Self-hydrolyzing maleimides for bioconjugation

Abstract

The present disclosure relates to novel compounds comprising an autohydrolyzing maleimide functional group, salts of these compounds, pharmaceutically acceptable salts of these compounds, methods of making these compounds, and methods of using these compounds for bioconjugation to antibodies. The present disclosure also relates to antibody-drug conjugates with improved stability under physiological conditions. The present disclosure also relates to antibody-drug conjugates comprising an autohydrolyzing maleimide functional group.

Description

[Technical Field]

[0001] The present disclosure relates to novel compounds comprising self-hydrolyzing maleimides for reaction with molecules containing thiol functional groups in thiol conjugation reactions. The present disclosure also relates to novel compounds comprising self-hydrolyzing maleimides for bioconjugation to antibodies or antibody portions. [Background technology]

[0002] Biomolecules, such as antibodies, often contain one or more thiol functional groups in cysteine ​​amino acids. Thiol functional groups derived from cysteine ​​amino acids are frequently used to attach drugs to antibodies, thereby forming antibody-drug conjugates (ADCs). The drug moiety of an ADC may contain a maleimide functional group, which reacts with a thiol functional group from a biomolecule in a bioconjugation reaction to form a thiosuccinimide bond between the antibody and the drug moiety. This thiosuccinimide bioconjugation reaction occurs rapidly under physiological conditions, achieves near-quantitative conjugation without a large excess of either species, and can be applied to a large number of molecules of biological interest. (See Nature Biotechnology, 2014, 32, 1059-1062.)

[0003] However, the formation of thiosuccinimide is reversible, and maleimide elimination occurs slowly under biologically relevant conditions. See Nature Biotechnology, 2014, 32, 1059-1062. Therefore, many ADCs experience measurable drug loss during prolonged circulation in the body, which can result in reduced activity. See Nature Biotechnology, 2014, 32, 1059-1062. However, maleimide functional group elimination can be mitigated if the thiosuccinimide instead undergoes a ring-opening hydrolysis reaction, resulting in a conjugation bond that no longer undergoes maleimide elimination. See Nature Biotechnology, 2014, 32, 1059-1062. See also Formula A.

[0004] [ka]

[0005] Formula A. Self-hydrolyzing maleimide functional group The amine adjacent to the maleimide group can induce hydrolysis of the thiosuccinimide bond after the antibody-maleimide conjugate is formed. This hydrolysis induced by the maleimide group is an example of autohydrolysis. The rate of hydrolysis generally correlates with the distance between the amine and the maleimide, with the closer the amine is to the maleimide, the faster the rate of autohydrolysis. See Nature Biotechnology, 2014, 32, 1059-1062.

[0006] Therefore, there is a need for novel compounds containing a maleimide functional group that (1) undergo bioconjugation reactions with thiol functional groups on biomolecules, (2) undergo rapid autohydrolysis of the formed thiosuccinimide, and (3) prevent loss of the maleimide functional group under desirable physiological conditions. See Formula A. Summary of the Invention

[0007] Disclosed herein are novel compounds containing a maleimide functional group that (1) undergo bioconjugation reactions with thiol functional groups, such as those found on biomolecules, and (2) undergo rapid autohydrolysis of the formed thiosuccinimide.

[0008] Also disclosed herein are autohydrolyzable maleimide-containing compounds that exhibit near-quantitative conjugation and autohydrolysis rates under desirable physiological conditions. Also disclosed herein are salts and / or pharmaceutically acceptable salts of these novel compounds. Also disclosed herein are methods for preparing antibody-maleimide conjugates and methods for making the autohydrolyzable maleimide-containing compounds.

[0009] As used herein, the formula

[0010] [ka] or a salt thereof, In the formula, R 1 teeth,

[0011] [ka] and R 2 PEG n , a bond and a peptide, or a combination thereof; n is 1 to 50; R 3 is a -phenyl-tetrazine group, or ligation group, where the tetrazine is optionally substituted with methyl.

[0012] As used herein, the formula

[0013] [ka] Also disclosed is a complex of, or a salt thereof, In the formula, R 1 teeth,

[0014] [ka] and R 2 PEG n , a bond and a peptide, or a combination thereof; n is 1 to 50; R 3 is a -phenyl-tetrazine group or linking group, wherein the tetrazine is optionally substituted with methyl; mAb is a monoclonal antibody S is a sulfur atom from a cysteine ​​residue on the monoclonal antibody.

[0015] As used herein, the formula

[0016] [ka] Also disclosed is a complex of the formula: 1 teeth,

[0017] [ka] and R 2 PEG n , a bond and a peptide, or a combination thereof; n is 1 to 50; R 3 is a -phenyl-tetrazine group or linking group, wherein the tetrazine is optionally substituted with methyl; mAb is a monoclonal antibody S is a sulfur atom from a cysteine ​​residue on the monoclonal antibody.

[0018] In one aspect, disclosed herein are compounds of Formula I or salts thereof, methods of making compounds of Formula I or salts thereof, and methods of using compounds of Formula I or salts thereof.

[0019] In one aspect, the present invention provides a compound of formula I

[0020] [ka] or a salt thereof, In the formula, R 1 teeth,

[0021] [ka] and R 2 PEG n , a bond and a peptide, or a combination thereof; n is 1 to 50; R 3 is a -phenyl-tetrazine group, or linking group, wherein the tetrazine is optionally substituted with methyl.

[0022] In one aspect, the present specification provides a compound:

[0023] [ka] Disclosed is 1-[3-[4-[3-[2-[2-[2-[2-[4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoyl]piperazin-1-yl]propyl]pyrrole-2,5-dione ("Compound A") or a salt thereof, which is selected from the group consisting of 1-(3-piperazin-1-ylpropyl)pyrrole-2,5-dione,

[0024] [ka] 3-[2-[2-[2-[2-[4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoic acid ("methyltetrazine-PEG4-acid");

[0025] [ka] It is obtained by combining with

[0026] In one aspect, provided herein is 1-[3-[4-[2-[4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl]acetyl]piperazin-1-yl]propyl]pyrrole-2,5-dione (“Compound B”), which has the formula:

[0027] [ka] or a salt thereof, (2,5-dioxopyrrolidin-1-yl)2-[4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl]acetate,

[0028] [ka] 1-(3-piperazin-1-ylpropyl)pyrrole-2,5-dione,

[0029] [ka]

[0030] and a base in the presence of a solvent to give compound B.

[0031] In one aspect, provided herein is N-[2-[2-(2,5-dioxopyrrol-1-yl)ethyl-methyl-amino]ethyl]-3-[2-[2-[2-[2-[4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanamide (“Compound C”), which has the following formula:

[0032] [ka] or a salt thereof, including 1-[2-[2-aminoethyl(methyl)amino]ethyl]pyrrole-2,5-dione trifluoroacetate,

[0033] [ka] Methyltetrazine-PEG4-acid,

[0034] [ka]

[0035] and an amide coupling reagent, and a base in a solvent.

[0036] In another embodiment, compound C or a salt thereof is (Z)-4-[2-[methyl-[2-[3-[2-[2-[2-[2-[4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoylamino]ethyl]amino]ethylamino]-4-oxo-but-2-enoic acid,

[0037] [ka] It is obtained by combining acetate and acetic anhydride.

[0038] In one aspect, provided herein is -[3-[2-[2-[2-[2-[4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoyl]piperazin-1-yl]ethyl]pyrrole-2,5-dione ("Compound D"):

[0039] [ka] 1-[2-[4 or a salt thereof is disclosed, which is 1-(2-piperazin-1-ylethyl)pyrrole-2,5-dione trifluoroacetate,

[0040] [ka] Methyltetrazine-PEG4-acid,

[0041] [ka] It is obtained by combining an amide coupling reagent and a base in a solvent.

[0042] In one aspect, provided herein is 2-[4-[2-(2,5-dioxopyrrol-1-yl)ethyl]-2-oxopiperazin-1-yl]-N-[2-[2-[2-[2-[4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenoxy]ethoxy]ethoxy]ethoxy]ethyl]acetamide (“Compound E”), which has the following formula:

[0043] [ka] or a salt thereof, 2-[4-[2-(2,5-dioxopyrrol-1-yl)ethyl]-2-oxo-piperazin-1-yl]acetic acid,

[0044] [ka] and 2-[2-[2-[2-[4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenoxy]ethoxy]ethoxy]ethoxy]ethanamine ("methyltetrazine-PEG4-amine");

[0045] [ka] It is obtained by combining an amide coupling reagent, an amide coupling solvent, and a base.

[0046] In one aspect, the present invention provides a compound of formula

[0047] [ka] or a salt thereof, In the formula, R 1 teeth,

[0048] [ka] and R 2 PEG n , a bond and a peptide, or a combination thereof; n is 1 to 50; R 3 is a -phenyl-tetrazine group or linking group, wherein the tetrazine is optionally substituted with methyl; mAb is a monoclonal antibody S is a sulfur atom from a cysteine ​​residue on the monoclonal antibody.

[0049] In one aspect, the present invention provides a compound of formula

[0050] [ka] or a salt thereof, In the formula, R 1 teeth,

[0051] [ka] and R 2 PEG n , a bond and a peptide, or a combination thereof; n is 1 to 50; R 3 is a -phenyl-tetrazine group or linking group, wherein the tetrazine is optionally substituted with methyl; mAb is a monoclonal antibody S is a sulfur atom from a cysteine ​​residue on the monoclonal antibody. DETAILED DESCRIPTION OF THE INVENTION

[0052] definition The term "salt" as used herein refers to a salt of a compound. The term "pharmaceutically acceptable salt" as used herein refers to a salt of a compound that is deemed acceptable for clinical and / or veterinary use. Examples of pharmaceutically acceptable salts and the general methodology for preparing them can be found in "Handbook of Pharmaceutical Salts: Properties, Selection and Use" P. Stahl, et al., 2nd Revised Edition, Wiley-VCH, 2011 and S. M. Berge, et al., "Pharmaceutical Salts", Journal of Pharmaceutical Sciences, 1977, 66(1), 1-19.

[0053] The compounds of Formula I can be readily converted into salts, such as pharmaceutically acceptable salts, and can be isolated as such salts. Salt formation can occur upon addition of a pharmaceutically acceptable acid to form an acid addition salt. Salts can also be formed simultaneously upon deprotection of nitrogen or oxygen, i.e., upon removal of a protecting group. Examples of reactions and conditions for salt formation can be found in Gould, PL, "Salt selection for basic drugs," International Journal of Pharmaceutics, 33:201-217 (1986); Bastin, RJ, et al., "Salt Selection and Optimization Procedures for Pharmaceutical New Chemical Entities," Organic Process Research and Development, 4:427-435 (2000); and Berge, SM, et al., "Pharmaceutical Salts," Journal of Pharmaceutical Sciences, 66:1-19, (1977). In one embodiment, acceptable counterions include trifluoroacetate or chloride.

[0054] [Table 1]

[0055] [Table 2-1]

[0056] [Table 2-2]

[0057] [Table 2-3]

[0058] [Table 2-4]

[0059] [Table 2-5]

[0060] [Table 2-6]

[0061] [Table 3]

[0062] In some embodiments, a combination of amide coupling reagents is used. In some embodiments, a combination of amide coupling solvents is used.

[0063] Formula I Compound and Salt Embodiments In one embodiment, R 1 teeth,

[0064] [ka] is.

[0065] In another embodiment, R 2 is PEG n and n is 1 to 10. In another embodiment, R 2 is PEG n and n is 2 to 6. In another embodiment, R 3 is a -phenyl-tetrazine group, wherein the tetrazine is optionally substituted with methyl. In another embodiment, the compound is

[0066] [ka]

[0067] In another embodiment, the compound is Compound A, or a salt thereof.

[0068] In another embodiment, compound A or a salt thereof is 1-(3-piperazin-1-ylpropyl)pyrrole-2,5-dione,

[0069] [ka] methyltetrazine-PEG4-acid,

[0070] [ka] In another embodiment, compound A or a salt thereof is obtained by mixing 1-(3-piperazin-1-ylpropyl)pyrrole-2,5-dione with 1-(3-piperazin-1-ylpropyl)pyrrole-2,5-dione in the presence of an amide coupling reagent, an amide coupling solvent, and a base.

[0071] [ka] Methyltetrazine-PEG4-acid,

[0072] [ka] In some embodiments, compound A or a salt thereof is obtained by mixing 1-(3-piperazin-1-ylpropyl)pyrrole-2,5-dione with 1-(3-piperazin-1-ylpropyl)pyrrole-2,5-dione in the presence of an amide coupling reagent and a solvent.

[0073] [ka] Methyltetrazine-PEG4-acid,

[0074] [ka]

[0075] and a methyl group in the presence of an amide coupling reagent and a base in a solvent. In one embodiment, Compound A can be obtained as a free base.

[0076] In another embodiment, a method of making Compound A or a salt thereof is disclosed. The method of making Compound A comprises reacting 1-(3-piperazin-1-ylpropyl)pyrrole-2,5-dione,

[0077] [ka] Methyltetrazine-PEG4-acid,

[0078] [ka] in the presence of an amide coupling reagent and a solvent.

[0079] In another embodiment, a maleic acid agent and tert-butyl 4-(3-aminopropyl)piperazine-1-carboxylate,

[0080] [ka] in acetic acid to give (Z)-4-[3-(4-tert-butoxycarbonylpiperazin-1-yl)propylamino]-4-oxo-but-2-enoic acid,

[0081] [ka] ("Preparation 1 Intermediate 1"). In another embodiment, the method of making Compound A also includes obtaining (Z)-4-[3-(4-tert-butoxycarbonylpiperazin-1-yl)propylamino]-4-oxo-but-2-enoic acid,

[0082] [ka] ("Preparation 1 Intermediate 1"), triethylamine, and a drying agent to combine tert-butyl 4-[3-(2,5-dioxopyrrol-1-yl)propyl]piperazine-1-carboxylate,

[0083] [ka] ("Preparation 1"). In another embodiment, the method of making Compound A also discloses that the drying agent comprises 4 Å molecular sieves. In another embodiment, the method of making Compound A also discloses that the drying agent comprises tert-butyl 4-[3-(2,5-dioxopyrrol-1-yl)propyl]piperazine-1-carboxylate,

[0084] [ka] ("Preparation 1") and a Boc deprotection reagent are combined to give 1-(3-piperazin-1-ylpropyl)pyrrole-2,5-dione,

[0085] [ka] The process also includes the step of obtaining

[0086] In another embodiment, the method of making compound A also discloses that the Boc deprotection reagent comprises trifluoroacetic acid in DCM, HCl in dioxane, HCl in methanol, or HCl in diethyl ether. In another embodiment, the method of making compound A also discloses that the Boc deprotection reagent comprises trifluoroacetic acid in DCM, and 1-(3-piperazin-1-ylpropyl)pyrrole-2,5-dione trifluoroacetate,

[0087] [ka] It also discloses that

[0088] In one embodiment, R 2 is a bond and R 3 is a -phenyl-tetrazine group, wherein the tetrazine is optionally substituted with methyl. In another embodiment, the compound is

[0089] [ka]

[0090] In another embodiment, the compound is Compound B, or a salt thereof.

[0091] In another embodiment, Compound B or a salt thereof is (2,5-dioxopyrrolidin-1-yl)2-[4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl]acetate,

[0092] [ka] and 1-(3-piperazin-1-ylpropyl)pyrrole-2,5-dione,

[0093] [ka] in the presence of a solvent and a base to give compound B.

[0094] In another embodiment, compound B or a salt thereof is N,N'-disuccinimidyl carbonate, methyltetrazine acid,

[0095] [ka] and a base in the presence of a solvent.

[0096] In another embodiment, the method for making Compound B or a salt thereof comprises reacting (2,5-dioxopyrrolidin-1-yl)2-[4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl]acetate,

[0097] [ka] and 1-(3-piperazin-1-ylpropyl)pyrrole-2,5-dione,

[0098] [ka] In another embodiment, the method comprises combining N,N'-disuccinimidyl carbonate and methyltetrazine acid,

[0099] [ka] and a base in the presence of a solvent to produce (2,5-dioxopyrrolidin-1-yl)2-[4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl]acetate

[0100] [ka] This includes obtaining

[0101] In one embodiment, R 1 teeth,

[0102] [ka] or a salt thereof. In another embodiment, R 2 is PEG n and n is 1 to 10. In another embodiment, R 2 is PEG n and n is 2 to 6. In another embodiment, R 3is a -phenyl-tetrazine group, wherein the tetrazine is optionally substituted with methyl. In another embodiment, the compound is

[0103] [ka]

[0104] In another embodiment, the compound is Compound C, or a salt thereof.

[0105] In another embodiment, compound C or a salt thereof is tert-butyl N-[2-[2-(2,5-dioxopyrrol-1-yl)ethyl-methyl-amino]ethyl]carbamate,

[0106] [ka] and a Boc-removal reagent in combination to give 1-[2-[2-aminoethyl(methyl)amino]ethyl]pyrrole-2,5-dione,

[0107] [ka] It is obtained by obtaining

[0108] In another embodiment, compound C or a salt thereof can be obtained when the Boc removal reagent comprises trifluoroacetic acid in DCM, HCl in dioxane, HCl in methanol, or HCl in diethyl ether. In another embodiment, compound C or a salt thereof can be obtained when the Boc removal reagent comprises trifluoroacetic acid in DCM and 1-[2-[2-aminoethyl(methyl)amino]ethyl]pyrrole-2,5-dione trifluoroacetate

[0109] [ka] can be obtained by giving

[0110] In another embodiment, compound C or a salt thereof is (Z)-4-[2-[methyl-[2-[3-[2-[2-[2-[2-[4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoylamino]ethyl]amino]ethylamino]-4-oxo-but-2-enoic acid,

[0111] [ka] In another embodiment, Compound C or a salt thereof, wherein the acetate comprises sodium acetate.

[0112] In another embodiment, a method of making Compound C or a salt thereof is disclosed. In another embodiment, the method of making Compound C or a salt thereof comprises: 1-[2-[2-aminoethyl(methyl)amino]ethyl]pyrrole-2,5-dione trifluoroacetate,

[0113] [ka] Methyltetrazine-PEG4-acid,

[0114] [ka] and an amide coupling reagent, and a base are combined in a solvent to produce N-[2-[2-(2,5-dioxopyrrol-1-yl)ethyl-methyl-amino]ethyl]-3-[2-[2-[2-[2-[4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanamide,

[0115] [ka] This includes obtaining

[0116] In another embodiment, the method of making Compound C, or a salt thereof, further comprises: tert-butyl N-[2-[2-(2,5-dioxopyrrol-1-yl)ethyl-methyl-amino]ethyl]carbamate,

[0117] [ka] and a Boc-removal reagent in combination to give 1-[2-[2-aminoethyl(methyl)amino]ethyl]pyrrole-2,5-dione,

[0118] [ka] This includes obtaining

[0119] In another embodiment, there is provided a method for making compound C or a salt thereof, wherein the Boc removal reagent comprises trifluoroacetic acid in DCM, HCl in dioxane, HCl in methanol, or HCl in diethyl ether. In another embodiment, there is provided a method for making compound C or a salt thereof, wherein the Boc removal reagent comprises trifluoroacetic acid in DCM, and 1-[2-[2-aminoethyl(methyl)amino]ethyl]pyrrole-2,5-dione trifluoroacetate,

[0120] [ka] get.

[0121] In another embodiment, the method of making compound C or a salt thereof comprises the step of: (Z)-4-[2-[methyl-[2-[3-[2-[2-[2-[2-[4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoylamino]ethyl]amino]ethylamino]-4-oxo-but-2-enoic acid;

[0122] [ka] In another embodiment, the method of making Compound C or a salt thereof, wherein the acetate comprises sodium acetate.

[0123] In another embodiment, the method of making Compound C, or a salt thereof, further comprises: tert-butyl N-[2-[2-aminoethyl(methyl)amino]ethyl]carbamate in acetic acid,

[0124] [ka] and a maleic acid agent in combination with (Z)-4-[2-[2-(tert-butoxycarbonylamino)ethyl-methyl-amino]ethylamino]-4-oxo-but-2-enoic acid,

[0125] [ka] The method includes the step of obtaining:

[0126] In another embodiment, there is provided a method of making compound C, or a salt thereof, wherein the maleic agent is selected from the group consisting of maleic anhydride, maleic acid, maleamic acid, unsubstituted maleimide, and N-(methoxycarbonyl)maleimide.

[0127] In one embodiment, R 1 teeth,

[0128] [ka] is.

[0129] In another embodiment, R 2 is PEG n and n is 1 to 10. In another embodiment, R 2 is PEG n and n is 2 to 6. In another embodiment, R 3is a -phenyl-tetrazine group, wherein the tetrazine is optionally substituted with methyl. In another embodiment, the compound is

[0130] [ka]

[0131] In another embodiment, the compound is Compound D, or a salt thereof.

[0132] In another embodiment, compound D or a salt thereof is 1-(2-piperazin-1-ylethyl)pyrrole-2,5-dione trifluoroacetate,

[0133] [ka] Methyltetrazine-PEG4-acid,

[0134] [ka] An amide coupling reagent and a base are combined in a solvent to produce 1-[2-[4-[3-[2-[2-[2-[2-[4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoyl]piperazin-1-yl]ethyl]pyrrole-2,5-dione,

[0135] [ka] It is obtained by obtaining

[0136] In another embodiment, compound D or a salt thereof is 1-(2-piperazin-1-ylethyl)pyrrole-2,5-dione trifluoroacetate,

[0137] [ka] Methyltetrazine-PEG4-acid,

[0138] [ka] An amide coupling reagent and N,N-diisopropylethylamine are combined in a solvent to give 1-[2-[4-[3-[2-[2-[2-[2-[4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoyl]piperazin-1-yl]ethyl]pyrrole-2,5-dione,

[0139] [ka] It is obtained by obtaining

[0140] In another embodiment, compound D or a salt thereof is tert-butyl 4-[2-(2,5-dioxopyrrol-1-yl)ethyl]piperazine-1-carboxylate,

[0141] [ka] and a Boc-removal reagent in a solvent. In another embodiment, compound D or a salt thereof, wherein the Boc-removal reagent comprises trifluoroacetic acid in DCM, HCl in dioxane, HCl in methanol, or HCl in diethyl ether. In another embodiment, compound D or a salt thereof, wherein the Boc-removal reagent comprises trifluoroacetic acid in DCM, 1-(2-piperazin-1-ylethyl)pyrrole-2,5-dione trifluoroacetate,

[0142] [ka] get.

[0143] In another embodiment, a method of making Compound D or a salt thereof is disclosed. In another embodiment, the method of making Compound D or a salt thereof comprises: 1-(2-piperazin-1-ylethyl)pyrrole-2,5-dione trifluoroacetate,

[0144] [ka] Methyltetrazine-PEG4-acid,

[0145] [ka] An amide coupling reagent and a base are combined in a solvent to produce 1-[2-[4-[3-[2-[2-[2-[2-[4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoyl]piperazin-1-yl]ethyl]pyrrole-2,5-dione,

[0146] [ka] This includes obtaining

[0147] In another embodiment, a method of making Compound D or a salt thereof is disclosed.

[0148] In another embodiment, the method of making compound D or a salt thereof comprises: 1-(2-piperazin-1-ylethyl)pyrrole-2,5-dione trifluoroacetate,

[0149] [ka] Methyltetrazine-PEG4-acid,

[0150] [ka] An amide coupling reagent and N,N-diisopropylethylamine are combined in a solvent to give 1-[2-[4-[3-[2-[2-[2-[2-[4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoyl]piperazin-1-yl]ethyl]pyrrole-2,5-dione,

[0151] [ka] This includes obtaining

[0152] In another embodiment, the method of making compound D or a salt thereof comprises: tert-butyl 4-[(2-(2,5-dioxopyrrol-1-yl)ethyl]piperazine-1-carboxylate,

[0153] [ka] and a Boc deprotection reagent in a solvent. In another embodiment, there is provided a method for making compound D or a salt thereof, wherein the Boc deprotection reagent comprises trifluoroacetic acid in DCM, HCl in dioxane, HCl in methanol, or HCl in diethyl ether. In another embodiment, there is provided a method for making compound D or a salt thereof, wherein the Boc deprotection reagent comprises trifluoroacetic acid in DCM, and 1-(2-piperazin-1-ylethyl)pyrrole-2,5-dione trifluoroacetate,

[0154] [ka] get.

[0155] In another embodiment, the method of making compound D or a salt thereof comprises: tert-butyl 4-(2-aminoethyl)piperazine-1-carboxylate,

[0156] [ka]

[0157] Furan-2,5-dione, acetic anhydride, and sodium acetate were added to the solvent to give tert-butyl 4-[2-(2,5-dioxopyrrol-1-yl)ethyl]piperazine-1-carboxylate,

[0158] [ka] This includes obtaining

[0159] In one embodiment, R 1 teeth,

[0160] [ka] is.

[0161] In another embodiment, R 2 is PEG n and n is 1 to 10. In another embodiment, R 2 is PEG n and n is 2 to 6. In another embodiment, R 3 is a -phenyl-tetrazine group, wherein the tetrazine is optionally substituted with methyl. In another embodiment, the compound is

[0162] [ka]

[0163] In another embodiment, the compound is Compound E, or a salt thereof.

[0164] In another embodiment, compound E or a salt thereof is 2-[4-[2-(2,5-dioxopyrrol-1-yl)ethyl]-2-oxo-piperazin-1-yl]acetic acid,

[0165] [ka] and methyltetrazine-PEG4-amine,

[0166] [ka] In another embodiment, compound E or a salt thereof is obtained by combining an amide coupling reagent and an amide coupling solvent and adding a base. In another embodiment, compound E or a salt thereof is obtained by combining an amide coupling reagent and an amide coupling solvent and adding a base. In another embodiment, compound E or a salt thereof is obtained by combining an amide coupling reagent and an amide coupling solvent and adding a base.

[0167] [ka] and methyltetrazine-PEG4-amine,

[0168] [ka] It is obtained by combining an amide coupling reagent and an amide coupling solvent and adding N,N-diisopropylethylamine.

[0169] In another embodiment, a method of making Compound E or a salt thereof is disclosed. In another embodiment, the method of making Compound E or a salt thereof comprises: 2-[4-[2-(2,5-dioxopyrrol-1-yl)ethyl]-2-oxo-piperazin-1-yl]acetic acid,

[0170] [ka] and methyltetrazine-PEG4-amine,

[0171] [ka] The method includes combining an amide coupling reagent and an amide coupling solvent and adding a base.

[0172] In another embodiment, the method of making Compound E or a salt thereof comprises: 2-[4-[2-(2,5-dioxopyrrol-1-yl)ethyl]-2-oxo-piperazin-1-yl]acetic acid,

[0173] [ka] and methyltetrazine-PEG4-amine,

[0174] [ka] The method includes combining an amide coupling reagent and an amide coupling solvent and adding N,N-diisopropylethylamine.

[0175] In another embodiment, when preparing Compound E or a salt thereof, 2-[4-[2-(2,5-dioxopyrrol-1-yl)ethyl]-2-oxo-piperazin-1-yl]acetic acid,

[0176] [ka] teeth, 2-[4-(2-aminoethyl)-2-oxopiperazin-1-yl]acetic acid,

[0177] [ka] and methyl 2,5-dioxo-2,5-dihydro-1H-pyrrole-1-carboxylate,

[0178] [ka] and a base in a solvent to produce 2-[4-[2-(2,5-dioxopyrrol-1-yl)ethyl]-2-oxo-piperazin-1-yl]acetic acid,

[0179] [ka] It is prepared by a process comprising obtaining

[0180] In another embodiment, when preparing Compound E or a salt thereof, 2-[4-(2-aminoethyl)-2-oxopiperazin-1-yl]acetic acid,

[0181] [ka] teeth, tert-butyl N-[2-(3-oxopiperazin-1-yl)ethyl]carbamate,

[0182] [ka] In another embodiment, when preparing Compound E or a salt thereof, the compound is prepared by a process comprising combining tert-butyl N-[2-(3-oxopiperazin-1-yl)ethyl]carbamate, sodium hydride, and tert-butyl bromoacetate in a solvent.

[0183] [ka] teeth, Piperazin-2-one, tert-butyl N-(2-oxoethyl)carbamate,

[0184] [ka] It is prepared by a process which includes combining acetic acid, and sodium triacetoxyborohydride.

[0185] In all of the above embodiments, it is understood that the base is a substance that reacts with the acid. In some embodiments, the base comprises a nitrogen-containing base. In some embodiments, the nitrogen-containing base comprises N,N-diisopropylethylamine, TEA, or pyridine. Examples of nitrogen-containing bases include, but are not limited to, diisopropylethylamine, TEA, and pyridine. More preferred nitrogen-containing bases include N,N-diisopropylethylamine and TEA. A single base or a mixture of two or more bases can be used.

[0186] Preferred embodiments of the amide coupling solvent include DCM, DMF, DMA, or NMP. A single solvent or a mixture of two or more solvents can be used.

[0187] In the above embodiments, the maleic agent is a compound comprising a dicarboxylic acid or an imide, where at least one of the carboxylic acids of the dicarboxylic acid is optionally substituted with an amide. In some embodiments, the maleic agent comprises maleic anhydride, maleic acid, maleamic acid, unsubstituted maleimide, or N-(methoxycarbonyl)maleimide.

[0188] R3 bond The distal methyltetrazine of the exemplified molecule is designed to conjugate to trans-cyclooctene (TCO)-functionalized biomolecules via inverse electron demand Diels-Alder (IEDDA) cycloaddition to form 4-linked 1-methyl-2,4a,5,6,7,8,9,10-octahydrocycloocta[d]pyridazine. 3 There are various other ligation techniques that can be used at the distal end of R3. There are various other ligation techniques that can be used at the distal end of R3. Examples of such ligation techniques are referred to herein as "linking groups." Linking groups that can be used include, but are not limited to: 1.Nitrogen-containing linking group Amide: Coupling with N-hydroxysuccinimide or other activated esters, optionally via amide coupling reagents ● Amines: Reductive amination with aldehydes / ketones • Imine: Condensation with aldehyde / ketone. ● Sulfonamide: sulfonyl fluoride mediated coupling ● Thiourea / urea:coupling with isothiocyanates / isocyanates 2. Sulfur-containing (typically thiol from cysteine) linking groups ● Disulfide bond: Coupling with pyridyl sulfides and other activating groups ● Aryl thioethers: Palladium-mediated coupling with aryl halides Native chemical ligation (NCL): Coupling of a C-terminal thioester with an N-terminal cysteine ● Thioether bond: ■ α-Thioether carbonyl: Coupling via α-halocarbonyl ■ β-Thioether sulfone: Coupling via monovinyl sulfone ■ β-Thioether ester: Coupling via acrylate ■ Thioether succinimide: Bonded via maleimide ■ Alternative traps to thiolates include bis-vinyl sulfones and vinyl sulfonamides. 3. Method for preparing linking groups ● Cycloaddition: ■ [3+2] Copper-Catalyzed Alkyne / Azide (Husgen Cycloaddition) ■ [3+2] Nitrone Cycloaddition to Olefins / Alkynes ● Strain-promoted alkyne / azide process: ■ Triazole products from azide-linked dibenzocyclooctyne (DBCO) ■ Octahydrocycloocta[d]pyridazine product from tetrazine coupled with TCO

[0189] Various modifying agents (R) are available for chemically modifying polypeptides such as antibodies to introduce novel active agents. 3 ) exists.

[0190] There are a variety of drug payloads that can be attached to R3, including, but not limited to, cytotoxins, immunostimulants, oligonucleotides, camptothecin analogs such as SN-38 and exatecan, maytansinoids such as maytansinoid DM1 and maytansinoid DM3, auristatins such as monomethyl auristatin E (MMAE) and monomethylauristatin F (MMAF), DNA damaging agents such as tubulysins and PBD dimers, and taxol derivatives such as docetaxel.

[0191] The present disclosure also relates to methods of making the compounds of formula I and salts thereof.

[0192] The present disclosure also relates to methods of using compounds of Formula I and salts thereof for antibody drug conjugation.

[0193] In one embodiment, the triple phosphate buffer comprises the following composition: 50 mM sodium phosphate monobasic monohydrate, 50 mM AMPSO, and 50 mM Na4P2O7.

[0194] The novel compounds containing maleimide functional groups can be conjugated to biological molecules such as antibodies, monoclonal antibodies, or antibody moieties.

[0195] Suitable biomolecules include monoclonal antibodies with engineered cysteines (or engineered cysteine-enabled mAbs) comprising IgG heavy and light chain constant regions, wherein the constant regions comprise at least one cysteine. In one embodiment, the mAb constant region comprises the following residues: C H Residue 124 in domain 1, C H Residue 157 in domain 1, C H Residue 162 in domain 1, C H Residue 262 in domain 2, C H Residue 375 in domain 3, C H Residue 373 in domain 3, C H Residue 397 in domain 3, C H Residue 415 in domain 3, C カッパ Residue 156 in the C domain カッパ Residue 171 in the C domain カッパ Residue 191 in the C domain カッパ Residue 193 in the C domain カッパ Residue 202 in the C domain カッパ In another embodiment, the constant region comprises at least one engineered cysteine ​​at one of residues 208 in the C domain. See U.S. Published Patent Application No. 2020 / 0155702, paragraphs

[0085] -

[0088] . In another embodiment, the constant region comprises at least one engineered cysteine ​​at one of the following residues: C H Residue 124 in domain 1, C H Residue 157 in domain 1, C H Residue 162 in domain 1, C H Residue 262 in domain 2, C H Residue 375 in domain 3, C H Residue 373 in domain 3, C H Residue 378 in domain 3, C H Residue 397 in domain 3, C H Residue 415 in domain 3, C カッパ Residue 156 in the C domain カッパ Residue 171 in the C domain カッパ Residue 191 in the C domain カッパResidue 193 in the C domain カッパ Residue 202 in the C domain カッパ It contains at least one engineered cysteine ​​at one of residues 208 in the domain.

[0196] In another embodiment, the mAb comprises an IgG heavy chain constant region, wherein the constant region is C H The cysteine ​​at residue 124 in the 1 domain and the C H Residues 157 and 162 in domain 1 and C H and a cysteine ​​at one but not all of residues 375 and 378 in the 3 domain. In another embodiment, the IgG heavy chain constant region is a human, mouse, rat, or rabbit IgG constant region. In another embodiment, the IgG heavy chain constant region is a human IgG1, human IgG2, or human IgG4 isotype, and even more particularly, is human IgG1 or human IgG4. In another embodiment, the IgG heavy chain constant region is a human IgG1 isotype. In another embodiment, the mAb comprises a human IgG1 heavy chain constant region, the constant region further comprising an isoleucine substituted at residue 247 and a glutamine substituted at residue 339. In another embodiment, the constant region comprises an isoleucine substituted at residue 247, a glutamine substituted at residue 339, and a glutamic acid substituted at residue 332. In another embodiment, the IgG heavy chain constant region is a human IgG4 isotype. In another embodiment, the mAb comprises a human IgG4 heavy chain constant region, wherein the constant region further comprises a proline substituted at residue 228, an alanine substituted at residue 234, and an alanine substituted at residue 235.

[0197] In another embodiment, the mAb comprises two heavy chain IgG constant regions, each IgG constant region comprising at least one cysteine. In another embodiment, each IgG constant region comprises the following residues: C H Residue 124 in domain 1, C H Residue 157 in domain 1, C H Residue 162 in domain 1, C H Residues 375 and C in domain 3H In another embodiment, the mAb comprises two heavy chain IgG constant regions, each IgG constant region comprising a cysteine ​​at one of residues 378 in the C3 domain. H cysteine ​​at residue 124 in the C1 domain and H Residues 157 and 162 in domain 1 and C H and a cysteine ​​at one but not all of residues 375 and 378 in the three domains. In another embodiment, each IgG constant region is a human, mouse, rat, or rabbit IgG. In another embodiment, each IgG constant region is a human IgG1, human IgG2, or human IgG4 isotype. In another embodiment, each IgG constant region is a human IgG1 or human IgG4. In another embodiment, each IgG heavy chain constant region is a human IgG1 isotype. In another embodiment, the two human IgG1 heavy chain constant regions further comprise an isoleucine substituted at residue 247 and a glutamine substituted at residue 339. In another embodiment, the constant region comprises an isoleucine substituted at residue 247, a glutamine substituted at residue 339, and a glutamic acid substituted at residue 332. In another embodiment, each IgG heavy chain constant region is a human IgG4 isotype. In another embodiment, the two human IgG4 heavy chain constant regions further comprise a proline substituted at residue 228, an alanine substituted at residue 234, and an alanine substituted at residue 235.

[0198] Unless otherwise specified herein, all references to residues appearing in the specification and examples are based on the EU index numbering system. See U.S. Published Patent Application No. 2020 / 0155702, paragraph

[0098] .

[0199] Preparation 0 Triple Phosphate Buffer Composition 13.8 grams of sodium phosphate monobasic monohydrate, 22.7 grams of APMSO, and 26.6 grams of NaPO were added to a 2 L flask and dissolved using 2 L of filtered deionized water. This 2 L solution was divided into 250 mL buffer solutions to produce solutions with various pHs ranging from 5 to 8 by adjusting the pH with either 1 M sodium chloride stock or 1 N sodium hydroxide.

[0200] Preparation 1 tert-Butyl 4-[(3-(2,5-dioxopyrrol-1-yl)propyl]piperazine-1-carboxylate

[0201] [ka] Maleic anhydride (789 mg, 7.97 mmol) was added to a solution of tert-butyl 4-(3-aminopropyl)piperazine-1-carboxylate (2.00 g, 7.97 mmol) in acetic acid (8 mL, 140 mmol). The mixture was stirred at room temperature for 12 minutes. A pale yellow solution was obtained. The volatiles were removed under reduced pressure to give a residue that was dried under vacuum. The crude intermediate (Z)-4-[3-(4-tert-butoxycarbonylpiperazin-1-yl)propylamino]-4-oxo-but-2-enoic acid,

[0202] [ka] ("Preparation 1 Intermediate 1") (2.72 g, 7.97 mmol) was dissolved in toluene (80 mL, 750 mmol). Triethylamine (5.6 mL, 40 mmol) and 4 Å molecular sieves (8.8 g) were added. The flask was fitted with a Dean-Stark trap and the mixture was heated at 120° C. for 48 h. After cooling to room temperature, the solid was removed by filtration and washed with DCM (40 mL). The volatiles were removed under reduced pressure to give a residue that was dried under vacuum. The thick residue was purified by normal phase chromatography ([10% MeOH / MTBE] / DCM). The title compound was isolated as a yellow flaky powder (353 mg, 1.09 mmol, 13.7% yield). MS m / z 324 (M+1).

[0203] Preparation 2 1-(3-piperazin-1-ylpropyl)pyrrole-2,5-dione, trifluoroacetic acid

[0204] [ka] Trifluoroacetic acid (2.0 mL, 26 mmol) was dissolved in DCM (5.0 mL) to tert-butyl 4-[3-(2,5-dioxopyrrol-1-yl)propyl]piperazine-1-carboxylate,

[0205] [ka] (“Preparation 1”) (353 mg, 1.09 mmol). A clear solution was obtained. The mixture was stirred for 2 hours, and the volatiles were removed under reduced pressure to give a residue that was dried under vacuum. The title compound was isolated as a white powder (325 mg, 0.964 mmol, 88.4% yield). MS m / z 224 (M+1).

[0206] Example 1 1-[3-[4-[3-[2-[2-[2-[2-[4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoyl]piperazin-1-yl]propyl]pyrrole-2,5-dione ("Compound A")

[0207] [ka] Under a nitrogen atmosphere, HATU (85 mg, 0.22 mmol) was dissolved in methyltetrazine-PEG4-acid (50 mg, 0.11 mmol) in DMF (0.5 mL),

[0208] [ka] and the resulting purple activated acid mixture was stirred at room temperature for 10 minutes. In a separate vessel, 1-(3-piperazin-1-ylpropyl)pyrrole-2,5-dione trifluoroacetic acid (27 mg, 0.080 mmol) in DMF (0.22 mL) was added.

[0209] [ka] and N,N-diisopropylethylamine (60 μL, 0.34 mmol) were mixed at room temperature. After 10 minutes, this mixture was added to the activated acid mixture via syringe. A purple solution resulted. After 90 minutes, additional 1-(3-piperazin-1-ylpropyl)pyrrole-2,5-dione (27 mg, 0.12 mmol) was added, and the mixture was stirred at room temperature for an additional 90 minutes, then placed in a −20° C. freezer and stored overnight. The reaction was diluted with water (20 mL) and DCM (40 mL). The layers were separated, and the organic layer was washed with water (2×20 mL), followed by saturated aqueous ammonium chloride, saturated aqueous sodium bicarbonate, and saturated aqueous sodium chloride. The organics were dried over magnesium sulfate, filtered, and concentrated to dryness to give a purple residue. The title compound was isolated as a purple residue (42.3 mg, 0.0659 mmol, 60.6% yield). MS m / z 642(M+1).

[0210] Example 2 1-[3-[4-[2-[4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl]acetyl]piperazin-1-yl]propyl]pyrrole-2,5-dione ("Compound B")

[0211] [ka] Under a N2 atmosphere, N,N'-disuccinimidyl carbonate (61 mg, 0.23 mmol) was added to methyltetrazine acid in dichloromethane (4.1 mL).

[0212] [ka] (50 mg, 0.21 mmol) and N,N-diisopropylethylamine (75 μL, 0.43 mmol) were added to a mixture of 2,5-dioxopyrrolidin-1-yl)-2-[4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl]acetate (99.7 mg, 0.305 mmol) was isolated as a purple solid. This material was dissolved in DCM (3.0 mL). Under a N atmosphere, this solution was slowly added to a solution of 1-(3-piperazin-1-ylpropyl)pyrrole-2,5-dione (49 mg, 0.22 mmol) and N,N-diisopropylethylamine (90 μL, 0.51 mmol) in THF (1.0 mL) and dichloromethane (3.0 mL). A purple solution resulted. After 2 min, additional 1-(3-piperazin-1-ylpropyl)pyrrole-2,5-dione (49 mg, 0.22 mmol) was added as a solid, and the mixture was stirred at room temperature for an additional 90 min. The volatiles were removed under reduced pressure to a residue that was absorbed onto silica gel (1 g) with DCM (10 mL). The volatiles were removed under reduced pressure to give a free-flowing residue, which was purified by chromatography (silica gel 12 g; 3 min DCM, then 6 min 5% [10% MeOH / MTBE] / DCM, then 5 min 10% [10% MeOH / MTBE] / DCM). The title compound was isolated as a purple foam (48 mg, 50% yield). MS m / z 436 (M+1).

[0213] Preparation 3 tert-Butyl N-[2-[2-(2,5-dioxopyrrol-1-yl)ethyl-methyl-amino]ethyl]carbamate

[0214] [ka] Maleic anhydride (564 mg, 5.75 mmol) was dissolved in acetic acid (4 mL, 140 mmol) to prepare tert-butyl N-[2-[2-aminoethyl(methyl)amino]ethyl]carbamate.

[0215] [ka] (1.25 g, 5.75 mmol). The mixture was stirred at room temperature for 7 hours. A pale yellow solution was obtained. The volatiles were removed under reduced pressure to give a residue which was dried under vacuum over the weekend. The crude intermediate, (Z)-4-[2-[2-(tert-butoxycarbonylamino)ethyl-methyl-amino]ethylamino]-4-oxo-but-2-enoic acid,

[0216] [ka] (1.81 g, 5.75 mmol) was dissolved in toluene (30 mL). Triethylamine (4 mL, 29 mmol) and 4 Å molecular sieves (3.0 g) were added. The mixture was heated to reflux for 6 h. After cooling to room temperature, the mixture was filtered through a pad of diatomaceous earth, and the pad was washed with DCM (3 × 5 mL). The filtrate was concentrated in vacuo to give a thick residue. The residue was dissolved in DCM (50 mL) and washed with water (20 mL). The organic layer was separated, dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The resulting residue was purified by normal phase chromatography (EtOAc / hexanes). The title compound was isolated as a pale yellow semi-solid (580 mg, 1.95 mmol, 33.9% yield). MS m / z 298 (M+1).

[0217] Example 3 N-[2-[2-(2,5-dioxopyrrol-1-yl)ethyl-methyl-amino]ethyl]-3-[2-[2-[2-[2-[4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanamide ("Compound C")

[0218] [ka] tert-butyl N-[2-[2-(2,5-dioxopyrrol-1-yl)ethyl-methyl-amino]ethyl]carbamate,

[0219] [ka] (75 mg, 0.25 mmol) was dissolved in trifluoroacetic acid (2.0 mL, 26 mmol) and DCM (4 mL) and stirred at room temperature for 1 h. The volatiles were removed under reduced pressure to give a residue which was dried under vacuum overnight to give 1-[2-[2-aminoethyl(methyl)amino]ethyl]pyrrole-2,5-dione trifluoroacetate,

[0220] [ka] was obtained as a white solid. 1-[2-[2-aminoethyl(methyl)amino]ethyl]pyrrole-2,5-dione trifluoroacetate,

[0221] [ka] Methyltetrazine-PEG4-acid,

[0222] [ka] (100 mg, 0.22 mmol) and HATU (200 mg, 0.51 mmol) were mixed together in DMF (2.0 mL) and THF (1.0 mL). To the solution, N,N-diisopropylethylamine (170 μL, 1.01 mmol) was added via syringe. The mixture was stirred at room temperature for 2 hours. The reaction was then diluted with DCM (50 mL) and washed with saturated aqueous ammonium chloride. The organic layer was separated, dried over sodium sulfate, filtered, and concentrated to dryness to give a dark red oil. Purification was performed by normal-phase flash column chromatography (MeOH / DCM). The first fraction afforded the title compound (74 mg, 0.25 mmol, 40.5% yield) as a red solid. MS m / z 616.2 (M+1). From the later fractions, the ring-opened product, (Z)-4-[2-[methyl-[2-[3-[2-[2-[2-[2-[4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoylamino]ethyl]amino]ethylamino]-4-oxo-but-2-enoic acid,

[0223] [ka] was obtained as a purple solid (65 mg, 26% yield). MS m / z 634 (M+1).

[0224] Example 3A N-[2-[2-(2,5-dioxopyrrol-1-yl)ethyl-methyl-amino]ethyl]-3-[2-[2-[2-[2-[4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanamide ("Compound C")

[0225] [ka] (Z)-4-[2-[methyl-[2-[3-[2-[2-[2-[2-[4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoylamino]ethyl]amino]ethylamino]-4-oxo-but-2-enoic acid (105 mg, 0.166 mmol),

[0226] [ka] was dissolved in acetic anhydride (2 mL). Sodium acetate (50 mg, 0.61 mmol) was then added. The mixture was heated at 70° C. for 4 h. Acetic anhydride was removed under reduced pressure. The residue was dissolved in DCM (50 mL) and washed with water (20 mL). The organic phase was separated, dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The resulting residue was purified by RP-HPLC (C18 column, A: 5% ammonium carbonate in water, B: ACN, 5-32% B over 6.5 min). The title compound was obtained as a purple solid (30 mg, 28% yield). MS m / z 616.6 (M+1).

[0227] Preparation 4 tert-Butyl 4-[2-(2,5-dioxopyrrol-1-yl)ethyl]piperazine-1-carboxylate

[0228] [ka] tert-butyl 4-(2-aminoethyl)piperazine-1-carboxylate,

[0229] [ka] (3.00 g, 13.1 mmol) was dissolved in acetic acid (6 mL). Furan-2,5-dione (1.28 g, 13.1 mmol) was added. Stirred at room temperature for 7 hours. The mixture was then stored in the refrigerator overnight. Most of the acetic acid was removed under vacuum (50 °C). Acetic anhydride (10 mL, 106 mmol) and sodium acetate (1.6 g, 20 mmol) were added. Heat to 80 °C for 2 hours. Most of the acetic anhydride was removed under vacuum (with toluene). The mixture was poured into saturated aqueous ammonium chloride (60 mL), adjusted to pH 5, and extracted with DCM (3 × 50 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to give the crude product as a dark oil. This material was purified by flash column chromatography (80 g silica gel, EtOAc / hexanes). The title compound was isolated (2.1 g, 6.8 mmol, 52% yield). MS m / z 310.3(M+1).

[0230] Example 4 1-[2-[4-[3-[2-[2-[2-[2-[4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoyl]piperazin-1-yl]ethyl]pyrrole-2,5-dione ("Compound D")

[0231] [ka] tert-butyl 4-[(2-(2,5-dioxopyrrol-1-yl)ethyl]piperazine-1-carboxylate,

[0232] [ka] (150 mg, 0.485 mmol) was dissolved in DCM (2 mL). Trifluoroacetic acid (1 mL, 13 mmol) was then added and stirred at room temperature for 1 hour. The solvent was removed under vacuum to dryness and further dried under high vacuum overnight to give the intermediate 1-(2-piperazin-1-ylethyl)pyrrole-2,5-dione trifluoroacetate,

[0233] [ka] obtained.

[0234] 1-(2-piperazin-1-ylethyl)pyrrole-2,5-dione trifluoroacetate, and methyltetrazine-PEG4-acid,

[0235] [ka] (130 mg, 0.283 mmol) was dissolved in DMF (2.0 mL) and THF (2 mL). HATU (380 mg, 0.969 mmol) was then added, followed by N,N-diisopropylethylamine (0.45 mL, 2.6 mmol). Stirred at room temperature for 2 h. Diluted with DCM (50 mL). Washed with saturated aqueous ammonium chloride (30 mL). The aqueous phase was pH 6. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to give the crude product as a red solid. Purification was performed by normal phase flash chromatography (12 g silica gel, 0% to 20% MeOH / EtOAc over 15 min). The title compound was isolated as a red solid (150 mg, 0.239 mmol, 49% yield). MS m / z 628.6 (M+1).

[0236] Preparation 5 tert-Butyl N-[2-(3-oxopiperazin-1-yl)ethyl]carbamate

[0237] [ka] Piperazin-2-one (510 mg, 5.10 mmol) and tert-butyl N-(2-oxoethyl)carbamate,

[0238] [ka] (0.91 g, 5.7 mmol) were mixed together in THF (20 mL) to form a clear solution. Acetic acid (0.6 mL, 10 mmol) was added and stirred at room temperature for 5 minutes. Sodium triacetoxyborohydride (3.33 g, 15.2 mmol) was added and stirred at room temperature under N2 for 4 hours. The mixture was then diluted with EtOAc (100 mL) and washed with brine and water. The organic layer was dried over anhydrous magnesium sulfate, filtered, and concentrated in vacuo. The resulting residue was purified by normal phase chromatography (0-50% MeOH / DCM). The title compound was isolated as a pale yellow solid (1.0 g, 4.1 mmol, 81% yield). MS m / z 243.9 (M+1).

[0239] Preparation 6 2-[4-(2-aminoethyl)-2-oxopiperazin-1-yl]acetic acid

[0240] [ka] tert-butyl N-[2-(3-oxopiperazin-1-yl)ethyl]carbamate,

[0241] [ka] (Preparation 5, 350 mg, 1.44 mmol) was dissolved in THF (3.0 mL). Sodium hydride (60% by weight in paraffin oil) (74 mg, 1.9 mmol) was added. The reaction was stirred at room temperature under N for 20 minutes. A solution of tert-butyl bromoacetate (364 mg, 1.87 mmol) in THF (0.5 mL) was then added via syringe, and stirring was continued for 3 hours. The mixture was then diluted with water (50 mL) and extracted with EtOAc (3 x 30 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to give the crude intermediate. The crude material was then dissolved in trifluoroacetic acid (3 mL, containing 30 μL of triisopropylsilane) and stirred at room temperature for 30 minutes. The majority of the trifluoroacetic acid was removed in vacuo. The residue was dissolved in methanol and passed through an SCX column (10 g), eluting with 2 M ammonia in methanol. The title compound was isolated as a dark solid (180 mg, 0.89 mmol, 62% yield). MS m / z 202 (M+1).

[0242] Preparation 7 2-[4-[2-(2,5-dioxopyrrol-1-yl)ethyl]-2-oxo-piperazin-1-yl]acetic acid

[0243] [ka] 2-[4-(2-aminoethyl)-2-oxopiperazin-1-yl]acetic acid,

[0244] [ka] (180 mg, 0.89 mmol) was dissolved in 1 M aqueous sodium bicarbonate (3 mL, 3 mmol) and cooled in an ice-water bath. Methyl 2,5-dioxo-2,5-dihydro-1H-pyrrole-1-carboxylate

[0245] [ka] (128 mg, 0.80 mmol) was added to the solution and stirred at 0° C. for 1 hour. The mixture was then diluted with water (10 mL) and washed with 3:1 chloroform:IPA (3×20 mL). The aqueous layer was adjusted to approximately pH 6 with 5 M aqueous HCl and extracted with 3:1 chloroform:IPA (3×20 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to give the title compound (35 mg, 0.12 mmol, 14% yield). MS m / z 282 (M+1). The aqueous phase was then frozen and lyophilized. This gave the crude title compound (200 mg, 0.36 mmol, 39%). MS m / z 282 (M+1).

[0246] Example 5 2-[4-[2-(2,5-dioxopyrrol-1-yl)ethyl]-2-oxopiperazin-1-yl]-N-[2-[2-[2-[2-[4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenoxy]ethoxy]ethoxy]ethoxy]ethyl]acetamide ("Compound E")

[0247] [ka] 2-[4-[2-(2,5-dioxopyrrol-1-yl)ethyl]-2-oxo-piperazin-1-yl]acetic acid,

[0248] [ka] (80 mg, 0.14 mmol), methyltetrazine-PEG4-amine,

[0249] [ka] (35 mg, 0.092 mmol) and HATU (71 mg, 0.18 mmol) were dissolved in DMF (1.0 mL, 13 mmol) and THF (1 mL) and stirred at room temperature for 5 min. N,N-diisopropylethylamine (80 μL, 0.46 mmol) was then added to the solution and stirred at room temperature for 1 h. The mixture was then diluted with water (50 mL) and extracted with 3:1 chloroform:IPA (2 × 20 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to give a dark red oil. Purification was performed twice by normal-phase flash chromatography (0–20% methanol / DCM). Compound E (10 mg, 0.015 mmol, 17% yield) was isolated. MS m / z 627.5 (M+1).

[0250] Example 6 Linker autohydrolysis evaluation Linker autohydrolysis was assessed by incubating the linkers in triple phosphate buffers with corresponding pH values ​​of 6.0, 6.5, 7.0, 7.5, and 8.0. Lyophilized linker powder was solubilized in 100% DMSO to create an 80 mM stock solution. Each linker was incubated in the buffer at a final linker concentration of 1.5 mM, and succinimide hydrolysis was measured at room temperature for 1 hour. The effect of pH on linker autohydrolysis was assessed by reverse-phase HPLC mass spectrometry at 0.6, 4, and 20 hour intervals. Table 4 provides the percentage hydrolysis at 0.6, 4, and 20 hour intervals. "nd" = no data.

[0251] Scheme 1: Maleimide autohydrolysis

[0252] [ka]

[0253] [Table 4]

[0254] Example 7 Linker conjugation evaluation The conjugation of these linkers via maleimide thiol chemistry was evaluated using a monoclonal antibody (mAb) with a site-specifically engineered cysteine. The mAb was diluted to 10 mg / mL in the above-mentioned phosphate buffers at pH 6.0, 6.5, 7.0, 7.5, and 8.0. Each of the linkers was added to the mAb at 20 molar equivalents and incubated at room temperature for 1 hour to allow conjugation. After the 1-hour incubation, excess linker was removed by desalting the solution back into buffers at pH 6.0, 6.5, 7.0, 7.5, and 8.0 using a spin desalting column using the standard manufacturer's protocol. The effect of pH on linker conjugation to the mAb and post-conjugation hydrolysis of the linker was assessed over time by reverse-phase LCMS.

[0255] Reverse-phase HPLC-mass spectrometry was used to assess linker conjugation to mAbs and post-conjugation hydrolysis of the linker. For this analysis, 10 μL of 1 mg / mL antibody-linker conjugate was prepared. Partial reduction was performed by adding 1.5 μL of 100 mM TCEP and 1 μL of 1 M Tris-HCl pH 8.0 to each sample and incubating at 37°C for 30 minutes. The antibody-linker conjugates were analyzed on days 0 and 3. The samples were pH-adjusted to pH 8.0 with 1 M Tris-HCl.

[0256] Table 5 provides the percentage conjugation rate and percentage post-conjugation hydrolysis rate at the 0 and 3 day intervals.

[0257] Scheme 2: Maleimide conjugation followed by autohydrolysis

[0258] [ka]

[0259] [Table 5]

Claims

1. formula 【Chemistry 1】 or a salt thereof (In the formula, R １ teeth, 【Chemistry 2】 and R ２ is PEG ｎ , a bond and a peptide, or a combination thereof; n is 1 to 50; R ３ is a -phenyl-tetrazine group, or linking group, said tetrazine optionally substituted with methyl).

2. R １ teeth, 【Transformation 3】 2. The compound of claim 1, wherein:

3. R ２ is PEG ｎ where n is 1 to 10, preferably n is 2 to 6, and more preferably R ３ The compound of claim 1 or 2, wherein is a -phenyl-tetrazine group, said tetrazine being optionally substituted with methyl.

4. The compound is 【Chemistry 4】 The compound according to any one of claims 1 to 3, which is a compound selected from the group consisting of benzophenone-3, benzophenone-4, benzophenone-5, benzophenone-6, benzophenone-7, benzophenone-8, benzophenone-9, benzophenone-10, benzophenone-11, benzophenone-12, benzophenone-13, benzophenone-14, benzophenone-15, benzophenone-1

5. R ２ The compound of claim 1 , wherein is a bond.

6. R ３ The compound of claim 5, wherein is a -phenyl-tetrazine group, said tetrazine optionally substituted with methyl.

7. The compound is 【Transformation 5】 or a salt thereof.

8. R １ teeth, 【Transformation 6】 2. The compound of claim 1, wherein:

9. R ２ is PEG ｎ where n is 1 to 10, preferably n is 2 to 6, and more preferably R ３ The compound of claim 8, wherein is a -phenyl-tetrazine group, said tetrazine optionally substituted with methyl.

10. The compound is 【Transformation 7】 or a salt thereof.

11. R １ teeth, 【Transformation 8】 2. The compound of claim 1, wherein:

12. R ２ is PEG ｎ where n is 1 to 10, preferably n is 2 to 6, and more preferably R ３ 12. The compound of claim 11, wherein is a -phenyl-tetrazine group, said tetrazine optionally substituted with methyl.

13. The compound is 【Chemistry 9】 or a salt thereof.

14. R １ teeth, 【Chemistry 10】 2. The compound of claim 1, wherein:

15. R ２ is PEG ｎ where n is 1 to 10, preferably n is 2 to 6, and more preferably R ３ 15. The compound of claim 14, wherein is a -phenyl-tetrazine group, said tetrazine optionally substituted with methyl.

16. The compound is 【Chemistry 11】 16. The compound of any one of claims 1, 14, or 15, which is:

17. 1. A method for preparing an antibody-maleimide conjugate, comprising:

17. A method comprising combining a monoclonal antibody (mAb) comprising at least one cysteine ​​and a compound of any one of claims 1 to 16, or a salt thereof, to form a reaction mixture.

18. 18. The method of claim 17, wherein the cysteine ​​is an engineered cysteine.

19. formula 【Chemistry 12】 or a salt thereof for preparing a pharmaceutical preparation, (In the formula, R １ teeth, 【Chemistry 13】 and R ２ is PEG ｎ , a bond and a peptide, or a combination thereof; n is 1 to 50; R ３ is a -phenyl-tetrazine group or linking group, said tetrazine optionally substituted with methyl; The use wherein the compound or a salt thereof is conjugated to a monoclonal antibody (mAb).

20. 20. The use of claim 19, wherein the cysteine ​​is an engineered cysteine.

21. formula 【Chemistry 14】 or a salt thereof (In the formula, R １ teeth, 【Chemistry 15】 and R ２ is PEG ｎ , a bond and a peptide, or a combination thereof; n is 1 to 50; R ３ is a -phenyl-tetrazine group or linking group, said tetrazine optionally substituted with methyl; mAb is a monoclonal antibody; S is a sulfur atom from a cysteine ​​residue on the monoclonal antibody).

22. The cysteine ​​is an engineered cysteine, preferably R ３ 22. The conjugate of claim 21, wherein said conjugate comprises a drug payload.

23. formula 【Chemistry 16】 or a salt thereof (In the formula, R １ teeth, 【Chemistry 17】 and R ２ is PEG ｎ , a bond and a peptide, or a combination thereof; n is 1 to 50; R ３ is a -phenyl-tetrazine group or linking group, said tetrazine optionally substituted with methyl; mAb is a monoclonal antibody; S is a sulfur atom from a cysteine ​​residue on the monoclonal antibody).

24. The cysteine ​​is an engineered cysteine, preferably R ３ The conjugate of claim 23 , wherein the

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