Synthesis of bicyclic toxin conjugates and their intermediates

Abstract

The present invention relates to a method for producing bicyclic toxin conjugates and intermediates thereof.

Description

[Technical Field]

[0001] Field of Invention The present invention relates to a method for synthesizing gvcMMAE, and to a method for synthesizing bicyclic toxin conjugates (BTCs) containing gvcMMAE (glutaryl-Val-Cit-PAB-MMAE), such as BT5528 and BT8009. [Background technology]

[0002] Background of the Invention Cyclic peptides can bind to protein targets with high affinity and target specificity, making them an attractive molecular class for therapeutic drug development. In fact, several cyclic peptides have already achieved successful clinical use, such as the antibacterial peptide vancomycin, the immunosuppressant drug cyclosporine, or the anticancer drug octreotide (Driggers et al. (2008), Nat Rev Drug Discov 7 (7), 608-24). The good binding properties stem from the relatively large interaction surface formed between the peptide and the target, as well as the reduced conformational flexibility of the cyclic structure. Typically, macrocyclic molecules bind to surfaces of several hundred square Å, such as the cyclic peptide CXCR4 antagonist CVX15 (400 Ų; Wu et al. (2007), Science 330, 1066-71), the cyclic peptide with an Arg-Gly-Asp motif that binds to integrin αVb3 (355 Ų) (Xiong et al. (2002), Science 296 (5565), 151-5), or the cyclic peptide inhibitor upain-1 (603 Ų; Zhao et al. (2007), J Struct Biol 160 (1), 1-10) that binds to urokinase-type plasminogen activators.

[0003] Due to their cyclic configuration, macrocyclic peptide molecules are less flexible than linear peptides, resulting in less entropy loss during binding to the target and higher potential binding affinity. Reduced flexibility also leads to target-specific conformation locking, further increasing binding specificity compared to linear peptides. This effect is exemplified by potent and selective inhibitors of matrix metalloproteinase 8 (MMP-8), which lose selectivity over other MMPs upon ring opening (Cherney et al. (1998), J Med Chem 41 (11), 1749-51). The favorable binding properties achieved through macrocyclization are even more pronounced in polycyclic peptides with more than one peptide ring, such as vancomycin, nisin, and actinomycin.

[0004] Various research teams have previously tethered polypeptides containing cysteine ​​residues to synthetic molecular structures (Kemp and McNamara (1985), J. Org. Chem; Timmerman et al. (2005), ChemBioChem). Meloen and collaborators have used tris(bromomethyl)benzene and related molecules for the rapid and quantitative cyclization of multiple peptide loops on synthetic scaffolds for structural mimicry of protein surfaces (Timmerman et al. (2005), ChemBioChem). Methods for producing candidate drug compounds, in which the compound is produced by tethering a cysteine-containing polypeptide to a molecular scaffold such as tris(bromomethyl)benzene, are described in WO2004 / 077062 and WO2006 / 078161.

[0005] Phage display-based combinatorial approaches are being developed for the production and screening of large libraries of bicyclic peptides against target organisms of interest (Heinis et al. (2009), Nat Chem Biol 5 (7), 502-7 and WO2009 / 098450). Briefly, a combinatorial library of linear peptides (Cys-(Xaa)6-Cys-(Xaa)6-Cys) containing two regions of three cysteine ​​residues and six random amino acids is presented on a phage and cyclized by covalent linkage of the cysteine ​​side chain to a small molecule (tris-(bromomethyl)benzene). [Overview of the project]

[0006] Summary of the Invention This invention provides a method for synthesizing glutaryl-Val-Cit-PAB-MMAE (gvcMMAE). [ka]

[0007] The present invention also provides a method for synthesizing a bicyclic toxin conjugate (BTC) containing gvcMMAE. In one embodiment, the bicyclic toxin conjugate (BTC) is BT5528 or a pharmaceutically acceptable salt thereof. In another embodiment, the bicyclic toxin conjugate (BTC) is BT8009 or a pharmaceutically acceptable salt thereof. [Modes for carrying out the invention]

[0008] Detailed description of the invention 1. General description of a certain aspect of the present invention The present invention has discovered that the method for synthesizing glutaryl-Val-Cit-PAB-MMAE (gvcMMAE) from Val-Cit-PAB-MMAE (vcMMAE), as shown in Scheme I, can improve large-scale production, including GMP production. For example, this method can be optimized as follows: The equivalent amount of glutaric acid anhydride can be reduced from 1.2 equivalents to approximately 1 to 1.1 equivalents (for example, approximately 1.1 equivalents); • 5.2 equivalents of DIEA can be changed to approximately 1.3 to 1.5 equivalents of TEA (for example, approximately 1.3 equivalents of TEA); and • A DMA / THF mixed solvent can be used as the reaction solvent.

[0009] In one embodiment, the reaction temperature may be approximately 15°C to 25°C. In another embodiment, the reaction temperature can be lowered to 0°C.

[0010] The optimization method was found to provide more stable reaction conditions. The resulting reaction solution can then be directly added to a poor solvent (e.g., THF:MTBE; e.g., approximately 20:39 to approximately 25:30 v / v THF:MTBE, e.g., approximately 1:2, e.g., approximately 75 v) to provide a good solid-state product (gvcMMAE). This optimization method was found to be more suitable for scale-up. Furthermore, the disclosed method was found to yield a gvcMMAE product with an improved purity profile.

[0011] Accordingly, in one embodiment, the present invention provides a method for synthesizing gvcMMAE, comprising reacting vcMMAE with a glutaric acid anhydride under the conditions shown herein in Scheme I or Scheme II.

[0012] In another embodiment, the present invention provides a method for synthesizing a bicyclic toxin conjugate (BTC), comprising reacting a gvcMMAE with a dicyclic compound.

[0013] 2. Compounds and Definitions The compounds of the present invention generally include those described above and further described by the classes, subclasses, and species disclosed herein. To the extent used herein, the following definitions should apply unless otherwise specified. For the purposes of the present invention, chemical elements are those defined in the periodic table, CAS version, Handbook of Chemistry and Physics, 75 thIt is identified according to Ed. Furthermore, the general principles of organic chemistry are described in “Organic Chemistry”, Thomas Sorrell, University Science Books, Sausalito: 1999, and “March's Advanced Organic Chemistry”, 5 th This is described in Ed., Ed.: Smith, MB and March, J., John Wiley & Sons, New York: 2001, and the contents of each of these are incorporated herein by reference.

[0014] To the extent used herein, the terms “aliphatic” or “aliphatic group” mean a linear (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain containing one or more fully saturated or unsaturated units, or a monocyclic or bicyclic hydrocarbon that is not aromatic but contains one or more fully saturated or unsaturated units having one bond point to the rest of the molecule (here meaning “carbocyclic”). Unless otherwise specified, an aliphatic group contains 1 to 6 aliphatic carbon atoms. In some embodiments, an aliphatic group contains 1 to 5 aliphatic carbon atoms. In other embodiments, an aliphatic group contains 1 to 4 aliphatic carbon atoms. In some embodiments, the aliphatic group contains 1 to 3 aliphatic carbon atoms, and in other embodiments, the aliphatic group contains 1 to 2 aliphatic carbon atoms. In some embodiments, “cycloaliphatic” (or “carbocyclic” or “cycloalkyl”) refers to a monocyclic C3-C6 hydrocarbon that is not aromatic but contains one fully saturated or one or more unsaturated units having one bond site to the rest of the molecule. Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl groups and hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.

[0015] To the extent used herein, the term “bridged bicyclic” refers to any bicyclic ring system having at least one bridge, i.e., carbocyclic or heterocyclic, saturated or partially unsaturated. As defined by IUPAC, a “bridge” is a multi-atom or single-atom unbranched chain or valence bond connecting two bridge heads, where “bridge heads” are any skeletal atoms of a ring system bonded to three or more skeletal atoms (excluding hydrogen). In some embodiments, a bridged bicyclic group has 7 to 12 ring members and 0 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Such bridged bicyclic groups are well known in the art and include the groups shown below, where each group is bonded to the rest of the molecule with any substituteable carbon or nitrogen atom. Unless otherwise specified, bridged bicyclic groups are optionally substituted with one or more substituents, as shown for aliphatic groups. In addition to or separately from this, any substituteable nitrogen in a bridged bicyclic group is optionally substituted. Examples of bridged bicyclic groups are: [ka] Includes.

[0016] The term "lower alkyl" refers to C 1-4 This refers to linear or branched alkyl groups. Examples of lower alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, and tert-butyl.

[0017] The term "lower haloalkyl" refers to a carbon atom substituted with one or more halogen atoms. 1-4 It is a linear or branched alkyl group.

[0018] The term "heteroatom" refers to oxygen, sulfur, nitrogen, phosphorus, or silicon (any oxidized form of nitrogen, sulfur, phosphorus, or silicon; any basic nitrogen in a quaternized form; or a substitute nitrogen in a heterocyclic ring, e.g., N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or NR + This means one or more (as in N-substituted pyrrolidinyls).

[0019] As used herein, the term "unsaturated" means that a part has one or more unsaturated units.

[0020] For the purposes of this text, the term “divalent hydrocarbon chain” refers to a divalent alkylene, alkenylene, and alkynylene chain that is straight or branched as defined herein.

[0021] The term "alkylene" refers to a divalent alkyl group. An "alkylene chain" is a polymethylene group, i.e., -(CH2) n -(where n is a positive integer, preferably 1-6, 1-4, 1-3, 1-2, or 2-3). A substituted alkylene chain is a polymethylene group in which one or more methylene hydrogen atoms are replaced by substituents. Suitable substituents for substituted aliphatic groups include the following:

[0022] The term "alkenylene" refers to a divalent alkenyl group. A substituted alkenylene chain is a polymethylene group containing at least one double bond, in which one or more hydrogen atoms are replaced by substituents. Suitable substituents for substituted aliphatic groups include the following:

[0023] The term "alkynylene" refers to a divalent alkynyl group. A substituted alkynylene chain is a polymethylene group containing at least one triple bond in which one or more hydrogen atoms are replaced by substituents. Suitable substituents for substituted aliphatic groups include the following:

[0024] For the purposes of this text, the term "cyclopropyrenyl" refers to a divalent cyclopropyl group with the following structure: [ka]

[0025] The term "halogen" refers to F, Cl, Br, or I.

[0026] The term “aryl,” used alone or as part of larger terms such as “aralkyl,” “aralkoxy,” or “aryloxyalkyl,” refers to a monocyclic or bicyclic ring system having a total of 5 to 14 ring members, where at least one ring in the system is aromatic and each ring in the system contains 3 to 7 ring members. The term “aryl” may be used interchangeably with the term “aryl ring.” In some embodiments of the present invention, “aryl” refers to aromatic ring systems, including but not limited to phenyl, biphenyl, naphthyl, and anthracyl, which may hold one or more substituents. Also included in the scope of the term “aryl,” as used herein, are groups in which an aromatic ring is condensed with one or more non-aromatic rings, such as indanyl, phthalimidyl, naphthoimidyl, phenantridinyl, or tetrahydronaphthyl.

[0027] The terms "heteroaryl" and "heteroaryl-", used alone or as part of a larger group, e.g., "heteroaryl" or "heteroaryl-", refer to a group having 5 to 10 ring atoms, preferably 5, 6, or 9 ring atoms; 6, 10, or 14 π electrons shared in a cyclic arrangement; and having 1 to 5 heteroatoms in addition to carbon atoms. The term "heteroatom" refers to nitrogen, oxygen, or sulfur, including nitrogen or sulfur in any oxidized form and basic nitrogen in any quaternized form. Heteroaryl groups include, but are not limited to, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridadinyl, pyrimidinyl, pyrazinyl, indolidinyl, prinyl, naphthilidinyl, and pteridinyl. To the extent used herein, the terms “heteroaryl” and “hetero-” also include groups in which a heteroaromatic ring is condensed to one or more aryl, cycloaliphatic, or heterocyclyl rings, and the radical or dot of the bond lies on the heteroaromatic ring. Non-limiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl, sinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolidinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyrido[2,3-b]-1,4-oxazine-3(4H)-one. Heteroaryl groups can be monocyclic or bicyclic. The term "heteroaryl" can be used interchangeably with the terms "heteroaryl ring," "heteroaryl group," or "heteroaromatic," all of which include a ring that is optionally substituted. The term "heteroaralkyl" refers to an alkyl group substituted with a heteroaryl group, where the alkyl and heteroaryl moieties are independently substituted as desired.

[0028] To the extent used herein, the terms “heterocyclic,” “heterocyclyl,” “heterocyclic radical,” and “heterocyclic ring” are interchangeable and refer to stable 5-7 member monocyclic or 7-10 member bicyclic heterocyclic moieties that are saturated or partially unsaturated and have one or more, preferably 1-4, heteroatoms as defined above, in addition to the carbon atom. When used in reference to the ring atoms of a heterocyclic ring, the term “nitrogen” includes substituted nitrogen. For example, in a saturated or partially unsaturated ring having 0-3 heteroatoms selected from oxygen, sulfur, or nitrogen, the nitrogen may be N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or + It can be NR (as in N-substituted pyrrolidinyl).

[0029] A heterocyclic ring can be bonded to its pendant group with any heteroatom or carbon atom that results in a stable structure, and any of the ring atoms can be substituted as desired. Examples of such saturated or partially unsaturated heterocyclic radicals include, but are not limited to, tetrahydrofuranyl, tetrahydrothiophenylpyrrolidinyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decadroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl. The terms "heterocyclic," "heterocyclyl," "heterocyclyl ring," "heterocyclic group," "heterocyclic moiety," and "heterocyclic radical" are used interchangeably here and also include groups in which a heterocyclyl ring, such as indolinyl, 3H-indolyl, chromanyl, phenantridinyl, or tetrahydroquinolinyl, is condensed with one or more aryl, heteroaryl, or cycloaliphatic rings. The heterocyclyl group can be monocyclic or bicyclic. The term "heterocyclylalkyl" refers to an alkyl group substituted with a heterocyclyl, where the alkyl and heterocyclyl moieties are independently substituted as desired.

[0030] As used herein, the term "partially unsaturated" refers to a ring moiety that contains at least one double or triple bond. The term "partially unsaturated" is intended to encompass rings having multiple unsaturation positions, but is not intended to include aryl or heteroaryl moieties as defined herein.

[0031] As described herein, the compounds of the invention may contain "optionally substituted" moieties. In general, the term "substituted" means that one or more hydrogens of the designated moiety are replaced with a suitable substituent, whether or not preceded by the term "optionally". Unless otherwise specified, an "optionally substituted" group may have suitable substituents at each substitutable position of the group, and when one or more positions in a structure are substituted with more than one substituent selected from the specified groups, the substituents may be the same or different at all positions. The combinations of substituents contemplated in this invention preferably result in the formation of stable or chemically feasible compounds. As used herein, the term "stable" refers to a compound that is substantially unchanged when subjected to the conditions necessary for its production, detection, and, in certain embodiments, recovery, purification, and use for one or more of the purposes disclosed herein.

[0032] Suitable monovalent substituents on a substitutable carbon atom of an "optionally substituted" group are, independently, halogen; -(CH2) 0-4 R 〇 ; -(CH2) 0-4 OR 〇 ; -O(CH2) 0-4 R o , -O-(CH2) 0-4 C(O)OR 〇 ; -(CH2) 0-4 CH(OR 〇 )2; -(CH2) 0-4 SR 〇 ; R 〇 that may be substituted with -(CH2) 0-4 Ph; R 〇 that may be substituted with -(CH2) 0-4 O(CH2) 0-1 Ph; R 〇 that may be substituted with -CH=CHPh; R 〇で can be replaced by -(CH2) 0-4 O(CH2) 0-1 -NO2;-CN;-N3;-(CH2) 0-4 N(R 〇 )2;-(CH2) 0-4 N(R 〇 )C(O)R 〇 ;-N(R 〇 )C(S)R 〇 ;-N(R 〇 )C(NR 〇 )N(R 〇 )2;-(CH2) 0-4 N(R 〇 )C(O)NR 〇 2;-N(R 〇 )C(S)NR 〇 2;-(CH2) 0-4 N(R 〇 )C(O)OR 〇 ;-N(R 〇 )N(R 〇 )C(O)R 〇 ;-N(R 〇 )N(R 〇 )C(O)NR 〇 2;-N(R 〇 )N(R 〇 )C(O)OR 〇 ;-(CH2) 0-4 C(O)R 〇 ;-C(S)R 〇 ;-(CH2) 0-4 C(O)OR 〇 ;-(CH2) 0-4 C(O)SR 〇 ;-(CH2) 0-4 C(O)OSiR 〇 3;-(CH2) 0-4 OC(O)R 〇 ;-OC(O)(CH2) 0-4 SR-、-SC(S)SR 〇 ;-(CH2) 0-4 SC(O)R 〇 ;-(CH2) 0-4 C(O)NR 〇 2;-C(S)NR 〇 2;-C(S)SR 〇 ;-(CH2) 0-4 OC(O)NR 〇2;-C(O)N(OR 〇 )R 〇 ;-C(O)C(O)R 〇 ;-C(O)CH2C(O)R 〇 ;-C(NOR 〇 )R 〇 [[ID=I2]];-(CH2) 0-4 SSR 〇 ;-(CH2) 0-4 S(O)2R 〇 ;-(CH2) 0-4 S(O)2OR 〇 ;-(CH2) 0-4 OS(O)2R 〇 ;-S(O)2NR 〇 2;-(CH2) 0-4 S(O)R 〇 ;-N(R 〇 )S(O)2NR 〇 2;-N(R 〇 )S(O)2R 〇 ;-N(OR 〇 )R 〇 ;-C(NH)NR 〇 2;-P(O)2R 〇 ;-P(O)R 〇 2;-OP(O)R 〇 2;-OP(O)(OR 〇 )2;-SiR 〇 3;-(C 1-4 linear or branched alkylene)O-N(R 〇 )2; or -(C 1-4 linear or branched alkylene)C(O)O-N(R 〇 )2, where each R 〇 is independently selected from hydrogen, C 1-6 aliphatic, -CH2Ph, -O(CH2) 0-1 Ph, -CH2-(5-6 membered heteroaryl ring), or a 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the above definition, two independently occurring R 〇These atoms, in conjunction with the atoms between them, form a 3- to 12-membered saturated, partially unsaturated, or aryl monocyclic or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, which may be substituted as follows.

[0033] R 〇 (or two R atoms exist independently, one with the atom in between) 〇 A suitable monovalent substituent on the ring formed by can be a halogen, -(CH2) 0-2 R ● ,-(HaroR ● ), -(CH2) 0-2 OH, -(CH2) 0-2 Ure ● ,-(CH2) 0-2 CH(OR ● )2;-O(HaroR ● ), -CN, -N3, -(CH2) 0-2 C(O)R ● ,-(CH2) 0-2 C(O)OH, -(CH2) 0-2 C(O)OR ● ,-(CH2) 0-2 SR ● ,-(CH2) 0-2 SH, -(CH2) 0-2 NH2, -(CH2) 0-2 NHR ● ,-(CH2) 0-2 NR ● 2, -NO2, -SiR ● 3. -OSiR ● 3. -C(O)SR ● ,-(C 1-4 (Linear or branched alkylene) C(O)OR ● , or -SSR ● And here, each R ● It is either unsubstituted or substituted by one or more halogens when preceded by "halo", and independently C 1-4 Aliphatic, -CH2Ph, -O(CH2) 0-1 Ph, or selected from 5-6 member saturated, partially unsaturated, or aryl rings having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. 〇Suitable divalent substituents on the saturated carbon atom include =O and =S.

[0034] Suitable divalent substituents on the saturated carbon atom of the "optionally substituted" group are: =O, =S, =NNR * 2. =NNHC(O)R * ,=NNHC(O)OR * ,=NNHS(O)2R * ,=NR * 、=NOR * , -O(C(R * 2)) 2-3 O-, or -S(C(R * 2)) 2-3 S- is included, where each R exists independently. * C is a hydrogen atom that can be substituted as defined below. 1-6 Selected from aliphatic or unsubstituted nitrogen, oxygen, or sulfur, the 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms. A suitable divalent substituent bonded to the vicinal substituteable carbon of the "optionally substituted" group is:-O(CR * 2) 2-3 It includes O-, where each R exists independently. * C is a hydrogen atom that can be substituted as defined below. 1-6 Selected from aliphatic or non-substituted nitrogen, oxygen, or sulfur, the 5-6 membered saturated, partially unsaturated, or aryl rings having 0-4 heteroatoms.

[0035] R * Suitable substituents on the aliphatic group include halogens, -R ● ,-(HaroR ● ), -OH, -OR ● ,-O(HaroR ● ), -CN, -C(O)OH, -C(O)OR ● -NH2, -NHR ● , -NR ● 2, or -NO2, where each R ● It is either unsubstituted or substituted by one or more halogens when preceded by "halo", and independently C 1-4Aliphatic, -CH2Ph, -O(CH2) 0-1 It is a 5-6 member saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from pH, nitrogen, oxygen, or sulfur.

[0036] A suitable substitutionable substituent on the nitrogen of the group "as desired" is -R † , -NR † 2, -C(O)R † , -C(O)OR † ,-C(O)C(O)R † -C(O)CH2C(O)R † -S(O)2R † -S(O)2NR † 2, -C(S)NR † 2, -C(NH)NR † 2, or -N(R † )S(O)2R † This includes; where each R † C can be substituted independently of hydrogen, as defined below. 1-6 It is a 5-6 member saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from aliphatic, unsubstituted-OPh, or unsubstituted nitrogen, oxygen, or sulfur, or, notwithstanding the above definition, two independently present R † These atoms, in conjunction with the atoms between them, form unsubstituted 3-12 member saturated, partially unsaturated, or aryl monocyclic or bicyclic rings having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0037] R † Suitable substituents on the aliphatic group are, independently, halogens, -R ● ,-(HaroR ● ), -OH, -OR ● ,-O(HaroR ● ), -CN, -C(O)OH, -C(O)OR ● -NH2, -NHR ● , -NR ● 2, or -NO2, where each R ●It is either unsubstituted or substituted by one or more halogens when preceded by "halo", and independently C 1-4 Aliphatic, -CH2Ph, -O(CH2) 0-1 It is a 5-6 member saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from pH, nitrogen, oxygen, or sulfur.

[0038] To the extent used herein, the term “pharmaceutically acceptable salt” means a salt that, within reasonable medical judgment, is suitable for use in contact with human and lower animal tissues without excessive toxicity, irritation, or allergic response, and is balanced by a reasonable benefit / risk ratio. pharmaceutically acceptable salts are well known in the art. For example, S.M. Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, which is incorporated herein by reference. Furthermore, pharmaceutically acceptable salts are described in detail in Pharmaceutical Salts: Properties, Selection, and Use, 2nd Revised Edition, (2011), P. Heinrich Stahl (Editor), Camille G. Wermuth (Editor), (ISBN: 978-3-906-39051-2), which is incorporated herein by reference in its entirety. pharmaceutically acceptable salts of the compounds of the present invention include those derived from appropriate inorganic and organic acids and bases. Examples of pharmaceutically acceptable, non-toxic acid addition salts are salts of amino groups formed by using inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or by other methods such as ion exchange.Other pharmaceutically acceptable salts include adipine, alginate, ascorbate, aspartate, benzenesulfonate, besilate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptone, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, and 2-hydroxy -Includes ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, mesylate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, etc.

[0039] Suitable base-derived salts include alkali metals, alkaline earth metals, ammonium, and N + (C 1-4 Contains alkyl) tetrasalts. Typical alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc. Further pharmaceutically acceptable salts, when appropriate, include non-toxic ammonium, quaternary ammonium, and halides, hydroxides, carboxylic acids, sulfuric acids, phosphoric acids, nitric acids, (C 1-6 It contains amine cations formed using counterions such as alkyl sulfonic acids and aryl sulfonic acids.

[0040] Unless otherwise specified, the structures described herein also mean all isomeric forms of the structure (e.g., enantiomers, diastereomers, and geometric (or conformational) forms); for example, R and S configurations, Z and E double bond isomers, and Z and E conformational isomers with respect to each chiral center. Accordingly, the single stereochemical isomers of the compound, as well as enantiomers, diastereomers, and geometric (or conformational) mixtures, are within the scope of the present invention. Unless otherwise specified, all tautomeric forms of the compound of the present invention are within the scope of the present invention.

[0041] To the extent used herein, “therapeutic dose” means the amount of a substance (e.g., a therapeutic agent, composition, and / or formulation) that elicits a desired biological response. In some embodiments, the therapeutic dose of a substance is an amount sufficient to treat, diagnose, prevent, and / or induce a disease, condition, or disorder when administered as part of a dosing regimen to a subject having or suspected of having the disease, condition, or disorder. As will be apparent to those skilled in the art, the effective dose of a substance can vary depending on factors such as the desired biological endpoint, the substance being delivered, and the target cells or tissues. For example, the effective dose of a compound in a formulation for treating a disease, condition, or disorder is an amount that reduces, improves, alleviates, inhibits, prevents, delays the onset, reduces the severity, and / or reduces the incidence of one or more symptoms or characteristics of the disease, condition, or disorder.

[0042] To the extent used herein, the terms “treatment” or “to treat” mean partially or completely reducing, preventing, delaying the onset of, preventing, improving, and / or mitigating one or more symptoms of a disease or disorder. To the extent used herein, the terms “treatment” and “to treat” mean partially or completely reducing, preventing, delaying the onset of, preventing, improving, and / or mitigating one or more symptoms of a disease or disorder as described herein. In some embodiments, a treatment may be administered after the onset of one or more symptoms. In some embodiments, the term “treatment” includes the prevention or cessation of the progression of a disease or disorder. In other embodiments, a treatment may be administered when symptoms are not present. For example, a treatment may be administered to a susceptible individual before the onset of symptoms (e.g., in light of the history of the symptoms and / or in light of genetic or other susceptibility factors). Treatment may continue after the symptoms have resolved, for example, to prevent or delay relapse. Thus, in some embodiments, the term “treatment” includes the prevention of relapse or recurrence of a disease or disorder.

[0043] To the extent used herein, the term “unit dosage form” refers to a physically distinct unit of a therapeutic formulation appropriate for the subject being treated. However, it is understood that the total daily dose of the compositions of the present invention is determined by the treating physician within reasonable medical judgment. The specific effective dose level for any particular subject or organism depends on a variety of factors, including the disorder being treated and its severity; the activity of the specific activator used; the specific composition used; the subject’s age, weight, general health, sex, and dietary habits; the time of administration and the rate of excretion of the specific activator used; the duration of treatment; drugs and / or additional treatments used in combination with or concurrently with the specific compound used; and analogous factors well known in the pharmaceutical field.

[0044] The bicyclic toxin conjugate BT8009 has the structure shown below, and the manufacture of BT8009 (BCY8245) is described in WO2019 / 243832, which is incorporated herein by reference in its entirety. [ka]

[0045] The bicyclic toxin conjugate BT5528 has the structure shown below, where the molecular scaffold is 1,1',1''-(1,3,5-triazinan-1,3,5-triyl)tripprop-2-en-1-one (TATA), and the peptide ligand is the amino acid sequence (β-Ala)-Sar 10 -A(HArg)DC i (HyP)LVNPLC ii LHP(D-Asp)W(HArg)C iii This includes, where Sar is sarcosine, HArg is homoarginine, and HyP is hydroxyproline. [ka]

[0046] 3. Description of one embodiment of the present invention In one embodiment, the present invention provides a method for synthesizing gvcMMAE, comprising reacting vcMMAE with glutaric anhydride. In one embodiment, the present invention provides a method for synthesizing gvcMMAE, comprising reacting vcMMAE with glutaric anhydride in a solvent containing N,N-dimethylacetamide (DMA) and tetrahydrofuran (THF). In one embodiment, the present invention provides a method for synthesizing gvcMMAE, comprising reacting vcMMAE with glutaric anhydride in a solvent containing N,N-dimethylacetamide (DMA) and tetrahydrofuran (THF), optionally comprising one or more further components such as methyl tert-butyl ether (MTBE).

[0047] In one embodiment, the method comprises adding glutaric anhydride to a solution containing vcMMAE in a solvent containing N,N-dimethylacetamide (DMA) and tetrahydrofuran (THF) to provide a reaction mixture. In one embodiment, the present invention provides a method for synthesizing gvcMMAE, comprising reacting vcMMAE with glutaric anhydride under the conditions shown herein in Scheme I or Scheme II.

[0048] In one embodiment, the provided method involves the reaction of vcMMAE with about 1 to about 1.1 equivalents of glutaric anhydride for about 1 equivalent of vcMMAE. In one embodiment, the glutaric anhydride is about 1.1 equivalents of vcMMAE. In another embodiment, the glutaric anhydride is about 1 equivalent, about 1.01 equivalents, about 1.02 equivalents, about 1.03 equivalents, about 1.04 equivalents, about 1.05 equivalents, about 1.06 equivalents, about 1.07 equivalents, about 1.08 equivalents, or about 1.09 equivalents of vcMMAE. In yet another embodiment, the glutaric anhydride is about 1.11 equivalents, about 1.12 equivalents, about 1.13 equivalents, about 1.14 equivalents, about 1.15 equivalents, about 1.16 equivalents, about 1.17 equivalents, about 1.18 equivalents, about 1.19 equivalents, or about 1.20 equivalents of vcMMAE. In one embodiment, vcMMAE is reacted with about 1 to about 1.20 equivalents of glutaric anhydride per about 1 equivalent of vcMMAE, for example, about 1 to about 1.1 equivalents of glutaric anhydride, for example, about 1.05 to about 1.15 equivalents of glutaric anhydride, for example, about 1.08 to about 1.12 equivalents of glutaric anhydride, for example, about 1.1 equivalents of glutaric anhydride.

[0049] In one embodiment, the amount of triethylamine (TEA) in the reaction mixture is about 1.3 equivalents of vcMMAE. In another embodiment, the amount of triethylamine (TEA) in the reaction mixture is about 1 to 1.6 equivalents of vcMMAE per 1 equivalent of vcMMAE. In another embodiment, the reaction mixture contains about 1.3 to 1.5 equivalents of triethylamine. In yet another embodiment, the amount of triethylamine (TEA) in the reaction mixture is about 1 equivalent, 1.05 equivalents, 1.10 equivalents, 1.15 equivalents, 1.2 equivalents, 1.25 equivalents, 1.3 equivalents, 1.35 equivalents, 1.4 equivalents, 1.45 equivalents, 1.5 equivalents, 1.55 equivalents, or 1.6 equivalents of vcMMAE. In one embodiment, the reaction mixture contains about 1 to about 1.6 equivalents of TEA, for example, about 1.3 to about 1.5 equivalents of TEA, for example, about 1.3 to about 1.4 equivalents of TEA, or for example, about 1.3 equivalents of TEA, per about 1 equivalent of vcMMAE. In one embodiment, the method involves adding about 1 to about 1.6 equivalents of TEA, for example, about 1.3 to about 1.5 equivalents of TEA, for example, about 1.3 to about 1.4 equivalents of TEA, or for example, about 1.3 equivalents of TEA, to the reaction mixture.

[0050] In one embodiment, the reaction of vcMMAE with glutaric anhydride takes place in a solvent containing N,N-dimethylacetamide (DMA) and tetrahydrofuran (THF). In another embodiment, the solvent contains DMA, THF, and one or more further components. In yet another embodiment, the solvent contains DMA, THF, and MTBE.

[0051] In one embodiment, the solvent comprises N,N-dimethylacetamide (DMA) and tetrahydrofuran (THF) in a volume ratio of about 1:15, or is a mixture of DMA and THF. In one embodiment, the solvent comprises DMA and THF in a volume ratio of about 1:10 to about 1:200, for example, about 1:50 to about 1:150, for example, about 1:100 to about 1:130, for example, about 1:110 to about 1:120, for example, about 1:115. In one embodiment, the solvent comprises DMA and THF in a volume ratio of about 1:10 to about 1:20, for example, about 1:12 to about 1:18, for example, about 1:14 to about 1:16, for example, about 1:15. In one embodiment, the solvent comprises N,N-dimethylacetamide (DMA) and tetrahydrofuran (THF) in a volume ratio of about 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:16, 1:17, 1:18, 1:19, or 1:20, or is a mixture of DMA and THF.

[0052] In one embodiment, THF is present at approximately 3 to 3.9 kg / kg. In one embodiment, THF is present at approximately 3 to 3.9 kg / kg relative to the mass of vcMMAE. In one embodiment, DMA is present at approximately 0.2 to 0.3 kg / kg. In one embodiment, DMA is present at approximately 0.2 to 0.3 kg / kg relative to the mass of vcMMAE. In one embodiment, the solvent contains, for example, approximately 0.2:3.9 to 0.3 to 3 v / v DMA / THF relative to vcMMAE. In one embodiment, the solvent is a mixture of approximately 0.25:3.75 v / v DMA and THF (0.25:3.75 v / v DMA / THF). In one embodiment, the solvent contains approximately 0.35:3.65 to 0.15:3.85 v / v DMA / THF. In one embodiment, the solvent contains approximately 0.3:3.7 to 0.2:3.8 v / v DMA / THF. In one embodiment, the solvent is a mixture of approximately 0.20:3.80 v / v DMA and THF. In another embodiment, the solvent is a mixture of approximately 0.30:3.70 v / v DMA and THF. In yet another embodiment, the solvent is a mixture of approximately 0.15:3.85 v / v DMA and THF. In yet another embodiment, the solvent is a mixture of approximately 0.35:3.65 v / v DMA and THF.

[0053] In one embodiment, the solvent comprises DMA, THF, and MTBE. In one embodiment, the solvent comprises DMA, THF, and MTBE in a volume ratio of about 1:A:B DMA:THF:MTBE, where A is about 10 to about 200, e.g., about 50 to about 150, e.g., about 100 to about 130, e.g., about 110 to about 120, e.g., about 115; and B is about 10 to about 500, e.g., about 100 to about 300, e.g., about 150 to about 250, e.g., about 180 to about 220, e.g., about 200.

[0054] In one embodiment, the solvent comprises DMA, THF, and MTBE; for example, relative to vcMMAE, DMA is present at approximately 0.2 to approximately 0.3 V; THF is present at approximately 25 to approximately 35 V; and DMA is present at approximately 30 to approximately 80 V. In another embodiment, for example relative to vcMMAE, DMA is present at approximately 0.23 to approximately 0.27 V (e.g., approximately 0.25 V); THF is present at approximately 26 to approximately 30 V (e.g., approximately 28 to 29 V, e.g., approximately 28.75 V); and DMA is present at approximately 40 to approximately 60 V / V (e.g., approximately 50 V).

[0055] In one embodiment, the DMA:THF ratio is approximately 0.2:35 to approximately 0.3:25 v / v. In another embodiment, the DMA:THF ratio is approximately 0.23:30 to approximately 0.27:26 v / v (for example, approximately 0.25:28 to 29 v / v, for example, 0.25:28.75 v / v). In another embodiment, the DMA:MTBE ratio is approximately 0.2:80 to approximately 0.3:30 v / v. In another embodiment, the DMA:MTBE ratio is approximately 0.23:60 to approximately 0.27:40 v / v (for example, approximately 0.25:50 v / v). In another embodiment, the THF:MTBE ratio is approximately 25:80 to approximately 35:30 v / v. In one embodiment, the THF:MTBE ratio is approximately 26:60 to 30:40 v / v (for example, approximately 28 to 29:50 v / v, or 28.75:50 v / v).

[0056] In one embodiment, the reaction between vcMMAE and glutaric anhydride takes place in a solvent containing dichloromethane (DCM).

[0057] In one embodiment, the reaction between vcMMAE and glutaric anhydride takes place in a solvent containing acetonitrile (MeCN).

[0058] In one embodiment, the reaction of vcMMAE and glutaric anhydride takes place in a solvent containing 2-methyltetrahydrofuran (2-MeTHF).

[0059] In one embodiment, the reaction between vcMMAE and glutaric anhydride takes place in a solvent containing N,N-dimethylacetamide (DMA).

[0060] In one embodiment, the reaction of vcMMAE with glutaric anhydride takes place in a solvent containing N,N-dimethylacetamide (DMA) and acetonitrile (MeCN). In another embodiment, the solvent contains N,N-dimethylacetamide (DMA) and acetonitrile (MeCN) in a volume ratio of about 1:3, or is a mixture of DMA and MeCN. In yet another embodiment, the solvent contains N,N-dimethylacetamide (DMA) and acetonitrile (MeCN) in a volume ratio of about 1:1, 1:2, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, or 1:15, or is a mixture of DMA and MeCN. In one embodiment, the solvent comprises N,N-dimethylacetamide (DMA) and acetonitrile (MeCN) in a volume ratio of about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, or 1.5:1, or is a mixture of DMA and MeCN.

[0061] In one embodiment, the reaction between vcMMAE and glutaric anhydride is carried out at a temperature of approximately -5 to approximately 25°C. In another embodiment, the reaction is carried out at a temperature of approximately 15°C to approximately 25°C. In yet another embodiment, the reaction is carried out at a temperature of approximately 18°C ​​to approximately 22°C, for example, approximately 20°C. In yet another embodiment, the reaction between vcMMAE and glutaric anhydride is carried out over a period of approximately 10 minutes to approximately 5 hours, for example, approximately 10 minutes to approximately 2 hours, for example, approximately 30 minutes to approximately 1.5 hours, for example, approximately 1 hour. In yet another embodiment, the reaction is carried out over a period of approximately 1 to approximately 3 hours. In yet another embodiment, the reaction is carried out until it is complete.

[0062] In one embodiment, the reaction involves stirring the reaction mixture. In one embodiment, the reaction involves stirring the reaction mixture for a period of about 10 minutes to about 5 hours, 10 minutes to about 2 hours, 30 minutes to about 1.5 hours, or about 1 hour at a temperature of about 15°C to about 25°C, for example, about 18°C ​​to about 22°C, for example, about 20°C; or for a period of about 1 to about 3 hours. In one embodiment, the reaction mixture is stirred until the reaction is complete. In one embodiment, the stirring speed is about 10 rpm to about 1000 rpm.

[0063] In one embodiment, the reaction between vcMMAE and glutaric anhydride is carried out at approximately 0°C. In another embodiment, the reaction between vcMMAE and glutaric anhydride is carried out at approximately -5°C to approximately 5°C. In another embodiment, the reaction between vcMMAE and glutaric anhydride is carried out at approximately -5°C, -4°C, -3°C, -2°C, or -1°C. In yet another embodiment, the reaction between vcMMAE and glutaric anhydride is carried out at approximately 1°C, 2°C, 3°C, 4°C, or 5°C.

[0064] In one embodiment, the present invention provides a method for synthesizing gvcMMAE, comprising reacting about 1 to about 1.1 equivalents of glutaric anhydride with about 1 equivalent of vcMMAE in a solvent containing about 0.2:3.9 to about 0.3 to 3 v / v DMA / THF at a temperature of about 15 to about 25°C.

[0065] In one embodiment, the present invention provides a method for synthesizing gvcMMAE, comprising adding about 1 to about 1.1 (e.g., about 1.1) equivalents of glutaric anhydride to a solution containing about 1 equivalent of vcMMAE and about 1.3 to about 1.5 equivalents of triethylamine in a solvent containing about 0.2:3.9 v / v to about 0.3 to 3 v / v DMA / THF (e.g., about 0.25:3.75 v / v DMA / THF), wherein the method comprises carrying out the reaction at a temperature of about 15 to about 25°C (e.g., about 20°C) for a period of about 10 minutes to about 2 hours.

[0066] In one embodiment, the present invention provides a method for synthesizing gvcMMAE, comprising: adding about 1 equivalent of vcMMAE to a solution containing about 1 equivalent of glutaric anhydride in a solvent containing about 0.2:3.9:0.3:3 v / v DMA / THF (e.g., about 0.25:3.75 v / v DMA / THF) to obtain a reaction mixture; adding about 1.3:0.5 equivalents of triethylamine; and stirring the reaction mixture at a temperature of about 15:0.25°C (e.g., about 20°C) for a period of about 10 minutes to about 2 hours (e.g., about 30 minutes to about 1.5 hours, e.g., about 1 hour).

[0067] In one embodiment, the present invention provides a method for synthesizing gvcMMAE, comprising reacting about 1 to about 1.1 equivalents of glutaric anhydride and about 1 equivalent of vcMMAE in a solvent containing DMA, THF, and MTBE in a volume ratio of about 1:A:B DMA:THF:MTBE (where A is about 10 to about 200 and B is about 10 to about 500) at a temperature of about 15 to about 25°C.

[0068] In one embodiment, the present invention provides a method for synthesizing gvcMMAE, comprising adding about 1 equivalent of vcMMAE and about 1.3 to about 1.5 equivalents of triethylamine to a solution containing about 1 equivalent of vcMMAE and about 1.5 equivalents of triethylamine in a solvent containing DMA, THF, and MTBE (where, for example, relative to vcMMAE, DMA is present at about 0.2 to about 0.3 V; THF is present at about 25 to about 35 V; and DMA is present at about 30 to about 80 V), wherein the method comprises carrying out the reaction for a period of about 10 minutes to about 5 hours at a temperature of about 15 to about 25°C (e.g., about 20°C).

[0069] In one embodiment, the present invention provides a method for synthesizing gvcMMAE, comprising adding about 1 to about 1.1 (e.g., about 1.1) equivalents of glutaric anhydride to a solution containing about 1 equivalent of vcMMAE in a solvent containing DMA, THF, and MTBE (where, for example, relative to vcMMAE, DMA is present at about 0.23 to about 0.27 V (e.g., about 0.25 V); THF is present at about 26 to about 30 V (e.g., about 28 to 29 V, e.g., about 28.75 V); and DMA is present at about 40 to about 60 V / v (e.g., about 50 V)) to obtain a reaction mixture; adding about 1.3 to about 1.5 equivalents of triethylamine; and stirring the reaction mixture for a period of about 1 to about 3 hours at a temperature of about 15 to about 25°C (e.g., about 20°C).

[0070] In one embodiment, the present invention provides a method for synthesizing gvcMMAE, comprising adding about 1.1 equivalents of glutaric acid anhydride to a solution containing about 1 equivalent of vcMMAE in a solvent of about 0.25:3.75 v / v DMA and THF (0.25:3.75 v / v DMA / THF) at about -5°C to about 5°C.

[0071] In one embodiment, the method includes stopping the reaction between vcMMAE and glutaric anhydride with water (H2O). In one embodiment, stopping the reaction includes adding about 0.002 to about 0.01 kg / kg of water to the reaction mixture. In one embodiment, stopping the reaction includes adding about 0.002 to about 0.01 kg / kg of water relative to the mass of vcMMAE. In one embodiment, stopping the reaction includes adding about 0.004 to about 0.008 kg / kg of water, for example, about 0.005 to about 0.007 kg / kg of water.

[0072] In one embodiment, stopping the reaction involves adding about 0.1 to about 1 equivalent of water to the reaction mixture. In another embodiment, stopping the reaction involves adding about 0.1 to about 1 equivalent of water (relative to the mass of vcMMAE) to the reaction mixture. In yet another embodiment, stopping the reaction involves adding about 0.2 to about 0.6 equivalents of water, for example, about 0.3 to about 0.5 equivalents of water to the reaction mixture.

[0073] In one embodiment, the reaction between vcMMAE and glutaric anhydride is stopped with water at a temperature of about 15 to about 25°C. In another embodiment, the reaction is stopped at a temperature of about 18°C ​​to about 22°C, for example, about 20°C. In another embodiment, the reaction is stopped for a period of about 1 minute to about 2 hours, for example, about 2 minutes to about 1 hour, for example, about 5 minutes to about 30 minutes, for example, about 8 minutes to about 20 minutes, for example, about 10 minutes.

[0074] In one embodiment, stopping the reaction involves stirring the reaction mixture. In one embodiment, stopping the reaction involves stirring the reaction mixture at a temperature of about 15 to about 25°C, for example, about 18°C ​​to about 22°C, for example, about 20°C. In one embodiment, stopping the reaction involves stirring the reaction mixture for a period of about 1 minute to about 2 hours, for example, about 2 minutes to about 1 hour, for example, about 5 minutes to about 30 minutes, for example, about 8 minutes to about 20 minutes, for example, about 10 minutes. In one embodiment, the stirring speed is about 10 rpm to about 1000 rpm.

[0075] In one embodiment, the present invention therefore provides a method for synthesizing gvcMMAE, comprising reacting about 1 to about 1.1 equivalents of glutaric anhydride and about 1 equivalent of vcMMAE in a solvent containing about 0.2:3.9 to about 0.3 to 3 v / v DMA / THF at a temperature of about 15 to about 25°C; and stopping the reaction with water.

[0076] In one embodiment, the present invention provides a method for synthesizing gvcMMAE, comprising adding about 1 to about 1.1 (e.g., about 1.1) equivalents of glutaric anhydride to a solution containing about 1 equivalent of vcMMAE and about 1.3 to about 1.5 equivalents of triethylamine in a solvent containing about 0.2:3.9 v / v to about 0.3 to 3 v / v DMA / THF (e.g., about 0.25:3.75 v / v DMA / THF), wherein the method includes stirring the reaction mixture at a temperature of about 15 to about 25°C (e.g., about 20°C); and stopping the reaction by adding about 0.002 to about 0.01 kg / kg of water or about 0.1 to about 1 equivalent of water to the reaction mixture.

[0077] In one embodiment, the present invention provides a method for synthesizing gvcMMAE, comprising: adding about 1 to about 1.1 (e.g., about 1.1) equivalents of glutaric anhydride to a solution containing about 1 equivalent of vcMMAE in a solvent containing about 0.2:3.9 to about 0.3 to 3 v / v DMA / THF (e.g., about 0.25:3.75 v / v DMA / THF) to obtain a reaction mixture; adding about 1.3 to about 1.5 equivalents of triethylamine; and stirring the reaction mixture at a temperature of about 15 to about 25°C (e.g., about 20°C) for a period of about 10 minutes to about 2 hours; and then adding to a reaction mixture of about 0.002 to about 0.01 kg / kg of water or about 0.1 to about 1 equivalent of water at a temperature of about 15 to about 25°C to quell the reaction.

[0078] In one embodiment, the present invention provides a method for synthesizing gvcMMAE, comprising adding about 1.1 equivalents of glutaric anhydride to a solution containing about 1 equivalent of vcMMAE in solvents of about 0.25:3.75 v / v DMA and THF (0.25:3.75 v / v DMA / THF) at about -5°C to about 5°C to obtain a reaction mixture, and then adding about 1.3 equivalents of TEA to the reaction mixture. In one embodiment, the reaction mixture is stirred for about 1 hour at about -5°C to about 5°C to complete the reaction (i.e., the reaction mixture becomes mainly gvcMMAE, or a mixture containing a gvcMMAE mixture). As used herein, unless otherwise implied by the context, the term “gvcMMAE mixture” typically refers to the product of the reaction of vcMMAE with glutaric anhydride, typically comprising gvcMMAE and optionally residual solvents, such as DMA, THF and / or TEA, and optionally any unreacted vcMMAE and / or glutaric anhydride.

[0079] In one embodiment, the gvcMMAE mixture is heated to approximately 15°C to approximately 25°C. In another embodiment, after the reaction is complete, the gvcMMAE mixture is heated to approximately 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, or 30°C. In another embodiment, the gvcMMAE mixture is heated until it becomes a clear solution.

[0080] In one embodiment, the gvcMMAE mixture is added to a poor solvent to obtain gvcMMAE as a precipitate. In another embodiment, the gvcMMAE mixture is added to a mixture of THF and MTBE to obtain gvcMMAE as a precipitate. In another embodiment, the gvcMMAE mixture is at approximately 15°C to approximately 25°C. In another embodiment, the gvcMMAE mixture is a clear solution. In another embodiment, the gvcMMAE mixture, which is a clear solution and is at approximately 15°C to approximately 25°C, is added to a mixture of THF and MTBE at approximately -5°C to approximately 5°C to obtain gvcMMAE as a precipitate.

[0081] In one embodiment, the THF and MTBE mixture contains about 20 to about 25 kg / kg of THF. In one embodiment, the THF and MTBE mixture contains about 20 to about 25 kg / kg of THF relative to the mass of vcMMAE. In one embodiment, the THF and MTBE mixture contains about 35 to about 39 kg / kg of MTBE. In one embodiment, the THF and MTBE mixture contains about 35 to about 39 kg / kg of MTBE relative to the mass of vcMMAE. In one embodiment, the THF and MTBE mixture contains about 20:39 to about 25:30 v / v THF / MTBE. In one embodiment, the THF and MTBE mixture is about 1:2 v / v THF and MTBE (1:2 v / v THF / MTBE). In one embodiment, the THF and MTBE mixture is approximately 0.5:2.5, 0.6:2.4, 0.7:2.3, 0.8:2.2, 0.9:2.1, 1.1:1.9, 1.2:1.8, 1.3:1.7, 1.4:1.6, or 1:1 v / v THF and MTBE. In one embodiment, the THF and MTBE mixture includes approximately 0.5:2.5 to approximately 1:1 v / v THF and MTBE.

[0082] In one embodiment, the gvcMMAE mixture is added to a mixture of THF and MTBE at approximately -5°C to approximately 5°C to obtain gvcMMAE as a precipitate. In another embodiment, the gvcMMAE mixture is added to a mixture of THF and MTBE containing approximately 20:39 to approximately 25:30 v / v THF / MTBE at approximately -5°C to approximately 5°C to obtain gvcMMAE as a precipitate. In yet another embodiment, the gvcMMAE mixture is added to a mixture of THF and MTBE containing approximately 20:39 to approximately 25:30 v / v THF / MTBE with stirring (e.g., dropwise) at approximately -5°C to approximately 5°C to obtain gvcMMAE as a precipitate, where the volume ratio of the DMA / THF mixture to the THF / MTBE mixture is approximately 4:20 to approximately 4:200, for example approximately 4:40 to approximately 4:100, for example approximately 4:50 to approximately 4:75, for example approximately 4:75.

[0083] In one embodiment, the gvcMMAE mixture is slowly added to a mixture of THF and MTBE at approximately -5°C to approximately 5°C to obtain gvcMMAE as a precipitate. In another embodiment, the gvcMMAE mixture is dropwise added to a mixture of THF and MTBE with stirring at approximately -5°C to approximately 5°C to obtain gvcMMAE as a precipitate. In another embodiment, the volume ratio of the DMA and THF mixture to the THF and MTBE mixture is approximately 4:20 to approximately 4:200, for example, approximately 4:40 to approximately 4:100, for example, approximately 4:50 to approximately 4:75, for example, approximately 4:75. In another embodiment, the volume ratio of the DMA and THF mixture to the THF and MTBE mixture is 4:75. In yet another embodiment, the volume ratio of the 0.25:3.75 v / v DMA / THF mixture to the 1:2 v / v THF / MTBE mixture is approximately 4:75. In one embodiment, the gvcMMAE mixture is slowly added to a mixture of approximately 1:2 v / v THF and MTBE at approximately -5°C to approximately 5°C to obtain gvcMMAE as a precipitate. In another embodiment, the gvcMMAE mixture is added dropwise to a mixture of approximately 1:2 v / v THF and MTBE with stirring at approximately -5°C to approximately 5°C to obtain gvcMMAE as a precipitate. In another embodiment, the volume ratio of the DMA and THF mixture to the THF and MTBE mixture is 4:75. In yet another embodiment, the volume ratio of the 0.25:3.75 v / v DMA / THF mixture to the 1:2 v / v THF / MTBE mixture is approximately 4:75.

[0084] In one embodiment, the gvcMMAE mixture in the THF / MTBE mixture is stirred for about 10 minutes to about 2 hours, for example, about 30 minutes to about 1.5 hours, for example, about 1 hour. In one embodiment, the stirring speed is about 10 rpm to about 1000 rpm.

[0085] In one embodiment, the gvcMMAE precipitate is filtered off as a wet filter cake and rinsed with MTBE. In one embodiment, the gvcMMAE precipitate is rinsed with about 3 to about 10 kg / kg of MTBE. In one embodiment, the gvcMMAE precipitate is rinsed with about 3 to about 5 kg / kg of MTBE. In one embodiment, the wet filter cake gvcMMAE is rinsed with 10 volumes of MTBE (where 4 volumes of the 0.25:3.75 v / v DMA / THF mixture).

[0086] In one embodiment, the gvcMMAE precipitate is rinsed with MTBE and then dried at approximately 35°C to approximately 45°C. In another embodiment, the gvcMMAE precipitate is rinsed with MTBE and then dried at 40°C. In yet another embodiment, the gvcMMAE precipitate is dried for approximately 1 hour to approximately 72 hours, for example, approximately 5 hours to approximately 48 hours, for example, approximately 15 hours to approximately 32 hours, for example, approximately 24 hours.

[0087] In one embodiment, therefore, the method of the present invention is: i) Dissolve 1 equivalent of vc-PAB-MMAE in approximately 3-3.9 kg / kg of THF and approximately 0.2-0.3 kg / kg of DMA at 15-25°C; ii) Add approximately 1 to 1.1 equivalents of glutaric acid anhydride and approximately 1.3 to 1.5 equivalents of TEA at 15 to 25°C; iii) Add approximately 0.002 to 0.01 kg / kg of water at 15 to 25°C to stop the reaction and obtain the product solution; iv) Transfer the product solution to a mixture of approximately 20-25 kg / kg of THF and approximately 35-39 kg / kg of MTBE at approximately -5 to approximately +5°C; v) Isolate the product by filtration; vi) Wash the product with 3-5 kg / kg of MTBE as desired and / or dry the product, for example, under vacuum. This includes the following.

[0088] One appeal, one method: i) Dissolve 1 equivalent of vc-PAB-MMAE in approximately 3-3.9 kg / kg of THF and approximately 0.2-0.3 kg / kg of DMA at 15-25°C while stirring; ii) Add approximately 1 to 1.1 equivalents (e.g., approximately 1.1 equivalents) of glutaric acid anhydride and approximately 1.3 to 1.5 equivalents (e.g., approximately 1.3 equivalents) of TEA at 15 to 25°C; and stir the reaction mixture for approximately 0.5 to 1.5 hours until the reaction is complete; iii) Add approximately 0.002 to 0.01 kg / kg of water while stirring at 15 to 25°C to stop the reaction and obtain the product solution; iv) Transfer the product solution dropwise to a mixture of approximately 20-25 kg / kg of THF and approximately 35-39 kg / kg of MTBE at approximately -5 to approximately +5°C; v) Isolate the product by filtration; vi) Wash the product with approximately 3-5 kg / kg of MTBE; and then dry the product under vacuum at approximately 35-45°C. This includes the following.

[0089] In one embodiment, the method of the present invention: i) Dissolve 1 equivalent of vc-PAB-MMAE in a solvent containing DMA, THF, and MTBE in a volume ratio of approximately 1:A:B DMA:THF:MTBE (where A is approximately 100 to approximately 130 (e.g., approximately 115); and B is approximately 100 to approximately 300 (e.g., approximately 200) at approximately 15 to 25°C; ii) Add approximately 1 to 1.1 equivalents (e.g., approximately 1.1 equivalents) of glutaric anhydride and approximately 1.3 to 1.5 equivalents (e.g., approximately 1.3 equivalents) of TEA at 15 to 25°C to obtain a product solution; iii) Precipitate gvcMMAE from the product solution; iv) Isolate the product by filtration; and v) Wash the product with approximately 3-5 kg / kg of MTBE, if desired; and / or dry the product, for example, under vacuum. This includes the following.

[0090] One appeal, one method: i) Dissolve 1 equivalent of vc-PAB-MMAE in a solvent containing DMA, THF, and MTBE (where, for example, DMA is present at approximately 0.2 to 0.3 V (e.g., approximately 25 V) relative to vcMMAE; THF is present at approximately 25 to 35 V (e.g., approximately 28 to 29 V); and DMA is present at approximately 30 to 80 V (e.g., approximately 50 V); ii) Add about 1 to 1.1 equivalents (e.g., about 1.1 equivalents) of glutaric acid anhydride and about 1.3 to 1.5 equivalents (e.g., about 1.3 equivalents) of TEA at 15 to 25°C; then stir the reaction mixture for, for example, about 1 to 3 hours until the reaction is complete to obtain a product solution; iii) Precipitate gvcMMAE from the product solution (for example, by adding the product solution dropwise to a mixture of approximately 20-25 kg / kg of THF and approximately 35-39 kg / kg of MTBE at approximately -5 to approximately +5°C); iv) Isolate the product by filtration; and v) Wash the product with approximately 3-5 kg / kg of MTBE; and then dry the product under vacuum at approximately 35-45°C. This includes the following.

[0091] In one embodiment, the present invention provides the compound gvcMMAE or a salt thereof. In another embodiment, the present invention provides the compound gvcMMAE obtained or obtainable by the method disclosed herein.

[0092] In one embodiment, the present invention relates to formula III: [ka] The present invention provides compounds or salts thereof.

[0093] In one embodiment, the present invention provides a composition comprising gvcMMAE or a salt thereof, and further comprising a compound of formula III or a salt thereof as an impurity. In one embodiment, the composition comprising gvcMMAE or a salt thereof contains less than about 1% of a compound of formula III or a salt thereof. In one embodiment, the composition comprising gvcMMAE or a salt thereof contains about 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or less than 0.1% of a compound of formula III or a salt thereof.

[0094] [ka] [ka]

[0095] In one embodiment, the present invention provides a method for synthesizing a bicyclic toxin conjugate (BTC), comprising reacting a gvcMMAE with a dicyclic compound.

[0096] In one embodiment, the dicyclic compound is a bicyclic peptide. In one embodiment, the dicyclic compound is a constrained bicyclic peptide that binds to nectin-4 with high affinity and specificity. In one embodiment, the bicyclic peptide is selected from those described in international patent application PCT / GB2019 / 051740 (international publication WO2019 / 243832), which is incorporated herein by reference in its entirety.

[0097] In one embodiment, the bicyclic peptide is a constrained bicyclic peptide that binds to Eph receptor tyrosine kinase A2 (EphA2) with high affinity and specificity. In one embodiment, the bicyclic peptide is selected from those described in international patent applications PCT / GB2018 / 053675 (International Publication WO2019 / 122860) and PCT / GB2018 / 053678 (International Publication WO2019 / 122863), which are incorporated herein by reference in their entirety.

[0098] In one embodiment, the bicyclic peptide is: [ka] [In the formula, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , and R 9 Each of them independently contains hydrogen or C 1-6 The group is optionally substituted with aliphatic, 3-8 member saturated or partially unsaturated monocyclic carbocyclic rings, phenyl, 8-10 member bicyclic aromatic carbocyclic rings, 4-8 member saturated or partially unsaturated monocyclic heterocyclic rings having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur, 5-6 member monocyclic heteroaromatic rings having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or 8-10 member bicyclic heteroaromatic rings having 1-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur. That is the case.

[0099] One reason, R 1 C is substituted with hydrogen or, if desired. 1-6 It is aliphatic. In one embodiment, R 1 teeth [ka] That is the case.

[0100] One reason, R 2 C is substituted with hydrogen or, if desired. 1-6 It is aliphatic. In one embodiment, R 2 teeth [ka] That is the case.

[0101] One reason, R 3 C is substituted with hydrogen or, if desired.1-6 It is aliphatic. In one embodiment, R 3 teeth [ka] That is the case.

[0102] One reason, R 4 C is substituted with hydrogen or, if desired. 1-6 It is aliphatic. In one embodiment, R 4 teeth [ka] That is the case.

[0103] One reason, R 5 C is substituted with hydrogen or, if desired. 1-6 It is aliphatic. In one embodiment, R 5 teeth [ka] That is the case.

[0104] One reason, R 6 C is substituted with hydrogen or, if desired. 1-6 It is aliphatic. In one embodiment, R 6 teeth [ka] That is the case.

[0105] One reason, R 7 C is substituted with hydrogen or, if desired. 1-6 It is aliphatic. In one embodiment, R 7 teeth [ka] That is the case.

[0106] One reason, R 8C is substituted with hydrogen or, if desired. 1-6 It is aliphatic. In one embodiment, R 8 teeth [ka] That is the case.

[0107] One reason, R 9 C is substituted with hydrogen or, if desired. 1-6 It is aliphatic. In one embodiment, R 9 teeth [ka] That is the case.

[0108] In one embodiment, the diring is given by formula II: [ka] It is a substance or a salt thereof, where R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , and R 9 Each of these, either individually or in combination, is defined below and described in the embodiments herein, where m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0109] In one embodiment, m is 0. In one embodiment, m is 1. In one embodiment, m is 2. In one embodiment, m is 3. In one embodiment, m is 4. In one embodiment, m is 5. In one embodiment, m is 6. In one embodiment, m is 7. In one embodiment, m is 8. In one embodiment, m is 9. In one embodiment, m is 10. In one embodiment, m is 11. In one embodiment, m is 12. In one embodiment, m is 13. In one embodiment, m is 14. In one embodiment, m is 15.

[0110] In one embodiment, the bicyclic toxin conjugate is of formula I: [ka] The substance thereof, or a pharmaceutically acceptable salt thereof, where R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 Each of , and m is defined below and described in the embodiments herein, both individually and in combination.

[0111] In one embodiment, the present invention provides a method for synthesizing a bicyclic toxin conjugate of formula I (BTC), comprising reacting gvcMMAE with a bicyclic compound of formula II.

[0112] In one embodiment, the bicyclic toxin conjugate of formula I is BT8009 or a pharmaceutically acceptable salt thereof.

[0113] In one embodiment, the bicyclic toxin conjugate of formula I is BT5528 or a pharmaceutically acceptable salt thereof.

[0114] In one embodiment, the present invention provides a bicyclic toxin conjugate (or a salt thereof) obtained or obtainable by the methods disclosed herein. Also provided are compositions comprising a bicyclic toxin conjugate or a salt thereof, comprising less than 1% (e.g., about 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or less than 0.1%) of a compound of formula III or a salt thereof. [Examples]

[0115] The following examples illustrate the present invention; however, they are not intended to limit the scope of the invention in any way. The beneficial effects of the pharmaceutical compounds, combinations, and compositions of the present invention can also be determined by other test models known to those skilled in the art.

[0116] A list of common abbreviations used in the experimental section. A%- Area% aq. - Water-based Con.-Dense hexane / EA - ethyl acetate DMA N,N-dimethylacetamide DIEA N,N-diisopropylethylamine TEA (Triethylamine) THF (Tetrahydrofuran) DCM Dichloromethane 2-MeTHF 2-methyltetrahydrofuran MeCN / Cacetonitrile MTBE methyl tert-butyl ether iPrOAc Isopropyl Acetate eq. - equivalent amount equip. - equipment g - gram GMP - Good Manufacturing Practice Non-GMP - Non-Good Manufacturing Practices h - time HCl - Hydrochloric acid HSGC - Headspace Gas Chromatography IC - Ion chromatography ICP-MS - Inductively Coupled Plasma Mass Spectrometry IMP - Impurities IPC - In-process control IR - Infrared absorption spectrum KF - Carl Fischer (Water selection) kg - kilogram L - liter LCMS - Liquid Chromatography Mass Spectrometry MBR - Manufacturing Batch Record mm - millimeter N-mol NaCl - Sodium Chloride NaHCO3- Sodium bicarbonate H2SO4- Sulfuric acid ND - Not detected PLM - Polarizing Microscope QC - Quality Control Spec. - Specifications STA - synTheAll H2O - Water a / a - per region COA - Certificate of Analysis Eq. - Equivalent FIO - Information only GC - Gas Chromatography HPLC - High-Performance Liquid Chromatography NaOH - Sodium hydroxide pH - Hydrogen ion concentration EtOH - Ethanol ML / ML'S mother liquor min - minutes mL - milliliter mol - mole NLT - and above NMT - below PO - Purchase Order ppm - parts per million RRT - Relative Retention Time RS - Residual solvent RT - room temperature STA - synTheAll Vol - volume w / w - weight ratio TP - Technical Package

[0117] Example 1: Production of gvcMMAE 1. Overview The method for gvcMMAE was well optimized, yielding 36.53 g of product with 98.56 A% HPLC purity and a corrected yield of 97.17%. All test items met the specifications.

[0118] 2. Introduction and Synthesis Scheme Regarding the reaction process: The reaction conditions were optimized as follows: the amount of glutaric acid anhydride was reduced from 1.2 equivalents to 1.1 equivalents; 5.2 equivalents of DIEA were replaced with 1.3 equivalents of TEA; a DMA / THF mixed solvent was used as the reaction solvent. In some studies, the reaction temperature was lowered to 0°C. The IPC under these conditions was the same as under the original conditions, but it was more stable and suitable for workup. In some studies, the reaction temperature was raised to approximately 15-25°C.

[0119] Regarding the post-treatment process: The reaction solution was directly placed in a 75V THF / MTBE (1:2) solution, and the solid precipitated in good condition.

[0120] The method is well-developed. For the system using TEA as the base, a clear solution was obtained at 0°C, so the room temperature reaction temperature was not tested. If the IPC is good at RT and the reaction mixture is stable, a reaction at 0°C may not be necessary.

[0121] 3. Laboratory work 3.1. Scheme 1: [ka]

[0122] 3.2. Overview In the original method, while IPC purity was excellent, the reaction mixture was found to be unstable when held for extended periods. Furthermore, the workup step in the original method was unsuitable for scale-up due to wall adhesion. After method optimization, more stable reaction conditions were developed. The reaction solution was directly added to a poor solvent (THF:MTBE = 1:2, 75V) to obtain a good solid state. The new method is suitable for scale-up.

[0123] 3.3. Method Development 3.3.1 Thorough knowledge of TP methods (Table 1) A 0.26 g vcMMAE (assay-corrected) reaction was performed to familiarize ourselves with the method. Excellent 99.5A% IPC purity was obtained after 1 hour of stirring. However, the IPC purity decreased to 94.5A% after 16 hours of stirring. The result indicated that the reaction mixture was not stable under these conditions. Further investigation of the reaction conditions (base / anhydrous packing, reaction temperature) resolved the reaction stability issue. If the results are reproducible and stable, the only concern is how to isolate the solid product.

[0124] Regarding work-up, after adding the reaction solution to an acidic saturated salt aqueous solution (70V), a solid precipitated, but it rapidly became viscous. The results indicated that DMA / brine was not a good crystallization system, and therefore, other solvents needed to be tried to find the best crystallization conditions. Since high-boiling point DMA can be removed when replaced with other solvents, low-boiling point solvents (DCM, MeCN, THF, and 2-MeTHF) were considered as reaction solvents to see if the same IPC results could be obtained. In addition, if DMA proved to be the only option for the reaction, a low volume of DMA (3V) was also tried, as it could be advantageous for efficient work-up (reduced water for precipitation, reduced organic solvent for extraction or precipitation). [Table 1]

[0125] 3.3.2. Solvent screening (Tables 2 and 3) Five different reaction solvents (DCM, MeCN, THF, 2-MeTHF, and DMA / 3v) were tested. Among them, DMA gave the best result with 98.98 A% IPC purity. DCM and THF also gave good IPC purity for gvcMMAE. For MeCN as the solvent, sticky solids were observed during the reaction. If it is proven that there is no good precipitation method when using DMA, DCM and THF can be tested as alternative solvents to DMA. Furthermore, mixed solvent systems (DMA:THF = 1:3, DMA:ACN = 1:3) were also tested (after basic equivalent and temperature screening), and acceptable IPC results were also obtained.

Table 2

[0126]

Table v

[0127] 3.3.3. Basic equivalent and temperature screening (Tables 4 and 5) Three reactions with different equivalents of base (1.3 equivalents, 2.4 equivalents, and 5.2 equivalents) were carried out at low temperature (-5 to 5 °C). The results showed that 1.3 equivalents of DIEA was sufficient to achieve the completion of the reaction. By extending the reaction time to 16 hours, the IPC purity decreased only slightly, indicating that the reaction was sufficiently stable at -5 to 5 °C for 16 hours.

[0128] The reactions in DCM and THF solvents were also tested at -5 to 5 °C. At low reaction temperatures, the IPC purity was still relatively high, and the mixture also had good stability. -5 to 5 °C and 1 hour were determined for the reaction.

Table 4

[0129]

Table 5

[0130] 3.3.4. Screening of glutaric acid anhydride filling (Tables 6 and 7) Different equivalents of glutaric anhydride (1.05 equivalents and 1.10 equivalents) were screened. 1.10 equivalents of glutaric anhydride is sufficient for complete conversion of the reaction. [Table 6]

[0131] [Table 7]

[0132] 3.3.5. First Round Method Optimization (Tables 8 and 9) We developed small variations of the method using a DMA / THF mixed solvent system and a sulfuric acid / 5% Na2SO4 solution termination system.

[0133] This method was validated by performing a batch reaction of 5 g of vcMMAE. After the reaction, the IPC purity was 99.37 A%. The reaction solution was inversely added to a 30V sulfuric acid (1.5 equivalents) / 5% Na2SO4 solution at -5 to 5°C. After filtration and drying, 7.0 g of crude product (containing Na2SO4) was obtained with 98.71 A% HPLC purity. Approximately 6.36 g of crude gvcMMAE was further diluted with 30V water to form a slurry to remove Na2SO4. After filtration and drying, 4.2 g of gvcMMAE was obtained with 98.89 A% HPLC purity and an 84% crude yield.

[0134] IC results show residual SO4 in gvcMMAE 2- This indicates that the concentration was 0.70%, which shows that the product is in a free acid state. Residual Na + The percentage was 0.37%. This indicated that X-RPD should be amorphous.

[0135] DVS testing showed that the product was hygroscopic at 25°C and 80% RH (adsorption curve between 40% RH and 80% RH at 25°C, showing 3.6% water uptake). [Table 8]

[0136] [Table 9]

[0137] 3.3.6. Solubility tests of gvcMMAE in different solvents (Table 10) The solubility of gvcMMAE (free acid state) was investigated in different solvents (THF, DCM, MTBE, n-heptane, iPrOAc, 1,4-dioxane). The assay results showed that gvcMMAE in its free base state is insoluble in most solvents. [Table 10]

[0138] 3.3.7. Salt formation test of free acid (Table 11) Five bases (calcium hydroxide, dicyclohexylamine, DABCO, tributylamine, and barium hydroxide) were tested for salt formation of gvcMMAE; THF was used as the solvent. For the dicyclohexylamine and tributylamine systems, the product was milky after base addition, and the purity of the mixture decreased to approximately 96A%, with 2A% impurities observed at RRT1.76 (the mixture was concentrated to approximately 30V, and MTBE at 60V was added, causing the solid to precipitate. The purity of the wet solid precipitated from the THF / MTBE / tributylamine system was 98.87A%, and the assay at ML was 0.01%. It was considered worthwhile to try adding MTBE to the THF / TEA or THF / tributylamine reaction solution to obtain a solid.) For the DABCO system, the product dissolved rapidly, but after stirring for 0.5 hours, it became jelly-like, and the purity of the mixture decreased significantly. In the barium hydroxide system, a solid precipitate formed after 1 hour of stirring, and the purity of the wet cake decreased to 96.32%. In the calcium hydroxide system, the product was milky after 1 hour of stirring.

[0139] Due to this decrease, tributylamine and TEA (similar properties) were considered as bases for the reaction.

Table 11

[0140] 3.3.8. Test of salt formation by using tributylamine or TEA (Tables 12 and 13) Four reactions with 0.3 g scale of vcMMAE were carried out using different base / solvent systems (base: tributylamine, TEA; solvent: THF, THF / DMA mixed solvent) to test the separation of gvcMMAE salts.

[0141] For the single solvent system (using THF as the solvent), the reaction solution became colloidal rapidly after the addition of the base (tributylamine as the base: turbid colloidal; TEA as the base: transparent colloidal).

[0142] For the THF / DMA / tributylamine system, the reaction solution was transparent. Then, 30 v of THF was added dropwise to the reaction solution for dilution, and the system became jelly-like (less severe than the single solvent).

[0143] For the THF / DMA / TEA system, the reaction solution was transparent. When 30 v of THF was added to the reaction solution and the solvent became concentrated, it began to become turbid. When 60 v of MTBE was added to the mixture, solids precipitated. After filtration, 0.275 g of solid was obtained with a crude yield of 78.4% and an HPLC purity of 98.64 A%.

[0144] The THF / DMA / TEA reaction system and the THF / MTBE post-treatment system were further investigated.

Table 12

[0145]

Table 13

[0146] 3.3.9. Testing DMA ratios to gain profit in the post-processing stage (Tables 14 and 15) Three reactions with different DMA:THF ratios were performed in a 0.3g vcMMAE to test suitable workup conditions for the isolation of the TEA salt. Good IPC results (99.14A%) were obtained when DMA:THF = 0.25v:3.75v.

[0147] Under the original reaction conditions (DMA:THF = 1:3), when 30V THF was added directly to the reaction solution, the solution became concentrated with the risk of colloidalization, making it unsuitable for scale-up. Therefore, the reverse addition of the reaction solution to the solvent was considered.

[0148] As a result, when the other two reaction solutions (DMA:THF = 0.5:3.5 and DMA:THF = 0.25:3.75) were added to a pre-mixed solvent at 90V (30V THF + 60V MTBE), a solid in good condition precipitated. [Table 14]

[0149] [Table 15]

[0150] 3.3.10 Testing of THF / MTBE volume and ratio (Tables 16 and 17) Three reactions were carried out with 1.0 g vcMMAE to test the volume of poor solvent (THF:MTBE = 1:2; 90v, 75v, and 60v were tested). After filtration and vacuum drying, 1.1 g of gvcMMAE TEA salt (90v post-treatment batch) was obtained with 98.98A% HPLC purity and 92.4% crude yield (residual solvent by HSGC: THF: 0.83%, MTBE: 5.23%, DMA: 1.35%; residual TEA by IC: 1.8%).

[0151] A moisture absorption experiment was conducted by leaving 100 mg of gvcMMAE standing under 60% RH conditions. The solid state remained unchanged after 3 days (the weight changed to 103 mg).

[0152] Various THF / MTBE ratios were also tried (THF:MTBE=1:3, THF:MTBE=1:4, 45V and 60V), but no favorable results were observed. The solid adhered to the wall after extending the stirring time (16 hours).

[0153] Based on volume and solid state, 75V THF / MTBE was determined to be the best option. [Table 16]

[0154] [Table 17]

[0155] 3.3.11. Testing of post-treatment at low temperatures (Tables 18 and 19) Workup of four batches was performed at -5 to 5°C in various volumes of poor solvent (THF:MTBE = 1:2; 30V, 45V, 60V, 75V). The 75V / -5 to 5°C condition yielded the best results with a good solid state, which did not adhere to the walls even after extended stirring time (16 hours). [Table 18] [Table 19]

[0156] 3.3.12. Demonstration conjugation study of gvcMMAE and peptides (Tables 20 and 21) Two demonstration reactions were performed using 0.1g peptide (BCY8234, 93.76% HPLC purity). For gvcMMAE prepared from a sulfur / 5% Na2SO4 solution, the IPC purity was 80.34%. After workup, the purity of the wet cake was 80.36%; for gvcMMAE prepared under TEA conditions, the IPC purity was 86.51%. After workup, the purity of the wet cake was 86.25%. The results indicate that gvcMMAE from TEA solution yielded good IPC purity. [Table 20] [Table 21]

[0157] 3.3.13. Stress tests of gvcMMAE at various drying temperatures (Tables 22 and 23) Stress tests were conducted on gvcMMAE at various drying temperatures. According to the results shown below, gvcMMAE was stable at 40°C for 3 days. [Table 22] [Table 23]

[0158] 3.3.14. Second Round Method Optimization (Tables 24 and 25) A robust method was developed using DMA / THF / TEA as the reaction system and THF / MTBE as the post-treatment system.

[0159] This method was validated by performing a single batch of 5g vcMMAE. The IPC purity was 98.37A%. After post-treatment, a good solid state was obtained, and the purity of the wet cake was 98.60A%. After vacuum drying, 6.03g (approximately 0.2g for stress testing, theoretical amount: 5.51g) of gvcMMAE was obtained, with an HPLC purity of 98.59A% (residual: TEA: 4.83%; THF: 0.01%; MTBE: 9.65%; DMA: 0.54%). [Table 24] [Table 25]

[0160] 3.4. Scaling up using optimization methods 3.4.1. Raw Material Usage Tests (Table 26) A vcMMAE usage test was conducted using 1g of vcMMAE. IPC was normal: gvcMMAE: 98.61A%. After post-treatment, a good solid state was obtained, and the purity of the wet cake was 98.45A%. After vacuum drying at 40°C for 16 hours, 1.12g (theoretical amount: 1.10g) of gvcMMAE was obtained, with an HPLC purity of 98.57A% (residual: TEA: 4.81%; THF: 0.07%; MTBE: 7.30%; DMA: 0.46%). After further vacuum drying at 40°C for 16 hours, the purity of gvcMMAE was 98.47A% (residual: TEA: 4.43%; THF: 0.05%; MTBE: 7.04%; DMA: 0.34%). [Table 26]

[0161] 3.4.2. Scaling up the first batch (Table 27) A single scale-up batch was performed using 10g of vcMMAE. IPC was normal: gvcMMAE: 98.53A%. After workup, 11.76g of gvcMMAE was obtained with a corrected yield of 95.64% at HPLC purity of 98.51A% (assay: 89.6%, residual: TEA: 3.10%; THF: 0.93%; MTBE: 3.1%; DMA: 1.4%; glutaric anhydride: 0.06%; KF: 0.57%). [Table 27]

[0162] 3.4.3. Scaling up the second batch (Tables 28 and 29) A single scale-up batch was performed with 30g of vcMMAE. IPC was normal: gvcMMAE: 98.51A%. After workup, 36.53g (approximately 0.3g for RS / IC testing) of gvcMMAE was obtained with a corrected yield of 97.17% at HPLC purity of 98.56A% (assay: 87.9%, residuals: TEA: 4.5%; THF: 1.8%; MTBE: 2.5%; DMA: 2.1%; glutaric anhydride: 0.09%; KF: 0.45%). [Table 28] [Table 29]

[0163] 3.5. Identification of Impurities LC-MS was performed to identify impurities. The possible structure of the impurity RRT 0.90 is shown below. The structures of other impurities remain unknown. [ka]

[0164] [Table 30]

[0165] 3.6. Typical method 1. Add DMAC / THF (120 mL, 0.25:3.75 v / v, 4.0 v) to R1 (reaction vessel 1). 2. Add vcMMAE (30g, assay corrected, 1.0 equivalent) to R1. 3. Adjust R1 to -5 to 5°C. 4. Stir R1 for 0.1 hours at -5 to 5°C. 5. Add glutaric acid anhydride (3.36 g, 1.1 equivalents) to R1. 6. Add TEA (3.54g, 1.3 equivalents) to R1. 7. Stir R1 for 1 hour at -5 to 5°C. 8. Sample collection for analysis (purity of IPCgvcMMAE). 9. Add THF (750 mL, 25V) to R2 (reaction vessel 2). 10. Add MTBE (1500 mL, 50V) to R2. 11. Adjust R2 to -5 to 5°C. 12. Stir R2 for 0.1 hours at -5 to 5°C. 13. Heat R1 to 15-25°C. (The solution will become clear when heated to room temperature.) 14. Transfer the solution from R1 to R2 by dropwise addition over 1 hour. 15. Stir R2 for 1 hour at -5 to 5°C. 16. Filter and rinse the wet cake with MTBE (300 mL, 10 V). 17. Dry the moist cake at 40°C for 16-32 hours. 18. Obtain samples for release testing.

[0166] 3.6. Further Method Development The reaction using 3.3 g of vcMMAE (1 equivalent) was carried out with 1.5 equivalents of glutaric acid anhydride. The impurity levels in the gvcMMAE product corresponding to the compound of formula (III) were quantified. Increasing the reaction temperature from 15 to 25°C resulted in an IPC purity of 99.13A% gvcMMAE in the reaction solution (row A; Table 31). Crystallization with THF / MTBE (row B; Table 31) resulted in a concentration of compound (III) of formula (III) in the product gvcMMAE of 0.33 A% (wet), which increased to 0.71 A% after drying. Experiments rinsing a dried cake with glutaric acid anhydride solution (THF / MTBE(1 / 2): 0.1 equivalents of anhydride in 3V) increased the level of compound III, suggesting that at least some of the impurities of formula III may be due to excess glutaric acid anhydride (though not bound by theory). However, experiments rinsing glutaric acid anhydride with solvent MTBE(10V), first MTBE / THF=2 / 1:5V, then MTBE:5V; or first THF:5V, then MTBE:5V, also increased the level of compound III. Termination of the reaction with water before crystallization (0.5 equivalents; 10 minutes; 15-25°C) yielded a significant improvement, with the crystallized gvcMMAE product containing only 0.52 A% of formula (III) (Table 31, row C). Although not bound by theory, this may demonstrate that termination of the reaction with water before crystallization prevents the reaction with excess glutaric anhydride.

[0167] [Table 31]

[0168] Further experiments were conducted using 1.4 g of vcMMAE (1 equivalent) and 1.15 equivalents of glutaric anhydride. The reaction was carried out using DMA / THF / MTBE (0.25 / 28.5 / 50V) as the reaction solvent. The impurity level in the gvcMMAE product corresponding to the compound of formula (III) was quantified. The suspension was in good condition, and the impurity level attributable to the compound of formula (III) did not increase with extended reaction time. The impurity levels corresponding to the further impurities shown in Table 31 were quantified. The impurity levels decreased significantly to approximately 0.17-0.18% and did not increase with extended reaction time. See Table 32.

[0169] [Table 32]

[0170] While several embodiments of the present invention are described, it is clear that modifications to the basic embodiments may provide other embodiments utilizing the compounds and methods of the present invention. Therefore, it is clear that the scope of the present invention is defined not by the specific embodiments represented in the exemplary methods, but by the appended claims.

[0171] The following are numbered embodiments of the present invention: 1. Glutaryl-Val-Cit-PAB-MMAE (gvcMMAE): [ka] or a method for synthesizing a salt thereof, wherein glutaric anhydride is dissolved in a solvent containing N,N-dimethylacetamide (DMA) and tetrahydrofuran (THF) in vcMMAE: [ka] A method comprising adding to a solution containing and obtaining a reaction reaction.

[0172] 2. The method of Embodiment 1, wherein the solvent is approximately 0.25:3.75 v / v DMA and THF (0.25:3.75 v / v DMA / THF).

[0173] 3. The method according to embodiment 1 or 2, wherein the amount of glutaric acid anhydride is about 1.1 equivalents and the amount of vcMMAE is about 1 equivalent.

[0174] 4. Any method according to embodiment 1 to 3, further comprising adding approximately 1.3 equivalents of TEA to the reaction mixture.

[0175] 5. Any of the methods described in Embodiments 1 to 4, wherein the reaction between vcMMAE and glutaric anhydride is carried out at approximately -5°C to approximately 5°C.

[0176] 6. The reaction mixture is stirred at approximately -5°C to approximately 5°C for about 1 hour to complete the reaction, thereby obtaining a mixture mainly consisting of gvcMMAE or a mixture of gvcMMAE, according to the method of embodiment 5.

[0177] 7. The method of embodiment 6, wherein the gvcMMAE mixture is heated to approximately 15°C to approximately 25°C.

[0178] 8. The method of embodiment 6, wherein the gvcMMAE mixture is heated until it becomes a clear solution.

[0179] 9. The method according to embodiment 7 or 8, wherein the gvcMMAE mixture is added to a mixture of THF and MTBE to obtain gvcMMAE as a precipitate.

[0180] 10. The method of embodiment 9, wherein the mixture of THF and MTBE is at approximately -5°C to approximately 5°C.

[0181] 11. The method according to embodiment 9 or 10, wherein the mixture of THF and MTBE is approximately 1:2 v / v THF and MTBE (1:2 v / v THF / MTBE).

[0182] 12. The method of embodiment 11, wherein the volume ratio of the 0.25:3.75 v / v DMA / THF mixture to the 1:2 v / v THF / MTBE mixture is 4:75.

[0183] 13. Any method according to embodiment 9 to 12, further comprising filtering off the gvcMMAE precipitate as a moist filter cake and rinsing the moist filter cake with MTBE.

[0184] 14. Furthermore, the diring of gvcMMAE and equation II: [ka] By reacting them, the bicyclic toxin conjugate of formula I is formed: [ka] [During the ceremony, R 1 , R 2 , R3 , R 4 , R 5 , R 6 , R 7 , R 8 , and R 9 Each of them independently contains hydrogen or C 1-6 A group optionally substituted from aliphatic, 3-8 member saturated or partially unsaturated monocyclic carbocyclic rings, phenyl, 8-10 member bicyclic aromatic carbocyclic rings, 4-8 member saturated or partially unsaturated monocyclic heterocyclic rings having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur, 5-6 member monocyclic heteroaromatic rings having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or 8-10 member bicyclic heteroaromatic rings having 1-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur; and m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. A method according to any one of embodiments 1 to 13, comprising forming a pharmaceutically acceptable salt thereof.

[0185] 15. The method of embodiment 14, wherein the bicyclic toxin conjugate of formula I is BT8009 or a pharmaceutically acceptable salt thereof.

[0186] 16. The method of embodiment 14, wherein the bicyclic toxin conjugate of formula I is BT5528 or a pharmaceutically acceptable salt thereof.

Claims

1. Glutaryl-Val-Cit-PAB-MMAE (gvcMMAE): 【Chemistry 1】 or a method for synthesizing a salt thereof, wherein glutaric anhydride is dissolved in a solvent containing N,N-dimethylacetamide (DMA) and tetrahydrofuran (THF) with vcMMAE: 【Chemistry 2】 A method comprising adding to a solution containing and obtaining a reaction reaction.

2. The method of claim 1, wherein the solvent comprises THF and DMA; therein, the THF is present in an amount of about 3 to about 3.9 kg / kg (relative to the mass of vcMMAE) and the DMA is present in an amount of about 0.2 to about 0.3 kg / kg (relative to the mass of vcMMAE).

3. The solvent contains approximately 0.2:3.9 to approximately 0.3 to 3 v / v DMA / THF. The method of claim 1 or 2, wherein, optionally, the solvent is approximately 0.25:3.75 v / v DMA and THF (0.25:3.75 v / v DMA / THF).

4. The method according to any one of claims 1 to 3, wherein the solvent further comprises methyl tert-butyl ether (MTBE).

5. The method of claim 1 or 4, wherein the solvent comprises DMA, THF and MTBE; where, for example, with respect to vcMMAE, DMA is present at about 0.2 to about 0.3 V; THF is present at about 25 to about 35 V; and DMA is present at about 30 to about 80 V.

6. This involves reacting vcMMAE with approximately 1 to approximately 1.1 equivalents of glutaric acid anhydride; The method according to any one of claims 1 to 5, wherein, if desired, the amount of glutaric anhydride is about 1.1 equivalents and the amount of vcMMAE is about 1 equivalent.

7. The reaction mixture contains about 1.3 to about 1.5 equivalents of triethylamine (TEA); The method of any one of claims 1 to 6, wherein the method optionally includes adding about 1.3 equivalents of TEA to the reaction mixture.

8. The method according to any one of claims 1 to 7, wherein the reaction between vcMMAE and glutaric anhydride is carried out at a temperature of about -5°C to about 25°C.

9. The method according to any one of claims 1 to 8, wherein the reaction between vcMMAE and glutaric anhydride is carried out at a temperature of about 15°C to about 25°C.

10. A method according to any one of claims 1 to 9, comprising stirring the reaction mixture during the reaction.

11. The method according to any one of claims 1 to 10, wherein the reaction mixture is stirred for about 1 hour at about -5°C to about 25°C to complete the reaction, and optionally a mixture mainly comprising gvcMMAE or a mixture of gvcMMAE is obtained.

12. The reaction between vcMMAE and glutaric acid anhydride is carried out in water (H ２ A method according to any one of claims 1 to 11, comprising stopping at O).

13. The reaction stops at approximately 0.002 to 0.01 kg / kg of water (H ２ A method according to any one of claims 1 to 12, comprising the addition of O).

14. The method of claim 12 or 13, wherein the reaction between vcMMAE and glutaric anhydride is stopped with water at a temperature of about 15 to about 25°C.

15. The method according to any one of claims 12 to 14, wherein stopping the reaction includes stirring the reaction mixture.

16. Heat the gvcMMAE mixture to approximately 15°C to 25°C; If desired, the gvcMMAE mixture is then heated until it becomes a clear solution. The method according to any one of claims 1 to 15.

17. The method according to any one of claims 1 to 16, wherein a gvcMMAE mixture is added to a mixture of THF and methyl tert-butyl ether (MTBE) to obtain gvcMMAE as a precipitate.

18. The method of claim 17, wherein the mixture of THF and MTBE is at a temperature of about -5°C to about 5°C.

19. The method of claim 17 or 18, wherein the mixture of THF and MTBE comprises about 20 to about 25 kg / kg of THF (relative to the mass of vcMMAE) and about 35 to about 39 kg / kg of MTBE (relative to the mass of vcMMAE).

20. The mixture of THF and MTBE contains approximately 20:39 to approximately 25:30 v / v THF / MTBE; If desired, the mixture of THF and MTBE is approximately 1:2 v / v THF and MTBE (1:2 v / v THF / MTBE). The method according to any one of claims 17 to 19.

21. The method according to any one of claims 17 to 20, wherein the volume ratio of the DMA / THF mixture to the THF / MTBE mixture is about 4:20 to about 4:200, for example, about 4:

75.

22. The method according to any one of claims 17 to 21, further comprising filtering off the gvcMMAE precipitate as a moist filter cake and rinsing the moist filter cake with MTBE.

23. Furthermore, gvcMMAE and the diring compound of formula II: 【Transformation 3】 By reacting them, the bicyclic toxin conjugate of formula I is formed: 【Chemistry 4】 [During the ceremony, R １ 、 R ２ 、 R ３ 、 R ４ 、 R ５ 、 R ６ 、 R ７ 、 R ８ 、 and R ９ each of which is independently hydrogen or a group optionally substituted from C １－６ aliphatic, a 3- to 8-membered saturated or partially unsaturated monocyclic carbocyclic ring, phenyl, an 8- to 10-membered bicyclic aromatic carbocyclic ring, nitrogen, oxygen, or sulfur, a 4- to 8-membered saturated or partially unsaturated monocyclic heterocyclic ring having 1 to 2 heteroatoms independently selected therefrom, a 5- to 6-membered monocyclic heteroaromatic ring having 1 to 4 heteroatoms independently selected therefrom, or an 8- to 10-membered bicyclic heteroaromatic ring having 1 to 5 heteroatoms independently selected therefrom; and m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. A method according to any one of claims 1 to 22, comprising forming a pharmaceutically acceptable salt thereof.

24. The method of claim 23, wherein the bicyclic toxin conjugate of formula I is BT8009 or a pharmaceutically acceptable salt thereof.

25. The method of claim 23, wherein the bicyclic toxin conjugate of formula I is BT5528 or a pharmaceutically acceptable salt thereof.

26. A compound that is a gvcMMAE obtainable by any of the methods of claims 1 to 22.

27. A bicyclic toxin conjugate of formula (I) that can be obtained by any of the methods of claims 22 to 25.

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