Synthesis of bicyclotoxin conjugates and intermediates thereof

Abstract

The invention relates to a method for preparing a bicyclotoxin conjugate and an intermediate thereof.

Description

Field of the invention

[0001] The present invention relates to methods of synthesizing gvcMMAE, and methods of synthesizing bicyclic toxin conjugates (BTCs) comprising gvcMMAE (glutaryl-Val-Cit-PAB-MMAE) (e.g. BT5528 and BT8009). BACKGROUND

[0002] Cyclic peptides are able to bind to protein targets with high affinity and target specificity, and are thus an attractive class of molecules for the development of therapeutics. In fact, several cyclic peptides have been successfully used in the clinic, like e.g. the antibacterial peptide vancomycin, the immunosuppressive drug cyclosporine or the anticancer drug octreotide (Driggers et al. (2008), Nat Rev Drug Discov 7(7), 608-24). The good binding properties are due to the relatively large interaction surface formed between the peptide and the target and the reduced conformational flexibility of the cyclic structure. Typically, macrocycles bind to surfaces of several hundred square angstroms, like e.g. the cyclic peptide CXCR4 antagonist CVX15 (Wu et al. (2007), Science 330, 1066-71), the cyclic peptide with the Arg-Gly-Asp motif that binds to integrin αVb3 (Xiong et al. (2002), Science 296(5565), 151-5) or the cyclic peptide inhibitor upain-1 (Zhao et al. (2007), J Struct Biol 160(1), 1-10) that binds to urokinase-type plasminogen activator.

[0003] Due to their cyclic conformation, peptide macrocycles are less flexible than linear peptides, leading to a smaller entropic loss upon binding to a target and to a higher potential binding affinity. The reduced flexibility also leads to a lock-in of the target-specific conformation, increasing the binding specificity compared to linear peptides. This effect has been exemplified for the potent and selective inhibitor of matrix metalloproteinase 8 (MMP-8), which loses its selectivity over other MMPs when its cycle is opened (Cherney et al. (1998), J Med Chem 41(11), 1749-51). The advantageous binding properties achieved by macrocyclization are even more pronounced in polycyclic peptides with more than one peptide cycle, like e.g. in vancomycin, nisin and actinomycin.

[0004] ​​​Different research groups have previously linked polypeptides with cysteine residues to synthetic molecular structures (Kemp and McNamara (1985), J. Org. Chem; Timmerman et al. (2005), ChemBioChem). Meloen and colleagues have used tris(bromomethyl)benzene and related molecules to rapidly and quantitatively cyclize multiple peptide loops onto synthetic scaffolds for structure modeling of protein surfaces (Timmerman et al. (2005), ChemBioChem). Methods for generating candidate drug compounds by linking cysteine-containing polypeptides to molecular scaffolds such as, for example, tris(bromomethyl)benzene are disclosed in WO 2004 / 077062 and WO 2006 / 078161.

[0005] Combinatorial approaches based on phage display have been developed to generate and screen large libraries of bicyclic peptides against target of interest (Heinis et al. (2009), Nat Chem Biol 5(7), 502-7 and WO 2009 / 098450). Briefly, combinatorial libraries of linear peptides containing three cysteine residues and two regions of six random amino acids (Cys-(Xaa)6-Cys-(Xaa)6-Cys) are displayed on phage and cyclized by covalently linking the cysteine side chains to a small molecule (tris(bromomethyl)benzene). SUMMARY

[0006] The present invention provides methods of synthesizing glutaroyl-Val-Cit-PAB-MMAE (gvcMMAE).

[0007] The present invention also provides methods of synthesizing bicyclic toxin conjugates (BTCs) comprising gvcMMAE. In some embodiments, the bicyclic toxin conjugate (BTC) is BT5528 or a pharmaceutically acceptable salt thereof. In some embodiments, the bicyclic toxin conjugate (BTC) is BT8009 or a pharmaceutically acceptable salt thereof. DETAILED DESCRIPTION 1. General Description of Certain Aspects of the Invention

[0008] It has now been found that the method of synthesizing glutaroyl-Val-Cit-PAB-MMAE (gvcMMAE) from Val-Cit-PAB-MMAE (vcMMAE) (as shown in Scheme I herein) can be improved for large scale production, including, for example, GMP production. For example, it has now been found that the method can be optimized by: • the equivalent of glutaric anhydride can be reduced from 1.2 eq. to about 1-1.1 eq. (e.g., about 1.1 eq.); • 5.2 eq. DIEA can be changed to about 1.3-1.5 eq. TEA (e.g., about 1.3 eq. TEA); and • DMA / THF mixed solvent can be used as the reaction solvent.

[0009] In some embodiments, the reaction temperature can be about 15 °C to about 25 °C. In some embodiments, the reaction temperature can be lowered to 0 °C.

[0010] This optimized process has been found to provide more stable reaction conditions. And the resulting reaction solution can be added directly to an anti-solvent (e.g., THF:MTBE; e.g., about 20:39 to about 25:30 v / v THF:MTBE, e.g., = about 1:2, e.g., about 75 v) to provide a product (gvcMMAE) in good solid state. This optimized process has been found to be more suitable for scale-up. Further, the disclosed process has been found to generate a gvcMMAE product with improved purity characteristics.

[0011] Thus, in one aspect, the present application provides a method of synthesizing gvcMMAE, the method comprising reacting vcMMAE with glutaric anhydride under conditions as shown in Scheme I or Scheme II herein.

[0012] In another aspect, the present application provides a method of synthesizing a Bicycle Toxin Conjugate (BTC), the method comprising reacting gvcMMAE with Bicycle. 2. Compounds and Definitions

[0013] The compounds of the present application include those generally described above, and are further illustrated by the classes, sub-classes, and species disclosed herein. The following definitions shall apply unless otherwise indicated. The following terms used in the present application shall have the definitions that follow, unless a different meaning is carried by context. thEd.) to identify chemical elements. In addition, general principles of organic chemistry are described in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999, and "March's Advanced Organic Chemistry", 5th Ed., Ed.: Smith, M.B. and March, J., John Wiley & Sons, New York: 2001, the entire contents of each of which are hereby incorporated by reference.

[0014] As used herein, the terms "aliphatic" or "aliphatic group" mean a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is completely saturated or that contains one or more units of unsaturation, or a monocyclic, bicyclic or polycyclic hydrocarbon moiety that is completely saturated or that contains one or more units of unsaturation, but not aromatic, or a combination thereof, wherein the moiety can be attached to the rest of the molecule by a single bond, such as would result from the removal of a hydrogens group, for example. Unless otherwise specified, an aliphatic group contains 1-6 aliphatic carbon atoms. In some embodiments, an aliphatic group contains 1-5 aliphatic carbon atoms. In other embodiments, an aliphatic group contains 1-4 aliphatic carbon atoms. In yet other embodiments, an aliphatic group contains 1-3 aliphatic carbon atoms, and in still other embodiments, an aliphatic group contains 1-2 aliphatic carbon atoms. In some embodiments, "cycloaliphatic" (or "carbocyclic" or "cycloalkyl") refers to a monocyclic, bicyclic or polycyclic hydrocarbon moiety that is completely saturated or that contains one or more units of unsaturation, but not aromatic, having from 3-10 ring members, preferably having 3-7 ring members, which is attached to the rest of the molecule by a single bond, such as would result from the removal of a hydrogen group. 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 groups.

[0015] As used herein, the term "bridged bicyclic" refers to any bicyclic ring system, i.e., carbocyclic or heterocyclic, saturated or partially unsaturated, having at least one bridge. As defined by IUPAC, a "bridge" is an unbranched chain of atoms or atoms or valence bonds connecting two bridgeheads, where a "bridgehead" is any skeletal atom of the ring system that is bonded to three or more skeletal atoms (not including hydrogen). In some embodiments, a bridged bicyclic group has 7-12 ring members and 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Such bridged bicyclic groups are well known in the art and include those described below, wherein each group is attached to the rest of the molecule at any substitutable carbon or nitrogen atom. Unless otherwise specified, a bridged bicyclic group can be optionally substituted with one or more substituents as described for aliphatic groups. Additionally or alternatively, any substitutable nitrogen of a bridged bicyclic group is optionally substituted. Exemplary bridged bicyclic groups include:

[0016] The term "lower alkyl" refers to a C 1-4 straight or branched chain alkyl group. Exemplary lower alkyl groups are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, and tert-butyl.

[0017] The term "lower haloalkyl" refers to a C 1-4 straight or branched chain alkyl group substituted with one or more halogen atoms.

[0018] The term "heteroatom" means one or more of oxygen, sulfur, nitrogen, phosphorus, or silicon (including any oxidized form of nitrogen, sulfur, phosphorus, or silicon; the quaternized form of any basic nitrogen; or an substitutable nitrogen of a heterocyclic ring, such as N (as in 3,4-dihydro-2H-pyranyl), NH (as in pyrrolidinyl), or NR + (as in N-substituted pyrrolidinyl)).

[0019] As used herein, the term "unsaturated" means having one or more units of unsaturation.

[0020] As used herein, the term "divalent hydrocarbon chain" refers to a straight-chain or branched- chain divalent alkylene, alkenylene, and alkynylene chain as defined herein.

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

[0022] The term "alkylene" refers to a divalent alkyl group. A substituted alkylene chain is a polymethylene group having at least one single bond in which one or more of the hydrogen atoms has been replaced with a substituent group. Suitable substituent groups include those described below for substituted aliphatic groups.

[0023] The term "alkynylene" refers to a divalent alkynyl group. A substituted alkynylene chain is a polymethylene group having at least one triple bond in which one or more of the hydrogen atoms has been replaced with a substituent group. Suitable substituent groups include those described below for substituted aliphatic groups.

[0024] As used herein, the term "cycloalkylene" refers to a divalent cycloalkyl group having the following structure:

[0025] The term "halogen" means F, CI, Br, or I.

[0026] The term "aryl" by itself or as part of a larger moiety such as in "aralkyl," "aralkoxy," or "aryloxyalkyl," means a monocyclic or bicyclic ring system having from 5 to 14 ring members in which at least one ring is aromatic and in which each ring in the ring system contains from 3 to 7 ring members. The term "aryl" can be used interchangeably with the term "aryl ring." In certain embodiments of the application, "aryl" refers to an aromatic ring system, including but not limited to phenyl, biphenyl, naphthyl, anthryl, and the like, which can bear one or more substituent groups. As used herein, groups in which an aromatic ring is fused to one or more non-aromatic rings, such as indanyl, phthalimidyl, naphthimidyl, phenanthridinyl, or tetrahydronaphthyl, and the like, are also included within the scope of the term "aryl."

[0027] The terms "heteroaryl" and "heteroar-", used alone or as part of a larger moiety (e.g., "heteroaralkyl" or "heteroaralkoxy"), refer to groups having 5 to 10 ring atoms, preferably 5, 6, or 9 ring atoms; having 6, 10, or 14 π electrons shared in a cyclic array; and, in addition to carbon atoms, having between one and five heteroatoms. The term "heteroatom" refers to nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur, and any quaternized form of a basic nitrogen. Heteroaryl groups include, without limitation, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, and pteridinyl. As used herein, the terms "heteroaryl" and "heteroar-", also include groups in which a heteroaromatic ring is fused to one or more aryl, cycloaliphatic, or heterocyclyl rings, where the radical or point of attachment is on the heteroaromatic ring. Non-limiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenoxazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyrido[2,3-b]-l,4-oxazin-3(4H)-one. A heteroaryl group can be mono- or bicyclic. The term "heteroaryl" can be used interchangeably with the terms "heteroaryl ring", "heteroaryl group", or "heteroaromatic", any of which includes rings that are optionally substituted.

[0028] As used herein, the terms "heterocycle", "heterocyclyl", "heterocyclic group", and "heterocyclic ring" are used interchangeably to mean a stable 5- to 7-membered monocyclic or 7- to 10-membered bicyclic heterocyclic moiety which is saturated or partially unsaturated and which, except for rings A and B, has between one and four heteroatoms per ring as set forth above. The term "nitrogen" when used in reference to the ring atoms of a heterocyclic ring means optionally substituted nitrogen. As an example, in a saturated or partially unsaturated ring having 0-3 heteroatoms selected from oxygen, sulfur, or nitrogen, the nitrogen can be N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or NR (as in N-substituted pyrrolidinyl). + NR (as in N-substituted pyrrolidinyl).

[0029] Heterocycles can be attached to their pendant groups at any heteroatom or carbon atom to produce a stable structure and any ring atom can be optionally substituted. Examples of such saturated or partially unsaturated heterocyclic groups include, but are not limited to, tetrahydrofuranyl, tetrahydrothiophenyl pyrrolidinyl, piperidinyl, pyrrolidinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl. The terms "heterocycle," "heterocyclyl," "heterocyclyl ring," "heterocyclic group," "heterocyclic moiety," and "heterocyclic radical" are used interchangeably herein, and also include groups in which the heterocyclyl ring is fused to one or more aryl, heteroaryl, or cycloaliphatic rings, such as indolinyl, 3H- indolyl, chroman, phenanthridinyl, or tetrahydroquinolinyl. The heterocyclyl radical can be mono- or bicyclic. The term "heterocyclylalkyl" means an alkyl group substituted with a heterocyclyl group, wherein the alkyl and heterocyclyl moieties are independently optionally substituted. As used herein, the term "partially unsaturated" means a ring moiety that includes at least one double or triple bond. The term "partially unsaturated" is intended to include rings that have multiple sites of unsaturation, but is not intended to include aryl or heteroaryl moieties, as defined herein.

[0030] As described herein, the compounds of the application can contain "optionally substituted" moieties. In general, the term "substituted", whether preceded by the term "optionally" or not, means that one or more hydrogens of the designated moiety are replaced by a suitable substituent. Unless otherwise indicated, an "optionally substituted" group can have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure can be substituted with more than one substituent, the substituents can be the same or different at each position. Combinations of substituents envisioned by this application are preferably those that result in the formation of stable or chemically feasible compounds. The term "stable", as used herein, refers to compounds that are not substantially altered when subjected to conditions that can

[0031] As described herein, the compounds of the application can contain "optionally substituted" moieties. In general, the term "substituted", whether preceded by the term "optionally" or not, means that one or more hydrogens of the designated moiety are replaced by a suitable substituent. Unless otherwise indicated, an "optionally substituted" group can have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure can be substituted with more than one substituent, the substituents can be the same or different at each position. Combinations of substituents envisioned by this application are preferably those that result in the formation of stable or chemically feasible compounds. The term "stable", as used herein, refers to compounds that are not substantially altered when subjected to conditions that can

[0032] Suitable monovalent substituents on substitutable carbon atoms of "optionally substituted" groups are independently halogen; -(CH2) 0-4 R o ; -(CH2) 0-4 OR o ; -O(CH2) 0-4 R o ; -O-(CH2) 0-4 C(O)OR o ​; -(CH2) 0-4 CH(OR o )2; -(CH2) 0- 4SR o ; -(CH2) 0-4 Ph, which can be substituted by R o ; -(CH2) 0-4 O(CH2) 0-1 Ph, which can be substituted by R o ; -CH=CHPh, which can be substituted by R o ; -(CH2) 0-4 O(CH2) 0-1 -pyridyl, which can be substituted by R o ; -NO2; -CN; -N3; -(CH2) 0-4 N(R o )2; -(CH2) 0-4 N(R o )C(O)R o ; -N(R o )C(S)R o ; -N(R o )C(NR o )N(R o )2; -(CH2) 0-4 N(R o )C(O)NR o 2; -N(R o )C(S)NR o 2; -(CH2) 0-4 N(R o )C(O)OR o ; -N(R o )N(R o )C(O)R o ; -N(R o )N(R o )C(O)NR o 2; -N(R o )N(R o )C(O)OR o ; -(CH2) 0-4 C(O)R o ; -C(S)R o ; -(CH2) 0-4 C(O)OR o ; -(CH2) 0-4 C(O)SR o ; -(CH2) 0-4 C(O)OSiR o 3; -(CH2) 0-4 OC(O)Ro ;-OC(O)(CH2) 0-4 SR-,-SC(S)SR o ; -(CH2) 0-4 SC(O)R o ; -(CH2) 0-4 C(O)NR o 2;-C(S)NR o 2;-C(S)SR o ; -(CH2) 0-4 OC(O)NR o 2;-C(O)N(OR o )R o ;-C(O)C(O)R o ;-C(O)CH2C(O)R o ;-C(NOR o )R o ; -(CH2) 0-4 SSR o ; -(CH2) 0-4 S(O)2R o ; -(CH2) 0-4 S(O)2OR o ; -(CH2) 0-4 OS(O)2R o ;-S(O)2NR o 2; -(CH2) 0-4 S(O)R o ;-N(R o )S(O)2NR o 2;-N(R o )S(O)2R o ;-N(OR o )R o ;-C(NH)NR o 2;-P(O)2R o ;-P(O)R o 2;-OP(O)R o 2;-OP(O)(OR o )2;-SiR o 3;-(C 1-4 linear or branched alkylene)ON(R o )2; or -(C 1-4 linear or branched alkylene) C(O)ON(R o )2, where each R o may be substituted as defined below and are independently hydrogen, C 1-6 Aliphatic groups, -CH2Ph, -O(CH2) 0-1Ph, -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 definitions, two independent occurrences of R o Together with the atoms between them, they form a 3-12 membered saturated, partially unsaturated or aromatic monocyclic or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur, which may be substituted as defined below.

[0033] R o (or by two independent occurrences of R o Suitable monovalent substituents on the ring formed by the atoms between them are independently halogen, -(CH2) 0-2 R · 、-(halogenated R · ), -(CH2) 0-2 OH, -(CH2) 0-2 OR · 、-(CH2) 0-2 CH(OR · )2;-O(halogenated R · )、-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 · , where each R · is unsubstituted or wherein the preceding "halo" is substituted only with one or more halogens, and is independently selected from C 1-4 Aliphatic groups, -CH2Ph, -O(CH2) 0-1 Ph, or a 5-6 membered saturated, partially unsaturated or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur. o Suitable divalent substituents on a saturated carbon atom of include =0 and =S.

[0034] Suitable divalent substituents on a saturated carbon atom of an "optionally substituted" group include the following: =0, =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-, where each independent occurrence of R* is selected from hydrogen, a substituted C 1-6 aliphatic group as defined below, or an unsubstituted 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents bound to an ortho substitutable carbon of an "optionally substituted" group include: -O(CR * 2) 2-3 O-, where each independent occurrence of R* is selected from hydrogen, a substituted C 1-6 aliphatic group as defined below, or an unsubstituted 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0035] Suitable substituents on an aliphatic group of R* include halogen, -R · , -(haloR · ), -OH, -OR · , -O(haloR · ), -CN, -C(O)OH, -C(O)OR · , -NH2, -NHR · , -NR · 2, or -NO2, where each R · is unsubstituted or where "halo" precedes only one or more halogens, and is independently a C 1-4 aliphatic group, -CH2Ph, -O(CH2) 0-1 Ph, or a 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0036] Suitable substituents on a substitutable nitrogen of an "optionally substituted" group include or where each is independently hydrogen, a substituted C 1-6aliphatic, unsubstituted -OPh, or an unsubstituted 5-6 membered saturated, partially unsaturated or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur, or, notwithstanding the above definitions, two independent occurrences of Together with the atoms between them, they form an unsubstituted 3-12 membered saturated, partially unsaturated or aromatic monocyclic or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur.

[0037] Suitable substituents on the aliphatic group are independently halogen, -R · 、-(halogenated R · ), -OH, -OR · 、-O(halogenated R · )、-CN、-C(O)OH、-C(O)OR · 、-NH2、-NHR · 、-NR · 2, or -NO2, where each R · is unsubstituted or wherein the preceding "halo" is substituted only with one or more halogens, and is independently C 1-4 Aliphatic groups, -CH2Ph, -O(CH2) 0-1 Ph, or a 5-6 membered saturated, partially unsaturated or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur.

[0038] As used herein, the term "pharmaceutically acceptable salt" refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with 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, incorporated herein by reference. Additionally, Pharmaceutical Salts: Properties, Selection, and Use, 2nd Revised Edition, (2011), P. Heinrich Stahl (Editor), Camille G. Wermuth (Editor), (ISBN: 978-3-906-39051-2) describes pharmaceutically acceptable salts in detail, incorporated herein by reference in its entirety. Pharmaceutically acceptable salts of the compounds of this application include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group with inorganic acids such as hydrochloric, hydrobromic, phosphoric, sulfuric, and perchloric acid, or with organic acids such as acetic, oxalic, maleic, tartaric, citric, succinic, or malonic acid, or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, besylate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like.

[0039] Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N + (C 1-4 Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, those derived from the crystallization of the compound with an organic or inorganic base, such as the trialkylamines, including triethylamine, tributylamine, pyridine, and the like; and those derived from the crystallization of the compound with an organic or inorganic acid, such as the hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, phosphate, nitrate, acetate, lactate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and the like.1-6 nontoxic ammonium, quaternary ammonium, and amine cations formed from the combination of a pharmaceutically acceptable anion with a basic amine.

[0040] Unless otherwise stated, structures depicted herein are also meant to include all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational)) forms of the structure; for example, the R and S configurations of each asymmetric center, Z and E double bond isomers, and Z and E conformational isomers. Therefore, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the present compounds are within the scope of the application. Unless otherwise stated, all tautomeric forms of the compounds of the application are within the scope of the application.

[0041] As used herein, “therapeutically effective amount” means an amount of a substance (e.g., a therapeutic agent, composition, and / or formulation) that elicits a desired biological response. In some embodiments, a therapeutically effective amount of a substance is an amount that is sufficient, when administered as part of a dosing regimen to a subject having or susceptible to a disease, condition, or disorder, to treat, diagnose, prevent, and / or delay the onset of the disease, condition, or disorder. As will be appreciated by persons of ordinary skill in the art, the effective amount of a substance can vary depending on such factors as the desired biological endpoint, the substance to be delivered, the target cell or tissue, etc. For example, an effective amount of a compound in a formulation to treat a disease, condition, or disorder is an amount that reduces, ameliorates, alleviates, inhibits, prevents, delays the onset of, reduces the severity of, and / or reduces the incidence of one or more symptoms or features of the disease, condition, or disorder.

[0042] As used herein, the term “treat” or “treating” means to partially or completely alleviate, inhibit, delay onset of, prevent, ameliorate, and / or alleviate a disease or disorder or one or more symptoms of a disease or disorder. As used herein, the term “treatment” or “treat” or “treating” means to partially or completely reduce, inhibit, delay onset of, prevent, ameliorate, and / or alleviate a disease or disorder or one or more symptoms of a disease or disorder, as described herein. In some embodiments, treatment can be administered after one or more symptoms have developed. In some embodiments, the term “treatment” includes preventing or arresting development of a disease or disorder. In other embodiments, treatment can be administered in the absence of symptoms. For example, treatment can be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and / or in light of genetic or other susceptibility factors). Treatment can also be continued after symptoms have resolved, for example to prevent or delay their recurrence. Accordingly, in some embodiments, the term “treatment” includes preventing relapse or recurrence of a disease or disorder.

[0043] As used herein, the expression "unit dosage form" refers to physically discrete units suitable for unitary dosing to a subject to be treated. It will be understood, however, that the total daily usage of the compositions of the present application will be decided by the attending physician within the scope of sound medical judgment. The specific effective dose level for any particular subject or organism will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific active agent employed; the specific composition employed; the age, body weight, general health, sex, and diet of the subject; the time of administration and rate of excretion of the specific active agent employed; the duration of the treatment; drugs and / or additional therapies used in combination or coincidentally with the specific compound employed, and like factors well known in the medical arts.

[0044] The bicyclomycin conjugate BT8009 has the structure shown below, and the formulation of BT8009 (BCY8245) is described in WO2019 / 243832, the entire contents of which are hereby incorporated by reference herein. BT8009

[0045] The bicyclomycin conjugate BT5528 has the structure shown below, where the molecular scaffold is 1,1',1"-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA), the peptide ligand comprises the amino acid sequence (beta-Ala)-Sar 10 -A(HArg)D-C i (HyP)LVNPLC ii LHP(D-Asp)W(HArg)C iii where Sar is sarcosine, HArg is homoarginine, and HyP is hydroxyproline. BT5528: 3. Description of Certain Embodiments of the Invention

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

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

[0048] In some embodiments, the provided method comprises reacting vcMMAE with about 1 to about 1.1 equivalents of glutaric anhydride (relative to about 1 equivalent of vcMMAE). In some embodiments, the glutaric anhydride is about 1.1 equivalents of vcMMAE. In some embodiments, the glutaric anhydride is about 1 equivalent, about 1.01 equivalent, about 1.02 equivalent, about 1.03 equivalent, about 1.04 equivalent, about 1.05 equivalent, about 1.06 equivalent, about 1.07 equivalent, about 1.08 equivalent, or about 1.09 equivalent of vcMMAE. In some embodiments, the glutaric anhydride is about 1.11 equivalent, about 1.12 equivalent, about 1.13 equivalent, about 1.14 equivalent, about 1.15 equivalent, about 1.16 equivalent, about 1.17 equivalent, about 1.18 equivalent, about 1.19 equivalent, or about 1.20 equivalent of vcMMAE. In some embodiments, the vcMMAE is reacted with about 1 to about 1.20 equivalents of glutaric anhydride, such as 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, such as about 1.1 equivalents of glutaric anhydride (relative to about 1 equivalent of vcMMAE).

[0049] In some embodiments, the triethylamine (TEA) in the reaction mixture is about 1.3 equivalents of vcMMAE. In some embodiments, the triethylamine (TEA) in the reaction mixture is about 1 equivalent to about 1.6 equivalents of vcMMAE, relative to about 1 equivalent of vcMMAE. In some embodiments, the reaction mixture comprises about 1.3 to about 1.5 equivalents of triethylamine. In some embodiments, the triethylamine (TEA) in the reaction mixture is about 1 equivalent, about 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 some embodiments, the reaction mixture comprises about 1 to about 1.6 equivalents of TEA, such as about 1.3 to about 1.5 equivalents of TEA, for example about 1.3 to about 1.4 equivalents of TEA, for example about 1.3 equivalents of TEA, relative to about 1 equivalent of vcMMAE. In some embodiments, the method comprises adding about 1 to about 1.6 equivalents of TEA, such as about 1.3 to about 1.5 equivalents of TEA, for example about 1.3 to about 1.4 equivalents of TEA, for example about 1.3 equivalents of TEA, to the reaction mixture.

[0050] In some embodiments, the reaction between vcMMAE and glutaric anhydride is in a solvent comprising N,N-dimethylacetamide (DMA) and tetrahydrofuran (THF). In some embodiments, the solvent comprises DMA, THF, and one or more additional components. In some embodiments, the solvent comprises DMA, THF, and MTBE.

[0051] In some embodiments, the solvent comprises N,N-dimethylacetamide (DMA) and tetrahydrofuran (THF), or is a mixture of DMA and THF in a volume ratio of about 1:15. In some embodiments, the solvent comprises DMA and THF in a volume ratio of about 1:10 to about 1:200, such as 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, such as about 1:115. In some embodiments, the solvent comprises DMA and THF in a volume ratio of about 1:10 to about 1:20, such as about 1:12 to about 1:18, for example about 1:14 to about 1:16, for example about 1:15. In some embodiments, the solvent comprises N,N-dimethylacetamide (DMA) and tetrahydrofuran (THF), or is a mixture of DMA and 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.

[0052] In some embodiments, THF is present at about 3 to about 3.9 kg / kg. In some embodiments, THF is present at about 3 to about 3.9 kg / kg relative to the mass of vcMMAE. In some embodiments, DMA is present at about 0.2 to about 0.3 kg / kg. In some embodiments, DMA is present at about 0.2 to about 0.3 kg / kg relative to the mass of vcMMAE. In some embodiments, the solvent comprises about 0.2:3.9 to about 0.3-3 v / v DMA / THF, e.g., relative to vcMMAE. In some embodiments, the solvent is a mixture of about 0.25:3.75 v / v DMA and THF (0.25:3.75 v / v DMA / THF). In some embodiments, the solvent comprises about 0.35:3.65 to about 0.15:3.85 v / v DMA / THF. In some embodiments, the solvent comprises about 0.3:3.7 to about 0.2:3.8 v / v DMA / THF. In some embodiments, the solvent is a mixture of about 0.20:3.80 v / v DMA and THF. In some embodiments, the solvent is a mixture of about 0.30:3.70 v / v DMA and THF. In some embodiments, the solvent is a mixture of about 0.15:3.85 v / v DMA and THF. In some embodiments, the solvent is a mixture of about 0.35:3.65 v / v DMA and THF.

[0053] In some embodiments, the solvent comprises DMA, THF, and MTBE. In some embodiments, the solvent comprises DMA, THF, and MTBE in a volume ratio of about 1 :A:B DMA:THF:MTBE, wherein A is about 10 to about 200, such as about 50 to about 150, e.g., about 100 to about 130, e.g., about 110 to about 120, such as 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 some embodiments, the solvent comprises DMA, THF, and MTBE; 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, e.g., relative to vcMMAE. In some embodiments, 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 28-29 v, e.g., about 28.75 v); and DMA is present at about 40 to about 60 v / v (e.g., about 50 v), e.g., relative to vcMMAE.

[0055] In some embodiments, the ratio of DMA:THF is about 0.2:35 to about 0.3:25 v / v. In some embodiments, the ratio of DMA:THF is about 0.23:30 to about 0.27:26 v / v (e.g., about 0.25:28-29 v / v, e.g., 0.25:28.75 v / v). In some embodiments, the ratio of DMA:MTBE is about 0.2:80 to about 0.3:30 v / v. In some embodiments, the ratio of DMA:MTBE is about 0.23:60 to about 0.27:40 v / v (e.g., about 0.25:50 v / v). In some embodiments, the ratio of THF:MTBE is about 25:80 to about 35:30 v / v. In some embodiments, the ratio of THF:MTBE is about 26:60 to about 30:40 v / v (e.g., about 28-29:50 v / v, e.g., 28.75:50 v / v).

[0056] In some embodiments, the reaction between vcMMAE and glutaric anhydride is in a solvent comprising dichloromethane (DCM). In some embodiments, the reaction between vcMMAE and glutaric anhydride is in a solvent that is dichloromethane (DCM).

[0057] In some embodiments, the reaction between vcMMAE and glutaric anhydride is in a solvent comprising acetonitrile (MeCN). In some embodiments, the reaction between vcMMAE and glutaric anhydride is in a solvent that is acetonitrile (MeCN).

[0058] In some embodiments, the reaction between vcMMAE and glutaric anhydride is in a solvent comprising 2-methyltetrahydrofuran (2-MeTHF). In some embodiments, the reaction between vcMMAE and glutaric anhydride is in a solvent that is 2-methyltetrahydrofuran (2-MeTHF).

[0059] In some embodiments, the reaction between vcMMAE and glutaric anhydride is in a solvent comprising N,N-dimethylacetamide (DMA). In some embodiments, the reaction between vcMMAE and glutaric anhydride is in a solvent that is N,N-dimethylacetamide (DMA).

[0060] In some embodiments, the reaction between vcMMAE and glutaric anhydride is in a solvent comprising N,N-dimethylacetamide (DMA) and acetonitrile (MeCN). In some embodiments, the solvent comprises N,N-dimethylacetamide (DMA) and acetonitrile (MeCN), or is a mixture of DMA and MeCN in a volume ratio of about 1 :3. In some embodiments, the solvent comprises N,N-dimethylacetamide (DMA) and acetonitrile (MeCN), or is a mixture of DMA and 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. In some embodiments, the solvent comprises N,N-dimethylacetamide (DMA) and acetonitrile (MeCN), or is a mixture of DMA and 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.

[0061] In some embodiments, the reaction between vcMMAE and glutaric anhydride is carried out at a temperature of about -5 to about 25 °C. In some embodiments, the reaction is carried out at a temperature of about 15 °C to about 25 °C. In some embodiments, the reaction is carried out at a temperature of about 18 °C to about 22 °C, such as about 20 °C. In some embodiments, the reaction between vcMMAE and glutaric anhydride is carried out for a time of about 10 minutes to about 5 hours, for example, about 10 minutes to about 2 hours, such as about 30 minutes to about 1.5 hours, for example, about 1 hour. In some embodiments, the reaction is carried out for a time of about 1 to about 3 hours. In some embodiments, the reaction is carried out until completion.

[0062] In some embodiments, the reaction comprises stirring the reaction mixture. In some embodiments, the reaction comprises stirring the reaction mixture at a temperature of about 15 °C to about 25 °C, for example, about 18 °C to about 22 °C, such as about 20 °C, for a time of about 10 minutes to about 5 hours, 10 minutes to about 2 hours, such as about 30 minutes to about 1.5 hours, for example, about 1 hour; or about 1 to about 3 hours. In some embodiments, the reaction mixture is stirred until the reaction is complete. In some embodiments, the stirring rate is about 10 rpm to about 1000 rpm.

[0063] In some embodiments, the reaction between vcMMAE and glutaric anhydride is carried out at about 0 °C. In some embodiments, the reaction between vcMMAE and glutaric anhydride is carried out at about -5 °C to about 5 °C. In some embodiments, the reaction between vcMMAE and glutaric anhydride is carried out at about -5 °C, -4 °C, -3 °C, -2 °C, or -1 °C. In some embodiments, the reaction between vcMMAE and glutaric anhydride is carried out at about 1 °C, 2 °C, 3 °C, 4 °C, or 5 °C.

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

[0065] In some embodiments, the present application provides a method of synthesizing gvcMMAE, the method comprising adding about 1 to about 1.1 (e.g., about 1.1) equivalents of glutaric anhydride to a solution comprising about 1 equivalent of vcMMAE in a solvent comprising about 0.2:3.9 v / v to about 0.3-3 v / v DMA / THF (e.g., about 0.25:3.75 v / v DMA / THF) and about 1.3 to about 1.5 equivalents of triethylamine, 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 time of about 10 minutes to about 2 hours.

[0066] In some embodiments, the present application provides a method of synthesizing gvcMMAE, the method comprising adding about 1 to about 1.1 (e.g., about 1.1) equivalents of glutaric anhydride to a solution comprising about 1 equivalent of vcMMAE in a solvent comprising about 0.2:3.9 to about 0.3-3 v / v DMA / THF (e.g., about 0.25:3.75 v / v DMA / THF) to provide 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 time 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 some embodiments, the present application provides a method of synthesizing gvcMMAE, the method comprising reacting about 1 to about 1.1 equivalents of glutaric anhydride with about 1 equivalent of vcMMAE in a solvent comprising DMA, THF, and MTBE in a volume ratio of about 1 :A:BDMA:THF:MTBE, wherein A is about 10 to about 200 and B is about 10 to about 500, at a temperature of about 15 to 25 °C.

[0068] In some embodiments, the present application provides a method of synthesizing gvcMMAE, the method comprising adding about 1 to about 1.1 (e.g., about 1.1) equivalents of glutaric anhydride to a solution comprising about 1 equivalent of vcMMAE in a solvent, the solvent comprising DMA, THF, and MTBE, wherein 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, e.g., relative to vcMMAE, and about 1.3 to about 1.5 equivalents of triethylamine, wherein the method comprises conducting the reaction at a temperature of about 15 to about 25 °C (e.g., about 20 °C) for a time of about 10 minutes to about 5 hours.

[0069] In some embodiments, the present application provides a method of synthesizing gvcMMAE, the method comprising adding about 1 to about 1.1 (e.g., about 1.1) equivalents of glutaric anhydride to a solution comprising about 1 equivalent of vcMMAE in a solvent, to provide a reaction mixture, the solvent comprising DMA, THF, and MTBE, wherein 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 30 v (e.g., about 28-29 v, e.g., about 28.75 v); and DMA is present at about 40 to about 60 v / v (e.g., about 50 v), e.g., relative to vcMMAE; 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 time of about 1 to about 3 hours.

[0070] In some embodiments, the present application provides a method of synthesizing gvcMMAE, the method comprising adding about 1.1 equivalents of glutaric anhydride to a solution comprising about 1 equivalent of vcMMAE in a solvent, the solvent comprising 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 some embodiments, the method comprises quenching the reaction between vcMMAE and glutaric anhydride with water (H2O). In some embodiments, quenching the reaction comprises adding about 0.002 to about 0.01 kg / kg water to the reaction mixture. In some embodiments, quenching the reaction comprises adding about 0.002 to about 0.01 kg / kg water relative to the mass of vcMMAE. In some embodiments, quenching the reaction comprises adding about 0.004 to about 0.008 kg / kg water, such as about 0.005 to about 0.007 kg / kg water.

[0072] In some embodiments, quenching the reaction includes adding about 0.1 to about 1 equivalents of water to the reaction mixture. In some embodiments, quenching the reaction includes adding about 0.1 to about 1 equivalents of water to the reaction mixture (relative to the mass of vcMMAE). In some embodiments, quenching the reaction includes adding about 0.2 to about 0.6 equivalents of water, such as about 0.3 to about 0.5 equivalents of water, to the reaction mixture.

[0073] In some embodiments, the reaction between vcMMAE and glutaric anhydride is quenched with water at a temperature of about 15 to about 25 °C. In some embodiments, the reaction is quenched at a temperature of about 18 °C to about 22 °C, such as about 20 °C. In some embodiments, the reaction is quenched for a time of about 1 minute to about 2 hours, such as 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, such as about 10 minutes.

[0074] In some embodiments, quenching the reaction includes stirring the reaction mixture. In some embodiments, quenching the reaction includes stirring the reaction mixture at a temperature of about 15 to about 25 °C, such as about 18 °C to about 22 °C, such as about 20 °C. In some embodiments, quenching the reaction mixture includes stirring the reaction mixture for a time of about 1 minute to about 2 hours, such as 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, such as about 10 minutes. In some embodiments, the stirring rate is about 10 rpm to about 1000 rpm.

[0075] Accordingly, in some embodiments, the present application provides a method of synthesizing gvcMMAE, the method including reacting about 1 to about 1.1 equivalents of glutaric anhydride with about 1 equivalent of vcMMAE in a solvent comprising about 0.2:3.9 to about 0.3-3 v / v DMA / THF at a temperature of about 15 to about 25 °C; and quenching the reaction with water.

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

[0077] In some embodiments, the present application provides a method of synthesizing gvcMMAE, the method comprising adding about 1 to about 1.1 (e.g., about 1.1) equivalents of glutaric anhydride to a solution comprising about 1 equivalent of vcMMAE in a solvent comprising about 0.2:3.9 to about 0.3-3 v / v DMA / THF (e.g., about 0.25:3.75 v / v DMA / THF) to provide 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 quenching the reaction by adding about 0.002 to about 0.01 kg / kg water or about 0.1 to about 1 equivalents of water to the reaction mixture at a temperature of about 15 to about 25 °C.

[0078] In some embodiments, the present application provides a method of synthesizing gvcMMAE, the method comprising adding about 1.1 equivalents of glutaric anhydride to a solution comprising about 1 equivalent of vcMMAE in a solvent comprising 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 some embodiments, the reaction mixture is stirred at about -5 °C to about 5 °C for about 1 hour to complete the reaction (i.e., the reaction mixture becomes a mixture comprising primarily gvcMMAE, or a gvcMMAE mixture). As used herein, the term “gvcMMAE mixture” generally relates to the reaction product of vcMMAE with glutaric anhydride, and generally comprises gvcMMAE and optionally residual reaction components, such as residual solvent, e.g., DMA, THF, and / or TEA, and further optionally can comprise any unreacted vcMMAE and / or glutaric anhydride.

[0079] In some embodiments, the gvcMMAE mixture is warmed to about 15 °C to about 25 °C. In some embodiments, the gvcMMAE mixture is warmed to about 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 after the reaction is complete. In some embodiments, the gvcMMAE mixture is warmed until it is a clear solution.

[0080] In some embodiments, the gvcMMAE mixture is added to a mixture of THF and MTBE to provide gvcMMAE in the form of a precipitate. In some embodiments, the gvcMMAE mixture is at about 15 °C to about 25 °C. In some embodiments, the gvcMMAE mixture is a clear solution. In some embodiments, the gvcMMAE mixture is added to a mixture of THF and MTBE at about -5 °C to about 5 °C, the gvcMMAE mixture is a clear solution and is at about 15 °C to about 25 °C, to provide gvcMMAE in the form of a precipitate.

[0081] In some embodiments, the mixture of THF and MTBE comprises about 20 to about 25 kg / kg THF. In some embodiments, the mixture of THF and MTBE comprises about 20 to about 25 kg / kg THF relative to the mass of vcMMAE. In some embodiments, the mixture of THF and MTBE comprises about 35 to about 39 kg / kg MTBE. In some embodiments, the mixture of THF and MTBE comprises about 35 to about 39 kg / kg MTBE relative to the mass of vcMMAE. In some embodiments, the mixture of THF and MTBE comprises about 20:39 to about 25:30 v / v THF / MTBE. In some embodiments, the mixture of THF and MTBE is about 1 :2 v / v THF and MTBE (1 :2 v / v THF / MTBE). In some embodiments, the mixture of THF and MTBE is about 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 some embodiments, the mixture of THF and MTBE comprises about 0.5:2.5 to about 1:1 v / v THF and MTBE.

[0082] In some embodiments, the gvcMMAE mixture is added to a mixture of THF and MTBE at about -5 °C to about 5 °C to provide gvcMMAE in the form of a precipitate. In some embodiments, the gvcMMAE mixture is added to a mixture of THF and MTBE comprising about 20:39 to about 25:30 v / v THF / MTBE at about -5 °C to about 5 °C to provide gvcMMAE in the form of a precipitate. In some embodiments, the gvcMMAE mixture is added (e.g., dropwise) to a mixture of THF and MTBE comprising about 20:39 to about 25:30 v / v THF / MTBE at about -5 °C to about 5 °C under stirring to provide gvcMMAE in the form of a precipitate, 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 4:40 to about 4:100, for example, about 4:50 to about 4:75, such as about 4:75.

[0083] In some embodiments, the gvcMMAE mixture is slowly added to a mixture of THF and MTBE at about -5 °C to about 5 °C to provide gvcMMAE in the form of a precipitate. In some embodiments, the gvcMMAE mixture is added dropwise to a mixture of THF and MTBE at about -5 °C to about 5 °C to provide gvcMMAE in the form of a precipitate. In some embodiments, the volume ratio of the mixture of DMA and THF to the mixture of THF and MTBE is about 4:20 to about 4:200, for example, about 4:40 to about 4:100, for example, about 4:50 to about 4:75, such as about 4:75. In some embodiments, the volume ratio of the mixture of DMA and THF to the mixture of THF and MTBE is 4:75. In some embodiments, the volume ratio of the 0.25:3.75 v / v DMA / THF mixture to the 1:2 v / v THF / MTBE mixture is about 4:75. In some embodiments, the gvcMMAE mixture is slowly added to a mixture of about 1:2 v / v THF and MTBE at about -5 °C to about 5 °C to provide gvcMMAE in the form of a precipitate. In some embodiments, the gvcMMAE mixture is added dropwise to a mixture of about 1:2 v / v THF and MTBE at about -5 °C to about 5 °C to provide gvcMMAE in the form of a precipitate. In some embodiments, the volume ratio of the mixture of DMA and THF to the mixture of THF and MTBE is 4:75. In some embodiments, the volume ratio of the 0.25:3.75 v / v DMA / THF mixture to the 1:2 v / v THF / MTBE mixture is about 4:75.

[0084] In some embodiments, the gvcMMAE mixture in THF / MTBE mixture is stirred for about 10 minutes to about 2 hours, such as about 30 minutes to about 1.5 hours, for example about 1 hour. In some embodiments, the stirring rate is about 10 rpm to about 1000 rpm.

[0085] In some embodiments, the gvcMMAE precipitate as described above is filtered as a wet cake, which is rinsed with MTBE. In some embodiments, the gvcMMAE precipitate is rinsed with about 3 to about 10 kg / kg MTBE. In some embodiments, the gvcMMAE precipitate is rinsed with about 3 to about 5 kg / kg MTBE. In some embodiments, the wet cake gvcMMAE is rinsed using 10 volumes of MTBE (where 0.25:3.75 v / v DMA / THF mixture is 4 volumes).

[0086] In some embodiments, following rinsing with MTBE, the gvcMMAE precipitate is dried at about 35 °C to about 45 °C. In some embodiments, following rinsing with MTBE, the gvcMMAE precipitate is dried at 40 °C. In some embodiments, the gvcMMAE precipitate is dried for about 1 hour to about 72 hours, such as about 5 hours to about 48 hours, for example about 15 hours to about 32 hours, for example about 24 hours.

[0087] Thus, in some embodiments, the process according to the present application comprises: i) dissolving 1 eq vc-PAB-MMAE in about 3-3.9 kg / kg THF and about 0.2-0.3 kg / kg DMA at 15-25 °C; ii) adding about 1-1.1 eq. glutaric anhydride and about 1.3-1.5 eq. TEA at 15-25 °C; iii) quenching the reaction by adding about 0.002-0.01 kg / kg water to the reaction at 15-25 °C to produce a product solution; iv) transferring the product solution to a mixture of about 20-25 kg / kg THF and about 35-39 kg / kg MTBE at about -5 to about +5 °C; v) isolating the product by filtration; vi) optionally washing the product with 3-5 kg / kg MTBE and / or drying the product, for example under vacuum.

[0088] In some embodiments, the process comprises: i) dissolving 1 eq vc-PAB-MMAE in about 3-3.9 kg / kg THF and about 0.2-0.3 kg / kg DMA at 15-25 °C under stirring; ii) adding about 1-1.1 eq. (e.g. about 1.1 eq.) glutaric anhydride and about 1.3-1.5 eq. (e.g. about 1.3 eq.) TEA at 15-25 °C; and stirring the reaction mixture until reaction completion, e.g. for about 0.5 to about 1.5 hours; iii) quenching the reaction by adding about 0.002-0.01 kg / kg water to the reaction at 15-25 °C under stirring to generate a product solution; iv) transferring the product solution drop-wise to a mixture of about 20-25 kg / kg THF and about 35-39 kg / kg MTBE at about -5 to about +5 °C; v) isolating the product by filtration; vi) washing the product with about 3-5 kg / kg MTBE; and drying the product under vacuum at about 35 to about 45 °C.

[0089] In some embodiments, the method according to the application comprises: i) dissolving 1 eq vc-PAB-MMAE in a solvent comprising DMA, THF and MTBE in a volume ratio of about 1 :A:B DMA:THF:MTBE, wherein A is about 100 to about 130 (e.g. about 115); B is about 100 to about 300 (e.g. about 200) at about 15-25 °C; ii) adding about 1-1.1 eq. (e.g. about 1.1 eq.) glutaric anhydride and about 1.3-1.5 eq. (e.g. about 1.3 eq.) TEA at 15-25 °C to generate a product solution; iii) precipitating gvcMMAE from the product solution; iv) isolating the product by filtration; and v) optionally washing the product with about 3-5 kg / kg MTBE; and / or drying the product, e.g. under vacuum.

[0090] In some embodiments, the method comprises: i) dissolving 1 eq vc-PAB-MMAE in a solvent comprising DMA, THF and MTBE, wherein DMA is present in about 0.2 to about 0.3 v (e.g. about 25 v); THF is present in about 25 to about 35 v (e.g. about 28-29 v); and DMA is present in about 30 to about 80 v (e.g. about 50 v), e.g. relative to vcMMAE; ii) adding about 1-1.1 eq. (e.g., about 1.1 eq.) glutaric anhydride and about 1.3-1.5 eq. (e.g., about 1.3 eq.) TEA at 15-25 °C; and stirring the reaction mixture, e.g., until the reaction is complete, e.g., for about 1 to about 3 hours, to produce a product solution; iii) precipitating gvcMMAE from the product solution (e.g., at about -5 to about +5 °C, by transferring the product solution dropwise into a mixture of about 20-25 kg / kg THF and about 35-39 kg / kg MTBE); iv) isolating the product by filtration; and v) washing the product with about 3-5 kg / kg MTBE; and drying the product under vacuum at about 35 to about 45 °C.

[0091] In some embodiments, the present application provides a compound gvcMMAE or a salt thereof. In some embodiments, the present application provides a compound gvcMMAE obtained or obtainable by a method as disclosed herein.

[0092] In some embodiments, the present application provides a compound of Formula III or a salt thereof:

[0093] In some embodiments, the present application provides a composition comprising gvcMMAE or a salt thereof, the composition further comprising a compound of Formula III or a salt thereof as an impurity. In some embodiments, a composition comprising gvcMMAE or a salt thereof comprises less than about 1% of a compound of Formula III or a salt thereof. In some embodiments, a composition comprising gvcMMAE or a salt thereof comprises less than about 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of a compound of Formula III or a salt thereof. Scheme I. Scheme II.

[0094] In some embodiments, the present application provides a method of synthesizing a bicyclic toxin conjugate (BTC), the method comprising reacting gvcMMAE with a bicyclic reaction.

[0095] In some embodiments, the bicyclic ring is a bicyclic peptide. In some embodiments, the bicyclic ring is a constrained bicyclic peptide that binds to Nectin-4 with high affinity and specificity. In some embodiments, the bicyclic peptide is selected from those described in International Patent Application No. PCT / GB2019 / 051740 (International Publication No. WO2019 / 243832), the entire contents of which are incorporated herein by reference.

[0096] In some embodiments, the bicyclic ring is a constrained bicyclic peptide that binds to Eph receptor tyrosine kinase A2 (EphA2) with high affinity and specificity. In some embodiments, the bicyclic peptide is selected from those described in International Patent Application Nos. PCT / GB2018 / 053675 (International Publication No. WO2019 / 122860) and PCT / GB2018 / 053678 (International Publication No. WO2019 / 122863), the entire contents of each of which are incorporated herein by reference.

[0097] In some embodiments, the bicyclic peptide is: wherein R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 and R 9 are each independently hydrogen or an optionally substituted group selected from C 1-6 aliphatic, 3-8 membered saturated or partially unsaturated monocyclic carbocyclic ring, phenyl, 8-10 membered bicyclic aromatic carbocyclic ring, 4-8 membered saturated or partially unsaturated monocyclic heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur, 5-6 membered monocyclic heteroaromatic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or 8-10 membered bicyclic heteroaromatic ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0098] In certain embodiments, R 1 is hydrogen or an optionally substituted C 1-6 aliphatic. In certain embodiments, R 1 is

[0099] In certain embodiments, R 2 is hydrogen or an optionally substituted C 1-6 aliphatic. In certain embodiments, R 2 is

[0100] In certain embodiments, R 3 is hydrogen or optionally substituted C 1-6 aliphatic. In certain embodiments, R 3 is

[0101] In certain embodiments, R 4 is hydrogen or optionally substituted C 1-6 aliphatic. In certain embodiments, R 4 is

[0102] In certain embodiments, R 5 is hydrogen or optionally substituted C 1-6 aliphatic. In certain embodiments, R 5 is

[0103] In certain embodiments, R 6 is hydrogen or optionally substituted C 1-6 aliphatic. In certain embodiments, R 6 is

[0104] In certain embodiments, R 7 is hydrogen or optionally substituted C 1-6 aliphatic. In certain embodiments, R 7 is

[0105] In certain embodiments, R 8 is hydrogen or optionally substituted C 1-6 aliphatic. In certain embodiments, R 8 is

[0106] In certain embodiments, R 9 is hydrogen or optionally substituted C 1-6 aliphatic. In certain embodiments, R 9 is

[0107] In some embodiments, the bicyclic ring is of Formula II: or salts thereof, wherein R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 and R9 each as defined below and described in embodiments herein, both individually and in combination, and m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0108] In some embodiments, m is 0. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, m is 5. In some embodiments, m is 6. In some embodiments, m is 7. In some embodiments, m is 8. In some embodiments, m is 9. In some embodiments, m is 10. In some embodiments, m is 11. In some embodiments, m is 12. In some embodiments, m is 13. In some embodiments, m is 14. In some embodiments, m is 15.

[0109] In some embodiments, the bicyclic toxin conjugate is of Formula I: or a pharmaceutically acceptable salt thereof, wherein R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 and R 9 each as defined below and described in embodiments herein, both individually and in combination.

[0110] In some embodiments, the present application provides a method of synthesizing a bicyclic toxin conjugate (BTC) of Formula I, the method comprising reacting gvcMMAE with a bicyclic of Formula II.

[0111] In some embodiments, the bicyclic toxin conjugate of Formula I is BT8009 or a pharmaceutically acceptable salt thereof.

[0112] In some embodiments, the bicyclic toxin conjugate of Formula I is BT5528 or a pharmaceutically acceptable salt thereof.

[0113] In some embodiments, the present application provides a bicyclic toxin conjugate (or a salt thereof) obtained or obtainable by a method as disclosed herein. Also provided are compositions comprising a bicyclic toxin conjugate or a salt thereof, and comprising less than 1% (e.g., less than about 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%) of a compound of Formula III or a salt thereof. Examples

[0114] The following examples illustrate the present invention described above; 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 relevant art.

[0115] List of common abbreviations used in the experimental section: A% - Area % aq.-water-containing / aqueous Con.-Concentration EtOAc / EA-ethyl acetate DMA N,N-dimethylacetamide DIEA N,N-Diisopropylethylamine TEA triethylamine THF Tetrahydrofuran DCM dichloromethane 2-MeTHF 2-Methyltetrahydrofuran MeCN / CAN Acetonitrile MTBE methyl tert-butyl ether iPrOAc Isopropyl acetate eq.-equivalent equip. g-gram GMP-Good Manufacturing Practice for Pharmaceuticals Non-GMP - does not comply with Good Manufacturing Practice (GMP) h-hour HCl-hydrochloric acid HSGC-Headspace Gas Chromatography IC-Ion Chromatography ICP-MS - Inductively Coupled Plasma Mass Spectrometry IMP-Impurities IPC-Process Control IR-infrared absorption spectrum KF - Karl Fischer (water determination) kg-kilogram L-liter LCMS-Liquid Chromatography Mass Spectrometry MBR - Manufacturing Batch Record mm-millimeter N-mole NaCl - Sodium Chloride NaHCO3-sodium bicarbonate H2SO4-sulfuric acid ND - Not Detected PLM - polarized light microscopy QC - quality control Spec. - specification STA - SynTheAll H2O - water a / a - area by area COA - certificate of analysis Eq. - equivalence FIO - for information only GC - gas chromatography HPLC - high performance liquid chromatography NaOH - sodium hydroxide pH - hydrogen ion concentration EtOH - ethanol M.L. / ML'S - mother liquor min - minute mL - milliliter mol - mole NLT - not less than NMT - not more than PO - purchase order ppm - parts per million RRT - relative retention time RS - residual solvent RT - room temperature STA - SynTheAll Vol - volume w / w - weight by weight TP - technical package Example 1: Preparation of gvcMMAE 1. SUMMARY

[0116] The process for gvcMMAE has been well optimized, resulting in 36.53 g of product with 98.56 A% HPLC purity and 97.17% corrected yield. All test items met specifications. 2. INTRODUCTION AND SYNTHESIS SCHEME

[0117] For reaction step: The reaction condition was optimized as follows: glutaric anhydride equivalent was reduced from 1.2 eq. to 1.1 eq.; 5.2 eq. of DIEA was changed to 1.3 eq. of TEA; DMA / THF mixed solvent was used as reaction solvent. In some studies, the reaction temperature was reduced to 0 °C. The IPC of this condition was the same as the original condition, but more stable and suitable for work-up. In some studies, the reaction temperature was increased to about 15-25 °C.

[0118] For the work-up step: the reaction solution was added directly to 75 v of THF / MTBE (1 :2) solution and the solid precipitated in good state.

[0119] The process has been well developed. For the system using TEA as base, no attempt was made to react at room temperature as a clear solution was obtained at 0 °C. If the IPC is good and the reaction mixture is stable at RT, it is not necessary to react at 0 °C. 3. Laboratory work 3.1. Scheme 1 3.2. Overview

[0120] In the original process, the IPC purity was excellent but proved to be unstable when the reaction mixture was kept for a long time. In addition, the work-up stage of the original process was not suitable for scale-up due to the wall-sticking phenomenon. After process optimisation, more stable reaction conditions were developed. The reaction solution was added directly to anti-solvent (THF:MTBE = 1 :2, 75 v) and good solid state was obtained. The new process is suitable for scale-up. 3.3. Process development 3.3.1 Familiarisation TP process (Table 1)

[0121] A one reaction of vcMMAE (assay corrected) at 0.26 g scale was performed to familiarise with the process. Excellent 99.5 A% IPC purity was obtained after 1 h stirring. However, after 16 h stirring, the IPC purity decreased to 94.5 A%. The results show that the reaction mixture is not stable under these conditions. Further investigation of the reaction conditions (base / acid anhydride loading, reaction temperature) was performed to address the instability problem of the reaction. If the results prove to be reproducible and stable, the only concern will be how to isolate the solid product.

[0122] For the work-up, the solid precipitated after the reaction solution was added to an acidic saturated brine solution (70 v) but quickly became sticky. The results show that DMA / brine is not a good crystallisation system and therefore other solvents need to be tried to find the best crystallisation conditions. When switching to other solvents, due to the high boiling point of DMA which is difficult to remove, low boiling point solvents (DCM, MeCN, THF and 2-MeTHF) were selected as reaction solvents to see if the same IPC results can be obtained. In addition, if DMA proves to be the only option for the reaction, a lower DMA volume (3 v) will also be tried as a lower volume of DMA can effectively benefit the work-up process (less water for precipitation, less organic solvent for extraction or precipitation). Table 1: Preparation of g vcMMAE 3.3.2. Solvent screening (Table 2 and Table 3)

[0123] Five different reaction solvents (DCM, MeCN, THF, 2-MeTHF and DMA / 3v) were tried. Among them, DMA gave the best results with an IPC purity of 98.98A%. DCM and THF also gave gvcMMAE of good IPC purity. For MeCN as a solvent, a sticky solid was found during the reaction. If it is proven that there is no good precipitation method when using DMA, DCM and THF can be tried as alternative solvents for DMA. In addition, a mixed solvent system (DMA:THF=1:3, DMA:ACN=1:3) was also tried (after basic equivalent and temperature screening) and acceptable IPC results were also obtained. Table 2: Solvent Screening Table 3. Impurity distribution of gvcMMAE 3.3.3. Alkali equivalent and temperature screening (Table 4 and Table 5)

[0124] Three reactions were performed at low temperatures (-5 to 5°C) using different equivalents of base (1.3 eq., 2.4 eq., and 5.2 eq.). The results showed that 1.3 eq. of DIEA was sufficient for complete reaction. Extending the reaction time to 16 h resulted in only a slight decrease in IPC purity, demonstrating that the reaction is stable for 16 h at -5 to 5°C.

[0125] The reaction was also attempted at -5 to 5°C using DCM and THF solvents. The IPC purity remained relatively high, and the mixture exhibited good stability at low reaction temperatures. For this reaction, -5 to 5°C and 1 h were clearly indicated. Table 4 Table 5. Impurity distribution of gvcMMAE 3.3.4. Screening of Glutaric Anhydride Loading (Tables 6 and 7)

[0126] Different equivalents of glutaric anhydride (1.05 eq. and 1.10 eq.) were screened. 1.10 eq. of glutaric anhydride was sufficient for complete conversion of the reaction. Table 6 Table 7 3.3.5. First round of process optimization (Table 8 and Table 9)

[0127] Minor changes to the process were developed using DMA / THF mixed solvent system and sulfuric / 5% Na2SO4 solution quenching system.

[0128] One batch of vcMMAE at 5 g scale was performed to validate the process. After reaction, the IPC purity was 99.37A%. The reaction solution was added inversely to 30 v of sulfuric acid (1.5 eq.) / 5% Na2SO4 solution at -5-5 °C. After filtration and drying, 7.0 g of crude product (containing Na2SO4) was obtained with HPLC purity of 98.71A%. About 6.36 g of crude gvcMMAE was further slurried in 30 v of water to remove Na2SO4. After filtration and drying, 4.2 g of gvcMMAE was obtained with HPLC purity of 98.89A% and crude yield of 84%.

[0129] IC results showed that the residual SO4 2- was 0.70% which meant that the product was in free acid state. The residual Na + was 0.37%. X-RPD showed that it could be amorphous.

[0130] DVS test showed that the product was hygroscopic at 25 °C at 80% RH (absorbed 3.6% water on the sorption curve between 40% RH and 80% RH at 25 °C). Table 8 Table 9 3.3.6. Study of solubility of gvcMMAE in different solvents (Table 10)

[0131] The solubility of gvcMMAE (free acid state) in different solvents (THF, DCM, MTBE, n-heptane, iPrOAc, 1,4-dioxane) was studied. According to the assay results, gvcMMAE in free base state was insoluble in most solvents. Table 10 3.3.7. Study of salt formation of free acid (Table 11)

[0132] Five bases (calcium hydroxide, dicyclohexylamine, DABCO, tributylamine and barium hydroxide) were tried for salt formation of gvcMMAE; THF was used as solvent. For dicyclohexylamine and tributylamine systems, the product was like milk after addition of base and the purity of the mixture decreased to about 96A% and 2A% of impurity @ RRT 1.76 was observed. (The mixture was concentrated to about 30 v and 60 v of MTBE was added to precipitate the solid. The purity of the wet solid precipitated from THF / MTBE / tributylamine system was 98.87A% with 0.01% assay in ML. It was thought that it was worth trying to get the solid by adding MTBE to THF / TEA or THF / tributylamine reaction solution.) For DABCO system, the product dissolved quickly but became jelly after stirring for 0.5 h and the purity of the mixture decreased significantly. For barium hydroxide system, the solid precipitated after stirring for 1 h and the purity of the wet cake decreased to 96.32A%. For calcium hydroxide system, the product was like milk after stirring for 1 h.

[0133] Based on this observation, tributylamine and TEA (similar in nature) were considered as bases for this reaction. Table 11 3.3.8. Study of salt formation by using tributylamine or TEA (Tables 12 and 13)

[0134] Four reactions of vcMMAE on 0.3 g scale were carried out with different base / solvent systems (base: tributylamine, TEA; solvent: THF, THF / DMA mixed solvent) to study the isolation of gvcMMAE salt.

[0135] For single solvent system (THF as solvent), the reaction solution became jelly quickly after addition of base (tributylamine as base: turbid jelly; TEA as base: transparent jelly).

[0136] For THF / DMA / tributylamine system, the reaction solution was clear. Then, 30 v of THF was added dropwise to the reaction solution for dilution, the system became jelly (milder than single solvent).

[0137] For THF / DMA / TEA system, the reaction solution was clear. When 30 v of THF was added dropwise to the reaction solution, the solvent became thick and then turbid. When 60 v of MTBE was added to the mixture, the solid precipitated. After filtration, 0.275 g of solid was obtained with HPLC purity of 98.64A% and crude yield of 78.4%.

[0138] THF / DMA / TEA reaction system and THF / MTBE work-up system were further studied. Table 12 Table 13 3.3.9. Investigation of the ratio of DMA to benefit the work-up stage (Tables 14 and 15)

[0139] Three reactions with different DMA:THF ratios were performed on a 0.3 g vcMMAE scale to investigate suitable work-up conditions for isolation of the TEA salt. Good IPC results (99.14 A%) were obtained when DMA:THF = 0.25 v:3.75 v

[0140] For the original reaction conditions (DMA:THF = 1 :3), when 30 v of THF was added directly to the reaction solution, the solution became thick with a risk of colloid, which was not suitable for scale-up. Therefore, it was considered to reverse-add the reaction solution to the solvent.

[0141] Therefore, the other two reaction solutions (DMA:THF = 0.5:3.5 and DMA:THF = 0.25:3.75) were dropped into 90 v (30 v THF + 60 v MTBE) of pre-mixed solvent and solids were precipitated in good condition. Table 14 Table 15 3.3.10 Investigation of the volume and ratio of THF / MTBE (Tables 16 and 17)

[0142] Three reactions of vcMMAE at 1.0 g scale were performed to investigate the volume of anti-solvent (THF:MTBE = 1 :2; 90 v, 75 v and 60 v were tried). After filtration and vacuum drying, 1.1 g of gvcMMAE TEA salt (90 v work-up batch) with 98.98 A% HPLC purity and 92.4% crude yield. (Residual solvents by HSGC: THF: 0.83%, MTBE: 5.23%, DMA: 1.35%; residual TEA by IC: 1.8%).

[0143] 100 mg of gvcMMAE was left under 60% RH conditions to perform a hygroscopicity experiment. The solid state did not change after 3 days (weight became 103 mg).

[0144] Different ratios of THF / MTBE (THF:MTBE=1:3, THF:MTBE=1:4, 45v and 60v) were also tried, but no better results were observed. After extended stirring time (16 hr), the solid adhered to the wall.

[0145] Considering both volume and solid state, 75v of THF / MTBE was determined to be the best choice. Table 16 Table 17 3.3.11. Study of post-treatment at low temperature (Tables 18 and 19)

[0146] Four batches of post-treatment were performed with different volumes of anti-solvent (THF:MTBE=1:2; 30v, 45v, 60v, 75v) at -5-5°C. 75v / -5-5°C conditions gave the best results, with a good solid state that did not stick to the wall after extended stirring time (16h). Table 18 Table 19 3.3.12. Demo Coupling Studies of gvcMMAE and Peptides (Tables 20 and 21)

[0147] Two demonstration reactions were performed on a 0.1 g scale of peptide (BCY8234, 93.76% HPLC purity). For gvcMMAE prepared from sulfuric acid / 5% NaSO solution, the IPC purity was 80.34%. After workup, the wet cake had an IPC purity of 80.36%. For gvcMMAE prepared from TEA conditions, the IPC purity was 86.51%. After workup, the wet cake had a purity of 86.25%. The results show that gvcMMAE from TEA solution gave better IPC purity. Table 20 Table 21 3.3.13 Stress Test of gvcMMAE at Different Drying Temperatures (Table 22 and Table 23)

[0148] Stress testing of gvcMMAE at different drying temperatures was performed. According to the results listed below, gvcMMAE was stable at 40°C for 3 days. Table 22 Table 23 3.3.14. Second round of process optimization (Tables 24 and 25)

[0149] A robust process was developed by using DMA / THF / TEA as the reaction system and THF / MTBE as the work-up system.

[0150] One batch of vcMMAE at 5 g scale was performed to validate the process. IPC purity was 98.37 A%. After work-up, good solid state was obtained and the purity of the wet cake was 98.60 A%. After vacuum drying, 6.03 g (about 0.2 g was taken for stress testing, theoretical amount: 5.51 g) of gvcMMAE was obtained with HPLC purity of 98.59 A% (residuals: TEA: 4.83%; THF: 0.01%; MTBE: 9.65%; DMA: 0.54%). Table 24 Table 25 3.4. Scale-up with the optimized process 3.4.1 Raw material usage test (Table 26)

[0151] A usage test of vcMMAE at 1 g scale was performed. IPC normal: gvcMMAE: 98.61 A%. After work-up, good solid state was obtained and the purity of the wet cake was 98.45 A%. After vacuum drying at 40°C for 16 h, 1.12 g (theoretical amount: 1.10 g) of gvcMMAE was obtained with HPLC purity of 98.57 A% (residuals: TEA: 4.81%; THF: 0.07%; MTBE: 7.30%; DMA: 0.46%). After vacuum drying at 40°C for another 16 h, the purity of gvcMMAE was 98.47 A% (residuals: TEA: 4.43%; THF: 0.05%; MTBE: 7.04%; DMA: 0.34%). Table 26 3.4.2. First scale-up (Table 27)

[0152] One scale-up of vcMMAE was performed at 10 g scale. IPC normal: gvcMMAE: 98.53 A%. After work-up, 11.76 g of gvcMMAE was obtained with HPLC purity of 98.51 A% and corrected yield of 95.64% (assay: 89.6%, residuals: TEA: 3.10%; THF: 0.93%; MTBE: 3.1%; DMA: 1.4%; glutaric anhydride: 0.06%; KF: 0.57%). Table 27 3.4.3. Second scale-up (Tables 28 and 29)

[0153] One scale-up of vcMMAE was performed at 30 g scale. IPC normal: gvcMMAE: 98.51 A%. After work-up, 36.53 g (about 0.3 g for RS / IC testing) of gvcMMAE was obtained with HPLC purity of 98.56 A% and corrected yield of 97.17% (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 3.5. Impurity identification

[0154] LC-MS has been performed to identify the impurities. The possible structure of the impurity RRT 0.90 is shown below. The structures of other impurities are still unknown. Table 30 3.6. Typical procedure 1. DMAC / THF (120 mL, 0.25:3.75 v / v, 4.0 v) was added to Rl (reaction vessel 1). 2. vcMMAE (30 g, assay corrected, 1.0 eq.) was added to Rl. 3. Rl was adjusted to -5~5 °C. 4. Stir Rl at -5 to 5°C for 0.1 h. 5. Add glutaric anhydride (3.36 g, 1.1 eq.) to Rl. 6. Add TEA (3.54 g, 1.3 eq.) to Rl. 7. Stir Rl at -5 to 5°C for 1 h. 8. Sample for analysis (IPC purity of gvcMMAE). 9. Add THF (750 mL, 25 v) to R2 (reaction vessel 2). 10. Add MTBE (1500 mL, 50 v) to R2. 11. Adjust R2 to -5 to 5°C. 12. Stir R2 at -5 to 5°C for 0.1 h. 13. Warm Rl to 15 to 25°C. (When warmed to room temperature, the solution will become clear) 14. Transfer the solution from Rl to R2 dropwise over 1 h. 15. Stir R2 at -5 to 5°C for 1 h. 16. Filter and rinse the wet cake with MTBE (300 mL, 10 v). 17. Dry the wet cake at 40°C for 16 to 32 h. 18. Sample for release testing. 3.6. Further Process Development A reaction was performed using 3.3 g of vcMMAE (1 eq.) with 1.5 eq. of glutaric anhydride. The level of impurity corresponding to the compound of formula (III) in the gvcMMAE product was quantified. Increasing the reaction temperature to 15-25°C resulted in an IPC purity of 99.13 A% gvcMMAE in the reaction solution (row A; Table 31). Crystallization with THF / MTBE (row B; Table 31) resulted in a concentration of 0.33 A% (wet) of the compound of formula (III) in the product gvcMMAE, which increased to 0.71 A% after drying. Experiments to rinse the dry cake with glutaric anhydride solution (0.1 eq. of anhydride in THF / MTBE (1 / 2): 3 v) resulted in an increase in the level of the compound of formula III, suggesting (without being bound by theory) that at least some of the impurity of formula III can have originated from the presence of excess glutaric anhydride. However, experiments to rinse off the glutaric anhydride using solvent MTBE (10 V), first MTBE / THF = 2 / 1 : 5 V, then MTBE: 5 V; or first THF: 5 V, then MTBE: 5 V; also resulted in an increase in the level of the compound of formula III. Quenching the reaction with water (0.5 eq; 10 min; 15-25 °C) prior to crystallization resulted in a significant improvement, with the crystallized gvcMMAE product containing only 0.52 A% of Formula (III) (Table 31, column C). Without being bound by theory, this can demonstrate that quenching the reaction with water prior to crystallization helps prevent reaction with excess glutaric anhydride. Table 31 Further experiments were performed using 1.4 g of vcMMAE (1 eq.) and 1.15 eq. of glutaric anhydride. The reaction was performed using DMA / THF / MTBE (0.25 / 28.5 / 50 v) as the reaction solvent. The level of Formula (III) compound corresponding impurity in the gvcMMAE product was quantified. The suspension was well behaved and the level of impurity originating from Formula (III) compound did not increase with the extension of the reaction time. The level of the additional impurity in Table 31 was quantified. The level of impurity was further significantly reduced to about 0.17-0.18% and did not increase with the extension of the reaction time. See Table 32. Table 32

[0155] While we have described a number of embodiments of the present application, it is apparent that our basic example can be altered to provide other embodiments utilising the compounds and methods of the present application. Thus, it will be appreciated that the scope of the present application is defined by the following claims and not by the example embodiments which are presented by way of illustration. The following are numbered aspects of the present application: 1. A method of synthesizing glutaroyl-Val-Cit-PAB-MMAE (gvcMMAE) or a salt thereof: The method comprises adding glutaric anhydride to a solution comprising vcMMAE in a solvent: to provide a reaction mixture, the solvent comprising N-dimethylacetamide (DMA) and tetrahydrofuran (THF). 2. The method according to aspect 1, wherein the solvent is about 0.25:3.75 v / v DMA and THF (0.25:3.75 v / v DMA / THF). 3. The method according to aspect 1 or 2, wherein glutaric anhydride is about 1.1 equivalents and vcMMAE is about 1 equivalent. 4. The method of any one of aspects 1-3, further comprising adding about 1.3 equivalents of TEA to the reaction mixture. 5. The method of any one of aspects 1-4, wherein the reaction between vcMMAE and glutaric anhydride is carried out at about -5 °C to about 5 °C. 6. The method of aspect 5, wherein the reaction mixture is stirred at about -5 °C to about 5 °C for about 1 hour to complete the reaction to provide a mixture comprising primarily gvcMMAE or a gvcMMAE mixture. 7. The method of aspect 6, wherein the gvcMMAE mixture is warmed to about 15 °C to about 25 °C. 8. The method of aspect 6, wherein the gvcMMAE mixture is warmed until it is a clear solution. 9. The method of aspect 7 or 8, wherein the gvcMMAE mixture is added to a mixture of THF and MTBE to provide gvcMMAE in the form of a precipitate. 10. The method of aspect 9, wherein the mixture of THF and MTBE is at about -5 °C to about 5 °C. 11. The method of aspect 9 or 10, wherein the mixture of THF and MTBE is about 1 :2 v / v THF and MTBE (1 :2 v / v THF / MTBE). 12. The method of aspect 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. 13. The method of any one of aspects 9-12, further comprising filtering out the gvcMMAE precipitate in the form of a wet cake and rinsing the wet cake with MTBE. 14. The method of any one of aspects 1-13, further comprising reacting gvcMMAE with a bicyclic of Formula II: to form a bicyclic toxin conjugate of Formula I: or a pharmaceutically acceptable salt thereof, wherein: R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 and R 9 are each independently hydrogen or an optionally substituted group selected from C1-6 aliphatic, 3-8 membered saturated or partially unsaturated monocyclic carbocyclic ring, phenyl, 8-10 membered bicyclic aromatic carbocyclic ring, 4-8 membered saturated or partially unsaturated monocyclic heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur, 5-6 membered monocyclic heteroaromatic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or 8-10 membered bicyclic heteroaromatic ring 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. 15. The method of aspect 14, wherein the bicyclomycin conjugate of Formula I is BT8009 or a pharmaceutically acceptable salt thereof. 16. The method of aspect 14, wherein the bicyclomycin conjugate of Formula I is BT5528 or a pharmaceutically acceptable salt thereof.

Claims

1. A method of synthesizing glutaryl-Val-Cit-PAB-MMAE (gvcMMAE) or a salt thereof: the method comprising adding glutaric anhydride to a solution comprising vcMMAE in a solvent: to provide a reaction mixture, the solvent comprising N-dimethylacetamide (DMA) and tetrahydrofuran (THF).

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

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

4. The method of any one of the preceding claims, 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; wherein the DMA is present at about 0.2 to about 0.3 v; the THF is present at about 25 to about 35 v; and the DMA is present at about 30 to about 80 v, for example, relative to vcMMAE.

6. The method of any one of the preceding claims, comprising reacting vcMMAE with about 1 to about 1.1 equivalents of glutaric anhydride; optionally, wherein glutaric anhydride is about 1.1 equivalents and vcMMAE is about 1 equivalent.

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

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

9. The method of any one of the preceding claims, wherein the reaction between vcMMAE and glutaric anhydride is conducted at a temperature of about 15 °C to about 25 °C.

10. The method of any one of the preceding claims, comprising stirring the reaction mixture during the reaction.

11. The method of any one of the preceding claims, wherein the reaction mixture is stirred at about -5 °C to about 25 °C for about 1 hour to complete the reaction, thereby optionally providing a mixture comprising primarily gvcMMAE, or a gvcMMAE mixture.

12. The method of any one of the preceding claims, comprising quenching the reaction between vcMMAE and glutaric anhydride with water (H2O).

13. The method of any one of the preceding claims, wherein quenching the reaction comprises adding about 0.002 to about 0.01 kg / kg water (H2O).

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

15. The method of any one of claims 12 to 14, wherein quenching the reaction comprises stirring the reaction mixture.

16. The method of any one of the preceding claims, wherein the gvcMMAE mixture is warmed to about 15 °C to about 25 °C; optionally, wherein the gvcMMAE mixture is warmed until it is a clear solution.

17. The method of any one of the preceding claims, wherein the gvcMMAE mixture is added to a mixture of THF and methyl tert-butyl ether (MTBE) to provide gvcMMAE in the form of a precipitate.

18. The method of claim 17, wherein the mixture of THF and MTBE is at 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 THF (relative to the mass of vcMMAE) and about 35 to about 39 kg / kg MTBE (relative to the mass of vcMMAE).

20. The method of any one of claims 17 to 19, wherein the mixture of THF and MTBE comprises about 20:39 to about 25:30 v / v THF / MTBE; optionally, wherein the mixture of THF and MTBE is about 1:2 v / v THF and MTBE (1:2 v / v THF / MTBE).

21. The method of 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 of any one of claims 17 to 21, further comprising filtering out the gvcMMAE precipitate in the form of a wet cake, and rinsing the wet cake with MTBE.

23. The method of any one of claims 1-22, further comprising reacting gvcMMAE with a bicyclic of Formula II: or a pharmaceutically acceptable salt thereof, wherein: m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. to form a bicyclic toxin conjugate of Formula I:

24. The method of claim 23, wherein the bicyclic toxin conjugate of Formula I is BT8009 or a pharmaceutically acceptable salt thereof. R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , and R 9 are each independently hydrogen or an optionally substituted group selected from C 1-6 aliphatic, 3-8 membered saturated or partially unsaturated monocyclic carbocyclic ring, phenyl, 8-10 membered bicyclic aromatic carbocyclic ring, 4-8 membered saturated or partially unsaturated monocyclic heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur, 5-6 membered monocyclic heteroaromatic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or 8-10 membered bicyclic heteroaromatic ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur; and 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, which is gvcMMAE, obtainable by the method of any one of claims 1 to 22.

27. A bicyclic toxin conjugate of Formula (I), obtainable by the method of any one of claims 22 to 25. ​ ​

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