CN103269721B - Pre-targeting test kit, method and the reagent wherein used - Google Patents

Abstract

The present invention describes for the medical imaging of targeting and/or the pre-targeting method for the treatment of and relevant test kit, wherein uses the abiogenic reaction chemical group demonstrating bio-orthogonal reaction each other.The present invention relates to use [4+2] inverse electrical requirements (inverse) Diels Alder chemistry to provide the coupling between pre-targeting probe and effector probe.To this end, one in these probes comprises electron deficiency tetrazine or other suitable diene, and another is the E-cyclo-octene with one or more upright substituent group.

Description

Pre-targeting test kit, method and the reagent wherein used

Technical field

The present invention relates to the pre-targeting method (pretargeting of the medical imaging for targeting and/or treatment Method), abiotic (abiotic) reaction demonstrating that bio-orthogonal (bio-orthogonal) is reactive each other is wherein used Property chemical group.The invention still further relates to containing at least one pre-targeting probe (pre-targeting probe) and at least one The pre-targeting test kit (kit) of effector probe (effector probe), wherein said pre-targeting probe comprises primary targeting Partly (primary targeting moiety) and the first bio-orthogonal reaction group, and wherein said effector probe bag Containing effector part, such as label or pharmaceutically active compound, and the second bio-orthogonal reaction group.The invention still further relates to The pre-targeting reagent used in said method and test kit.Present invention is particularly directed to nuclear imaging and X-ray therapy.

Background technology

In many fields of medical diagnosis and treatment, it is desirable to optionally by reagent, such as therapeutic agent (medicine) or examine Disconnected (such as imaging) agent is delivered to the specific site in experimenter (subject) such as patient body or finite region.

The active targeting of organ or tissue is by desired active part (such as contrast-enhancing agent or cytotoxin chemical combination Thing) being directly or indirectly implemented in combination with targeting structure, its be combined with cell surface or promote at target site interested or Neighbouring cellular uptake.Targeting moiety for this type of reagent of targeting is typically cell surface target (such as membrane receptor), knot Structure albumen (such as amyloid plaque) or cell internal target (such as RNA, DNA, enzyme, cell-signaling pathways) have the structure of affinity. These parts can be antibody (fragment), protein, fit (aptamers), oligopeptide, oligonucleotide, oligosaccharide and peptide, peptidomimetic (peptoids) and known at specified disease or obstacle accumulation organic drug compound.As selection, contrast/therapeutic agent can With targeting metabolic pathway, it is expressed in disease (such as infect or cancer) period and raises, such as DNA, protein and film synthesis and Carbohydrate absorbs.In illing tissue, diseased cells can be distinguished from health tissues and provide in early days by above-mentioned mark Detection, specific diagnosis and unique probability of (targeting) treatment.

It is it generally for successful molecular imaging/therapeutic agent with especially for one major criterion of nuclear imaging/therapeutic agent Present the picked-up of high target, demonstrate from non-target tissue simultaneously and quickly remove (by kidney and/or liver and gall system) from blood. But, this is frequently present of problem: such as, and imaging research in people is it has been shown that through radiolabeled antibody at knub position In 24 hours, can reach Cmax, but need the concentration of other several days described labeled antibody in the circulating cycle just to decline To being as little as enough to occur the level of successful imaging.

Slow or insufficient accumulation in target tissue and slowly remove these problems brought from non-target region (special It is not for nuclear imaging and treatment) already lead to the application of pre-targeting method.

Pre-targeting refers to the step in targeted approach, is wherein provided with pre-targeting to primary target (such as cell surface) and visits Pin.The latter includes secondary target, and it is finally by further probe (the described effect physical prospecting by being equipped with secondary targeting moiety Pin) carry out targeting.

So, in pre-targeting, pre-targeting probe is attached to primary target.Described pre-targeting probe also carries secondary target, It promotes diagnosis (imaging) and/or therapeutic agent, described effector probe specific binding.Forming described pre-targeting probe Structure have been located at target site after (spend such as 24 hours time), if removing is insufficient naturally, then can make Remove the part of excess from blood by scavenger.The second incubation step (preferably take relatively short period of time, such as 1-6 hour) In, described effector probe is attached to described by its secondary targeting moiety (secondary targeting moiety) The pre-targeting probe that (in advance) combines.Described secondary target (being present on described pre-targeting probe) and described secondary targeting moiety (are deposited It is on described effector probe) quickly should combine with high specific and high-affinity, and should be stable in vivo.

For imaging, the general concept of pre-targeting is drawn in FIG.Here, described effector probe be comprise for The image probe of the detectable of imaging pattern.Described effector probe is attached to described by its secondary targeting group The pre-targeting probe that (in advance) combines.

The general example of secondary target/secondary targeting moiety pair is biotin/streptavidin or antibody/antigen body System.In order to effectively, described effector probe must quickly from internal discharge (such as passing through kidney) with having desired by providing The high tumor accumulation of relatively low non-target accumulation.Therefore, these probes are the least.

In nuclear imaging and X-ray therapy, the concept of pre-targeting has further benefit, because spending time taking pre-target Radionuclide can be required for step to implement, and use the secondary targeting step of radionuclide can be real quickly Execute.The latter makes it possible for more short-life radionuclide, has the advantage that will minimize the radiological dose of patient, example As used PET reagent to replace SPECT reagent.In conjunction with multidentate ligand system (streptavidin, dendritic (dendrimers)) pre-targeting method is used signal can be provided at target site to amplify in mri.Additionally, usual the method Be conducive to the use of general contrast medium.

Generally (such as antibody-antigene) and especially (biotin-streptavidin, anti-in pre-targeting in biology Body/hapten, antisense oligonucleotide) implement high selectivity interact entity the biggest.Therefore, peptide and little organic is used Part is as the pre-targeting of primary targeting group, and metabolic imaging and cell internal target imaging, owing to the size of secondary target makes The use of little primary group is meaningless and still cannot realize.

And, existing pre-targeting system is limited by the factor relevant to their biological nature.Biotin is endogenous Molecule, and its conjugate (conjugates) can be disconnected by sero-enzyme biotin enzyme.When using antisense pre-targeting, Described oligonucleotide can be attacked by RNAse and DNAse.Protein and peptide also experience natural decomposition approach.These phase interactions With being weakened further by residence time in the non-covalent of them and dynamic characteristic and limited target.And, endogenous biological Element is combined with biotin conjugate competition streptavidin.Finally, streptavidin is high degree of immunogenicity.

A recent development is to avoid and be based only upon natural/biological targeting structure (i.e. biotin/avidin chain bacterium Element, antibody/hapten, antisense oligonucleotide) the relevant shortcoming of pre-targeting.

One document of this respect is WO 2010/051530, wherein based on some diene such as tetrazine class and dienophile Such as trans-cyclooctene alcohol (trans-cyclooctenol, TCO) between reactivity pre-targeting is discussed.

One further document of this respect is Li etc., Chemical Communications, 2010,46 (42), p. 8043-8045, which depict based on 3 ,-diaryl-s-tetrazine and¹⁸Between the trans-cyclooctene of F-labelling The radiolabeling procedure for bioconjugation of Diels-Alder reaction.

Rossin etc., Angew. Chem. Int., Ed 2010,49, p. 3375-3378 relate to by using inverse The tumor of Diels-Alder reaction (inverse-electron-demand Diels-Alder reaction) of electrical requirements Pre-targeting.

Blackman etc., J. Am. Chem. Soc., 2008,130, p. 13518-13519 describe based on inverse Quick bio reactive for the Diels-Alder of electrical requirements combines.

Royzen etc., J. Am. Chem. Soc., 2008,130, p. 3760-3761 relate to passing through metal complex The photochemical syntheses of the trans-cyclooctene of the functionalization driven.

Although being obtained in that relatively quick reaction on the basis of these systems, but this also can not show a candle to above-mentioned biotin- The reactivity of streptavidin system.Therefore, it is to avoid the shortcoming of the latter, the major requirement of described reaction is but sacrificed, i.e. Speed.Accordingly, it is desirable to provide a kind of system, it is not based on biomolecule as discussed above, and also has the fastest Speed reaction rate.

Summary of the invention

In order to preferably meet above-mentioned hope, one aspect of the present invention provides the medical imaging for targeting and/or treatment Test kit (kit), it comprises at least one pre-targeting probe and at least one effector probe, wherein said pre-targeting probe Comprise primary targeting moiety and the first bio-orthogonal reaction group, and wherein said effector probe comprises effector part (such as label or pharmaceutically active compound) and the second bio-orthogonal reaction group, wherein said first and second biologies are just Any one of friendship reactive group is dienophile (dienophile), and described first and second bio-orthogonal reaction groups In another be diene, wherein said dienophile is the 8 ring dienophiles meeting formula (1):

Wherein the position of R is calm (equatorial) and R_aPosition be upright (axial), wherein X, Y, R and R_aEach represent H independently, or most six times (at most six instances) represents choosing freely following group The substituent group of the group become: alkyl, aryl, O-aryl, O-alkyl, S-aryl, S-alkyl, S (O)-aryl, S (O)-alkyl, S (O)₂-aryl, S (O)₂-alkyl, Si-aryl, Si-alkyl, Si-O-alkyl, OCO-alkyl, OCO-aryl, SCO-alkyl, SCO- Aryl, OCS-alkyl, OCS-aryl, SCS-alkyl, SCS-aryl, F, Cl, Br, I, N₃、SO₂H、SO₃H、SO₄H、PO₄H、OH、 SH、NO₂、NO、CN、OCN、SCN、NCO、NCS、CF₃, NR ' R " wherein R ' and R ' ' be independently of one another H, alkyl or aryl, C (= O) O-alkyl, C (=O) O-aryl, C (=S) O-alkyl, C (=S) O-aryl, C (=O) S-alkyl, C (=O) S-aryl, C (=S) S- Alkyl, C (=S) S-aryl, C (=O) NR ' R ' ' wherein R ' and R ' ' be independently of one another H, aryl or alkyl, NR ' CO-alkyl its Middle R ' be H, alkyl or aryl, NR ' CO-aryl wherein R ' be H, alkyl or aryl, NR ' C (=O) O-alkyl wherein R ' be H, alkane Base or aryl, NR ' (C=O) O-aryl wherein R ' be H, alkyl or aryl, OCONR '-alkyl wherein R ' be H, alkyl or aryl, OCONR '-aryl wherein R ' be H, alkyl or aryl, NR ' CONR ' '-alkyl wherein R ' and R ' ' be independently of one another H, alkyl or Aryl, NR ' CONR ' '-aryl wherein R ' and R ' ' be independently of one another H, alkyl or aryl, NR ' CSNR ' '-alkyl wherein R ' and R ' ' be independently of one another H, alkyl or aryl and NR ' CSNR ' '-aryl wherein R ' and R ' ' be H, alkyl or virtue independently of one another Base, CR ' NR ' ' wherein R ' and R ' ' are H, alkyl or aryl independently of one another；Wherein R_aIn one be included in optionally by In the connector part (Linker Moiety) of interval body extremely described pre-targeting probe or effector probe；Two of which R or R_a Part can form ring together；With at least one of which and most four R_aIt not hydrogen.

On the other hand, the invention provides pre-targeting method, and the pre-targeting reagent wherein used, and wherein use should The medical imaging of the targeting of test kit or therapy.

Another further aspect, the present invention is to meet the compound of above-mentioned formula (1), its pre-targeting method in animals or humans In.

Another aspect, the invention reside in and have the anti-of one or more upright substituent group (axial substituents) Formula-cyclo-octene in the pre-targeting method reacted based on inverse Diels-Alder as the purposes of dienophile reactant.

Accompanying drawing explanation

Fig. 1 depicts the general approach of pre-targeting concept as above.

Fig. 2 provides the reaction scheme for [4+2] Diels-Alder reaction；At (3,6)-two-(2-pyridine radicals)-s- Between tetrazine and E-cyclo-octene, it is followed by wherein being formed the inverse Diels-Alder reaction of product and dinitrogen.Because described instead Formula-Cyclooctene derivative comprises electron withdraw group unlike in classical Diels-Alder reaction, thus such Diels-Alder reaction reacts different from classical Diels-Alder, and commonly referred to " inverse electrical requirements Diels-Alder Reaction ".Hereinafter, the Diels-Alder cycloaddition reaction that two reactions steps sequences are the most initial (typically needs against electronics Ask Diels-Alder cycloaddition) and follow-up inverse Diels-Alder reaction will be simply referred to as " inverse Diels-Alder reacts " or " inverse DA”.Fig. 3 (a and b) depicts the general approach utilizing inverse Diels-Alder chemistry for pre-targeting.

Fig. 4 provides the pre-target of tumor utilizing the inverse-DA relating to mAb (B) modified for TCO-and radiolabeled tetrazine (A) To scheme.

Fig. 5 to Figure 10 shows the synthetic schemes of the compound related in embodiment.

Figure 11 shows that the E-discussed below of cyclo-octene alcohol is secondary and E-main isomer, and the contrast of Z isomer, Show spatial chemistry.

Figure 12 shows the structure of tetrazine-DOTA probe 28.

Figure 13 depicts three kinds of different cyclo-octene dienophiles internal stability in mice.

Figure 14 shows the SPECT/CT projection of mice alive.

Figure 15 depicts the replacement building-up process of tetrazine probe 28, and corresponding Gd-complex, 28-Gd^IIISynthesis.

Figure 16 shows the synthetic route of tetrazine model probe 35,38,40 and 42.

Figure 17 shows the synthetic route of novel TCOs.

Figure 18 provides (the carrier added) that TCO (20a or 20b) and carrier add at low concentrations in PBS¹⁷⁷Normalization reaction yield between Lu-tetrazine 28 (1eq.).

Figure 19 provides the internal stability that blood is removed the TCO 20b that the CC49-being corrected combines.Described data Point represents meansigma methods and described error bar represents a standard deviation (n=3).

Figure 20 shows the blood profile of CC49-TCO (20b) structure.Data point represents meansigma methods and error bar represents one Individual standard deviation (n=3).

Figure 21 provides the bio distribution without CC49-TCO in mice with tumor (20b) structure.Post represents meansigma methods and error Rod represents a standard deviation (n=3).

Figure 22 shows¹²⁵I-CC49 and¹²⁵I-CC49-TCO 44b (7.5) is moving without the blood in mice with tumor (n=3) Mechanics.Data are given with percentage injected dose/gram (%ID/g) with a standard deviation (error bar).

After Figure 23 provides mAb injection 4 days¹²⁵I-CC49 (open tubular column) and¹²⁵I-CC49-TCO 44b (7.5) is (solid Post) without the bio distribution in (n=3) in mice with tumor.Described post represents the percentage ratio with a standard deviation (error bar) Injection dosage/gram (%ID/g).

Figure 24 shows the internal stability of the TCO 44b being attached to CC49.Data point is to have a standard deviation (by mistake Difference rod) three times measurement average, fit to second order polynomial (Prism GraphPad v. 5.01).

Detailed description of the invention

Tension link octene (strained cyclooctene) parent used in the present invention is hereinafter represented with E-cyclo-octene Divinyl macromer.According to usual nomenclature, it will be appreciated that the result of alternatively X or Y, depend on position and the molecular weight of substituent group, phase Can become on same cyclo-octene isomeric forms and be expressed as Z-isomer.In the present invention, any replacement variant of the present invention, Regardless of whether be " E " or " Z " in form, or " cis " or " trans " isomer, will be considered as all that unsubstituted trans-ring is pungent Alkene, or the derivant of unsubstituted E-cyclo-octene.Term " trans-cyclooctene " (TCO) and E-cyclo-octene can exchange use And for all dienophiles according to the present invention, and for substituent group in form by when needing contrary name, It is all suitable for.I.e. the present invention relates to the carbon atom 1 and 6 of wherein following numbering be positioned at E (entgegen) or the cyclo-octene of trans position.

In general sense, the present invention is based on such a understanding: using derivative trans-cyclooctene, such as with instead The form of formula-cyclo-octene alcohol, as in the system of dienophile, selects the hydroxyl being wherein used for being derivatized to connector structure to be in The isomer of stand up position.E-cyclo-octene alcohol, it is in so-called crown conformation (crown conformation), has two kinds Isomer, the one at equatorial position (equatorial position) with OH is main isomer and has in stand up position The one having OH is secondary isomer.Described latter isomer is selected according to the present invention, and below with " E-is secondary " table Show.

It is not intended to be limited by theory, it is believed by the inventors that based on this discovery, the upper one or more upright substituent groups of TCO The demand solved in pre-targeting based on described inverse Diels-Alder reaction for more high response that exists for provide and answer Case.

Broadly, therefore the present invention expands to beyond the substituent group definition be given about formula (1) as provided above.Have one The fact that individual or multiple upright substituent group it is believed that and cause higher HOMO energy.HOMO, as it is known by the man skilled in the art, represent Highest occupied molecular orbital.According to the present invention, MOPAC simulation is used to determine the HOMO energy of described TCO.MOPAC (Molecular Orbital Package, molecular orbit bag) is to calculate known software in chemical field.Described MOPAC is soft Part bag includes several well-known semiempirical MO methods, including AM1 and PM3.Term AM1 and PM3 refers to different Hamiltonian function (Hamiltonians), i.e. Austin Model 1 and parameterized model 3 Hamiltonian function are (for AM1, also Ask for an interview M. J. S. Dewar etc., J. Am. Chem. Soc., 107,3902 (1985)；For PM3, also ask for an interview J. J. P. Stewart .J. Comput. Chem., 10,209 (1989) and J. Comput. Chem., 10,221 (1989))。

In the present invention, use comprises following method: in computer environment, it is provided that the molecule of the derivant of cyclo-octene Structure, is determined by the energy-optimised each molecular structure of minimum formation, determine described AM1 and PM3 Hamiltonian function and so that it is determined that Highest occupied molecular orbital (HOMO).

MOPAC data in table 6 show that the presence service of upright substituent group is in providing increasing in described trans-cyclooctene ring The purpose of the HOMO energy added.

Put up with specific embodiment and further describe the present invention with reference to some accompanying drawing, but the present invention is not by these institutes Limit and only limited by claim.Any reference in claim should be construed as restriction scope.Described Accompanying drawing is only schematic rather than determinate.In the drawing, the size of some key elements may quilt for purpose of explanation Exaggerate and not to scale (NTS) draws.In the present description and claims when using term " comprising ", it is not arranged Except other key element or step.When relating to singular noun and using indefinite article or definite article such as " a " or " an ", " the ", unless Additionally illustrate that otherwise it includes multiple (kind) described noun.

Additionally, be also noted that in description and claims that the term " comprising " used should not be construed as limited to The equipment enumerated subsequently；It is not excluded for other key element or step.Therefore, the scope of statement " comprising the device of device A and B " should not It is defined as the device being only made up of assembly A and B.It represents, for the present invention, the associated component that only has of described device is A And B.

" alkyl " and " aryl " is related in several chemical formulas.In this respect, " alkyl " represents independently of one another and is less than The acyclic straight of ten carbon atoms, side chain or cyclic alkyl radical, it can include 1-3 hetero atom such as O, N or S, preferably Having 1-6 carbon atom, and " aryl " represents the aryl less than ten carbon atoms or heteroaryl independently of one another, it can include 1-3 hetero atom such as N or S.In several chemical formulas, about " R " of letter such as " A ", " B ", " X ", " Y " and various numbering Group illustrates group or substituent group.The definition of these letters will be understood with reference to various, i.e. in different formulas, unless separately External declaration, otherwise these letters can have different meanings independently of one another.

In the embodiment of present invention further optimization, following chemical formula relates to " alkyl " and " virtue Base ".In this respect, what " alkyl " represented less than 10 carbon atoms independently of one another is aliphatic, straight chain, side chain, full Sum, undersaturated and/or ring-type alkyl, it can include 1-10 hetero atom such as O, N or S, and " aryl " is each independent Ground represents the aryl less than 20 carbon atoms or heterocyclic aryl, and it can be replaced, and it can include 1-10 Hetero atom such as O, N, P or S." aryl " also includes " alkaryl " or " aralkyl " (simply example: benzyl)." alkyl ", " virtue Base ", " alkaryl " and " aralkyl " carbon number of comprising can represent (i.e. C by the title before this type of term₁-C₁₀Alkane Base refers to that described alkyl can comprise 1 to 10 carbon atoms).Some compound of the present invention has chiral centre and/or tautomerism Body, and all of enantiomer, diastereomer (diasteriomers) and tautomer, and theirs is mixed Compound is within the scope of the present invention.

Inverse Diels-Alder reacts

Inverse Diels-Alder coupling chemistry generally includes and couples to form the reactant pair of unstable intermediate, described in Mesosome eliminates the little molecule as unique by-product by inverse Diels-Alder reaction, and (it can be to depend on initial compounds Such as N₂、CO₂, RCN) to form stable product.Described paired reactant includes as a kind of reactant (i.e. a kind of bio-orthogonal Reactive group) be suitable for diene, such as tetrazine derivatives such as electron deficiency tetrazine, and as another kind reactant (i.e. another Kind of bio-orthogonal reaction group) tension force (strained) cyclo-octene according to formula (1).

Such as electron deficiency (substituted) tetrazine quickly reacts, with the abnormal of the tension force E-cyclo-octene of the present invention, the company of result in Connecing reaction intermediate (ligation intermediate), it is made by elimination in [4+2] inverse Diels-Alder cycloaddition N for unique by-product₂It is rearranged to stable dihydrogen dazin.This figure 2 illustrates.

Both reactive species are abiotic, therefore do not suffer from quick internal metabolism.They are bio-orthogonal, Such as they the most optionally react in Physiological Medium.It is that described diene and described ring are pungent about this advantage put Alkene is the most substantially non-reacted to the biomolecule on intracellular or cell surface and other regions all such as serum etc..Cause This, the Compounds and methods for of the present invention can use in living cells, tissue or organism (organism).And, described instead Answering property group is relatively small and can introduce and does not significantly change size biology in Biosample or live organism.Utilize institute State [4+2] inverse Diels-Alder to react, it is possible to use little reaction participant such as tetrazine or cyclo-octene are by large-sized primary Targeting moiety such as antibody is combined with label or other molecule.Even more desirably, the reaction that utilization (coupling) is relatively small Participant such as tetrazine and cyclo-octene, can make relatively small primary targeting moiety such as peptide and label or other molecule knot Close.Described pre-targeting probe and the size of effector probe and character be not by described secondary target and the big shadow of secondary targeting moiety Ring so that (in advance) targeting scheme can be used in little targeting moiety.Because in such manner, it is possible to other tissue of targeting, the most described probe Destination be not limited to vascular system (vascular system) and intercellular space (currently used antibody-streptavidin Targeting is usually limited to vascular system and intercellular space).

Document about inverse electrical requirements Diels-Alder reaction and the behavior of reactive species pair includes: Thalhammer, F; Wallfahrer, U; Sauer, J, Tetrahedron Letters, 1990, 31 (47), 6851-6854; Wijnen, JW; Zavarise, S; Engberts, JBFN, Journal Of Organic Chemistry, 1996, 61, 2001-2005; Blackman, ML; Royzen, M; Fox, JM, Journal Of The American Chemical Society, 2008, 130 (41), 13518-19), R. Rossin, P. Renart Verkerk, Sandra M. van den Bosch, R. C. M. Vulders, l. Verel, J. Lub, M. S. Robillard, Angew Chem Int Ed 2010, 49, 3375, N. K. Devaraj, R. Upadhyay, J. B. Haun, S. A. Hilderbrand, R. Weissleder, Angew Chem Int Ed 2009,48,7013 and Devaraj etc., Angew.Chem.Int.Ed., 2009,48,1-5.

It should be understood that broadly, substantially can apply to pre-targeting can make according to the above-mentioned coupling chemistry of the present invention Any molecule, group or part right.I.e. one such to comprising primary targeting moiety, and it can be attached to primary Target, and comprise at least one secondary target further.Another will be the secondary targeting portion being suitable for being attached to described secondary target Point, and comprise the applicable therapeutical effect (typically pharmaceutically active compound) that plays further, or it is fixed to fit through imaging technique Position (i.e. label) or both parts.

Therefore, arbitrary by defined above straight according in the present invention, described pre-targeting probe and described effector probe Vertical substituted cyclo-octene functionalization, and another is by tetrazine or other diene functionalization being suitable for.This figure 3 illustrates.Top Scheme (Fig. 3 a) show pre-targeting probe, containing by connector part (optionally comprising flexible spacer body) with as primary The bipyridyl tetrazine that the antibody of targeting moiety connects, and effector probe, containing by connector (flexible spacer body) with can The cyclo-octene (as secondary targeting moiety) that detection label connects.Following scheme (Fig. 3 b) shows the side of contrast Case, the pre-targeting probe i.e. comprising cyclo-octene and the effector probe comprising tetrazine.

Although described figure the most clearly illustrates spatial chemistry, it should be appreciated that the most described cyclo-octene be according to The upright substituted cyclo-octene of formula (1) as defined above.

Dienophile

One the basic one-tenth of the present invention is that selection dienophile, i.e. according to the upright replacement of formula as defined above (1) TCO, it makes to be obtained in that up to 10 times or the more reaction rate increased for described bio-orthogonal coupled reaction.Or Person is said differently, and the response time is only the 10% of the original time needed.Or it is said differently again, a kind of reactant dense Degree can be low 10 times.

Described dienophile, broadly, is the trans-cyclooctene with at least one upright substituent group, i.e. at least a part of which At least one stand up position of one saturated carbon atom is not hydrogen.

As explained above, at least one and most four R_aIt not hydrogen, it is meant that this R_aIt is substituent group or company A part for interface structure.Preferably, non-hydrogen R_aQuantity be 1 or 2.

It is highly preferred that described at least one and most four described non-hydrogen R_aIt is positioned at the free R of choosing² _a、R³ _a、R⁴ _aAnd R⁵ _aComposition The position of group.Still more preferably, R² _a、R³ _a、R⁴ _aAnd R⁵ _aIn one or two be not hydrogen.Most preferably, substituent group or connection Body structure is as R³ _aAnd R⁴ _aIn one or both exist.

Preferably, described substituent group is selected from the group defined above with respect to formula as defined above (1).It is highly preferred that it is above-mentioned R_aIt is alkyl or O-alkyl, more preferably methyl or the O-tert-butyl group.

It should be noted that R_aSelection and preferred option and another carbon atom on or identical carbon atoms at equatorial position Whether there is any substituent group (R group in formula (1) i.e. as defined above) unrelated.Preferably, upright except one or two Beyond substituent group, there is also one or two calm substituent group, described substituent group preferably includes to be the part of connector structure R or R_a.But, in another preference, it is contemplated that reach balance between synthetic work and reactivity, preferably one or two Individual R_aIt not hydrogen, and other R and R all_aIt is all hydrogen.

In a further preference, X and/or Y is O-alkyl or alkyl, more preferably methyl.

Diene

Those skilled in the art will know that a large amount of diene in described inverse Diels-Alder reacts with reactivity.Preferably Diene with reference to formula (2)-(5) given below.

Wherein R¹Group selected from consisting of: H, alkyl, aryl, CF₃、CF₂-R’、OR’、SR’、C(=O)R’、C(=S) R’、C(=O)O-R’、C(=O)S-R’、C(=S)O-R’、C(=S)S-R’、C(=O)NR’R’’、C(=S)NR’R’’、NR’R’’、NR’ C(=O)R’’、NR’C(=S)R’’、NR’C(=O)OR’’、NR’C(=S)OR’’、NR’C(=O)SR’’、NR’C(=S)SR’’、NR’C (=O) NR ' ' R ' ' ', NR ' C (=S) N ' R ' ' R ' ' ' wherein R ', R ' ' and R ' ' are H, aryl or alkyl independently of one another；A and B is each From independently selected from by the substituted carbon of alkyl, the substituted carbon of aryl, nitrogen, N⁺O^-、N⁺R wherein R is the group of alkyl composition, and condition is A It is not all carbon with B；X selects free O, N-alkyl and the group of C=O composition, and Y is that CR wherein R is selected from the group that consists of: H, alkane Base, aryl, C (=O) OR ', C (=O) SR ', C (=S) OR ', C (=S) SR ', C (=O) NR ' R ' ' wherein R ' and R ' ' are the most only It is on the spot H, aryl or alkyl；

Diene cyclo-octene being particularly suitable as reacting to participant is:

Wherein R¹And R²It is each independently selected from the group consisted of: H, alkyl, aryl, CF₃、CF₂-R’、NO₂、OR’、 SR’、C(=O)R’、C(=S)R’、OC(=O)R’’’、SC(=O)R’’’、OC(=S)R’’’、SC(=S)R’’’、S(=O)R’、S(= O)₂R’’’、S(=O)₂NR’R’’、C(=O)O-R’、C(=O)S-R’、C(=S)O-R’、C(=S)S-R’、C(=O)NR’R’’、C(=S) NR’R’’、NR’R’’、NR’C(=O)R’’、NR’C(=S)R’’、NR’C(=O)OR’’、NR’C(=S)OR’’、NR’C(=O)SR’’、 NR’C(=S)SR’’、OC(=O)NR’R’’、SC(=O)NR’R’’、OC(=S)NR’R’’、SC(=S)NR’R’’、NR’C(=O)NR’ R ' ', NR ' C (=S) N ' R ' R ' ', wherein R ' and R ' ' is H, aryl or alkyl independently of one another, and R ' ' ' be independently aryl or Alkyl；A selects the group of free N-alkyl, N-aryl, C=O and CN-alkyl composition；B is O or S；X selected from the group that consists of: N, CH, C-alkyl, C-aryl, CC (=O) R ', CC (=S) R ', CS (=O) R ', CS (=O)₂R’’’、CC(=O)O-R’、CC(=O)S- R ', CC (=S) O-R ', CC (=S) S-R ', CC (=O) NR ' R ' ', CC (=S) NR ' R ' ', R ' and R ' ' are H, aryl independently of one another Or alkyl and R ' ' ' are aryl or alkyl independently；Y selects free CH, C-alkyl, C-aryl, N and N⁺O^-The group of composition；

Diene cyclo-octene another kind being particularly suitable as reacting to participant is:

Wherein R¹And R²It is each independently selected from the group consisted of: H, alkyl, aryl, CF₃、CF₂-R’、NO₂、OR’、 SR’、C(=O)R’、C(=S)R’、OC(=O)R’’’、SC(=O)R’’’、OC(=S)R’’’、SC(=S)R’’’、S(=O)R’、S(= O)₂R’’’、S(=O)₂NR’R’’、C(=O)O-R’、C(=O)S-R’、C(=S)O-R’、C(=S)S-R’、C(=O)NR’R’’、C(=S) NR’R’’、NR’R’’、NR’C(=O)R’’、NR’C(=S)R’’、NR’C(=O)OR’’、NR’C(=S)OR’’、NR’C(=O)SR’’、 NR’C(=S)SR’’、OC(=O)NR’R’’、SC(=O)NR’R’’、OC(=S)NR’R’’、SC(=S)NR’R’’、NR’C(=O)NR’ R ' ', NR ' C (=S) N ' R ' R ' ', wherein R ' and R ' ' is H, aryl or alkyl independently of one another, and R ' ' ' be independently aryl or Alkyl；A selects free N, C-alkyl, C-aryl and N⁺O^-The group of composition；B is N；X is selected from the group consisted of: N, CH, C-alkane Base, C-aryl, CC (=O) R ', CC (=S) R ', CS (=O) R ', CS (=O)₂R’’’、CC(=O)O-R’、CC(=O)S-R’、CC(=S) O-R ', CC (=S) S-R ', CC (=O) NR ' R ' ', CC (=S) NR ' R ' ', wherein R ' and R ' ' is H, aryl or alkane independently of one another Base and R ' ' ' are aryl or alkyl independently；Y selects free CH, C-alkyl, C-aryl, N and N⁺O^-The group of composition；

Useful especially tetrazine derivatives is electron deficiency tetrazine class, is i.e. not generally considered the group of supplied for electronic or part Substituted tetrazine, preferably with electron-withdrawing substituent.

These electron deficiency tetrazine classes are typically compliant with following structural formula:

Here, R¹And R²Representative is selected from the substituent group of the group consisted of independently of one another: H, 2-pyridine radicals, 3-pyridine Base, 4-pyridine radicals, 2,6-pyrimidine radicals, 2,5-pyrimidine radicals, 3,5-pyrimidine radicals, 2,4-pyrimidine radicals or phenyl, optionally by one or Multiple electron withdraw groups such as NO₂、F、Cl、CF3、CN、COOH、COOR、CONH₂、CONHR、CONR₂、CHO、COR、SO₂R、 SO₂OR, NO, Ar replace, and wherein R is C₁-C₆Alkyl and Ar represent aryl, particularly phenyl, pyridine radicals or naphthyl.

According in each compound of formula (2)-(5), described R¹And R²Group (being included in those on X or Y) is permissible It is further provided with the as described below connector being suitable for or interval body part.Similarly, and with it independently, as defined above The dienophile of formula (1) the as described below connector being suitable for or interval body part can also be further provided with.

According to an embodiment, the present invention is used for the imaging of targeting.

According to this embodiment, the imaging of specific primary target is by the spy of the primary targeting moiety of described pre-targeting probe Anisogamy and utilize that the detectable label analyte detection comprised in described effector probe is this to be implemented in combination with.

Primary target

" primary target (the primary target) " used in the present invention relates to diagnosing and/or treating tested in formation method Survey, and/or treat the target modulated by pharmaceutically active compound or other form of therapy, combine or address.

Described primary target can any applicable target in the mankind or animal body or on pathogen or parasite, such as Comprise following group: cell such as cell membrane and cell wall, receptor such as cell-membrane receptor, intracellular structure such as Golgi body Or mitochondrion, enzyme, receptor, DNA, RNA, virus or virion, antibody, protein, carbohydrate, monosaccharide, polysaccharide, cell Cytokines, hormone, steroid, somatostatin receptor, monoamine oxidase, MAO, muscarinic receptor, myocardial sympathetic system (myocardial sympatic nerve system), leukotriene receptor (the most on the leukocytes), urokinase fibrin Dissolved preferment activator receptor (uPAR), folate receptor, apoptosis mark, (resisting) angiogenesis mark, gastrin are subject to Body, dopaminergic system, serotonergic system, GABA energy system (GABAergic system), epinephrine reaction are System, cholinergic system, opiate receptor, GPIIb/IIIa receptor and other thrombosis associated receptor, fibrin, calcitonin receptor, Stimulin receptor, integrin receptor, VEGF/EGF receptor, EGF, matrix metalloproteinase (MMP), P/E/L-select element to be subject to Body, ldl receptor, P-glycoprotein, neurotensin receptor, neuropeptide receptor, Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 receptor, NK receptor, cck receptor, sigma-receptor, Interleukin-1 receptor, herpes simplex virus tyrosine kinase, human tyrosine kinase.

A particular according to the present invention, described primary target is protein such as receptor.As optional selection, Described primary target can be metabolic pathway, and it such as infects or up-regulated during cancer in disease, such as DNA synthesis, albumen Matter synthesis, film synthesis and carbohydrate picked-up.In illing tissue, above-mentioned mark can be different to that health tissues and provides The uniqueness of the treatment that detection, specific diagnosis and treatment are especially targeted may in early days.

Pre-targeting probe

Pre-targeting probe comprises the part can being combined with primary target interested.

Targeting moiety is typically cell surface target (such as membrane receptor), structural protein (such as amyloid plaque) or cell Internal target (such as RNA, DNA, enzyme, cell-signaling pathways) has the structure of affinity.These parts can be antibody (fragment), egg White matter, fit, oligopeptide, oligonucleotide, oligosaccharide and peptide, peptidomimetic and the known organic medical of accumulation at specific disease or obstacle Compounds.

This document describes the particular of the applicable primary targeting moiety for test kit of the present invention, and include receptor Binding peptide and antibody.One particular of the present invention relates to the use of little targeting moiety such as peptide, thus acquisition can Targeted probes through cell.

" the primary targeting moiety " that use in the present invention relates to the targeted probes part being attached to primary target.Primary targeting portion The specific examples divided is bonded to peptide or the protein of receptor.Other example of primary targeting moiety is bonded to cell compound Antibody or its fragment.Antibody can generate for non-protein compound and protein or peptide.Other primary targeting moiety can To be made up of fit, oligopeptide, oligonucleotide, oligosaccharide and peptidomimetic and organic drug compound.Primary targeting moiety is preferably with height Specificity, high-affinity, the most even covalent bond, and be stable the most in vivo with the combination of described primary target.

So that selectively targeted primary target listed above, the primary targeting moiety of described targeted probes can contain Compound, it includes but not limited to: antibody, antibody fragment such as Fab2, Fab, scFV, bivalent antibody (diabodies), polymerization Thing (relying on the cancer target of EPR effect), protein, peptide such as octreotide and derivant, VIP, MSH, LHRH, Chemotactic Peptide, bell Toad peptide, elastin laminin, peptide analogues, carbohydrate, monosaccharide, polysaccharide, virus, full cell (whole cells), phage, Medicine, chemotherapeutics, receptor stimulating agent and antagonist, cytokine, hormone, steroid.Imagination in present disclosure The example of organic compound be or derived from: estrogens such as estradiol, androgens, progestogens, cortical steroid, Paclitaxel, etoposide, doxorubricin, methotrexate, folic acid and cholesterol.

A particular according to the present invention, described primary target is receptor, and the primary targeting moiety being suitable for Include but not limited to the part of this receptoroid or still with the ligand moiety to this receptor, such as in receptor binding albumen plasmogamy Receptor binding peptide in the case of body.

Other examples of proteinaceous primary targeting moiety include interferon, such as α, β and IFN-γ, leukocyte Interleukin and protein growth factor, such as tumor growth factor, such as α, β tumor growth factor, platelet derived growth factor (PDGF), uPAR targeting proteins, apolipoprotein, LDL, annexin V, Endostatin and angiogenesis inhibitor element.

The selectable example of primary targeting moiety includes DNA, RNA, PNA and LNA, and it is such as mutual with described primary target Mend.

A particular according to the present invention, employs little lipotropy primary targeting moiety, and it can be in conjunction with To intracellular primary target.

A further particular according to the present invention, select described primary target and primary targeting moiety thus Cause tissue that is specific or that increase or the targeting of disease, described tissue or disease such as cancer, inflammation, infection, cardiovascular Disease such as thrombosis, atherosclerotic lesion, anoxia position such as apoplexy, tumor, cardiovascular disorder, brain disorder, cell wither Die, angiogenesis, organ and reporter gene/enzyme.At the beginning of this can have tissue by selection, cell or disease specific express Level target realizes.Such as, the intracellular accumulation of folacin receptor mediated folate of film and the like such as methotrexate.Normal structure Interior expression is limited, but receptor is overexpressed in various tumor cell types.

Can be multimeric compounds according to an embodiment, described pre-targeting probe and described effector probe (multimeric compounds), it comprises multiple primary and/or secondary target and/or targeting moiety.These multimeric compounds Can be polymer, dendritic, liposome, polymer beads or other polymer architecture.For amplification detection signal It is particularly interesting that have the targeted probes of more than one secondary target, it makes it possible to combine several effector probe.

Described pre-targeting probe contains above-mentioned first bio-orthogonal reaction group further.This group serves as " secondary Target ", i.e. as the targeted probes part of the first reaction participant provided for described inverse Diels-Alder coupling chemistry.

As it has been described above, described secondary target can be arbitrary participant of described coupled reaction.That is, an embodiment In, it is electron deficiency tetrazine.In another embodiment, it is the upright substituted TCO of above-mentioned formula (1).

In described pre-targeting probe, described primary targeting moiety and described first bio-orthogonal reaction group can be with those This is joined directly together.They can also be bonded to each other by connector, in addition they can all with primary targeting support (scaffold) such as biopolymer such as polypeptide connects.The most in the simplest situations, described connector part is key.It is suitable for Connector part farther include, but be not limited to, have from 2 to 200, particularly 3 to 113 and preferred 5-50 repetitive Polyethylene Glycol (PEG) chain.By regulation PEG chain length, it is possible to affect described probe circulation time in physiological systems.This is right It is especially relevant (because described primary targeting to be attached partially to the initial targeting step of described primary target in pre-targeting probe May relate to relatively slow process, need relatively long circulation time).Connector part optionally includes biopolymer sheet Section, such as widow-or polypeptide or polyactide (polylactides).

It should be understood that the present invention includes wherein said diene and described dienophile and described pre-targeting or effector probe Arbitrary connection any it is contemplated that mode.The method implementing the combination to these probes, such as by reactive amino acid example Such as lysine or cysteine, dawn the most known to those skilled in the art.

Effector probe

Effector probe comprises the effector part that can provide diagnosis, imaging and/or the therapeutic effect wanted.Described effect Physical prospecting pin is answered to comprise secondary targeting moiety further.

Described secondary targeting moiety relates to being formed for available secondary target (comprised in the most described pre-targeting probe Or multiple bio-orthogonal reaction group) the effector probe portion of reaction participant.It will be appreciated that described secondary target be as In the case of the upright substituted TCO of the formula (1) of upper definition, described secondary targeting moiety will be diene such as tetrazine, otherwise also So.

Described effector part can be the most detectable label." detectable " used herein relates to making Obtain the effector probe portion that such as described probe can be detected when being present in cell, tissue or organism.At this In bright content, a type of contemplated detectable is that contrast provides agent.Present disclosure contemplates Different types of detectable is the most described below.

Therefore, according to a particular of the present invention, the pre-targeting test kit of the present invention is used for into method Picture, particularly medical imaging.In order to identify described primary target, use the image probe comprising one or more detectable As described effector probe.The specific examples of the detectable of described image probe is right for conventional imaging system Partly such as can the structure of MRI-imaging, spin label, optical markings thing, ultrasonic response structure, X-ray response portion than providing Point, radionuclide, (biological) be luminous and FRET type dye.Exemplary detecting contemplated in present disclosure Label include but may be not necessarily limited to fluorescence molecule (such as auto-fluorescence molecule (autofluorescent molecules), The molecule etc. fluoresced when contacting with reagent), radio-labeled thing；Biotin (such as will combine via with avidin Biotin carries out the biotin detected)；Fluorescence labels, for the imaging arrangement of MRI, including paramagnetic metal, imaging agents, example Described at U.S. Pat. No. 4,741,900 and 5,326,856) etc..Radionuclide for imaging can With e.g. selected from the isotope of the group consisted of:³H、¹¹C、¹³N、¹⁵O、¹⁸F、¹⁹F、⁵¹Cr、⁵²Fe、⁵²Mn、⁵⁵Co、⁶⁰Cu、⁶¹Cu、⁶²Zn、⁶²Cu、⁶³Zn、⁶⁴Cu、⁶⁶Ga、⁶⁷Ga、⁶⁸Ga、⁷⁰As、⁷¹As、⁷²As、⁷⁴As、⁷⁵Se、⁷⁵Br、⁷⁶Br、⁷⁷Br、^8OBr、⁸²Br、⁸²Rb、⁸⁶Y、⁸⁸Y、⁸⁹Sr、⁸⁹Zr、⁹⁷Ru、⁹⁹Tc、¹¹⁰In、¹¹¹In、¹¹³In、¹¹⁴In、¹¹⁷Sn、¹²⁰I、¹²²Xe、¹²³I、¹²⁴I、¹²⁵I、¹⁶⁶Ho、¹⁶⁷Tm、¹⁶⁹Yb、¹⁹³Pt、¹⁹⁵Pt、²⁰¹Tl and²⁰³Pb。

Element or isotope that other Element and isotope can also such as be used for treatment in some applications are used for Imaging.

Described can the part of MRI imaging can be such as paramagnetic ion or supperparamagnetic particles.Described paramagnetic ion Can be selected from the element of group consisted of: Gd, Fe, Mn, Cr, Co, Ni, Cu, Pr, Nd, Yb, Tb, Dy, Ho, Er, Sm, Eu、Ti、Pa、La、Sc、V、Mo、Ru、Ce、Dy、Tl.Described ultrasonic response part can comprise microvesicle, its shell by phospholipid and/or (biodegradable) polymer and/or human serum albumin are constituted.Described microvesicle can be filled with fluorinated gas or liquid.

Described X-ray response part includes but not limited to that iodine, barium, barium sulfate, gastrografin maybe can comprise and uses iodine Vesicle, liposome or the polymer capsule that compound and/or barium sulfate are filled.

Additionally, detectable contemplated in present disclosure also includes to be detected by antibodies Peptide or polypeptide, such as by combining detectable labeled antibody or anti-by combine via sandwich assay detection Body.In one embodiment, described detectable is small size organic PET and SPECT label, such as¹⁸F、¹¹C Or¹²³I.Due to their small size, organic PET or SPECT label is perfect for for monitoring intracellular event, because of Performance and particularly its film conveying of targeting device typically will not be affected greatly for them.Comprise PET label and conduct The image probe of the arbitrary inverse Diels-Alder active part of secondary targeting moiety is lipophilic and can passively diffuse Enter and diffuse out cell until it finds its combination participant.And, two kinds of components the most do not hinder cross blood brain barrier and from And make it possible in brain inner region imaging.

When described effector probe intends to comprise based on metal such as the lanthanide series metal (such as Gd) of MRI Contrast enhanced Detectable time, it preferably provides with the form of chelate.In this case, described effector probe preferably comprises Can be with the structure division of this metalloid formation co-ordination complex.A good example about this is derived from 1,4,7,10-tetra- Azacyclo-dodecane-1,4,7,10-tetraacethyl (H₄And Cyclen-α dota), α ', α, " α ' "-four Methyl isophthalic acid, 4,7,10-tetraacethyl (H₄Dotma) macro ring lanthanide series (III) chelate.

Described effector part can also is that treatment part such as pharmaceutically active compound.There is provided herein pharmaceutically active The example of compound.Treatment probe can also optionally comprise detectable.

Therefore, according to another embodiment, the pre-targeting test kit of the present invention and method are used for the treatment of targeting.This leads to Cross utilization containing secondary targeting moiety and one or more pharmaceutically active agent (i.e. medicine or same for radiotherapeutic radioactivity Position element) effector probe realize.Medicine for targeting delivers the applicable medicine of situation and is known in the art.Optionally, Described treatment probe can also include detectable label, such as one or more preparations.Radioactive nucleus for treatment Element can be the isotope of group being selected from consisting of:²⁴Na、³²P、 ³³P、 ⁴⁷Sc、 ⁵⁹Fe、⁶⁷Cu、 ⁷⁶As、 ⁷⁷As、⁸⁰Br、 ⁸²Br、 ⁸⁹Sr、 ⁹⁰Nb、 ⁹⁰Y、 ¹⁰³Ru、 ¹⁰⁵Rh、 ¹⁰⁹Pd、 ¹¹¹Ag、¹¹¹In、 ¹²¹Sn、 ¹²⁷Te、 ¹³¹I、 ¹⁴⁰La、¹⁴¹Ce、 ¹⁴²Pr、 ¹⁴³Pr、 ¹⁴⁴Pr、 ¹⁴⁹Pm、 ¹⁴⁹Tb、 ¹⁵¹Pm、 ¹⁵³Sm、 ¹⁵⁹Gd、 ¹⁶¹Tb、 ¹⁶⁵Dy、 ¹⁶⁶Dy、 ¹⁶⁶Ho、¹⁶⁹Er、 ¹⁷²Tm、 ¹⁷⁵Yb、 ¹⁷⁷Lu、 ¹⁸⁶Re、 ¹⁸⁸Re、 ¹⁹⁸Au、 ¹⁹⁹Au、 ²¹¹At、 ²¹¹Bi、 ²¹²Bi、 ²¹²Pb、 ²¹³Bi、²¹⁴Bi、 ²²³Ra and²²⁵Ac。

As selection, the medicine in described treatment probe is selected from the photosensitizer (sensitizers) for photodynamic therapy.

As selection, described treatment probe comprises and is attached to interior therapeutic entity, such as T cell, natural killer cell or Other endogenous structure such as protein, identification division.

In described effector probe, the described secondary the most described second bio-orthogonal reaction group of targeting moiety and described Effector part can be connected directly to one another.They can also be bonded to each other by connector, in addition they can all with secondary Targeting support connects.Described connector can be independently selected from same section as above, such as Polyethylene Glycol.Described secondary Targeting support can be such as biopolymer such as polypeptide.

The invention still further relates to utilize the pre-targeting method of inverse Diels-Alder reaction.Here, primary targeting portion will be comprised The pre-targeting probe dividing (such as antibody and antibody fragment or receptor binding peptide) is injected in experimenter, wherein said pre-targeting Probe is respectively with the diene being suitable for, preferably according to any one compound of above-mentioned formula (2)-(5), or with according to above-mentioned formula (1) Cyclo-octene functionalization.Be attached to target (the most primary or metastatic tumo(u)r pathological changes, atheromatous plaque, infarcted region, Inflammation or infection position etc.) and from blood circulation with after non-target tissue's (such as blood, liver, spleen, kidney etc.) removes, Injection effect physical prospecting pin, it contains secondary targeting moiety and carries E-cyclo-octene the most respectively or tetrazine derivatives (is i.e. present in institute State the reactive homologue of bio-orthogonal reaction group in pre-targeting probe) and medicine or can imaging tags.Described effect Physical prospecting pin is answered to be attached to described primary targeting moiety and provide disease site described in high contrast or selective therapy.

The invention still further relates to the targeting of general metabolic pathway, described general metabolic pathway disease (such as infect or Cancer) period up-regulated such as DNA, protein and film synthesize and carbohydrate absorbs.The probe being suitable for comprises by diene Or the aminoacid of dienophile labelling, sugar, nucleic acid and choline, metabolic tracer analog currently used in this area, [¹¹C]- Methionine, [¹⁸F]-fluorodeoxyglucose (FDG), deoxidation-[¹⁸F]-fluorothymidine (FLT) and [¹¹C]-choline.There is hypermetabolism Or the cell of propagation has the higher picked-up to these construction units (building block).In the method, such as tetrazine Or E-Cyclooctene derivative enters these or other path and accumulates in and/or on cells.Fully gathering and removing After free probe, by through detectably labelling or the tetrazine probe of (cell permeable) that carry medicine or E-cyclo-octene probe (or carrying the probe of other diene/dienophile according to the present invention) is sent into respectively in connection with the E-cyclo-octene accumulated or tetrazine Metabolite.As the benefit being better than common FDG (fluorine 18 fluorodeoxyglucose) type imaging, it is possible to obtain time enough Make it possible to targeting moiety described in elevated concentrations before sending into radioactivity, thus increase target to non-target ratio.As selection, permissible The metabolic pathway of targeting disease specific and/or metabolite.

The invention still further relates to the pre-targeting of cell internal target.Due to their small size, organic PET label (¹⁸F, ¹¹C) It is highly suitable for monitoring intracellular events, because they generally will not affect the performance of described targeting device greatly, especially It is that its film carries (contrary with big and polarity radiation metal chelate structure conjugate).Although the quilt used in the present invention Substituted tetrazine part and described E-cyclo-octene are not necessarily little, but they are relative non-polarity, and can be used in The intramolecular imaging of protein, mRNA, signal path etc..Described secondary (such as PET labelling) substituted tetrazine part or E- Cyclo-octene probe (the most described effector probe) can passively diffuse into and diffuse out cell until it finds its combination to join With person or experience activity uptake mechanism.These performances also allow for being used for inverse Diels-Alder reaction pre-targeting in brain, because two Kind component does not the most hinder crosses blood brain barrier.

The signal that the invention still further relates to pre-targeting amplifies and/or multivalence assembling.At least one primary targeting device with contain The dendritic (dendrimer) of multiple tetrazine parts, polymer or nano-particle combine.After receptor combines, inject One or more with for nuclear imaging (such as radiating metallo-chelate, radiohalogen etc.) or MRI (such as Gd chelate) (one or more) cyclo-octene that contrast part combines.Inverse Diels-Alder reaction subsequently causes MRI contrast at target tissue The high concentration of agent.Additionally, the multivalence at target site will increase the reaction power with described upright substituted TCO effector conjugates Learn, it is provided that effective target accumulation of such as MRI contrast agent.Certainly, described upright substituted TCO can be used for described targeting device In conjugate, described tetrazine (or other diene of the present invention) is connected to reporter (reporter).

Connection approach and test kit

The invention further relates to inverse Diels-Alder reaction as preparation and medicine are connected to targeting structure The purposes of the approach of such as peptide.Described effector can comprise the isotopically labeled prothetic group of organic PET or SPECT, for PET/ The metal complex of SPECT/MRI and the microvesicle for ultra sonic imaging, the fluorogen for optical imagery and be used for radiating treatment α and the β radiation source of method and usual cytotoxic anticancer agent.Described imaging/therapeutic agent can with side base tetrazine or other be suitable for Diene portions functionalization and by upright substituted TCO derivant by targeting group functionalization, vice versa.

The approach of the present invention is to advantageous particularly for nuclear imaging and radiotherapeutic reagent: decline in view of radionuclide Becoming, it is useful for implementing the most time-consuming step (the actual targeting in subject) as pre-targeting step.According to the present invention, choosing Select upright substituted TCO, to obtain above-mentioned very quickly inverse Diels-Alder chemistry for secondary targeting, enabling make Use multiple radionuclide, including shorter those in service life compared with the conventional method.The effect of upright substituted TCO functionalization Answer physical prospecting pin and be suitable for diene such as carry tetrazine pre-targeting probe can extremely low concentration couple in vivo without The sustained blood circulation of effector part (the most described radionuclide).It will be appreciated that this to diene particularly tetrazine official energy The pre-targeting probe with upright substituted TCO that the effector probe changed combines is equally applicable.And, described reactive group The most stable, and therefore present the reactivity of longer life and side reaction the most easily occurs.

It will be appreciated that foregoing provide benefit such as minimize the radiological dose to patient.And, it causes utilizing The i.e. positron emission tomography agent of PET replaces SPECT i.e. single photon emission computerized tomography agent (single photon emission computerized tomography agents).And, the reactivity of increase makes it possible in vivo with relatively Low concentration application.

Present invention is especially suitable for multi-modal imaging, optionally use different preparations so that identical target is visual Change.As selection, described image probe comprises the different label of at least two and enables to realize multi-modal imaging.

The application in molecular imaging of [4+2] of the improvement of the present invention inverse Diels-Alder chemistry is to all types and chi Very little targeting structure opens the door of pre-targeting.This permission is intracellular and metabolic imaging is with from passing through what pre-targeting accumulation obtain The accumulation of high target and low background are benefited.Equally, for less and more kinds of various targeting devices, the signal amplification side of pre-targeting The most tetrazines of case and/or many dendritics or liposome become feasible.

Owing to described reaction participant is abiotic and bio-orthogonal, therefore uses and utilize upright substituted TCO to make Pre-targeting for [4+2] inverse Diels-Alder reaction of above-mentioned dienophile is not hindered by endogenous competition and metabolism/decomposition, And stable covalent bond is provided.Select target metabolic pathway, and corresponding tetrazine-metabolite derivative, thin with normal by it Born of the same parents compare the high flux in such as tumor cell, it is provided that in cell or on target cells, the artificial tetrazine of high density is subject to Body or the assembling of other chemistry handle, it is to avoid there may come a time when to be in the use of low-level endogenous cell surface receptor.

The further particular of the present invention relates to comprising metabolic precursor thereof and image probe, more particularly comprises The test kit of the image probe of detectable, described detectable is the contrast medium for conventional imaging system.This Class detectable can be but not limited to choosing freely can the structure of MRI imaging, spin label, optical markings thing, ultrasonic The label of the group of response reagent, X-ray response reagent, radionuclide and FRET class dyestuff composition.In the present invention one In particular, employ reporter probe.This reporter probe can be the substrate of enzyme, more particularly for institute It is not endogenic for stating cell, but the enzyme having infected by gene therapy or use external agent and having introduced.Relate in this article And the non-endogenous of the gene in cell or tissue is used for representing that described gene does not naturally occur in described cell or tissue And/or express.As selection, this type of reporter probe is the molecule being incorporated in cell by the way of receptor or pump, and it can To be endogenic or to be incorporated into intracellular by gene therapy or use external agent infection.As selection, described reporter Probe is the molecule reacting some (change) condition in cell or tissue environment.

Present invention additionally comprises the reagent in mentioned reagent box.This type of reagent a kind of is to comprise primary targeting moiety and life The pre-targeting agent of thing orthogonal reaction group, wherein said bio-orthogonal reaction group is that [4+2] inverse Diels-Alder reacts Reaction participant.Specific reaction participant described above, the most above-mentioned electron deficiency tetrazine or other be suitable for diene, Or the upright substituted cyclo-octene according to the present invention.The invention still further relates to these reagent at the medical imaging of targeting or targeting Purposes in treatment, and the described reagent related in this method.Especially, the present invention relates to these reagent in pre-targeting These purposes in method, and relate to these reagent used in this approach.This type of reagent another kind of is to comprise to detect Label and the image probe of bio-orthogonal reaction group, wherein said bio-orthogonal reaction group is that [4+2] is inverse The reaction participant of Diels-Alder reaction.

The invention still further relates to comprise detectable and the image probe of bio-orthogonal reaction group, wherein said life Thing orthogonal reaction group is the reaction participant of [4+2] inverse Diels-Alder reaction.The invention further relates to comprise pharmacy The treatment probe of reactive compound and bio-orthogonal reaction group, wherein said bio-orthogonal reaction group is that [4+2] is inverse The reaction participant of Diels-Alder reaction.

The part of the present invention or pre-targeting method, give experimenter including by pre-targeting reagent as above and make Described reagent circulates in the system of experimenter and effectively realizes described primary targeting moiety a period of time to the combination of primary target, Then unconjugated reagent is removed from health.Typical time period for it is 12 to 96 hours, specifically about 48 hours.

Additionally, the invention provides formation method, including implementing pre-targeting method as mentioned above, then give also according to The image probe of the present invention, in wherein said pre-targeting reagent together with the bio-orthogonal reaction group in described image probe Form the reaction participant of [4+2] inverse Diels-Alder reaction.Similarly, the invention provides the medical science of targeting in experimenter Therapeutic Method, it includes implementing pre-targeting method as mentioned above, then gives the treatment probe also according to the present invention, Qi Zhongsuo State and pre-targeting reagent is formed together with the bio-orthogonal reaction group in described image probe [4+2] inverse Diels-Alder The reaction participant of reaction.

The invention still further relates to the above-mentioned pre-targeting reagent for imaging as above or Therapeutic Method.

For summary, on the basis of inverse Diels-Alder chemistry, the molecular imaging of bio-orthogonal pre-targeting and treatment are used In bringing great advantage to patient.On the one hand, it is for undertaking the excellent figure obtaining target tissue such as cancer and cardiovascular pathological changes Picture.On the other hand, it is possible to greatly reduce and be derived from radioactive compound and intrinsic side effect that usual genotoxic potential medicine gives, with Shi Zengjia arrives the effective dose of illing tissue.Additionally, it will be greatly expanded the collection of the traceable molecular events causing disease. Especially, this technology can allow for using the target tissue away from blood vessel and promoting the one-tenth of informative intracellular environment Picture.

With reference to following nonlimiting examples and the indefiniteness accompanying drawing enclosed, the present invention will be described.

Embodiment

Material

All reagent and solvent all obtain (from Sigma-Aldrich, Acros, ABCR, Invitrogen from commercial source Obtain reagent with Merck, obtain common and deuterium from Biosolve, Merck and Cambridge Isotope Laboratories For solvent), and the most all i.e. use without further purification.1-amino-3,6,9,12,15,18,21,24, 27,30,33,36-ten dioxa nonatriacontane-39-acid (21) obtains from Polypure.[¹¹¹In] indium chloride and [¹²⁵I] iodate Sodium solution is purchased from PerkinElmer.By milli-Q water filtering system (Millipore) distillation and de-ionized water (18M Ω cm).Process labelling buffer overnight with Chelex-100 resin (BioRad Laboratories), be then passed through 0.22 μm mistake Filter and 4 DEG C of storages.Indogen iodate pipe, the test kit checked for bicinchoninic acid (BCA), gelcode blue protein dye Color solution and Zeba desalination centrifugal column (spin columns) (40 kDa MW cut-offs, 0.5-2 mL) are purchased from Pierce Protein Research (Thermo Fisher Scientific).For preparing phosphate buffered saline (PBS) (PBS) pH7.4 Tablet from Calbiochem (Merck) obtain.Amicon Ultra-4 and Ultra-15 centrifugal filter unit (50 kDa MW by) purchased from Millipore.Mice serum is purchased from Innovative Research.The synthesis of tetrazine 28 and radio-labeled are such as Implement described in Rossin etc., Angew Chem Int Ed 2010,49,3375.

Method

Use Bruker DPX300 spectrometer or Bruker Avance600 spectrometer at CDCl₃Or [D₆] record in DMSO H NMR spectroscopy.DEPT pulse train is used to distinguish¹³(q=quadruple, t=be triple, s=double sum p=mono-weight for C-NMR multiplicity (primary)).Agilent ESI-TOF mass spectrograph have recorded high-resolution ESI mass spectrum (HRMS), with cation mode Measure.

System is implemented at Combiflash Companion equipment (Teledyne Isco) upper use SiliCycle silicagel column Standby column chromatography.Use is equipped with Agilent 1200 instrument of C18 Zorbax post (21.2 × 150 mm, 5 μm granules), application Water containing 0.1% TFA and MeCN gradient, implement preparation HPLC.It is being equipped with Gabi radioactivity seeker (Raytest) Implement in Agilent 1100 system to analyze radiation-HPLC.Sample is carried in Agilent Eclipse XDB-C18 post (4.6 × 150mm, 5 μm granules) on, its by the linear gradient in water of the MeCN containing 0.1% TFA with 1 mL/min eluting (10% MeCN 2 min, then increases to 45% MeCN in 11 min).UV wavelength is preset in 254 nm.Put being equipped with Gabi Volume-exclusion (SEC) HPLC is implemented in Agilent 1200 system of penetrating property detector.Sample is carried in BioSep-SEC-S 2000 posts (300 × 7.8 mm, 5 μm granules, Phenomenex) are above and with 20 mM phosphate, 150 mM NaCl, pH 6.8 with 1 ML/min eluting.UV wavelength is preset in 260 and 280 nm.

The 200 mM EDTA eluting that are used in 0.9% NaCl aqueous solution and phosphor imager (FLA-7000, Fujifilm) the ITLC-SG bar (Pall) of upper imaging is determined by radiation-TLC¹¹¹In and¹⁷⁷Lu labelling productivity.At these Under the conditions of, free¹¹¹In and¹⁷⁷Lu is with R_f=0.9 migrates, and¹¹¹In/¹⁷⁷Lu-tetrazine is retained in situ.¹²⁵I-labelling productivity also leads to Cross radiation-TLC to measure, use use 1:1 MeOH/ ethyl acetate mixture eluting the ITLC-of imaging on phosphor imager SG bar.Under these conditions, free¹²⁵I and¹²⁵I-SHPP is with R_f=0.9 migrates, and¹²⁵I-mAbs is retained in situ.

Phastgel system uses IEF-3-9 gel and 7.5% PAGE homogeneous gel (homogeneous respectively Gels) (GE Healthcare Life Sciences) implements isoelectrofocusing (IEF) analysis and SDS-PAGE.IEF calibrates solution (wide PI, pH 3-10) is purchased from purchased from GE Healthcare and protein MW standard solution (the double-colored standard of Precision Plus) BioRad.When electrophoresis, by gelcode indigo plant by described gel-colored 2 hours, decolour in water and the most then sweep with conventional panel Retouch instrument digitized.

By the NanoDrop 1000 spectrophotometer (absorptance at 280 nm；Thermo Fisher Scientific) Or use the concentration of BCA measurements determination CC49 solution.

LS 174T tumor model (tumor model).People clone cancerous cell line LS174T obtain from ATCC and be saved in 10% heat-inactivated hyclone (Gibco), penicillin (100 U/mL), streptomycin (100 μ g/mL) and 2mM Glutamax In the Eagle ' s MEM (Sigma) supplemented.With 5 × 10 in the 100 aseptic PBS of μ L⁶Cell is to naked female Balb/C mice (20-25 gram of body weight, Charles River Laboratories) carries out subcutaneous vaccination.

Embodiment 1

As shown in fig. 3a, as by the example being attached partially to antibody derivative for tetrazine, be prepared for molecule 1 (see Fig. 5).One example of corresponding probe 2 (derived from E-cyclo-octene) figure 6 illustrates.Two kinds of molecules all contain PEG chain.Point Son 1 comprises the N-hydroxy-succinamide base section coupled for amino group present in described molecule and antibody.In 2 Part derivative for DOTA may be used for carrying rare earth ion such as the Gd of MR imaging or for nuclear imaging and treatment (SPECT) Lu-177.

Fig. 5 shows the synthesis of 1.According to (Blackman, ML such as Blackman; Royzen, M; Fox, JM, Journal of The American Chemical Society, 2008,130 (41), 13518-19) it is prepared for initiateing The derivative molecule 5 of tetrazine.Converting it into acid 6 by reacting with glutaric anhydride, the N-hydroxysuccinimidyl acyl being subsequently formed it is sub- Amido ester 7.This N-hydroxy-succinamide base ester is for by anti-with the PEG derivant 8 of commercially available (IRIS biochem) Acid 9 should be formed, itself so that be converted to its N-hydroxy-succinamide base ester 1.

Fig. 6 shows the synthesis of 2.According to (Yap, GPA such as Yap; Royzen, M; Fox, JM, Journal of The American Chemical Society, 2008,130 (12), 3,760 61) it is prepared for (E)-ring octyl-4-enol (10).It is converted to ester 12 with the help of commercially available (Aldrich) isocyanate derivates 11, is then saponified into acid 13.Make to be reacted with the amine 18 derived from DOTA and PEG by the 13 N-hydroxy-succinamide base esters 14 formed, to form end-product 2.Make to prepare after 17 deprotections DOTA derivant 18, described 17 and then (the most commercially available by DOTA derivant 15 and PEG derivant 16 Obtain) prepared by (respectively from Macrocyclics and IRIS Biotech).

Embodiment 2

This example illustrates the opposition mutually of the molecule of embodiment 1, Fig. 7 shows formation institute after being attached to antibody State the described E-Cyclooctene derivative 3 of pre-targeting part.Tetrazine/the DOTA that can act as the effector probe shown in Fig. 3 b spreads out Raw probe 4 figure 8 illustrates.

E-Cyclooctene derivative 3 is formed in the following way: the PEG derivant 8 of commercially available (IRIS biochem) is (also See Fig. 5) it is reacted to form acid 19 with N-hydroxy-succinamide base ester 14, it is subsequently formed the N-hydroxysuccinimidyl acyl being derived from this acid Imido grpup derivant (Fig. 7).

Fig. 8 shows the synthesis of probe 4 derivative for described tetrazine/DOTA.Described probe is derived by DOTA and PEG Prepared by the reaction of amine 18 (see Fig. 6) and N-hydroxy-succinamide base ester 7 (see Fig. 5).

Embodiment 3

The present embodiment is shown in Figure 9, and it provides for synthesizing (E)-2,5-dioxo pyrrolidin-1-base 1-(4-((ring Octyl-4-alkene-1-base epoxide) methyl) phenyl)-1-oxo-5,8,11,14,17,20,23,26,29,32,35,38-ten dioxy Miscellaneous-2-azepine hentetracontane-41-acid esters (TCO-O-PEG10-N-N-Hydroxysuccinimide (NHS), main and secondary isomer 23a and 23b respectively) scheme.

Compounds represented E-compounds represented E-that is main and that be labeled as (digital) b being labeled as (digital) a is secondary.

(E-is main)-2,5-dioxo pyrrolidin-1-base 4-((ring octyl-4-thiazolinyl epoxide) methyl) benzoate (20a)。

According to literature procedure (M. Royzen, G. P. A. Yap, J. M. Fox, J. Am. Chem. Soc. 2008,130,3760) (E)-ring octyl-4-enol (10a, main isomer, containing about Z-isomer described in 13%) has been synthesized.

The 10a (1.70 g, 13.5 mmol) that 60% sodium hydride dispersion (1.8 g, 45 mmol) adds ice bath cooling exists In solution in 60 mL DMF.After being stirred at room temperature 4 hours, by 4-bromo methyl acid (3.85 g, 17.9 mmol) portioning Add and in room temperature, suspension be stirred overnight.Pour the mixture in water (100 mL), add t-butyl methyl ether (100 ML), 37% hydrochloric acid (5 mL) it is subsequently added into.After separation, extract water layer by t-butyl methyl ether (2 × 100 mL).With water (25 ML) organic layer that washing merges, uses MgSO₄It is dried and evaporates.Residue is made to pass through thin silicon glue-line by 4:1 hexane/ethyl acetate. The residue obtained after evaporation is dissolved in heptane (50 mL) at 70 DEG C, then cools down, it is provided that 19a.Described product is dissolved In dichloromethane (40 mL), add N-hydroxy-succinamide (0.57 g, 4.9 mmol), by mixture in ice bath cold But, N, N '-dicyclohexylcarbodiimide (1.03 g, 4.99 mmol) it is subsequently added into.Remove ice bath after 30 minutes and stir in room temperature Mix described reactant mixture 18 hours.After filtering and evaporating, use ethyl acetate at heptane (0-on silica gel by column chromatography 15%) the gradient-purified residue in.Then, described residue it is dissolved in t-butyl methyl ether (20 mL) and pours heptane into In (50 mL), obtain the 20a (1.42 g, 29%) of white solid forms.

(E-is main)-2,5-dioxo pyrrolidin-1-base 1-(4-((ring octyl-4-alkene-1-base epoxide) methyl) phenyl)-1- Oxo-5,8,11,14,17,20,23,26,29,32,35,38-ten dioxa-2-azepine hentetracontane-41-acid esters (23a)。

The 20a (100 mg, 0.280 mmol) solution in dichloromethane (2 mL) is added dropwise in ice bath stirring In 21 (175 mg, 0.283 mmol) and the triethylamine (290 μ L, 2.08 mmol) solution in dichloromethane (2 mL).Will be anti- Mixture is answered to be stirred at room temperature 16 hours.The thick intermediate 22a obtained after evaporation is dissolved in dichloromethane (5 mL) also Ice bath cools down.Add double (2,5-dioxo pyrrolidin-1-base) carbonic ester (170 mg, 0.664 mmol) and pyridine (28 μ L, 0.35 mmol), and in room temperature, reactant mixture is stirred 3 hours.Filtering mixt also evaporates, by column chromatography at silica gel The upper use methanol gradient-purified product in dichloromethane (5-10%), obtains the 23a (119 mg, 39%) of viscous oil.

(E-is secondary)-2,5-dioxo pyrrolidin-1-base 4-((ring octyl-4-thiazolinyl epoxide) methyl) benzoate (20b)。

According to above-mentioned literature procedure synthesis (E)-ring octyl-4-enol (10b, secondary isomer).By 60% sodium hydride dispersion (2.1 g, 53 mmol) add in the 10b (2.53 g, 20.1 mmol) of ice bath cooling solution in 50 mL oxolanes. After being stirred at room temperature 4 hours, mixture is cooled down again and adds 4-bromo methyl acid (4.53 with the time portioning of 5 minutes G, 21.1 mmol).Add 25 mL oxolanes and described suspension is being stirred at room temperature 4 days.Add ice, be subsequently added into 12.0 g citric acid.Described mixture is extracted twice by 150 mL t-butyl methyl ether.Organic layer washs with 25 mL water, is dried And evaporate.By using the heptane chromatography as eluent of 80 g silica gel and the ethyl acetate containing the amount being stepped up Purification residue.Merge product fraction (not being kept completely separate between product and initial alcohol) and from (the cooling of about 30 mL heptane recrystallization To-15 DEG C), obtain the 19b (0.86 g, 17%) of white solid forms.It is dissolved in 40 mL dichloromethane.Add N-hydroxyl Base butanimide (0.48 g, 4.17 mmol) also cools down mixture in ice.Add N, N '-dicyclohexylcarbodiimide Mixture is also stirred 30 minutes in ice by (0.80 g, 3.88 mmol), is then stirred at room temperature 18 hours.By filtering, There is provided as the chromatography of eluent with dichloromethane washing, rotary evaporation and on 25 g silica gel with heptane-ethyl acetate Product.Product fraction and the 75 mL t-butyl methyl ether of evaporation are mixed and mixture are warmed to 60 DEG C to provide solution. Described solution is concentrated to 20 mL.It is gradually added 50 mL heptane and obtains the precipitation of described product.It is collected by filtration and uses heptan Alkane washing provides 1.05 g 20b (15%).

(E-is secondary)-2,5-dioxo pyrrolidin-1-base 1-(4-((ring octyl-4-alkene-1-base epoxide) methyl) phenyl-1- Oxo-5,8,11,14,17,20,23,26,29,32,35,38-ten dioxa-2-azepine hentetracontane-41-acid esters (23b).

This compound starts preparation from 20b in the way of similar to 23a.Productivity with 93% obtains viscous oil 23b。

Embodiment 4

The present embodiment is shown in Figure 10, which show for synthesizing (E)-2,5-dioxo pyrrolidin-1-base 1-(4- (((ring octyl-4-alkene-1-base epoxide) carbonyl) amino) phenyl)-1-oxo-5,8,11,14,17,20,23,26,29,32,35, The reaction scheme of 38-ten dioxa-2-azepine hentetracontane-41-acid esters.

Compounds represented E-compounds represented E-that is main and that be labeled as (digital) b being labeled as (digital) a is secondary.

(E-is main)-2,5-dioxo pyrrolidin-1-base 4-(((ring octyl-4-alkene-1-base epoxide) carbonyl) amino) benzene first Acid esters 14a.

N, N '-dicyclohexylcarbodiimide (9.50 g, 0.046 mol) portioning is added in ice the 4-aminobenzene of cooling Formic acid (5.84 g, 0.043 mol), N-hydroxy-succinamide (5.0 g, 0.044 mmol) and the mixing of 60 mL isopropanols In thing.Filter suspension after being stirred at room temperature 16 hours and with 60 mL isopropanols, the solid obtained be stirred for one hour. Cooling it is incorporated in ice by mixed for thick the solid 24 and 100 mL dichloromethane filtered and obtain after drying under vacuum.Add 20% carbon Acyl chlorides solution (26 mL, 49.4 mmoL) in toluene also obtains solution after stirring 30 minutes.The solid that will obtain after evaporation Wash twice with 100 mL toluene and be dried under a high vacuum.Obtain the 25 (36%) of 4.03 g white solid forms.By described solid Body portioning adds in the 10a (1.40 g, 11.1 mmol) solution in 35 mL dichloromethane.By stirred for described suspension Night, and then heat 2 hours at 40 DEG C.On silica with dichloromethane eluent, then after re crystallization from toluene, it is thus achieved that The 14a (41%) of 1.77 g solid forms.Described product comprises the Z-isomer of about 12%, and it can not be removed by chromatography or crystallization Go.

(E-is main)-2,5-dioxo pyrrolidin-1-base 1-(4-(((ring octyl-4-alkene-1-base epoxide) carbonyl) amino) Phenyl)-1-oxo-5,8,11,14,17,20,23,26,29,32,35,38-ten dioxa-2-azepine hentetracontane-41-acid Ester 27a.

The 14a (100 mg, 0.26 mmol) solution in dichloromethane (2 mL) is added in ice bath stirring In 21 (160 mg, 0.26 mmol) and the triethylamine (400 μ L, 2.9 mmol) solution in dichloromethane (2 mL).By institute State reactant mixture and be stirred at room temperature 16 hours.The thick intermediate 26a obtained after evaporation is dissolved in dichloromethane (5 mL), And cool down in ice bath.Add double (2,5-dioxo pyrrolidin-1-base) carbonic ester (89 mg, 0.35 mmol) and pyridine (28 μ L, 0.35 mmol) and described reactant mixture is stirred at room temperature 16 hours.Filtering mixt, with 3 mL water extraction 3 times and With 3 mL saline water extraction once.The 27a (47%) of 1.23 g viscous oil is obtained after being dried with magnesium sulfate and evaporate.

(E-is secondary)-2,5-dioxo pyrrolidin-1-base 4-(((ring octyl-4-alkene-1-base epoxide) carbonyl) amino) benzene first Acid esters 14b.

But start to obtain this compound with 32% productivity from 10b in the way of similar to preparation 14a.Pure owing to obtaining 10b, therefore this product do not contain described Z-isomer.

(E-is secondary)-2,5-dioxo pyrrolidin-1-base 1-(4-(((ring octyl-4-alkene-1-base epoxide) carbonyl) amino) Phenyl)-1-oxo-5,8,11,14,17,20,23,26,29,32,35,38-ten dioxa-2-azepine hentetracontane-41-acid Ester 27b:

In the way of similar to preparation 27a, but start to obtain this compound with 89% productivity from 14b.

Embodiment 5

Tetrazine radio-labeled

The tetrazine (28 that DOTA-is combined；Figure 12, at Angew Chem Int Ed 2010,49,3375 such as Rossin Described in) dissolve (1 mg/mL) in 0.2 M ammonium acetate pH 7.0 and before use-80 DEG C of storages.By the 28 of aliquot with Suitable amount [¹¹¹In] indium chloride or [¹⁷⁷Lu] lutecium chloride group be incorporated in 37 DEG C under gentle agitation cultivate 10 minutes.It is subsequently adding Described solution is also the most additionally cultivated 5 minutes by 5 μ L 10mm DTPA.By adding 0.9 molar equivalent for described tetrazine InCl₃Or LuCl₃Implement the labelling reaction that carrier adds.Typically, use the method to obtain the quantitative mark more than 98% to produce Rate and radiochemical purity.

Be combined with the mAb of trans cyclo-octene (TCO) NHS ester 14,20,23,27

Here, and in subsequent step, all compound number are all referring to described a and b isomer.

Typically, 0.6 mole (for kinetic measurement) or 10 moles with cumulative volume is 250 μ L PBS are (for internal Research) the structurally-modified 1 mg CC49 of TCO-NHS (5 mg/mL solution in PBS) of equivalent.Will with 1M sodium carbonate buffer PH regulator is to 9.The most under agitation room temperature implements reaction 30 minutes.Subsequently, Amicon Ultra-15 centrifugal device is used The modified mAbs of TCO-is thoroughly washed with PBS.The TCO radical amount of each antibody is determined by tetrazine titration (described below).

With carrier addition¹⁷⁷Lu radio-labeled tetrazine-DOTA 28.By the mAb modified with the TCO of about 0.6 molar equivalent (25 μ g) and 3 molar equivalents¹⁷⁷Lu-28 (0.5 nM) reacts.By the mAb (25 μ g) modified with the TCO of about 10 molar equivalents With 15 molar equivalents¹⁷⁷Lu-28 (2.5 nM) reacts.Reaction 10 minutes is implemented at 37 DEG C in 50 μ L PBS pH 7.4.Logical Cross SDS-PAGE and phosphor imager (phosphor imager) analytical reactions mixture, and from the bands of a spectrum of corresponding described mAb In radioactivity determine reaction yield.Counting is quantitative with AIDA Image Analyzer software (Raytest).Find TCO-mAb It is 80-90% in conjunction with productivity.

MAb radio-labeled

Enough in 50 μ L PBS^[125I^]Sodium iodide (5-15 MBq) adds the 1 mg/mL Bolton-of 1 μ L The Hunter reagent (N-succinimido-3-[4-hydroxy phenyl] propionic ester (SHPP)) solution and the 4 of 25 μ L in DMSO The mg/mL chloramine-T (N-chlorine 4-methyl benzenesulfonamide, sodium salt) solution in PBS.The solution mixing 10-20 second that will be obtained, 5 μ L DMF and 100 μ L toluene are added in bottle,¹²⁵I-SHPP is extracted in organic facies, transfers it to glass In bottle.At gentle N₂Flowing down and blown out by toluene, (0.1-0.5 mg is at 50-250 μ L PBS to add CC49-TCO solution afterwards In), with 1M carbonate buffer solution by pH regulator to 9, and cultivate reactant mixture 30 minutes at RT under gentleness is vibrated.Cultivate After, determine labelling productivity by radio-ITLC.Then described crude product mixture is loaded into Zeba to be centrifuged on desalting column, institute State Zeba and be centrifuged desalting column saline solution pre-balance (pre-equilibrated).Rinse described with 20 μ L saline solutions Described flushing liquor is also also loaded on described post by reaction bottle.Zeba after purification, by radiation-ITLC, radiation-HPLC and SDS-PAGE determines¹²⁵The radiochemical purity of I-CC49-TCO solution；Protein concentration is determined by BCA detection.

Typically, this process is used, for purification¹²⁵I-CC49-TCO species obtain more than 70%¹²⁵I-SHPP MAb combine and > radiochemical purity of 98%.

Reaction rate

With carrier addition¹⁷⁷Lu is with the specific activity radio-labeled tetrazine-DOTA 28 of 3 MBq/ μ g.Make¹⁷⁷Lu-28(33 NM) with the CC49 (0.33,1 and 1.67 μM) of the increase concentration modified with the TCO of 1eq. in 200 μ L PBS, pH 7.4 37 DEG C are reacted 5 minutes.20 μ L samples are extracted and with four in the selected time (15,30,45,60,90,120,180 and 300 seconds) Piperazine 6 (Fig. 5；Described in the Angew Chem Int Ed 2010,49,3375 such as Rossin) (1.5 L, 5mg/mL exist In DMF) quencher.Each mixture of aliquot is analyzed by SDS-PAGE and phosphor imager, and from corresponding described mAb's Radioactivity in bands of a spectrum determines cycloaddition productivity.Counting uses AIDA Image Analyzer software (Raytest) quantitative.

Table 1:CC49-TCO structure and¹⁷⁷The second-order kinetics constant of reaction between Lu-28.O=ether connects；C=amino Formic acid esters (carbamate) connects

Embodiment 6

Stability in trans-cyclooctene body

Use CC49-PEG₁₀Main (the CC49-23a of-TCO-O；The 8.4 every CC49 of TCO；The 300 μ g/100 every mices of μ L), CC49-PEG₁₀Secondary (the CC49-27b of-TCO-C；The 3.3 every CC49 of TCO, the μ g/100 every mice of μ L) and the CC49-of spacer-free Main (the CC49-20a of TCO-O；The 8.0 every CC49 of TCO, the 300 μ g/100 every mices of μ L) enter without mice with tumor (n=3 often group) Row vein is injected.Extract from saphena (vena saphena) at selected time point (from injecting latter 1 hour to less than 4 days) Blood sample is also collected in the bottle comprising heparin.Weigh described blood sample and add excess¹¹¹In-tetrazine 28.37 DEG C cultivate after 20 minutes, with PBS, blood aliquot diluted 10 times and analyzed by SDS-PAGE.From corresponding described mAb's Radioactivity in bands of a spectrum determines cycloaddition productivity, and with AIDA Image Analyzer software quantitative counting.At three individually The assessment amount (%ID/g blood) of mAb, wherein said mice quilt present in the blood of each time point in mice group (n=3) Injection is corresponding¹²⁵I-CC49-TCO (the 300 μ g/100 every mices of μ L, about 0.2 MBq).Described data (are removed for mAb and are carried out Correction) normalize to the 100%ID TCO when t=0.

The result of this experiment shows, except removing the TCO physiology minimizing caused from blood due to mAb, is being injected Wherein said TCO passes through PEG₁₀Interval body is incorporated in two groups of mices of the structure of CC49, the amount of circular response TCO group Exist over time and reduce (Figure 13 A and B) further.It is connected to institute without interval body for being injected wherein said TCO The mice of the structure stating mAb is not the most such situation (Figure 13 C).This demonstrate spacer-free CC49-TCO structure phase in vivo For described CC49-PEG₁₀The more high stability of-TCO structure.

Embodiment 7

Biodistribution experiments

By with having and not there is PEG₁₀Interval body¹²⁵(100 μ g/100 μ L are the least for the CC49-TCO structure of I-labelling Mus, about 0.2 MBq), and used after 24 or 72 hours¹¹¹In-tetrazine 28 (the 21 μ g/75 every mices of μ L, about 0.8 MBq) is to taking Mice (n=3) with tumor carries out intravenous injection and implements dual-isotope biodistribution experiments.After giving tetrazine three hours, use different fluorine Animal described in etherization is also put to death by neck dislocation.Extract blood by cardiac puncture, and gather, peel off dry (blotted And weigh organ interested and tissue dry).The described sample radioactivity together with reference material is measured with really in γ-enumerator Fixed described %ID/ gram.For¹²⁵I and¹¹¹Described energy window is set as 10-80 keV and 100-510 keV by In respectively.Radiation mark Note mAbs and¹¹¹Being distributed in shown in table 2-5 of In-tetrazine.

Table 2: dual-isotope Biodistribution data is injected¹⁷⁷Lu-tetrazine 28 (the 21 μ g/75 every mices of μ L, about 0.5 MBq), after 3 hours, give¹²⁵I-CC49-PEG₁₀Main (the CC49-23a of-TCO-O；The 100 μ g/100 every mices of μ L, about 0.2 MBq) after 27 hours or 99 hours.Data are given with %ID/ gram ± SD.

Table 3: single isotope Biodistribution data is injected¹¹¹In-tetrazine 28 (the 21 μ g/75 every mices of μ L, about 0.5 MBq), after 3 hours, the main (CC49-20a of CC49-TCO-O is given；The 100 μ g/100 every mices of μ L) 27 hours or after 75 hours. Data are given with %ID/ gram ± SD.

Table 4: dual-isotope Biodistribution data is injected¹¹¹In-tetrazine 28 (the 21 μ g/75 every mices of μ L, about 0.5 MBq) After 3 hours, give¹²⁵Secondary (the CC49-14b of I-CC49-TCO-C；The 100 μ g/100 every mices of μ L, about 0.2 MBq) 27 hours Or after 75 hours.Data are given with %ID/ gram ± SD.

Table 5: dual-isotope Biodistribution data is injected¹¹¹In-tetrazine 28 (the 21 μ g/75 every mices of μ L, about 0.5 MBq) After 3 hours, give¹²⁵Secondary (the CC49-20b of I-CC49-TCO-O；The 100 μ g/100 every mices of μ L, about 0.2 MBq) 27 hours Or after 75 hours.Data are given with %ID/ gram ± SD.

Described Biodistribution data confirm the CC49-TCO structure (constructs) lacking described interval body relative to First generation CC49-PEG₁₀The highest internal stability of the main 23a of-TCO-O.CC49-PEG is used before injecting described tetrazine₁₀- In main (CC49-23a) pretreatment mice of 24 hours of TCO-O, due to the reaction between described TCO and described tetrazine, institute State a large amount of mAb (15.81 ± 0.22%ID/ gram) present in tumor and cause 3.09 ± 0.01%ID/ gram¹⁷⁷Lu accumulates (table 3). Unfortunately,¹⁷⁷Lu-tetrazine also results in low with the reaction (5.55 ± 1.85%ID/ gram) of a large amount of mAb-TCO of circulation in blood Tumor is to blood ratio (T/B=3.1 ± 1.0).When giving described radiolabeled tetrazine after described mAb4 days, due to mAb's Removing, described T/B ratio significantly improves (21.3 ± 4.0).But, the most also observe about four times low¹⁷⁷Lu-tetrazine is swollen Accumulation (0.86 ± 0.39%ID/ gram) in tumor.Definitely this minimizing not mAb-TCO of tetrazine accumulation is from the knot of tissue release Really (tumor still suffers from 10.99 ± 4.24%ID/ gram¹²⁵I-mAb), it is and reasonably by the body of 4 day period TCO between injecting Interior degraded is caused.

On the contrary, when with lacking described PEG₁₀Described in the CC49-TCO structure pretreatment of interval body during mice, it was observed that mAb notes After penetrating 3 days¹¹¹The tumor uptake of In-tetrazine does not reduce (table 4-6) after injecting 1 day relative to mAb.Simultaneously as mAb-TCO Removing from blood, described T/B increases to above 4 than from about 2.During this period of time tumor combine TCO be maintained anti- Answering property shows that it is relative to described first generation CC49-PEG₁₀The more high stability of-TCO.

Embodiment 8

Imaging experiment

Accept 100 μ g without PEG₁₀The CC49-TCO structure 3 of interval body (spacer) or after 4 days use¹¹¹In-tetrazine 28 The mice of tumor is carried in (the 21 μ g/75 every mices of μ L, 20-50 MBq) injection.After about 1 hour, anaesthetize described mice and be placed on It is equipped with for the nose cone of anesthesia and the animal beds of the sensor for respiration monitoring control.Injection tetrazine uses four-head after 2 hours Many pin holes toy SPECT/CT imaging system (NanoSPECT, Bioscan Inc.) implement single photon emission computed tomography Imaging (Single photon emission computed tomography, SPECT).Use the pin hole of 1.4 mm diameters Implement described SPECT with the acquisition time/view (24 projections) of 120-140 second and gather (1 hour altogether).For¹¹¹In will Described energy window is set in 245 keV ± 15% and 171 keV ± 20%.After scanning for the first time two to four days, anaesthetized by excess Described mice is carried out euthanasia and overnight obtains second time SPECT/CT scanning.Implemented CT scan (2 before the SPECT phase every time Second/projection, 360 projections) to obtain the anatomic information about increased radioactivity.After described collection, use manufacturer Software (InVivoScope 1.39, patch 1) iterative approximation data.For tumor, liver, kidney and leg muscle, manual Draw region (ROIs) interested, in triplicate.With being filled with known quantity¹¹¹The body mould of In is demarcated for tissue radiation The scanner that property is quantitative.Figure 13 A-C respectively illustrates and has injected CC49-TCO-O secondary (CC49-20b), CC49-TCO-O master Want three mices of (CC49-20a) and CC49-TCO-C secondary (CC49-14b) image in representational time scale.

Figure 13: (left) gives (A) CC49-TCO-O secondary (CC49-20b), and (B) CC49-TCO-O is main (CC49-20a) Or (C) CC49-TCO-C secondary (CC49-14b) (the 100 μ g/100 every mice of μ L) is after 3-4 days, injection¹¹¹In-tetrazine 28 (21 The μ g/75 every mice of μ L, about 40 MBq) the SPECT/CT projection of the mice that lives after 2 hours.(right), described imaging first time, 2-4 phase The after death SPECT/CT scanning of same mouse after it.White arrow represents tumor.

After in mice, longitudinal SPECT/CT studies display CC49-TCO-O (main and secondary two kinds of forms) pretreatment 3 days Height in tumor¹¹¹In-tetrazine absorbs, and it is therefore evident that the internal stability (Figure 13 A and B) of these TCO-structures.Now (left Figure), most of non-tumors combine mAb-TCO from loop cleaning, and therefore due to¹¹¹The homaluria of In-tetrazine is except institute Stating unique visible organ beyond tumor is kidney and bladder.It is essential that in the organ such as liver of blood and rich blood Do not observe radioactivity.Low-down background radiation is relative to using first generation CC49-PEG₁₀-TCO (see R. Rossin, P. Renart Verkerk, Sandra M. van den Bosch, R. C. M. Vulders, l. Verel, J. Lub, M. S. Robillard, Angew Chem Int Ed 2010,49,3375) the basic improvement of result that obtains, And translate into bone marrow in patient and the lower dosage of other dose limitation organ.

It should be noted that the tumor of mice of described CC49-TCO-O pretreatment is in injection¹¹¹In-tetrazine 72-96 is little It is still highly radioactive time after, shows the high internal stability of described TCO-tetrazine ring addition compound product.Evening time point, Still more visible activity in described mouse kidney, and some accumulate in liver, be reasonably due to antigen come off and due to During injection tetrazine, still in blood, the removing of the described mAb of circulation is caused.The RIT of the pre-targeting in target is cancer patient Time, in tumor, radioactivity keeps the time extended also to be very important.It practice, Therapeutic radionuclides is incorporated into target The time of tissue is the longest, and the dosage being delivered to tumor is the highest.

Unexpectedly, the secondary rear injection of CC49-TCO-C is being given¹¹¹The mice of In-tetrazine observe at a fairly low Radioactive uptake (uptake), it may be possible to delays between twice process 4 days are caused (Figure 13 C).But, also it is this In situation, after scanning 2 days in early days, the signal in tumor still suffers from, it was demonstrated that obtained by described internal Diels-Alder reaction The internal stability of cycloaddition product.

Embodiment 9

This embodiment relate to the various substituent groups on the diverse location of trans cyclo-octene computer simulation (in silico) Test.For different substituted cyclo-octene, use MOPAC software (Cambridge stware Mopac Pro version 8.03) HOMO energy is calculated.Result provides in table 6 below.

Table 6

HOMO energy different substituents determined by Mopac.Here (a) represents upright；E () represents calm.

Carbon atom is numbered according to following structure:

Embodiment 10

The substituting building-up process of tetrazine 28, and corresponding Gd-complex, the synthesis of 28-Gd.

With reference to Figure 15, it illustrates synthetic route.

5-oxo-5-(6-(6-(pyridine-2-base)-1,4-dihydro-1,2,4,5-tetrazine-3-base) pyridin-3-yl amino) Valeric acid (30).

According to literature procedure (M. L. Blackman, M. Royzen, M. J. Fox,J. Am. Chem. Soc. 2008, 130, 13518-13519) and synthesize 6-(6-(pyridine-2-base)-1,4-dihydro-1,2,4,5-tetrazine-3-base)-pyrrole Pyridine-3-amine (29).By 29 (428 mg, 1.69 mmol) and glutaric anhydride (231 mg, 2.03 mmol) in THF (10 mL) Mixture under argon inert atmosphere 60 DEG C heat 40 hours.After cooling, wash orange precipitation with THF (5 mL) and be dried To obtain 30 (537 mg, 87%) of orange solids form.

5-oxo-5-(6-(6-(pyridine-2-base)-1,2,4,5-tetrazine-3-base) pyridin-3-yl amino) valeric acid (6)

By compound 30 (166 mg；0.452 mmol) it is suspended in acetic acid (3 mL) and adds sodium nitrite (93.5 mg；1.36 mmol).Observe fast colorizing purple suspension.After stirring 15 minutes, filter described reactant mixture, use water (2 × 6 mL) and acetone (3 mL) wash, and are dried, to obtain product (152 mg of violet solid form；92%).

(37,41-dioxo-41-((6-(6-(pyridine-2-base)-1,2,4,5-tetrazine-3-base) pyridin-3-yl) ammonia Base)-3,6,9,12,15,18,21,24,27,30,33-11 oxa--36-azepine hentetracontane base) t-butyl carbamate (31)。

PyBOP (148 mg, 0.284 mmol) is added in 6 (94.4 mg, 0.258 mmol), the amino-PEG of 0 DEG C₁₀- Amino-Boc (150 mg, 0.233 mmol) and N, N-diisopropyl ethyl amine (newly distilling, 100 mg, 0.774 mmol) exist In stirring mixture in DMF (2 mL).Described mixture is made to be heated up to room temperature and continue to stir 15 minutes.Evaporate described transparent , dark red solution to being dried, and product is re-dissolved in chloroform (5 mL), with 0.2 M KH₂PO₄(pH=4.5, 3×3 And saturated Na mL)₂CO₃(2 × 3 mL) washs, then precipitation in Anaesthetie Ether (20 mL).It is collected by by centrifugal Use on silicon dioxide and precipitate described in the column chromatography purification of methanol gradient (0-10%) in chloroform, it is provided that purple waxy solid 31 (148 mg, 64%) of form.

2,2 ', 2 ' '-(10-(2,40,44-trioxy--44-((6-(6-(pyridine-2-base)-1,2,4,5-tetrazine-3-base) Pyridin-3-yl) amino)-6,9,12,15,18,21,24,27,30,33,36-11 oxa--3,39-diaza tetratetracontane Base)-Cyclen-1,4,7-three base) triacetic acid (28).

Product 31 (72.3 mg, 0.0727 mmol) is dissolved in DCM (1 mL), and adds TFA (1 mL).In room temperature Stir described mixture 1 hour.After evaporation, residue is dissolved in acetonitrile (1.5 mL) and in Anaesthetie Ether (15 mL) Precipitation.It is isolated by filtration purple precipitation thus the TFA-salt of 32 is provided with quantitative productivity.It is dissolved in DMF (1.5 mL) In, and add DOTA-NHS (69.6 mg, 0.075 mmol) and N, N-diisopropyl ethyl amine (newly distills, 44 mg, 0.341 mmol).Described mixture is stirred at room temperature 30 minutes.Evaporate transparent, after dark red/purple solution, roughage is dissolved in water In and by preparation HPLC purification.28 (80.5 mg, 87% productivity) are obtained after lyophilization.

The Gd of 28^III-complex, (28-Gd^III)

Compound 28 (204 mg, 0.160 mmol) is dissolved in ammonium acetate aqueous solution (0.1 M, pH=5.5,5 mL) In, and add gadolinium acetate (III) hydrate (96.8 mg, 0.239 mmol).Described mixture is stirred at room temperature 30 minutes, connects Immediately by preparation HPLC purification.After lyophilization, it is thus achieved that the Gd of violet solid form^III(188 mg, 82% produces-complex Rate).MS (ESI, m/z): value of calculation C₅₇H₈₉N₁₃O₂₀Gd⁺ ([M +H]⁺): 1433.56, measured value: 1433.58.

Embodiment 11

The chemosynthesis of new model tetrazine probe

Several tetrazine model probes comprising selectable tetrazine part are synthesized.With reference to Figure 16, it illustrates described conjunction Become route.

3-(5-butyrylamino-2-pyridine radicals)-6-(5-(trifluoromethyl)-2-pyridine radicals)-1,2,4,5-tetrazine (35)

By 2-cyano group-5-trifluoromethylpyridin (200 mg, 1.16 mmol), 2-cyano group-5-under argon inert atmosphere The stirring in ethanol (2 mL) of amino-pyridine (300 mg, 2.52 mmol) and sulfur (80 mg, 2.52 mmol).Add hydration Hydrazine (0.60 g；12.0 mmol) and by described mixture 80 DEG C of heated overnight.Make reactant mixture cool down and add water (2 mL).Centrifugal acquisition solid, washs described solid with water/ethanol=1/2 and is dried to provide 135 mg crude products 33.Xiang He And centrifuged supernatant add water, cause the precipitation of the roughage (188 mg) of further batch, be isolated also by centrifugal It is dried.

By these thick amine product (33) and butyryl oxide. (285 mg；1.80 mmol) stir in THF (5 mL) and add at 65 DEG C Heat is overnight.Concentrate described reactant mixture, and in hexane/Anaesthetie Ether=3/1, stir residue.Filter with glass filter This suspension by using hexane/acetone mixture as the silica column chromatography purification residue of eluent, it is thus achieved that 90 Mg crude product 34 (about 60% purity).

Unpurified amidated dihydro tetrazine 34 (62 mg) is suspended in the mixed of THF (1.5 mL) and water (2.0 mL) In compound.While stirring, add NaNO₂(88 mg；1.28mmol), and then at 0 DEG C sulphuric acid (130 mg are dripped；1.33 Mmol) solution in water (1 mL).Observe that fast colorizing becomes red suspension.After stirring 3 minutes, dilute with chloroform and water Described reactant mixture, stays purple to precipitate, and described purple precipitation separates by filtering with glass filter.In concentrating filtrate Organic layer and with Anaesthetie Ether dilution with induce violet solid after-crop precipitation.With chloroform and the mixing of Anaesthetie Ether Thing grinds the solid that (triturated) merges, and be isolated by filtration to provide pure tetrazine 35 (about 25 mg, 8% gross production rate, from 2-cyano group-5-trifluoromethylpyridin calculates).

¹H NMR (CDCl₃/CD₃OD): δ=9.2 (s, 1H), 8.9 (d, 1H), 8.75 (multiple signal, 3H), 8.3 (d, 1H), 2.45 (t, 2H), 1.8 (m, 2H), 1.05 (t, 3H) ppm. ¹⁹F NMR (CDCl₃/ CD₃OD): δ=-62.9 ppm. LC-MS/PDA: a peak in chromatograph, m/z=390.2 (M+H⁺) and 800.8 (2M+Na⁺), λ_max=329 and 526 nm.

3-(5-fluoro-2-pyridine radicals)-6-(5-amide-based small-2-pyridine radicals)-1,2,4,5-tetrazine (38)

Under argon inert atmosphere in the ethanol (1.5 mL) the stirring 2-fluoro-pyridine of cyano group-5-(100 mg, 0.82 Mmol), 2-cyano group-5-amino-pyridine (200 mg, 1.68 mmol) and sulfur (55 mg, 1.72 mmol).Add hydrazine hydrate (0.35 g；7.0 mmol) and heat described mixture overnight at 90 DEG C.Make described reactant mixture cool down and add ethanol (5 mL).Filter with glass filter and obtain solid, wash with hexane and be then dried, to provide 90 mg crude products 36.

Then, THF (1.5 mL) stirs this thick amine product 36 and butyryl oxide. (93 mg；0.59 mmol) and at 65 DEG C Heating.After reacting overnight under argon gas, described reactant mixture is cooled down, dilute with some hexanes (about 3 mL) and use glass mistake Filter (glass filter) filters.By applying hexane/acetone mixture as the silica column chromatography purification of eluant Residue, it is thus achieved that pure amidated product 37 (about 28 mg, 10% gross production rate calculate from the 2-fluoro-pyridine of cyano group-5-).

¹H NMR (CDCl₃/CD₃OD): δ = 9.25 (bs, 1H, NH), 8.7 (s, 1H), 8.5 (s, 1H, NH), 8.4 (multiple signal, 2H), 8.2 (m, 1H), 8.1 (m, 1H), 7.95 (m, 1H), 7.5 (m, 1H), 2.4 (t, 2H), 1.75 (m, 2H), 1.05 (t, 3H) ppm. ¹³C NMR (CDCl₃/CD₃OD): δ = 172.8, 161.6, 159.0, 146.6, 146.1, 143.5 (d), 141.6, 139.3, 136.8, 136.6, 136.4, 127.0,124.0,123.8,122.6,121.5,38.9,18.8,13.5 ppm (owing to C-F couples, some carbon Signal is doublet).LC-MS/PDA: a peak in chromatograph, m/z=342.1 (M+H⁺), λ_max = 288 nm。

By described amidated dihydro tetrazine 37 (28 mg；0.082 mmol) it is suspended in THF (2 mL) and water (2 mL) In mixture.While stirring, drip NaNO at 0 DEG C₂(85 mg；1.23 mmol), and sulphuric acid (120 mg；1.23 mmol) Solution in water (2 mL).Observe fast colorizing purple suspension.After stirring 3 minutes, add chloroform and water.Wash with water Wash described purple chloroform layer twice, then concentrate.In a small amount of chloroform, stir solid residue, be then added thereto to hexane. Being poured out by almost colourless supernatant, drying solid is to obtain 21 mg purple powder 38 (productivity: 75%).

¹H NMR (CDCl₃/CD₃OD): δ=8.8 (multiple signal, 5H), 7.75 (m, 1H), 2.45 (t, 2H), 1.8 (m, 2H), 1.05 (t, 3H) ppm. ¹³C NMR (CDCl₃/CD₃OD): δ = 173.4, 162.9, And 139.4,138.8,126.9,125.8 162.6,162.4,159.8,146.0,143.2,141.3,139.7 D (), (owing to C-F couples, the signal of some carbon is double to 125.2,124.4,124.2,39.0,18.7,13.5 ppm Weight peak).¹⁹F NMR (CDCl₃/CD₃OD): δ=-120.4 ppm. LC-MS/PDA: a peak in chromatograph, m/z= 340.2 (M+H⁺), λ_max=324 and 529 nm.

3-(2-pyridine radicals)-6-methyl isophthalic acid, 2,4,5-tetrazine (40)

In ethanol (5 mL), 2-cyanopyridine (500 mg, 4.8 mmol), ethanamidine salt is stirred under argon inert atmosphere Hydrochlorate (2.00 g, 21.2 mmol) and sulfur (155 mg, 4.8 mmol).Add hydrazine hydrate (2.76g；55.2 mmol) and 20 DEG C are stirred described mixture overnight.Filter muddy mixture and evaporate filter liquor to being dried, it is thus achieved that the thick product that 2.9 g are orange Thing 39.

Then, this crude product (800 mg) is suspended in the mixture of THF (3 mL) and acetic acid (4 mL).Add at 0 DEG C Enter NaNO₂(2.0g；29.0 mmol) solution in water (3 mL).Observe and be coloured to redness/red suspension at once.0 After DEG C stirring 5 minutes, add chloroform and water.Wash described purple chloroform layer with water twice, then concentrate.At chloroform and hexane 1:1 mixture stirs solid residue, then filters.Concentrating filtrate and by application chloroform/acetone mixture as washing Crude product described in the silica column chromatography purification of de-liquid, it is thus achieved that (48 mg, 21% gross production rate, from 2-cyanopyridine for pure products 40 Calculate).

¹H NMR (CDCl₃): δ = 8.96 (d, 2H), 8.65 (d, 2H), 7.99 (t, 2H), 7.56 (dd, 3H), 3.17 (s, 3H) ppm. ¹³C NMR (CDCl₃): δ = 168.1. 163.6, 150.9, 150.3, 137.4,126.3,123.9,21.4 ppm. LC-MS/PDA: a peak in chromatograph, m/z=174.3 (M+H⁺), λ_max=274 and 524 nm.

Double (4-the pyridine radicals)-1,2,4,5-tetrazine (42) of 3,6-

4-cyanopyridine (858 mg are heated at 90 DEG C under argon inert atmosphere；8.24 mmol) and a hydrazine hydrate (1.24 g；24.7 mmol) 16 hours.Described mixture is made to be cooled to room temperature and then dilute with water (3 mL).Filter orange precipitation (41) and with water (3 mL) wash, be dissolved in subsequently in DMSO (10 mL).DDQ (372 mg are added to this solution；1.64 mmol).Observe and be coloured to dark red solution immediately.After 60 minutes, add saturated sodium bicarbonate solution (20 mL) and use chloroform (3 30 mL) extract product.Use Na₂SO₄It is dried the organic layer merged and is evaporated to be dried to obtain pink solid form Title compound (52 mg；5% gross production rate, calculates from 4-cyanopyridine).

¹H-NMR (CDCl₃): δ 8.97 (d, 4H), 8.52 (d, 4H) ppm. LC-MS/PDA: in chromatograph One peak, m/z=237.2 (M+H⁺), λ_max=271 and 523 nm.

Embodiment 12

The chemosynthesis of extra trans-cyclooctene structure

With reference to Figure 17, it describes synthetic route in detail.

(E-is main)-2,5-dioxo pyrrolidin-1-base 2-(ring octyl-4-alkene-1-base epoxide) acetate (44a)

To the 10a (1.73 g, 13.73 mmol comprise about 10% cis-isomer) of ice cooling in THF (40 mL) The sodium hydride (60%, 2.60 g, 65.0 mmol) that solution is added in oil.Stir described mixture 15 minutes at RT, then will It is heated to 50 DEG C and reaches 1 hour.In ice, cool down described mixture, add bromoacetic acid (2.50 g, 17.9 mmol).In ice Stir described suspension 1 hour, then stir 64 hours at about 25 DEG C, add more THF to keep suspension capable of stirring (cumulative volume about 100 mL).After 50 DEG C of heating 1 hour, cool down described mixture, be slowly added to water.Removed by rotary evaporation Major part THF also adds more water (cumulative volume 50 mL).Extract aqueous mixture with MTBE (2 × 75 mL), and use 25 mL Water washing organic layer.The water layer merged with 16 g acidified with citric acid, and extract product with 2 × 75 mL MTBE.It is dried and rotates Evaporation leaves residue, by column chromatography (40 g SiO₂) be purified.Merge product fraction, from the heptan containing trace MTBE Alkane recrystallization residue.This provides the product 43a of 810 mg, and (810 mg, 4.40 mmol, 32%, containing a small amount of syn-isomerism Body).¹H-NMR (CDCl₃): δ 1.4 – 2.45 (m, 10H), 3.1 – 3.2 (m, 1H), 3.9 – 4.1 (AB, 2H), 5.3 – 5.65 (m, 2H)。

Product 43a is dissolved in 30 mL dichloromethane.Add N-hydroxy-succinamide (715 mg, 6.22 mmol) And in ice, cool down mixture.Add DCC (1.42 g, 6.89 mmol) and in ice, stir described mixture 30 minutes, then Stir 4 hours at RT.Filter, rotary evaporation and carry out on 40 g silica gel use heptane/EtOAc gradient chromatograph provide 44a. Merge product fraction, and from the heptane recrystallization containing a small amount of MTBE.The afterproduct crystallization of scraping.Obtain 180 mg's after filtration Product (0.64 mmol, 15%, containing about 20% cis-isomer).

(E-is secondary)-2,5-dioxo pyrrolidin-1-base 2-(ring octyl-4-alkene-1-base epoxide) acetate (44b)

The sodium hydride being added in oil to the 10b (0.78 g, 6.19 mmol) of ice cooling solution in THF (30 mL) (60%, 0.94 g, 23.5 mmol).Stir described mixture 15 minutes at RT, be then heated to 50 DEG C and reach 1 hour.In ice Cool down described mixture, add bromoacetic acid (1.41 g, 10.14 mmol).At 20 hours (sample tables of about 25 DEG C of stirred suspensions Bright have not occurred coupling), then stir 6 hours at 55 DEG C, stir 3 days at 25 DEG C, and be stirred for 6 hours at 55 DEG C.By rotation Turn evaporation and remove major part THF, and add 50 mL MTBE, be subsequently added into ice and 25 mL water.Stratum disjunctum, and use 30 mL MTBE extracts water layer.With 25 mL water washings organic layer in succession.In ice, cool down the water layer of merging, add 50 mL MTBE, connect Addition 5.1 g citric acids.Stratum disjunctum, and extract water layer with 50 mL MTBE.It is dried and rotary evaporation leaves residue (43b), it is used as such for next step.¹H-NMR (CDCl₃): δ 1.2 – 2.45 (m, 10H), 3.65 – 3.75 (m, 1H), 4.1 (s, 2H), 5.45 – 5.65 (m, 2H)。

Product 43b is dissolved in 30 mL dichloromethane.Addition N-hydroxy-succinamide (1.60 g, 13.91 And in ice, cool down described mixture mmol).Add DCC (3.11 g, 15.10 mmol) and in ice, stir 30 points of mixture Clock, then stirs 3 hours at RT.Filter, rotary evaporation and carry out on 40 g silica gel use toluene followed by dichloromethane make Chromatograph for eluent provides 44b, it is mixed with the NHS ester of bromoacetic acid.Mixture is dissolved in 25 mL MTBE, to Described solution adds 25 mL heptane.After stirring 2 hours, the filtering mixt NHS ester of bromoacetic acid (solid be).Rotary evaporation is filtered Go out liquid, residue is dissolved in temperature MTBE, adds some heptane, then solution is cooled to RT.It is settled out product.Filter 30 mg product 44b (0.11 mmol, 2%) are provided.

(E-is main)-2,5-dioxo pyrrolidin-1-base 2-(ring octyl-4-alkene-1-base epoxide)-2-phenylacetic acid ester (46a)

It is added in oil to the 10a (3.0 g, 23.8 mmol, containing < 10% cis-isomer) solution in THF (40 mL) In 60% NaH (3.0 g, 75.0 mmol).At RT stirring mixture 10 minutes, being then heated to 50 DEG C, to reach 1.5 little Time.In ice after cooling, portioning adds DL-2-bromo-acid (3.87 g, 18.0 mmol).THF (20 is added to thick paste thing ML) and 25 DEG C of stirred suspensions 18 hours.By rotary evaporation removing major part THF and 50 mL MTBE are added at 55 DEG C. In cold water, cool down mixture and add some ice, being subsequently added into 50 mL water.Stratum disjunctum also extracts water layer with 50 mL MTBE. With 25 mL water washings organic layer in succession.In ice, cool down the water layer of merging, add 50 mL MTBE, be subsequently added into 10 g lemons Lemon acid.Stratum disjunctum also extracts water layer with 50 mL MTBE.It is dried and rotary evaporation leaves 5.05 g product 45a, by it like this For next step.

Product 45a is dissolved in 50 mL dichloromethane.Add N-hydroxy-succinamide (2.51 g, 21.8 mmol) And in ice, cool down described mixture.Add DCC (5.03 g, 24.4 mmol) and in ice, stir mixture 15 minutes, then Stir 3 hours at RT.Filter, rotary evaporation and carry out on 55 g silica gel use toluene/dichloromethane gradient chromatograph provide 2.8 g 46a (7.83 mmol, 33% based on 10a, comprises about 5% cis-isomer).

(E-is secondary)-2,5-dioxo pyrrolidin-1-base 2-(ring octyl-4-alkene-1-base epoxide)-2-phenylacetic acid ester (46b)

60% NaH being added in oil to the solution in THF (60 mL) of the 10b (1.0 g, 7.9 mmol) at 0 DEG C (1.26 g, 31.5 mmol), and heat the mixture to 50 DEG C 1.5 hours.After being cooled to 0 DEG C, by DL-2-bromo-acid (2.22 g, 10.3 mmol) add with the solution form in THF (5 mL), and it is little to be stirred vigorously viscous suspension 16 at RT Time.It is heated to 40 DEG C after 24 hours, pours the mixture in the citric acid (9.2 g) solution in water (100 mL).With MTBE (3 × 50 mL) extracts aqueous mixture, uses Na₂SO₄It is dried and evaporates solvent to obtain yellow oil.Pass through column chromatography (SiO₂, CH₂Cl₂/ MeOH 2%) purification provides product, unreacted TCO and the mixture of by-product.By be dissolved in MTBE and Water (it alkalizes by 33% NaOH solution) achieves and is further purified.With acidified with citric acid and then water layer MTBE washs, Extract with MTBE (3 ×).Use Na₂SO₄It is dried the organic layer merged, after solvent evaporation, obtains the compound 45b of yellow oil (463 mg, 1.8 mmol, 23% productivity).

To at the 45b (463 mg, 1.8 mmol) of 0 DEG C and N-hydroxy-succinamide (250 mg, 2.2 mmol) at THF Solution in (9 mL) adds the DCC (372 mg, 1.8 mmol) solution in THF (2 mL).Stir described reaction at RT to mix Compound 16 hours, is filtered to remove precipitation afterwards.Concentrating filtrate by column chromatography (SiO in a vacuum₂, heptane/ EtOAc gradient 25%-40%) purification to be to provide without the 46b (451 mg, 1.3 mmol, 71% productivity) of mill base form.

(E-is main)-2,5-dioxo pyrrolidin-1-base 2-(ring octyl-4-alkene-1-base epoxide)-2 Methylpropionic acid ester (49a)

It is added in oil to the 10a (3.0 g, 23.8 mmol) of cooling solution in THF (120 mL) in ice bath 60% sodium hydride (3.8 g, 95 mmol).Remove ice bath and stir described mixture 30 minutes at RT, then little 50 DEG C of stirrings 1 Time.In ice after cooling, it is slowly added to DL-2-bromo-propionic acid (3.3 mL, 35.6 mmol).During described addition, mixture becomes Obtain very thickness, diluted with THF (50 mL).After described interpolation completes, remove ice bath and in RT stirring described reaction mixing Thing 16 hours.Followed the tracks of the progress of reaction by NMR, it presented the conversion ratio of 40% after 16 hours.Then at 35 DEG C by mixture It is stirred for 24 hours, it is thus achieved that the conversion ratio of 84%.Concentrated reaction mixture in a vacuum, with MTBE dilution and then with water (200 ML) quencher (quenched).Stratum disjunctum with the citric acid (22.5 g) solution Acidified aqueous layer in water (60 mL).Use MTBE (3 × 200 mL) extracts described water layer.Use Na₂SO₄Be dried merge organic layer and evaporate described solvent with provide comprise 47a's Mixture (5.19 g).Described material is without being further purified for following step.

It is slowly added at hexane to the solution in THF (200 mL) of the diisopropylamine (13 mL, 92 mmol) at-70 DEG C In 2.5 M n-BuLis (32 mL, 80 mmol).Then described mixture is slowly warmed to-20 DEG C and be again cooled to- 70℃.Add compound 47a (5.19 g) with the solution form in THF and make described mixture be warmed to-20 DEG C.In this temperature Degree adds iodomethane (10.7 mL, 172 mmol) and described mixture is warmed to 5 DEG C.Materials and analyze according to NMR described Reaction completes.Reactant mixture is poured the citric acid (40 g) solution in water (200 mL) into and with MTBE (3 × 150 ML) extract.The organic layer merged is washed with aqueous citric acid solution and saline.Use Na₂SO₄Obtain after being dried and evaporating described solvent Yellow oil (9.4 g).By column chromatography (SiO₂, CH₂Cl₂/ MeOH gradient 1%-4%) purification provide yellow pulp form pure 48a (1.15 g, 5.4 mmol, two step 23% productivity).¹H-NMR (CDCl₃): δ 1.1 2.45 (m) and 1.4 (2s) (16H), 3.2 – 3.3 (m, 1H), 5.3 – 5.7 (m, 2H).To at the 48a (1.15 g, 5.4 mmol) and N-of 0 DEG C The N-Hydroxysuccinimide (622 mg, 5.4 mmol) solution in THF (27 mL) adds DCC (1.12 g, 5.4 mmol) and exists Solution in THF (5 mL).After addition, described mixture is heated up to RT and stirs 16 hours in this temperature.Dilute with MTBE Release mixture, and filter out precipitation.After evaporation solvent, by column chromatography (SiO₂, heptane/EtOAc gradient 20%-40%) purification remain Excess.The 49a (707 mg, 2.3mmol, 42% productivity) of clear crystal form is provided from heptane/EtOAc crystallization.

(E-is secondary)-2,5-dioxo pyrrolidin-1-base 2-(ring octyl-4-alkene-1-base epoxide)-2 Methylpropionic acid ester (49b)

It is added in oil to the 10b (1.5 g, 11.9 mmol) of cooling solution in THF (60 mL) in ice bath 60% sodium hydride (1.9 g, 48 mmol).Remove ice bath and stir described mixture 1.5 hours at 50 DEG C.In ice after cooling, It is slowly added to DL-2-bromo-propionic acid (1.7 mL, 17.8 mmol).During interpolation, mixture becomes very sticky thick and with THF (35 ML) dilution.Remove ice bath after having added and stir described reactant mixture 16 hours at RT.Then described mixture is heated Keep 24 hours to 43 DEG C.Concentrate described reactant mixture in a vacuum, with MTBE dilution and then with water (200 mL) quencher. Stratum disjunctum, and with citric acid solution Acidified aqueous layer in water.Described water layer is extracted with MTBE (3 × 100 mL).Use Na₂SO₄Dry The organic layer of dry merging also evaporates the mixture (2.26 g) that solvent comprises 47b with offer.Described material is without being further purified For following step.

It is slowly added to the solution in THF (100 mL) of the diisopropylamine (5.6 mL, 39.6 mmol) at-70 DEG C 2.5 M n-BuLis (13.5 mL, 33.8 mmol) in hexane.Then slowly described mixture is warmed to 0 DEG C and again It is cooled to-70 DEG C.Add compound 47b (2.26 g) with the form of solution in THF and described mixture is warmed to-20 ℃.Add iodomethane (4.6 mL, 73.9 mmol) at such a temperature and make described mixture be warmed to 15 DEG C.Sampling basis NMR analyzes described reaction and completes.Reactant mixture poured in the citric acid (20 g) solution in water (140 mL) and use MTBE (3 × 100 mL) extracts.The organic layer merged is washed with aqueous citric acid solution and saline.Use Na₂SO₄It is dried and evaporates molten Yellow pulp (4.6 g) is obtained after agent.By column chromatography (SiO₂, CH₂Cl₂/ MeOH gradient 1%-4%) purification offer white knot is provided The pure 48b (181 mg, 0.85mmol, 2 step 7% productivity) of brilliant solid form.

Exist to 48b (181 mg, 0.85 mmol) and the N-hydroxy-succinamide (98 mg, 0.85 mmol) at 0 DEG C Solution in THF (5 mL) adds the DCC (175 mg, 0.85 mmol) solution in THF.After addition, described mixture is added Temperature stirs 5 hours to RT and in this temperature.With MTBE dilution mixture thing and filter out precipitation.After evaporating described solvent, pass through post Chromatograph (SiO₂, heptane/EtOAc gradient 25%) purification residue with provide white solid forms 49b (257 mg, 0.83 Mmol, 98% productivity).

(E-is main)-2,5-dioxo pyrrolidin-1-base 4-(ring octyl-4-alkene-1-base epoxide) benzoate (52a)

The sodium hydride being added in oil at the solution of THF (50 mL) to the 10a (3.45 g, 27.38 mmol) of ice cooling (60%, 2.5 g, 62.5 mmol).Described mixture is stirred 15 minutes by ice, is then heated to 50 DEG C and reaches 1 hour.At ice The described mixture of middle cooling, and add with time of 5 minutes be dissolved in 5 mL THF 4-fluorobenzoyl chloride (1.72 g, 10.84 mmol).(NMR of sample showed to exist a lot of product, but there is also 4-fluorobenzene in 2 days to stir described mixture at 25 DEG C The TCO-ester of formic acid), then stir 3 hours at 50 DEG C.It is water-cooled described mixture, and is slowly added to 10 mL water, then add Enter 2 g sodium hydroxide and 5 mL water.Remove major part solvent by rotary evaporation, add THF and 2 g sodium hydroxide and by described Mixture is heated 4 hours at 50 DEG C.Remove major part solvent by rotary evaporation and add methanol to remaining pastel.By institute State mixture to heat 2 hours at 50 DEG C, then at 55 DEG C of rotary evaporations.Remaining suspension is diluted with 50 mL water.Add MTBE (100 mL), stratum disjunctum also washs organic layers with 25 mL water.Described organic layer comprises a.o. trans cyclo-octene alcohol.With 20 g lemons The water layer of the ice cooling that lemon acid treatment merges, and extract product with 2 × 75 mL MTBE.It is dried and rotary evaporation stays solid to remain Excess, it is made up of the mixture of product and 4-fluobenzoic acid.Described solid is heated with 40 mL methanol.By water (15-20 ML) it is slowly added in described temperature solution until it becomes muddy.Filter and cool down filter liquor and be settled out product.Filter, use 1/1 first Alcohol/water washing and be dried under vacuum the product 51a that provides 0.827 g to want (3.36 mmol based on 4-fluorobenzoyl chloride are 30%)。

With DCC (1.46 g, 7.08 mmol) process ice cooling main trans acids 51a (1.14 g, 4.63 mmol) and The N-hydroxy-succinamide (725 mg, 6.30 mmol) mixture in 50 mL dichloromethane.Ice stir described mixed Compound 1 hour, then stirs 3 hours at RT.Filter, rotary evaporation and be implemented on 40 g silica gel use heptane/EtOAc gradient Chromatograph provide 52a fraction (it is by syn-isomerism body pollution).Merge ensuing product fraction, and with heptane and ethyl acetate Mixture be warmed to 60 DEG C.Make suspension be cooled to RT, then filter.This provides 0.973 g product 52a (2.83 Mmol, 61%).

(E-is secondary)-2,5-dioxo pyrrolidin-1-base 4-(ring octyl-4-alkene-1-base epoxide) benzoate (52b)

The hydrogenation being added in oil to the 10b (2.82 g, 22.38 mmol) of ice cooling solution in THF (40 mL) Sodium (60%, 2.0 g, 50 mmol).In ice, stir described mixture 15 minutes, stir 30 minutes at RT, be then heated to 50 DEG C reach 1 hour.In ice, cool down described mixture, and add, with the time of 15 minutes, the 4-fluorobenzene first being dissolved in 8 mL THF Acyl chlorides (1.72 g, 10.84 mmol).(NMR of sample showed to there is many trans rings in 18 hours to stir described mixture at RT Matsutake alcohol), then stir 6 hours at 50 DEG C, stir 3 days at 25 DEG C, and be stirred for 1 hour at 50 DEG C.It is slowly added to 2.0 g hydrogen Sodium oxide solution in 5 mL water, is subsequently added into 5 mL water.Major part THF is removed and to the paste obtained by rotary evaporation Shape thing adds 40 mL methanol.Mixture is heated 3 hours at 50 DEG C, adds 20 mL THF and continue to heat 4 hours.At 25 DEG C Stir described mixture overnight (NMR shows to still suffer from a small amount of ester), then heat 4 hours at 50 DEG C, then rotary evaporation.With 50 mL water dilute remaining suspension.Adding MTBE (100 mL), stratum disjunctum also washs organic layers with 25 mL water.Described organic Layer comprises a.o. trans cyclo-octene alcohol.The water layer of ice cooling that merges by 15 g citric acid treatment also carries with 2 × 75 mL MTBE Take product.Being dried and rotary evaporation leaves solid residue, it is made up of the mixture of product and 4-fluobenzoic acid.Use 40 mL Methanol is heated described solid.It is slowly added to water (25 mL) to described temperature solution, is then allowed to cool to RT the most stirred Night.Filter, with 1/1 methanol/water wash and be dried under vacuum offer 0.76 g want product 51b (3.09 mmol, based on 4-fluorobenzoyl chloride is 28%).

With DCC (0.95 g, 4.61 mmol) process ice cooling described secondary trans acids 51b (0.66 g, 2.68 And the N-hydroxy-succinamide (460 mg, 4.0 mmol) mixture in 50 mL dichloromethane mmol).Ice stirs Described mixture 1 hour, then stirs 16 hours at RT.Filter, rotary evaporation and be implemented on 30g silica gel use dichloromethane Chromatograph as eluent.Product is dissolved in 5 mL ethyl acetates.Add heptane and described in 60 DEG C of part rotary evaporations Solution is until there is precipitation.Add heptane and stir described mixture 5 minutes at 60 DEG C, being then allowed to cool to RT.Filtration carries For 0.437 g product 52b (1.27 mmol, 47%).

(E-is main, secondary)-2,5-dioxo pyrrolidin-1-base 2-(1-methyl ring octyl-4-alkene-1-base) acetate (58)

Hydrogenperoxide steam generator (25 mL, 300 mmol) by 35% adds palladium (45% Engelhard, 1.0 g, 2 Mmol), benzoquinone (432 mg, 4mmol) and 1, in the mixture of 5-cyclo-octadiene (27 mL, 200 mmol).Institute is stirred at 30 DEG C State mixture 5 days until NMR analyzes the conversion ratio showing described parent material 95%.Pour described mixture into Et₂In O (1 L) And add water (1 L).NaOH solution with the 33% described mixture that slowly alkalizes is cooled with ice simultaneously.Stratum disjunctum also uses Et₂O(2 ×) extract water layer.Wash, with 1 N NaOH, the organic layer twice merged and use Na₂SO₄It is dried.Careful evaporation solvent provides yellow Thick 53 (16.1 g, 130 mmol, 65% productivity) of oil form.

1.6 M n-BuLis solution (45 mL, 72 mmol) in hexane is added diisopropylamine (14 mL, 100 Mmol), in the solution in THF (250 mL), it is cooled to-80 DEG C.Described mixture is gradually warmed to 0 DEG C and then cools down To-80 DEG C.Add phosphine acyl acetic acid three ethyl (triethylphosphonoacetate) (15 mL, 75 mmol) at THF Solution in (100 mL), and stir described mixture 45 minutes at-70 DEG C.It is subsequently adding 53 (6.21 g, 50 mmol) to exist Mixture is also slowly warmed to RT by solution in THF (50 mL).After 16 hours, described mixture is further heated to backflow 8 hours until NMR analyzes display and converts completely.Described mixture poured in water (250 mL) and extract with MTBE (3 ×).With Saline washs the organic layer merged and uses Na₂SO₄It is dried.Rotary evaporation of solvent also passes through column chromatography (SiO₂, heptane/EtOAc 5%) 54 (4.74 g, 24 mmol, 49% productivity) of colourless oil are obtained after purification.

The methyUithium solution (59 mL, 94 mmol) of 1.6 M is added in ice bath cooling Copper diiodide (I) (9.53 g, 50 mmol) at Et₂In suspension in O (21 mL).Concentrate grey solution in a vacuum at 0 DEG C and use CH₂Cl₂Peel off (stripped) twice.Residue is suspended in cold CH₂Cl₂In (100 mL) and be slowly added to TMSCl (4.0 mL, 46.5 Mmol) front it is cooled to-80 DEG C.Then by the way of dropping, 54 (4.74 g, 24 mmol) are added at CH₂Cl₂In (60 mL) Solution, and made described mixture be warmed to RT with 2 hours.Being quenched into by described mixture, (quenched into) is saturated NH₄Before in Cl aqueous solution (150 mL), it is further stirred for 16 hours at RT.Stir mixture at RT and add ammonia (50 mL).Filtering mixt, uses CH₂Cl₂(3 × 75 mL) extracts filter liquor.Wash with water and use Na₂SO₄It is dried the organic of merging Layer.Rotary evaporation of solvent also passes through column chromatography (SiO₂, heptane/EtOAc 3%) purification provide colourless oil 55 (4.47 g, 21 mmol, 89% productivity).¹H-NMR (CDCl₃): δ 0.8 1.9 (m), 1.05 (s) and 1.25 (t) (16H), 2.15 – 2.3 (m, 2H), 4.0 – 4.2 (q, 2H), 5.4 – 5.55 (m, 1H), 5.65 – 5.75 (m, 1H)。

With heptane/Et₂The mixture of O 3:1v/v (about 500 mL) fills flask, described flask be equipped with mercury lamp and with Pump and the post being filled with the silica gel (36 g have some common grade silica gel in bottom) that 10% silver nitrate (I) impregnates connect.Add Enter 55 (4.47 g, 21.3 mmol) and essence of Niobe (2.7 mL, 21.3 mmol) at a small amount of Et₂After solution in O, open Mercury lamp.After making irradiated solution continuously flow through the irradiation of described post 20 hours, do not have in the NMR of reactor content analyzes Observing parent material, 30% MTBE being used in heptane washs described post.With ammonia treatment post content and use CH₂Cl₂(3 ×) extract.Use Na₂SO₄It is dried the organic layer merged, at evaporation solvent and by column chromatography (SiO₂, heptane/EtOAc gradient 3% > 4%) compound 56 (1.41 g, 6.7 mmol, 31% productivity) of colourless oil is obtained after purification.Described main and secondary isomery Body cannot separate, and the most described isomer is used further as a mixture.

¹H-NMR (CDCl₃): δ 0.8 1.9 (m), 1.0 (s) and 1.25 (t) (16H), 2.1 2.4 (m, 2H), 4.0 – 4.2 (q, 2H), 5.5 – 5.65 (m, 2H)。

The lithium hydroxide monohydrate (84 mg, 2.0 mmol) solution in water (1mL) and EtOH (1 mL) is added 56 In (210 mg, 1.0 mmol).In order to preferably dissolve described compound, add THF (1 mL) and MeOH (2 mL).Stir at RT Convert the most complete after mixing 16 hours.Add extra Lithium hydrate (85 mg) and described mixture is heated to 45 DEG C 4 little Time, 30 DEG C 16 hours and 50 DEG C 4 hours until converting completely.Concentrate described mixture molten with citric acid in a vacuum Liquid neutralizes.Extract with MTBE (3 ×) and use Na₂SO₄After drying, rotary evaporation of solvent provide yellow oil 57 (150 mg, 0.82 mmol, 82% productivity).

¹H-NMR (CDCl₃): δ 1.0 (2s, 3H), 1.3 – 2.0 (m, 10H), 2.1 – 2.45 (m, 2H), 5.5 – 5.65 (m, 2H)。

Exist to 57 (150 mg, 0.82 mmol) and the N-hydroxy-succinamide (113 mg, 0.98 mmol) of ice cooling Solution in THF (5 mL) adds the DCC (170 mg, 0.82 mmol) solution in THF (1 mL).Described mixed in RT stirring Then compound 16 hours also dilutes with MTBE.Precipitation is filtered to remove and at evaporation solvent and by column chromatography (SiO₂, heptan Alkane/EtOAc gradient 10% > 30%) (197 mg, 0.71 mmol, 86% produce to obtain the compound 58 of colourless oil after purification Rate).

¹H-NMR (CDCl₃): δ 1.05 and 1.1 (2s, 3H), 1.35 2.35 (m, 10H), 2.4 (s, 1H), 2.5- 2.75 (AB, 1H), 2.85 (s, 4H), 5.5 – 5.65 (m, 2H)。

Embodiment 13

The kinetics of new TCOs

About it, reactivity of tetrazine 28 be have evaluated described newly synthesized TCO structure.Embodiment 5 describes process.

Table 7:CC49-TCO structure and¹⁷⁷The second-order kinetics constant of the reaction between Lu-28.

For all TCOs, all exist far-reaching and one between described main (calm) and described secondary (uprightly) isomer The reactive difference caused, the latter has much bigger reactivity than the former.Additionally, the high response of TCO 58 supports the most extremely Substituent group in the stand up position of the connector of antibody still provides the reactivity of the increase of described trans cyclo-octene ring.

Embodiment 14

The stability of new model tetrazine probe and reactivity

Several tetrazine moulds of selectable tetrazine part are comprised about they reactivities in water and estimation of stability Type probe.

The hydrolytic stability test of tetrazine class

The specific tetrazine of 10 μ L solution (25 mM) in DMSO, and filtering solution is diluted with PBS (3 mL). Use the reduction of UV spectrum monitoring absorption band at 525 nm, and from this data determination hydrolysis rate and half-life.

The tetrazine class reactivity to trans-ring octyl-4-alkene-1-alcohol (secondary isomer, 10b)

Implement competitive assay to determine specific tetrazine and 3-(5-acetamido-2-pyridine radicals)-6-(2-pyridine radicals)-1,2, 4,5-tetrazines (by it selected as reference tetrazine) are at the inverse electronics with trans-ring octyl-4-alkene-1-alcohol (secondary isomer, 10b) Reactivity ratio in the reaction of demand Diels-Alder.

Add to acetonitrile (0.100 mL) and join described in the solution in DMSO (25 mM) of the specific tetrazine described in 5 μ L and 5 μ L Than tetrazine solution in DMSO (25 mM).Dilute this mixture with water (0.9 mL), analyze mensuration two by LC-MS/PDA Plant the absolute magnitude of tetrazine.Then, trans-ring octyl-4-alkene-1-alcohol (secondary isomer, 10b) is added at DMSO (25 μ L 2.5 MM) solution in, and stir described mixture 5 minutes.The absolute magnitude measuring two kinds of tetrazines is analyzed again by LC-MS/PDA, And calculate the conversion ratio of two kinds of tetrazines.The reactivity ratio of two kinds of tetrazines is determined from these conversion ratios.

Table 8: the reactivity of model tetrazine probe and stability

The substituent group being clear that on tetrazine from table 8 has far-reaching influence to stability and reactivity, therefore can It is enough in the design for the special probe of application-specific.

Embodiment 15

The main secondary TCO of vs, 20a vs 20b and the vitro reactions sex differernce of tetrazine probe 28

Implement this experiment to prove that higher TCO reactivity is converted into and radiolabeled four under the low concentration of two kinds of reagent The higher reaction yield of piperazine.For the purpose of it, we used the TCO main 20a (k being connected to CC49₂=19600±1400 M^-1s^-1) and TCO secondary 20b (k₂=136700±2300 M^-1s^-1) (for main and secondary respectively 6.1 and 6.6 TCOs/ Molecule).Two kinds of mAb solution are diluted to obtain 1 × 10 in PBS^-5M to 1 × 10^-8The TCO concentration of M, and make it add with carrier Enter¹⁷⁷Lu-tetrazine (being 1 equivalent relative to mAb) reacts 1 minute at 37 DEG C.The reactant mixture (20 μ L) of aliquot is then With excess tetrazine 6 quencher, add the assay buffer of irreducibility and analyzed by SDS-PAGE.Spectrum from corresponding described mAb Radioactivity determination cycloaddition productivity in band with AIDA Image Analyzer software quantitative counting.Repeat described experiment three Secondary.As to desired by bimolecular reaction, the reduction of the concentration of two kinds of reactive species described in solution after cultivating 1 minute It is converted into lower reaction yield (Figure 18).But, when using described upright TCO 20b, at micro-molar concentration and the lowest dense Under degree, due to the faster kinetics with described tetrazine, reaction yield apparently higher than use that calm TCO 20a obtains that A bit.

Embodiment 16

The internal stability of TCO 20b

Implement this experiment with extension embodiment 6 described in series internal TCO stability measurement.In that series, send out Now it is attached to PEG interval body that the TCOs of CC49 is main and secondary (23a and 27b) is unstable in vivo, finds without PEG simultaneously The TCO of interval body main (20a) is stable at least 4 days (Figure 13) in vivo.In described new experiment, it then follows identical process, I Test the internal stability of the TCO secondary (20b, 6.9 equivalents combine) that CC49-without PEG interval body combines.In Figure 19 Show and mAb is removed the described internal stability data being corrected.Find that TCO 20b that described CC49-combines is in vivo The most slowly degrade, support further and there is the TCOs of the short connector to antibody in vivo than being combined by longer connector The discovery that TCOs is more stable.

Embodiment 17

By hematodinamics and the bio distribution of the CC49 antibody of the most commensurability TCO 20b part functionalization

Implement a series of research with TCO (20b) radical amount on each CC49 molecule of assessment to blood circulation and tumor The impact of targeting.It is expected with the partially modified antibody of too much TCO to cause the decline of blood circulatory half-life and CC49-will be affected The tumor uptake of TCO structure.

The hematodinamics (see Figure 20 and 21) of CC49-TCO structure

With carrying 0,4.9,9.3 or 15.8 TCO (20b) group/mAb's¹²⁵I-CC49 is to female naked Balb/C mice (20-25 g body weight, Charles River Laboratories, n=3) carry out injecting (the 100 μ g/100 every mices of μ L, about 0.5 MBq).Extract blood sample at the time point (5 minutes, 3,6 hours, 1,2,3 days) selected from saphena, weigh and with 1 ML PBS dilutes.MAb is after tetra-days in injection, anaesthetizes described animal and is put to death by neck dislocation, collecting blood also by cardiac puncture Gather, peel off dry, weigh organ interested and tissue, and add 1 mL PBS.γ-enumerator (gamma-counter, Wizard II, Perkin Elmer) in measure the radioactivity in all samples to determine the percentage of per gram of tissue together with reference material Than injection dosage (%ID/g).The CC49-TCO structure half-life in blood is with GraphPad Prism (version 5.01) Calculate (by blood profile being fitted to two-phase decay).

The kinetics blood parameters of table 9:CC49-TCO (20b).

The cancer target of CC49-TCO (20b) structure

With using 0,8.5,12.7 or 18.7 TCO 20b group/mAb functionalizations¹²⁵I-CC49 is to carrying the little of tumor Mus (square method part；100 mm³Tumor size) carry out injecting (the 100 μ g/100 every mices of μ L, 0.2-0.4 MBq, n=4). Put to death after injecting mAb4 days and receive¹²⁵The mice of I-CC49.Injection mAb used after 72 hours¹¹¹In-DOTA-tetrazine 28 is to acceptance ?¹²⁵The mice of I-CC49-TCO 20b carries out injecting (being 25 equivalents relative to mAb, about 0.8 MBq) and after injection tetrazine 3 Within individual hour, put to death.During execution, collect blood by cardiac puncture and gather, peel off dry, weigh organ interested and group Knit, and add 1 mL PBS.γ-enumerator (Wizard 3, Perkin Elmer) uses dual-isotope scheme (for¹²⁵I and¹¹¹In energy window is respectively set as 10-80 keV and 100-510 keV) measure putting in all samples together with reference material Penetrating property is to determine the percentage injected dose (%ID/g) of per gram of tissue.

Table 10: mAb is after 3 days (except *) in injection¹²⁵I-CC49-TCO 20b structure biology in the mice carrying tumor Distribution.Data are given with average %ID/g ± SD (n=4)

* mice is put to death after mAb injects 4 days

The low immunoreactivity of *.

Table 11:¹¹¹The In-tetrazine 28 (after injecting 3 hours) life in the mice with CC49-TCO 20b structure pretreatment Thing is distributed.Data are given with average %ID/g ± SD (n=4)

Data from table 9-11 are it is apparent that TCO modification ratio is not to be exceeded 9.

Embodiment 18

The hematodinamics of CC49-TCO 44b and internal stability

Hematodinamics

With¹²⁵Tumor free mice (n=3/ group) is injected (the 100 every mices of μ g, 0.2-0.4 by the CC49 of I-labelling MBq), CC49 is group modified with 7.5 TCO 44b.At the time point (1,3,6,24,48 and 72 hours) selected from saphena Extraction blood sample is also collected in the bottle containing heparin.Injection mAb after tetra-days, anaesthetize described mice and by neck dislocation at Extremely.Extract blood by cardiac puncture and gather, peel off dry, weigh organ interested and tissue, adding 1 mL PBS, γ-enumerator (Wizard 3, Perkin Elmer) carries out the percentage ratio note counting to determine each organ together with reference material Penetrate dosage (%ID).

In this study, modified for TCO-CC49 presents the removing (Figure 22) faster than unmodified CC49.For CC49-TCO and CC49, the half-life (T from the blood that area under a curve calculates_1/2=ln2×AUC/C₀) it is respectively 22.2 Hour and 26.3 hours.We by this owing to the functionalization of Lys residue on described mAb.As shorter sanguimotor result, At the end of described experiment, it was additionally observed that less amount of in most of organs¹²⁵I-CC49-TCO, described organ is at counting Before do not irrigate (perfused) (Figure 23).

Internal stability

With¹²⁵The CC49-TCO 44b (the 220 every mices of μ g, 0.4 MBq) of I-labelling to individually group without mice with tumor (n= 3) inject.At selected time point (1,3,6,24,48 and 72 hours) from saphena extraction blood sample (about 50 microlitre) And be collected in the bottle comprising heparin.MAb is after tetra-days in injection, anaesthetizes described mice and is put to death by neck dislocation.Pass through heart Puncture extraction blood and remove harmonization of the stomach thyroid, peeling off dry doubling and count to determine often together with reference material in γ-enumerator The percentage injected dose (%ID) of individual organ.Low in these organs¹²⁵I picked-up (is 0.17 ± 0.03%ID and in first under one's belt Shape gland is 0.88 ± 0.33%ID) confirm that described radiolabeled mAb maintains internal label during 4 days evaluate.

Weigh described blood sample, with PBS be diluted to 100 μ L and add excess with 0.1 MBq/ μ g specific activity radiation The carrier of labelling adds¹⁷⁷Lu-tetrazine 28.37 DEG C cultivate described mixture 20 minutes and with 400 × g be centrifuged 5 minutes with Washed corpuscles.Then 30 μ L of supernatant are applied to Zeba Desalt centrifugal column (0.5 ml, 40 kDa MW cut-offs, Pierce) Liquid.After Li Xin, from cylinder eluting height MW Diels-Alder product, retain the tetrazine of excess simultaneously.γ-enumerator is adopted With dual-isotope scheme (for¹²⁵I be 10-80 keV window and for¹⁷⁷Lu is 155-380 keV window) measure in described eluate The radioactivity comprised.To only comprise¹⁷⁷The Serum samples of Lu-tetrazine is for correcting from described cylinder¹⁷⁷Lu reveals.About radiation Property correction for attenuation¹²⁵Then I counting also calculates¹⁷⁷Lu/¹²⁵I ratio.¹⁷⁷Lu/¹²⁵The reduction of I ratio shows to exist in mouse blood sample Excess¹⁷⁷Lu-tetrazine and¹²⁵Reaction yield lower between I-CC49-TCO, and therefore show the internal of described TCO group Inactivation.Figure 24 shows as the intact TCO of %¹⁷⁷Lu/¹²⁵I is than change (being normalized to 100% during t=0) over time.It is worth note Meaning, after mAb injection less than 48 hours described TCO 44b groups seem be in vivo completely stable (97.8 ± 1.6% intact TCO), and observe that some degrade (being 80.4 ± 1.8% intact TCO after injecting mAb4 days) at more late time point.

Embodiment 19

¹⁷⁷The Lu-tetrazine 28 bio distribution in the mice carrying tumor by CC49-TCO 44b pre-targeting

With¹²⁵I-CC49 mice (the square method part to carrying tumor；100 mm³Tumor size；N=4) inject (the 100 every mices of μ g, about 0.2 MBq), described¹²⁵I-CC49 is with 7.5 TCO44b group functionalizations.MAb 30 and 48 is little in injection Shi Hou, mice accepts the scavenger (galactose-MSA-tetrazine, 160 μ g/ dosage) of a dosage then makes it connect after 2 hours It is subject to¹⁷⁷Lu-tetrazine 28 (being 10 equivalents relative to described mAb, about 0.5 MBq).Injection tetrazine, after three hours, anaesthetizes described mice And put to death by neck dislocation, extract blood by cardiac puncture, gather organ interested and tissue peel off dry.Weigh all Collect sample and add 1 mL PBS.In γ-enumerator use dual-isotope scheme (for¹²⁵I is 10-80 keV window With for¹⁷⁷Lu is 155-380 keV window) measure sample radioactivity to determine the percentage ratio note of per gram of tissue together with reference material Penetrate dosage (%ID/g).

Biodistribution data shows¹²⁵The high tumor uptake of I-CC49-TCO 44b.Described tumor uptake is higher than using other TCO structure (seeing below) obtain those, the long blood being reasonably because the CC49 with 7.5 TCO-44b group functionalizations follows Ring.Conversely, because give the scavenger of two dosage, described scavenger catches circulation CC49-TCO and is directed to liver, It is by rapid metabolization there, and therefore in other organs all, mAb retains is all low.As higher mAb picked-up in tumor As a result,¹⁷⁷The picked-up of Lu-tetrazine is also apparently higher than those (the seeing below) of acquisition in previously experiment.It is additionally, since and is giving Tracing step (chase step) before tetrazine eliminates the CC49-TCO that non-tumor combines, in other organs all 's¹⁷⁷Lu picked-up is insignificant.Kidney is only had to present as the result of tetrazine homaluria at a relatively high¹⁷⁷Lu retains.This causes Target high in the organ of all considerations in addition to kidney is to non-target ratio.It should be noted that present in tumor and blood 34 ± 4% and 10 ± 1% TCOs react with tetrazine the most respectively.

Table 12: injection¹⁷⁷Lu-tetrazine 28 (the 8.5 μ g/80 every mices of μ L, about 0.5 MBq), after 3 hours, gives¹²⁵I- The CC49-TCO-44b (the 100 μ g/ 100 every mices of μ L, about 0.2 MBq) dual-isotope Biodistribution data after 50 hours. Data are given with %ID/ gram ± SD or tumor/organ ratio ± SD (n=4).

Claims (16)

1. for the medical imaging of targeting and/or the test kit for the treatment of, its comprise at least one pre-targeting probe and at least one Effector probe, wherein said pre-targeting probe comprises primary targeting moiety and the first bio-orthogonal reaction group, and wherein Described effector probe comprises effector part, and the second bio-orthogonal reaction group, and wherein said first and second is biological Any one of orthogonal reaction group is another of dienophile and described first and second bio-orthogonal reaction groups Being diene, wherein said dienophile is the 8 ring dienophiles meeting formula (1):

Wherein the position of R is calm and R_aPosition be upright, wherein X, Y, R and R_aEach represent H independently, or The substituent group that person represents selected from the group consisted of for most six times: alkyl, aryl, O-aryl, O-alkyl, OCONR '-alkyl Wherein R ' be H, alkyl or aryl, OCONR '-aryl wherein R ' be H, alkyl or aryl；Wherein R_aIn one be included in, appoint Selection of land passes through interval body, to the connector part of described pre-targeting probe or described effector probe；Two of which R or R_aPortion Divide and can form ring together；With at least one of which and most four R_aIt not hydrogen,

Wherein said 8 ring dienophiles do not include following compound:

Wherein said diene is selected from the compound of formula (5) defined below:

Wherein R¹And R²Representative is selected from the substituent group of the group consisted of independently of one another: H, 2-pyridine radicals, 3-pyridine radicals, 4- Pyridine radicals, 2,6-pyrimidine radicals, 2,5-pyrimidine radicals, 3,5-pyrimidine radicals, 2,4-pyrimidine radicals or phenyl, optionally by one or more suctions Electron group replaces, and described electron withdraw group is selected from NO₂、F、Cl、CF₃、CN、COOH、COOR、CONH₂、CONHR、CONR₂、 CHO、COR、SO₂R、SO₂OR, NO or Ar, wherein R is C₁-C₆Alkyl and Ar represent aryl；Comprise at least with wherein said diene One, optionally by interval body, to described pre-targeting probe or the key of described effector probe.

Test kit the most according to claim 1, the dienophile of wherein said formula (1) meets following one or more requirement:

A) X is methyl；

B) Y is methyl.

Test kit the most according to claim 1, wherein Ar represents phenyl, pyridine radicals or naphthyl.

4. pre-targeting reagent, it comprises primary targeting moiety and bio-orthogonal reaction group, wherein said bio-orthogonal reaction Property group be according to the dienophile of formula (1) limited in claim 1 or 2 any one.

5. image probe, it comprises detectable label and bio-orthogonal reaction group, and this detectable label is choosing The isotope of free group consisting of:³H、¹¹C、¹³N、¹⁵O、¹⁸F、¹⁹F、⁵¹Cr、⁵²Fe、⁵²Mn、⁵⁵Co、⁶⁰Cu、⁶¹Cu、⁶²Zn、⁶²Cu、⁶³Zn、⁶⁴Cu、⁶⁶Ga、⁶⁷Ga、⁶⁸Ga、⁷⁰As、⁷¹As、⁷²As、⁷⁴As、⁷⁵Se、⁷⁵Br、⁷⁶Br、⁷⁷Br、^8OBr、⁸²Br、⁸²Rb、⁸⁶Y 、⁸⁸Y、⁸⁹Sr、⁸⁹Zr、⁹⁷Ru、⁹⁹Tc、¹¹⁰In、¹¹¹In、¹¹³In、¹¹⁴In、¹¹⁷Sn、¹²⁰I、¹²²Xe、¹²³I、¹²⁴I、¹²⁵I、¹⁶⁶Ho、¹⁶⁷Tm、¹⁶⁹Yb、¹⁹³Pt、¹⁹⁵Pt、²⁰¹Tl and²⁰³Pb, wherein said bio-orthogonal reaction group is according in claim 1 or 2 The dienophile of the formula (1) limited.

6. treatment probe, it comprises pharmaceutically active compound and bio-orthogonal reaction group, and this pharmaceutically active compound is choosing The isotope of free group consisting of:²⁴Na、³²P、 ³³P、 ⁴⁷Sc、 ⁵⁹Fe、⁶⁷Cu、 ⁷⁶As、 ⁷⁷As、 ⁸⁰Br、 ⁸²Br、 ⁸⁹Sr 、 ⁹⁰Nb、 ⁹⁰Y、 ¹⁰³Ru、 ¹⁰⁵Rh、 ¹⁰⁹Pd、 ¹¹¹Ag、 ¹²¹Sn、 ¹²⁷Te、 ¹³¹I、 ¹⁴⁰La、 ¹⁴¹Ce、 ¹⁴²Pr、 ¹⁴³Pr、 ¹⁴⁴Pr 、 ¹⁴⁹Pm、 ¹⁴⁹Tb、 ¹⁵¹Pm、 ¹⁵³Sm、 ¹⁵⁹Gd、 ¹⁶¹Tb、 ¹⁶⁵Dy、 ¹⁶⁶Dy、 ¹⁶⁶Ho、 ¹⁶⁹Er、 ¹⁷²Tm、 ¹⁷⁵Yb、 ¹⁷⁷Lu、 ¹⁸⁶Re、 ¹⁸⁸Re、 ¹⁹⁸Au、 ¹⁹⁹Au、 ²¹¹At、 ²¹¹Bi、 ²¹²Bi、 ²¹²Pb、 ²¹³Bi、 ²¹⁴Bi、 ²²³Ra and²²⁵Ac, wherein said Bio-orthogonal reaction group is the dienophile according to the formula (1) limited in claim 1 or 2.

The pre-targeting reagent the most according to claim 4 purposes in preparing test kit, this test kit is used for pre-targeting method, should Method includes giving pre-targeting reagent according to claim 4 to experimenter and making described reagent in the system of described experimenter Circulation effectively realizes described primary targeting moiety a period of time to the combination of primary target, then from the health unconjugated examination of removing Agent.

The image probe the most according to claim 5 purposes in preparing test kit, this test kit is used for formation method, the method Including:

-implement pre-targeting method according to claim 7, then give image probe, it comprises detectable label and life Thing orthogonal reaction group, this detectable label is the isotope selected from the group consisted of:³H、¹¹C、¹³N、¹⁵O、¹⁸F、¹⁹F、⁵¹Cr、⁵²Fe、⁵²Mn、⁵⁵Co、⁶⁰Cu、⁶¹Cu、⁶²Zn、⁶²Cu、⁶³Zn、⁶⁴Cu、⁶⁶Ga、⁶⁷Ga、⁶⁸Ga、⁷⁰As、⁷¹As、⁷²As、⁷⁴As、⁷⁵Se、⁷⁵Br、⁷⁶Br、⁷⁷Br、^8OBr、⁸²Br、⁸²Rb、⁸⁶Y、⁸⁸Y、⁸⁹Sr、⁸⁹Zr、⁹⁷Ru、⁹⁹Tc、¹¹⁰In、¹¹¹In、¹¹³In、¹¹⁴In、¹¹⁷Sn、¹²⁰I、¹²²Xe、¹²³I、¹²⁴I、¹²⁵I、¹⁶⁶Ho、¹⁶⁷Tm、¹⁶⁹Yb、¹⁹³Pt、¹⁹⁵Pt、²⁰¹Tl and²⁰³Pb, wherein said life Thing orthogonal reaction group is the diene according to the formula (5) limited in claim 3, or

-implement pre-targeting method, it includes giving pre-targeting reagent to experimenter, and described pre-targeting reagent comprises primary targeting Part and bio-orthogonal reaction group, wherein said bio-orthogonal reaction group is according to the formula limited in claim 3 (5) diene, and make described reagent circulate in the system of described experimenter effectively to realize described primary targeting moiety to primary A period of time of the combination of target, then remove unconjugated reagent from health, then give imaging according to claim 5 and visit Pin,

Wherein said pre-targeting reagent forms described [4+ together with the described bio-orthogonal reaction group in described image probe 2] the reaction participant of inverse Diels-Alder reaction.

The treatment probe the most according to claim 6 purposes in preparing test kit, this test kit targeting in experimenter Medical therapy, the method includes:

-implement pre-targeting method according to claim 7, then give to treat probe, it comprises pharmaceutically active compound and life Thing orthogonal reaction group, this pharmaceutically active compound is the isotope selected from the group consisted of:²⁴Na、³²P、 ³³P、 ⁴⁷Sc、 ⁵⁹Fe、⁶⁷Cu、 ⁷⁶As、 ⁷⁷As、 ⁸⁰Br、 ⁸²Br、 ⁸⁹Sr、 ⁹⁰Nb、 ⁹⁰Y、 ¹⁰³Ru、 ¹⁰⁵Rh、 ¹⁰⁹Pd、 ¹¹¹Ag、 ¹²¹Sn 、 ¹²⁷Te、 ¹³¹I、 ¹⁴⁰La、 ¹⁴¹Ce、 ¹⁴²Pr、 ¹⁴³Pr、 ¹⁴⁴Pr、 ¹⁴⁹Pm、 ¹⁴⁹Tb、 ¹⁵¹Pm、 ¹⁵³Sm、 ¹⁵⁹Gd、 ¹⁶¹Tb、 ¹⁶⁵Dy、 ¹⁶⁶Dy、 ¹⁶⁶Ho、 ¹⁶⁹Er、 ¹⁷²Tm、 ¹⁷⁵Yb、 ¹⁷⁷Lu、 ¹⁸⁶Re、 ¹⁸⁸Re、 ¹⁹⁸Au、 ¹⁹⁹Au、 ²¹¹At、 ²¹¹Bi、 ²¹²Bi、 ²¹²Pb、 ²¹³Bi、 ²¹⁴Bi、 ²²³Ra and²²⁵Ac, wherein said bio-orthogonal reaction group is according in claim 3 The diene of the formula (5) limited, or

-implement pre-targeting method, it includes giving pre-targeting reagent to experimenter, and described pre-targeting reagent comprises primary targeting Part and bio-orthogonal reaction group, wherein said bio-orthogonal reaction group is according to the formula limited in claim 3 (5) diene, and make described reagent circulate in the system of described experimenter effectively to realize described primary targeting moiety to primary A period of time of the combination of target, then remove unconjugated reagent from health, then give treatment according to claim 6 and visit Pin,

Wherein said pre-targeting reagent forms described [4+ together with the described bio-orthogonal reaction group in described treatment probe 2] the reaction participant of inverse Diels-Alder reaction.

The reagent the most according to claim 4 purposes in preparing test kit, this test kit is for according to claim 7 to 9 times Anticipate in the method for.

11. meet the compound of the formula (1) defined in claim 1 or 2 any one purposes in preparing test kit, this examination In agent box pre-targeting method in animal or people.

12. have the trans cyclo-octene of one or more upright substituent group, meet the compound of claim 1 or 2 Chinese style (1), As the purposes of dienophile reactant in the pre-targeting method reacted based on inverse Diels-Alder.

13. for the medical imaging of targeting and/or the test kits for the treatment of, its comprise at least one pre-targeting probe and at least one Effector probe, wherein said pre-targeting probe comprises primary targeting moiety and the first bio-orthogonal reaction group, and wherein Described effector probe comprises effector part, and the second bio-orthogonal reaction group, and wherein said first and second is biological Any one of orthogonal reaction group is that another of dienophile and described first and second bio-orthogonal reaction groups is Diene, wherein said dienophile is the 8 ring dienophiles meeting formula (1):

Wherein the position of R is calm and R_aPosition be upright, wherein X, Y, R and R_aEach represent H independently, or The substituent group that most six times represent selected from the group consisted of: alkyl, O-alkyl, O-aryl, OCONR ' alkyl wherein R ' are H Or alkyl, OCONR ' aryl wherein R ' are H or alkyl；Wherein R_aIn one be included in, optionally by interval body, to the most described In the connector part of pre-targeting probe or described effector probe；Two of which R or R_aPart can form ring together；And its In at least one and most four R_aIt not hydrogen,

Wherein said 8 ring dienophiles do not include following compound:

Wherein said diene is selected from the compound of formula (5) defined below:

Wherein R¹And R²Represent independently of one another selected from the substituent group of group consisted of: 2-pyridine radicals, phenyl or by one or The substituted phenyl of multiple electron withdraw groups, described electron withdraw group is selected from NO₂、F、Cl、CF₃、CN、COOH、COOR、CONH₂、 CONHR、CONR₂、CHO、COR、SO₂R、SO₂OR, NO or Ar, wherein R is C₁-C₆Alkyl and Ar represent aryl.

14. test kits according to claim 13, wherein Ar represents phenyl, pyridine radicals or naphthyl.

15. pre-targeting reagent, it comprises primary targeting moiety and bio-orthogonal reaction group, wherein said bio-orthogonal reaction Property group be the diene according to the formula (5) defined in claim 13 and the parent according to the formula (1) defined in claim 13 double The reaction participant of the inverse Diels-Alder reaction of [4+2] between alkene body.

16. according to the test kit of claim 1 or 13, and wherein said effector part is label or pharmaceutically active compound.