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] This disclosure relates to tetrazines having improved properties. Compositions and combinations comprising the tetrazines of this disclosure, as well as methods for using and producing the tetrazines, are also provided. [Background technology]

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

[0003] Compound 2.1 and some of its properties are described in the following further publications: 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] While this ligation works well both in vitro and in vivo, identifying novel compounds that are optimal for clinical use remains a major research challenge. Therefore, it is desirable to identify novel tetrazines that possess one or more of the following overall favorable properties in both in vitro and in vivo conditions: good stability, good reactivity with transcyclooctenes (especially in vivo) and / or high payload release from transcyclooctenes (especially in vivo), low cell membrane permeability, low cytotoxicity, and low genotoxicity. [Overview of the project] [Problems that the invention aims to solve]

[0005] Therefore, there is a need for novel tetrazines that can solve one or more of the problems and / or requests mentioned above. [Means for solving the problem]

[0006] In one respect, the present disclosure relates to a compound, or a salt, hydrate, or solvate thereof, wherein the compound has a structure according to the following formula (1). [ka] Here, L 1A and L 1B is an independently selected linker; E 1A The group is selected from the following: [ka] Here, MMC + DMC is a monovalent metal cation; 2+ is a divalent metal cation; preferably, MMC + is Na + And; preferably, DMC 2+ is Ca2+ It 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, the composition is a pharmaceutical composition.

[0008] In yet another aspect, the present disclosure (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, relates to a combination with.

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

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

[0011] In a further aspect, still, the present disclosure (ia) a compound according to formula (1), or a salt, hydrate or solvate thereof; and / or, (iia) a composition according to the present disclosure with a dienophile, or a salt, solvate or hydrate thereof is a non - therapeutic method of reacting, 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 in the non-therapeutic method.

[0012] Furthermore, from a more detailed perspective, this disclosure relates to click response, (a) Compounds according to formula (1), or their salts, hydrates, or solvates; (b) Compositions relating to the present disclosure; and / or, (c) Combinations of the Disclosure Regarding its non-therapeutic use.

[0013] In a further respect, the present disclosure relates to a method for preparing the compound described in claim 7, the method being: (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), subject the reaction product of step (a) to an oxidation reaction; (c) Optionally, subject the reaction product of step (a) or step (b) to a salt formation reaction. This process includes, 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 respect, this disclosure relates to a method for treating a disease in the subject, and said method is (a) Compounds according to formula (1), or their salts, hydrates, or solvates; (b) Compositions relating to the present disclosure; and / or, (c) Combinations of the Disclosure The process includes administering the substance to the subject, Preferably, the subject is a human; preferably, the disease is cancer. Regarding the above method...

[0015] In a final respect, 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 pharmaceutical product for the treatment of a disease in a subject, preferably the subject being human; preferably the disease being cancer. [Modes for carrying out the invention]

[0016] Compound 2.1, described above, was first reported by Rossin et al., Angew. Chem. Int. Ed. 2010, volume 49, pages 3375-3378. In that report, the compound was used in relation to 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 conjugate and detect unreacted trans-cyclooctene, as reported by Rossin et al., Bioconjugate Chem. 2016, volume 27, pages 1697-1706; and Rossin et al., Nature Commun. 2018, volume 9, article 1484.

[0018] In addition, compound 2.1 possesses several properties that are of great interest for the in vivo release of a payload, typically a drug, attached to trans-cyclooctene. Compound 2.1 is more reactive to trans-cyclooctene than other tetrazines and has a good clearance rate (Rossin et al., Nature Commun. 2018, volume 9, article 1484, left column of page 6). Compound 2.1, like most bis-(2-pyridyl)-tetrazines, has a low release rate with most trans-cyclooctenines, although the release rate of bis-(2-pyridyl)-tetrazines is (almost) quantitative when trans-cyclooctenines with certain substituents are used (International Publication WO2020 / 256546, in particular, pages 294-296 of Example 5). Therefore, compound 2.1 is also a promising candidate for use in in vivo payload delivery. Consequently, compound 2.1 has been reported in the literature as the best-studied tetrazine for in vivo use and a promising candidate for clinical use.

[0019] However, the inventors have identified and, for the first time herein, improveable properties of compound 2.1. Such properties may occur 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 compared to when compound 2.1 is used for radiographic and / or radiotherapy.

[0020] Firstly, compound 2.1 was found to strongly inhibit the physiologically important enzymes cyclooxygenase (COX-1), acetylcholinesterase (ACES), monoamine oxidase B (MAO-B), and L-type dihydropyridine calcium channel. Each of these proteins is important for maintaining health in the subject, and undesirable inhibition of these enzymes and / or transporters can lead to side effects. Therefore, it is desirable to provide a tetrazine compound that exhibits low inhibitory activity against one or more of these enzymes and / or transporters. Based on this, it is desirable to provide tetrazines that show lower inhibition of one or more of these enzymes and / or transporters.

[0021] Secondly, 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 in which compound 2.1 can be safely used in vivo. Therefore, it is also desirable to provide novel tetrazines with a higher maximum tolerated dose.

[0022] Furthermore, the synthesis of compound 2.1 involves multiple steps, and it is desirable to provide a tetrazine that can be synthesized in fewer steps and / or by a simpler method.

[0023] Therefore, certain aspects and embodiments of this disclosure are based on qualified insight that, in a broad sense, tetrazines of formula (1) may satisfy one or more of the desired conditions described above.

[0024] In particular, the 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 of this specification.

[0025] Furthermore, the tetrazines of formula (1) typically have a relatively higher maximum tolerable dose in mice, as shown in Example 4 of this specification.

[0026] Furthermore, the tetrazines of formula (1) can be easily synthesized in fewer steps than required for compound 2.1, as shown in Example 2 of this specification.

[0027] Finally, in many embodiments, the tetrazines of formula (1) also exhibit even better in vitro and in vivo properties, as shown in Example 5 of this specification.

[0028] Preferred embodiments of formula (1) are further described below. All of these embodiments can be combined, provided they are not mutually exclusive.

[0029] Compound of formula (1)

[0030] The compounds of this disclosure conform to formula (1) described above. For ease of reference, the compounds of formula (1) are referred to below, for example, as “the compounds of this disclosure.” It will be understood that such notation includes salts, hydrates, or solvates of the compounds by such reference.

[0031] Regarding equation (1), MMC + Preferably, Na + , K + and Cu + Selected from the group consisting of the following. Most preferably, MMC+ is Na + That is the case.

[0032] Regarding equation (1), DMC 2+ 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+ Selected from the group consisting of the following. Most preferably, DMC 2+ is Ca 2+ That is the case.

[0033] In equation (1), L 1A and L 1B This is an independently selected linker.

[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 Selected from the group consisting of C(O)S-. More preferably, L 1A -NR L1A It is C(O)-, and most preferably L 1A It is -NHC(O)-.

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

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

[0037] Preferably, L 1B -CH2-, [ka] Selected from the group consisting of, Here, the wavy line is L 1A It indicates a bond to, and the asterisk is E 1A This indicates a connection to [the specified location].

[0038] L 1B With respect to x, x is an integer in the range of 1 to 12; preferably x is an integer in the range of 2 to 7; and more preferably x is an integer in the range of 2 to 4. Most preferably x is 3.

[0039] Preferably, L 1B and E 1A These components together form residues selected from the following groups. [ka]

[0040] With respect to equation (1), y is an integer in the range of 1 to 13; preferably, y is an integer in the range of 1 to 11; more preferably, y is an integer in the range of 2 to 9; more preferably, y is an integer in the range of 2 to 7; more preferably, y is an integer in the range of 2 to 6; more preferably, y is an integer in the range of 2 to 4. Most preferably, y is 3.

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

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

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

[0044] More preferably, the compound of formula (1) is as follows. [ka]

[0045] More preferably, the compound of formula (1) is as follows. [ka]

[0046] More preferably, the compound of formula (1) is as follows. [ka]

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

[0048] Composition of the present disclosure

[0049] This disclosure also relates to compositions 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 this disclosure further comprises a pharmaceutically acceptable carrier. If a salt of the compound of formula (1) is included in the composition of this disclosure, it is also preferable that a pharmaceutically acceptable salt is used.

[0050] Combinations of this disclosure

[0051] The disclosure also relates to (A1) compounds according to formula (1), or salts, hydrates or solvates thereof; and / or (A2) compositions according to the disclosure; and (B) dienophiles, or combinations thereof with salts, solvates or hydrates 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 dienophiles used herein comprise an eight-membered ring non-aromatic cyclic monoalkenylene residue comprising at least one allyl carbon and optionally one or more heteroatoms, preferably the heteroatoms being N, O, or Si. The eight-membered ring non-aromatic cyclic monoalkenylene residue may optionally be substituted. Preferably, the eight-membered ring non-aromatic cyclic monoalkenylene residue is a cyclooctene residue, more preferably a trans-cyclooctene (TCO) residue. Most preferably, the trans-cyclooctene residue is a whole carbon ring.

[0054] Preferably, at least five members, more preferably at least six, and most preferably at least seven members, of the eight-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 that is an entire carbocyclic, 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 three, more preferably at least four, and most preferably at least five CH2 residues. The substituted members are preferably N or C, more preferably C.

[0055] Preferably, the dienophile contains a payload released in response to a reaction with tetrazine of formula (1). The payload is preferably a drug or chelate residue containing a radionuclide for imaging. When the dienophile contains a payload, at least one allyl carbon of the eight-membered ring non-aromatic cyclic monoalkenylene residue is directly bonded to a cleavable bond. The cleavable bond contains 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 carbamate bond, ether bond and 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, any spacer is a self-immolative linker. Self-immolative linkers are well known to those skilled in the art.

[0056] Preferably, the dienophiles relating to this disclosure are 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 according to any embodiment thereof, more preferably those described on page 44, line 5 to the last line of page 74 of the same specification, or according to any embodiment thereof.

[0057] While not intended to be theoretically constrained, other substituents that may optionally be present on the 8-membered non-aromatic cyclic monoalkenylene residue are not considered to qualitatively affect the release of the payload in response to the diene. In other words, the payload is released in response to the diene regardless of whether other substituents are present on the 8-membered non-aromatic cyclic monoalkenylene residue. Several mechanisms for the release of the payload are known to those skilled in the art. For example, they are described on page 39 of International Publication WO2020 / 256546, particularly in Scheme 2 on page 9.

[0058] Dienophiles can be synthesized by those skilled in the art based on known synthetic routes of cyclooctene and corresponding rings containing one or more heteroatoms. Those skilled in the art are also aware of the diversity of cyclooctene derivatives that can be synthesized via ring-closing metathesis reactions using Grubbs catalysts. As mentioned above, TCO may contain one or more heteroatoms in the ring. This is readily apparent to those skilled in the art (e.g., International Publication WO2016 / 025480). For example, the presence of thioethers in TCO is referred to below: [Cere et al. J.Org.Chem.1980,45,261]. Also, for example, the -O-SiR2-O residue in TCO is referred to below: [Prevost et al. J.Am.Chem.Soc.2009,131,14182]. The allylic release group (R 48For TCO synthesis 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 the following formula (2). [ka] Here, L 1 C4~C4 is a straight or branched chain. 12 Alkylene, C3-C8 (hetero)cycloalkylene, C6-C 12 Arylene and C4~C 11 Selected from the group consisting of heteroarylenes; preferably, L 1 C4~C4 is a straight or branched chain. 12 It is alkylene, more preferably L 1 C4~C4 is a straight or branched chain. 10 It is an alkylene, most preferably L 1 It is a straight-chain C5-C6 alkylene; L 2a , L 2b and L 2d Each is independent of the other, -C(O)NL 2T -, -NL 2T C(O)-, -O-, -S-, -NL 2T -, -N=N- and -C(O)- are selected from the group; where L 2T is a hydrogen atom or a methyl atom, preferably L 2T is a hydrogen atom; L 2c The is selected from the group consisting of C1-C8 (hetero)alkantriyl, C5-C6 (hetero)alentriyl, C3-C7 cycloalkantriyl and C2-C7 heterocycloalkantriyl; preferably, L2c is C1-C8 (hetero)alkanetriyl, more preferably, L 2c is C1-C8 alkanetriyl, most preferably, L 2c is C4-C6 alkanetriyl; T 1 is -OT 1A 、a 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 、-O-C(O)T 1A 、-C(O)N(T 1A )2, -N(T 1A )2-CO-T 1A and -Si(T 1A )3 selected from the group consisting of; preferably, T 1 is -OT 1A ; most preferably, T 1 is -OH; each T 1A is independently selected from the group consisting of a hydrogen atom, (hetero)alkyl, (hetero)alkenyl, (hetero)alkynyl, (hetero)aryl and an amino acid residue; preferably, T 1A is a hydrogen atom or methyl, more preferably, T 1A is a hydrogen atom; T 2 is a bioconjugation moiety or -L 3 -C B group; preferably, the bioconjugation moiety is N-maleimidyl; L 3 is the residue of a bioconjugation moiety; preferably, L 3 is the residue of a maleimidyl group or the residue of an N-hydroxysuccinimidyl group; C BThis is selected from the group consisting of proteins, nucleic acids, peptides, carbohydrates, aptamers, lipids, low molecular weight organic compounds, polymers, LNA, PNA, amino acids, peptoids, chelate moieties, fluorescent dyes, phosphorescent dyes, organic particles, gels, cells, and combinations thereof; preferably, C B It is a protein, more preferably C B is an antibody or a bispecific antibody (diabody), and more preferably C B It is a bispecific antibody, most preferably C B AVP0458 consists of two monomers, each of which has an amino acid sequence according to sequence ID number: 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, most preferably an integer in the range of 23 to 25; and, R 48 is a detachable group, preferably R 48 is -O-CO-C A And here, C A This is the payload, preferably C A Preferably, the agent is a drug that binds to a -O-CO- residue via a secondary or tertiary nitrogen atom that is part of the drug to form a carbamate; preferably, the agent is monomethyl auristatin E (MMAE).

[0060] More preferably, the compound according to formula (2) is the compound according to the following formula (3). [ka] Here, x is an integer in the range of 4 to 12; preferably, x is an integer in the range of 4 to 8; more preferably, x is an integer in the range of 4 to 6.

[0061] In equations (2) and (3), T2 However, it is preferable to select from the following groups. [ka] Here, C B It is a protein.

[0062] More preferably, the dienophile is one of the following, or a salt, hydrate, or solvate thereof. [ka]

[0063] When used herein with respect to dienophiles in this disclosure, E 1 It is either -H or -CH3.

[0064] More preferably, the dienophile is one of the following, or a salt, hydrate, or solvate thereof. [ka] JPEG0007848417000018.jpg54170

[0065] More preferably, the dienophile is as follows: [ka] Here, C B AVP0458 is composed of two monomers, each of which has an amino acid sequence according to sequence ID number: 1; preferably, C B C B It is bonded to the malemidyl group via a sulfur atom that is part of the cysteine ​​group, preferably the sulfur atom is part of the cysteine ​​group.

[0066] Most preferably, the dienophile is one of the following, or a salt, hydrate, or solvate thereof. [ka] Here, C B AVP0458 is composed of two monomers, each of which has an amino acid sequence according to sequence ID number: 1; preferably, C B C B It is bonded to the malemidyl group via a sulfur atom that is part of the cysteine ​​group, preferably the sulfur atom is part of the cysteine ​​group.

[0067] In other preferred embodiments, the dienophile is a conjugate, or a salt, hydrate, or solvate thereof. Preferably, the conjugate is: [ka] Here, CJ is in the range of 1 to 12; where C B AVP0458 consists of two monomers, each of which has an amino acid sequence according to sequence ID number: 1; preferably, CJ is 2-10, more preferably 2.5-8, even more preferably 3-6, still more preferably 3.5-4, most preferably about 4; preferably, C B It is bonded to each maleidyl group via a sulfur atom, preferably the sulfur atom being part of cysteine.

[0068] More preferably, the conjugate is one of the following, or a salt, hydrate, or solvate thereof. [ka] JPEG0007848417000023.jpg131170 Here, CJ is in the range of 1 to 12; where C B AVP0458 consists of two monomers, each of which has an amino acid sequence according to sequence ID number: 1; preferably, CJ is 2-10, more preferably 2.5-8, even more preferably 3-6, even more preferably 3.5-4, most preferably about 4; preferably, CB It is bonded to each maleidyl group via a sulfur atom, preferably the sulfur atom being part of cysteine.

[0069] AVP0458

[0070] As used herein, AVP0458 refers to a TAG72-binding bispecific antibody obtained from a CC49 antibody. AVP0458 is a bispecific antibody composed of two monomers, each monomer having an amino acid sequence according to sequence ID number 1 below. Sequence ID number: 1 (Amino acid sequence of AVP0458 bispecific antibody monomer): SVQLQQSDAELVKPGASVKISCKASGYTFTDHAIHWVKQNPEQGLEWIGYFSPGNDDFKYNERFKGKATLTADKSSSTAYLQLNSLTSEDSAVYFCTRSLNMAYWGQGTSVTVSSGGGGSDIVMTQS C SS C PVSVGEKVTLSCKSSQSLLYSGNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLSISSVETEDLAVYYCQQYYSYPLTFGAGTKLVLKR

[0071] Here, the underlined part indicates cysteine, and when AVP0458 is itself a part of the dienophile of the Disclosure, the cysteine ​​preferably indicates a cysteine ​​modified with or conjugated to the dienophile of the Disclosure or a residue thereof.

[0072] Therefore, in sequence ID number 1, it is preferable that at least one of the underlined cysteines, more preferably both of the underlined cysteines, is modified with or bonded to a dienophile according to this disclosure. In other words, the sulfur atom of the underlined cysteine ​​is T as defined herein. 2 Preferably, it is bound to a residue, preferably, T 2This is a residue of the N-maleimidyl group.

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

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

[0075] For the non-therapeutic methods of disclosure, it is preferable 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 know suitable solvents for the reaction between tetrazine and dienophile. Preferably, the solvent includes water, and more preferably, the solvent is water.

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

[0077] Method for preparing the compound of formula (1)

[0078] This disclosure also relates to a method for preparing preferred compounds of formula (1). 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] If SM1a is used in step (a), then step (b) is performed 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 nitrate in the presence of an acid, preferably formic acid. If SM1b is used in step (a), then step (b) does not need to be performed.

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

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

[0082] While this disclosure describes specific embodiments herein, it is not limited to those embodiments and is limited only by the claims. When an indefinite or definite article is used to refer to a singular noun, such as "a" or "an," or "the," it encompasses the plural form of that noun unless otherwise specified.

[0083] As used herein and in the claims, the verb "including" and its conjugations are used in a non-restrictive sense, meaning that the item following the word is included, but not excluded from items not specifically mentioned.

[0084] Furthermore, the use of the indefinite articles "a" or "an" to refer to an element does not rule out the possibility that there are two or more of that element, unless the context explicitly requires that there be only one. Therefore, the indefinite articles "a" or "an" usually mean "at least one."

[0085] Therefore, the expression "apparatus comprising means A and B" should not be limited to an apparatus consisting solely of components A and B. In the context of the present invention, this means that the relevant components of the apparatus are only A and B.

[0086] The compounds disclosed herein may arise in various tautomers. Unless otherwise specified, the compounds disclosed herein are understood to include all tautomers. Where the structure of a compound is shown as a specific tautomer, unless otherwise specified, it should be understood that this disclosure is not limited to that specific tautomer.

[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 comprising an amine and a carboxylic acid, the amine may be protonated and the carboxylic acid may be deprotonated at the same time.

[0088] In some formulas, groups or substituents are indicated by letters, e.g., "A", "B", "X", "Y", and various (numbered) "R" groups. In addition, the number of repeating units is indicated by letters, e.g., -(CH2) n - can be denoted by n. The definitions of these letters should be read by referring to each expression, that is, in different expressions, these letters may have different meanings independently unless otherwise indicated.

[0089] In this specification, terms such as "alkyl" are used. The number of carbon atoms in these groups (excluding carbon atoms in any arbitrary substituents described later) can be indicated by prefixing such terms with a number (for example, "C1-C8 alkyl" means that the alkyl group may have 1 to 8 carbon atoms). For clarification, a butyl group substituted with an -OCH3 group is written as C4 alkyl because the carbon atoms in the substituent are not included in the carbon atom count.

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

[0091] The term "salt" refers to a compound formed when an acidic proton, typically an acid proton, is substituted with a cation, such as a metal cation or an organic cation. The term "salt" also refers to a compound formed when an amine is protonated. Where applicable, the salt is pharmaceutically acceptable, although this is not required for salts not intended for administration to a patient. For example, in a salt of a 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 as 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 may be derived from pharmaceutically acceptable inorganic salts or organic bases, and from pharmaceutically acceptable inorganic or organic acids.

[0093] "Medically acceptable salt" refers to a pharmaceutically acceptable salt of a compound, where such salt is derived from various 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, also includes salts of organic or inorganic acids, such as hydrochloride, hydrobromide, formate, tartrate, besylate, mesylate, acetate, maleate, oxalate, etc.

[0094] In this specification, the terms “residue” and “group” are to be used interchangeably when referring to a part of a molecule.

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

[0096] The base is, for example, -((R 51 )2-R 52 )2-(Here, R 51 and R 52 When indicated as (where represents a certain residue) or by a similar notation, each R 51 and R 52 Before the residue is selected, it is -R 51 -R 51 -R 52 -R 51 -R 51 -R 52 -It should be written as such, R 51 and R 52 It will become clear that you do not first select the residues and then write down the formula.

[0097] Examples

[0098] Example 1: General Method

[0099] All reagents, chemicals, materials, and solvents, including unlisted nitrile-initiated compounds, were obtained from commercial suppliers and used as received. All solvents were AR quality. Thin-layer chromatography (TLC) was performed on Kieselgel F-254 pre-coated silica plates. Column chromatography was performed using 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 The C-NMR spectrum is from Bruker Avance III HD. 1 Regarding H-NMR, 400 MHz and 13 For 1C-NMR, use a 100MHz spectrometer or JEOL( 1 ¹H-NMR was reported at 500 MHz and 298 K. Chemical shifts were reported in ppm from TMS to low field 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 connected 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 procedure was performed at 298K using a 3μF column, with an injection volume of 1–4μL, a flow rate of 0.2mL / min, and a gradient of acetonitrile (MeCN) in H2O (both containing 0.1% formic acid) (5%–100%, for 10 minutes, followed by a 3-minute 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-(pyridine-2-yl)-1,2,4,5-tetrazin-3-yl)pyridine-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 in Rossin et al., Angew. Chem. Int. Ed. 2010, 49, 3375~3378.

[0103] Example 2.2: 2,2'-((2-((carboxymethyl)(2-oxo-2-((6-(6-(pyridine-2-yl)-1,2-dihydro-1,2,4,5-tetrazin-3-yl)pyridine-3-yl)amino)ethyl)amino)ethyl)azanejyl)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 acid 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, turbid mixture was stirred at room temperature under an argon atmosphere for 5 hours. 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 value C 22 H 25 Observed value for N9O7: 527.19 Da [M+H] + 528.42Da, and [MH + ] - 526.50 Da.

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

[0106] The crude reaction mixture of compounds 2 and 3 was diluted with water (15 mL) and acidified by adding formic acid (0.2 mL). Sodium nitrite (400 mg, 5.79 mmol) was added, and the pink mixture was stirred in a sealed flask at room temperature for 1 hour. 1 M aqueous ammonium acetate solution (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, 5% MeCN / 0.1 M aqueous ammonium acetate gradient to 25%). The combined product fraction was lyophilized, redissolved in water (25 mL), lyophilized again, and redissolved in water. Formic acid (0.25 mL) was added to the pink solution, thereby precipitating the product. 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 centrifugation was repeated twice, then washed with MeCN, and centrifugation was repeated twice. The remaining pink solid was vacuum-dried to obtain 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 for N9O7: 525.17Da [M+H]+ 526.33 Da and [MH] - 524.42 Da.

[0107] Example 2.4 :2-({2-[bis(carboxylatomethyl)amino]ethyl}[({6-[6-(pyridine-2-yl)-1,2,4,5-tetrazin-3-yl]pyridine-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-H2O (pH approximately 9.0) and subsequently acidifying it to pH 6.4 with glacial acetic acid. To an acidic suspension of 2.4 (172 mg, 0.33 mmol; 10 mg / mL) of the compound in milliQ-H2O, CaCO3 (1.67 mL, 0.53 mmol of a homogeneous suspension at 32 mg / mL) was added dropwise. As the pH increased, tetrazine dissolved at pH 5.8, and the addition of CaCO3 was stopped at pH 6.5. Subsequently, 1.0 M sodium acetate (pH 6.4) was added to the tetrazine to finally obtain a 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 rinsing with milliQ-H2O (6 volumes), and then eluted with milliQ-H2O:MeOH (1:1) with the assistance of vacuum. The tetrazine-containing fractions were combined, reduced to 80% of their volume under vacuum, diluted with milliQ-H2O, and then lyophilized after micropore filtration. The lyophilized residue was redissolved in milliQ-H2O at 50 mg / mL, filtered again through micropore, and lyophilized to obtain 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 values ​​for CaN9NaO7: Composition: C (45.1%), Ca (6.8%), N (21.5%), Na (3.9%). Measured values: C (44.0%), Ca (7.2%), N (20.5%), Na (3.1%).

[0109] Example 2.5 :3-(2-(2-(3-oxo-3-((6-(6-((pyridine-2-yl)-1,2-dihydro-1,2,4,5-tetrazin-3-yl)pyridine-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 under an argon atmosphere at room temperature 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 under an argon atmosphere at room temperature for 90 minutes, and then washed twice with 0.5 M aqueous citrate (60 mL). The combined organic layers were dried over sodium sulfate, filtered, and concentrated. Crude product 2.6 was used without further purification. ESI-MS: m / z calculated value C 22 H 27 Observed value for N7O6: 485.20 Da [M+H] + 486.25Da and [MH + ] - 484.33Da.

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

[0112] Crude product 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 minutes, 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 solution to 30%). The combined product fraction was lyophilized to obtain 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 for N7O6: 483.19 Da [M+H] + 484.50 Da and [MH] - 482.33Da.

[0113] Example 2.7 :3-(2-{2-[2-({6-[6-(pyridine-2-yl)-1,2,4,5-tetrazin-3-yl]pyridine-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-H2O, NaHCO3 (13 μmol from a 0.13 mL, 8.4 mg / mL solution) was added dropwise. As the pH increased, the tetrazine dissolved at pH=4.5, and the addition of NaHCO3 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 at the top of the column and was rinsed with milliQ-H2O (6 times the volume), and then eluted with milliQ-H2O:MeOH (1:1) with the assistance of vacuum. The tetrazine-containing fractions were combined, reduced to 50% of their volume under vacuum, and lyophilized after micropore filtration. The freeze-dried residue was redissolved in milliQ-H2O at 15 mg / mL and then freeze-dried to obtain compound 2.8 as a homogeneous pink, fluffy solid.

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

[0116] A solution of compound 2.2 (168 mg, 0.66 mmol) in DMF (5 mL) was slowly added to 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 separated by centrifugation and precipitation. The precipitate was washed with MeCN (30 mL) and dried under reduced pressure to obtain 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 For N8O5, the observed value is 426.14 Da [M+H]. + 427.33Da and [MH] - 425.42Da.

[0117] Example 2.9: 2,2'-((2-oxo-2-((6-(6-((pyridine-2-yl)-1,2,4,5-tetrazin-3-yl)pyridine-3-yl)amino)ethyl)azanegiyl)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 then 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, subsequently washed with water (20 mL) and acetonitrile (25 mL), and dried under reduced pressure to obtain 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, the inhibition of several physiologically relevant target proteins by tetrazines (via enzymes and transporters) was tested in vitro. The enzymes tested were cyclooxygenase (COX-1), acetylcholinesterase (ACES), and monoamine oxidase (MAO-B), and the transporter tested was 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. Target proteins were mixed in appropriate buffer with tetrazine 2.1 (reference), 2.4, 2.5, 2.7, or 2.10 (final tetrazine concentration 10 μM). The final concentrations 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 appropriate incubation time at the appropriate temperature, the enzyme activity of the enzyme or, in the case of the transporter, ligand binding was tested according to standard procedures from the literature. From this residual enzyme activity or ligand binding, the percentage of inhibition was determined.

[0121] The results of these experiments are shown in Table 1 below. According to this, reference compound 2.1 typically exhibits a higher inhibitory effect on target protein activity compared to the tetrazines of formula (1), thereby showing lower inhibition. This advantageous effect of the compounds of formula (1) is particularly evident when considering the inhibition of COX-1, ACES, and calcium channel L-type dihydropyridine.

[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 males and females) were subjected to single doses of tetrazine 2.1 (reference), 2.4, 2.5, 2.7, or 2.10. Different groups were used for different dosages. For example, three different mouse groups were used for dosages of 8, 39, and 78 μmol / kg of reference compound 2.1. A control group of mice administered only with vehicle (phosphate-buffered saline (PBS), pH 7.4) without tetrazine was also used (vehicle control group).

[0125] The mice were handled in accordance with ethical guidelines. For example, based on the judgment of the veterinarian specializing in laboratory animals, the mice were housed in appropriate environments, their health was regularly monitored, they were given free access to certified mouse feed, and they had free access to water. Environmental controls for the animal housing rooms 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] The dose formulation was prepared as follows: Tetradicin 2.1 (reference), 2.4, 2.5, 2.7, or 2.10 was dissolved in PBS (pH 7.4) to prepare a storage solution for each tetrazine. The pH of the above solutions was adjusted to 7.17 using 2 M sodium carbonate. Further dilution of the tetrazine storage solution was performed in PBS (pH 7.4) until the desired concentration was achieved.

[0127] On day 1, the mice were administered the above-mentioned formulation (expressed in μmoles of tetrazine per kg of mouse body weight) intravenously. The mice were then observed for any signs of death for 72 hours after administration. The number of mice that died after 72 hours ("mortality rate") was calculated by dividing it by the original total number of mice.

[0128] The results of these experiments are summarized in Table 2 below. Table 2 clearly shows that the maximum permissible dose of reference compound 2.1 is approximately 39 μmol / kg. In contrast, for the tetrazines of formula (1) (2.4, 2.5, 2.7, and 2.10), the maximum permissible dose is at least 57 μmol / kg. The improvement is particularly significant for compounds 2.5, 2.7, and 2.10, which have an MTD value of at least 236 μmol / kg compared to reference compound 2.1.

[0129] [Table 2]

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

[0131] Compounds 2.4, 2.5, 2.7, and 2.10 of formula (1) were also tested for the following: i. Reactivity with trans-cyclooctene in vitro and / or in vivo, related to payload release from 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 study using LS174T colon cancer cells; v. Passive membrane permeability at pH 7.4 using a parallel artificial membrane permeability assay; and / or, vi. Genotoxicity (i.e., mutagenicity) in the presence or absence of S9 in Salmonella typhimurium strains TA98, TA100, TA1535, and 1537, and in Escherichia coli strain WP2 uvrA[pKM101].

[0132] All experiments were conducted using standard procedures known in the art. In all cases, the compounds of formula (1) showed the expected results. In particular, these results were almost the same as those obtained for compound 2.1 when compound 2.1 was subjected to the same tests i - vi. Furthermore, the present invention also provides the following: [Section 1] A compound, or a salt, hydrate, or solvate thereof, wherein the compound has a structure according to the following formula (1):

Chemical Structure

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Claims

1. A compound, or a salt, hydrate, or solvate thereof, wherein the compound has a structure according to the following formula (1): 【Chemistry 1】 Here, L 1A is -NHC(O)-; L 1B is -CH 2-, 【Chemistry 11】 Selected from the group consisting of; Here, the wavy line indicates a bond to L 1A, and the asterisk indicates a bond to E 1A; x is an integer in the range of 2 to 4; E 1A The group is selected from the following: 【Chemistry 2】 Here, MMC + is a monovalent metal cation; DMC 2+ It is a divalent metal cation, The aforementioned compound, or its salt, hydrate, or solvate.

2. MMC + Na + The compound according to claim 1, or a salt, hydrate, or solvate thereof.

3. DMC 2+ Ca 2+ The compound according to claim 1, or a salt, hydrate, or solvate thereof.

4. L 1B and E 1A together form residues selected from the group consisting of the following: 【Chemistry 4】 Here, y is an integer in the range of 2 to 4. The compound according to claim 1, or a salt, hydrate, or solvate thereof.

5. The aforementioned compound is selected from the group consisting of the following: 【Transformation 5】 The compound according to claim 1, or a salt, hydrate, or solvate thereof.

6. The aforementioned compound, 【Transformation 6】 The compound according to claim 1, or a salt, hydrate, or solvate thereof.

7. The aforementioned compound, 【Transformation 7】 The compound according to claim 1, or a salt, hydrate, or solvate thereof.

8. The aforementioned compound, 【Transformation 8】 The compound according to claim 1, or a salt, hydrate, or solvate thereof.

9. The compound is 【Chemistry 12】 The compound according to claim 1, or a salt, hydrate, or solvate thereof.

10. A composition comprising the compound described in claim 1, or a salt, hydrate, or solvate thereof.

11. The composition according to claim 10, wherein the composition is a pharmaceutical composition.

12. A compound according to claim 1, or a salt, hydrate, or solvate thereof, for use in the treatment of a disease in a subject, wherein the subject is human and the disease is cancer.

13. The composition according to claim 10, for use in the treatment of a disease in a subject, wherein the subject is a human and the disease is cancer.

14. A method for preparing the compound described in claim 5, wherein the method is: (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), subject the reaction product of step (a) to an oxidation reaction; (c) Optionally, subject the reaction product of step (a) or step (b) to a salt formation reaction. This process includes, Here, SM1a, SM1b, SM2, SM3, and SM4 are as follows: 【Chemistry 9】 The aforementioned method.

15. The method according to claim 14, wherein SM1a is used in step (a).

16. In click response, (a) The compound described in claim 1, or a salt, hydrate or solvate thereof; and / or (b) The composition according to claim 10; Non-therapeutic use of it.

17. An agent used for treating a disease in a subject, wherein the agent is (a) The compound described in claim 1, or a salt, hydrate or solvate thereof; and / or (b) The composition according to claim 10; Includes, Here, the subject is a human being; and the disease is cancer. The aforementioned agent.

18. A method for manufacturing a pharmaceutical product for the treatment of a disease in a subject, using the compound described in claim 1, or a salt, hydrate, or solvate thereof; and / or the composition described in claim 10, Here, the subject is a human being; and the disease is cancer. The aforementioned method.

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