Tetrazines with improved properties

Abstract

The disclosure herein may encompass tetrazines that may have improved properties, for example, in clinical applications. In some embodiments, the compositions and methods herein may advantageously reduce the inhibitory effects of physiologically important enzymes compared to other tetrazines. Moreover, the tetrazines of the present disclosure may also, in some embodiments, exhibit a higher maximum tolerated dose compared to other tetrazine candidates. In addition, the tetrazines of the present disclosure may, in some embodiments, be readily synthesizable and exhibit additional desirable properties in vitro and in vivo.

Description

[Technical Field]

[0001] The present disclosure relates to tetrazines with improved properties. Compositions and combinations include the disclosed tetrazines, as well as methods of using and making the tetrazines. [Background technology]

[0002] In the field of bioorthogonal chemistry, the ligation between tetrazines and dienophiles, particularly trans-cyclooctenes, has been extensively studied. Rossin et al., Angew. Chem. Int. Ed. 2010, volume 49, pages 3375-3378, describe a tetrazine, referred to herein as compound 2.1, having the following structure: [ka]

[0003] Compound 2.1 and some of its properties are described in the following further papers: pretargeting, viz. Rossin et al., J. Nucl. Med. 2013, volume 54; pages 1989-1995; Rossin et al., Bioconjugate Chem. 2013, volume 24, pages 1210-1217; Rossin et al., Mol. Pharm. 2014, volume 11, pages 3090-3096; Van Duijnhoven et al., J. Nucl. Med. 2015, volume 56, pages 1422-1428; and Edem et al. Molecules 2020, volume 25, page 463.

[0004] Although the ligation works well both in vitro and in vivo, identifying optimal new compounds for clinical use remains a major research challenge. Therefore, it is desirable to identify novel tetrazines that have overall favorable in vitro and in vivo properties, as well as one or more of the following: good stability, good reactivity with and / or high payload release from transcyclooctenes (particularly in vivo), low cell membrane permeability, low cytotoxicity, and low genotoxicity. Summary of the Invention [Problem to be solved by the invention]

[0005] Thus, there is a need for new tetrazines that address one or more of the problems and / or needs discussed above. [Means for solving the problem]

[0006] In one aspect, the disclosure relates to a compound, or a salt, hydrate, or solvate thereof, wherein the compound has a structure according to formula (1): [ka] where: L 1A and L 1B are independently selected linkers; E 1A is selected from the group consisting of: [ka] Here, MMC + is a monovalent metal cation; DMC 2+ is a divalent metal cation; preferably MMC + Na + and preferably DMC 2+ Ca2+ is.

[0007] In another aspect, the present disclosure relates to a composition comprising a compound according to Formula (1), or a salt, hydrate or solvate thereof, preferably wherein the composition is a pharmaceutical composition.

[0008] In yet another aspect, the present disclosure provides a method for manufacturing a semiconductor device comprising: (A1) a compound according to formula (1) or a salt, hydrate or solvate thereof; and / or (A2) a composition according to the present disclosure; (B) a dienophile, or a salt, solvate, or hydrate thereof; preferably, the dienophile comprises a trans-cyclooctene residue; This relates to a combination of

[0009] In a further aspect, the present disclosure relates to a compound according to formula (1), or a salt, hydrate or solvate thereof; a composition according to the present disclosure; or a combination according to the present disclosure, for use as a medicament.

[0010] In still further aspects, the present disclosure relates to a compound according to formula (1), or a salt, hydrate or solvate thereof; a composition according to the present disclosure; or a combination according to the present disclosure, for use in treating a disease in a subject, wherein preferably the subject is a human; and preferably the disease is cancer.

[0011] In a further aspect, the present disclosure still further provides a method for manufacturing a semiconductor device comprising: (ia) a compound according to formula (1) or a salt, hydrate or solvate thereof; and / or (iia) a composition according to the present disclosure, comprising a dienophile, or a salt, solvate or hydrate thereof; a non-therapeutic method of reacting with The method relates to the above non-therapeutic method, wherein the method comprises the step of contacting (ia) and / or (iia) with the dienophile, or a salt, solvate, or hydrate thereof; preferably, the non-therapeutic method is an in vitro method; and preferably, the dienophile comprises a trans-cyclooctene residue.

[0012] In yet a further aspect, the present disclosure provides a method for preparing a compound comprising the steps of: (a) a compound according to formula (1), or a salt, hydrate, or solvate thereof; (b) a composition according to the present disclosure; and / or (c) Combinations in accordance with the present disclosure Concerning the non-therapeutic use of

[0013] In yet a further aspect, the present disclosure provides a method for preparing a compound of claim 7, the method comprising: (a) reacting SM1a or SM1b with a reagent selected from the group consisting of SM2, SM3 and SM4; (b) when SM1a is used in step (a), subjecting the reaction product of step (a) to an oxidation reaction; (c) optionally subjecting the reaction product of step (a) or step (b) to a salt-forming reaction. The process includes the steps of: Here, SM1a, SM1b, SM2, SM3 and SM4 are as follows: [ka] Preferably, in step (a), SM1a is used; preferably, in step (b), the reaction product of step (a) is contacted with sodium nitrite.

[0014] In a further aspect, the disclosure provides a method of treating a disease in a subject, the method comprising: (a) a compound according to formula (1), or a salt, hydrate, or solvate thereof; (b) a composition according to the present disclosure; and / or (c) Combinations in accordance with the present disclosure to the subject, Preferably, the subject is a human; and preferably, the disease is cancer. However, regarding the above method.

[0015] In a final aspect, the present disclosure relates to a method of using a compound of formula (1), a composition of the present disclosure, or a combination of the present disclosure in the manufacture of a medicament for treating a disease in a subject, preferably wherein the subject is a human; preferably wherein the disease is cancer. DETAILED DESCRIPTION OF THE INVENTION

[0016] The above-mentioned compound 2.1 was first reported by Rossin et al., Angew. Chem. Int. Ed. 2010, volume 49, pages 3375-3378, where it was used for tumor pretargeting and was selected for its favorable pharmacokinetic properties, reactivity, and sufficient stability in vivo.

[0017] Furthermore, compound 2.1 has also been used in vivo to bind and detect unreacted trans-cyclooctene by Rossin et al., Bioconjugate Chem. 2016, volume 27, pages 1697–1706; and Rossin et al., Nature Commun. 2018, volume 9, article 1484.

[0018] Additionally, compound 2.1 possesses several highly attractive properties for the in vivo release of payloads, typically drugs, attached to trans-cyclooctene. Compound 2.1 exhibits higher reactivity with trans-cyclooctene than other tetrazines and a favorable clearance rate (Rossin et al., Nature Commun. 2018, Volume 9, Article 1484, left column, page 6). Compound 2.1, like most bis-(2-pyridyl)-tetrazines, exhibits a low release rate with most trans-cyclooctenes. However, when trans-cyclooctenes with certain substituents are used, the release rate of bis-(2-pyridyl)-tetrazines is (almost) quantitative (WO 2020 / 256546, especially pages 294-296 of Example 5). Therefore, compound 2.1 is also a strong candidate for use in in vivo payload delivery. Consequently, compound 2.1 is the most well-studied tetrazine for in vivo use in the literature and has been reported to be a promising candidate for clinical use.

[0019] However, the inventors have identified, and are reported herein for the first time, potentially improvable properties of compound 2.1 that may arise under certain conditions when compound 2.1 is used in vivo as an activator for payload release from trans-cyclooctene, which typically requires higher doses than when compound 2.1 is used for diagnostic radioimaging and / or radiotherapy.

[0020] First, compound 2.1 was found to potently inhibit the physiologically important enzymes cyclooxygenase (COX-1), acetylcholinesterase (ACES), monoamine oxidase B (MAO-B), and the L-type dihydropyridine calcium channel. Each of these proteins is important in maintaining the health of a subject, and undesired inhibition of these enzymes and / or transporters can result in side effects. Therefore, it would be desirable to provide tetrazine compounds that exhibit reduced inhibitory activity against one or more of these enzymes and transporters. Based on this, it would be desirable to provide tetrazines that exhibit reduced inhibition of one or more of these enzymes and / or transporters.

[0021] Second, compound 2.1 was found to have a maximum tolerated dose (MTD) of approximately 39 μmol / kg in mice. This may limit the therapeutic window during which compound 2.1 can be safely used in vivo. Therefore, it would also be desirable to provide novel tetrazines with higher maximum tolerated doses.

[0022] Moreover, the synthesis of compound 2.1 involves multiple steps, and it would be desirable to provide tetrazines that can be synthesized in fewer steps and / or by more convenient methods.

[0023] Therefore, some aspects and embodiments of the present disclosure are based on the insight that tetrazines of formula (1), in their broadest sense, may satisfy one or more of the above-mentioned desires.

[0024] In particular, tetrazines of formula (1) can exhibit low inhibition of cyclooxygenase (COX-1), acetylcholinesterase (ACES), monoamine oxidase (MAO-B), and / or L-type dihydropyridine calcium channels, as shown in Example 3 herein.

[0025] Moreover, tetrazines of formula (1) typically have a relatively higher maximum tolerated dose in mice, as shown in Example 4 herein.

[0026] Moreover, tetrazines of formula (1) can be readily synthesized in fewer steps than those required for compound 2.1, as shown in Example 2 herein.

[0027] Finally, in many embodiments, the tetrazines of Formula (1) also exhibit superior in vitro and in vivo properties, as demonstrated in Example 5 herein.

[0028] Preferred embodiments of formula (1) are further described below: All of these embodiments can be combined, unless they are mutually exclusive.

[0029] Compound of formula (1)

[0030] The compounds of the present disclosure are those according to formula (1) shown above. For ease of reference, compounds of formula (1) will be referred to below as, for example, "compounds of the present disclosure." It will be understood that such designations include salts, hydrates, or solvates of the compounds by such reference.

[0031] Regarding equation (1), MMC + is preferably Na + , K. + and Cu + Most preferably, MMC+ Na + is.

[0032] Regarding equation (1), DMC 2+ is preferably Ca 2+ , Mg 2+ , Mn 2+ , Zn 2+ , Cu 2+ , Cd 2+ , Cr 2+ , Co 2+ , Fe 2+ , Pb 2+ , Ni 2+ , Hg 2+ , Sn 2+ and Pt 2+ Most preferably, DMC is selected from the group consisting of 2+ Ca 2+ is.

[0033] In formula (1), L 1A and L 1B are independently selected linkers.

[0034] Preferably, L 1A is a linker containing 10 or fewer atoms. More preferably, L 1A -O-, -S-, -SS-, -NR L1A -, -N=N-, -C(O)-, -C(O)NR L1A -, -OC(O)-, -C(O)O-, -OC(O)NR L1A -, -NR L1A C(O)-, -NR L1A C(O)O-, -NR L1A C(O)NR4-, -SC(O)-, -C(O)S-, -SC(O)O-, -OC(O)S-, -SC(O)NR L1A - and -NR L1A Even more preferably, L is selected from the group consisting of C(O)S—. 1A Ha-NR L1A C(O)—, and most preferably L 1A is -NHC(O)-.

[0035] Regarding equation (1), R L1Ais a hydrogen atom or C 1~3 Preferably, R L1A is a hydrogen atom.

[0036] Preferably, L 1B is a linker containing 50 atoms or less; more preferably, L 1B is a linker containing 30 atoms or less; even more preferably, L 1B is a linker containing 25 atoms or less; and most preferably, L 1B is a linker containing 10 or fewer atoms.

[0037] Preferably, L 1B is -CH2-, [ka] is selected from the group consisting of Here, the wavy line is L 1A and an asterisk indicates binding to E 1A The bond to

[0038] L 1B wherein x is an integer ranging from 1 to 12; preferably, x is an integer ranging from 2 to 7; and more preferably, x is an integer ranging from 2 to 4. Most preferably, x is 3.

[0039] Preferably, L 1B and E 1A together form residues selected from the group: [ka]

[0040] With respect to formula (1), y is an integer ranging from 1 to 13; preferably, y is an integer ranging from 1 to 11; more preferably, y is an integer ranging from 2 to 9; more preferably, y is an integer ranging from 2 to 7; more preferably, y is an integer ranging from 2 to 6; more preferably, y is an integer ranging from 2 to 4. Most preferably, y is 3.

[0041] Preferably, the compound of formula (1) 18 It does not contain F, and more preferably the compound of formula (1) does not contain radionuclides.

[0042] Preferably, the compound of formula (1), or a salt, solvate or hydrate thereof, is not: [ka]

[0043] Preferably, the compound of formula (1) is selected from the group consisting of: [ka]

[0044] More preferably, the compound of formula (1) is: [ka]

[0045] More preferably, the compound of formula (1) is: [ka]

[0046] More preferably, the compound of formula (1) is: [ka]

[0047] Most preferably, the compound of formula (1) is: [ka]

[0048] Compositions of the present disclosure

[0049] The present disclosure also relates to a composition comprising a compound according to formula (1), or a salt, hydrate, or solvate thereof. Preferably, the composition is a pharmaceutical composition. Preferably, the composition of the present disclosure further comprises a pharmaceutically acceptable carrier. When a salt of a compound of formula (1) is included in the composition of the present disclosure, it is also preferred to use a pharmaceutically acceptable salt.

[0050] Combinations of the present disclosure

[0051] The present disclosure also relates to (A1) a compound according to formula (1), or a salt, hydrate, or solvate thereof; and / or (A2) a composition according to the present disclosure; in combination with (B) a dienophile, or a salt, solvate, or hydrate thereof.

[0052] Preferably, the combination of the present disclosure is a kit. More preferably, the combination of the present disclosure is a kit in which (A1) and / or (A2) are physically separated from (B).

[0053] Preferably, the dienophile used herein comprises an 8-membered non-aromatic cyclic monoalkenylene residue containing at least one allylic carbon and optionally one or more heteroatoms, preferably N, O, or Si. The 8-membered non-aromatic cyclic monoalkenylene residue may be optionally substituted. Preferably, the 8-membered non-aromatic cyclic monoalkenylene residue is a cyclooctene residue, more preferably a trans-cyclooctene (TCO) residue. Most preferably, the trans-cyclooctene residue is an all-carbon ring.

[0054] Preferably, at least 5, more preferably at least 6, and most preferably at least 7 members of the 8-membered non-aromatic cyclic monoalkenylene residue are unsubstituted. Preferably, the vinyl carbon is unsubstituted, i.e., CH. Thus, when the dienophile contains a trans-cyclooctene residue, which is an all-carbon ring, the trans-cyclooctene residue is preferably a ring having one double bond between two CH residues, i.e., -CH=CH-, and the ring further contains at least 3, more preferably at least 4, and most preferably at least 5 CH residues. The substituted ring members are preferably N or C, more preferably C.

[0055] Preferably, the dienophile comprises a payload that is released upon reaction with the tetrazine of formula (1). The payload is preferably a drug or chelating residue containing a radionuclide for imaging. When the dienophile comprises a payload, at least one allylic carbon of the 8-membered non-aromatic cyclic monoalkenylene residue is directly bonded to a cleavable bond. The cleavable bond comprises at least one S, N, NH, or O and is selected from the group consisting of carbamate, thiocarbamate, carbonate, thiocarbonate, ether, ester, thioether, and thioester bonds. More preferably, the cleavable bond is selected from the group consisting of a carbamate bond, an ether bond, and an ester bond. Most preferably, the cleavable bond is a carbamate. At least one S, N, NH, or O of the cleavable bond is part of the payload or part of any spacer between the cleavable bond and the payload. Preferably, the optional spacer is a self-immolative linker. Self-immolative linkers are well known to those skilled in the art.

[0056] Preferably, the dienophiles according to the present disclosure are those described in International Publication No. WO2020 / 256546 (which is incorporated herein by reference), in particular those described in any one of the appended claims 1 to 4 or any embodiment thereof, more preferably those described in the same specification, from page 44, line 5 to page 74, last line, or any embodiment thereof.

[0057] Without intending to be bound by theory, it is believed that the other substituents that may be optionally present in the 8-membered non-aromatic cyclic monoalkenylene residue do not qualitatively affect the release of the payload upon reaction with diene.In other words, regardless of whether other substituents exist in the 8-membered non-aromatic cyclic monoalkenylene residue, the payload is released upon reaction with diene.Some of the mechanisms of the release of the payload are known to those skilled in the art.For example, they are described in WO2020 / 256546, page 39, particularly in scheme 2 on page 9.

[0058] Dienophiles can be synthesized by those skilled in the art based on known synthetic routes for cyclooctene and corresponding rings containing one or more heteroatoms. Those skilled in the art are also aware of the variety of cyclooctene derivatives that can be synthesized via ring-closing metathesis reactions using Grubbs catalysts. As mentioned above, TCOs may contain one or more heteroatoms in the ring. This is well understood by those skilled in the art (e.g., International Publication No. WO 2016 / 025480). For example, see Cere et al. J. Org. Chem. 1980, 45, 261 for the presence of thioethers in TCOs. Also, for example, see Prevost et al. J. Am. Chem. Soc. 2009, 131, 14182 for the -O-SiR2-O residue in TCOs. Leaving groups (R) located at the allylic position can be used. 48For TCO syntheses where ) is an ether, ester, carbonate, carbamate, or thiocarbamate see [Versteegen et al Angew. Chem. Int. Ed. 2018, 57, 10494] and [Steiger et al Chem Comm 2017, 53, 1378].

[0059] Preferably, the dienophile is a compound, or a salt, hydrate, or solvate thereof, wherein the compound has a structure according to formula (2): [ka] where: L 1 is a straight or branched chain C4-C 12 Alkylene, C3-C8 (hetero)cycloalkylene, C6-C 12 Arylene and C4-C 11 heteroarylene; preferably, L 1 is a straight or branched chain C4-C 12 alkylene, and more preferably, L 1 is a straight or branched chain C4-C 10 alkylene, most preferably L 1 is a straight-chain C5-C6 alkylene; L 2a , L 2b and L 2d are each independently -C(O)NL 2T -, -NL 2T C(O)-, -O-, -S-, -NL 2T -, -N=N-, and -C(O)-; 2T is a hydrogen atom or methyl, and preferably, L 2T is a hydrogen atom; L 2c is selected from the group consisting of C1-C8 (hetero)alkanetriyl, C5-C6 (hetero)arenetriyl, C3-C7 cycloalkanetriyl, and C2-C7 heterocycloalkanetriyl; preferably, L2c is C1-C8 (hetero)alkanetriyl, more preferably L 2c is C1-C8 alkanetriyl, and most preferably, L 2c is a C4-C6 alkanetriyl; T 1 -OT 1A , hydrogen atom, C2-C6 alkyl, C6 aryl, C4-C5 heteroaryl, C3-C6 cycloalkyl, C5-C 12 Alkyl (hetero)aryl, C5-C 12 (Hetero)arylalkyl, C4-C 12 Alkylcycloalkyl, -N(T 1A )2, -ST 1A , -SO3H, -C(O)T 1A , -C(O)OT 1A , -OC(O)T 1A , -C(O)N(T 1A )2, -N(T 1A )2-CO-T 1A and -Si(T 1A ) 3; preferably, T 1 Ha-OT 1A and most preferably, T 1 is -OH; Each T 1A are independently selected from the group consisting of a hydrogen atom, a (hetero)alkyl, a (hetero)alkenyl, a (hetero)alkynyl, a (hetero)aryl, and an amino acid residue; preferably, T 1A is a hydrogen atom or methyl, and more preferably, T 1A is a hydrogen atom; T 2 is a bioconjugation moiety or -L 3 -C B group; preferably, the bioconjugation residue is N-maleimidyl; L 3 is the residue of a bioconjugation reaction residue; preferably, L 3 is a residue of a maleimidyl group or a residue of an N-hydroxysuccinimidyl group; C Bis selected from the group consisting of proteins, nucleic acids, peptides, carbohydrates, aptamers, lipids, small organic compounds, polymers, LNA, PNA, amino acids, peptoids, chelating moieties, fluorescent dyes, phosphorescent dyes, organic particles, gels, cells, and combinations thereof; preferably, C B is a protein, more preferably C B is an antibody or a diabody, and even more preferably C B is a bispecific antibody, most preferably C B is AVP0458 consisting of two monomers, each of the two monomers having an amino acid sequence according to SEQ ID NO: 1; y is an integer in the range of 1 to 50; preferably, y is an integer in the range of 10 to 40; more preferably, y is an integer in the range of 12 to 37, even more preferably, an integer in the range of 15 to 35, even more preferably, an integer in the range of 20 to 30, and most preferably, an integer in the range of 23 to 25; and R 48 is a releasable group, preferably R 48 -O-CO-C A where C A is the payload, preferably C A is preferably a drug that is attached to the —O—CO— residue via a secondary or tertiary nitrogen atom that is part of the drug to form a carbamate; preferably, the drug is monomethyl auristatin E (MMAE).

[0060] More preferably, the compound according to formula (2) is a compound according to formula (3) below: [ka] Here, x is an integer ranging from 4 to 12; preferably, x is an integer ranging from 4 to 8; more preferably, x is an integer ranging from 4 to 6.

[0061] In equations (2) and (3), T2 is preferably selected from the group consisting of: [ka] where: C B is a protein.

[0062] Even more preferably, the dienophile is: or a salt, hydrate or solvate thereof: [ka]

[0063] As used herein with respect to dienophiles of the present disclosure, E 1 is —H or —CH3.

[0064] Even more preferably, the dienophile is: or a salt, hydrate or solvate thereof: [ka] JPEG2026507549000018.jpg54170

[0065] Even more preferably, the dienophile is: [ka] where C B is AVP0458, which is composed of two monomers, each of which has an amino acid sequence according to SEQ ID NO: 1; preferably, C B is C B Preferably, the sulfur atom is part of a cysteine.

[0066] Most preferably, the dienophile is: or a salt, hydrate, or solvate thereof: [ka] where C B is AVP0458, which is composed of two monomers, each of which has an amino acid sequence according to SEQ ID NO: 1; preferably, C B is C B Preferably, the sulfur atom is part of a cysteine.

[0067] In another preferred embodiment, the dienophile is a conjugate, or a salt, hydrate, or solvate thereof. Preferably, the conjugate is: [ka] where CJ ranges from 1 to 12; B is AVP0458 consisting of two monomers, each of the two monomers having an amino acid sequence according to SEQ ID NO: 1; preferably, CJ is 2 to 10, more preferably 2.5 to 8, even more preferably 3 to 6, even more preferably 3.5 to 4, and most preferably about 4; preferably, C B is attached to each maleimidyl group via a sulfur atom, which is preferably part of a cysteine.

[0068] More preferably, the conjugate is: or a salt, hydrate or solvate thereof: [ka] JPEG2026507549000023.jpg131170 where CJ ranges from 1 to 12; where C B is AVP0458 consisting of two monomers, each of the two monomers having an amino acid sequence according to SEQ ID NO: 1; preferably, CJ is 2 to 10, more preferably 2.5 to 8, even more preferably 3 to 6, even more preferably 3.5 to 4, and most preferably about 4; preferably, CB is attached to each maleimidyl group via a sulfur atom, which is preferably part of a cysteine.

[0069] AVP0458

[0070] As used herein, AVP0458 refers to a TAG72-binding bispecific antibody derived from the CC49 antibody. AVP0458 is a bispecific antibody composed of two monomers, each having an amino acid sequence according to SEQ ID NO: 1 below. SEQ ID NO: 1 (amino acid sequence of AVP0458 bispecific antibody monomer): SVQLQQSDAELVKPGASVKISCKASGYTFTDHAIHWVKQNPEQGLEWIGYFSPGNDDFKYNERFKGKATLTADKSSSTAYLQLNSLTSEDSAVYFCTRSLNMAYWGQGTSVTVSSGGGGSDIVMTQS C SS C PVSVGEKVTLSCKSSQSLLYSGNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLSISSVETEDLAVYYCQQYYSYPLTFGAGTKLVLKR

[0071] Here, the underline indicates a cysteine, and when AVP0458 is itself part of a dienophile of the present disclosure, the cysteine ​​preferably indicates a cysteine ​​modified with or bound to a dienophile of the present disclosure or a residue thereof.

[0072] Thus, in SEQ ID NO: 1, it is preferred that at least one of the underlined cysteines, and more preferably both of the underlined cysteines, is modified with or bound to a dienophile in accordance with the present disclosure. In other words, the sulfur atom of the underlined cysteine ​​is modified with or bound to a dienophile in accordance with the present disclosure. 2 It is preferably attached to a residue, preferably T 2is the residue of an N-maleimidyl group.

[0073] Non-therapeutic methods and uses of compounds of formula (1)

[0074] In some embodiments, the present disclosure relates to non-therapeutic methods and uses. Preferably, the dienophiles used herein are as described for the combinations of the present disclosure.

[0075] For the disclosed non-therapeutic method, it is preferred that the compound of formula (1) (i.e., (ia)) and / or the composition of the present disclosure (i.e., (iia)) and the dienophile are further contacted with a solvent. Those skilled in the art will know suitable solvents for the reaction between tetrazine and dienophile. Preferably, the solvent comprises water, and more preferably, the solvent is water.

[0076] For non-therapeutic uses, the click reaction is preferably a bioorthogonal click reaction. Preferably, the click reaction is performed in vitro, although non-therapeutic reactions can be performed in vivo as well.

[0077] Methods for the preparation of compounds of formula (1)

[0078] The present disclosure also relates to a method for preparing a preferred compound of formula (1), wherein in step (a) of the method, SM1a or SM1b is reacted with a reagent selected from the group consisting of SM2, SM3 and SM4. Preferably, SM1a is used in step (a).

[0079] When SM1a is used in step (a), then step (b) is carried out after step (a). In step (b), the reaction product of step (a) is subjected to an oxidation reaction. Preferably, in step (b), the oxidation reaction is carried out by adding sodium nitrite, optionally in the presence of an acid, preferably formic acid. When SM1b is used in step (a), step (b) does not need to be carried out.

[0080] In a completely optional step (c), the reaction product of step (a) or step (b) is subjected to a salt formation reaction. Those skilled in the art are aware of standard procedures for forming salts from compounds containing one or more carboxyl groups. Preferably, sodium and / or calcium salts are formed in step (c).

[0081] In all cases of steps (a), (b) and (c), it is preferred that the reagents are contacted with a solvent. Those skilled in the art will know suitable solvents to be used in the above steps.

[0082] While the present disclosure is described herein with reference to particular embodiments, the disclosure is not limited to those embodiments, but rather only by the claims. When an indefinite or definite article is used in reference to a singular noun, e.g., "a" or "an," "the," it includes the plural of that noun unless otherwise specified.

[0083] As used in this specification and claims, the verb "comprise" and its conjugations are used in an open-ended sense meaning that items following the word are included, but items not specifically mentioned are not excluded.

[0084] In addition, the reference to an element by the indefinite article "a" or "an" does not exclude the possibility that there is more than one of that element, unless the context clearly requires that there is only one of that element. Thus, the indefinite article "a" or "an" usually means "at least one."

[0085] Therefore, the scope of the expression "a device comprising means A and B" should not be limited to a device consisting only of component A and component B. It means that, in the context of the present invention, the relevant components of the device are only A and component B.

[0086] The compounds disclosed herein can occur in various tautomeric forms.Unless otherwise specified, the compounds disclosed herein are meant to include all tautomeric forms.When the structure of a compound is shown as a specific tautomeric form, it should be understood that the present disclosure herein is not limited to that specific tautomeric form unless otherwise specified.

[0087] Unless otherwise specified, the compounds and / or groups thereof disclosed herein may be protonated or deprotonated. It will be understood that a compound may have multiple charges, which may be of opposite signs. For example, in a compound containing an amine and a carboxylic acid, the amine may be protonated, and the carboxylic acid may be deprotonated.

[0088] In some formulas, groups or substituents are indicated by reference to letters, such as "A," "B," "X," "Y," and various (numbered) "R" groups. Additionally, the number of repeat units is indicated by reference to letters, such as -(CH) n The definitions of these letters should be read with reference to the respective formulae, i.e., in different formulae, these letters may each independently have different meanings unless otherwise indicated.

[0089] In this specification, "alkyl" and the like are referred to. The number of carbon atoms contained in these groups (excluding carbon atoms contained in any optional substituents described below) can be indicated by adding a number before such a term (e.g., "C1-C8 alkyl" means that the alkyl group can have 1 to 8 carbon atoms). As a precaution, a butyl group substituted with an -OCH3 group is represented as a C4 alkyl because the carbon atoms in the substituent are not included in the carbon atom count.

[0090] As used herein, an alkyl group is unsubstituted and has the general formula C n H 2n+1 and may be linear or branched. Examples of alkyl groups include methyl, ethyl, propyl, 2-propyl, t-butyl, 1-hexyl, 1-dodecyl, etc. Thus, C 1~3 The alkyl groups are methyl, ethyl, 1-propyl and 2-propyl.

[0091] The term "salt thereof" refers to a compound formed when an acidic proton, typically an acid proton, is replaced by a cation, such as a metal cation or an organic cation. The term "salt thereof" also refers to a compound formed when an amine is protonated. Where applicable, the salt is a pharmaceutically acceptable salt, although this is not required for salts not intended for administration to patients. For example, in the salt of the compound, the compound may be protonated with an inorganic or organic acid to form a cation, with the conjugate base of the inorganic or organic acid being the anionic component of the salt.

[0092] The term "pharmaceutically acceptable" salt means a salt that is acceptable for administration to a patient, e.g., a mammal (a salt having a counterion that has acceptable mammalian safety for a given administration regimen). Such salts can be derived from pharmaceutically acceptable inorganic or organic bases and pharmaceutically acceptable inorganic or organic acids.

[0093] "Pharmaceutically acceptable salt" refers to pharmaceutically acceptable salts of a compound, where the salts are derived from a variety of organic and inorganic counterions known in the art, including, for example, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, etc., and, if the molecule contains a basic functional group, includes salts of organic or inorganic acids, such as hydrochloride, hydrobromide, formate, tartrate, besylate, mesylate, acetate, maleate, oxalate, etc.

[0094] It will be understood that the terms "residue" and "group" are used interchangeably herein when referring to portions of a molecule.

[0095] It will be understood that when a heteroatom is represented as -X(R')2- (where X is a heteroatom and R' is a residue), this means that two R' residues are attached to the heteroatom.

[0096] For example, the group -((R 51 )2-R 52 )2-(where R 51 and R 52 represents a residue) or similar notation, individual R 51 and R 52 Before the residue is selected, it is 51 -R 51 -R 52 -R 51 -R 51 -R 52 - Should be described as described as R 51 and R 52 It will be understood that one does not first select the residues of and then write down the formula.

[0097] Example

[0098] Example 1: General Methods

[0099] All reagents, chemicals, materials, and solvents, including undescribed nitrile starting compounds, were obtained from commercial sources and used as received. All solvents were of AR quality. Analytical thin-layer chromatography (TLC) was performed on Kieselgel F-254 precoated silica plates. Column chromatography was performed on Screening Devices BV silica gel (flash: 40-63 μm mesh; normal: 60-200 μm mesh). Reverse-phase chromatography was performed using a Buchi Reveleris C18 column (80 g). 1 H-NMR and 13 C-NMR spectra were obtained using a Bruker Avance III HD ( 1 H-NMR at 400MHz and 13 C-NMR (100 MHz) spectrometer or JEOL ( 1 H-NMR was performed at 500 MHz and reported at 298 K. Chemical shifts were reported in ppm downfield from TMS at room temperature. Abbreviations used for splitting patterns are s = singlet, d = doublet, dd = double doublet, t = triplet, q = quartet, m = multiplet, and br = broad. HPLC-PDA / MS was performed using a Shimadzu LC-10 AD VP series HPLC coupled to a diode array detector (Finnigan Surveyor PDA Plus detector, Thermo Electron Corporation) and an Ion-Trap (LCQ Fleet, Thermo Scientific). HPLC analysis was performed using an Alltech Alltima HP C 18The analysis was performed at 298 K using a 3 μ column, injection volumes of 1–4 μL, a flow rate of 0.2 mL / min, and a gradient of acetonitrile (MeCN) in HO (both containing 0.1% formic acid) typically from 5% to 100% over 10 min, followed by a 3 min hold at 100%.

[0100] Example 2: Synthesis of tetrazines

[0101] Example 2.1 :Reference compound 2.1 [ka]

[0102] The synthesis of 2,2',2''-(10-(2,40,44-trioxo-44-((6-(6-(pyridin-2-yl)-1,2,4,5-tetrazin-3-yl)pyridin-3-yl)amino)-6,9,12,15,18,21,24,27,30,33,36-undecaoxa-3,39-diazatetratetracontyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (2.1) has been reported by Rossin et al., Angew. Chem. Int. Ed. 2010, 49, 3375-3378.

[0103] Example 2.2: 2,2'-((2-((carboxymethyl)(2-oxo-2-((6-(6-(pyridin-2-yl)-1,2-dihydro-1,2,4,5-tetrazin-3-yl)pyridin-3-yl)amino)ethyl)amino)ethyl)azanediyl)diacetic acid (2.3) [ka]

[0104] Compound 2.2 was prepared according to Blackman et al. J. Am. Chem. Soc. 2008, 130, 13518-13519. Ethylenediaminetetraacetic dianhydride (1.86 g, 7.26 mmol) was dissolved in dry DMSO (3 mL) by gentle heating. The mixture was cooled to room temperature, and a solution of compound 2.2 (450 mg, 1.78 mmol) dissolved in DMSO (11 mL) was slowly added. The orange, cloudy mixture was stirred at room temperature for 5 hours under an argon atmosphere. Subsequently, water (0.1 mL) was added, and the mixture was stirred for 30 minutes. The compound 2.3 mixture was used without further purification. ESI-MS: m / z calculated C 22 H 25 Observed value [M+H]: 527.19 Da for N9O7 + 528.42Da, and [MH + ] - 526.50Da.

[0105] Example 2.3 2,2'-((2-((carboxymethyl)(2-oxo-2-((6-(6-(pyridin-2-yl)-1,2,4,5-tetrazin-3-yl)pyridin-3-yl)amino)ethyl)amino)ethyl)azanediyl)diacetic acid (2.4) [ka]

[0106] The crude reaction mixture of compound 2.3 was diluted with water (15 mL) and acidified by the addition of formic acid (0.2 mL). Sodium nitrite (400 mg, 5.79 mmol) was added, and the pink mixture was stirred at room temperature in a sealed flask for 1 hour. 1 M aqueous ammonium acetate (30 mL) was added, and the pink suspension was centrifuged at 3000 rpm for 10 minutes. The clear, dark pink supernatant was separated and purified by reverse-phase chromatography (C18 column, gradient from 5% MeCN / 0.1 M aqueous ammonium acetate to 25%). The combined product fractions were lyophilized, redissolved in water (25 mL), lyophilized again, and redissolved in water. Formic acid (0.25 mL) was added to the pink solution, causing the product to precipitate. The suspension was centrifuged at 3000 rpm for 10 minutes, after which the clear, pale pink supernatant was discarded. The pink solid was washed with water (25 mL) and centrifuged twice, and then washed with MeCN and centrifuged twice. The remaining pink solid was dried under vacuum to give 486 mg of compound 2.4 (52% overall yield). 1 H-NMR(400MHz,DMSO-d6):δ 12.41(br.s,3H),10.78(s,1H),9.13(d,J=2.5Hz,1H),8.94(dd,J=4.8,1.7Hz,1H),8.62(m,2H),8.52(dd,J=8. 7,2.5Hz,1H),8.16(td,J=7.8,1.8Hz,1H),7.73(dd,J=7.8,4.7Hz,1H),3.53(m,J=10.4Hz,8H),2.85(s,4H)ppm. 13 C-NMR(101MHz,DMSO-d6):δ 173.40,172.97,171.94,163.52,163.27,151.08,150.67,144.47,142.13,138.49 ,138.28,127.05,126.75,125.28,124.66,58.55,55.68,55.37,52.58,52.22ppm. ESI-MS: m / z calculated value C 22 H 23 Observed value [M+H]: 525.17 Da for N9O7+ 526.33Da and [MH] - 524.42Da.

[0107] Example 2.4 :2-({2-[bis(carboxylatomethyl)amino]ethyl}[({6-[6-(pyridin-2-yl)-1,2,4,5-tetrazin-3-yl]pyridin-3-yl}carbamoyl)methyl]amino)sodium calcium acetate (2.5) [ka]

[0108] 1 M sodium acetate was prepared by dissolving sodium acetate trihydrate in milliQ-HO (pH 9.0) and subsequently acidifying with glacial acetic acid to pH 6.4. To an acidic suspension of compound 2.4 (172 mg, 0.33 mmol; 10 mg / mL) in milliQ-HO, CaCO (1.67 mL, 0.53 mmol of a 32 mg / mL homogeneous suspension) was added dropwise. Upon increasing the pH, the tetrazine dissolved at pH 5.8, and the addition of CaCO was stopped at pH 6.5. Subsequently, 1.0 M sodium acetate (pH 6.4) was added to the tetrazine to obtain a final sodium acetate concentration of 0.1 M. The solution was applied to a Sep-Pak column (10 g, Waters) for purification (i.e., removal of excess calcium and sodium acetate components). The tetrazine remained at the top of the column and was rinsed once with 0.1 M sodium acetate, followed by milliQ-HO (6 volumes), and eluted with milliQ-HO:MeOH (1:1) with vacuum assistance. The tetrazine-containing fractions were combined, reduced to 80% of their volume under vacuum, diluted with milliQ-HO, and lyophilized after micropore filtration. The lyophilized residue was redissolved in milliQ-HO at 50 mg / mL, micropore filtered again, and lyophilized to give compound 2.5 as a homogeneous, pink, fluffy powder. 1H-NMR(400MHz,D2O) δ 8.89(dd,J=2.6,0.6Hz,1H),8.79(ddd,J=4.8,1.7,0.9Hz,1H),8.63-8.55(m,2H),8.37(dd,J=8.7,2.6Hz,1H),8.1 6(td,J=7.8,1.7Hz,1H),7.74(ddd,J=7.7,4.7,1.1Hz,1H),3.58(s,2H),3.32-3.06(m,6H),2.77-2.50(m,4H)ppm. 13 C-NMR(100MHz,D2O) δ 179.9,179.3,174.1,162.9,162.5,150.3,148.3,143.9,142.0,139.0,137.3,128.9,127.6,125.3,124.7,60.5,59.8,54.8ppm. ESI-MS calculated value C 22 H 23 For N9O7, 525.17 (excluding sodium and calcium), observed value M+H + 526.25. Elemental analysis C 22 H 20 Calculated for CaN9NaO7: Composition: C (45.1%), Ca (6.8%), N (21.5%), Na (3.9%). Found: C (44.0%), Ca (7.2%), N (20.5%), Na (3.1%).

[0109] Example 2.5 3-(2-(2-(3-oxo-3-((6-(6-(pyridin-2-yl)-1,2-dihydro-1,2,4,5-tetrazin-3-yl)pyridin-3-yl)amino)propoxy)ethoxy)ethoxy)propanoic acid (2.6) [ka]

[0110] 3,3'-((oxybis(ethane-2,1-diyl))bis(oxy))dipropionic acid (5.90 g, 23.6 mmol) was dissolved in chloroform (100 mL), and pyridinium p-toluenesulfonate (0.15 g, 0.597 mmol) and EDC HCl (1.13 g, 5.92 mmol) were added. The solution was stirred at room temperature under argon for 30 minutes. Subsequently, compound 2.2 (1.50 g, 5.92 mmol) was added, followed by DMAP (0.36 g, 2.95 mmol). The orange solution was stirred at room temperature under argon for 90 minutes and then washed twice with 0.5 M aqueous citric acid (60 mL). The combined organic layers were dried over sodium sulfate, filtered, and concentrated. The crude product 2.6 was used without further purification. ESI-MS: m / z calculated value C 22 H 27 Observed value [M+H]: 485.20 Da for N7O6 + 486.25Da and [MH + ] - 484.33Da.

[0111] Example 2.6 3-(2-(2-(3-oxo-3-((6-(6-(pyridin-2-yl)-1,2,4,5-tetrazin-3-yl)pyridin-3-yl)amino)propoxy)ethoxy)ethoxy)propanoic acid (2.7) [ka]

[0112] Crude 2.6 was dissolved in MeCN (30 mL) and water (30 mL), and formic acid (1.5 mL) was added, followed by sodium nitrite (1.23 g, 17.8 mmol). The pink / red solution was stirred in a sealed flask at room temperature for 30 min and then diluted with water (90 mL). The mixture was filtered and purified by reverse-phase chromatography (C18 column, gradient from 15% MeCN / 0.1% formic acid aqueous to 30%). The combined product fractions were lyophilized to give compound 2.7 as a pink fluffy solid (1.32 g, 46% overall yield). 1 H-NMR(400MHz, CDCl3):δ 9.61(br.s,1H),8.97(dt,J=4.6,1.4Hz,1H),8.79(m,J=6.3,2.6Hz,2H),8.77-8.62(m,2H),8.01(td,J=7.8,1.8Hz,1H),7.58(ddd,J=7.6,4. ppm. 13 C-NMR(101MHz,CDCl3):δ 174.18,171.32,163.53,163.02,150.91,150.07,143.56,141.93,138.55,137.57,1 27.36,126.55,125.27,124.38,70.70,70.48,70.20,67.02,66.78,37.91,35.40ppm. ESI-MS: m / z calculated value C 22 H 25 Observed value [M+H]: 483.19 Da for N7O6 + 484.50Da and [MH] - 482.33Da.

[0113] Example 2.7 :3-(2-{2-[2-({6-[6-(pyridin-2-yl)-1,2,4,5-tetrazin-3-yl]pyridin-3-yl}carbamoyl)ethoxy]ethoxy}ethoxy)sodium propanoate (2.8) [ka]

[0114] To an acidic suspension of compound 2.7 (5.65 mg, 10 μmol, 10 mg / mL) in milliQ-HO, NaHCO (0.13 mL, 13 μmol from an 8.4 mg / mL solution) was added dropwise. Upon increasing the pH, the tetrazine dissolved at pH 4.5, and NaHCO addition was stopped at pH 7.2. The solution was applied to a C18 Sep-Pak column for purification (i.e., removal of excess sodium). The tetrazine remained on top of the column, was rinsed with milliQ-HO (6 volumes), and eluted with milliQ-HO:MeOH (1:1) with the aid of vacuum. The tetrazine-containing fractions were combined, reduced to 50% of their volume under vacuum, and lyophilized after micropore filtration. The lyophilized residue was redissolved in milliQ-H2O at 15 mg / mL and lyophilized to give compound 2.8 as a homogeneous pink fluffy solid.

[0115] Example 2.8: 2,2′-((2-oxo-2-((6-(6-(pyridin-2-yl)-1,2-dihydro-1,2,4,5-tetrazin-3-yl)pyridin-3-yl)amino)ethyl)azanediyl)diacetic acid (2.9) [ka]

[0116] To a solution of compound 2.2 (168 mg, 0.66 mmol) in DMF (5 mL) was slowly added a solution of nitrilotriacetic anhydride (114 mg, 0.66 mmol) in DMF (1 mL). The solution was stirred at room temperature under an argon atmosphere for 20 hours. Water (30 mL) and formic acid (0.3 mL) were added to precipitate the product, which was then isolated by centrifugation and settling. The precipitate was washed with MeCN (30 mL) and dried under reduced pressure to give compound 2.9 as an orange powder (217 mg, 77% yield). 1H-NMR(400MHz,DMSO-d6):δ 12.63(br.s,2H),10.71(br.s,1H),8.95(s,1H),8.88(s,1H),8.84(d,J=2.4Hz,1H),8.64(dd,J=4.8,1.6Hz,1H) ,8.19(dd,J=8.7,2.5Hz,1H),8.07-7.83(m,3H),7.53(ddd,J=6.9,4.8,1.6Hz,1H),3.58(s,4H),3.55(s,2H)ppm. ESI-MS: m / z calculated value C 18 H 18 Observed value [M+H]: 426.14 Da for N8O5 + 427.33Da and [MH] - 425.42Da.

[0117] Example 2.9: 2,2′-((2-oxo-2-((6-(6-(pyridin-2-yl)-1,2,4,5-tetrazin-3-yl)pyridin-3-yl)amino)ethyl)azanediyl)diacetic acid (2.10) [ka]

[0118] Compound 2.9 (120 mg, 0.282 mmol) was suspended in water (20 mL) and sodium nitrite (97 mg, 1.41 mmol) was added. The suspension was stirred at room temperature under an argon atmosphere and became pink and clear within 10 minutes. After 1 hour, a precipitate formed again and the mixture was stored at 4 °C for 1 hour. The precipitate was separated by centrifugation, washed with water (20 mL) and acetonitrile (25 mL), and dried under reduced pressure to give compound 2.10 as a pink powder (110 mg, 92%). 1H-NMR(400MHz,DMSO-d6):δ 12.53(br.s,2H),10.92(br.s,1H),9.07(d,J=2.5Hz,1H),8.94(d,J=4.2Hz,1H),8.66(d,J=8.7Hz,1H),8.60(d,J=7 9Hz,1H), 8.47(dd,J=8.7,2.6Hz,1H),8.16(td,J=7.8,1.8Hz,1H),7.73(ddd,J=7.7,4.7,1.2Hz,1H),3.62(m,6H)ppm. 13 C-NMR(101MHz,DMSO-d6):δ 173.86,172.77,171.68,163.55,163.24,151.09,150.71,144.69,141.66.13 8.33,138.28,127.08,126.50,125.46,124.69,59.58,56.45ppm ESI-MS:m / z Description C 18 H 16 N8O5 contains 424.12Da; + 425.33Day[MH] - 423.42Da.

[0119] Example 3: Inhibition of physiologically relevant target proteins by tetrazines

[0120] In this example, tetrazines were tested in vitro for the inhibition of multiple physiologically relevant target proteins (via enzymes and transporters). The enzymes tested were cyclooxygenase (COX-1), acetylcholinesterase (ACES), and monoamine oxidase (MAO-B), and the transporter tested was the calcium channel L-type dihydropyridine. Human-derived COX-1, ACES, and MAO-B were used, while rat-derived calcium channel L-type dihydropyridine was used. Standard literature protocols were used, and relevant controls were included to ensure the validity of the results. The target proteins were mixed with tetrazines 2.1 (reference), 2.4, 2.5, 2.7, or 2.10 (final tetrazine concentration 10 μM) in the appropriate buffer. The final concentration for each target protein depended on the standard protocol used, but for each individual target protein, this concentration was the same for the different tetrazines tested. After an appropriate incubation time at the appropriate temperature, the enzymatic activity of the enzyme or the ligand binding in the case of the transporter was tested according to standard procedures from the literature. From this residual enzymatic activity or ligand binding, the percentage of inhibition was determined.

[0121] The results of these experiments are shown in Table 1 below, which reveals that Reference Compound 2.1 typically provides a higher inhibitory effect on target protein activity, thereby exhibiting lower inhibition, compared to the tetrazines of Formula (1). This advantageous effect of the compounds of Formula (1) is particularly evident when looking at the inhibition of COX-1, ACES, and calcium channel L-type dihydropyridines.

[0122] [Table 1]

[0123] Example 4: Maximum tolerated dose (MTD) of tetrazines in mice

[0124] In this example, groups of Swiss albino mice (equal numbers of animals of both sexes) were administered a single dose of tetrazine 2.1 (reference), 2.4, 2.5, 2.7, or 2.10. Different groups were used for different doses. For example, three different groups of mice were used for doses of 8, 39, and 78 μmol / kg of reference compound 2.1. A control group of mice was also used (vehicle control group) that received only vehicle (phosphate-buffered saline (PBS), pH 7.4) without tetrazine.

[0125] Mice were treated in accordance with ethical guidelines. For example, based on the judgment of a laboratory animal veterinarian, mice were housed in an appropriate environment, their health was regularly checked, and they were fed certified mouse chow ad libitum and had free access to water. Environmental controls for the animal room were set to maintain a temperature of 22–25°C, humidity of 30–70% RH, and a 12-hour light / 12-hour dark cycle.

[0126] Dose formulations were prepared as follows: Tetrazine 2.1 (reference), 2.4, 2.5, 2.7, or 2.10 was dissolved in PBS (pH 7.4) to prepare a stock solution of each tetrazine. The pH of the above solution was adjusted to 7.17 using 2 M sodium carbonate. Further dilutions of the tetrazine stock solution were made with PBS (pH 7.4) until the desired concentration was achieved.

[0127] Mice were given the above-mentioned dosage formulation (expressed in μmol of tetrazine per kg of mouse body weight) intravenously on day 1. Mice were then observed for any signs of mortality up to 72 hours after administration. The number of mice that died after 72 hours ("mortality rate") was calculated by dividing by the original total number of mice.

[0128] The results of these experiments are summarized in Table 2 below. In Table 2, it is clear that the maximum tolerated dose of reference compound 2.1 is about 39 μmol / kg. In contrast, for the tetrazines of formula (1) (2.4, 2.5, 2.7 and 2.10), the maximum tolerated dose is at least 57 μmol / kg. The improvement is particularly notable for compounds 2.5, 2.7 and 2.10, which have MTD values ​​of at least 236 μmol / kg compared to reference compound 2.1.

[0129] [Table 2]

[0130] Example 5: General in vitro and in vivo properties

[0131] Compounds 2.4, 2.5, 2.7 and 2.10 of formula (1) were also tested for: i. reactivity with trans-cyclooctene in vitro and / or in vivo, in relation to payload release from the trans-cyclooctene; ii. In vitro stability in mouse, rat, and human plasma; iii. In vitro stability in the presence of mouse, rat, and human microsomes; iv. Cytotoxicity test using LS174T colon cancer cells; v. Passive membrane permeability at pH 7.4 using the Parallel Artificial Membrane Permeability Assay; and / or vi. Genotoxicity (i.e., mutagenicity) in S. typhimurium strains TA98, TA100, TA1535, and 1537 and in E. coli strain WP2 uvrA[pKM101] in the presence or absence of S9.

[0132] All experiments were performed using standard procedures known in the art. In each case, the compound of formula (1) exhibited the expected results. In particular, these results were nearly identical to those obtained for compound 2.1 when compound 2.1 was subjected to the same tests i-vi.

Claims

1. A compound, or a salt, hydrate, or solvate thereof, wherein the compound has a structure according to formula (1): 【Chemistry 1】 where: L 1A and L 1B are independently selected linkers; E 1A is selected from the group consisting of: 【Chemistry 2】 where: MMC + is a monovalent metal cation; DMC 2+ is a divalent metal cation; Preferably, MMC + Na + and Preferably, DMC 2+ Ca 2+ is.

2. L 1A 2. The compound of claim 1, or a salt, hydrate or solvate thereof, wherein is a linker containing 10 or fewer atoms.

3. L 1A -O-, -S-, -SS-, -NR L1A -, -N=N-, -C(O)-, -C(O)NR L1A -, -OC(O)-, -C(O)O-, -OC(O)NR L1A -, -NR L1A C(O)-, -NR L1A C(O)O-, -NR L1A C(O)NR 4 -, -SC(O)-, -C(O)S-, -SC(O)O-, -OC(O)S-, -SC(O)NR L1A - and -NR L1A C(O)S—; where: R L1A is a hydrogen atom or C 1～3 is alkyl; Preferably, L 1A Ha-NR L1A C(O)—; Preferably, R L1A is a hydrogen atom, 3. A compound according to claim 1 or 2, or a salt, hydrate or solvate thereof.

4. L 1B is a linker containing 50 or fewer atoms; Preferably, L 1B is a linker containing 30 or fewer atoms; More preferably, L 1B is a linker containing 25 or fewer atoms; Most preferably, L 1B is a linker containing 10 or fewer atoms, The compound according to any one of claims 1 to 3, or a salt, hydrate or solvate thereof.

5. L 1B is -CH 2 -, 【Transformation 3】 selected from the group consisting of where: The wavy line is L 1A and an asterisk indicates binding to E 1A indicates a bond to x is an integer ranging from 1 to 12; Preferably, x is an integer ranging from 2 to 7; More preferably, x is an integer ranging from 2 to 4. The compound according to any one of claims 1 to 4, or a salt, hydrate or solvate thereof.

6. L 1B and E 1A together form residues selected from the group 【Chemistry 4】 where: y is an integer ranging from 1 to 13; Preferably, y is an integer ranging from 2 to 6; More preferably, y is an integer ranging from 2 to 4.

6. The compound according to any one of claims 1 to 5, or a salt, hydrate or solvate thereof.

7. The compound is selected from the group consisting of: 【Transformation 5】 The compound according to any one of claims 1 to 6, or a salt, hydrate or solvate thereof.

8. The compound is 【Transformation 6】 The compound according to any one of claims 1 to 7, or a salt, hydrate or solvate thereof, wherein

9. The compound is 【Transformation 7】 The compound according to any one of claims 1 to 8, or a salt, hydrate or solvate thereof, wherein

10. The compound is 【Transformation 8】 The compound according to any one of claims 1 to 9, or a salt, hydrate or solvate thereof, wherein

11. A composition comprising the compound according to any one of claims 1 to 10, or a salt, hydrate or solvate thereof, preferably said composition being a pharmaceutical composition.

12. (A1) A compound according to any one of claims 1 to 10, or a salt, hydrate or solvate thereof; and / or (A2) the composition according to claim 11; (B) Dienophile, or its salt, solvate, or hydrate wherein preferably the dienophile comprises a trans-cyclooctene residue; A combination with.

13. A compound according to any one of claims 1 to 10, or a salt, hydrate or solvate thereof; a composition according to claim 11; or a combination according to claim 12, for use as a pharmaceutical.

14. A compound according to any one of claims 1 to 10, or a salt, hydrate or solvate thereof, for use in treating a disease in a subject, wherein preferably the subject is a human; preferably the disease is cancer; a composition according to claim 11; or a combination according to claim 12.

15. 8. A method for preparing a compound according to claim 7, said method comprising: (a) reacting SM1a or SM1b with a reagent selected from the group consisting of SM2, SM3 and SM4; (b) when SM1a is used in step (a), subjecting the reaction product of step (a) to an oxidation reaction; (c) optionally subjecting the reaction product of step (a) or step (b) to a salt-forming reaction. The process includes the steps of: Here, SM1a, SM1b, SM2, SM3 and SM4 are as follows: 【Chemistry 9】 Preferably, in step (a), SM1a is used; Preferably, in step (b), the reaction product of step (a) is contacted with sodium nitrite. The method.

16. (ia) a compound according to any one of claims 1 to 10, or a salt, hydrate or solvate thereof; and / or (iia) The composition of claim 11 is a dienophile, or a salt, solvate, or hydrate thereof. a non-therapeutic method of reacting with The method comprises the step of contacting (ia) and / or (iia) with the dienophile, or a salt, solvate or hydrate thereof; Preferably, the non-therapeutic method is an in vitro method; and Preferably, the dienophile comprises a trans-cyclooctene residue. The non-therapeutic method.

17. In the click reaction, (a) a compound according to any one of claims 1 to 10, or a salt, hydrate, or solvate thereof; (b) the composition of claim 11; and / or (c) the combination according to claim 12 Non-therapeutic use of.

18. 1. A method of treating a disease in a subject, the method comprising: (a) a compound according to any one of claims 1 to 10, or a salt, hydrate, or solvate thereof; (b) the composition of claim 11; and / or (c) the combination according to claim 12 to said subject, Preferably, the subject is a human; Preferably, the disease is cancer. The method

19. 13. A method of using a compound according to any one of claims 1 to 10, or a salt, hydrate or solvate thereof; a composition according to claim 11; and / or a combination according to claim 12, in the manufacture of a medicament for the treatment of a disease in a subject, comprising: Preferably, the subject is a human; Preferably, the disease is cancer. The method.

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