blood protein such as HSA, which increases the half time of the conjugate in the blood compared to a comparable agent unable to bind a blood protein. The increased blood half time results in conjugates better able to accumulate at target tissue. Additionally, where the conjugates of the invention are bound to a paramagnetic metal, the binding to a blood protein such as HSA results in an increase of the relaxivity of the chelator which is commonly referred to as enhanced relaxivity. Such conjugates, when bound to the blood proteins diffuse into the extravascular space much more slowly than those which cannot bind to proteins in the blood. Therefore their effective residence time in the blood is much longer and because they are bound to blood proteins such as HSA they exhibit enhanced relaxivity and enhanced signal vs chelators which are not protein bound, permitting improved imaging of the intravascular space and facilitating CVMRI.
[0021] In one embodiment, such conjugates also include a chelating moiety Ch whose relaxivity changes to a greater degree then that of a standard chelator when a molecule to which it is conjugated to binds proteins in the blood. Such agents provide a Tl shortening (<50 ms) of only the intravascular space for an extended period of time, further facilitating CVMRI. Such chelating moieties, referred to herein as high or enhanced relaxivity chelators, include for example, : CM-DOTMA, CM-DOTA, CEMP-D03A, 7MP-DO3A, 7MP-CM- DO3 A, TE-DOTA and 4-CEMP-DTTA whose structures are shown in Chart 4 . [0022] The present invention is also directed to a method of preparing a conjugate capable of binding a blood protein such as human serum albumin (HSA), wherein said method comprises the steps of: attaching a chelating moiety to the first linker, attaching said first linker to the hydrophobic group R via a protecting group having a carboxylic group thereto, attaching an optional second linker to said hydrophobic group, and attaching an optional targeting vector Q to said optional second linker. The conjugate may then be optionally complexed with M, a metal/radionuclide. The present invention is also directed to a method of preparing a conjugate capable of binding a blood protein such as human serum albumin wherein said method comprises attaching a hydrophobic group R which is capable of binding HSA to a first linker, then attaching the first linker to the chelating moiety Ch, then attaching another conjugatable group in the first linker to a second linker followed by attachment of the second linker to an optional molecule which can serve as a targeting vector. The conjugate may then be optionally complexed with M, a metal/radionuclide.
[0023] The present invention is also directed to a method of preparing a conjugate capable of binding a blood protein such as HSA, wherein said method comprises preparing the components of the entire construct as modules such as Ch-Ll, R-P (where P is an optional protecting group for a reactive function other than that to be employed for assembling the construct) and L2-Q and then attaching the modules in any desired order to obtain constructs such as Ch - Ll - R - L2 - Q, Ch-L1(L2-Q)-R or Ch(L2-Q)-Ll-R. The conjugate may then be optionally complexed with M, a metal/radionuclide.
[0024] The present invention is also directed to assembly on solid phase of the entire conjugate or a subset of the entire conjugate described above using orthogonal or quasiorthogonal series of protecting groups which allow creation of any of the conjugate Ch - Ll - R - L2 - Q, Ch-L1(L2-Q)-R, Ch(L2-Q)-Ll-R or any part of the conjugate on solid phase in fully or partially protected form followed by simultaneous cleavage from the solid phase and deprotection of the desired construct. The conjugate may then be optionally complexed with M, a metal/radionuclide.
[0025] The present invention is further directed to compounds which provide a high fraction bound to serum. For example, compounds of the invention may have a fraction bound to serum greater than about 0.87 (87%, expressed as a percentage). The present invention is also directed to compounds which have high measured relaxivity in Seronorm™. For example, compounds of the invention may have a measured relaxivity in Seronorm™ of greater than about 36.57. Still further, the present invention is also directed to compounds which have both high fraction bound to serum and high measured relaxivity. For example, compounds of the invention may have fraction bound to serum greater than about 87% and a measured relaxivity in Seronorm™ of greater than about 36.57.
[0026] The present invention is also directed to methods of using the conjugates of the invention. For example, conjugates of the invention may be used in methods of treating a disease in a mammal comprising the step of administering a composition comprising the conjugates of the present invention. Similarly, conjugates of the invention may be used in methods of imaging a site in a mammal, comprising the steps of administering a composition comprising the conjugates of the present invention and imaging the mammal.
DEFINITIONS
[0027] As used herein, the term "therapeutic" includes at least partial alleviation of symptoms of a given condition. For example, treatment of an individual can result in a decrease in the size of a tumor or diseased area, or prevention of an increase in size of the tumor or diseased area or partial alleviation of other symptoms. Treatment can result in reduction in the number of blood vessels in an area of interest or can prevent an increase in the number of blood vessels in an area of interest. Treatment can also prevent or lessen the number or size of metastic outgrowths of the main tumor(s).
[0028] As used herein, the term "polypeptide" is used to refer to a compound of two or more amino acids joined through the main chain (as opposed to side chain) by a peptide amide bond (-C(=O)NH-). The term "peptide" is used interchangeably herein with "polypeptide" but is generally used to refer to polypeptides having fewer than 40, and preferably fewer than 25 amino acids.
[0029] As used herein, the term "relaxivity" means the ability of a compound to shorten the Tl (longitudinal relaxation time) of water protons or other atoms which can be detected by Tl- weighted MRI imaging. The symbol for relaxivity is r and where the Tl relaxivity is quoted the symbol is rl . Usually this value is quoted as being measured at a given field strength, such as 20MHz, for example, and the value is given in units of mM^sec"1. In the case of MRI agents based on gadolinium chelates the value is assumed to be per gadolinium atom. In cases where there is more than one gadolinium atom in a MRI imaging agent the aggregate relaxivity may be quoted and often the relaxivity per gadolinium atom is also quoted in order to clarify the interpretation of the data.
[0030] As used herein, the term "high relaxivity" refers to values of rl provided by MRI agents of a given class, particularly MRI agents based on gadolinium chelates, which are significantly greater than those provided by typical gadolinium chelate-based MRI agents under the same conditions. By significantly greater is meant at least 10% greater than the relaxivity of the comparison gadolinium chelate. By 'same conditions' is meant that the chelators in question are employed under the same conditions of measurement and if conjugated to another molecular species such as a targeting vector or a protein binding moiety, they are conjugated to the same molecular species.
[0031] As used herein the term 'enhanced relaxivity' refers to an increase in relaxivity, rl, which is obtained when a MRI agent, typically based on a gadolinium chelator, binds to a macromolecular species such as a blood protein. The comparison is made between the relaxivity of the MRI agent measured in water or optionally in aqueous solutions of a buffer such a pH 7 phosphate buffer or phosphate buffered saline solution and the relaxivity of the MRI agent in the presence of a defined concentration of a macromolecular target. In one preferred embodiment the macromolecular target is human serum albumin (HSA). [0032] The term "binding" refers to the determination by standard assays, including those described herein, that a binding moiety such as a hydrophobic group, a small molecule, a peptide, or a polypeptide binds reversibly to a given target. Such standard assays include, but are not limited to, equilibrium dialysis, gel filtration, surface plasmon resonance and the monitoring of spectroscopic changes, such as increased or decreased absorption, a bathochromic or hypsochromic shift, an increase or decrease or an elimination of or an induction of fluorescence, for example, that result in the bound state. [0033] "Homologues" of the binding species disclosed herein (including hydrophobic groups, small molecules, peptides or polypeptides) may be produced using any of the modification or optimization techniques described herein or known to those skilled in the art. Such homologues will be understood to fall within the scope of the present invention and the definition of "binding species" so long as the substitution, addition, or deletion of functionality, or in the case of peptidic species, amino acids or other such modification does not eliminate its ability to bind to the target. The term "homologous," as used herein, with regard to peptides and polypeptides refers to the degree of sequence similarity between two polymers (i.e. polypeptide molecules or nucleic acid molecules). When the same nucleotide or amino acid residue or one with substantially similar properties (i.e. a conservative substitution) occupies a sequence position in the two polymers under comparison, then the polymers are homologous at that position. For example, if the amino acid residues at 60 of 100 amino acid positions in two polypeptide sequences match or are homologous, then the two sequences are 60% homologous. The homology percentage figures referred to herein reflect the maximal homology possible between the two polymers, i.e., the percent homology when the two polymers are so aligned as to have the greatest number of matched (homologous) positions. Polypeptide homologues within the scope of the present invention will be at least 70% and preferably greater than 80% homologous to at least one of the binding sequences disclosed herein.
[0034] The term "specificity" refers to a binding polypeptide having a higher binding affinity for one target over another. Binding specificity may be characterized by a ratio of the dissociation equilibrium constants (KD) or association equilibrium constants (Ka) for the two tested target materials.
[0035] The term "patient" as used herein refers to any mammal, especially humans. [0036] The term "pharmaceutically acceptable" carrier or excipient refers to a non-toxic carrier or excipient that may be administered to a patient, together with a compound of this invention, and which does not destroy the pharmacological activity thereof. [0037] The term "target" or "target molecule" refers to any substance that a binding moiety or binding polypeptide (including for example a targeting vector or hydrophobic compound) can bind to, such as a protein or polypeptide, a cell, a receptor, a carbohydrate, a lipid, an oligonucleotide, etc. As used herein, "target" also includes a family of receptors, such as, for example, protein-tyrosine kinase receptors.
[0038] The terms "therapeutic agent" or "therapeutic" refer to a compound or an agent having a beneficial, therapeutic or cytotoxic effect in vivo. Therapeutic agents include those compositions referred to as, for example, bioactive agents, cytotoxic agents, drugs, chemotherapy agents, radiotherapeutic agents, genetic material, etc. [0039] The following common abbreviations are used throughout this specification: 9- fluorenylmethyloxycarbonyl (fmoc or Fmoc), 1-hydroxybenozotriazole (HOBt), N5N'- diisopropylcarbodiimide (DIC), acetic anhydride (Ac2O), (4,4-dimethyl-2,6-dioxocyclohex-l- ylidene)-3-methylbutyl (ivDde), trifluoroacetic acid (TFA), Reagent B (TFA:H2O:phenol:triisopropylsilane, 88:5:5:2), N,N-dϋsopropylethylamine (DIEA), O-(1H- benzotriazole-l-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), O-(7- azabenzotriazol-l-yl)-l,l,3,3-tetramethyluronium hexafluorphosphate (HATU), N- hydroxysuccinimide (NHS), solid phase peptide synthesis (SPPS), dimethyl sulfoxide (DMSO), dichloromethane (DCM), dimethylformamide (DMF), and N-methylpyrrolidinone (NMP).
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0040] The present invention is directed, in one embodiment, to novel conjugates that exhibit superior ability to bind a blood protein such as Human Serum Albumin (HSA) and which may be used in diagnostic and/or therapeutic applications.
[0041] The present invention is further directed, in another embodiment, to novel conjugates that exhibit the ability to bind human serum at greater than about 87% and which may be used in diagnostic and/or therapeutic applications.
[0042] The present invention is further directed, in still another embodiment, to novel conjugates that exhibit a measured relaxivity in Seronorm™ of greater than about 36.57 and which may be used in diagnostic and/or therapeutic applications.
[0043] The present invention is further directed, in another embodiment, to novel conjugates that exhibit the ability to bind human serum at greater than about 87% and a measured relaxivity in Seronorm™ (a solution of human serum albumin commercially available from
Ny corned- Amersham) of greater than about 36.57 which may be used in diagnostic and/or therapeutic applications.
[0044] The present invention is further directed, in yet another embodiment, to compounds that comprise one or more moieties that bind a blood protein such as HSA, to which are conjugated to a metal chelator. Most preferably, the compounds comprise one or more agents that bind the protein HSA, as well as a metal chelator linked to the HSA-binding portion by one or more linkers.
[0045] In one preferred embodiment the conjugates have the structure:
Ch- Ll - R - L2 - Q
wherein Ch is a chelating moiety, optionally complexed with M, a metal or a radionuclide; Ll is a first linker; R is a hydrophobic amino acid or a hydrophobic non-amino acid compound which renders the entire construct capable of binding a blood protein, particularly of binding human serum albumin (HSA); and
L2 is an optional second linker that is further linked to an optional targeting vector Q, which can direct the entire construct to a tissue bearing the target.
[0046] Note that the components CH, Ll, R, L2 and Q need not be attached in the order shown but may also be attached according to, for example: Ch-L1(L2-Q)-R ; and Ch(L2-Q)-Ll-R.
[0047] In another preferred embodiment the conjugates comprise : Ch-Ll-R, wherein Ch, Ll and R have the above meanings.
Chelating Moieties
[0048] Ch is a chelating moiety, which is optionally complexed with a metal or a radionuclide. In one embodiment, Ch is a paramagnetic metal chelate, which, when complexed with paramagnetic metal may serve as a contrast agent for use in MRI. Suitable chelators are known in the art and include, e.g. , acids with methylene phosphonic acid groups, methylene carbohydroxamine acid groups, carboxyethylidene groups, carboxymethylene groups or methylene carboxamide groups. Examples of useful chelators include, but are not limited to, diethylenetriamine pentaacetic acid (DTPA), l,4,7,10-tetraazacyclotetradecane-l,4,7,10-tetraacetic acid (DOTA), 1 -substituted 1,4,7,- tricarboxymethyl 1,4,7,10 teraazacyclododecane triacetic acid (DO3A), ethylenediaminetetraacetic acid (EDTA), and 1,4,8,11-tetraazacyclotetradecane-l, 4, 8,11- tetraacetic acid (TETA).
[0049] Additional useful chelating ligands are ethylenebis-(2-hydroxy-phenylglycine) (EHPG), and derivatives thereof, including 5-Cl-EHPG, 5Br-EHPG, 5-Me-EHPG, 5- t-Bu-EHPG, and 5-sec-Bu-EHPG; benzodiethylenetriamine pentaacetic acid (benzo-DTPA) and derivatives thereof, including dibenzo-DTPA, phenyl-DTPA, diphenyl-DTPA, benzyl-DTPA, and dibenzyl DTPA; bis-2 (hydroxybenzyl)-ethylene-diaminediacetic acid (HBED) and derivatives thereof; the class of macrocyclic compounds which contain at least 3 carbon atoms, more preferably at least 6, and at least two heteroatoms (O and/or N), which macrocyclic compounds can consist of one ring, or two or three rings joined together at the hetero ring elements, e.g., benzo-DOTA, dibenzo-DOTA, and benzo-NOTA, where NOTA is 1,4,7-triazacyclononane N,N',N"-triacetic acid, benzo-TETA, benzo-DOTMA, where DOTMA is l,4,7,10-tetraazacyclotetradecane-l,4,7, 10-tetra(methyl tetraacetic acid), and benzo-TETMA, where TETMA is 1,4,8,11- tetraazacyclotetradecane- 1,4, 8, 11 -(methyl tetraacetic acid); derivatives of 1,3-propylenediaminetetraacetic acid (PDTA) and triethylenetetraaminehexaacetic acid (TTHA); derivatives of l,5,10-N,N',N"-tris(2,3-dihydroxybenzoyl)-tricatecholate (LICAM) and l,3,5-N,N',N"-tris(2,3-dihydroxybenzoyl) aminomethylbenzene (MECAM). A preferred chelator for use in the present invention is DTPA.
[0050] In one embodiment, Ch comprises DTPA, DOTA, EDTA, TETA, EHPG, HBED, NOTA, DOTMA, TETMA, PDTA, TTHA, LICAM, MECAM or derivatives thereof. In a preferred embodiment Ch comprises DOTA or a derivative thereof. In a more preferred embodiment Ch comprises D03A or DO3A10CM.
[0051] In another embodiment Ch is a chelator for a radioactive metal or radionuclide. Such chelators include, for example, linear, macrocyclic, terpyridine, and N3S, N2S2, or N4 chelants (see also U.S. 5,367,080, U.S. 5,364,613, U.S. 5,021,556, U.S. 5,075,099, U.S. 5,886,142), and other chelators known in the art including, but not limited to, HYNIC, DTPA, EDTA, DOTA, TETA, and bisamino bisthiol (BAT) chelators (see also U.S. 5,720,934). For example, N4 chelators are described in U.S. Patent Nos. 6,143,274; 6,093,382; 5,608,110; 5,665,329; 5,656,254; and 5,688,487. Certain N3S chelators are described in PCT/CA94/00395, PCT/CA94/00479, PCT/CA95/00249 and in U.S. Patent Nos. 5,662,885; 5,976,495; and 5,780,006. The chelator may also include derivatives of the chelating ligand mercapto-acetyl-acetyl-glycyl-glycine (MAG3), which contains an N3S, and N2S2 systems such as MAMA (monoamidemonoaminedithiols), DADS (N2S diaminedithiols), CODADS and the like. These ligand systems and a variety of others are described in Liu and Edwards, Chem Rev 99, 2235-2268 (1999) and references therein.
[0052] The chelator may also include complexes containing ligand atoms that are not donated to the metal in a tetradentate array. These include the boronic acid adducts of technetium and rhenium dioximes, such as are described in U.S. Patent Nos. 5,183,653; 5,387,409; and 5,118,797, the disclosures of which are incorporated by reference herein, in their entirety.
[0053] In another embodiment, disulfide bonds of a binding polypeptide of the invention are used as two ligands for chelation of a radionuclide such as 99mTc. In this way the peptide loop is expanded by the introduction of Tc (peptide-S-S-peptide changed to peptide-S-Tc-S- peptide). This has also been used in other disulfide containing peptides in the literature (J. Q. Chen, A. Cheng, N. K. Owen, T. H. Hoffman, Y. Miao, S. S. Jurisson, T. P. Quinn. J. Nucl. Med. 42, 1847-1855 (2001)) while maintaining biological activity. The other chelating groups for Tc can be supplied by amide nitrogens of the backbone, another cystine amino acid or other modifications of amino acids.
[0054] Particularly preferred metal chelators include those of Formula 20, 21, and 22 (for 111In and lanthanides such as paramagnetic gadolinium (Gd3+) and radioactive lanthanides, such as, for example 177Lu, 90Y, 153Sm, and 166Ho) and those of Formula 23, 24, and 25 (for radioactive 99mTc, 186Re, and 188Re) set forth below. These and other metal chelating groups are described in U.S. Patent Nos. 6,093,382 and 5,608,110, which are incorporated by reference herein in their entirety. Additionally, the chelating group of formula 22 is described in, for example, U.S. Patent No. 6,143,274; the chelating group of formula 24 is described in, for example, U.S. Patent Nos. 5,627,286 and 6,093,382, and the chelating group of formula 25 is described in, for example, U.S. Patent Nos. 5,662,885; 5,780,006; and 5,976,495.
Figure imgf000016_0001
(22)
Figure imgf000016_0002
Figure imgf000017_0001
(23b)
Figure imgf000017_0002
(24a) (24b)
Figure imgf000018_0001
(25)
[0055] In the above Formulas 20 and 21, R is alkyl, preferably methyl. In the above Formula 24, X is either CH2 or O, Y is either C1-C10 branched or unbranched alkyl; Y is aryl, aryloxy, arylamino, arylaminoacyl; Y is arylkyl - where the alkyl group or groups attached to the aryl group are C1-C10 branched or unbranched alkyl groups, C1-C10 branched or unbranched hydroxy or polyhydroxyalkyl groups or polyalkoxyalkyl or polyhydroxy-polyalkoxyalkyl groups, J is C(=O)-, OC(=O)-, SO2, NC(=O)-, NC(=S)-, N(Y), NC(=NCH3)-, NC(=NH)-, N=N-, homopo Iy amides or heteropolyamines derived from synthetic or naturally occurring amino acids; all where n is 1-100. Other variants of these structures are described, for example, in U.S. Patent No. 6,093,382. The disclosures of each of the foregoing patents, applications and references are incorporated by reference herein, in their entirety. [0056] The chelators may be covalently linked to the HSA-binding moiety via Ll, or directly to the HSA binding moiety which is optionally further linked to L2 and/or Q, the targeting vector, as described previously, and then directly labeled with the radioactive metal of choice (see, e.g., WO 98/52618, U.S. 5,879,658, and U.S. 5,849,261). Further examples of representative chelators and chelating groups contemplated by the present invention are described in, e.g., WO 98/18496, WO 86/06605, WO 91/03200, WO 95/28179, WO 96/23526, WO 97/36619, WO 01/82974, WO 01/064708, WO 00/38738, PCT/US98/01473, PCT/US98/20182, and U.S. 4,899,755, U.S. 5,474,756, U.S. 5,846,519 and U.S. 6,143,274, each of which is hereby incorporated by reference in its entirety. [0057] A preferred chelator used in conjugates of the present invention is 1 -substituted 4,7,10-tricarboxymethyl 1,4,7,10 tetraazacyclododecane triacetic acid (DO3 A). Another preferred chelating moiety is l,4,7,10-tetraazacyclotetradecane-l,4,7,10-tetraacetic acid (DOTA). The present invention also contemplates chelators comprising DOTA derivatives; e.g., substituted derivatives of DOTA having 4 carboxyl groups chelating gadolinium or another metal and wherein the linkage of the macrocyclic system to the linker Ll is via either a group emanating from one of the alpha-carbon atoms of one of the chelating sidearms or from the macrocyclic carbon backbone.
[0058] Yet another chelator moiety is DO3A10CM, an abbreviation of 10-carbonylmethyl- l,4,7,10-tetraazacyclododecane-l,4,7-triacetic acid. This designation indicates that the compound has 3 carboxylate moieties chelating the gadolinium or other metal, and a fourth chelating group one component of which is a carbonyl group which is not the carbonyl group of a carboxylic acid, but rather the carbonyl group of another functionality such as a carboxamide or a hydroxamate, or an acylhydrazine, or of a semicarbazide, or a semicarbazone for example.
[0059] It has been found that high relaxation rates provide increased contrast in the image. Increased contrast makes it possible to obtain better physiological information in a shorter period of time. This leads to significant advantages in terms of image quality and cost. Therefore, compounds with high relaxivity (as defined above) and a greater enhanced relaxivity upon binding to a macromolecular target provide a stronger signal enhancing effect per molecule than can be obtained with more typical relaxation agents that are used in contrast enhancement. One application that may take advantage of an enhanced relaxivity chelate is linking a compound or complex to a bioactive compound or targeting moiety that targets a particular tissue. Localization at the target via the targeting moiety can result in a higher signal enhancement than could be obtained if a comparable compound were linked that had normal relaxivity. Therefore, in a preferred embodiment Ch is a chelating moiety having high or enhanced relaxivity. Examples of high or enhanced relaxivity chelators that are useful for the present invention are described in, e.g., WO 99/45967, WO 99/45968 (manganese chelates), WO 98/05625 and WO 98/05626, each of which is hereby incorporated by reference in its entirety. For example, Ch may be selected from the following table: Chart 4. Enhanced Relaxivity (ER) Chelate Systems
Figure imgf000020_0001
7MP-CM-DO3A 4-CE-DTPA TE-DOTA 4-CEMP-DTTA In a more preferred embodiment Ch is a chelator with enhanced relaxivity selected from CE-DTPA; CM-DOTMA; CM-DOTA; 7MP-DO3A and TE-DOTA.
[0060] A most preferred high relaxivity chelator for use in contrast agents for MRI is an aza ligand that is able to complex metal ions, in particular paramagnetic ions. Such an aza ligand should be able to form chelates with the transition metals or metals of the Lanthanide series, exhibiting a high level of affinity and selectivity for the relevant paramagnetic ions, as well as suitable pharmacokinetic properties such as excretion, binding to plasma proteins, metabolical inertia, and the like. Aza ligands that are particularly useful for the present invention are described in WO 03/008390, which is hereby incorporated by reference in its entirety.
[0061] Structures of some preferred chelating moieties of the present invention are shown in the charts below, and further discussed in the Examples. DO3A10CM was the standard chelating function that was employed for screening the candidate conjugates. Other chelators were employed in an attempt to optimize the conjugates for use in MRI. These chelates were chosen because they were found to display a greater increase in relaxivity than standard chelators in an assay that measures the relaxivity of the Gd-chelate when its motion is restricted artificially. The artificial restriction of the motion of the chelate is designed to mimic the binding of a chelator containing molecule to a blood protein, such as HSA (Human Serum Albumin). As described above the increase in relaxivity is often referred to as enhanced relaxivity - ER for short - by comparison with the unbound state. Thus, pairing up of one of the ER chelating molecules with a strong binder of HSA is expected to provide a greater increase in relaxivity upon binding the HSA than would be afforded by the compound comprising the same HSA binding molecule with the standard chelating function DO3A10CM. Structures of the standard chelates of lanthanides and other metals
Figure imgf000021_0001
Structures of optimized chelates of lanthanides and other metals
Figure imgf000022_0001
CM-DOTMA CM-DOTA
Figure imgf000022_0002
Figure imgf000022_0003
7MPCMDO3A 4-CE-DTPA TE-DOTA 4-CEMP-DTTA
[0062] Other chelators useful for the present invention will be apparent to those skilled in the art and/or on review of this application or references cited in this application.
Metals
[0063] Optionally the chelating moiety Ch is complexed with a metal M or a radionuclide. M may be a metal ion such as, for example, a paramagnetic or radioactive metal ion. In one embodiment M is a paramagnetic metal ion. Preferred paramagnetic metal ions have atomic numbers 21-29, 42, 44, or 57-83, including ions of the transition metal or lanthanide series which have one, and more preferably five or more, unpaired electrons and a magnetic moment of at least 1.7 Bohr magneton. Preferred paramagnetic metals include, but are not limited to, chromium (III), manganese (II), manganese (III), iron (II), iron (III), cobalt (II), nickel (II), copper (II), praseodymium (III), neodymium (III), samarium (III), gadolinium (III), terbium (III), dysprosium (III), holmium (III), erbium (III), europium (III) and ytterbium (III). Additionally, compounds of the present invention may also be conjugated with one or more superparamagnetic particles. Preferably in this embodiment M comprises
Λ M /rn 2+ , -NN Ti-2+ , /G-ΛdJ3+ , nDy 3+ , nPr 3+ , PCr 3+ , rCio 3+ , ΓFe 3+ , rTpi-3+ , mTib 3+ , -NN TdJ3+ , O Sm 3+
Figure imgf000022_0004
,
T HTo 3+ , TE-.r 3+ , τ P>a 4+ or T E-1u 2+ . [0064] One skilled in the art will be able to select a metal according to the dose required to detect target containing tissue, as well as considering other factors such as, e.g., toxicity of the metal to the subject. See, Tweedle et al., Magnetic Resonance Imaging (2nd ed.), vol. 1, Partain et al., eds. (W.B. Saunders Co. 1988), pp. 796-97. Generally, the desired dose for an individual metal will be proportional to its relaxivity, modified by factors such as the biodistribution, pharmacokinetics and metabolism of the metal.
[0065] The trivalent cation of gadolinium, referred to as Gd(III) or Gd3+, is particularly preferred for MRI contrast agents, due to its high relaxivity and low toxicity, with the further advantage that it exists in only one biologically accessible oxidation state. This minimizes undesired metabolization of the metal by a patient. Gd(III) chelates have been used for clinical and radiologic MR applications since 1988, and approximately 30% of all MR examinations currently employ a gadolinium-based contrast agent. Another preferred metal is Cr +, which is advantageous and relatively inexpensive.
[0066] In another embodiment M is a radioactive metal ion or a radionuclide. Preferably, in this embodiment M is selected from 18F, 1241, 1251, 1311, 1231, 77Br, 76Br, 99mTc, 51Cr, 67Ga, 68Ga, 47Sc, 51Cr, 167Tm, 141Ce, 111In, 168Yb, 175Yb, 140La, 90Y, 88Y, 153Sm, 166Ho, 165Dy, 166Dy, 62Cu, 64Cu, 67Cu, 97Ru, 103Ru, 186Re, 188Re, 203Pb, 211Bi, 212Bi, 213Bi, 214Bi, 105Rh, 109Pd, 117mSn, 149Pm, 161Tb, 177Lu, 198Au or 199Au and the like. Preferably M is 99mTc, 67Ga, 68Ga, 90Y, 88Y, 111In Or 177Lu.
Linkers
[0067] As explained infra, one or more linkers (e.g. Ll) may be present between the chelating agent and the hydrophobic compound, and optionally, one or more additional linkers (L2) may be present between the targeting vector and the remainder of the conjugate. Use of such linkers/spacers may improve the relevant properties of the conjugates (e.g., by increasing serum stability, etc.). Linkers that are useful for the present invention may include, but are not restricted to, substituted or unsubstituted alkyl chains, polyethylene glycol derivatives, amino acid spacers, sugars, aliphatic or aromatic spacers, lipid molecules, or combinations of any of the foregoing.
[0068] In a particularly preferred embodiment, the conjugates of the invention include Ll and optionally L2. In a more preferred embodiment Ll (and L2 if present) comprise at least one amino acid. For example, Ll may comprise GIy or Lys. Similarly, in a preferred embodiment Ll comprises trans-4-methylaminocyclohexanecarboxylic acid (referred to as a). In a more preferred embodiment Ll comprises two amino acids. Preferably for compounds of Class 2 where (see Table 2) Ll can be Gly-Lys (referred to as GK), alternatively Ll can be Lys(Gly) which indicates that the GIy is appended to the epsilon nitrogen of the Lys component of Ll instead of the alpha nitrogen of the Lys. In general when the linker comprises Lys attachment can be at the alpha and epsilon amines, at the alpha amine and carboxylic acid or at the carboxylic acid and epsilon amine. For compounds of Class 1 Ll in another more preferred embodiment is Gly-Gly (referred to as GG); Pro- trans-4 aminomethylcyclohexanecarbonyl (referred to as Pa); Asp- trans-4- aminomethylcyclohexanecarbonyl (referred to as Da); isonipecotic acid- trans-4- aminomethylcyclohexanecarbonyl (referred to as ia); GIy- Asp (referred to as GD); GIy- trans-4-aminomethylcyclohexanecarbonyl (referred to as Ga); GIu- trans-4- aminomethylcyclohexanecarbonyl (referred to as Ea); trans-4- aminomethylcyclohexanecarbonyl-Gly (referred to as aG); Asp-Asp (referred to as DD); Glu-Leu (referred to as EL); Asp-Leu (referred to as DL) or Leu-Leu (referred to as LL) . For compounds of Class 1 the most preferred are a, Ga and aa.
[0069] In a preferred embodiment L2 is present and includes sufficient functional groups to link the targeting vector Q to the remainder of the conjugate. Preferably L2 comprises one or more amino acids. In a preferred embodiment L2 comprises GIy or oligoglycines (GIy)n where n = 1-10, or Adoa (where Adoa refers to 8-amino, 3-6-dioxaoctanoyl) or (Adoa)n where n = 1-10, or combinations of GIy and Adoa or combinations of (GIy)n and Adoa, or combinations of (GIy)n and (Adoa)n or combinations of GIy and (Adoa)n in the order given or in the reverse order wherein the Adoa or (Adoa)n precedes the GIy or (GIy)n or in any other combination of the two linkers.
[0070] In general, methods disclosed herein as well as other known methods can be used to couple the metal chelate and the linker. See, e.g., WO 95/28967, WO 98/18496, WO 98/18497 and discussions therein. The present invention contemplates linking of the chelate(s) on any position, provided the metal chelate retains the ability to bind the metal tightly in order to minimize toxicity. Similarly, a component of a compound of this invention may be modified or elongated in order to generate a locus for attachment to a metal chelate, provided such modification or elongation does not eliminate its ability to bind the target.
The Hydrophobic Group R
[0071] R is a hydrophobic moiety which renders the entire conjugate able to bind to a blood protein such as HSA. Preferably R is a hydrophobic amino acid, a hydrophobic moiety derived from a hydrophobic amino acid or a hydrophobic organic group. In a preferred embodiment of the present invention, R comprises a hydrophobic amino acid. In a preferred embodiment the hydrophobic amino acid is comprised of an entity containing an indole ring substituted at the indole nitrogen with a hydrophobic substituent such as a substituted benzene sulfonamide or substituted benzamide moiety. In a particularly preferred embodiment R is tryptophan or a derivative thereof; /-biphenylalanine (referred to as B); or /- 3,3-diphenylalanine (referred to as e). In a preferred embodiment both R and Ll comprise amino acids (or are derived from amino acids). In an even more preferred embodiment, Ll comprises two amino acids and R comprises a hydrophobic amino acid which binds HSA. For example, in preferred compounds of the invention Ll-R comprises glycyl-trans-4- aminomethylcyclohexanecarbonyl-/-4-biphenylalanyl (referred to herein as GaB) or trans-4- aminomethylcyclohexanecarbonyl-/-4-biphenylalanyl (referred to herein as aB) . [0072] In another embodiment R is a hydrophobic organic group. Preferably in this embodiment R is a hydrophobic organic group of similar size to a hydrophobic amino acid. More preferably, R is a hydrophobic molecule that contains one or more benzene rings or 6- membered carbon aryl groups. Preferably R is a hydrophobic aliphatic or aromatic carboxylic acid. In a preferred embodiment R is selected from:
Figure imgf000026_0001
* Indicates point of attachment of G in the general structure.
[0073] In the compositions of the present invention, the purpose of the hydrophobic, blood protein binding portion is to prolong circulation of the conjugate in the bloodstream, in contrast with non-protein binding molecules of similar molecular weight. Typically, these non-protein binding molecules would be expected to be rapidly distributed from the blood stream to the extravascular space and then excreted from the body, e.g. , through the urine or the feces. In contrast, in the conjugates of the present invention, binding of the molecule to a protein in the blood, such as HSA, increases the blood residence time of the compound, because the molecular size of the bound blood protein is such that it is only very slowly excreted from the bloodstream, as compared to smaller molecules. When the compounds of the present invention are bound to the blood protein, they are unavailable to the pathways for excretion of small molecules such as, for example, passive diffusion across the endothelial cell junctions into the extravascular space, or uptake by the kidneys or the liver via a first pass effect.
Targeting Moieties
[0074] The conjugates of the present invention may comprise an optional targeting vector Q, which directs the entire conjugate to tissue bearing the target. Thus, Q directs the conjugate to the appropriate site, or involves the compounds in a reaction, where the desired diagnostic or therapeutic activity will occur. Q may be a targeting protein, antibody, peptide or equivalent, derivative or analog thereof; Q may also be, e.g., an enzyme or a molecule that binds to an enzyme or it may be an antibiotic. Q may be a protein such as an antibody, antibody fragment, diabody, or triabody, a small molecule which functions as a targeting vector a carbohydrate or an aptamer. In one embodiment, the targeting vector Q may be a peptide, equivalent, derivative or analog thereof which functions as a ligand that binds to a particular site, or which has binding affinity for a particular site or a specific metabolic function. The targeting vector may be a peptide that binds to a receptor or enzyme of interest. In another embodiment, the targeting vector may be an enzyme, or a molecule that binds to an enzyme. In another embodiment, the targeting moiety may be an antibiotic. [0075] In a preferred embodiment, the targeting moiety is a peptide. For example, the targeting moiety may be a peptide hormone such as, for example, luetinising hormone releasing hormone (LHRH) such as that described in the literature [e.g. , Radiometal-Binding Analogues of Leutenizing Hormone Releasing Hormone PCT/US96/08695; PCT/US97/ 12084 (WO 98/02192)]; insulin; oxytosin; somatostatin; Neuro kinin-1 (NK-I); Vasoactive Intestinal Peptide (VIP) including both linear and cyclic versions as delineated in the literature, [e.g., Comparison of Cyclic and Linear Analogs of Vasoactive Intestinal Peptide. D. R. Bolin, J. M. Cottrell, R. Garippa, N. Rinaldi, R. Senda, B. Simkio, M. O'Donnell. Peptides: Chemistry, Structure and Biology Pravin T. P. Kaumaya, and Roberts S. Hodges (Eds)., Mayflower Scientific LTD., 1996, pp. 174-175]; gastrin releasing peptide (GRP); bombesin and other known hormone peptides, as well as analogues and derivatives thereof.
[0076] Other useful targeting peptides include analogs of somatostatin which, for example, are Lanreotide (Nal-Cys-Thr-DTrp-Lys-Val-Cys-Thr-N^), Octreotide (Nal-Cys-Thr-DTrp- Lys-Val-Cys-Thr-ol), and Phe-Cys-Thr-DTrp-Lys-Val-Cys-Thr-ol. These analogues are described in the literature [e.g. , Potent Somatostatin Analogs Containing N-terminal Modifications, S. H. Kim, J. Z. Dong, T. D. Gordon, H. L. Kimball, S. C. Moreau, J.-P. Moreau, B.A. Morgan, W. A. Murphy and J. E. Taylor; Peptides: Chemistry, Structure and Biology Pravin T. P. Kaumaya, and Roberts S. Hodges (Eds)., Mayflower Scientific LTD., 1996, pp. 241-243.]
[0077] Still other useful targeting peptides include Substance P agonists [e.g., G. Bitan, G. Byk, Y. Mahriki, M. Hanani, D. Halle, Z. Selinger, C. Gilon, Peptides: Chemistry, Structure and Biology, Pravin T. P. Kaumaya, and Roberts S. Hodges (Eds), Mayflower Scientific LTD., 1996, pp. 697-698; G Protein Antagonists A novel hydrophobic peptide competes with receptor for G protein binding, Hidehito Mukai, Eisuke Munekata, Tsutomu Higashijima, J. Biol. Chem. 1992, 267, 16237-16243]; NPY(Yl) [e.g., Novel Analogues of Neuropeptide Y with a Preference for the Yl -receptor, Richard M. Soil, Michaela, C. Dinger, Ingrid Lundell, Dan Larhammer, Annette G. Beck-Sickinger, Eur. J. Biochem. 2001, 268, 2828-2837; 99mTc-Labeled Neuropeptide Y Analogues as Potential Tumor Imaging Agents, Michael Langer, Roberto La Bella, Elisa Garcia-Garayoa, Annette G. Beck-Sickinger, Bioconjugate Chem. 2001, 12, 1028-1034; Novel Peptide Conjugates for Tumor-Specific Chemotherapy, Michael Langer, Felix Kratz, Barbara Rothen-Rutishauser, Heidi Wnderli- Allenspach, Annette G. Beck-Sickinger, J. Med. Chem. 2001, 44, 1341-1348]; oxytocin; endothelin A and endothelin B; bradykinin; Epidural Growth Factor (EGF); Interleukin-1 [Anti-IL-1 Activity of Peptide Fragments of IL-I Family Proteins, I. Z. Siemion, A. Kluczyk, Zbigtniew Wieczorek, Peptides 1998, 19, 373-382]; and cholecystokinin (CCK-B) [Cholecystokinin Receptor Imaging Using an Octapeptide DTPA-CCK Analogue in Patients with Medullary Thyroid Carcinoma, Eur. J. Nucl Med. 200, 27, 1312-1317]. [0078] Literature which gives a general review of targeting peptides, can be found, for example, in the following: The Role of Peptides and Their Receptors as Tumor Markers, Jean-Claude Reubi, Gastrointestinal Hormones in Medicine, p. 899-939; Peptide Radiopharmaceutical in Nuclear Medicine, D. Blok, R. I. J. Feitsma, P. Vermeij, E. J. K. Pauwels, Eur. J. Nucl Med. 1999, 26, 1511-1519; and Radiolabeled Peptides and Other Ligands for Receptors Overexpressed in Tumor Cells for Imaging Neoplasms, John G. McAfee, Ronald D. Neumann, Nuclear Medicine and Biology, 1996, 23, 673-676 (somatostatin, VIP, CCK, GRP, Substance P, Galanin, MSH, LHRH, Arginine-vasopressin, endothelin). All of the literature and references cited are herein incorporated by reference in their entirety.
[0079] Other targeting peptides are disclosed in the following: Co-expressed peptide receptors in breast cancer as a molecular basis of in vivo multireceptor tumour targeting. Jean Claude Reubi, Mathias Gugger, Beatrice Waser. Eur. J. Nucl Med. 2002, 29, 855-862, (includes NPY, GRP); Radiometal-Binding Analogues of Leutenizing Hormone Releasing Hormone PCT/US96/08695 (LHRH); PCT/US97/12084 (WO 98/02192) (LHRH); PCT/EP90/01169 (radiotherapy of peptides); WO 91/01144 (radiotherapy of peptides); and PCT/EPOO/01553 (molecules for the treatment and diagnosis of tumours), all of which are herein incorporated by reference in their entirety.
[0080] Additionally, analogs of a targeting peptide can be used. These analogs include molecules that target a desired site receptor with avidity that is greater than or equal to the targeting peptide itself, as well as muteins, retropeptides and retro-inverso-peptides of the targeting peptide. One of ordinary skill will appreciate that these analogs may also contain modifications which include substitutions, and/or deletions and/or additions of one or several amino acids, insofar that these modifications do not negatively alter the biological activity of the peptides described therein. These substitutions may be carried out by replacing one or more amino acids by their synonymous amino acids. Synonymous amino acids within a group are defined as amino acids that have sufficiently similar physicochemical properties to allow substitution between members of a group in order to preserve the biological function of the molecule. Synonymous amino acids as used herein include synthetic derivatives of these amino acids (such as for example the D-forms of amino acids and other synthetic derivatives), and may include, for example, those listed in the following Table. In the chart and throughout this application amino acids are abbreviated interchangeably either by their three letter or single letter abbreviations, which are well known to the skilled artisan. Thus, for example, T or Thr stands for threonine, K or Lys stands for lysine, P or Pro stands for proline and R or Arg stands for arginine.
Figure imgf000029_0001
[0081] Deletions or insertions of amino acids may also be introduced into the defined sequences provided they do not alter the biological functions of said sequences. Preferentially such insertions or deletions should be limited to 1, 2, 3, 4 or 5 amino acids and should not remove or physically disturb or displace amino acids which are critical to the functional conformation. Muteins of the peptides or polypeptides described herein may have a sequence homologous to the sequence disclosed in the present specification in which amino acid substitutions, deletions, or insertions are present at one or more amino acid positions. Muteins may have a biological activity that is at least 40%, preferably at least 50%, more preferably 60-70%, most preferably 80-90% of the peptides described herein. However, they may also have a biological activity greater than the peptides specifically exemplified, and thus do not necessarily have to be identical to the biological function of the exemplified peptides. Analogues of targeting peptides also include peptidomimetics or pseudopeptides incorporating changes to the amide bonds of the peptide backbone, including thioamides, methylene amines, and E-olefins. Also peptides based on the structure of a targeting peptide or its peptide analogues with amino acids replaced by N-substituted hydrazine carbonyl compounds (also known as aza amino acids) are included in the term analogues as used herein.
[0082] The targeting peptide may be attached to the linker via the N or C terminus or via attachment to the epsilon nitrogen of lysine, the gamma nitrogen or ornithine or the second carboxyl group of aspartic or glutamic acid.
[0083] Most preferably, the targeting peptide may be LHRH, insulin, oxytosin, somatostatin, NK-I, VIP, GRP, bombesin or any other hormone peptides known in the art, as well as analogs and derivatives thereof. For a discussion of other targeting peptides that are useful in the present invention, see, e.g., WO 04/062574, incorporated herein by reference in its entirety.
Constructs of the Present Invention
[0084] Table 1, Table 2 and Table 2a list conjugates of the present invention complexed with Gd. The complexes are divided into two "classes" based on the structure of the HSA binding portion of the conjugates that were evaluated.
[0085] The first class includes conjugates of the formula: chelator-linker Ll- hydrophobic AA, complexed with Gd and the tested compounds in this class are listed in Table 1. "Class 2" includes compounds having the formula: chelator-linkerLl-hydrophobic non-AA, complexed with Gd and the tested compounds in this class are listed in Tables 2 and 2a. The distinction between the linkerLl and the hydrophobic AA or hydrophobic non-AA is somewhat formalistic as it was understood that all of these might contribute to binding of the constructs to HSA. The tables also include and evaluation of the enhanced relaxivity of the constructs. The Examples provide details regarding the synthesis, screening and evaluation of representative compounds of the two classes.
[0086] The following explains the column headings in Table 1 and Table 2:
[0087] Chelate class refers to the type of chelating function that is present in the compound:
S stands for standard, and ER stands for the chelating moieties expected to provide enhanced
Tl relaxivity by comparison with the standard chelating function. Not all cases provided enhanced Tl relaxivity, by comparison with their standard chelator analogs..
[0088] The parameter gi refers to the relaxation gain, which is the ratio of the Tl relaxivity of the substance in a solution of HSA called Seronorm™ (commercially available from
Ny corned- Amersham) to the relaxivity of the same compound in water at a specified concentration. Values close to 1 indicate that no binding to protein is occurring and that the compound is unsuitable. On the other hand, higher values (generally, about 3 and above) are more desirable because they highlight, in an MRI image, the bloodstream (intravascular compartment) due to the much higher Tl relaxivity (which is the source of the MRI signal) when bound to the HSA in the blood.
[0089] The parameter rl§ is the Tl relaxivity of the substance in a solution of human serum albumin called Seronorm™ (commercially available from Ny corned- Amersham).
[0090] The parameter rlw is the Tl relaxivity of the compound in water, where no protein is present.
[0091] The parameter Fb is the fraction of the compound bound to human serum albumin
(HSA) under standard conditions of defined concentration and temperature. A value of 0.90 indicates that 90% of the molecules of the test compound are bound to HSA. A higher fraction bound (Fb) means that more of the compound should provide the higher signal characteristic of the bound molecule due to immobilization of the chelating function, or significant slowing of the chelating function upon binding of the chelate-HSA binding ligand to the HSA. Compounds in Class 1 and Class 2 were evaluated, and the results are as follows. The abbreviations used in these tables and throughout the application are known to those skilled in the art and/or set forth herein, particularly in the sections regarding chealators, linking groups and hydrophobic groups.
Table 1: Gd Complexes of Conjugates of the present invention in Class 1: Chelator- Linker-Hydrophobic Amino Acid
Figure imgf000031_0001
Figure imgf000032_0001
Table 2: Gd Complexes of Conjugates of the present invention in Class 2: Structure Chelator-Linker-Hydrophobic Acyl Group
Figure imgf000032_0002
Figure imgf000033_0001
[0092] The following shows the hydrophobic R groups of carboxylic acids that were selected for preparation and testing as the R (hydrophobic non-amino acid) portion of conjugates of the invention :
Structures of Selected Hydrophobic R Groups Constituting the Class 2
Compound Library
GENERAL STRUCTURE
Figure imgf000034_0001
Figure imgf000034_0002
Figure imgf000034_0003
8 10 11 12
Figure imgf000034_0004
13 14 15 16 17 18
*
Figure imgf000034_0005
19 20 21 22 23
Figure imgf000034_0006
24 25 26 27 28
* Indicates point of attachment of R in the general structure.
[0093] Table 2b shows the primary screening results for the Gd complexes of conjugates of the invention in Class 2 with the structure chelator-linker-hydrophobic carboxylic acid. In particular, the listed conjugates of the general structure DO3A10CM-G-K-hydrophobic carboxylic acid were prepared and evaluated. The "Group Number" refers to the groups as labeled in the chart above.
Figure imgf000035_0001
Figure imgf000036_0001
I ND = not determined
[0094] Conjugates DO3A10CM-D-a-B; DO3A10CM-G-a-B(Meg); DO3 Al OCM-G-L-B; CM-DOTMA-a-B; CE-DTP A-a-B; 7MP-DO3A-G-a-B and CM-DOTA-a-B, optionally conjugated to a second linker L2 and/or a targeting vector Q are preferred. CM-DOTMA-a-B; CE-DTP A-a-B; 7MP-DO3A-G-a-B and CM-DOTA-a-B, optionally conjugated to a second linker L2 and/or a targeting vector Q are particularly preferred. As described herein such conjugates may be complexed with a metal, or a paramagnetic metal such as for example, Gd, or others described above or with a radionuclide from amongst those described above. [0095] Compounds of the invention with fractions bound to serum of at least 0.87 are particularly preferred. A higher fraction bound to serum will result in a longer lifetime in blood, thus providing a more persistant signal. Similarly, compound so f the invention with higher relaxivity values are also particularly preferred. Higher relaxivity value will result in more signal for a given compound after administration. Table 6 in Example 8 shows fraction bound to serum and relaxivity data collected for representative compounds of the present invention compared to a reference compound, MS-325, whose structure is presented below. As shown in Table 6, Gd-(TE-DOTA)-aB Meg provides a relaxivity (ri) in Seronorm™ (HSA) of 42.09, well above the comparator MS-325 (T1 Seronorm = 36.57). Such a high relaxivity will provide more signal for a given compound after administration. Furthermore, Gd-(TE-DOTA)-aB Meg provides an Fb of 0.996, well above the Fb of the comparator MS- 325 (Fb = 0.87)
MS-325 (Gadofosveset Trisodium)
Figure imgf000037_0001
MS-325
[0096] As further shown in Table 6, the following compounds each provide higher bound fractions than the MS325 comparator: Gd-( 10CM-DO3 A)-G-G- W-(Nin-2,4,6- trimethylbenzenesulfonyl)-O"Meg; Gd-(10CM-DO3A)-aaB a; Gd-(10CM-DO3A)-aak a,b; Gd-(10CM-DO3 A)-DDk a; Gd-(10CM-DO3 A)-EEB a,b; Gd-(10CM-DO3A)-Pak a,c; Gd- (10CM-DO3A)-Pak a,b,c; Gd-(10CM-DO3A)-Dak a,b meg; Gd-(10CM-DO3 A)-DDj a,c; Gd-(10CM-DO3 A)-DDX c; Gd-(10CM-DO3 A)-PaJ a,c; Gd-(10CM-DO3A)-aaX; Gd- (10CM-DO3 A)-GGm Meg; Gd-(10CM-DO3 A)-Ga0 Meg; Gd-(10CM-DO3 A)-GGo Meg a; Gd-(10CM-DO3 A)-Pa0 Meg; Gd-(10CM-DO3A)-Pam Meg; Gd-(10CM-DO3A)-Gam Meg a; Gd-(10CM-DO3A)-aao Meg a; Gd-(10CM-DO3A)-aam Meg a;Gd-(10CM-DO3A)-Gan Meg; Gd-(10CM-DO3A)-Ga-(p-Cl)-Bip; Gd-(CEMP-DO3 A)-GaB; Gd-(CEMP-DO3A)-aB Na; Gd-4-CEMP-DTTA-GaB Meg; Gd-(CE-DTPA)-GaB 3Meg; Gd-(CE-DTP A)-aB 3Na; Gd-(CE-DTPA)-aB 3Meg; Gd-(CM-DOTA)-aB Meg; Gd-(CM-DOTA)-aB Na; Gd-(7MP- DO3A)-GaB; Gd-(7MP-DO3A)-GaB Meg; Gd-(7MP-CM-DO3A)-aB Meg; Gd-(TE- DOTA)-aB Meg; Gd-(10CM-DO3A)-K-(l-fluorenecarboxamido)O- Na+; Gd-(IOCM- DO3A)-K-(l-fluorenecarboxamido)- OH Meg; [(Fl-l)-K{G-[Gd-(10CM-DO3A)]}- OH](Meg); and [(Fl-l)-K[G-{Gd-(10CM-DO3A)]}-O-](Na+).
Uses of Conjugates of the Invention
[0097] The conjugates of the present invention are useful for to detect and treat various medical conditions. For example, the conjugates of the present invention can be employed for diagnostic imaging. In particular, because the conjugates bind a blood protein such as HSA they are useful for prolonged imaging of the intravascular space, or simply the vasculature, because of their ability to be confined to that region for longer periods of time. Thus compounds of the present invention are useful for imaging the vascular space by, for example, MRI or nuclear medicine imaging, depending on the type of metal complexed with the chelator. Methods of the invention include the steps of administering to a patient a pharmaceutical preparation that includes a conjugate of the invention complexed with a metal or a radionuclide and imaging the compound after administration to the patient. In a preferred embodiment, the imaging step includes magnetic resonance imaging, or nuclear imaging. In these methods, the administering step may include, e.g., inhalation, transdermal absorption, intramuscular injection, subcutaneous injection, intravenous injection, intraperitoneal injection, intraarterial injection or parenteral administration. [0098] Even in the case where a fraction of the compound is in the extravascular space, it is still possible to image the vasculature, because the extravascular space contains little HSA. Therefore, for conjugates useful for MRI (e.g. those complexed with a paramagnetic metal ion ), the Tl relaxivity, rl, of the compound in the extravascular space will be similar to that found in water, whereas the rl of the compound in the vascular space, which contains HSA, will be high because the compound is bound to HSA in that region. [0099] In one embodiment, conjugates of the present invention which are useful for MRI (e.g. those that are complexed with a paramagnetic metal ion) are useful as CVMRA agents in therapeutic and diagnostic applications. The invention further includes novel methods for preparation of such agents.
[00100] In one embodiment, the conjugates of the present invention useful for MRI exhibit superior relaxivity. The term rls is defined as the relaxivity of the compound measured in Seronorm™ (a commercially available preparation of human serum albumin) at 20 MHz in mM'V1. The term rlw is defined as the relaxivity of the compound measured in water at 20 MHz in mM'V1. Agents of the invention were shown to have superior properties using the methods of parallel synthesis and mass primary screening of candidate compounds, as described in the Examples.
[00101] The total signal in blood symbolized as Sbiood is a function of the relaxivity- concentration product for the bound and unbound test compound in the blood. Expressing this as a function of rlw gives:
Sbiood = (l-Fb)*rlw*Cblood+ gi*rlw*Fb*Cblood (I) where:
• Fb is the fraction of the construct bound to protein in blood
• rlw is the Tl relaxivity of the test compound measured in water at a standard concentration of the test compound
• rls is the Tl relaxivity of the test compound measured in Seronorm solution at a Standard concentration of the test compound
• Cbiood is the concentration of the test compound in blood
• gi is the Standard relaxation gain or the relaxation enhancement defined as the ratio rls/rlw under a defined standard of concentration and temperature.
[00102] The first term of the addition equation, (l-Fb)*rlw*Cbiood, gives the contribution of the unbound compound to the total signal in blood. The second term, gi*rlw*Fb*Cbiood, which contains the multiplier gi, gives the contribution of the HSA-bound compound to the total signal in blood. It can be seen that when Fb is high, most of the signal in the blood is due to the bound fraction of the compound. This signal is magnified by the factor of gi (the relaxation gain). This parameter is also referred to as the enhanced relaxivity or relaxation enhancement which is realized upon binding of the contrast agent to the macromolecule. In the regime of MRI imaging field strengths generally employed (0.3 T to 1.5 T) this increase of relaxivity is realized by a significant increase in the rotational correlation time τr, which has a effect dominant effect on the Tl relaxation rate of protons by the gadolinium atom in the MRI contrast agent.
[00103] For example, if the compound is 80% bound (Fb = 0.8) to HSA in blood, most of the gadolinium atoms in the solution and their associated bound water molecules are rotating at a significantly reduced rate (longer τr). This leads to an increase in the relaxation rate of the associated water and the water in the inner and out sphere of the gadolinium atoms of the bound chelate. If the relaxation gain gi is assumed to be 3.0, which is a modest value (see Table 1, infra) and rlw = 6 (an arbitrarily chosen value), substitution in the equation above gives:
Sbiood = (l-0.8)*6*Cblood + 3.0*6*0.8*Cblood
Further combining terms gives:
Sbiood = 1 -2 Cblood + 14.4 Cblood = 15.6Cblood
[00104] Now the ratio of the signal due to the bound fraction of the test molecule in blood to the unbound fraction is 14.4/1.2 = 12 (the concentration terms cancel). By comparison, the available signal with a compound of the same concentration in blood that does not bind HSA and possesses the same rlw is:
Sbiood = (l -0)*rlw*Cbiood
because Fb is 0 (the compound is 0% bound) and the second term is therefore zero.
Hence,
Sbiood = 6* Cblood
[00105] The ratio of the signal for the two compounds is, therefore:
^ protein binding compound/ ^ protein non-binding compound ~~ 1 J -J^blood/θCblood ~~ 2. JOJ Or 2jδ /o
This ratio is time dependent and, except in the period immediately after administration, is a likely underestimate (the concentration of the non-protein binding compounds in blood is reduced versus their protein binding congeners with time).
[00106] Thus, it can be seen that there are many advantages of the blood protein-bound conjugates of the invention, including confinement to the vascular compartment for a longer period, and the higher signal strength available due to protein binding of the chelate functionalized protein binding molecule.
[00107] In a preferred embodiment of the present invention, the compound is first administered, and clearance of unbound compound from blood is then allowed to occur before the vascular space is imaged. However, this process is not always strictly necessary. In the case where imaging is conducted after a waiting period, the administrator must account for the fraction of compound that may migrate to the extravascular space, which also contains HSA.
[00108] If the concentration of the compound in the extravascular space were to be equal to that in the intravascular space (an unlikely circumstance), both areas would provide signal in the MRI, and the ability to distinguish the extravascular space from the intravascular space might thus be diminished. However, even in such a case, signal intensity in the intravascular space would still be greater in most instances. This is because the concentration of HSA in the extravascular spaces (which has been measured) is generally between 10-16 g/L, except in the gut, where it is 25-30 g/L. These values should be compared with the value of ~25g/L in the vascular space (which includes both the volume of plasma and the other blood components, e.g., erythrocytes and white cells). Therefore, independent of the fractional distribution of the compound in the vascular space versus the extravascular space, the present invention provides an advantage for signal strength in the blood due to the higher concentration of HSA in blood, except in the case of the gut.
[00109] Imaging according to the present invention should preferably be conducted at time point early enough to allow binding of the constructs of the present invention to intravascular HSA (on the order of one circulation time, i.e. 1-2 min) and clearance of unbound compound to the extravascular space (Ty2 ~ 16 min). Both of these rates of clearance are much faster than the rate of clearance of intravascular HSA (with or without the molecule bound) to the extravascular space (4%/h).
[00110] Thus, utilizing the conjugates of the invention diagnostic imaging methods, and in particular, MRI imaging, can be employed to provide morphological imaging of the vasculature for detection of a variety of conditions, including, for example, vascular abnormalities, stenosis or even sites of angiogenesis that can be characterized. The morphology and permeability of tumor vasculature can be similarly assessed in accordance with the present invention. The constructs of the present invention are applicable to MRI; they are also applicable to other forms of imaging such, as e.g., ultrasound imaging, wherein metal chelators facilitate the process, and/or light imaging; and additionally to other diagnostic and therapeutic applications that may be contemplated by those skilled in the art. [00111] The conjugates of the present invention are applicable to other modes of diagnostic imaging as well as to therapeutic applications. For example, where the conjugates of the invention are complexed with metals having different properties than, for example, paramagnetic metals, other applications can be contemplated by those of ordinary skill in the art. For example, the HSA- binding portion of the molecule may be used to prolong the residence time in blood of the constructs such as, e.g., targeted pharmaceuticals, including therapeutics and diagnostics. Also, radiometals may be chelated by the chelating function and a construct that utilizes the HSA binding sequence and the chelator bound to a targeting function which directs the construct to tumor sites or to other diseased tissues. The radiometal may also be useful for diagnosis or radiotherapy. Because the conjugates of the invention bind to a blood protein such as HAS, the blood residence time is prolonged, allowing more opportunity for binding of the radiopharmaceutical to the target site. [00112] The present invention also contemplates the delivery of other compounds such as, e.g., chemotherapeutics. For example, a conjugate of the invention which includes a targeting vector that targets a specific type of tumor can be linked to a chemotherapeutic. Thus, the conjugate, by virtue of the fact that it binds to the intravascular HSA, can prolong the blood residence time of the construct. Such an increase of blood residence time is advantageous for many reasons; for example, it allows more opportunity for binding of the conjugate to the target site because the HSA-bound construct is essentially in a depot form, where it is slowly released to the target, as opposed to being cleared in a first pass clearance. [00113] The constructs of the present invention may also be used to target genetic material to specific cells. The conjugates of the invention may further include genetic material and may be used to localize genetic material to cells or tissue containing the desired target. Thus such constructs may be useful in gene therapy. The genetic material may include nucleic acids, such as RNA or DNA, of either natural or synthetic origin, including recombinant RNA and DNA and antisense RNA and DNA. Types of genetic material that may be used include, for example, genes carried on expression vectors such as plasmids, phagemids, cosmids, yeast artificial chromosomes (YACs) and defective or "helper" viruses, antigene nucleic acids, both single and double stranded RNA and DNA and analogs thereof, such as phosphorothioate and phosphorodithioate oligodeoxynucleotides. Additionally, the genetic material may be combined, for example, with lipids, proteins or other polymers. Delivery vehicles for genetic material may include, for example, a virus particle, a retroviral or other gene therapy vector, a liposome, a complex of lipids (especially cationic lipids) and genetic material, a complex of dextran derivatives and genetic material, etc. [00114] The present invention is directed, in another embodiment, to novel methods for preparing and using such conjugates in diagnostic and therapeutic applications. Magnetic Resonance Imaging (MRI)
[00115] MRI contrast agents including conjugates of the invention may be used in the same manner as conventional MRI contrast reagents. When imaging target-containing tissue such as, for example, a site of angiogenesis, certain MR techniques and pulse sequences may be preferred to enhance the contrast of the site to the background blood and tissues. These techniques include (but are not limited to), for example, black blood angiography sequences that seek to make blood dark, such as fast spin echo sequences (see, e.g., Alexander et al., Magnetic Resonance in Medicine, 40(2): 298-310 (1998)) and flow-spoiled gradient echo sequences (see, e.g., Edelman et al., Radiology, 177(1): 45-50 (1990)). These methods also include flow independent techniques that enhance the difference in contrast, such as inversion-recovery prepared or saturation-recovery prepared sequences that will increase the contrast between target containing tissue, such as an angiogenic tumor, and background tissues. Finally, magnetization transfer preparations may also improve contrast with these agents (see, e.g., Goodrich et al., Investigative Radiology, 31(6): 323-32 (1996)). [00116] The labeled reagent may be administered to the patient in the form of an injectable composition. The method of administering the MRI contrast agent is preferably parenterally, meaning intravenously, intraarterially, intrathecally, interstitially, or intracavitarilly. For imaging active angiogenesis, intravenous or intraarterial administration is preferred.
[00117] For MRI, it is contemplated that the subject will receive a dosage of contrast agent sufficient to enhance the MR signal at the target (e.g. , a site of angiogenesis) at least 10%. After injection of the composition including the MRI reagent, the patient is scanned with a MRI machine to determine the location of any sites containing the target. In therapeutic settings, upon target localization, a cytotoxic or therapeutic agent can be immediately administered, if necessary, and the patient can be subsequently scanned to determine the location of any targeted sites.
Nuclear Imaging (Radionuclide Imaging) and Radiotherapy
[00118] The present invention may also be useful in the field of nuclear medicine.
Conjugates of the present invention may be complexed with a radioactive metal or other radionuclide appropriate for scintigraphy, SPECT or PET imaging or with a radionuclide appropriate for radiotherapy.
[00119] For use as a PET agent, a conjugate of the present invention may be complexed with one of the various positron emitting metal ions, such as 51Mn, 52Fe, 60Cu, 68Ga, 72As, 94mTc, or 110In. The constructs of the present invention can also be labeled by halogenation using radionuclides, such as 18F, 124I, 125I, 131I, 123I, 77Br, and 76Br. Preferred metal radionuclides for scintigraphy or radiotherapy include 99mTc, 51Cr, 67Ga, 68Ga, 47Sc, 51Cr, 167Tm, 141Ce, 111In, 168Yb, 175Yb, 140La, 90Y, 88Y, 153Sm, 166Ho, 165Dy, 166Dy, 62Cu, 64Cu, 67Cu, 97Ru, 103Ru, 186Re, 188Re, 203Pb, 211Bi, 212Bi, 213Bi, 214Bi, 105Rh, 109Pd, 117mSn, 149Pm, 161Tb, 177Lu, 198Au and 199Au. The choice of metal or halogen will be determined based on the desired therapeutic or diagnostic application. For example, for diagnostic purposes the preferred radionuclides include 64Cu, 67Ga, 68Ga, 99mTc, and 111In. For therapeutic purposes, the preferred radionuclides include 64Cu, 90Y, 105Rh, 111In, 117mSn, 149Pm, 153Sm, 161Tb, 166Dy, 166Ho, 175Yb, 177Lu, 1867188Re, and 199Au.
[00120] Preferably, a radioactive lanthanide, such as, for example, 177Lu, 90Y, 153Sm,
111In, or 166Ho is used with DOTA or D03A in radiopharmaceutical conjugates of the invention.
[00121] 99mTc is preferred for diagnostic applications because of its low cost, availability, imaging properties, and high specific activity. The nuclear and radioactive properties of 99mTc make this isotope an ideal scintigraphic imaging agent. This isotope has a single photon energy of 140 keV and a radioactive half- life of about 6 hours, and is readily available from a 99Mo-99mTc generator. Other appropriate radionuclides for preferred embodiments of the present invention will be easily determined by one of ordinary skill in the art.
[00122] The metal radionuclides may be chelated by any of the chelating moieties discussed herein, supra. One skilled in the art can select an appropriate chelator for a desired radionuclide.
[00123] Complexes of radioactive technetium are particularly useful for diagnostic imaging and complexes of radioactive rhenium are particularly useful for radiotherapy. In forming a complex of radioactive technetium with the reagents of this invention, the technetium complex, preferably a salt of 99mTc pertechnetate, is reacted with the reagent in the presence of a reducing agent. Preferred reducing agents are dithionite, stannous and ferrous ions; the most preferred reducing agent is stannous chloride. Means for preparing such complexes are conveniently provided in a kit form comprising a sealed vial containing a predetermined quantity of a reagent of the invention to be labeled and a sufficient amount of reducing agent to label the reagent with 99mTc. Alternatively, the complex may be formed by reacting a conjugate of the invention with a pre-formed labile complex of technetium and another compound known as a transfer ligand. This process is known as ligand exchange and is well known to those skilled in the art. The labile complex may be formed using such transfer ligands as tartrate, citrate, gluconate or mannitol, for example. Among the 99mTc pertechnetate salts useful with the present invention are included the alkali metal salts such as the sodium salt, or ammonium salts or lower alkyl ammonium salts.
[00124] Preparation of the complexes of the present invention where the metal is radioactive rhenium may be accomplished using as starting materials rhenium compounds wherein the metal is in the +5 or +7 oxidation state. Examples of compounds in which rhenium is in the Re(VII) state are NH4ReO4 or KReO4. Re(V) is available as, for example,
[ReOCl4](NBu4), [ReOCl4](AsPh4), ReOCl3(PPh3)2 and as ReO2(pyridine)4 + (Ph is phenyl;
Bu is n-butyl). Other rhenium reagents capable of forming a rhenium complex may also be used.
[00125] Radioactively-labeled scintigraphic imaging agents provided by the present invention must contain a suitable amount of radioactivity. In forming 99mTc complexes, it is generally preferred to form radioactive complexes in solutions containing radioactivity at concentrations of from about 0.01 millicurie (mCi) to 100 mCi per mL.
[00126] Generally, the unit dose to be administered has a radioactivity of about 0.01 mCi to about 100 mCi, preferably 1 mCi to 20 mCi. The solution to be injected at unit dosage is from about 0.01 mL to about 10 mL.
[00127] After injection of the radionuclide imaging agent into the patient, a PET camera or a gamma camera calibrated for the gamma ray energy of the nuclide incorporated in the imaging agent is used to image areas of uptake of the agent and quantify the amount of radioactivity present in the site. Imaging of the site in vivo can take place in a matter of a few minutes. However, imaging can take place, if desired, in hours or even longer, after the radiolabeled peptide is injected into a patient. In most instances, a sufficient amount of the administered dose will accumulate in the area to be imaged within about 0.1 of an hour to permit the taking of scintiphotos.
[00128] The radiotherapeutic and radiodiagnostic compositions of the invention can include physiologically acceptable buffers, and can require radiation stabilizers to prevent radiolytic damage to the compound prior to injection. Radiation stabilizers are known to those skilled in the art, and may include, for example, para-aminobenzoic acid, ascorbic acid, gentistic acid and the like.
[00129] The present invention also contemplates a single, or multi-vial kit that contains all of the components needed to prepare the radiopharmaceuticals of this invention, other than the radionuclide. A single-vial kit preferably contains a conjugate of the invention, a source of a stannous salt (if reduction is required, e.g., when using technetium), or other pharmaceutically acceptable reducing agent, and is appropriately buffered with pharmaceutically acceptable acid or base to adjust the pH to a value of about 3 to about 9. The quantity and type of reducing agent used would depend highly on the nature of the exchange complex to be formed. The proper conditions are well known to those who are skilled in the art. It is preferred that the kit contents be in lyophilized form. Such a single vial kit may optionally contain labile or exchange ligands such as glucoheptonate, gluconate, mannitol, malate, citric or tartaric acid and can also contain reaction modifiers such as diethylenetriamine-pentaacetic acid (DPTA), ethylenediamine tetraacetic acid (EDTA), or α-, β-, or γ-cyclodextrin that serve to improve the radiochemical purity and stability of the final product. The kit may also contain stabilizers, bulking agents such as mannitol, that are designed to aid in the freeze-drying process, and other additives known to those skilled in the art.
[00130] A multi-vial kit preferably contains the same general components but employs more than one vial in reconstituting the radiopharmaceutical. For example, one vial may contain all of the ingredients that are required to form a labile Tc(V) complex on addition of pertechnetate (e.g., the stannous source or other reducing agent). Pertechnetate is added to this vial, and after waiting an appropriate period of time, the contents of this vial are added to a second vial that contains the conjugate of the invention, as well as buffers appropriate to adjust the pH to its optimal value. After a reaction time of about 5 to 60 minutes, the complexes of the present invention are formed. It is advantageous that the contents of both vials of this multi-vial kit be lyophilized. As above, reaction modifiers, exchange ligands, stabilizers, bulking agents, etc. may be present in either or both vials.
Pharmaceutical Compositions and Administration
[00131] Conjugates of the invention may be administered by any convenient route customary for diagnostic or therapeutic agents; e.g. , parenterally, enterally or intranasaly, and preferably by infusion or bolus injection, or by depot or slow release formulation. In a preferred embodiment, the composition may be formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Other pharmaceutically acceptable carriers include, but are not limited to, sterile water, saline solution, buffered saline (including buffers like phosphate or acetate), alcohol, vegetable oils, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, paraffin, etc. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as, e.g., lidocaine to ease pain at the site of the injection; and further may include preservatives, stabilizers, wetting agents, emulsifϊers, salts, lubricants, etc. as long as they do not react deleteriously with the active compounds. Similarly, the composition may comprise conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for parenteral, enteral or intranasal application which do not deleteriously react with the active compounds. Generally, the ingredients will be supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as, e.g. , an ampoule or sachette indicating the quantity of active agent in activity units. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade "water for injection" or saline. Where the composition is to be administered by injection, an ampoule of sterile water for injection or saline may be provided so that the ingredients may be mixed prior to administration.
EXAMPLES
[00132] In the following examples, the synthesis and evaluation of several representative conjugates of the general formula chelator-linkerLl-hydrophobic compound is included. The conjugates and their Gd complexes were prepared and screened for enhanced relaxivity (ER). Points of interest for the experiments were the hydrophobic compound/HSA binder, the linker, and the gadolinium chelator.
EXAMPLE 1 Synthesis and Screening of Gd-(DO3A10CM)-Ga(0B Meg (Class 1)
[00133] The compound (final compound referred to as compound (12)) was prepared as follows:
Figure imgf000047_0001
[00134] To a solution of JV-t-butoxycarbonyl-L-biphenylalanine (1) (10.00 g, 30.55 mmol) in methanol-water (100 mL, 1 : 1) was added cesium carbonate (9.95 g, 30.54 mmol), and the mixture was stirred for 20 min at room temperature. The reaction mixture was evaporated to dryness and the evaporation was continued with dry DMF (20 mL, twice) to dryness. The solid material was dissolved in dry DMF (100 mL), and benzyl bromide (5.75 g, 33.62 mmol) was added dropwise from an additional funnel. After the addition, the reaction mixture was stirred at room temperature for 15 h. The salts were removed by filtration, and the filtrate was evaporated to a paste which was taken-up in ethyl acetate (200 mL). The organic layer was washed with water (2 x 200 mL), dried (Na2SO4) and evaporated to a semi-solid which was then triturated with hexane to provide a colorless solid. The material was filtered, washed with hexane and dried to providel2.3 g (96.4% yield) of N-t- Butyloxycarbonyl-L-biphenylalanine benzyl ester (2). [00135]
HPLC: tR 22.15 min Column: YMC C18, 4.6 mm x 250 mm, S-IOP μm, 12θA pore; Eluents: A: H2O (0.1% TFA), B: CH3CN (0.1% TFA); Elution: Initial condition: 45% B, then linear gradient to 95% B over 25 min; Flow rate: 1.0 mL/min, Detection: UV @230 nm).
1H-NMR (500 MHz, CDCl3): δ 1.42 (s, 9 H, t-C4H9), 3.15 (m, 2 H, CH2CH), 4.67 (m, 1 H, CH), 5.04 (d, 2 Η, NH), 5.18 (dd, 2 Η, CH2C6H5) and 7.08-7.55 (m, 14 H, ArH).
MS: 432.2 (M+Η)+.
Figure imgf000048_0001
[00136] To a solution of N-t-Butyloxycarbonyl-L-biphenylalanine benzyl ester (2) (10 g, 23.17 mmol) in CH2Cl2 (50 mL) was added phenol (1.5 g) followed by trifluoroacetic acid (50 mL), and the mixture was stirred at room temperature for 30 min. Volatiles were removed by evaporation and the paste thus obtained was dissolved in ether (50 niL), and allowed to stand at room temperature. A white crystalline solid separated out and was filtered, washed with ether and dried. This provided 9.89 g (96% yield)of L-biphenylalanine benzyl ester trifluoroacetate salt (L-Bip-OBzl»TFA) (3).
HPLC: tR 8.12 min Column: YMC C18, 4.6 mm x 250 mm, S-IOP μm, 12θA; Eluents: A: H2O (0.1% TFA), B: CH3CN (0.1% TFA);Elution: Initial condition: 45% B, linear gradient to 95% B over 25 min; Flow rate: 1.0 niL/min, Detection: UV @230 nm.
1H-NMR (500 MHz, DMSO-de): δ 3.05-3.21 (2dd, 2 H, CH2CH), 4.41 (t, 1 H, CH2CH),
5.12 (dd, 2 Η, CH2C6H5), 7.21-7.65 (m, 14 H, ArH) and 8.58 (bs, 3Η, NH3 +).
MS: 332.2 (M+H)+.
Figure imgf000049_0001
[00137] A mixture of L-Bip-OBzl»TFA (3) (9.00 g, 20.21 mmol), HATU [O-(7- azabenzotriazol- 1 -yl)- 1 , 1 ,3 ,3-tetramethyluronium hexafluorophosphate, PerSeptive Biosystems] (11.52 g, 30.32 mmol) and trans-4-N-^butoxycarbonylaminomethyl- cyclohexanecarboxylic acid (Boc-a) (5.72 g, 21.23 mmol) in dichloromethane (30 mL) was added diisopropylethylamine (8.00 g, 61.90 mmol), and the solution was stirred at room temperature for 2 h. The reaction mixture was then evaporated on a rotary evaporator to remove dichloromethane and the residue was taken-up in ethyl acetate (200 mL). The organic layer was washed sequentially with dilute HCl (0.05 N, 2 x 200 mL), NaHCO3 (5%, 2 x 200 mL) and water (2 x 200 mL). The organic layer was then dried (Na2SO4), filtered and evaporated to provide trαns-4-N-^butoxycarbonylaminomethylcyclohexane-carboxyl-L- biphenylalanine benzyl ester (Boc-a-Bip-OBzl) (4) (11.0 g ~ quantitative yield) as a light brown foamy solid. HPLC: tR 20.14 min (Column: YMC-C18, 4.6 mm x 250 mm, S-IOP μm, 12θA; Eluents: A: H2O (0.1% TFA), B: CH3CN (0.1% TFA);; Elution:I Initial condition: 45% B, then linear gradient to 95% B over 25 min; Flow rate: 1.0 mL/min, Detection: UV @ 230 nm).
1H-NMR (500 MHz, CDCl3): δ 0.92 (m, 2 H, cyclohex-CH2), 1.45 (s & m, 11 H, J-C4H9 & cyclohex-CH2), 1.60-2.09 (m, 6 H, cyclohex-CH2), 2.95 (m, 2 H, bip-CH2CH), 3.15 (m, 2 H, bocNHCH2), 4.55 (m, 1 H, bip-αH), 4.95 (m, 1 H, bocNH), 5.18 (m, 2 H, CH2C6H5), 5.92 (d, 1 H, CHNHCO) and 7.01-7.60 (m, 14 Η, ArH).
MS: 571.4 (M+Η)+
Figure imgf000050_0001
[00138] A solution of Boc-a-Bip-OBzl (4) (11.00 g, 20.20 mmol) in dichloromethane (50 mL) was treated with phenol (1.5 g) followed by trifluoroacetic acid (50 mL), and the mixture was stirred at room temperature for 30 min. The volatiles were removed by rotary evaporation and the residue was triturated with ether. The solid was filtered and washed with ether to provide trα/?5-4-aminomethylcyclohexanecarboxyl-L-biphenylalanine benzyl ester trifluoro acetate salt (a-Bip-OBzl»TFA) (5) in nearly quantitative yield (11.80 g) as a colorless solid..
HPLC: tR 9.50 min. Column: YMC C18 4.6 mm x 250 mm, S-IOP μm, 12θA; Eluents: A: H2O (0.1% TFA), B: CH3CN (0.1% TFA); Elution: Initial condition: 45% B, then linear gradient to 95% B over 25 min; Flow rate: 1.0 mL/min; Detection: UV @ 230 nm). 1H-NMR (500 MHz, CDCl3): δ 0.94 (m, 2 H, cyclohex-CH2), 1-45 (m, 2 H, cyclohex-CH2), 1.60-2.15 (m, 6 H, cyclohex-CH2), 2.75 (m, 2 H, bip-CH2CH), 3.15 (m, 2 H, bocNHCH2), 4.85 (m, 1 H, bip-αH), 5.18 (m, 2 H, CH2C6H5), 5.92 (d, 1 H, CHNHCO) and 6.91-7.71 (m, 14 Η, ArH).
MS: 471.3 (M+Η)+
HH
Figure imgf000051_0001
[00139] To a mixture of a-Bip-OBzMFA (5) (11.80 g, 20.20 mmol), Boc-Gly (5.31 g, 30.31 mmol), and HATU (11.52 g, 30.32 mmol) in dichloromethane (50 mL) was added N,N-diisopropylethylamine (7.88, 60.98 mmol). The mixture was stirred at room temperature for 2 h. The volatiles were removed on a rotary evaporator, and the residue obtained was taken up in ethyl acetate (200 mL). The organic layer was washed sequentially with dilute HCl (0.5 N, 2 x 150 mL), aqueous sodium carbonate (5%, 2 x 150 mL) and water (150 mL). The organic layer was then separated, dried (Na2SO4), filtered and evaporated on a rotary evaporator to provide N-£-Butoxycarboxylglycyl-£ra/?s-4- aminomethylcyclohexanecarbonyl-L-biphenylalanine benzyl ester (Boc-Gly-a-Bip-OBzl) (6) (12.60 g, quantitative yield) as an off-white foam .
HPLC: tR 16.33 min; Column: YMC C18 4.6 mm x 250 mm, S-IOP μm, 12θA; Eluents: A: H2O (0.1% TFA), B: CH3CN (0.1% TFA); Elution: Initial condition: 45% B, then linear gradient to 95% B over 25 min; Flow rate: 1.0 mL/min, Detection: UV @ 230 nm. 1H-NMR (CDCl3): δ 0.94 (m, 2 H, cyclohex-CH2), 1-45 (s & m, 12 H, C4H9 & cyclohex- CH2), 1.70-2.15 (m, 5 Η, cyclohex-CH2), 3.15 (m, 4 Η, bip-CH2CΗ & bocNHCH2), 3.75 (d, 2 Η, gly-CH2), 4.95 (m, 1 Η, bip-αH), 5.18 (m, 3 Η, bocNH & CH2C6H5), 5.92 (d, 1 H, CHNHCO), 6.32 (t, 1 Η, CH2CONHCH2) and 6.91-7.59 (m, 14 H, ArH).
MS: 628.6 (M+Η)+
BOCHN" —
Figure imgf000052_0001
[00140] A solution of Boc-Gly-a-Bip-OBzl (6) (12.5 g, 19.91 mmol) and phenol (0.5 g) in dichloromethane (50 mL) was treated with trifluoroacetic acid (50 mL) and stirred at room temperature for 30 min. The volatiles were removed and the residual trifluoroacetic acid was co-evaporated with chloroform to provide Gly-a-Bip-OBzl (TFA salt) (T) (13.00 g, quantitative yield) as a light brown foam. This was triturated and washed with ether, filtered and dried. [00141]
HPLC: tR 9.41 min; Column: YMC-C18, 4.6 mm x 250 mm, S-IOP μ, 12θA; Eluents: A: H2O (0.1% TFA), B: CH3CN (0.1% TFA); Elution: Initial condition: 45% B, then linear gradient to 95% B over 25 min; Flow rate: 1.0 mL/min, Detection: UV @ 230 nm).
1H-NMR (500 MHz, CDCl3): δ 0.88 (m, 2 H, cyclohex-CH2), 1.25 to 2.1 l(m, 8 Η, C4H9 & cyclohex-CH2 & CH), 3.12 (m, 4 Η, bip-CH2CΗ & CONHCH2), 3.79 (d, 2 Η, gly-CH2), 4.85 (m, 1 Η, bip-αH), 5.18 (m, 2 Η, CH2C6H5), 6.55 (d, 1 H, CHNHCO), 6.32 (t, 1 Η, CH2CONHCH2), 6.91-7.89 (m, 15 H, CH2CONHCH2 & ArH) and NH3 +.
MS: 528.3 (M+Η)+.
Figure imgf000053_0001
Figure imgf000053_0002
[00142] To a mixture ofGly-a-Bip-OBzl«TFA (7) (13.00 g, 20.32 mmol),
DO3A10CM-1 ,4,7-tris-f-butyl ester (8) (19.26 g, 30.48 mmol) and HATU (11.58 g, 30.48 mmol) in dichloromethane (50 mL) was added N,N-diisopropylethylamine (7.98 g, 60.98 mmol). The mixture was stirred at room temperature for 2 h. The volatiles were removed on a rotary evaporator and the residue was taken up in ethyl acetate (200 mL). The organic layer was washed sequentially with dilute HCl (0.05 N, 2 x 150 mL), aqueous sodium carbonate (5%, 2 x 150 mL) and water (150 mL). The organic layer was then separated, dried (Na2SO4), filtered and evaporated on a rotary evaporator to provide DO3A10CM-(l,4,7-tris- t-butyl ester)-glycyl-trα/?5-4-aminomethylcyclohexanecarbonyl-L-biphenyl-alanine benzyl ester (9) (19.80 g, 90.40 % yield) as an off-white foam.
TLC: Rf 0.42 (silica gel, 5% methanol in dichloromethane) HPLC: tR 13.44 min; Column: YMC-C18, 4.6 mm x 250 mm, S-IOP μm, 12θA; Eluents: A: H2O (0.1% TFA), B: CH3CN (0.1% TFA); Elution: Initial condition: 45% B, then linear gradient to 95% B over 25 min; Flow rate: 1.0 mL/min, Detection: UV @ 230 nm.
1H-NMR (500 MHz, CDCl3): δ 0.95 (m, 2 H, cyclohex-CH2), 1.48 ( m & s, 29 H, C4H9 & cyclohex-CH2), 1.75 to 2.11 (m, 6 H, cyclohex-CH2 & CH), 2.15 to 3.85 (m, 30 H, macrocyclic-H, bip-CH2CH, CONHCH2 and gly-CH2), 4.95 (m, 1 Η, bip-αH), 5.18 (2d, 2 Η, CH2C6H5), 6.05 (d, 1 H, CHNHCO), 6.55 (t, 1 Η, CH2CONHCH2), 6.85 (t, 1 H, NCH2CONH), 7.09-7.59 (m, 14 Η, ArH).
MS: 1082.7 (M+Η)+
Figure imgf000054_0001
DO3A10CM (tri-f-butyl ester)-Gly-a-Bip-OBzl (9) (19.5 g, 18.03 mmol) in ethanol (100 mL) was mixed with Pd-C catalyst (5 g, Degussa type, 10%) and hydrogenated on a Parr hydrogenator at 40 psi for 5 h. Filtration through Celite removed the catalyst, and the filtrate was evaporated to provide DO3A10CM (1,4,7-tris-f-butyl ester)-Gly-a-Bip-OH (10) (17.0 g, 95% yield) as an off- white solid.
HPLC: tR 8.10 min; Column: YMC C18, 4.6 mm x 250 mm, S-IOP μm, 12θA; Eluents: A: H2O (0.1% TFA), B: CH3CN (0.1% TFA);Elution: Initial condition: 45% B, linear gradient to 95% B in 25 min; Flow rate: 1.0 niL/min, Detection: UV @ 230 nm.
1H-NMR (500 MHz, CDCl3): δ 0.91 (m, 2 H, cyclohex-C/f2), 1.48 (m & s, 29 H, C4H9 & cyclohex-C/f2), 1.65 to 4.22 (m, 36 H, cyclohex-C/f2, macrocyclic-H, bip-CH2CH, CONHCH2 and gly-CH2), 4.75 (m, 1 Η, bip-αH), 6.51 (bd, 1 Η, CΗNHCO), 6.69 (bt, 1 Η, CH2CONHCH2), 7.12 (bt, 1 H, NCH2CONH), 7.23-7.57 (m, 9 Η, ArH).
MS: 992.7 (M+Η)+
[00143]
Figure imgf000056_0001
[00144] DOTA(tri-t-butyl ester)-Gly-a-Bip-OH (10) (17.0 g, 17.15 mmol) was treated with trifluoroacetic acid:water:phenol:triisopropylsilane (88:5:5:2) (Reagent B) (300 rnL) with stirring for 4 h under a nitrogen atmosphere. After removal of the solvent the paste thus obtained was triturated with ether (50 mL), and the mixture was centrifuged and the ether layer was decanted. This process was repeated twice to completely remove phenol. The resulting solid was dried to provide 13.5 g (95.5% yield) of DO3A10CM-Gly-a-Bip-OH as an off-white solid. The material, was purified by anion exchange (Bio-Rad, AG1-X2 resin, 100-200 mesh, formate form) by elution of the column with 4 L each of 0.1M, 0.2M and 0.3M aqueous formic acid followed by elution with 0.4M formic acid (~ 12 L) whereupon the desired compound was eluted (eluant monitored by UV detector at 230 nm). Product- containing fractions were combined and the volatiles were removed by rotary evaporation to provide a foam The foam was dissolved in water and freeze-dried to provide DO3A10CM- Glycyl-trans-4-aminomethylcyclohexanecarbonyl-L-Biphenylalanine (DO3A10CM-Gly-a- Bip-OH) (11) (12.2 g, 86.3% yield) DO3A10CM- as a colorless crystalline solid. HPLC: R, 39.35 min (Column: YMC-C18, 4.6 mm x 250 mm, S-IOP μm, 12θA; Eluents: A: H2O (0.1% TFA), B: CH3CN (0.1% TFA); Elution: Initial condition: 0% B, then linear gradient to 50% B in 50 min; Flow rate: 1.0 mL/min, Detection: UV @ 230 nm).
1H-NMR (500 MHz, DMSO-de): δ 0.82-2.08 (m, 10 H, cyclohex-CH2), 2.60-4.25 (m & s, 30 H, macrocyclic-H, bip-CH2CH, CONHCH2 and gly-CH2), 4.45 (m, 1 Η, bip-αH), 7.23- 7.67 (m, 9 Η, ArH), 8.15 (m, 2 Η, CΗNHCO & CH2CONHCH2) and 8.51 (bt, 1 H, CH2CONHCH2).
MS: 824.5 (M+H)+
resin column
Figure imgf000057_0001
11
[00145] DO3A10CM-Gly-a-Bip-OH (11) (6 g, 7.28 mmol) was dissolved in water (150 mL), gadolinium acetate tetrahydrate (4.43 g, 10.91 mmol) was added and the mixture was stirred for 30 min. The pH of the solution was adjusted to 4.50 with IN NaOH, and the mixture was stirred at ambient temperature for 16 h. The excess gadolinium was precipitated as gadolinium oxide by raising the pH of the reaction mixture to 8.50 (addition of 0.5N NaOH) and stirring overnight. The gadolinium oxide was filtered and the product-containing filtrate was treated with 0.1N HCl to lower the pH to 4.50. The product was purified in three portions by reverse phase HPLC as follows: A reversed phase C18 preparative column (Rainin, 41.4 x 250 mm, 8μ, 6θA), was equilibrated with water (no TFA). The sample was applied to the column, and the column was eluted with water-acetonitrile (8:2 v/v), and then a linear gradient elution from 20% acetonitrile to 50% acetonitrile in 60 min was performed. The fractions were analyzed on an analytical reversed phase Cl 8 column, and the fractions containing the product in >99.5% purity were pooled and freeze-dried to provide the Gd- DO3A10CM-G-a-B-OH as a colorless fluffy solid. The gadolinium chelate was converted into its meglumine salt by passing it through a cation exchange column (Bio-Rad, AG 50W- X8 resin, sulfonic acid, meglumine form). The fractions containing the product (detected by UV at 230 nm) were collected and lyophilized to provide Gd-DO3 Al OCM-GIy cyl-trans -4- aminomethylcyclohexanecarboxyl-L-Biphenylalanine (Gd-DO3A10CM-Gly-a-Bip- OH»Meg) (12) as a colorless fluffy solid. The entire sample purified in this manner provided 5.49 g (82% yield) of compound 12. [00146]
HPLC: tR 39.82 min; Column: YMC C18 4.6 mm x 250 mm, S-IOP μm, 12θA; Eluents: A: H2O (0.1% TFA), B: CH3CN (0.1% TFA); Elution: Initial condition: 0% B, linear gradient to 50% B in 50 min; Flow rate: 1.0 niL/min, Detection: UV @ 230 nm.
MS: 979.6 with an isotopic pattern for Gd (M+H)+ ; 196.0 (M+l)+ for meglumine
Elemental Analysis: Calcd for C4iH54N7OnGd'C7Hi7NO5'4.0 H2O: C, 46.29; H, 6.39; N, 9.00; Gd, 12.63.
Found: C, 46.13; H, 6.52; N, 8.78; Gd, 12.48; %Na: <0.02; % H2O: 6.12.
[00147] The product (12) was then screened using an IBM PC20-minispec relaxometer for relaxivity in water and Seronorm™, and for HSA binding by the centrifree micro partition method.
Materials and Methods
[00148] Determination of20rls, 20rlw and calculation ofgj32 Seronorm™ (Batch # 508471, Nycomed, distributed in the U.S. by Accurate Chemical and Scientific Corp., Westbury, NY) was reconstituted with 80% of the recommended volume of deionized, distilled water. 0.4 mL of 2.5 mM Gd-conjugate and 1.6 mL of Seronorm™ or water were added to an NMR Tube, 7.5 mm x 180mm, (Bruker, Spectrospin, Milton, Ontario, Canada, Cat# 022-0910) and mixed (Seronorm™ now diluted to 100% of the recommended volume). The Seronorm™/Gd-chelate-peptide and water-peptide mixtures were warmed for not longer than 20 min at 380C prior to the relaxivity measurement, so that the sample was at the same temperature as the PC20 relaxometer (also referred to as the NMR). Prior to measurement of sample Tl 's (in triplicate), the NMR signal was optimized (SOP 830-210 IBM PC/20 Multispec Relaxometer 12/18/97). Tl is also measured for a pure water sample and a fresh aqueous Seronorm™ sample. The average Tl for each Seronorm™/Gd-conjugate sample was used in the equation below to calculate relaxivity/mM in Seronorm™ rls. rls = (1/Tl sample in Seronorm™ - 1/Tl Seronorm™)/[Gd-conjugate] The average Tl for each water/peptide sample was used in the equation below to calculate water rl relaxivity/mM.
rlw = (1/Tl sample in water - 1/Tl water)/[Gd-conjugate-peptide]
Where [Gd-conjugate] is measured in the Seronorm™ sample by ICP.
The standard relaxation gain of a peptide in Seronorm™ is defined by the equation below: gi = rls / rlw
Determination of Fraction Bound to Seronorm™ by the Centrifree Method
After the PC20 relaxivity measurements, the samples were cooled to room temperature, the
Seronorm™/Gd-conjugate mixture was removed from the NMR tubes and 1 mL was placed in a Centrifree Micropartition Device (Millipore, Beverly, MA). The device was centrifuged at 500xg for 45 min in a fixed angle rotor (Beckman Model J2-21M, JA-20 rotor) and 0.5 mL was removed from below the filter for Gd measurement by ICP. The [Gd] in 0.5 mL of the uncentrifuged Seronorm™/peptide mixture was measured as a control. The fraction of Gd- chelate -peptide bound was calculated using the equation below:
Fraction Bound (Fb) = [Gd] control - [Gd]Seronorm™/Gd-conjugate) / [Gd] control
Results [00149] It was found that the peptidic moiety g\ycy\-trans-4- aminomethylcyclohexanecarbonyl - / - 4 - biphenylalanyl (GaB) is a useful linker- hydrophobic compound moiety, and this compound or its des-glycyl congener aB was appended to several gadolinium chelating systems in an attempt to provide a compound with high relaxivity.
[00150] The following were evaluated for Gd-(D(BAl OCM)-Ga(I)B Meg: mouse tolerance, MRA in rabbits, blood Tl and imaging in rabbits and plasma Tl lowering ex vivo in monkeys. The compound tested displayed an acceptable tolerance profile in mice (MLD = 3 mmol/kg). It is surmised that the buildup of too much hydrophobic functionality in a local region of the molecule will inevitably lead to a suboptimal tolerance profile. Results are shown below, in Table 3 :
Figure imgf000060_0001
b = 0 05 mmol/Kg dose, , e - 0 15 mM, g - 0 12 mM Fb s-Fb r-Fb m refer to the fraction bound in human, rabbit and monkey serum albumin respectively
[00151] The compound was optimized using ligand diversity, i.e., the d and / isomers of the compound were prepared as their meglumine salts. The d,l mixture was constituted from the individual compounds. HPLC purification gave the pure ligand which was converted to the chelate and purified by prep HPLC. The results of primary screening for the new compounds prepared using the strategy of ligand diversity are shown in Table 4.
Figure imgf000060_0002
3MLD = 3 0 mmol/Kg, MLD evaluation not conducted [00152] In each case the optimization strategy met its goal. The racemic compound
Gd-DO3A10CM-G-a-(d,/)-Bip displayed gl5 rls and rlw values essentially identical to those found for either the d- or 1-isomer alone. This indicates that the binding of this compound does not appear to be chiroselective. Also indicated is that methods for synthesis of racemic amino acids can be employed to prepare the <i,/-Bip, thus reducing the cost of a Bip-based compound.
[00153] Optimization of the compound was further conducted via chelate diversity. A number of enhanced relaxivity gadolinium chelates were identified, which, when conjugated to HSA binding compounds, were hypothesized to provide higher relaxivity than Gd- DO3A10CM-monoamides. The structures of these are given below:
Chart 4. Enhanced Relaxivity (ER) Chelate Systems
Figure imgf000061_0001
CM-DOTMA CM-DOTA CEMP-DO3A 7MP-DO3A COO
Figure imgf000061_0003
Figure imgf000061_0002
7MP-CM-DO3A 4-CE-DTPA TE-DOTA 4-CEMP-DTTA
[00154] Results of the optimization are shown above, in Table 1 and below, in Table 5.
Figure imgf000061_0004
Figure imgf000062_0001
* neutralization of the Na salt to pH 4 5 was followed by prep HPLC
EXAMPLE 2 Synthesis and Screening of Gd-(DO3A10CM)-GK(Fl-9)-OH Meg (Class
2)
[00155] The strategy for preparing the lysine-based compounds required the use of the
Dde protecting group for the quasi-orthogonal deprotection of the ω-amino group of lysine (to which the hydrophobes would be appended). The compound was prepared as follows:
Fmoc-Lys-(Boc)-OBn-Nα-(9-Fluorenylmethoxycarbonyl)-Nω-(Boc)-lysine benzyl ester
A 250 mL round bottomed flask equipped with a magnetic stir bar and septum capped inlet was charged with water (30 mL), sodium bicarbonate (4.20 g, 50 mmol, 2.38 equiv) and tetrabutylammonium hydrogen sulfate (3.60 g, 10.62 mmol, 0.504 equiv). The mixture was stirred (gas evolution). Then dichloromethane (45 mL) was added to the stirred mixture followed by Fmoc-Lys-(Boc)-OH (9.87 g, 21.06 mmol, Advanced ChemTech) which was added in small portions over a 5 minute period. Then benzyl bromide (3.602 g, 21.06 mmol, Aldrich) was added via syringe dropwise over a 1 minute period. The mixture was stirred rapidly for 18h at ambient temperature using a vent needle in the septum cap to relieve pressure from evolved carbon dioxide. TLC analysis was performed using both dichloromethane/ethylacetate 9/1 v/v and dichloromethane/methanol 9/1 v/v; this showed that the reaction was essentially complete. (The former system allows us to detect the consumption of benzyl bromide (solvent front) and formation of the product (R/ = 0.5); the latter system allows us to detect the tetrabutylammonium salt of Fmoc-Lys(Boc)-OH
(Ry=0.3).)
The organic layer was separated and the aqueous layer was extracted with 50 mL of dichloromethane. The dichloromethane layers were combined, volatiles were removed and the residue was redissolved in 200 mL of ethyl acetate. The organic phase was washed with water (4 x 200 mL) to remove tetrabutylammonium salts (visible by development in the iodine chamber) and then dried (sat. brine, then MgSO4). The volatiles were removed to give a residue which was applied (as a solution in 60 mL of dichloromethane) to a 35 cm high x 54 mm wide column of Merck 9385 silica gel packed in dichloromethane. The column was eluted with dichloromethane (1300 mL), dichloromethane-ethyl acetate 95/5 v/v (1000 mL), dichloromethane-ethyl acetate 90/10 v/v (1000 mL) and then dichloromethane-ethyl acetate 85/15 v/v for the remainder of the elution. The fraction size was 50 mL and the desired compound was obtained in 48-84. Fractions 56-74 were taken as the heart cut. The volatiles were removed to provide a foam which was triturated with ether-hexanes to initiate precipitation. Then the volatiles were removed on the rotary evaporator employing an ambient temperature water bath and the resulting solid was pumped on at 7 mm Hg for two days. This provided the desired material (10.66 g) in 90.6% yield as an amorphous white solid.
This compound is known in the literature (Matsoukas, John M.; Hondrelis, John; Agelis, George; Barlos, Kleomenis; Gatos, Dimitrios; Ganter, Renee; Moore, Diana; Moore, Graham J. Novel Synthesis of Cyclic Amide-Linked Analogs of Angiotensins II and III. J. Med. Chem. (1994), 37(18), 2958-69.) but the 1H-NMR obtained for this sample is given for reference. Registry Number: 159541-28-3.
1H-NMR (500 MHz, DMSO-de} δ 7.86 (d, 2H), 7.82 (d, IH), 7.71 (d, 1.77H), 7.58 (app t, 0.25H), 7.4 (app t, 2H), 7.33 (m, 7H), 6.77 (app t, 0.66H), 5.13 (s with superimposed mult, 2H), 4.30 (m, 2H), 4.21 (t, IH), 4.07 (m, IH), 2.89 (m, 2H), 1.68 (m, 2H), 1.36 s, with superimposed multiplet). On larger scale after it is ascertained that all of the starting material is consumed the organic layer was separated and the volatiles were removed by rotary evaporation and pumping at high vacuum. Then the residue was taken up in ethyl acetate and extracted (6X) with water (to remove all tetrabutylammonium salts) and the organic layer was dried. The volatiles were removed and the residue triturated with ether-hexanes and vigorously agitated by stirring overnight. Filtration gave the desired material, NαBoc-Nω-Fmoc-Lys-OBn which was isolated in high yield and purity.
Nα-Fmoc-Lys-OBn»TFA
Nα-(9-Fluorenylmethoxycarbonyl)-Nω-(Boc)-lysine benzyl ester (10.5 g, 18.79 mmol) was dissolved in CF^Cb/anisole 45/5 v/v (50 mL) with stirring. To this was added trifluoroacetic acid (50 mL) and the mixture was stirred 1.5 h at ambient temperature. The volatiles were removed on the rotary evaporator and then by pumping at 0.1 mm Hg for 0.5 h. Then anhydrous diethyl ether (250 mL) was added, the mixture was agitated and the side of the flask was scratched with the oil using a glass rod. This led to formation of a solid which was broken up and well agitated. The flask was cooled in the freezer for 1 h and the solid was collected. Pumping overnight removed residual ether. This gave 10.53 g (97.87% yield) of the desired product as a white solid. The HCl salt (which we have prepared on a small scale previously) is a known compound: (Matsoukas, John M.; Hondrelis, John; Agelis, George; Barlos, Kleomenis; Gatos, Dimitrios; Ganter, Renee; Moore, Diana; Moore, Graham J. Novel Synthesis of Cyclic Amide-Linked Analogs of Angiotensins II and III. J. Med. Chem. (1994), 37(18), 2958-69.)
1H-NMR (500 MHz, DMSO-de,): δ 7.9 (overlapping d, 5.4H), 7.7 (br d, 1.85H), 7.58 (app t, 0.25 H), 7.40 (app t, 2.12 H), 7.33 (m, 6.95H), 6.91 (m, 0.09H), 5.12 (s with shallow overlapping m, 2H), 4.31 (m, 2H), 4.21 (app t, 1.0H), 4.07 (m, 1.0H), 3.95 (m, 0.13H), 1.68 (m, 1.93H), 1.53(m, 2.14H), 1.36 (m, 2.04H), 1.07 (t, 0.07H, residual ether CH3, totals to ~1% diethyl ether). Note: Non-integer integrals and fractional integrals result from conformational isomerism.
Figure imgf000065_0001
Nα-Fmoc-Nω-(Fluorene-9-Carbonyl)-Lysine Benzyl Ester (3).
A 100 niL round-bottomed flask equipped with magnetic stir bar and septum capped inlet was charged with Fmoc-Lys-O-Bz*TFA (T) (3.00 g, 5.24 mmol), HATU (2.097 g, 5.502 mmol, 1.05 equiv., Perseptive Biosystems), fluorene-9-carboxylic acid (2) (1.156g, 5.502 mmol, 1.05 equiv, Aldrich Chemical) and dichloromethane (25 mL) with stirring in that order. The mixture was stirred at ambient temperature and diisopropylethylamine (2.099 g, 16.244 mmol, 3.10 equiv. Aldrich) was added dropwise over a period of 30 seconds. The mixture was stirred 5 min and then DMF (2 mL) was added. The mixture was stirred an additional 3 h; during this time a copious precipitate formed and the mixture could not be stirred. Then dichloromethane (70 mL) was added, the solid was broken up and the mixture was vigorously stirred for 3h. The solid was collected and dried by pumping at high vacuum to give 2.26 g of the product as a white solid.
The deep yellow mother liquor was examined by TLC employing E. Merck silica gel plates and dichloromethane-ethyl acetate 9/1 v/v as the developing solvent. This showed the presence of the major product (same R/ as the collected solid) and a higher R/ impurity later identified as 9-fluorenone; the starting acid was consumed. The mother liquor was processed by washing with 0.5M NaHSO4 (200 mL), saturated NaHCO3 (2 x 200 mL), 5% Na2CO3 (2 x 200 mL), 0.5M NaHSO4 (200 mL), saturated brine (200 mL) and dried (MgSO4). Removal of the drying agent and evaporation gave 1.11 g of a yellow solid. This was dissolved in 25 mL of dichloromethane and applied to a 29 mm diameter x 25 cm height column of Merck 9385 silica gel packed in dichloromethane. The column was eluted with 400 mL of dichloromethane, 95/5 dichloromethane-ethyl acetate 600 mL and then with dichloromethane-ethyl acetate 9/1 v/v (600 mL) in 20 mL fractions. Fractions 7-9 contained 9-fluorenone (0.070 g). This is a contaminant in the starting 9-fluorene carboxylic acid. Fractions 47-90 contained the desired product 0.84 g. The combined yield of product was 3.1O g (90.9% yield). TLC: (E Merck Silica gel 60 GF, CH2Cl2-EtOAc 9/1 v/v) R7 = 0.56, R/ of 9-fluorenone = 0.98, R/fluorene-9-carboxylic acid = 0.26 (trails from 0.12 to 0.44).
1H-NMR (500 MHz, DMSO-d6) δ 8.38 (t, IH), 7.87 and 7.86 (overlapping app doublets, 5H), 7.72 (d, 1.88 H), 7.59 (app t, 0.27H), 7.51 (d, 2H), 7.43-7.26 (13H), 5.14 (s with low intensity m overlapping, 2H), 4.79 (s, IH), 4.33 (m, 2H), 4.23 (t, IH), 4.12 (m, IH), 3.40 (br s, IH may be due to residual water), 3.12 (m, 2H), 1.76 and 1.70 (pair of m, 2H), 1.46 and 1.39 (pair of m, 2H) ppm.
13 C-NMR (125 MHz, DMSO-d6) δ 172.4, 169.4, 156.3, 143.8, 143.7, 143.0, 141.3, 140.7, 136.0, 128.4, 128.0, 127.8, 127.60, 127.64, 127.2, 127.1, 125.24, 125.21, 124.8, 120.12, 120.07, 65.89, 65.71, 54.74, 54.03, 46.66, 38.57, 30.34, 28.71, 22.98 ppm.
Mass Spectrum ESI+ 783.2 (M+Cs)+ (Cs from residual CsI used by the vendor for machine calibration), 689.3 (M+K)+, 674.2 (M+1+Na)+, 673.3 (M+Na)+, 652.3 (M+l+H)+, 651.3 (M+H)+, 567.2 (unassigned), 489.2 (unassigned), 456.2, 455.2 (M - 9-fluorene-methyleneoxy radical)+, 441 (estimated, not delineated by the operator, unassigned), 429.3 (M-Fmoc+2H)+.
Elemental analysis: Found: C, 76.14%; H, 5.69%; N, 4.06%.
CaIc. for C42H38N2O5: C, 77.52%; H, 5.89%; N, 4.30%. 1H-NMR indicates 0.51 mol water.
Calc'd for C42H38N2O5 «0.51 H2O C, 76.44%; H, 5.96%; N, 4.24 %.
Figure imgf000066_0001
[00156] To a suspension of the Fmoc derivative (3) (10 g, 15 mmol) in acetonitrile (50 mL) was added 25 % piperidine in acetonitrile (150 mL, final concentration of piperidine 20%), and the mixture was stirred at room temperature for 60 min. Acetonitrile was removed in vacuo; the residue was stirred with hexane (150 rnL) for 10 min and the hexane layer was decanted. This process was repeated four times. The resulting solid was filtered, washed once more with hexane (50 mL) and dried to afford Nε-(9-fluorenecarbonyl)-lysine benzyl ester (4) (6.25 g, 93 % yield).
TLC: R/ 0.45 (silica gel, CH2Cl2:Me0H, 95:5 v/v)
HPLC: tR: 5.24 min Column: YMC ODS-A 4.6 mm x 250 mm, S-IOP μm, 12θA particle; Eluents: A: H2O (0.1% TFA), B: CH3CN (0.1% TFA); Elution: Initial condition: 50 % B, then linear gradient to 100% B in 20 min; Flow rate: 1.0 mL/min, Detection: UV @ 254 nm.
1H-NMR (500 MHz, CDCl3): δ 1.2-1.65 (8H, NH2 and 3CH2), 3.05 (bs, 2H, CH2), 3.33 (m, IH, CH), 4.76 (s, IH, CH), 5.09 (s, 2H, benzylic), 5.28 (bs, IH, NH), 7.3 l(s, 7H, ArH), 7.42 (m, 2H, ArH), 7.65 (m, 2H, ArH), 7.75 (m, 2H, ArH).
MS: 429.2 (M+H)+
CFXOO-
Figure imgf000067_0001
t-Bu-OOC *"* ' * ^ COO-t-Bu r ~I t-Bu-OOC ^ N^N ^^ OH HATU/DIEA/CH2CI2
A I π o
Figure imgf000067_0002
[00157] To a mixture of DO3A10CM-(tri-t-butyl ester)-glycine (5) (8.8 g, 14 mmol) and Nε-(9-fluorenecarbonyl)- lysine benzyl ester (3) (5.13 g, 12 mmol) in anhydrous methylene chloride (100 mL) was added HATU (5.32 g, 14 mmol), and the mixture was stirred for 5 min. Diisopropylethyl amine (5.46 g, 42 mmol) was added to the reaction mixture and stirring was continued for 6 h. Methylene chloride was removed in vacuo, the residue was dissolved in ethyl acetate (150 mL), washed with saturated sodium bicarbonate solution (3 x 100 mL), sodium bisulfate solution (0.5 molar, 2 x 100 mL) and finally with brine (100 mL). The ethyl acetate layer was dried and solvent removal afforded the coupled product as a light yellow glassy solid (13g). This was purified by column chromatography over silica gel (Merck 9385, 350 g) using methylene chloride and methanol to afford the required product (8.6 g, 69 % yield) DO3A10CM-(tri-t-butyl ester)-glycinyl-Nε-(9-fluorenecarbonyl)-lysine benzyl ester (6).
TLC: R/, 0.6 (silica gel, 6 % MeOH/ CH2Cl2)
HPLC: tR 9.37 min. (Column: YMC ODS-A, 4.6 mm x 250 mm, S-IOP μm, 12θA; Eluents: A: H2O (0.1% TFA), B: CH3CN (0.1% TFA);; Elution:: Initial condition: 50 % B, then linear gradient to 100% B in 20 min; Flow rate: 1.0 ml/min, Detection: UV @ 254 nm).
1H-NMR (500 MHz, CDCl3): δ 1.385-1.436 (m, 27H, t-C4H9), 1.4-1.8 (m, 6H, CH2), 4.35 (bs, IH, CHCOOCH2C6H5), 4.88s (s, IH, CH), 5.12 (s, CH2, benzylic), 6.6, 6.8 and 7.1 (broad singlets, 3H, NH), 7.3-7.8(m, 13H, ArH).
MS: (M+H)+ 1040.6, (M+Na)+ 1062.5.
Figure imgf000069_0001
[00158] To a solution of (6) (8.30 g, 8 mmol) in methanol (80 mL) was added palladium catalyst (3.0 g, 10% DeGussa type), and the mixture was hydrogenated at 50 psi for 16 h. The catalyst was filtered off, the volatiles were removed in vacuo and the residue was dried. This was treated with TFAiH2O :triisopropylsilane (88:5.5:2 v/v/v) (120 mL) for 4h under a nitrogen atmosphere. The volatiles were removed in vacuo, the residue was triturated with ether (2 x 100 mL) to provide a solid which was collected, washed with ether (50 mL) to afford DO3A10CM-glycinyl-Nε -(9-fluorenecarbonyl) -lysine (7) as the TFA salt (6.9 g, yield 86 %). This material was further purified on a reversed phase Cl 8 preparative LC column (Rainin, 41.4 x 250 mm, 8μ particle, 6θA pore size) which was equilibrated with 0.1% aqueous TFA. The column was then eluted with water-acetonitrile solvent mixture (both containing 0.1 % TFA), starting a linear gradient from 10% acetonitrile to 80% acetonitrile in 80 min. The fractions were analyzed on an analytical reversed phase Cl 8 column, and those containing the pure product were pooled and freeze-dried to provide DO3A10CM-glycinyl-N ε-(9-fluorenecarbonyl)-lysine (7), (4.7 g, yield, 58 %) as a colorless fluffy solid.
HPLC: tR 13.14 min; Column: YMC ODS-A 4.6 mm x 250 mm, S-IOP μm, 12θA; Eluents: A: H2O (0.1% TFA), B: CH3CN (0.1% TFA); Elution: Initial condition: 10 % B, linear gradient to 100% B over 20 min; Flow rate: 1.0 mL/min, Detection: UV @ 254 nm. 1H-NMR (500 MHz, DMSO): δ 1.32-1.74 (m, 6H, CH2), 2.80-3.95 (m, 28H5CH2), 4.20 (bs, IH, CHCOOH), 5.12 (s, CH2, benzylic), 4.76 (s, IH, CH), 7.25-7.55 (m, 6H, ArH), 7.85 (m,2H, ArH), 8.30, 8.40 and 8.62 (broad singlets, 3H, NH).
MS: 782.3 (M+H)+, (M+Na)+ 804.3, 590.3, 391.7.
Figure imgf000070_0001
L) Gd(OAc)3, pH 4.5 2.) Prep.HPLC
3.) AG50-X2 ion exchange resin Meglumine form
Figure imgf000070_0002
[00159] DO3A10CM-glycinyl-Nε-(9-fluorenecarbonyl)-lysine (7) (4.3 g, bis- trifluoroacetate salt, 5.5 mmol) was dissolved in water (100 mL) and gadolinium acetate tetrahydrate (2.43 g, 6 mmol) was added. The mixture was stirred for 30 min. The pH of the solution was adjusted to 4.50 with IN NaOH, and the mixture was stirred at ambient temperature for 24 h. HPLC analysis indicated the completion of the reaction. The solution containing the product was loaded onto a reversed phase Cl 8 preparative column (Rainin, 41.4 x 250 mm, 8μ, 60 A) which was equilibrated with water. The column was then eluted with 10% aqueous acetonitrile and then a linear gradient from 10% acetonitrile to 80% acetonitrile in 80 min was run. The fractions were analyzed on an analytical reversed phase Cl 8 column and the fractions containing the product in > 99.5% purity were pooled and freeze-dried to provide (Gd-DOTA)-Glycinyl-Nε-(9-fluorenecarbonyl)-lysine, meglumine salt (8) as a colorless fluffy solid. To convert this Gd-chelate into its meglumine salt form, it was applied to a sulfonic acid cation exchange column (Bio-Rad, AG 50W-X8 resin)which had been converted into the meglumine form and the column was washed with water. The fractions containing the product (the eluent was monitored with an UV detector at 254 nm) were collected and lyophilized to provide Gd-DO3A10CM-Glycinyl-Nε-(9- fluorenecarbonyl)-lysine meglumine salt (8) (4.2g, 63 % yield) as a colorless fluffy solid..
HPLC: tR 10.40 min (Column: YMC ODS-A, 4.6 mm x 250 mm, S-IOP μm, 12θA; Eluents: A: H2O (0.1% TFA), B: CH3CN (0.1% TFA); Elution: initial condition: 10 % B, linear gradient to 80 % B over 20 min; Flow rate: 1.5 ml/min, Detection: UV @ 220 nm).
MS: 937.2 isotopic pattern for Gd (M+H)+ and 195.9 meglumine (M+l)4
Elemental Analysis: CaIc. for C38H48N7Oi iGd»C7Hi7NO5 »5.5 H2O: C, 44.32; H, 6.28; N, 9.11; Gd, 12.78 Found: C, 44.18, H,5.96; N, 9.01; Gd, 12.01; %Na: <0.24;
[00160] The compound was screened as described above in Example 1. Results showed that MDL > 3.0 mmol/Kg in mice. Other results are shown in Table 2.
EXAMPLE 3 - Synthesis of Gd(CM-DOTMA)-aB Na+ (Class 1)
Figure imgf000071_0001
[00161] The compound was prepared as follows: a suspension of L-malic acid (1) (Aldrich Chemical Company, 120.0 g, 0.895 mol) in 2,2-dimethoxyethane (1200 mL) was heated under reflux with stirring until all of the material went into solution (2 h). The solution was concentrated under reduced pressure using a rotary evaporator and the resulting oil was dissolved in about 1000 mL of 7:3 hot hexane/isopropyl ether and filtered. The solid that separated from the cooled filtrate was collected and washed with 7:3 hexane/isopropyl ether (2 x 50 mL). The combined filtrates were again concentrated to about 250 mL, yielding more of the product L-malic acid acetonide (2) as colorless crystalline solid. Yield: 131.O g (84%).
m. p. 114-1160 C.
1H-NMR (500 MHz, CDCl3): δ 1.5 and 1.55 (2s, 6H, -C-CH3), 2.8 (AB qd, 2H, -CH2- COOH), 4.6( dd, IH, -CH-CH2COOH) and 11.1 (bs, IH -COOH). 13C-NMR (CDC13) δ
25.46, 26.37, 35.63, 70.08, 111.1, 171.7 and 174.9.
MS: 197 (M+Na)4
Elemental Analysis: CaIc. for C7Hi0O5: C, 48.28; H, 5.79; O, 45.94%. Found: C, 48.18; H, 5.69.
Figure imgf000072_0001
[00162] A solution of the acetonide 2 (30.0 g, 0.172 mol) in dry acetonitrile was treated with 18-crown-6 (0.462 g, 1.75 mmol) and powdered potassium carbonate (23.8 g, 0.172 mol) and stirred under nitrogen with an overhead stirrer. Benzyl bromide (29.0 g, 0.17 mol) was added neat, and stirring was continued under nitrogen at RT for an additional 20 h. Acetonitrile was evaporated and the paste obtained was dissolved in water (300 mL). The aqueous solution was extracted with ethyl acetate (3 x 200 mL) and the organic layer was washed with water (3 x 200 mL), saturated sodium chloride (1 x 300 mL) and dried. Removal of the solvent furnished a colorless solid. The solid was recrystallized from hexane: isopropyl ether (7:3) to yield the benzyl ester of acetonide of L-malic acid 3 as colorless needles. Yield: 41.7 g (91.8%).
m. p. 62-63° C. 1H-NMR (500 MHz, CDCl3): δ 1.5 and 1.55 (2s, 3H, -C-CH3), 2.9 (ABqd, 2H, -CH2- COOBn), 4.7 (dd, IH, -CH-CH2 COOBn), 5.15 (q, 2H, -CH2-Ph) and 7.3 (m, 5H, Ar-H).
13C-NMR (125 MHz, CDCl3): δ 26.58, 27.4, 67.74, 71.36, 111.9, 129.1, 129.2, 136.0, 169.8 and 172.7.
MS: 287.2 (M+Na)+
Elemental Analysis: CaIc. for Ci4Hi6O5: C, 63.63; H, 6.1; 0, 30.27%.
Found: C, 63.62; H, 6.03.
Figure imgf000073_0001
[00163] A solution of the acetonide 3 (100.Og, 0.378 mol) in dioxane (250 rnL) was treated with 2N H2SO4 and stirred vigorously at RT. Initially, the reaction mixture was cloudy and after about 30 min. the solution became clear. TLC indicated that the reaction was complete. The crude reaction mixture was saturated with sodium sulfate and extracted with ethyl acetate (5 x 300 mL). The combined organic layers were washed with saturated sodium chloride and dried (Na2SO4). Filtration followed by evaporation of the solvent yielded a colorless oil which solidified slowly on standing. The solid was again recrystallized from isopropyl ether/hexane (2:1) to yield the 3-benzyloxycarbonyl-2-hydroxy propionic acid [the monobenzyl ester of (L)-malic acid] 4 as a colorless solid. Yield: 77.0 g (90.9%).
m. p. 68-690 C.
1H-NMR (500 MHz, CDCl3): δ 2.9 (m, 2H, -CH2-COOBn), 4.5 (d, IH, -CHOH), 5.15 (q, 2H, -CH2Ph) and 7.3 (m, 5H, Ar-H). 13C-NMR (500 MHz, CDCl3): δ 38.03, 66.69, 66.90, 128.1, 128.2, 128.4, 134.9, 170.7 and 176.8.
MS: 247.1 (M+Na)+, 225.2 (M+H)+
Elemental Analysis: CaIc. for CnHi2O5: C, 58.93; H, 5.39; O, 35.68%. Found: C, 59.4; H, 5.41%.
Figure imgf000074_0001
4 5
[00164] A solution of hydroxy acid 4 (77.0 g, 0.34 mol) in dry CH2Cl2 was treated with anhydrous triethylamine (70.7 g, 0.7 mol) and was cooled to 0° C. Acetic anhydride was added with stirring while maintaining the reaction temperature below 10° C. After the addition (about 10 min), the reaction mixture was allowed to come to RT and stirred for 20 h. Water (200 mL) was added and the mixture stirred for 1 h. The reaction mixture was cooled in ice and treated with 4N HCl (200 mL) and stirred for 30 min more. The organic layer was separated and the aqueous layer was extracted with CH2Cl2 (3 x 200 mL). The combined organic layer was washed with water (2 x 300 mL), saturated NaCl (1 x 500 mL) and dried (Na2SO4). Evaporation of the solvent under reduced pressure yielded 2-acetoxy-3- benzyloxycarbonyl propionic Acid (5) as a pale brown oil. This was dried overnight under vacuum (<0.1 mm) and employed immediately thereafter for the next step in the sequence. Yield: 90.5g (100%).
1H-NMR (500 MHz, CDCl3): δ 2.1(s, 3H, -0-CO-CH3), 2.9 (d, 2H, -CH2-COOBn), 5.15(q, - CH2-Ph), 5.5 (t, IH-CH-OAc), 7.3 (m, Ar-H) and 10.6 (bs, IH, -COOH).
MS: 267.2 (M+H)+
Figure imgf000075_0001
5 6
[00165] To a solution of the acid (5) (94.0 g, 0.35 mol) in dry CH2Cl2 (1400 niL), anhydrous MgSO4 (168.5 g, 1.5 mol) was added and stirred. To the above solution, t-BuOH (130.Og, 1.77 mol) was added followed by concentrated. H2SO4 in portions to maintain the temperature at about 25° C. After the addition, the flask was tightly stoppered and stirred at RT for 24 h. The reaction mixture was poured into saturated NaHCO3 (68.Og, 0.8 mol in 1500 mL of water) and stirred vigorously to neutralize the acid. The organic layer was separated, washed with water (1 x 500 mL), and then with saturated NaCl (1 x 500 mL). The dried (Na2SO4) organic layer was filtered and evaporated to give the crude t-butyl-2-acetoxy- 3-benzyloxycarbonyl propionate product (6) as a pale brown oil. The oil was kept under high vacuum for 20 h (< 0.1 mm) and was then taken on to the next step without purification. Yield: 100.0 g (89%).
1H-NMR (500 MHz, CDCl3): δ 2.1(s, 3H, -0-CO-CH3), 2.9 (d, 2H, -CH2-COOBn), 5.15(q, - CH2-Ph), 5.5 (t, IH-CH-OAc), 7.3 (m, Ar-H) and 10.6 (bs, IH, -COOH)
MS: 345.4 (M+Na)+, 323.4 (M+H)+, 267.2 (M+H -C4Hg)+
Figure imgf000075_0002
[00166] A solution of the acetate 6 (100.0 g, 0.31 mol) in a mixture of z-PrOH/t-BuOH (400 mL/100 mL) was cooled in an ice-bath and was treated with neat anhydrous hydrazine (12.7 g, 0.37 mol). After 30 min an additional amount of hydrazine (3.5 g, 0.1 mol) was added and the mixture was stirred for 30 min. Then another portion of hydrazine (0.34 g, 0.01 mol) was added and the mixture was stirred for 30 min. Silica gel TLC analysis indicated the completion of the reaction. The total reaction time was 2 h. The volatiles were removed under reduced pressure and the residue was taken up in water (500 mL). The oil that separated was extracted with ethyl acetate (3 x 250 mL) and the combined organic layers were washed with water (3 x 200 mL), saturated NaCl (1 x 250 mL) and dried (Na2SO4). Evaporation of the solvent yielded a pale yellow oil. The oil was dissolved in hot hexane (500 mL) and was filtered. The filtrate was cooled in an ice-bath. The crystals that separated were filtered and washed with ice-cold hexane (2 x 100 mL). The resultant compound t- butyl-3-benzyloxycarbonyl-2-hydroxy propionate,(7) obtained as a colorless solid, was dried at RT under vacuum for 20 h. Yield: 53.8 g (62%).
m. p. 67-68° C.
1H-NMR (500 MHz, CDCl3): δ 1.42 (9s, 9H, -C-CH3), 2.8 (m, 2H, -CH2-COOBn), 4.35 (t, IH, -CH-OH), 5.15 (q, 2H, -CH2Ph) and 7.3 9m, 5H, Ar-H).
13C-NMR (125 MHz, CDCl3): δ 27.82, 38.93, 66.6, 67.34, 82.95, 128.3, 128.5, 135.5, 170.2 and 172.5.
MS: 303.0, (M+Na)+
Elemental Analysis: CaIc. for Ci5H20O5: C, 64.27; H, 7.19; O, 28.54%. Found: C, 64.27; H, 7.35%
Figure imgf000076_0001
Z S
[00167] To a solution of the alcohol 7 (21.0 g, 0.075 mol) in dry dichloromethane (100 mL), 2,6-lutidine (9.1 g, 0.085 mol) was added and the mixture was cooled to -10° C. Trifluoromethanesulfonic anhydride (23.3 g, 0.0825 mol) was added dropwise with stirring under nitrogen over a period of about 15-20 min. After the addition, the reaction mixture was stirred at 0° C for 1 h. The reaction mixture was poured into crushed ice (250 g) and the organic layer was washed with ice-cold 2N HCl (2 x 100 niL), ice-cold water (2 x 100 rnL) and dried with anhydrous calcium sulfate in a freezer before its use. The t-butyl-3- benzyloxycarbonyl-2-O-[trifluoromethanesulfonyl) propionate (8) was kept as a solution in dichloromethane. The triflate 8 was found to be very unstable at room temperature and therefore was used immediately as a solution in dichloromethane as soon as it was prepared.
1H-NMR (500 MHz, CDCl3): δ 1.5 (s, 9H, f-butyl CH3), 3.2(d, 2H, -CH2-COOBn), 5.2 (q, 2H, -COOCH2Ph), 5.4 (triplet, IH, -CH-), and 7.4 (m, 5H, Ar-H).
Figure imgf000077_0001
10
[00168] A solution of 1,4,7, 10-tetraazacyclododecane (TACD) (9) (31.54 g, 0.18 mol) in dry dichloromethane (100 mL) was cooled in an ice-bath and a solution of the triflate 8 (nominally 0.075 mol ) in dry dichloromethane (filtered from the drying agent directly into a cold-jacketed addition funnel that was kept at 0° C) was added rapidly with vigorous stirring under nitrogen. The reaction mixture was stirred at 0° C for 30 min and then allowed to come to RT and stirred overnight. The reaction mixture was diluted with 200 mL of ethyl acetate and filtered through a bed of celite and washed with 1 : 1 dichloromethane/ethyl acetate (2 x 50 mL). The combined organic layers were washed with water (3 x 200 mL) and dried (Na2SO4). Removal of the solvent yielded the crude monoalkylated TCAD (10) as a pale brown paste. This was used directly in the next step in order to avoid decomposition by adventitious moisture. Yield: 28.57 g (87.8%, crude).
TLC: Rf 0.18 (1 :1 :0.1 -CHCl3ZI-PrOHZNH4OH).
1H-NMR (500 MHz, CDCl3): δ 1.48 (s, 9H, -C-CH3), 2.5-3.0 (m, 21H, -N-CH2 and -CH2- COOBn), 3.8 (t, IH, -CH-COO-t-Bu), 5.15 (q, 2H, -CH2-Ph) and 7.3 (m, 5H, Ar-H). MS: 435.3 (M+H)+.
μ r ft
HAOH
Figure imgf000078_0001
11 12
[00169] To a suspension of 4A molecular sieves (50.0 g) in dry dichloromethane (200 niL), f-butyl-(D)-lactate (11) (Fluka, 51.4 g, 0.352 mol) and 2,6-lutidine (46.0 g, 0.43 mol) were added with stirring until the solids dissolved. The resulting solution was cooled to -20° C and trifluoromethanesulfonic anhydride (119.1 g, 0.42 mol) in dry dichloromethane (50 mL) was added drop wise under nitrogen with stirring over a period of -30 min. The reaction mixture was stirred for 1 h more at -20° C, decanted into a separatory funnel, washed with 2N ice-cold HCl (2 x 200 mL), water (2 x 200 mL) and dried. The solvent was evaporated under reduced pressure at RT and the resulting brown liquid was filtered through a 300 g bed of flash silica gel packed in a sintered glass funnel which was washed with IL of 20% ethyl acetate in hexane. The filtrate was concentrated under reduced pressure at RT to constant weight. The product, /-butyl-O-(trifluoromethanesulfonyl)-(D)-lactate (12) was obtained as pale yellow oil. The purity of the product was established by silica gel TLC and 1H-NMR analysis. Yield: 97.8 g (100%). [00170]
1H-NMR (500 MHz, CDCl3): δ 1.5 (s, 9H, -C-CH3), 1.65 (d, 3H, -CH-CH3) and 5.2 (q, IH, - CH-Me).
Figure imgf000078_0002
[00171] A solution of the monoalkylated TACD 10 (38.2 g, 0.063 mol) in dry acetonitrile (300 mL) was treated with 150.0 g of 4A molecular sieves and stirred while being cooled in an ice-bath. The triflate 12 (97.0 g, 0.35 mol) was added dropwise from an addition funnel under nitrogen with stirring. After the addition, the reaction mixture was allowed to come to RT and stirred for an additional 20 h under nitrogen. The solvent was evaporated under reduced pressure at RT and the residue was dissolved in 500 mL of ethyl acetate and treated with saturated sodium carbonate solution (500 mL). The organic layer was separated and washed with water (2 x 250 mL) and dried (Na2SO4). Evaporation of the solvent yielded a brown oil which was chromatographed over flash silica gel (2 Kg) using chloroform- methanol as the eluent. The product was eluted with 2% methanol in chloroform to provide fully protected CM-DOTMA 13 as a golden-yellow foam. Yield: 72.2 g (82.8%). [00172]
1H-NMR (500 MHz, CDCl3): δ 1.3 (bs, 9H, -CH-CH3), 1.48 (s, 36H, -C-CH3), 2.4-2.8 (m, 18H, -CH2), 3.5 (m, 3H, -CH-Me), 4.2 (d, IH, -CH-CH2COOBn), 5.2 (AB q, 2H, -CH2-Ar) and 7.4 (m, 5H, Ar-H).
MS: 819.9 (M+H)+.
Elemental Analysis: CaIc. C45H74N4F3Oi3SNa: C, 54.53; H, 7.53; N, 5.65; F 5.75; S, 3.24, Na, 2.32%. Found: C, 54.75; H, 7.57; N, 5.54; F, 5.36; S, 3.11; Na, 2.48%.
Figure imgf000079_0001
[00173] A solution of the fully protected CM-DOTMA 13 (25.0 g, 0.025 mol) in 90% methanol-water (50 mL) was hydrogenated in the presence of Pd/C (5.0 g, 10%; 50% by weight water) at 50 psi for 4 h. The catalyst was filtered off through a pad of Celite™ and washed with 90% methanol-water (2 x 25 mL). The combined filtrates were concentrated under reduced pressure to obtain CM-DOTMA tetra-t-butyl ester, monoacid (14) as a colorless foam. The product was thoroughly dried under vacuum overnight (< 0.1 mm) and used in the next step without further purification. Yield: 19.45 g (85%).
1H-NMR (500 MHz, CDCl3): δ 1.2 (m, 9H, CH-CH3), 1.5 (s, 9H, C-CH3), 2.2-3.2 (m, 18H, -CH2), 3.7 (m, 3H, -CH-Me) and 4.2 (m, IH, -CH2-CH-COO-t-Bu).
MS: 729.5 (M+H)+.
Figure imgf000080_0001
[00174] A solution of N-Boc-(L)-biphenylalanine (15) (Advanced Chem Tech, 46.0 g,
0.135 mol) in methanol (250 mL) stirred and cesium carbonate (43.88 g, 0.135 mol) in water (250 mL) was added. The solid slowly dissolved to give a pale yellow homogeneous solution. The water and methanol were removed under reduced pressure to give a colorless solid that was dried under vacuum for 2 h. The solid was suspended in dry DMF (500 mL) and cooled in ice. Benzyl bromide (23.0 g, 0.135 mol) was added with stirring under nitrogen and the mixture was stirred for 20 h at RT. DMF was removed under reduced pressure and the residue was taken up in 500 mL of water and extracted with ethyl acetate (5 x 200 mL). The organic layer was washed with saturated aqueous sodium carbonate (I x 200 mL), water (2 x 200 mL), and dried (Na2SO4). Removal of the solvent yielded N-Boc-(L)- biphenyl alanine benyl ester (16) (56.0 g; 96% yield) as a colorless solid, which did not require further purification. [00175] TLC: R/ 0.75 (silica gel, 2% methanol in CH2Cl2).
1H-NMR (500 MHz, CDCl3): δ 1.42 (s, 9 H, t-C4HP), 3.15 (m, 2 H, CH2CH), 4.67 (m, 1 H, CH), 5.04 (d, 2 Η, NH), 5.18 (dd, 2 Η, CH2C6H5) and 7.08-7.55 (m, 14 H, ArH).
MS: 432.2 (M+Η)4
Figure imgf000081_0001
[00176] trans-4-Aminomethylcyclohexanecarboxylic acid (17) (31.0 g, 0.197 mol) was added to a solution of sodium carbonate (23.32 g, 0.22 mol) in water (350 mL) with stirring. Upon dissolution of the solid the solution was cooled in an ice bath and a solution of di-t- butyldicarbonate (48.0 g, 0.22 mol in 350 mL of THF). was added dropwise with rapid stirring. After the addition, the ice bath was removed and the solution was allowed to stir at RT for 4 h. The volatiles were removed under reduced pressure, the residue was taken up in 500 mL of water, the aqueous solution was extracted with ethyl acetate (4 x 100 mL) and the organic layer was discarded. The pH of the solution was adjusted to ~3.0 by addition of saturated aqueous KHSO4 solution; this operation resulted in the formation of a precipitate. The precipitated solid was filtered off and washed with water (4 x 100 mL) and air-dried. The solid was dissolved in ethyl acetate (500 mL) and washed sequentially with water (3 x 200 mL) and saturated sodium chloride solution (300 mL) and dried (Na2SO4). Evaporation of the volatiles provided 4-Boc-aminomethylcyclohexane-carboxylic acid (18) as a colorless fluffy solid. Yield: 50.67 g (100%).
Figure imgf000081_0002
[00177] Boc-biphenylalanine (16a) (13.93 g, 0.03 mol) was added to a stirred solution of phenol (2.5 g) in TFA (50 mL). The mixture was stirred for 30 min at RT and then the TFA was removed under reduced pressure. The residue was triturated with 100 mL of anhydrous ether and the solid that separated was filtered and washed with anhydrous ether (2 x 25 niL). The solid, L-biphenylalanine benzyl ester»TFA (16b) was dried under vacuum (<
0.1 mm Hg) for 2 h before use.
[00178]
[00179] HPLC: tR 8.72 min; Column: YMC C18-ODS 4.6 mm x 250 mm; S-IOP μ particle; 12θA pore; Eluents: A: H2O (0.1% TFA), B: CH3CN (0.1% TFA); Elution: Initial condition: 45% B, then linear gradient 45-95% B over 25 min; Flow rate: 1 mL/min;
Detection: UV @ 230 nm.
1H-NMR (500 MHz, DMSO-de): δ 3.05-3.21 (2dd, 2 H, CH2CH), 4.41 (t, 1 H, CH2CH),
5.12 (dd, 2 Η, CH2C6H5), 7.21-7.65 (m, 14 H, ArH) and 8.58 (bs, 3Η, NH3 +).
MS: 332.2 (M+H)+.
[00180]
/rans-4-N-^Butyloxycarbonylaminomethylcyclohexanecarboxyl-L-biphenylalanine benzyl ester (Boc-a-Bip-OBn) (19).
Figure imgf000082_0001
A mixture of Bip-OBn»TFA (16b) (9.00 g, 20.21 mmol), HATU [O-(7-azabenzotriazol-l-yl)- 1,1,3,3-tetramethyluronium hexafluorophosphate] (11.52 g, 30.32 mmol) and trans-4-N-t- butyloxy-carbonylaminomethylcyclohexane-carboxylic acid (Boc-a) (17) (5.72 g, 21.23 mmol) in dichloromethane (30 mL) was added diisopropylethylamine (8.00 g, 61.90 mmol) and the solution was stirred at room temperature for 2 h. The reaction mixture was then evaporated on a rotary evaporator to remove dichloromethane and the residue was taken-up in ethyl acetate (200 mL). The organic layer was washed with dil.HCl (0.05 N, 2 x 200 mL) followed by washing with NaHCO3 (5%, 2 x 200 mL) and water (2 x 200 mL). The organic layer was then dried (Na2SO4), filtered and evaporated to provide a light brown foamy solid in nearly quantitative yield (11.00 g). The material was sufficiently pure for use in the following step. TLC: Rf 0.55 (silica gel, 5% methanol in dichloromethane).
MS: 571.4 (M+H)+.
1H-NMR (500 MHz, CDCl3): δ 0.92 (m, 2 H, cyclohex-C/f2), 1.45 (s & m, 11 H, J-C4H9 & cyclohex-C/f2), 1.60-2.09 (m, 6 H, cyclohex-C/f2), 2.95 (m, 2 H, bip-CH2CH), 3.15 (m, 2 H, bocNHCH2), 4.55 (m, 1 H, bip-αH), 4.95 (m, 1 H, bocNH), 5.18 (m, 2 H, CH2C6H5), 5.92 (d, 1 H, CHNHCO) and 7.01-7.60 (m, 14 Η, ArH).
Figure imgf000083_0001
[00181] N-(trα/?5-4-aminomethylcyclohexanecarbonyl)-L-biphenylalanine benzyl ester trifluoroacetic acid salt (20).
[00182] To a solution of phenol (4. Ig, 0.0432 mol) in anhydrous TFA (150 niL), N- {trans -4-N-Boc-aminomethylcyclohexanecarbonyl)-L-biphenylalanine benzyl ester (19) (12.32 g, 0.0216 mol) was added and the mixture was stirred at RT for 30 min. TFA was removed under reduced pressure and the residue was triturated with anhydrous ether (250 mL). Compound (20) that separated as a solid was filtered, washed with anhydrous ether (4 x 50 mL) and dried at RT under high vacuum (4 h, 0.05 mm). It was used in the next step without purification.
HPLC: tR 9.50 min (Column: YMC C18, 4.6 mm x 250 mm, S-IOP μm particle, 12θA pore; Eluents: A: H2O (0.1% TFA), B: CH3CN (0.1% TFA); Elution: Initial condition: 45% B, then linear gradient to 95% B over 25 min; Flow rate: 1.0 mL/min, Detection: UV @ 230 nm.
1H-NMR (500 MHz, CDCl3): δ 0.94 (m, 2 H, cyclohex-CH2), 1.45 (m, 2 Η, cyclohex-CH2), 1.60-2.15 (m, 6 Η, cyclohex-CH2), 2.75 (m, 2 Η, bip-CH2CΗ), 3.15 (m, 2 H, bocNHCH2), 4.85 (m, 1 H, bip-αH), 5.18 (m, 2 Η, CH2C6H5), 5.92 (d, 1 H, CHNHCO) and 6.91-7.71 (m, 14 Η, ArH).
MS: 471.3 (M+Η)+.
Figure imgf000084_0001
[00183] CM-DOTMA acid (14) (19.45 g, 0.0216 mol) was dissolved in anhydrous dichloromethane (100 mL) and cooled to -10° C under a nitrogen atmosphere. HATU (9.12 g, 0.024 mol) was added followed by diisopropylethylamine (3.1 g, 0.024 mol) and the mixture was stirred for 10 min at -10° C. A suspension of N-(trans-4- aminomethylcyclohexanecarbonyl)-L-biphenylalanine benzyl ester trifluoroacetic acid salt (20) and diisopropylethylamine (3.1 g, 0.024 mol) in anhydrous dichloromethane (50 mL) was added in one lot to the activated acid above and the reaction was continued for 1 h at - 10° C. The solution was cloudy after the addition of the amine but gradually clarified in about 5 min. At the end of the reaction the solvent was removed under reduced pressure and saturated aqueous sodium carbonate (500 mL) was added to the residue.. The aqueous mixture was extracted with ethyl acetate (5 x 200 mL) and then combined. The organic layer was washed with water (3 x 200 mL), saturated sodium carbonate (2 x 200 mL), and dried (Na2SO4). Removal of the solvent yielded a brown paste (25.0 g) which was chromatographed over flash silica gel (2 Kg). Elution with 2% methanol in chloroform yielded the product as a golden yellow paste (22 g, 86% yield). Though the resultant product CE-DOTMA-a-B (21) was homogeneous by silica gel TLC analysis, HPLC analysis indicated a purity of only 80%. Repeated attempts to further purify the material were unsuccessful. It was decided to purify at the next stage in the sequence. TLC: Rf 0.41 (9:1, CHCl3/MeOH).
1H-NMR (500 MHz, CDCl3): δ 0.92, (m, 2H), 1.23 (d, 9H, CH-CH3), 1.48 (2s and a m, 38H, -f-butyl methyls and -CH2), 1.6-3.2 (m, 28H, -N-CH2 and other-CH^s), 3.6 (m, 3H, -CH - (Me)-COO-^-Bu), 4.2 (dd, IH, -CH-CH2-CO-NH), 4.9 (m, IH, -CH-COOBn), 5.2 (Abq, 2H, -CH2-Ph), 5.9 (d, IH, -NH-CH-COOBn), 6.5 (t, IH, -NH-CO-CH2), 7.1 (d, 2H, Ar-H), 7.3 (m, 6H, Ar-H), 7.4 (m, 4H, Ar-H) and 7.55 (m, 2H, Ar-H).
MS: 1181.8 (M+H)+.
HH 8:1, v/v
Figure imgf000085_0002
Figure imgf000085_0001
[00184] A hydrogenation bottle was charged with a solution of fully protected CM- DOTMA-a-B (21) (19.0 g, 0.016 mol) in CH3OH:Water 9:lv/v , (100 mL) and Pd/C (10% Pd content, 50% water by weight, 3.8 g). The vessel was evacuated and refilled with hydrogen several times. Then the hydrogenation was conducted for 4 h at 50 psi at ambient temperature. . The catalyst was filtered off through a bed of Celite™ and washed with CH3OHiH2O 9:1 v/v (2 x 20 mL). The combined filtrate was concentrated under reduced pressure to a foam which was kept under high vacuum (< 0.05 mm) for 4 h. The foam was then dissolved in anhydrous TFA (200 mL) containing anisole (25 mL) and stirred for 20 h at RT. The volatiles were removed under reduced pressure and the residue was triturated with 500 mL of anhydrous ether. The solid that precipitated was filtered off and washed with anhydrous ether (2 x 100 mL). The analytical HPLC chromatogram of the product CE- DOTMA-a-B ligand (22) (14.1 g; > 100%) displayed two peaks when a linear gradient system or an isocratic elution method was employed. Attempted purification by preparative HPLC (several runs) provided only low yields of the predominant product.. Hence it was decided to form the Gd chelate and then purify the chelate.
[00185]
HPLC: tR 17.15 min (major isomer), 15.27 (minor isomer); Column: YMC C18-ODS; 4.6 mm x 250 mm, S-IOP μ; 12θA; . Eluents: A: H2O (0.1% TFA), B: CH3CN (0.1%
TFA);Elution: Isocratic 30% B.
13C-NMR (DMSO-de): δ 28.65, 29.53, 29.65, 36.36, 37.03, 43.68, 44.86, 44.98, 45.39, 45.62, 45.71, 45.77, 45.81, 45.9, 45.93, 45.98, 47.09, 47.2, 47.31, 47.37, 47.47, 47.64, 53.18, 57.09, 58.41, 126.4, 126.5, 127.3, 129.8, 137.2, 138.2, 140.0, 158.1, 158.3, 169.8, 171.8, 173.3 and 175.3.
MS: 867.6 (M+H)+.
Elemental Analysis: CaIc. C44H62N6Oi2' 1.8 TFA«4 H2O ; C, 49.96; H, 6.32; F, 8.97; N,
7.34; O, 27.40%. Found: C, 50.15; H, 6.02; F, 8.49; N, 7.06%.
[00186]
Figure imgf000086_0001
A solution of the ligand (22) (14.1 g, 0.012 mol; calculated based on CMDOTA-a-B»2 TFA»2H2O) in water (200 mL) was treated with solid Gd (OAc)3 tetrahydrate (6.09 g, 0.015 mol) and a saturated solution of N-methylglucamine was added with stirring until the pH of the solution was adjusted to 6.0. The reaction mixture was then allowed to stir at RT and the pH of the reaction mixture was monitored hourly. Initially, the pH dropped to about 4 and then was brought back to 6 by the addition of meglumine; after 8 h the pH of the reaction mixture was stable (pH 6.0). The reaction was continued for 20 h more at RT and then the pH of the reaction was adjusted to 9.0 addition of aqueous meglumine. The precipitated gadolinium oxide was filtered off using a 0.22 μ filter and the filtrate was purified by preparative HPLC to obtain Gd-CEDOTMA-a-B (23) (4.2 g, 34% yield of the major isomer).
HPLC: Analytical: tR 19.89 min (purity >99%). Column: YMC C18-ODS 250 mm x 4.6 mm; S-IOP μ; 12θA; Eluents: A: H2O (0.1% TFA), B: CH3CN (0.1% TFA); Elution: Isocratic 30% B for 30 min; Flow Rate: lmL/min; Detection: UV @ 230 nm; Fluorescence: 280 Ex, Em 320 nm.
Preparative: Column: 50 mm X 250 mm YMC C18- ODS; S-IOP μ; 12θA Eluents: A: H2O (0.1% TFA), B: CH3CN (0.1% TFA); Elution: Initial condition: 0% B, then linear gradient 0- 60% B over 60 min.Flow rate: 75 mL/min, Detection: UV @ 230 nm;
MS: 1022.4 (M+H)+.
Elemental Analysis: CaIc. for C44H59N6Oi2Gd'2(C7Hi7NO5>3.2H2O: C, 47.41; H, 6.82; N, 7.63; O, 27.44; Gd, 10.70%. Found: C, 47.89; H, 6.68; N, 7.60; Gd, 11.02; H2O, 4.09%.
[00187] The final product (23) was then screened using an IBM PC20-minispec relaxometer for relaxivity in water and Seronorm™, and for HSA binding by the centrifree micro partition method. As in Example 1, the following were tested: mouse tolerance, MRA in rabbits, blood Tl and imaging in rabbits and plasma Tl lowering ex vivo in monkeys. The compound tested displayed an acceptable tolerance profile in mice (MLD = 3 mmol/kg) Results appear in Table 1 and Table 5.
EXAMPLE 4 -Synthesis and Screening of Gd-TE-DOTA-a-B Meg (Class 1)
Figure imgf000088_0001
[00188] The compound was prepared as follows: To a solution of 1 (100 mg, 0.192 mmol) in H2O (1.5 niL) was added Gd(OAc)3^H2O (85.7 mg, 0.21 mmol, Aldrich) in H2O (1.5 mL). The pH of the solution was maintained at 4.5 by adding IN NaOH and it was stirred at RT for 2 days. It was directly used for the next step without further purification. [For related compounds see: Jacques, Vincent; Gilsoul, Dominique; Comblin, Vinciane; Desreux, Jean F. Rigidified macrocyclic lanthanide chelates for magnetic resonance imaging. Journal of Alloys and Compounds (1997), 249(1-2), 173-177. Desreux, Jean F.; Tweedle, Michael F.; Ratsep, Peter C; Wagler, Thomas R.; Marinelli, Edmund R. Preparation of hepatobiliary tetraazamacrocyclic magnetic resonance contrast agents. U.S. (1994), 12 pp. US 5358704 A 19941025]
HPLC: tR 6.5 min. Column: YMC C18 4.6 mm x 250 mm, S-IOP μm, 12θA; Eluents: A: H2O (0.1% TFA), B: CH3CN (0.1% TFA); Elution: Isocratic 4% B; Flow rate: 1.0 ml/min; Detection: UV @ 230 nm.
MS: 677.1 (M+H)+.
[00189] To a crude mixture of (2) in H2O (3 mL) was added thiophosgene (44 mg, 0.384 mmol) in CHCl3 (1.5 mL) in one portion. The resulting mixture was stirred at RT for 2h. The process was monitored by HPLC. The layers were separated and the pH of the aqueous layer was adjusted from 1.8 to 7 by adding IN NaOH. This solution was directly used for the next step without further purification.
HPLC: tR: 6.3 min; Column: YMC C18 4.6 mm x 250 mm, S-IOP μm, 12θA; Eluents: A: H2O (0.1% TFA), B: CH3CN (0.1% TFA), Elution: Isocratic 20% B; Flow rate 1.0 ml/min.; Detection: UV @ 230 nm.
MS: 719.1 (M + H)+.
[00190] To a solution of 4 (40 mg, 0.081 mmol) in MeOH (5 mL) was added Et3N (8.2 mg, 0.081 mmol). The solvent was evaporated and THF (2 mL) was added. This suspension was added to the above crude 3 in H2O and the suspension was stirred at RT for 16 h. CH3CN/H2O (1 :1, v/v; 2 mL) was added and the solution became homogeneous. The pH of the solution was adjusted from 6 to 9 and it was stirred at RT for 16 h (final pH of the solution was 7). It was purified by prep. HPLC using a YMC C- 18 (250 mm x 20mm) reverse phase HPLC column. The column was eluted with 5% CH3CN/H2O (both containing 0.05% TFA) for 5 min., 5-20% in 10 min., and finally 20-50% in 60 min. The desired fractions were collected and lyophilized. This gave 25 mg of the product with purity of 89% (HPLC). The material was then purified by DEAE-sephadex resin. The compound in 5 mM triethylammonium bicarbonate solution was loaded onto the reconditioned resin and it was eluted with a gradient of aqueous triethylammonium bicarbonate solution (5 mM-800 mM, ) total volume of 360 mL). The desired fractions were combined and lyophilized to give 25 mg of (5) (97% pure as determined by analytical HPLC), as a white fluffy solid.
HPLC:_fo: 6.7 min; Column: YMC C18 4.6 mm x 250 mm, S-IOP μm, 12θA; Eluents: A: H2O (0.1% TFA), B: CH3CN (0.1% TFA);Elution: Isocratic 40% B, Flow rate: 1.0 niL/min; Detection: UV at 230 nm.
MS: 1099.4 (M + H)+.
[00191] BioRad AG 50-X8 ion exchange resin (100 mL) in a glass column fitted with a conductivity meter at the column outlet, was reconditioned as follows: The column was washed with 400 mL of 3 N HCl, H2O (to pH 7, 400 mL of 2 N NaOH, H2O to pH 7, then 400 mL of 3 N HCl and finally rinsed with H2O to pH 7. N-methyl-D-glucamine (35 g in 100 mL of H2O) was passed through the column; then the column was washed with a large excess of H2O until the conductivity was <0.9μS/cm The Gd-chelate (5) (25 mg) in 20 mL of H2O was applied to the column and the eluate was monitored by UV at 230 nm, employing a Knauer Variable Wavelength UV Detection using a 0.4 mm flow cell. The fractions containing the compound were combined and lyophilized. A total of 25 mg of the compound 6 was obtained as a white fluffy solid.
HPLC: tR 6.8 min; Column: YMC C18 4.6 x 250 mm, S-IOP μm, 12θA pore; Eluents: A: H2O (0.1% TFA), B: CH3CN (0.1% TFA); Elution: 40% B; Flow rate: 1.0 niL/min; UV @ 230 nm.
MS: (M+H)+ 1099.4.
[00192] After synthesis, the compound was evaluated as described in Examples 1-3 above. Results are shown in Table 1.
EXAMPLE 5 - Synthesis and Screening of Gd(CE-DTP A)-a-B 3Meg (Class 1)
Figure imgf000090_0001
The compound was prepared as follows: To a solution of 1 (7.0 g, 11.75 mmol) in CH2Cl2 (120 mL) was added 2 (11.6 g, 15.3 mmol,) (L-Glutamic acid and L-lysine as useful building blocks for the preparation of bifunctional DTP A- like ligands. Anelli, Pier Lucio; Fedeli, Franco; Gazzotti, Ornella; Lattuada, Luciano; Lux, Giovanna; Rebasti, Fabrizio. Bioconjugate Chemistry (1999), 10(1), 137-140), HATU [O-(7-azabenzotriazol-l-yl)- 1,1,3,3-tetramethyluronium hexafluorophosphate] (5.95 g, 15.3 mmol), and Et3N (3.1 g, 30.9 mmol, Aldrich). The clear solution was stirred at room temperature for 3 h. The volatiles were evaporated and the residue was dissolved in EtOAc (1000 mL). It was washed with 5 % NaHCO3 (2 x 400 mL), 0.05 N HCl (2 x 400 mL), H2O (1 x 200 mL), and dried (MgSO4). Evaporation of solvent afforded 16.7 g (excess of theory) of 3 (containing some ethyl acetate).
MS: 1199 (M+H)+.
HPLC: tR 27.2 min; Column: YMC C18 4.6 x 250 mm, S-IOP μm, 12θA pore; Eluents: A: H2O (0.1% TFA), B: CH3CN (0.1% TFA); Elution: Initial condition: 45% B, then linear gradient to 95% B over 25 min.; Flow rate: 1.0 ml/min.; Detection: UV @ 230 nm.
To a solution of 3 (16.7 g, 13.9 mmol) in EtOH (80 mL) was added palladium, on carbon (10 wt. % (dry basis), wet, Degussa type ElOl NE/W, 1.4 g, Aldrich). The mixture was hydrogenated at 45 psi for 20 h. The catalyst was filtered through a Celite™ cake and the volatiles were evaporated to provide 15.3 g (99% yield) of compound 4.
HPLCrfe: 21.3 min. Column: YMC C18, 4.6 x 250 mm, S-IOP μm, 12θA pore; Eluents: A: H2O (0.1% TFA), B: CH3CN (0.1% TFA); Elution: Initial condition:45% B, then linear gradient to 95% B over 25 min.;; Flow rate: 1.0 ml/min.; Detection: UV @ 230 nm. [00193]
1H-NMR (500 MHz, CDCl3): δ 0.95-1.85 (m, 8H, CH2's on cyclohexyl); 1.40 (s, 45H, t- Bu's; 2.05 (m, 2H, CH2CH2CONH-); 2.15-2.25 (m, IH, CONHCH2CH on cyclohexyl); 2.40- 2.50 (m, IH, CHCONH on cyclohexyl); 2.55-2.65 (m, 2H, CH2CH2CONH-); 2.65-2.80 (m, 8H, NCH2's); 3.00-3.15 (m, 2H, CH2 on biphenyl); 3.15-3.38 (m, 2H, CH7CH7CONHCH2); 3.48-3.32 (m, 9H, NCH2C02tBu & NHCO2tBu); 4.79-4.85 (m, IH, NHCHCO2Bn); 6.19 (d, IH, CONHCH); 6.87 (m, IH, CONHCH2); 7.00-7.53 (m, 14H, CH's on biphenyl and benzyl).
MS: 1109 (M+H)+. [00194] Compound 4 (15.3 g, 13.8 mmol) was dissolved in 400 mL of TFA/H2θ/triisopropylsilane (95:2.5:2.5, v/v/v). The mixture was stirred at room temperature for 4 h. The volatiles were evaporated to provide a pasty residue. Ether (400 mL) was added, the mixture was stirred for 10 min and the precipitate was filtered. This was repeated 3X to remove all the organic impurities. The grayish solid was dried in vacuo to give 12.5 g of a crude material. This was dissolved in CHsC]NkH2O: IN HCl 1 :4: 1 v/v/v and purified by preparative HPLC employing a Rainin Dynamax-60 A C-18 column 250 mm x 41.4 mm equipped with a guard column.. The column was eluted at 50 mL/min and a gradient elution (CH3CN/H2O (both containing 0.1 % TFA) 15 % to 75 % over 120 min) was employed. The product-containing fractions were lyophilized to give pure 5 as a white fluffy solid (7.3 g, 53% yield).
HPLC: tR 12.3 min; Column: YMC C18 4.6 x 250 mm, S-IOP μ, 12θA pore; Eluents: A: H2O (0.1% TFA), B: CH3CN (0.1% TFA); Elution: Initial condition: 25% B , then linear gradient to 65% B over 20 min.; Flow rate: 1.0 mL/min.; Detection: UV at 230 nm.
1H-NMR (500 MHz, D2O): δ 0.85-1.70 (m, 9H, CH2's on cyclohexyl & CONHCH2CH on cyclohexyl); 1.85-1.92 (m, IH, CHCONH on cyclohexyl); 1.85-1.95 (m, 2H, CH2CH2CONH-); 2.30-2.40 (m, 2H, CH2CH2CONH-); 2.90-3.10 (m, 8H, NCH2's); 3.35- 3.40 (m, 4H, CH2 on biphenyl & CH2CH2CONHCH2); 3.48-3.52 (m, IH, NHCHCO7H); 3.92-3.40 (s, 8H, NCH2COOH); 4.60-4.65 (m, IH, CONHCHCOOH); 7.30-7.65 (m, 9H, CH's on biphenyl).
MS: 828.4 (M+H)+.
[00195] To a suspension of 5 (6 g, 6.0 mmol) in H2O (300 mL) was added IN NaOH solution to adjust the pH to 5. Then a solution of Gd(OAc)3 (3.3 g, 8.0 mmol) in H2O (10 mL) was added and the pH of the mixture was maintained at 5 by adding IN NaOH. The cloudy solution was stirred at room temperature for 16 h. This solution was purified by preparative HPLC employing a Rainin Dynamax-60 A C-18 column (250 mm x 41.4 mm) equipped with guard column.. The column was eluted at 50 mL/min and a gradient elution (0% CH3CN:H2O v/v over 10 min., then ramp from 0-20 % over 10 min., then ramp to 75 % over 30 min) was employed. Pure product-containing fractions were lyophilized to give compound 6 as a white fluffy solid (4.8 g, 81.3% yield).
HPLC: tR 12.2 min; Column: YMC C18 4.6 x 250 mm, S-IOP μm, 12θA pore; Eluents: A: H2O (0.1% TFA), B: CH3CN (0.1% TFA); Elution: Initial condition: 25% B, then linear gradient to 65% B over 20 min., Flow rate: 1.0 ml/min.; Detection: UV at 230 nm.
MS: 983.3 (M+H)+.
Elemental Analysis: CaIc. for C40H47N5O14GdNa3^H2O: C, 39.70; H, 5.41; N, 5.79; Na, 5.70; Gd 12.99. Found: C, 39.75; H, 5.29; N, 5.72; Na, 5.45; Gd, 13.15.
[00196] A glass column was packed with 100 mL of BioRad AG50W-X8 ion exchange resin and the outlet was connected to a conductivity meter. The column was washed with 400 mL of 3N HCl, rinsed with H2O to pH 7, washed with 400 mL of 2N NaOH, rinsed with H2O to pH 7, then washed with 400 mL of 3N HCl and then rinsed with H2O until pH 7. Next, the column was washed with a solution of 70 g of N-methyl-D-glucamine in 300 mL of H2O followed by distilled deionized water until the conductivity was <0.9 μS/cm. The Gd- chelate 6 (3.6 g, 3.7 mmol) in 400 mL of H2O/CH3CN 80/20 (v/v) was applied to the column and the eluate was monitored by UV at 230 nm. The product-containing fractions were combined and lyophilized. A total of 5.3 g (88% yield) of the desired meglumine salt 7 was obtained as a white fluffy solid.
HPLC: tR 12.3 min.Column: YMC C18, 4.6 x 250 mm, S-IOP μm, 12θA pore; Eluents: A: H2O (0.1% TFA), B: CH3CN (0.1% TFA); Elution: Initial condition: 25% B, then linear gradient to 65% B in 20 min., Flow rate: 1.0 mL/min; Detection: UV @ 230 nm.
MS: 983.4 (M+H)+.
Elemental Analysis: CaIc. for C6IHiOiN8O29GdOH2O: C, 45.18; H, 6.65; N, 6.91; Gd, 9.70. Found: C, 44.88; H, 6.59; N, 6.72; Gd, 9.95. [00197] The compound was analyzed as described above in Examples 1-4. Results are shown in Tables 1 and 5.
EXAMPLE 6 - Synthesis of Gd-(7-MP-DO3 A)-GaB (Meglumine salt)
(Class 1)
Figure imgf000094_0001
[00198] To a solution of 1,4,7, 10-tetraazacyclododecane (TACD) (21.5 g, 0.125 mol) in CHCl3 (200 ml) was added benzyl-2-bromoacetate (11.45 g, 7.92 mL, 0.05 mol) in CHCl3 (50 mL) dropwise over a period of 2 h and the reaction mixture was stirred at 40C for 24 h. Chloroform was evaporated and the residue was treated with a saturated solution of sodium bicarbonate (200 mL). The mixture was extracted with methylene chloride and the organic phase was washed with water. The organic layer was dried with sodium sulfate. Methylene chloride was removed to give a viscous oil, which was vacuum-dried for 24 h to provide crude 1-benzyloxycarbonylmethy- 1,4, 7, 10-tetraazacyclododecane (1) (12.0 g, 75% yield) which was used in the next step without further purification. 1H-NMR (500 MHz, CDCl3): δ 2.55 and 2.75 (m, 16 H, NCH2CH2N), 3.48 (s, 2 H, NCH2CO2CH2Ph),5.12(s, 2 H, CH2Ph), 7.32 (m, 5 Η, ArH).
MS: 321 (M+Η)+.
[00199] A round-bottomed flask was charged with the benzyl ester l (11.0 g, 0.034 mol) in toluene (300 mL) and dimethylformamide diethyl acetal (7.3 g, 8.5 mL, 0.049 mol). The mixture was heated at 850C for 12 h with stirring. The volatiles were evaporated on a rotary evaporator to provide a yellow oil which was treated with dry ether (200 mL). A gummy solid formed during the addition of ether and the yellow ether solution was filtered through Celite™. The filter cake was washed with ether (2 x 100 mL). The combined ether washings were concentrated to give crude 1-benzyl (1,4,7, 10-tetraazatricyclo[5.5.1.0<4,l, 3>-tridec-10- yl)acetate (2) (10.2 g, 89% yield) as a thick yellow oil which was used in the next step without further purification.
1H-NMR (500 MHz, CDCl3): δ 2.60 and 3.09 (m, 16 H, NCH2CH2N), 3.52 (s, 2 Η, NCH2CO2CH2Ph), 4.81 (s, 1 H, CH), 5.12 (s, 2 H, CH2Ph), 7.32 (m, 5 Η, ArH).
MS: 331 (M+Η)+.
[00200] A solution of benzyl (l,4,7,10-tetraazatricyclo[5.5.1.0<4,l,3>-tridec-10- yl)acetate (2) (10.0 g, 0.06 mol) in THF (100 mL) and water (25 mL) was stirred at room temperature for 18 h. The volatiles were removed under vacuum and the light yellow oil obtained was dried under vacuum to provide compound 3 (9.0 g, 85.7%) which was used in the next step without further purification.
1H-NMR (500 MHz, CDCl3): δ 2.60-2.9 and 3.4-3.9 (m and s, 18 H, NCH2CH2N and NCH2CO2CH2Ph ), 5.12 (s, 2 H, CH2Ph), 7.32 (m, 5 Η, ArH), 8.12 (s, 1Η, CΗO).
MS: 349.2 (M+Η)+. [00201] A stirred solution of l-benzyloxycarbonylmethy-7-formyl -1,4,7,10- tetraazacyclododecane (3) (9.0 g , 0.0258 mol) in acetonitrile (80 rnL) was treated with diisopropylethylamine (13.0 g , 17.6 rnL, 0.1 mol) and t-butyl bromoacetate (11.6 g, 8.8 mL, 0.06 mol) RT for 24 h. The volatiles were removed on a rotary evaporator and the residue was treated with water (150 mL) and the entirety of the contents of the flask was extracted with ethyl acetate (250 mL). The ethyl acetate layer was separated and washed sequentially with aqueous NaHCO3 solution (200 mL), water (200 mL) and dried (Na2SO4). The volatiles were removed and the oil obtained was chromatographed over silica gel. The column was packed with hexane-ethyl acetate (7:3, v/v) and eluted sequentially with hexane-ethyl acetate (7:3, v/v; 600 mL), hexane-ethylacetate (1 : 1 v/v; 600 mL), and finally with pure ethyl acetate. The product-containing fractions were collected, the volatiles removed and the residue was dried under vacuum to give 4 (8.7 g, 59% yield) as a thick yellow oil. [00202]
1H-NMR (500 MHz, CDCl3): δ 1.45 (s, 18 H, C4H9), 2.65-2.95 (m, 12 H, NCH2CH2N), 3.42 and 3.49 (m, 2H, NCH2CH2N, NCH2CO2CH2Ph and NCH2CO2C4H9), 5.12 (s, 2 H, CH2Ph), 7.32 (m, 5 Η, ArH), 8.02 (s, 1 Η, CΗO).
MS: 577.5 (M+Η)+.
HRMS (FAB): m/z, CaIc. for C30H48N4O7 599.3421 (M+Na+); Found: 599.3422.
[00203] A mixture of 1 -benzyloxycarbonylmethy-7-formyl- 4, 10-di-t- butoxycarbonylmethyl-l,4,7,10-tetraazacyclododecane (4) (5.0 g, 8.6 mmol), hydroxylamine hydrochloride (0.8 g, 11.5 mmol) and water (0.3 mL) in isopropanol (35 mL) was refluxed for 12 h. The progress of the reaction was monitored by TLC using CH2C12:CH3OH (95:5, v/v). At completion of the reaction the isopropanol and water were removed on a rotary evaporator and the resulting viscous oil was dried under vacuum. This was treated with chloroform (200 mL) and the light precipitate that formed was filtered and the chloroform solution was concentrated to a viscous oil. The oil was dried under vacuum to give a foamy solid. This was triturated with ether (75 mL) and the precipitated white solid was filtered and dried to provide 3.52 g (74% yield) of benzyl ester 5. 1H-NMR (500 MHz, CDCl3): δ 1.49 (s, 18 H, C4H9), 2.60-2.9 (m and 16 H, NCH2CH2N), 3.45 (s, 4 H, NCH2CO2C4H9), 3.52 (s, 2 H, NCH2CO2CH2Ph), 5.12 (s, 2 H, CH2Ph), 7.32 (m, 5 Η, ArH).
MS: 549 (M+Η)+.
[00204] To a mixture of the benzyl ester 5 (3.0 g,5.47 mmol) and diisopropylethylamine (3.25 g, 4.38 mL, 25 mmol) in acetonitrile (10 mL) was added the triflate A (Phillion, D. P., US 4740608 (1988)), (3.0 g, 8.42 mmol) in acetonitrile (5 mL). The mixture was stirred at 4O0C for 6 h. After the reaction, acetonitrile was evaporated and the residue was treated with a saturated solution of sodium carbonate (25 mL). The mixture was extracted with ethyl acetate (2 x 100 mL), washed with water (100 mL) and dried (Na2SO4). The ethyl acetate was evaporated and the oil obtained was dried under vacuum to provide a foam which was triturated with hexane (3 x 75 mL) at 5O0C to give 1- benzyloxycarbonymethyl-4,10-di-t-butoxycarbonymethyl-7-di-t-butoxyphosphinylmethyl- 1,4,7,10-tetraazacyclododecane (6) (3.0 g, 73% yield) as a white solid. An analytical sample was obtained by crystallization from hexane-ethyl acetate.
1H-NMR (500 MHz, CDCl3): δ 1.42 (s, 18 H, C4H9), 1.51(s, 18 Η, O=PC4H9), 2.10-2.95 (m, 24 Η, NCH2CH2N, NCH2CO2 and NCH2PO), 5.10 (s, 2 Η, CH2C6H5), 7.35 (m, 5 H,
CH2C6Hs).
MS: 755.5 (M+Η)+.
HRMS (FAB): m/z, Calcd. for C38H67N4O9PNa 777.4543 (M+Na); Found: 777.4599.
[00205] To a solution of 1 -benzyloxycarbonymethyl-4, 10-di-t-butoxycarbonymethyl-
7-di-t-butoxyphosphinylmethyl-l,4,7,10-tetraazacyclododecane (6) (1.0 g, 0.0078 mmol) in THF (20 mL) was added Pd-C 10% (0.5 g, Degussa type ~ 50% water), and the mixture was hydrogenated at 45 psi for 12 h. The catalyst was filtered through Celite™ and the filter cake was washed with THF (2 x 30 niL). The combined THF washings were concentrated on a rotary evaporator to give the acid as a thick, viscous oil which was dried under vacuum for 24 h to give l-carboxymethyl-4,10-di-t-butoxycarbonymethyl-7-di-t-butoxyphosphinylmethyl- 1,4,7,10-tetraazacyclododecane (7) (0.84 g, 95% yield) as a foamy solid which was used without further purification.
1H-NMR (500 MHz, CDCl3): δ 1.42 (s, 18 H, C4H9), 1.51(s, 18 H, O=PC4H9), 2.50-2.95 (m, 24 Η, NCH2CH2N and NCH2CO2 and NCH2PO).
MS (FAB): 687 (M+Na)+.
ΗRMS (FAB): m/z, CaIc. for C3iΗ6iN4O9P 665.4254 (M+H+); Found: 665.4256.
[00206] To a cooled (O0C) mixture of 1 -carboxymethyl-4, 10-di-t- butoxycarbonymethyl-7-di-t-butoxyphosphinylmethyl- 1,4,7,10-tetraazacyclododecane (7), (0.832 g; 1.0 mmol) and HATU (0.38 g, 1.0 mmol) in CH2Cl2 (10 mL) was added DIEA (0.39 g, 0.55 mL, 3.0 mmol) and the mixture was stirred at O0C for 5 min. The tripeptide 8 (0.51 g, 0.967 mmol) was added and the mixture was stirred at O0C for 1 h. The volatiles were evaporated and the residue was treated with NaHCO3 solution (10 mL) and the resulting mixture was extracted with CH2Cl2 (2 x 15 mL). The organic phase was washed with water and dried (Na2SO4). Evaporation of the volatiles gave an oil, which was vacuum-dried to give a foamy solid which was triturated with water to provide a granular solid which was filtered. The solid was again triturated with water, filtered and air-dried to provide compound 9 (1.12 g, 97% yield) which was used directly in the next step.
1H-NMR (500 MHz, CDCl3): δ 0.942 (m, 2 H, cyclohex ), 1.42 (s, 18 H, C4H9), 1.51 (s, 18 Η, O=PC4H9), 1.62-3.4 (m, 32 Η, NCH2CH2N, cyclohex, NCH2CO2 and NHCOCH2NH), 4.92 (m, 1 H, CHCH2Ar), 5.15 (q, 2 H, CH2C6H5), 6.0 (d, 1 H, CONHCH), 6.75 and 6.9 (2 x bt, 2 H, CONHCH2), 7.0-7.55 (m, 14 H, ArH).
MS: 1197 (M+Na)+. [00207] To a solution of compound 9 (1.12 g, 0.95 mmol) in THF (15 niL) was added Pd-C 10% (0.5 g, Degussa type ~ 50% water), and the mixture was hydrogenated at 45 psi for 12 h. The catalyst was filtered through Celite™ and the filter cake was washed with THF (2 x 30 mL). The combined THF washings were concentrated on a rotary evaporator to give the product 10 as a thick, viscous oil which was vacuum-dried for 24 h to give 10 (0.92 g, 85% yield) as a foamy solid..
1H-NMR (500 MHz, CDCl3): δ 0.94 (m, 4 H, cyclohex ), 1.42 (s, 18 H, C4H9), 1.51(s, 18 H, O=PC4H9), 1.62-3.4 (m, 32 H, NCH2CH2N, cyclohex, NCH2CO2, NCH2PO and NHCOCH2NH), 4.80 (m, 1 H, CHCH2Ar), 6.45 (m, 1 H, CHNHCO) 6.75 and 7.10 (2 x bt, 2 H, CONHCH2), 7.28-7.62 (m, 9 H, ArH).
MS: 1107 (M+Na)+.
[00208] TFA (1.5 mL) was added to compound 10 (0.35 g, 0.32 mmol) and the mixture was stirred at RT for 6 h. Ether (50 mL) was then added and the precipitated solid was filtered and dried under vacuum to give compound 11 (0.25 g, 72% yield) as a white solid.
HPLC: tR 25.8 min Column: YMC C18, 4.6 x 250 mm, S-IOP μm, 12θA pore; Eluents: A: H2O (0.1% TFA), B: CH3CN (0.1% TFA); Elution: Initial condition: 0% B, then linear gradient to 80% B over 40 min; Flow rate: 1.0 mL/min, Detection: UV @ 230 nm.
1H-NMR (500 MHz, DMSOd6): δ 0.81-2.1 (m, 14 H, cyclohex, NHCH2CO and CONHCH2CO) 2.75-3.7 (m, 16 Η, NCH2CH2N), 3.48 (s, 2 Η, NCH2CO2CΗ2Ph),5.12 (s, 2 H, CH2Ph), 7.32 (m, 5 Η, ArH).
MS: 860 (M+Η)4
[00209] To a solution of compound 11 (0.25 g, 0.23 mmol) in water (5 mL) Gd(OAc)3
(0.125 g, 0.3 mmol) in water (2 mL) was added. The initial pΗ was 2.57 and was adjusted to 5.0 by addition of IN NaOH solution. The solution became turbid and the mixture was allowed to stir for 24 h. The progress of the reaction was followed by HPLC. Upon completion of the reaction the solution was centrifuged to allow decantation of the liquid layer as a clear solution. This was purified by preparative HPLC. Fractions containing the pure product were collected and lyophilized to give the product 12 (0.125 g, 52% yield) as a white solid.
HPLC: tR 25.80 min Column: YMC C18 4.6 x 250 mm, S-IOP μm, 12θA pore; Eluents: A: H2O (0.1% TFA), B: CH3CN (0.1% TFA); Elution: Initial condition: 0% B, then linear gradient to 60% B over 30 min; Flow rate: 1.0 mL/min, Detection: UV @ 230 nm.
MS: 1015 (M+H)+.
Elemental Analysis: CaIc. for C40H55N7O12PGd^ H2O: C, 45.75; H, 5.66; N, 9.34; Gd 14.97; O, 21.33; P, 2.95%.
To a solution of compound 11 (108 mg, 1.0 mmol) in water (5 mL), Gd(OAc)3 (48 mg, 1.2 mmol) in water (2 mL) was added. The initial pH was 2.50 and the pH of the solution was adjusted to 5.0 by the addition of aqueous meglumine solution. The solution became turbid and was stirred for 24 h. The progress of the reaction was followed by HPLC. The solution was centrifuged in order to allow decantation of the liquid phase as a clear solution. The decanted solution was purified by preparative HPLC. Fractions containing the pure product were collected and freeze dried to give compound 12a (50 mg) as a white solid.
HPLC: tR 25.25 min; Column: YMC C18, 4.6 x 250 mm, S-IOP μm, 12θA pore; Eluents: A: H2O (0.1% TFA), B: CH3CN (0.1% TFA); Elution:: Initial condition: 0% B, then linear gradient to 80% B over 40 min; Flow rate: 1.0 mL/min, Detection: UV @ 230 nm.
Elemental Analysis: CaIc. for
Figure imgf000100_0001
C, 42.88; H,6.73; N,
8.33; Gd, 10.40; 0,29.62, P, 2.05%.
Found: C, 43.21; H, 6.56; N, 8.10; Gd, 8.80%.
[00210]
[00211] The product was analyzed as described above in Examples 1-5. Results are shown in Tables 1 and 5. EXAMPLE 7: Preparation of [(Fl-l)-K(Gd-DOTA)-OH]Meg
(Class 2)
Figure imgf000101_0001
A 500 niL round-bottomed flask equipped with a stir bar was charged with tetrabutylammonium hydrogen sulfate (25 mmol, 8.5 g, 0.5 equiv), NaHCO3 (139.3 mmol, 11.7 g, 2.8 equiv) and water (80 mL)(caution, gas evolution). The mixture was stirred for 30 min and Boc-Lys(Fmoc)-OH (50 mmol, 23.46 g, 1 equiv) in CH2Cl2 (80 mL) was added. The mixture was stirred for 20 min, and benzyl bromide (8.9 g, 52 mmol, 6.2 mL, 1.04 equiv) was added in one portion.. After 18.5 h, HPLC analysis showed that the reaction was complete. The organic layer was separated and the aqueous layer was extracted with EtOAc (150 mL). The organic layers were combined, the volatiles were removed and the residue was dissolved in EtOAc (250 mL). The organic phase was washed with water (3 x 250 mL) to remove the tetrabutylammonium salts, and then dried with saturated brine followed by Na2SO4). The drying agent was filtered and the volatiles were removed to provide the crude product 1 as an oil in 100% yield. [00212]
[00213] Ether (300 mL) was added to the crude oil 1 with agitation. The solution turned cloudy. Hexane (50 mL) was added to the solution. This resulted in formation of a white precipitate. The whole solution was kept in a refrigerator for 15 h. This resulted in the formation of a well-defined solid which was collected and dried overnight. The yield of pure solid product 1 was 26.7g (95.6%).
[00214]
1H-NMR (500 MHz, CDCl3): δ 1.5 (s, 9H, -C(CH3)3 on Boc group), 1.2-1.9 ( m, CH2 on
Lysine), 3.2 (s, 2H, CH2-N(FmOc)) 4.17-4.82 (m, 4H, CH2 -O-CO) CH-CH2, and CH-CO).
5.2 (dd, 2H, CH2-Ph), 7.3-7.8 (m, 13H all aromatic H).
MS: 597.2 (M+K)+, 581.3 (M+Na)+, 559.3 (M+H)+, 459.3 (M-Boc+H)+.
[00215] Boc-Lys(Fmoc)-benzyl ester (1) (14.5 g, 26.0 mmol) was dissolved in CH2Cl2 (80 mL) and anisole (6 mL). The mixture was stirred for 5 min. TFA (80 mL) was added and the mixture was stirred for 1.5 h at ambient temperature. HPLC analysis indicated complete reaction. The volatiles were removed, and ether (200 mL) was added. A solid formed after 15 min of stirring and was collected after 3h. This was dried overnight to give pure Lys(Fmoc)-OBn»TFA (14.4 g, 97% yield).
HPLC: tR 8.44 min; Column: YMC C18 4.6 x 250 mm, S-IOP μm, 12θA pore; Eluents: A: H2O (0.1% TFA), B: CH3CN (0.1% TFA); Elution: Isocratic 50% B; Flow rate: 1.0 niL/min; Detection: UV @ 254 nm.
1H-NMR (500 MHz, CDCl3): δ 1.5 (s, 9H), 1.2-1.9 (m, CH2 on Lysine), 3.1 (s, 2H, CH2- N(Fmoc)), 3.95-4.40 (m, 4H, CH2 -O-CO, CH-CH2, and CH-CO ), 5.1 (dd, 2H, CH2-Ph),
7.3-7.8 (m, 13H all aromatic H).
MS: 481.2 (M+Na)+, 459.3 (M+l)+.
[00216] Compound 2 (13.0 g, 22.7 mmol, 1.0 equiv), 1-fluorenecarboxylic acid (5.0 g, 23.8 mmol,1.05 equiv) and HATU (9.0 g, 23.8 mmol, 1.05 equiv) were charged to a 500 mL round-bottomed flask. Then CH2Cl2 (135 mL) was added. The resulting suspension was stirred for 5 min after which DIEA (6.14 g, 8.28 rnL, 23.8 mmol, 2.10 equiv) was added, followed by DMF (5 rnL). After 30 min, stirring became difficult and an additional 50 mL portion Of CH2Cl2 was added. The mixture was stirred for 3 h upon which HPLC analysis indicated completion of the reaction. The flask was cooled to ~10°C, the solid was collected, and was washed with cold CH2Cl2 and 50/50 hexane/CH2Cl2. The solid was dried overnight to give ~ 18 g of crude product. HPLC and NMR analysis indicated the presence of impurities. Therefore the product was washed with EtOAc/hexane 50/50 (3 x 200 mL), EtOAc (2 x 200 mL) andether (1 x 200 mL). This provided the pure product 3 (14.5 g, 98% yield).
HPLC: tR 36.4 min. Column: YMC C18, 4.6 mm x 250 mm, S-IOP μm, 12θA pore; Eluents: A: H2O (0.1% TFA), B: CH3CN (0.1% TFA); Elution: Initial condition: 30% B, then linear gradient to 100% B over 35 min, hold 10 min at 100% B, then ramp to 30% B over 5 min; Flow rate: 1.0 mL/min; Detection: UV @ 254 nm.
1H-NMR (500 MHz, DMSO-d6): δ 1.2-1.9 (m, CH2 on Lysine), 3.0 (s, 2H, CH2-N(FmOc)), 4.0-4.5 (m, 6H, CH2 -O-CO, CH-CH2, -C-CH2-C-and CH-CO), 5.2 (q, 2H, CH2-Ph), 7.3-8.1 (m, all aromatic H).
MS: 696.4 (M+K)+, 573.4 (M+Na)+, 651.4 (M+l)+.
[00217] Compound 3 (11.62 g) was suspended in CH3CN (100 mL)and the stirred suspension was cooled to O0C in an ice bath. A solution of 40% piperidine in CH3CN (100 mL) was added to the mixture and the mixture was stirred for 40 min. The flask was allowed to rise to room temperature and the reaction mixture was stirred for 3 h. The volatiles were removed; hexane was added and the suspension was stirred for 10 min. The solid was collected by filtration and washed with hexane (2 x 200 mL). Then the solid was dissolved in CHCl3 (45 mL), and applied to a 300 g silica gel column which was eluted with CHCl3, 1% MeOH in CHCl3, 2% MeOH in CHCl3, 5% MeOH in CHCl3 and 10% MeOH in CHCl3. The product-containing fractions were combined and the volatiles were removed to provide compound 4 (5.8 g ,74% yield). HPLC: tR 16.69 min. Column: YMC C18, 4.6 mm x 250 mm, S-IOP μm, 12θA pore; Eluents: A: H2O (0.1% TFA), B: CH3CN (0.1% TFA); Elution: Initial condition: 30% B, then linear gradient to 100% B over 35 min, hold 10 min at 100% B, then ramp to 30% B over 5 min; Flow rate: 1.0 mL/min; Detection: UV @ 254 nm.
1H-NMR (500 MHz, CDCl3): δ 1.3-1.9(m, CH2 on Lysine), 2.7(m, 2H, CH2-NH2), 4.2 (s, 2H, C-CH2-C), 4.9 (m, IH, CH), 5.2 (q, 2H, CH2-Ph), 6.8-8.0 (m, all the aromatic H).
MS: 429.4 (M+H)+.
[00218] Compound 4 (4.60 g, 10.75 mmol, 1.0 equiv.), HATU (6.13 g, 16.13 mmol, 1.5 equiv.), CH2Cl2 (80 mL) and DO3A10CM-l,4,7-tris t-butyl-ester»NaCl (10.5 g, 16.13 mmol, 1.5 equiv.) were added to a 500 mL round-bottomed flask. The flask was cooled to O0C and DIEA (4.17 g, 32.25 mmol, 3.0 equiv.) was added. The reaction mixture was stirred at O0C for 15 min and then at room temperature for 19 h. HPLC analysis indicated complete reaction. The volatiles were removed, the residue was dissolved in EtOAc and washed with saturated NaHCO3 solution (3 x 250 mL), 3% NaHCO3 solution (2 x 250 mL), and saturated brine (2 x 250 mL). The organic phase was dried over Na2SO4. After removal of the volatiles, a 14 g portion of a pale yellow glass was isolated. The weight of material was greater than that calculated based on quantitative yield due to the presence of the excess DO3A10CM-l,4,7-tris-t-butyl-ester»NaCl employed in the synthesis. This crude product 5 was directly used without further purification.
HPLC: tR 13.7 min; Assay 72% purity (largest impurity peak 9%); YMC C 18, 4.6 mm x 250 mm, S-IOP μm, 12θA pore; Eluents: A: H2O (0.1% TFA), B: CH3CN (0.1% TFA); Elution: Initial condition: 50% B, then linear gradient to 100% B over 25 min, hold for 10 min at 100% B then ramp to 50% B over 5 min; Flow rate: l.OmL/min; Detection: UV @ 254 nm;
1H-NMR (500 MHz, CDCl3): δ 1.3-1.6 (m, CH2 on Lysine), 1.5 (m, 27 H, C(CH3J3), 2.0- 4.0 (m, macrocycle, CH2-NH, CH2-COO, C-CH2-C), 4.5 (IH, CH), 5.2 (q, 2H, CH2-Ph), 7.0- 8.0 (m, all aromatic H).
MS: 983.6 (M+H)+. [00219] A hydrogenation bottle was charged with crude compound 5 (14 g), CH3OH (100 rnL) and ~3 g of 10% Pd/C catalyst. The bottle was evacuated and refilled several times with hydrogen. Then compound 5was subjected to hydrogenolysis at 50 psi H2 for 3.5 h at ambient temperature. HPLC analysis indicated complete hydrogenolysis. After removal of the catalyst and solvent, 12 g (94% yield) of the crude product 6 was obtained. This was used without further purification.
HPLC: tR 6.77 min; Assay 94% purity; Column: YMC C18, 4.6 mm x 250 mm, S-IOP μm, 12θA pore; Eluents: A: H2O (0.1% TFA), B: CH3CN (0.1% TFA); Elution: Initial condition: 50% B, then linear gradient to 100% B over 25 min, hold for 10 min at 100% B then ramp to 50% B over 5 min; Flow rate: l.OmL/min; Detection: UV @ 254 nm.
1H-NMR (500 MHz, CDCl3): (Crude compound): δ 1.3-1.6 (m, CH2 on Lysine), 1.5 (m, 27 H, C(CH3J3), 2.0-4.0 (m, macrocycle, CH2-NH, CH2-COO, C-CH2-C), 4.5 (IH, CH), 7.0-8.0 ( m, all aromatic H).
MS: 915.5 (M+Na)+, 893.5 (M+H)+.
[00220] Compound 6 (12 g) was treated with Reagent B (TFA : H2O : Phenol : i- (C3Hy)3SiH, 88:5:5:2, v/v/w/v) (200 mL) for 18h. HPLC analysis indicated complete reaction. The volatiles were removed and ether was added to precipitate the product. The precipitated product was collected and washed with ether and the resulting material was purified by preparative reversed phase HPLC using a Cl 8 preparative column. This provided the pure product ligand 7 (4.1 g, 53% yield). An additional 1.1 g of 95% pure product was also obtained.
HPLC: tR 9.64 min; Assay: 99.6% purity; Column: YMC C 18, 4.6 mm x 250 mm, S-IOP μm, 120A pore; Eluents: A: H2O (0.1% TFA), B: CH3CN (0.1% TFA); Elution: Isocratic 28% B; Flow rate: 1.0 niL/min; Detection: UV @ 254 nm. 1H-NMR (500 MHz, CDCl3): δ 1.3-1.8 (m, 6H, CH2 on Lysine), 3.0-4.2 (m, macrocycle, CH2-NH, CH2-COO, C-CH2-C), 4.5 (IH, CH), 7.3-8.5 (m, all aromatic H).
MS: 747.4 (M+Na)+, 725.4 (M+l)+.
Elemental Analysis: CaIc. for C38H51N7On-2.1 H2O-U TFA: C, 49.48; H, 5.68; N, 8.79;O, 25.93; F, 10.13. Found: C, 49.66; H, 5.73; N, 8.79; F, 9.73.
[00221] (Fl- 1)-K(DO3A1 OCM)-OH 7 (3.9 g, 5.37 mmol) was dissolved in water (135 mL), and Gd(OAc)3»4H2O (2.38 g, 5.86 mmol, 1.09 equiv.) was added. The mixture was stirred for 20 min. The pH of the solution was adjusted to ~4.5 with IN NaOH. The mixture was stirred at ambient temperature for 18h. Analytical reverse phase HPLC showed the reaction was completed. The product was purified by reversed phase preparative HPLC using a preparative scale C18 column. A total of 3.9 g pure product was obtained. A cation exchange column (Bio-Rad AG50W-X8 resin in the meglumine form) was prepared. A dilute aqueous solution of the free acid 8a was applied to the column and the column was washed with water and fractions were collected. The fractions containing the desired product, compound 8b, were collected, combined and lyophilized to give the pure product (3.95 g) as a colorless fluffy solid. The yield from compound 7 was 91%. HPLC analysis indicated no free gadolinium ion in the final product.
HPLC: tR 8.18 min; Assay: >99% purity; Column: YMC C18, 4.6 mm x 250 mm, S-IOP μm, 12θA pore; Eluents: A: H2O (0.1% TFA), B: CH3CN (0.1% TFA); Elution: Isocratic 28% B; Flow rate: 1.0 mL/min; Detection: UV @ 254 nm.
MS: (M+Na)+ 902.3, (M+l)+ for Gd-chelate 880.3, (M+Meg)+ 1075.4, (M+l)+ 880.3, (Meg+1)+ 195.9.
Elemental Analysis: Calc.for GdC36H45N6O10-C7H17NO5-SOH2O: C, 45.77; H, 6.08; N, 8.69; O, 25.52; Gd, 13.94 %. Found: C, 46.32; H, 6.14; N, 8.63; Gd, 13.80; Na, 0.08; % H2O 5.05.
[00222] The compound was analyzed as described above in Examples 1-5. Results are shown in Table 2. EXAMPLE 8: Compounds Having High Fraction Bound to Serum and/or Higher Measured Relaxivity in Seronorm™ Versus Comparator MS325 (Gadofosveset
Trisodium),
[00223] Compounds of the present invention were analyzed to compare binding to human serum and measured relaxivity in Seronorm™ versus the reference compound MS-
325.
[00224] A higher fraction bound to serum will result in a longer lifetime in blood, thus providing a more persistant signal. Higher relaxivity value will result in more signal for a given compound after administration.
[00225] Table 6 below shows fraction bound to serum and relaxivity data collected for representative compounds of the present invention:
Table 6: Fraction Bound to Serum and Relaxivity
Figure imgf000107_0001
Figure imgf000108_0001
Figure imgf000109_0001
Figure imgf000110_0001
Key for bound fraction (Fb) values: a = bound fraction > MS325, b = bound fraction = MS325, c = bound fraction < MS325
Key for Rl Seronorm™ values: a = relaxivity in Seronorm™ > MS325, b = relaxivity in Seronorm™ below but within 10% of MS325 value, c = relaxivity in Seronorm™ <90% of MS325 value
[00226] The following explains the column headings in Table 6:
[00227] Chelator type refers to the chelator used in the compound.
[00228] Relaxation gain determined refers to the relaxation gain, which is the ratio of the Tl relaxivity of the substance in a solution of HSA called Seronorm™ (commercially available from Nycomed-Amersham) to the relaxivity of the same compound in water at a specified concentration. Values close to 1 indicate that no binding to protein is occurring and that the compound is unsuitable. On the other hand, higher values are more desirable because they highlight, in an MRI image, the bloodstream (intravascular compartment) due to the much higher Tl relaxivity (which is the source of the MRI signal) when bound to the HSA in the blood.
[00229] The parameter rls is the Tl relaxivity of the substance in a solution of human serum albumin called Seronorm™ (commercially available from Nycomed- Amersham)
[00230] The parameter rlw is the Tl relaxivity of the compound in water, where no protein is present.
[00231] The parameter Fb is the fraction of the compound bound to human serum albumin (HSA) under standard conditions of defined concentration and temperature. A value of 0.90 indicates that 90% of the molecules of the test compound are bound to HSA. A higher fraction bound (Fb) means that more of the compound should provide the higher signal characteristic of the bound molecule due to immobilization of the chelating function, or significant slowing of the chelating function upon binding of the chelate-HSA binding ligand to the HSA.
[00232] As shown in Table 6, Gd-(TE-DOTA)-aB Meg provides a relaxivity (ri) in
Seronorm™ (HSA) of 42.09, well above the comparator MS-325 (n Seronorm = 36.57). Such a high relaxivity will provide more signal for a given compound after administration. Furthermore, Gd-(TE-DOTA)-aB Meg provides an Fb of 0.996, well above the Fb of the comparator MS-325 (Fb = 0.87).
[00233] As further shown in Table 6, the following compounds each provide higher bound fractions than the MS325 comparator: Gd-(10CM-DO3 A)-G-G- W-(Nin-2,4,6- trimethylbenzenesulfonyl)-Cr Meg; Gd-(10CM-DO3A)-aaB Na;Gd-(10CM-DO3A)-aak Meg; Gd-(10CM-DO3 A)-DDk Meg; Gd-(10CM-DO3 A)-EEB Meg; Gd-(10CM-DO3A)-Pak Na; Gd-(10CM-DO3A)-Pak Meg; Gd-(10CM-DO3A)-Dak Meg; Gd-(10CM-DO3 A)-DDj Na; Gd-(10CM-DO3 A)-DDX Na; Gd-(10CM-DO3 A)-PaJ Na; Gd-(10CM-DO3A)-aaX Na; Gd- (10CM-DO3 A)-GGm Meg; Gd-(10CM-DO3 A)-Ga0 Meg; Gd-(10CM-DO3 A)-GGo Meg; Gd-(10CM-DO3 A)-Pa0 Meg; Gd-(10CM-DO3A)-Pam Meg; Gd-(10CM-DO3A)-Gam Meg; Gd-(10CM-DO3A)-aao Meg; Gd-(10CM-DO3A)-aam Meg; Gd-(10CM-DO3A)-Gan Meg; Gd-(10CM-DO3 A)-Ga-(p-Cl)-Bip Na; Gd-(CEMP-DO3 A)-GaB; Gd-(CEMP-DO3A)-aB Na; Gd-4-CEMP-DTTA-GaB Meg; Gd-(CE-DTPA)-GaB 3Meg; Gd-(CE-DTP A)-aB 3Na; Gd- (CE-DTP A)-aB 3Meg; Gd-(CM-DOTA)-aB Meg; Gd-(CM-DOTA)-aB Na; Gd-(7MP- DO3A)-GaB; Gd-(7MP-DO3A)-GaB Meg; Gd-(7MP-CM-DO3A)-aB Meg; Gd-(TE- DOTA)-aB Meg; Gd-(10CM-DO3A)-K-(l-fiuorenecarboxamido)O- Na+; Gd-(IOCM- DO3A)-K-(l-fiuorenecarboxamido)-O" Meg; [(Fl-l)-K{G-[Gd-(10CM-DO3A)]}-O"](Meg); and [(Fl- l)-K[G-{Gd-(10CM-DO3A)]} -CT] (Na+).
[00234] Definitions of abbreviations for unnatural amino acids as used herein are presented below: c = 8-aminocaprylic acid j=L-O-benzyl-tyrosine, k=L-2,6-dichlorobenzyl-tyrosine. m = L- (Nin-2,4,6-trimethybenzensulphonyl)-tryptophan, n = L-(Nin-2,4,6- triisopropybenzensulphonyl)-tryptophan, o = L-(Nin-2,4,6-trimethylbenzoyl)-tryptophan, p = O-benzyl-L-threonine, X= L-2-naphthylalanine.
Figure imgf000112_0001

Claims

We claim:
1. A composition for the imaging and/or treatment of a disease in a mammal, comprising a compound having the formula:
Ch - Ll - R - L2 wherein Ch is a chelating moiety optionally complexed with M, a metal or a radionuclide;
Ll is a first linker;
R is a hydrophobic compound; and
L2 is an optional second linker wherein said compound is selected from the group consisting of: Gd-(10CM-DO3 A)-G-G- W- (Nin-2 ,4,6-trimethylbenzenesulfonyl)-Cr Meg; Gd-(10CM-DO3A)-aaB Na;Gd-(10CM- DO3A)-aak Meg; Gd-(10CM-DO3 A)-DDk Meg; Gd-(10CM-DO3 A)-EEB Meg; Gd-(IOCM- DO3A)-Pak Na; Gd-(10CM-DO3A)-Pak Meg; Gd-(10CM-DO3A)-Dak Meg; Gd-(IOCM- DO3 A)-DDj Na; Gd-(10CM-DO3 A)-DDX Na; Gd-(10CM-DO3 A)-PaJ Na; Gd-(IOCM- DO3A)-aaX Na; Gd-(10CM-DO3 A)-GGm Meg; Gd-(10CM-DO3A)-Gao Meg; Gd-(IOCM- DO3 A)-GGo Meg; Gd-(10CM-DO3 A)-Pa0 Meg; Gd-(10CM-DO3A)-Pam Meg; Gd-(IOCM- D03A)-Gam Meg; Gd-(10CM-DO3A)-aao Meg; Gd-(10CM-DO3A)-aam Meg; Gd-(IOCM- DO3 A)-Gan Meg; Gd-(10CM-DO3A)-Ga-(p-Cl)-Bip Na; Gd-(CEMP-DO3 A)-GaB; Gd- (CEMP-DO3A)-aB Na; Gd-4-CEMP-DTTA-GaB Meg; Gd-(CE-DTPA)-GaB 3Meg; Gd- (CE-DTP A)-aB 3Na; Gd-(CE-DTP A)-aB 3Meg; Gd-(CM-DOTA)-aB Meg; Gd-(CM- DOTA)-aB Na; Gd-(7MP-DO3A)-GaB; Gd-(7MP-DO3A)-GaB Meg; Gd-(7MP-CM- DO3A)-aB Meg; Gd-(TE-DOTA)-aB Meg; Gd-(10CM-DO3 A)-K-(I - fluorenecarboxamido)O- Na+; Gd-(10CM-DO3A)-K-(l-fluorenecarboxamido)-O" Meg; [(Fl-I )-K (G- [Gd-( 10CM-DO3 A)] } -O~](Meg); and [(Fl- 1 )-K[G- (Gd-( 10CM-DO3 A)] } -O ] (Na+) and salt or alternative salt forms thereof; and optionally further comprises a targeting vector Q.
2. The composition of claim 1 wherein the compound having the formula: Ch - Ll - R - L2 is Gd-(TE-DOTA)-aB Meg or alternative salt forms thereof, optionally further comprising a targeting vector Q.
3. The composition of claim 1 wherein the relaxivity in human serum albumin is 10% greater than the relaxivity of gadofosveset trisodium.
4. The composition of claim 1 wherein the fraction bound to serum is greater than the fraction of gadofosveset trisodium bound to serum.
5. The composition of claim 1 complexed with a metal.
6. The composition of claim 5 wherein the metal is a paramagnetic metal ion.
7. The composition of claim 6 wherein the paramagnetic metal ion is selected from the group consisting of Mn2+, Cu2+, Fe2+, Co2+, Ni2+, Gd3+, Eu3+, Dy3+, Pr3+, Cr3+, Co3+,
T F^e 3+ , rTpi-3+ , rTp-bi 3+ , τNvτdj3+ , α Sm 3+ , THTo 3+ , TE-.r 3+ , τP>a 4+ or T E-1u 2+.
8. The composition of any one of claims 1 or 2 complexed with gadolinium.
9. The composition of claim 1 complexed with a radionuclide.
10. A method of imaging a tissue or organ of a mammal comprising the steps of: administering a composition of claim 1 complexed with a metal useful for magnetic resonance imaging and imaging a tissue or organ by magnetic resonance imaging.
11. A method of imaging at least a portion of the cardiovascular system of a mammal comprising the steps of: administering a composition of claim 1 complexed with a metal useful for magnetic resonance imaging and imaging a tissue or organ by cardiovascular magnetic resonance angiography.
12. A method of imaging a tissue or organ of a mammal comprising the steps of: administering a composition of claim 1 complexed with a radionuclide suitable for nuclear imaging and imaging a tissue or organ by nuclear imaging.
13. The method of claim 12 where in the nuclear imaging is selected from the group consisting of: scintigraphy, SPECT and PET imaging.
14. The composition of claim 9 wherein the radionuclide is selected from the group consisting Of 51Mn, 52Fe, 60Cu, 68Ga, 72As, 94mTc, or 110In, 99mTc, 51Cr, 67Ga, 68Ga, 47Sc, 51Cr, 167Tm, 141Ce, 111In, 168Yb, 175Yb, 140La, 90Y, 88Y, 153Sm, 166Ho, 165Dy, 166Dy, 62Cu, 64Cu, 67Cu, 97Ru, 103Ru, 186Re, 188Re, 203Pb, 211Bi, 212Bi, 213Bi, 214Bi, 105Rh, 109Pd, 117mSn, 149Pm, 161Tb, 177Lu, 198Au and 199Au.
15. The method of claim 12, wherein the metal is selected form the group consisting Of64Cu, 67Ga, 68Ga, 99mTc, and 111In.
16. A method of treating a patient in need thereof with radiotherapy comprising administering a compound of claim 1 complexed with a radionuclide suitable for radiotherapy.
17. The method of claim 16, wherein the radionuclide is selected from the group consisting Of64Cu, 90Y, 105Rh, 111In, 117mSn, 149Pm, 153Sm, 161Tb, 166Dy, 166Ho, 175Yb, 177Lu, 1867188Re, and 199Au.
18. The composition of claim 1, wherein Q and Ll both comprise amino acids.
19. The composition of claim 1 further comprising a chemotherapeutic.
20. The composition of claim 1 , where in the targeting vector is a peptide.
21. The composition of claim 20, where in the targeting peptide is selected from the group consisting of: peptide hormones, analogs of somatostatin, Substance P agonists, oxytocin; endothelin A and endothelin B; bradykinin; Epidural Growth Factor (EGF); Interleukin-1; and cholecystokinin (CCK-B).
22. A method of preparing a composition of claim 1 , wherein said method comprises the steps of: attaching a chelating moiety to the first linker, attaching said first linker to the hydrophobic group R via a protecting group having a carboxylic group thereto, attaching an optional second linker to said hydrophobic group, and attaching an optional targeting vector Q to said optional second linker.
23. A method of preparing a composition of claim 1, wherein said method comprises attaching a hydrophobic group R which is capable of binding HSA to a first linker, then attaching the first linker to the chelating moiety Ch, then attaching another conjugatable group in the first linker to a second linker followed by attachment of the second linker to an optional molecule which can serve as a targeting vector.
24. A method of preparing a composition of claim 1 , wherein said method comprises preparing the components of the entire construct as modules such as Ch-Ll, R-P (where P is an optional protecting group for a reactive function other than that to be employed for assembling the construct) and L2-Q and then attaching the modules in any desired order to obtain constructs such as Ch - Ll - R - L2 - Q, Ch-L1(L2-Q)-R or Ch(L2- Q)-Ll-R.
25. The method of any one of claims 22-24 further comprising complexing the composition with a metal or a radionuclide.
26. A composition for the imaging and/or treatment of a disease in a mammal, comprising a compound having the formula:
Ch - Ll - R - L2 - Q wherein Ch is a chelating moiety optionally complexed with M, a metal or a radionuclide;
PCT/US2008/064359 2007-05-21 2008-05-21 Conjugates which bind a blood protein such as human serum albumin and methods of using the same in diagnostic and therapeutic applications WO2008144728A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US93109007P 2007-05-21 2007-05-21
US60/931,090 2007-05-21

Publications (1)

Publication Number Publication Date
WO2008144728A1 true WO2008144728A1 (en) 2008-11-27

Family

ID=40122211

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2008/064359 WO2008144728A1 (en) 2007-05-21 2008-05-21 Conjugates which bind a blood protein such as human serum albumin and methods of using the same in diagnostic and therapeutic applications

Country Status (1)

Country Link
WO (1) WO2008144728A1 (en)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104072607A (en) * 2013-03-29 2014-10-01 中国科学院化学研究所 Organic metal ruthenium conjugated protein and preparation method and application thereof
WO2016083605A1 (en) * 2014-11-28 2016-06-02 Ge Healthcare As Lanthanide complex formulations
JP2017538685A (en) * 2014-11-28 2017-12-28 ジーイー・ヘルスケア・アクスイェ・セルスカプ Metal complex preparation
CN108288072A (en) * 2018-01-26 2018-07-17 深圳市唯特视科技有限公司 A kind of facial expression synthetic method based on generation confrontation network
WO2018200615A3 (en) * 2017-04-27 2019-01-10 The Regents Of The University Of California Modified mri contrast agents and uses thereof
CN110426464A (en) * 2019-07-08 2019-11-08 苏州天马药业有限公司 The HPLC detection method of isomers in a kind of meglumine

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060241018A1 (en) * 2003-01-13 2006-10-26 De Haen Christoph Linkers for radiopharmaceutical compounds
US20060269479A1 (en) * 2005-04-19 2006-11-30 Colton Clark K Amphiphilic polymers and methods of use thereof
US20070003479A1 (en) * 1995-02-01 2007-01-04 Epix Pharmaceuticals, Inc., A Delaware Corporation Diagnostic imaging contrast agents with extended blood retention

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070003479A1 (en) * 1995-02-01 2007-01-04 Epix Pharmaceuticals, Inc., A Delaware Corporation Diagnostic imaging contrast agents with extended blood retention
US20060241018A1 (en) * 2003-01-13 2006-10-26 De Haen Christoph Linkers for radiopharmaceutical compounds
US20060269479A1 (en) * 2005-04-19 2006-11-30 Colton Clark K Amphiphilic polymers and methods of use thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
COMBLIN ET AL.: "Designing New MRI Contrast Agents: a Coordination Chemistry Challenge", COORDINATION CHEMISTRY REVIEWS, vol. 185-186, October 1999 (1999-10-01), pages 451 - 470 *

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104072607B (en) * 2013-03-29 2019-01-15 中国科学院化学研究所 A kind of Organometallic ruthenium compound protein and the preparation method and application thereof
CN104072607A (en) * 2013-03-29 2014-10-01 中国科学院化学研究所 Organic metal ruthenium conjugated protein and preparation method and application thereof
CN107001293B (en) * 2014-11-28 2023-09-19 通用电气医疗集团股份有限公司 Lanthanide complex formulations
JP2017535553A (en) * 2014-11-28 2017-11-30 ジーイー・ヘルスケア・アクスイェ・セルスカプ Lanthanide complex preparation
JP2017538685A (en) * 2014-11-28 2017-12-28 ジーイー・ヘルスケア・アクスイェ・セルスカプ Metal complex preparation
US12070511B2 (en) 2014-11-28 2024-08-27 Ge Healthcare As Lanthanide complex formulations
US11186553B2 (en) 2014-11-28 2021-11-30 Ge Healthcare As Metal complex formulations
CN107001293A (en) * 2014-11-28 2017-08-01 通用电气医疗集团股份有限公司 Lanthanide complexes preparation
US11400172B2 (en) 2014-11-28 2022-08-02 Ge Healthcare As Lanthanide complex formulations
RU2707070C2 (en) * 2014-11-28 2019-11-22 ДжиИ Хелткер АС Compositions containing complexes of metals of series of lanthanides
US10576169B2 (en) 2014-11-28 2020-03-03 Ge Healthcare As Lanthanide complex formulations
AU2015352426B2 (en) * 2014-11-28 2020-03-05 Ge Healthcare As Lanthanide complex formulations
WO2016083605A1 (en) * 2014-11-28 2016-06-02 Ge Healthcare As Lanthanide complex formulations
JP2020517584A (en) * 2017-04-27 2020-06-18 ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア Modified MRI contrast agents and uses thereof
WO2018200615A3 (en) * 2017-04-27 2019-01-10 The Regents Of The University Of California Modified mri contrast agents and uses thereof
CN108288072A (en) * 2018-01-26 2018-07-17 深圳市唯特视科技有限公司 A kind of facial expression synthetic method based on generation confrontation network
CN110426464A (en) * 2019-07-08 2019-11-08 苏州天马药业有限公司 The HPLC detection method of isomers in a kind of meglumine

Similar Documents

Publication Publication Date Title
AU2003299957B2 (en) Improved gastrin releasing peptide compounds
EP2097438B1 (en) Fibrin binding peptide conjugates for diagnostic and therapeutic applications
EP2790732B1 (en) Clicked somatostatin conjugated analogs for biological applications
JP5676673B2 (en) Cancer imaging and treatment
US20110280802A1 (en) Linkers For Radiopharmaceutical Compounds
WO2022098745A1 (en) Compositions, delivery systems, and methods useful in tumor therapy
US7922998B2 (en) Gastrin releasing peptide compounds
US8420050B2 (en) Gastrin releasing peptide compounds
WO2008144728A1 (en) Conjugates which bind a blood protein such as human serum albumin and methods of using the same in diagnostic and therapeutic applications
EP1706154A1 (en) Improved gastrin releasing peptide compounds
MXPA06007322A (en) Improved gastrin releasing peptide compounds

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 08756047

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 08756047

Country of ref document: EP

Kind code of ref document: A1