Tetrazines with improved properties

Abstract

Disclosed herein include tetrazines that may have improved properties, for example, in clinical use. In certain embodiments, the compostions and methods herein may advantageously decrease inhibition of physiologically relevant enzymes as compared to other tetrazines. Moreover, the tetrazines of the disclosure, in some embodiments, may also show a higher maximum tolerated dose as compared other tetrazine candidates. Additionally, the tetrazines of the disclosure may in certain embodiments be readily synthesized and display further desired properties both in vitro and in vivo.

Description

Compositions of the disclosureThe disclosure also pertains to a composition comprising a compound according to Formula (1), or the salt, hydrate, or solvate thereof. Preferably, the composition is a pharmaceutical composition. Preferably, the composition of the disclosure further comprises a pharmaceutically acceptable carrier. It is also preferred that if a salt of a compound of Formula (1) is included in the composition of the disclosure, a pharmaceutically acceptable salt is used.Combinations of the disclosureThe disclosure also relates to a combination of (Al) a compound according to Formula (1), or the salt, hydrate, or solvate thereof; and / or (A2) a composition according to the disclosure; with (B) a dienophile or a salt, solvate, or hydrate thereof.Preferably, the combination of the disclosure is a kit. More preferably, the combination of the disclosure is a kit wherein (Al) and / or (A2) is / are physically separated from (B).Preferably, a dienophile as used herein comprises an eight-membered non-aromatic cyclic mono-alkenylene moiety comprising at least one allylic carbon, and optionally comprising one or more heteroatoms, preferably the heteroatom is N, O, or Si. The eightmembered non-aromatic cyclic mono-alkenylene moiety is optionally substituted. Preferably, the eight-membered non-aromatic cyclic mono-alkenylene moiety is a cyclooctene moiety, more preferably a Zraws-cyclooctene (TCO) moiety. Most preferably, the Zraws-cyclooctene moiety is an all-carbon ring.Preferably, at least five, more preferably at least six, and most preferably at least seven, members of the eight-membered non-aromatic cyclic mono-alkenylene moiety are notsubstituted. Preferably, the vinylic carbons are not substituted, i.e. are CH. As such, if the dienophile comprises a Zraws-cyclooctene moiety that is an all-carbon ring, then the trans-cyclooctene moiety preferably is a ring with one double bond between two CH moieties, i.e. -CH=CH-, and the ring further comprises at least three, more preferably at least four, and most preferably at least five CH2 moieties. The substituted members are preferably N or C, more preferably C.Preferably, the dienophile comprises a payload that is released upon reacting with a tetrazine of Formula (1). The payload is preferably a drug or a chelating moiety comprising a a radionuclide for imaging. If the dienophile comprises a payload, then at least one allylic carbon of the eight-membered non-aromatic cyclic mono-alkenylene moiety is directly linked 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 carbamate, ether, and ester bonds. Most preferably, the cleavable bond is a carbamate. The at least one S, N, NH, or O of the cleavable bond is part of the payload, or part of an optional spacer between the cleavable bond and the payload. Preferably, the optional spacer is a self-immolative linker. Self-immolative linkers are well-known in the art.Preferably, the dienophile in relation to the disclosure is as described in WO 2020 / 256546, which is incorporated herein by reference, in particular in accordance with any one of claims 1 to 4 thereof, or any embodiment thereof, more preferably as described at page 44, line 5, to page 74, last line, or any embodiment thereof.Without wishing to be bound by theory, it is believed that optionally present other substituents on the eight-membered non-aromatic cyclic mono-alkenylene moiety do not qualitatively influence the release of the payload upon reaction with a diene. In other words, the payload will be released upon reaction with a diene, regardless of whether other substituents are present on the eight-membered non-aromatic cyclic mono-alkenylene moiety. Several mechanisms for the release of the payload are known in the art. These are for example described in WO 2020 / 256546, in particular in Scheme 2 on page 39.Dienophiles can be synthesized by the skilled person on the basis of known synthesis routes to cyclooctenes and corresponding hetero atom(s)-containing rings. The skilled person further is aware of the wealth of cyclooctene derivatives that can be synthesized via the ring closing metathesis reaction using Grubbs catalysts. As mentioned above, the TCO possibly includes one or more heteroatoms in the ring. This is as such sufficiently accessible to the skilled person [e.g. WO2016025480], Reference is made, e.g., to the presence of a thioetherin TCO: [Cere et al. J. Org. Chem. 1980, 45, 261], Also, e.g., an - 0-SiR2-0 moiety in TCO: [Prevost et al. J. Am. Chem. Soc. 2009, 131, 14182], References to TCO syntheses wherein the allylic positioned leaving group (R4s) is an ether, ester, carbonate, carbamate or a thiocarbamate are: [Versteegen et al Angew. Chem. Int. Ed. 2018, 57, 10494], and [Steiger et al Chem Comm 2017, 53, 1378],Preferably, the dienophile is a compound or a salt, hydrate, or solvate thereof; wherein said compound has a structure according to Formula (2):X Formula (2); whereinL1 is selected from the group consisting of linear or branched C4-C12 alkylene, C3-C8 (hetero)cycloalkylene, C6-C12 arylene, and C4-C11 heteroarylene; preferably L1 is linear or branched C4-C12 alkylene, more preferably L1 is linear or branched C4-C10 alkylene, and most preferably L1 is linear C5-C6 alkylene;L2a, L2b, and L2d are each independently selected from the group consisting of -C(O)NL2T-, -NL2TC(O)-, -O-, -S-, -NL2T-, -N=N-, and -C(O)-; wherein L2T is hydrogen or methyl, preferably L2T is hydrogen;L2c is selected from the group consisting of Ci-Cs (hetero)alkanetriyl, C5-C6 (hetero)arenetriyl, C3-C7 cycloalkanetriyl, and C2-C7 heterocycloalkanetriyl; preferably L2c is Ci-Cs (hetero)alkanetriyl, more preferably L2c is Ci-Cs alkanetriyl, and most preferably L2c is C4-C6 alkanetriyl;T1 is selected from the group consisting of -OT1A, hydrogen, C2-C6 alkyl, Ce aryl, C4-C5 heteroaryl, C3-C6 cycloalkyl, C5-C12 alkyl(hetero)aryl, C5-C12 (hetero)arylalkyl, C4-C12 alkylcycloalkyl, -N(T1A)2, -ST1A, -SO3H, -C(O)T1A, -C(O)OT1A, -O-C(O)T1A -C(O)N(T1A)2, -N(T1A)2-CO-T1A, and -Si(T1A)3; preferably T1 is -OT1A; and most preferably T1 is -OH; each T1A is independently selected from the group consisting of hydrogen, (hetero)alkyl, (hetero)alkenyl, (hetero)alkynyl, (hetero)aryl, and an amino acid residue; preferably T1A is hydrogen or methyl, more preferably T1Ais hydrogen;T2 is a bioconjugation moiety or a group -L3-CB; preferably the bioconjugation moietyis N-maleimidyl; L3 is a residue of a bioconjugation moiety; preferably L3 is a residue of a maleimidyl moiety or a residue of an N-hydroxysuccinimidyl moiety;CB is selected from the group consisting of proteins, nucleic acids, peptides, carbohydrates, aptamers, lipids, small organic molecules, polymers, LNA, PNA, amino acids, peptoids, chelating moieties, fluorescent dyes, phosphorescent dyes, organic particles, gels, cells, and combinations thereof; preferably CB is a protein, more preferably CB is an antibody or a diabody, even more preferably CB is a diabody, and most preferably CB is AVP0458 consisting of two monomers, wherein each of the two monomers has an amino acid sequence according to SEQ ID NO: 1;y is an integer in a range of from 1 to 50; preferably y is an integer in a range of from 10 to 40, more preferably in a range of from 12 to 37, even more preferably in a range of from 15 to 35, more preferably still in a range of from 20 to 30, and most preferably in a range of from 23 to 25; andR48 is a releasable group, preferably R48 is -O-CO-CA; wherein CA is a payload, preferably CA is a drug; preferably the drug is linked to the moiety -O-CO- via a secondary or tertiary nitrogen atom that is part of the drug, forming a carbamate; preferably the drug is monomethyl auristatin E (MMAE).More preferably, the compound according to Formula (2) is a compound is accordingto Formula (3):from 4 to 12; preferably x is an integer in a range of from 4 to 8, more preferably x is an integer in a range of from 4 to 6.In Formula (2) and Formula (3), it is preferred that T2 is selected from the group consisting ofCB is a protein.Even more preferably, the dienophile is:As used herein in relation to dienophiles of the disclosure E1 is -H or -CH3.More preferably still, the dienophile is:Yet more preferably, the dienophile is:consisting of two monomers, wherein each of the two monomers has an amino acid sequence according to SEQ ID NO: 1; preferably CB is linked to the maleimidyl group via a sulfur atom5 that is part of CB, preferably the sulfur atom is part of a cysteine.Most preferably, the dienophile is:wherein CB is AVP0458 consisting of two monomers, wherein each of the two monomers has an amino acid sequence according to SEQ ID NO: 1; preferably CB is linked to the maleimidyl group via a sulfur atom that is part of CB, preferably the sulfur atom is part of acysteine.In other preferred embodiments, the dienophile is a conjugate, or a salt, hydrate, or solvate thereof. Preferably, the conjugate is:wherein CJ is in a range of from 1 to 12; wherein CB is AVP0458 consisting of two monomers, wherein each of the two monomers has an amino acid sequence according to SEQ ID NO: 1; preferably CJ is of from 2 to 10, more preferably of from 2.5 to 8, even more5 preferably of from 3 to 6, even more preferably still of from 3.5 to 4, and most preferably about 4; preferably CB is linked to each maleimidyl group via a sulfur atom, preferably the sulfur atom is part of a cysteine.More preferably, the conjugate is:thereof; wherein CJ is in a range of from 1 to 12; wherein CB is AVP0458 consisting of twomonomers, wherein each of the two monomers has an amino acid sequence according to SEQID NO: 1; preferably CJ is of from 2 to 10, more preferably of from 2.5 to 8, even more preferably of from 3 to 6, even more preferably still of from 3.5 to 4, and most preferably about 4; preferably CB is linked to each maleimidyl group via a sulfur atom, preferably thesulfur atom is part of a cysteine.AVP0458As used herein, AVP0458 refers to a TAG72-binding diabody derived from the CC49 antibody. AVP0458 is a diabody consisting of two monomers, each monomer having an amino acid sequence according to SEQ ID NO: 1:SEQ ID NO:1 (amino acid sequence of AVP0458 diabody monomer):SVQLQQSDAELVKPGASVKISCKASGYTFTDHAIHWVKQNPEQGLEWIGYFSPGNDD FKYNERFKGKATLTADKSSSTAYLQLNSLTSEDSAVYFCTRSLNMAYWGQGTSVTV SSGGGGSDIVMTQSCSSCPVSVGEKVTLSCKSSQSLLYSGNQKNYLAWYQQKPGQSP KLLIYWASTRESGVPDRFTGSGSGTDFTLSISSVETEDLAVYYCQQYYSYPLTFGAGT KLVLKRHerein, the underlining indicates the cysteines that are preferably modified with or linked to a dienophile of the disclosure or the remainder thereof if AVP0458 is itself part of the dienophile of the disclosure.Thus, in SEQ ID NO: 1 it is preferred that at least one of the underligned cysteines, more preferably both underlined cysteines, is modified with or linked to a dienophile according to the disclosure. In other words: it is preferred that the sulfur atom of the underlined cysteines is coupled to a moiety T2 as defined herein, preferably T2 is the residue of an N-maleimidyl group.Non-therapeutic methods using and uses for using compounds of Formula (1)In some embodiments, the disclosure pertains to non-therapeutic methods and non-therapeutic uses. Preferably, the dienophile used therein is as described in relation to the combination of the disclosure.For the non-therapeutic method of the disclosure it is preferred that the compound of Formula (1) (viz. (ia)), and / or the composition of the disclosure (viz. (iia)), and the dienophile are further contacted with a solvent. The skilled person is aware of suitable solvents for a reaction between a tetrazine and a dienophile. Preferably, the solvent comprises water, and more preferably the solvent is water.For the non-therapeutic use, the click reaction is preferably a bioorthogonal click reaction. Preferably, the click reaction is performed in vitro, although non-therapeutic reactions in vivo can be carried out as well.Methods for preparing compounds of Formula (1)The disclosure also relates to a method for preparing preferred compounds of Formula (1). In step (a) thereof, SMla or SMlb is reacted with a reagent selected from the group consisting of SM2, SM3, and SM4. Preferably, SMla is used in step (a).If SMla 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 oxidation. Preferably, in step (b) the oxidation is performed by adding sodium nitrate, optionally in the presence of an acid, preferably formic acid. If SMlb is used in step (a), then step (b) does not need to be carried out.In the entirely optional step (c) the reaction product of step (a) or step (b) is subjected to salt formation. The skilled person is aware of standard procedures to form salts fromcompounds comprising one or more carboxylic acid groups. Preferably, sodium and / or calcium salts are formed in step (c).For all of steps (a), (b), and (c), it is preferred that the reagents are contacted with a solvent. The skilled person is aware of suitable solvents to be used in said steps.The present disclosure is herein described with respect to particular embodiments, but the disclosure is not limited thereto but only by the claims. Where an indefinite or definite article is used when referring to a singular noun e.g. "a" or "an", "the", this includes a plural of that noun unless something else is specifically stated.The verb "to comprise", and its conjugations, as used in this description and in the claims is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded.In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there is one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".Thus, the scope of the expression "a device comprising means A and B" should not be limited to devices consisting only of components A and B. It means that with respect to the present disclosure, the only relevant components of the device are A and B.The compounds disclosed herein may occur in different tautomeric forms. The compounds disclosed herein are meant to include all tautomeric forms, unless stated otherwise. When the structure of a compound is depicted as a specific tautomer, it is to be understood that the disclosure of the present application is not limited to that specific tautomer, unless stated otherwise.Unless stated otherwise, the compounds disclosed herein and / or groups thereof may be protonated or deprotonated. It will be understood that it is possible that a compound may bear multiple charges which may be of opposite sign. For example, in a compound containing an amine and a carboxylic acid, the amine may be protonated while simultaneously the carboxylic acid is deprotonated.In several formulae, groups or substituents are indicated with reference to letters such as “A”, “B”, “X”, “Y”, and various (numbered) “R” groups. In addition, the number of repeating units may be referred to with a letter, e.g. n in -(CH2)n-. The definitions of these letters are to be read with reference to each formula, i.e. in different formulae these letters, each independently, can have different meanings unless indicated otherwise.Herein, reference is made to "alkyl", and the like. The number of carbon atoms that these groups have, excluding the carbon atoms comprised in any optional substituents as defined below, can be indicated by a designation preceding such terms (e.g. “Ci-Cs alkyl” means that said alkyl may have from 1 to 8 carbon atoms). For the avoidance of doubt, a butyl group substituted with a -OCH3 group is designated as a C4 alkyl, because the carbon atom in the substituent is not included in the carbon count.As used herein, alkyl groups are unsubstituted and have the general formula CnH2n+i and may be linear or branched. Examples of alkyl groups include methyl, ethyl, propyl, 2-propyl, t-butyl, 1-hexyl, 1-dodecyl, etc. Thus, C1.3 alkyl groups are methyl, ethyl, 1-propyl, and 2-propyl.The term "salt thereof’ means a compound formed when an acidic proton, typically a proton of an acid, is replaced by a cation, such as a metal cation or an organic cation and the like. The term "salt thereof’ also means a compound formed when an amine is protonated. Where applicable, the salt is a pharmaceutically acceptable salt, although this is not required for salts that are not intended for administration to a patient. For example, in a salt of a compound the compound may be protonated by 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.The term "pharmaceutically accepted salt” means a salt that is acceptable for administration to a patient, such as a mammal (salts with counter-ions having acceptable mammalian safety for a given dosage regime). Such salts may be derived from pharmaceutically acceptable inorganic or organic bases and from pharmaceutically acceptable inorganic or organic acids."Pharmaceutically acceptable salt" refers to pharmaceutically acceptable salts of a compound, which salts are derived from a variety of organic and inorganic counter ions known in the art and include, for example, sodium, potassium, calcium, magnesium, ammonium, tetraalkyl ammonium, etc., and when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, formate, tartrate, besylate, mesylate, acetate, maleate, oxalate, etc.It will be understood that herein, the terms “moiety” and “group” are used interchangeably when referring to a part of a molecule.It will be understood that when a heteroatom is denoted as -X(R’)2-, wherein X is the heteroatom and R’ is a certain moiety, then this denotes that two moieties R’ are attached to the heteroatom.It will be understood that when a group is denoted as, for example,-((Rs 1)2^52)2- or a similar notation, in which R51 and R52 are certain moieties, then this denotes that first, it should be written as -R51-R51-R52-R51-R51-R52- before the individual R51 and R52 moieties are selected, rather than first selecting moieties R51 and R52 and then writing out the formula.ExamplesExample 1: General methodsAll reagents, chemicals, materials and solvents were obtained from commercial sources and were used as received, including nitrile starting compounds that have not been described. All solvents were of AR quality. Analytical thin layer chromatography (TLC) was performed on Kieselgel F-254 precoated silica plates. Column chromatography was carried out on Screening Devices B.V. silica gel (flash: 40-63 pm mesh; normal: 60-200 pm mesh). Reverse phase chromatography was performed using a Eiichi Reveleris Cl8 column (80 g). 'H NMR and 13C NMR spectra were recorded on a Bruker Avance III HD (400 MHz for 1H NMR and 100 MHz for 13C NMR) spectrometer or a JEOL (500 MHz for 'H NMR) at 298 K. Chemical shifts are reported in ppm downfield from TMS at rt. 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-analyses were performed using a Alltech Alltima HP Cis 3p column using an injection volume of 1-4 pL, a flow rate of 0.2 mL / min and typically a gradient (5 % to 100 % in 10 min, held at 100 % for a further 3 min) of acetonitrile (MeCN) in H2O (both containing 0.1 % formic acid) at 298 K.Example 2: Synthesis of tetrazinesExample 2.1: Reference compound 2.12.1The synthesis of 2,2’,2”-(10-(2,40,44-Trioxo-44-((6-(6-(pyridine-2-yl)-l,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-l,4,7-triyl)triacetic acid (2.1) was reported in Rossin et al., Angew. Chern. Int. Ed. 2010, 49, 3375 -3378.Example 2.2: 2,2 '-((2-((Carboxymethyl)(2-oxo-2-(6-(6-(pyridin-2-yl)-l,2-dihydro-l, 2,4,5-tetrazin-3-yl)pyridin-3-yl)amino)ethyl)amino)ethyl)azanediyl)diacetic acid (2.3)o2.2                                         2.3Compound 2.2 was prepared according to Blackman et al. J. Am. Chern. 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 allowed to cool to room temperature and a solution of 2.2 (450 mg, 1.78 mmol) in DMSO (11 mL) was slowly added. The orange, hazy mixture was stirred at room temperature under an atmosphere of argon for 5 h.Subsequently water (0.1 mL) was added and the mixture was stirred for 30 min. 2.3 mixture was used without further purification. ESI-MS: m / z Calc, for C22H25N9O7 527.19 Da; Obs. [M+H]+ 528.42 Da and [M-H+]' 526.50 Da.Example 2.3: 2,2 '-((2-((Carboxymethyl)(2-oxo-2-(6-(6-(pyridin-2-yl)-l,2,4,5-tetrazin-3-yl)pyridin-3-yl)amino)ethyl)amino)ethyl)azanediyl)diacetic acid (2.4)2.32.4The crude reaction mixture of 2.3 was diluted with water (15 mL) and acidified by addition of formic acid (0.2 mL). Sodium nitrite (400 mg, 5.79 mmol) was added and the pink mixture was stirred in a closed flask at room temperature for 1 h. An aqueous solution of 1 Mammonium acetate (30 mL) was added and the pink suspension was centrifuged at 3000 rpm for 10 min. The clear, dark pink supernatant was isolated and purified by reversed-phase chromatography (Cl8 column, gradient of 5% MeCN / 0.1 M aqueous ammonium acetate to 25%). The combined product fractions were freeze dried, redissolved in water (25 mL), and again freeze dried and redissolved in water. To the pink solution was added formic acid (0.25 mL), which caused the product to precipitate. The suspension was centrifuged at 3000 rpm for 10 min, after which the clear, faint pink supernatant was discarded. The pink solid was washed with water (25 mL) and centrifuged, for two more times, and then washed with MeCN and centrifuged, for two more times. The remaining pink solid was dried in vacuo to give 486 mg (52% overall yield) of 2.4. 'H NMR (400 MHz, DMSO-d6): 8 12.41 (br.s, 3H), 10.78 (s, 1H), 9.13 (d, J = 2.5 Hz, 1H), 8.94 (dd, J = 4.8, 1.7 Hz, 1H), 8.62 (m, 2H), 8.52 (dd, J = 8.7, 2.5 Hz, 1H), 8.16 (td, J = 7.8, 1.8 Hz, 1H), 7.73 (dd, J = 7.8, 4.7 Hz, 1H), 3.53 (m, J = 10.4 Hz, 8H), 2.85 (s, 4H) ppm. °CNMR(101 MHz, DMSO-d6): 8 173.40, 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, 52.22 ppm. ESI-MS: m / z Calc, for C22H23N9O7 525.17 Da; Obs. [M+H]+ 526.33 Da and [M-H]’ 524.42 Da.Example 2.4: Calcium sodium 2-({2-[bis(carboxylatomethyl)amino] ethyl} [({6-[6-(pyridin-2-yl)-l,2,4,5-tetrazin-3-yl]pyridin-3-yl}carbamoyl)methyl]amino)acetate (2.5)2.4                       2.5IM Sodium acetate was prepared by dissolving sodium acetate trihydrate in milliQ-H20 (pH ca. 9.0) followed by acidification to pH=6.4 acidic with AcOH glacial. To an acidic suspension of 2.4 (172 mg, 0.33 mmol; 10 mg / mL) in milliQ-H20 was added dropwise CaCOs (1.67 mL, 0.53 mmol of a 32 mg / mL homogenous suspension). Upon increasing pH, the tetrazine dissolved at pH=5.8 and CaCOs addition was halted at pH=6.5. Subsequently, 1.0 M sodium acetate (pH=6.4) was added to the tetrazine to obtain a final 0.1 M sodium acetate concentration. The solution was applied to a Sep-Pak column (10 gr, Waters) forpurification (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-H20 (6 volumes) prior to elution with milliQ-H2O:MeOH (1:1) aided by vacuum pull. Tetrazine containing fractions were combined, reduced 80% by volume in vacuo, diluted with milliQ-H20, and freeze-dried after micropore filtration. The freeze-dried residue was redissolved in milliQ-H20 at 50 mg / mL, micropore filtered once more, and freeze-dried to obtain 2.5 as a homogenous pink fluffy powder. 1H NMR (400 MHz, D2O) 6 8.89 (dd, J= 2.6, 0.6 Hz, 1H), 8.79 (ddd, J= 4.8, 1.7, 0.9 Hz, 1H), 8.63 - 8.55 (m, 2H), 8.37 (dd, J= 8.7, 2.6 Hz, 1H), 8.16 (td, J= 7.8, 1.7 Hz, 1H), 7.74 (ddd, J = 7.7, 4.7, 1.1 Hz, 1H), 3.58 (s, 2H), 3.32 - 3.06 (m, 6H), 2.77 - 2.50 (m, 4H) ppm. 13C NMR (100 MHz, D2O) 6 179.9, 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, 54.8 ppm. ESI-MS cal. for C22H23N9O7 525.17 (excl. sodium calcium), found. M+H+ 526.25. Elemental anal, calcd for C22H2oCaN9NaO?: Composition: C (45.1%), Ca (6.8%), N (21.5%), Na (3.9%). Measured: C (44.0%), Ca (7.2%), N (20.5%), Na (3.1%).Example 2.5: 3-(2-(2-(3-Oxo-3-((6-(6-(pyridin-2-yl)-l, 2-dihydro-l, 2,4,5-tetrazin-3-yl)pyridin-3-yl)amino)propoxy)ethoxy)ethoxy)propanoic acid (2.6)2.2                                            2.63,3'-((Oxybis(ethane-2,l-diyl))bis(oxy))dipropionic acid (5.90 g, 23.6 mmol) was dissolved in chloroform (100 mL), and pyridinium / ?-toluenesulfonate (0.15 g, 0.597 mmol) and EDC HC1 (1.13 g, 5.92 mmol) were added. The solution was stirred at room temperature under an atmosphere of argon for 30 min. 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 an atmosphere of argon for 90 min, and then washed twice with 0.5 M aqueous citric acid (60 mL). The combined organic layers were dried with sodium sulfate, filtered, and concentrated. Crude 2.6 was used without further purification. ESI-MS: m / z Calc, forC22H27N7O6 485.20 Da; Obs. [M+H]+ 486.25 Da and [M-H+]' 484.33 Da.Example 2.6: 3-(2-(2-(3-Oxo-3-((6-(6-(pyridin-2-yl)-l, 2,4,5-tetrazin-3-yl)pyridin-3-yl)amino)propoxy)ethoxy)ethoxy)propanoic acid (2.7)2.72.6The 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 closed flask at room temperature for 30 min, and then diluted with water (90 mL). The mixture was filtered and purified by reversed-phase chromatography (Cl8 column, gradient of 15% MeCN / 0.1% aqueous formic acid to 30%). The combined product fractions were freeze-dried to give 2.7 as a pink, fluffy solid (1.32 g, 46% overall yield). XH NMR (400 MHz, CDCL): 8 9.61 (br.s, 1H), 8.97 (dt, J = 4.6, 1.4 Hz, 1H), 8.79 (m, J = 6.3, 2.6 Hz, 2H), 8.77 - 8.62 (m, 2H), 8.01 (td, J = 7.8, 1.8 Hz, 1H), 7.58 (ddd, J = 7.6, 4.7, 1.2 Hz, 1H), 3.85 (t, J = 5.5 Hz, 2H), 3.80 (t, J = 5.8 Hz, 2H), 3.74 (s, 4H), 3.72 - 3.59 (m, 4H), 2.76 (t, J = 5.5 Hz, 2H), 2.65 (t, J = 5.8 Hz, 2H) ppm. 13CNMR(101 MHz, CDCL): 6 174.18, 171.32, 163.53, 163.02, 150.91, 150.07, 143.56, 141.93, 138.55, 137.57, 127.36, 126.55, 125.27, 124.38, 70.70, 70.48, 70.20, 67.02, 66.78, 37.91, 35.40 ppm. ESI-MS: m / z Calc, for C22H25N7O6 483.19 Da; Obs. [M+H]+ 484.50 Da and [M-H]’ 482.33 Da.Example 2.7: Sodium 3-(2-{2-[2-({6-[6-(pyridin-2-yl)-1,2,4,5-tetrazin-3-yl]pyridin-3-yl}carbamoyl)ethoxy]ethoxy}ethoxy)propanoate (2.8)2.72.8To an acidic suspension of 2.7 (5.65 mg, 10 pmol, 10 mg / mL) in milliQ-H2O was added dropwise NaHCOs (0.13 mL, 13 pmol of a 8.4 mg / mL solution). Upon increasing pH, the tetrazine dissolved at pH=4.5 and NaHCOs addition was halted 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-H20 (6 volumes) prior to elution with milliQ-H20:Me0H (1:1) aided by vacuum pull. Tetrazine containing fractions were combined, reduced 50% by volume in vacuo and freeze-dried after micropore filter filtration. The freeze-dried residue was redissolved in milliQ-H20 at 15 mg / mL and freeze-dried to obtain 2.8 as homogenous pink fluffy solid.Example 2.8: 2,2 '-((2-Oxo-2-(66-(6-(pyridin-2-yl)-l, 2-dihydro-l, 2,4,5-tetrazin-3-yl)pyridin-3-yl)amino)ethyl)azanediyl)diacetic acid(2.9)2.2                                         2.9To a solution of 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 atmosphere of argon for 20 h. Water (30 mL) and formic acid (0.3 mL) were added to precipitate the product, which was isolated by centrifugation and decantation. The precipitate was washed with MeCN (30 mL), and dried in vacuo to yield 2.9 as orange powder (217 mg, 77% yield). 'H NMR (400 MHz, DMSO-d6): 8 12.63 (br.s, 2H), 10.71 (br.s, 1H), 8.95 (s, 1H), 8.88 (s, 1H), 8.84 (d, J = 2.4 Hz, 1H), 8.64 (dd, J = 4.8, 1.6 Hz, 1H), 8.19 (dd, J = 8.7, 2.5 Hz, 1H), 8.07 - 7.83 (m, 3H), 7.53 (ddd, J = 6.9, 4.8, 1.6 Hz, 1H), 3.58 (s, 4H), 3.55 (s, 2H) ppm. ESI-MS: m / z Calc, for CisHisNsOs 426.14 Da; Obs. [M+H]+ 427.33 Da and [M-H]’ 425.42 Da.Example 2.9: 2,2 '-((2-Oxo-2-(-6-(6-(pyridin-2-yl)-l, 2,4,5-tetrazin-3-yl)pyridin-3-yl)amino)ethyl)azanediyl)diacetic acid (2.10)O                              02.9                                 2.10Compound 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 atmosphere of argon, and turned pink and became clear within 10 min. After 1 h, a precipitate was formed again and the mixture was stored at 4°C for 1 h. The precipitate was isolated by centrifugation, washed with subsequently water (20 mL) and acetonitrile (25 mL), and dried in vacuo, to yield 2.10 as a pink powder (110 mg, 92%). 'H NMR (400 MHz, DMSO-d6): 6 12.53 (br.s, 2H), 10.92 (br.s, 1H), 9.07 (d, J = 2.5 Hz, 1H), 8.94 (d, J = 4.2 Hz, 1H), 8.66 (d, J = 8.7 Hz, 1H), 8.60 (d, J = 7.9 Hz, 1H), 8.47 (dd, J = 8.7, 2.6 Hz, 1H), 8.16 (td, J = 7.8, 1.8 Hz, 1H), 7.73 (ddd, J = 7.7, 4.7, 1.2 Hz, 1H), 3.62 (m, 6H) ppm. 13C NMR (101 MHz, DMSO-d6): 8 173.86, 172.77, 171.68, 163.55, 163.24, 151.09, 150.71, 144.69, 141.66, 138.33, 138.28, 127.08, 126.50, 125.46, 124.69, 59.58, 56.45 ppm. ESI-MS: m / z Calc, for Ci8Hi6N8O5 424.12 Da; Obs. [M+H]+ 425.33 Da and [M-H]’ 423.42 Da.Example 3: Inhibition of physiologically relevant target proteins by tetrazinesIn this Example, the inhibition of several physiologically relevant target proteins (viz. enzymes and a transporter) by tetrazines were tested in vitro. The enzymes tested were cyclooxygenase (COX-1), acetyl cholinesterase (ACES), and monoamine oxidase (MAO-B), and the tested transporter was calcium channel L-type, dihydropyridine. COX-1, ACES, and MAO-B from humans was used, while calcium channel L-type, dihydropyridine from rats 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 tetrazine 2.1 (reference), 2.4, 2.5, 2.7, or 2.10 (final concentration of tetrazine 10 pM) in appropriate buffers. The final concentrations for the respective target proteins 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 an appropriate temperature, the enzymatic activity of the enzyme or the ligand binding in case of the transporter wastested in accordance with standard procedures from literature. From this remaining enzymatic activity or ligand binding, the percentage of inhibition was determined.The results of these experiments are presented in Table 1 below. Therein, it is clear that reference compound 2.1 typically leads to a higher inhibition of the target protein activity as compared to the tetrazines of Formula (1), which show a lower inhibition. This advantageous effect of the compounds of Formula (1) is especially apparent when looking at the inhibition of COX-1, ACES, and calcium channel L-type, dihydropyridine.Table 1. Results from the enzyme inhibition studies.Tetrazine % COX-1 inhibition % MAO-B inhibition % ACES inhibition % Calcium channel L-type dihydropyridine inhibition 2.1 (reference) 59 36 44 44 2.4 53 20 11 10 2.5 18 9 -1 1 2.7 37 38 22 -17 2.10 14 22 5 -3 Example 4: maximum tolerated dose (MTD) of tetrazines in miceIn this Example, groups of Swiss albino mice (an equal number of male and female animals) were subjected to 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, for the doses 8, 39, and 78 pmol / kg of reference compound 2.1 three different groups of mice were used. Control groups were also used, wherein mice were only administered vehicle (phosphate-buffered saline (PBS) pH 7.4) without tetrazine (“vehicle control”).Mice were treated in line with ethical guidelines. For example, mice were maintained and monitored for good health at the discretion of a laboratory animal veterinarian, certified rodent diet was provided ad libitum, and water was available ad libitum. Environmental controls for the animal room were set to maintain a temperature of 22 to 25°C, humidity of 30-70% RH, and a 12-h light / 12-h dark cycle.The 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 separate stock solutions of each tetrazine. The pH of the above solutions was adjusted with 2M sodium carbonate to 7.17. Furtherdilutions of the tetrazine stock solutions were done with PBS pH 7.4 to achieve a desired concentration.Mice were dosed on day 1 by intravenously administering the dose formulation (expressed in pmol of tetrazine per kg body weight of the mouse). Thereafter, the mice were 5 observed up to 72 hours post-dose for any signs of mortality. The number of dead mice after 72 hours (“mortality”) was then divided by the original number of mice.The results of these experiments are summarized in Table 2 below. Therein, it is clear that the maximum tolerated dose of reference compound 2.1 is about 39 pmol / kg. By contrast, for the tetrazines of Formula (1) (2.4, 2.5, 2.7, and 2.10) the maximum tolerated dose is at10 least 57 pmol / kg. The improvement as compared to reference compound 2.1 is especially evident for compounds 2.5, 2.7, and 2.10 which have MTD’s of at least 236 pmol / kg.Table 2. Results from maximum tolerated dose studies in mice.Tetrazine Tetrazine dose (gmol / kg) No. of mortality vs. total no. of mice No tetrazine (vehicle control) N / A 0 out of 6 2.1 (reference) 8 0 out of 6 39 (MTD) 0 out of 6 78 4 out of 6 2.4 19 0 out of 6 57 (MTD) 0 out of 6 95 4 out of 4 2.5 85 0 out of 6 171 0 out of 6 512 (MTD) 0 out of 6 2.7 103 0 out of 6 207 0 out of 6 621 (MTD) 0 out of 6 2.10 118 0 out of 6 177 0 out of 6 236 (MTD) 0 out of 6 Example 5: general in vitro and in vivo propertiesCompounds 2.4, 2.5, 2.7, and 2.10 of Formula (1) were also tested for their:i. in vitro and / or in vivo reactivity towards Zraws-cyclooctenes in relation to payload release from said Zraws-cyclooctene;ii.   in vitro stability in mouse, rat, and human plasma;iii.   in vitro stability in presence of mouse, rat, and human microsomes;iv. cell toxicity using LS174T colon carcinoma cells;v. passive membrane permeability at pH 7.4 using a Parallel Artificial Membrane Permeability Assay; and / orvi. genotoxicity (i.e. mutagenecitiy) in S. typhimurium strains TA98, TA100, TA1535 and 1537; and in E. coli strain WP2 uvrA[pKM101] in presence or absence of S9.All experiments were carried out using standard protocols known in the art. In all cases, the compounds of Formula (1) showed desired results. In particular, these results were comparable to those obtained for 2.1 when 2.1 was subjected to the same tests i-vi.

Claims

Claims:

1.       A compound or a salt, hydrate, or solvate thereof; wherein saidcompound has a structure according to Formula (1):Formula (1);whereinL1A and L1B are independently selected linkers;E1A is selected from the group consisting of:_ + MMC+whereinMMC+ is a monovalent metal cation; optionally Na+;DMC2+ is a divalent metal cation; optionally Ca2+; and with the proviso that the compound of Formula (1) is notNHOOH- 12or a salt, solvate, or hydrate thereof.

2.       The compound according to claim 1, or the salt, hydrate, or solvatethereof; wherein L1A is a linker containing at most 10 atoms.

3.      The compound according to any one of the preceding claims, or thesalt, hydrate, or solvate thereof; wherein L1A is selected from the group consisting of -O-, -S-, -SS-, -NRL1A-,-N=N-, -C(O)-, -C(O)NRL1A-, -OC(O)-, -C(O)O-, -OC(O)NRL1A-, -NRL1AC(O)-, -NRL1AC(O)O-, -NRL1AC(O)NR4-, -SC(O)-, -C(O)S-, -SC(O)O-, -OC(O)S-, -SC(O)NRL1A-, and -NRL1AC(O)S-;whereinRL1A is hydrogen or C1-3 alkyl;optionally wherein L1A is -NRL1AC(O)-;optionally wherein RL1A is hydrogen.

4.      The compound according to any one of the preceding claims, or thesalt, hydrate, or solvate thereof; wherein L1B is a linker containing at most 50 atoms; at most 30 atoms; at most 25 atoms; or at most 10 atoms.

5.      The compound according to any one of the preceding claims, or thesalt, hydrate, or solvate thereof; wherein L1B is selected from the group consisting of -CH2-,*; andwhereinthe wiggly line indicates a bond to L1A and the asterisk a bond to E1A; x is an integer in a range of from 1 to 12; from 2 to 7; or from 2 to 4.

6.       The compound according to any one of the preceding claims,or the salt, hydrate, or solvate thereof; wherein L1B and E1A together form amoiety selected from the group consisting of:O\^)^ / OH' y UO_ + MMC+;whereiny is an integer in a range of from 1 to 13; a range of from 2 to 6; or a range of from 2 to 4.7.The compound according to any one of the preceding claims,or the salt, hydrate, or solvate thereof, wherein said compound is selected from the group consisting of:; and.

8.      The compound according to any one of the preceding claims, or thesalt, hydrate, or solvate thereof, wherein said compound is:

9.       The compound according to any one of claims 1 to 7, or the salt,hydrate, or solvate thereof, wherein said compound is:

10.      The compound according to any one of claims 1 to 7, or the salt,hydrate, or solvate thereof, wherein said compound is:

11.     A composition comprising a compound according to any one of claims1 to 10, or the salt, hydrate, or solvate thereof; or a pharmaceutical composition.

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

13.      The compound according to any one of claims 1 to 10, or thesalt, hydrate, or solvate thereof; the composition according to claim 11; or the combination according to claim 12; for use as a medicament.

14.      The compound according to any one of claims 1 to 10, or the salt,hydrate, or solvate thereof; the composition according to claim 11; or the combination according to claim 12; for use in the treatment of a disease in a non-human subject,optionally the disease is cancer.

15.     A method for preparing a compound according to claim 7,wherein said method comprises the steps of:(a) reacting SM1a or SM1b with a reagent selected from the group consisting of SM2, SM3, and SM4;(b) if SM1a is used in step (a), subjecting the reaction product of step (a) to oxidation;(c) optionally, subjecting the reaction product of step (a) or step (b) to salt formation;wherein SM1a, SM1b, SM2, SM3, and SM4 are:SM1aSM2OOHOOHONOSM3optionally in step (a) SM1a is used;optionally in step (b) the reaction product of step (a) is contacted withsodium nitrite.

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

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

18.     A method of treating a disease in a non-human subject, wherein saidmethod comprises the step of administering to said subject:(a) the compound according to any one of claims 1 to 10, or the salt, hydrate, or solvate thereof;(b) the composition according to claim 11; and / or(c) the combination according to claim 12; optionally the disease is cancer.

19.      Use of a compound according to any one of claims 1 to 10, or the salt,hydrate, or solvate thereof; the composition according to claim 11; and / or the combination according to claim 12; in the manufacture of a medicament for the treatment of a disease in a subject;wherein the disease is cancer.

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