Synthesis of bicyclic toxin conjugates and intermediates

Abstract

The present invention relates to bicyclic toxin conjugates, methods for their preparation and methods of use to treat cancer.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Patent Application No. 63 / 260,878, filed September 3, 2021, the entire contents of which are incorporated herein by reference.

[0002] FIELD OF THEINVENTION The present invention relates to methods for synthesizing bicyclic toxin conjugates (BTCs), such as BT8009, which comprise a constrained bicyclic peptide covalently linked to the potent anti-tubulin agent MMAE, and intermediates thereto. [Background technology]

[0003] Cyclic peptides are an attractive class of molecules for the development of therapeutic drugs, since they can bind to protein targets with high affinity and target specificity. Indeed, several cyclic peptides have already been successfully used in the clinic, for example, the antimicrobial peptide vancomycin, the immunosuppressant cyclosporine, and 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 surfaces of several hundred square angstroms, such as the cyclic peptide CXCR4 antagonist CVX15 (400 Å2; Wu et al. (2007), Science 330, 1066-71), a cyclic peptide with an Arg-Gly-Asp motif that binds to integrin αVb3 (355 Å2) (Xiong et al. (2002), Science 296 (5565), 151-5), or the cyclic peptide inhibitor upain-1 that binds to urokinase-type plasminogen activator (603 Å2; Zhao et al. (2007), J Struct Biol 160 (1), 1-10).

[0004] Due to their cyclic configuration, peptide macrocycles are less flexible than linear peptides, which leads to less entropy loss upon binding to the target, resulting in higher potential binding affinity. The reduced flexibility also leads to target-specific conformational locking, which increases binding specificity compared to linear peptides. This effect has been demonstrated by potent and selective inhibitors of matrix metalloproteinase 8 (MMP-8), which lost selectivity over other MMPs when the ring was opened (Cherney et al. (1998), J Med Chem 41 (11), 1749-51). The favorable binding properties achieved by macrocyclization are even more pronounced in polycyclic peptides with more than one peptide ring, such as vancomycin, nisin, and actinomycin.

[0005] Previously, various research teams have conjugated polypeptides containing cysteine ​​residues to synthetic molecular structures (Kemp and McNamara (1985), J. Org. Chem; Timmerman et al. (2005), ChemBioChm). Meloen and coworkers have used tris(bromomethyl)benzene and related molecules to rapidly and quantitatively cyclize multiple peptide loops onto synthetic scaffolds to mimic the structure of protein surfaces (Timmerman et al. (2005), ChemBioChem). Methods for the generation of drug candidates generated by conjugating cysteine-containing polypeptides to molecular scaffolds such as tris(bromomethyl)benzene are disclosed in WO 2004 / 077062 and WO 2006 / 078161.

[0006] A phage display-based combinatorial approach has been developed to generate and screen large libraries of bicyclic peptides against targets of interest (Heinis et al. (2009), Nat Chem Biol 5 (7), 502-7 and WO2009 / 098450). Briefly, a combinatorial library of linear peptides containing three cysteine ​​residues and two regions of six random amino acids (Cys-(Xaa)6-Cys-(Xaa)6-Cys) was displayed on phage and cyclized by covalently linking the cysteine ​​side chain to a small molecule (tris-(bromomethyl)benzene). Summary of the Invention

[0007] The present invention provides a bicyclic toxin conjugate and its preparation method.In some embodiments, the bicyclic toxin conjugate of the present invention comprises a constrained bicyclic peptide covalently linked to a potent antitubulin agent MMAE.In some embodiments, the bicyclic toxin conjugate comprises a constrained bicyclic peptide that binds to nectin-4 with high affinity and specificity.

[0008] In some embodiments, the present invention provides a compound of formula I: [ka] [In the formula, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 wherein m, n, and n are each defined below and as described in the embodiments (both alone and in combination) herein, or a pharma- ceutically acceptable salt thereof.

[0009] In some embodiments, the present invention provides methods for preparing a bicyclic toxin conjugate of the present invention, or a synthetic intermediate thereof, according to the schemes and steps described herein.

[0010] In some embodiments, the present invention provides a method of preventing and / or treating a cancer described herein, comprising administering to a patient a bicyclic toxin conjugate of the present invention.

[0011] In some embodiments, the present invention provides synthetic intermediates or compositions thereof useful for the preparation of the bicyclic toxin conjugates of the present invention. [Brief description of the drawings]

[0012] [Figure 1] Figure 1 shows the factorial regression: +56 (%) vs. TFA (%), DTT (%). CenterPt is shown in Figure 1 showing a normal plot of the effect on the +56 impurity.

[0013] [Diagram 2] FIG. 2 shows a Pareto plot of the effect on the +56 impurity.

[0014] [Diagram 3] Figure 3 shows the factorial regression: +163 (%) vs. TFA (%), DTT (%). CenterPt is shown in Figure 3 showing a normal plot of the effect on the +163 impurity.

[0015] [Figure 4] FIG. 4 shows a Pareto plot of the effect on the +163 impurity.

[0016] [Diagram 5] FIG. 5 shows response optimization of the cleavage cocktail.

[0017] Detailed Description of the Invention 1. General Description of Certain Aspects of the Invention Numerous bicyclic toxin conjugates and methods for their synthesis are described in International Patent Application No. PCT / GB2019 / 051740 (International Publication No. WO 2019 / 243832), the entire contents of which are incorporated herein by reference. For example, bicyclic toxin conjugate BCY8245 (BT8009) is described as being synthesized as follows: step 1) solid phase synthesis of Fmoc-Val-Cit; step 2) Fmoc deprotection; step 3) amide formation with monomethyl glutarate; step 4) cleavage of glutaryl-Val-Cit methyl ester from the resin under mildly acidic conditions; step 5) formation of an amide at the C-terminus with p-aminobenzyl alcohol; step 6) formation of p-nitrophenyl carbamate using bis(4-nitrophenyl)carbonate; step 7) M step 8) hydrolysis of the glutaryl methyl ester to form the acid; step 9) activation of the acid and treatment with N-hydroxysuccinimide to form the activated NHS ester; step 10) treatment of the NHS ester with BCY8234 in the presence of base (DIEA) in DMA to form BCY8245, followed by standard reverse phase purification using a C18 semi-preparative column (TFA conditions) and lyophilization to obtain the pure bicyclic toxin complex BCY8245 (BT8009).

[0018] Here, we show that treating Val-Cit-PAB-MMAE with glutaric anhydride and directly forming the amide with the resulting acid and BCY8234 reduces the number of steps in the synthetic route and improves the impurity profile and yield.

[0019] The improved process of BCY8245 includes, but is not limited to, the following features: Simplified two-step process; · Improved yield (44% yield in two steps, LC purity 96.9%); Using 1 equivalent of gvcMMAE / TBTU in the coupling step (second step) to reduce the identified RRT0.93 impurity; and - Optimized filtration and column purification steps.

[0020] Furthermore, we improved the synthesis of the bicyclic peptide BCY8234.

[0021] The improved process of BCY8234 includes, but is not limited to, the following features: A reduction in the amount of aspartimide impurities formed; · Optimization of the deblocking cocktail containing 3% Oxyma in 10% piperidine / DMF; · Using DITU in coupling reactions to help inhibit oxidation of cysteine; · Using sarcosine dipeptide derivatives in sarcosine coupling; Use of high-loading (>0.8mmol / g) resin; During the bicyclic peptide formation process, TATA was reduced to 1.3 eq., the reaction time was shortened to 4 h, and the ACN content was reduced to 20%; Identification of optimal pH values ​​for column packing (pH = 6.8) and long-term crude product storage (pH = 4.5); and The purified TFA salt is desalted and then lyophilized to improve its long-term stability.

[0022] Thus, in one aspect, the present invention provides a compound of formula I: [ka] [In the formula, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , and R 11 are each independently hydrogen or C 1-6an optionally substituted group selected from an aliphatic, a 3-8 membered saturated or partially unsaturated monocyclic carbocyclic ring, a phenyl, an 8-10 membered bicyclic aromatic carbocyclic ring, a 4-8 membered saturated or partially unsaturated monocyclic heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur, a 5-6 membered monocyclic heteroaromatic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an 8-10 membered bicyclic heteroaromatic ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur; m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15; and n is 0, 1 or 2. or a pharma- ceutically acceptable salt thereof.

[0023] In another aspect, the present invention provides a method of preparing a bicyclic toxin conjugate of formula I or a salt thereof. In certain embodiments, the compounds are generally prepared according to Scheme I set forth below, where each of the variables, reagents, intermediates and reaction steps are as defined below and described in the embodiments (both alone and in combination) herein. [ka] [ka]

[0024] Variable R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , m and n are as defined above and are classes and subclasses described herein.

[0025] In one embodiment, the present invention provides a method for preparing a bicyclic toxin conjugate (BTC) of formula I from homochiral starting materials of high enantiomeric and diastereomeric purity according to the steps shown in Scheme I above. 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 and R 11 are as defined above for compounds of formula I, each independently being hydrogen or C 1-6 an optionally substituted group selected from an aliphatic, a 3-8 membered saturated or partially unsaturated monocyclic carbocyclic ring, a phenyl, an 8-10 membered bicyclic aromatic carbocyclic ring, a 4-8 membered saturated or partially unsaturated monocyclic heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur, a 5-6 membered monocyclic heteroaromatic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an 8-10 membered bicyclic heteroaromatic ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0026] In compounds of this formula, m is as defined above for compounds of formula I and is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

[0027] In compounds of this formula, n is as defined above for compounds of formula I and is 0, 1 or 2.

[0028] In step S-1, a fragment of formula F-1 is coupled to an anhydride of formula A via ring-opening addition to the anhydride of formula A to form a fragment of formula F-2.

[0029] In step S-2, the F-2 fragment is coupled to the F-3 fragment via amide formation to form a compound of formula I. Amide formation can be achieved using a wide variety of coupling agents known in the art, including but not limited to: ·N,N'-dicyclohexylcarbodiimide (DCC); ·N,N'-Diisopropylcarbodiimide (DIC); ·N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC); ·N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU); ·N,N,N',N'-Tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU); ·O-(1H-6-chlorobenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU); ·(Benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP); ·(7-Azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP); · Bromotripyrrolidinophosphonium hexafluorophosphate (PyBrOP); · Benzotriazol-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate (BOP); ·Bis(2-oxo-3-oxazolidinyl)phosphine chloride (BOP-Cl); ·3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one (DEPBT); · 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphorinane-2,4,6-trioxide (T3P); ·1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide tetrafluoroborate (TATU); ·N,N,N',N'-Tetramethyl-O-(benzotriazol-1-yl)uronium tetrafluoroborate (TBTU); ·2-(endo-5-norbornene-2.3-dicarboxylimido)-1,1,3,3-tetramethyluronium tetrafluoroborate (TNTU); ·O-[(ethoxycarbonyl)cyanomethyleneamino]-N,N,N',N'-tetramethyluronium tetrafluoroborate (TOTU); ·O-(2-oxo-1(2H)pyridyl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TPTU); N,N,N',N'-Tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (TSTU); or ·O-(3,4-Dihydro-4-oxo-1,2,3-benzotriazin-3-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TDBTU).

[0030] In another aspect, the present invention provides a method for preparing fragment F-3 or a salt thereof. In certain embodiments, the compounds of the present invention are generally prepared according to Scheme II as set forth below, in which the variables, reagents, intermediates and reaction steps are each as defined below and as described in the embodiments (both alone and in combination) herein. [ka] [ka] [ka]

[0031] In one embodiment, the present invention provides a method for preparing a fragment of formula F-3 in enantiomerically enriched form according to the steps shown in Scheme II above. 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 and R 11 are as defined above for compounds of formula I, and each independently is hydrogen or C 1-6 an optionally substituted group selected from an aliphatic, a 3-8 membered saturated or partially unsaturated monocyclic carbocyclic ring, a phenyl, an 8-10 membered bicyclic aromatic carbocyclic ring, a 4-8 membered saturated or partially unsaturated monocyclic heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur, a 5-6 membered monocyclic heteroaromatic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an 8-10 membered bicyclic heteroaromatic ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0032] In compounds of this formula, m is as defined above for compounds of formula I and is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

[0033] In compounds of this formula, n is as defined above for compounds of formula I and is 0, 1 or 2.

[0034] In step S-1′, the compound of formula G is deprotected to form a nitrogen protecting group PG 3 and then coupled to a protected amino acid of formula F, followed by PG 3 is removed to form a compound of formula E via amide formation.

[0035] Those skilled in the art can easily determine the PG concentration using various conditions. 3It will be appreciated that in some embodiments, PG 3 Removal can be accomplished by treatment with 20% piperidine in DMF (deblocking step). 3 Removal may be followed by a washing cycle with DMF prior to the coupling / recoupling step.

[0036] In step S-2', the compound of formula E is reacted with PG 3 Iterative coupling to protected amino acids followed by PG 3 to form a compound of formula D via amide formation. Amide formation can be achieved using a wide variety of coupling agents known in the art, such as, but not limited to, DCC, DIC, EDC, HATU, HBTU, HCTU, PyBOP, PyAOP, PyBrOP, BOP, BOP-Cl, DEPBT, T3P, TATU, TBTU, TNTU, TOTU, TPTU, TSTU, or TDBTU. Those skilled in the art will recognize that amide formation can be achieved using the above coupling agents.

[0037] In some embodiments, amide formation is accomplished using DIC / Oxyma to give the compound of formula D.

[0038] In step S-3', the compound of formula D is a) cleaved from the solid phase resin and b) globally deprotected (i.e., the indicated PG 2 and P.G. 1 Protecting groups, as well as R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , and R 11(removal of any additional protecting groups on the groups) to obtain a compound of formula C. Those skilled in the art will recognize that cleavage from the solid phase and global deprotection can be achieved by treatment with acid. Those skilled in the art will also recognize that cleavage from the solid phase and global deprotection can be achieved in one step by treatment with an acid such as TFA and a TFA cocktail containing a cation scavenger, including but not limited to DTT, TIS and NHI, in a solvent such as water.

[0039] In step S-4', the compound of formula C is cyclized onto compound B (TATA) to give a compound of formula F-3. One skilled in the art will recognize that the reaction can proceed via three Michael additions of the cysteine ​​residues of the compound of formula C to TATA, which can be accomplished under basic conditions to give a cyclic product.

[0040] PG of formula D 1 Each group is independently a suitable alcohol protecting group. Suitable alcohol protecting groups are well known in the art and are described in detail in Protecting Groups in Organic Synthesis, TW Greene and PGM Wuts, 4th Edition, John Wiley & Sons, 2006, the entirety of which is incorporated herein by reference. Suitable alcohol protecting groups, taken together with the --O-- moiety to which they are attached, include, but are not limited to, ethers, substituted methyl ethers, substituted ethyl ethers, substituted benzyl ethers, and the like. PG of formula D 1 Exemplary groups include t-butyl (tBu), methyl, ethyl, methoxymethyl, tetrahydrofuranyl, allyl, benzyl (Bn), acetate, 2-hydroxyethyl, and the like. In certain embodiments, PG in the compound of formula D is 1 The PG group in the compound of formula D is t-butyl (tBu), methyl, acetate, or ethyl. 1 The group is t-butyl (tBu).

[0041] PG of formulas D, E and G2 Each group is independently a suitable thiol protecting group. Suitable thiol protecting groups are well known in the art and are described in Protecting Groups in Organic Synthesis, TW Greene and PGM Wuts, 4 th Edition, John Wiley & Sons, 2006, the entirety of which is incorporated herein by reference. Suitable thiol protecting groups, taken together with the --S-- moiety to which they are attached, include, but are not limited to, ethers, substituted methyl ethers, substituted ethyl ethers, substituted benzyl ethers, and the like. PGs of formula D, E and G 2 Exemplary groups include t-butyl (tBu), methyl, ethyl, methoxymethyl, tetrahydrofuranyl, allyl, benzyl (Bn), diphenylmethyl, triphenylmethyl (Tr), adamantyl, etc. In certain embodiments, the PG group of the compounds of formulas D, E, and G is 2 The PG group in the compounds of formulas D, E, and G is triphenylmethyl (Tr), t-butyl (tBu), methyl, diphenylmethyl, or adamantyl. 2 The group is triphenylmethyl (Tr).

[0042] PG of formula F and F' 3 Each group is independently a suitable amino protecting group. Suitable amino protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, TW Greene and PGM Wuts, 4th Edition, John Wiley & Sons, 2006, which is incorporated herein by reference in its entirety. Suitable amino protecting groups, taken together with the --NH-- moiety to which they are attached, include, but are not limited to, aralkylamines, carbamates, allylamines, amides, and the like. PG of formula F and F' 3Exemplary groups include t-butyloxycarbonyl (BOC), ethyloxycarbonyl, methyloxycarbonyl, trichloroethyloxycarbonyl, allyloxycarbonyl (Alloc), benzyloxycarbonyl (CBZ), allyl, benzyl (Bn), fluorenylmethylcarbonyl (Fmoc), acetyl, chloroacetyl, dichloroacetyl, trichloroacetyl, phenylacetyl, trifluoroacetyl, benzoyl, pivaloyl, and the like. In certain embodiments, the PG of the compounds of formula F and F' is 3 The PG group in the compounds of formula F and F' is t-butyloxycarbonyl, ethyloxycarbonyl, fluorenylmethylcarbonyl (Fmoc), or acetyl. 3 The group is fluorenylmethylcarbonyl (Fmoc).

[0043] Those skilled in the art will recognize that the iterative amide coupling and deprotection protocols using homochiral building blocks described herein can be adapted to provide compounds of formulas E, D, C, and F-3 with high enantiomeric and diastereomeric purity. In certain embodiments, one diastereomer of the compounds of formulas E, D, C, and F-3 is formed substantially free of other stereoisomers. As used herein, "substantially free" means that the compound is made up of a significantly higher proportion of one diastereomer. In other embodiments, at least about 98% by weight of the desired diastereomer is present. In yet other embodiments of the present invention, at least about 99% by weight of the desired diastereomer is present. Such diastereomers can be isolated from the diastereomeric mixture by any method known to those skilled in the art, including high performance liquid chromatography (HPLC) and crystallization, or can be prepared by the methods described herein.

[0044] 2. Compounds and Definitions The compounds of the present invention include those generally described above, and are further exemplified by the classes, subclasses, and species disclosed herein. As used herein, the following definitions shall apply unless otherwise specified. For purposes of the present invention, chemical elements are defined as defined in the Periodic Table of the Elements, CAS edition, Handbook of Chemistry and Physics, 75 th Ed. In addition, the general principles of organic chemistry are defined in “Organic Chemistry”, Thomas Sorrell, University Science Books, Sausalito: 1999, and “March's Advanced Organic Chemistry”, 5 th Ed., Ed.: Smith, MB and March, J., John Wiley & Sons, New York: 2001, the entire contents of each of which are incorporated herein by reference.

[0045] As used herein, the term "aliphatic" or "aliphatic group" refers to a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is fully saturated or contains one or more units of unsaturation, or a monocyclic or bicyclic hydrocarbon that is fully saturated or contains one or more units of unsaturation but is not aromatic (also referred to herein as "carbocyclic," "cycloaliphatic," or "cycloalkyl"), having a single point of attachment to the remainder of the molecule. 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 still 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 "carbocycle" or "cycloalkyl") refers to a monocyclic C3-C6 hydrocarbon that is fully saturated or contains one or more units of unsaturation, but is not aromatic, and has a single point of attachment to the rest of the molecule. Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl groups, and hybrids thereof, such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl, or (cycloalkyl)alkenyl.

[0046] As used herein, the term "bridged bicyclic" refers to any bicyclic ring system having at least one bridge, i.e., saturated or partially unsaturated carbocyclic or heterocyclic. As defined by IUPAC, a "bridge" is an unbranched chain of atoms or an atom or valence bond connecting two bridgeheads, where a "bridgehead" is any skeletal atom of the ring system that is connected to three or more skeletal atoms (except hydrogen). In some embodiments, the 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 the groups defined below, where each group is attached to the remainder of the molecule at any substitutable carbon or nitrogen atom. Unless otherwise specified, bridged bicyclic groups may be optionally substituted by one or more substituents as defined for aliphatic groups. Additionally or alternatively, the substitutable nitrogen of the bridged bicyclic group may be optionally substituted. Exemplary bridged bicyclic groups include: [ka] Examples include:

[0047] The term "lower alkyl" refers to C 1-4 It means a straight or branched alkyl group. Exemplary lower alkyl groups are methyl, ethyl, propyl, isopropyl, butyl, isobutyl and tert-butyl.

[0048] The term "lower haloalkyl" refers to a C substituted with one or more halogen atoms. 1-4 It means a straight or branched alkyl group.

[0049] The term "heteroatom" refers to any of oxygen, sulfur, nitrogen, phosphorus, or silicon (in the oxidized form of nitrogen, sulfur, phosphorus, or silicon; in the quaternized form of a basic nitrogen; or in the substitutable form of a heterocyclic ring, such as N (in the case of 3,4-dihydro-2H-pyrrolyl), NH (in the case of pyrrolidinyl), or NR +(including in the case of N-substituted pyrrolidinyl) means one or more of the following:

[0050] As used herein, the term "unsaturated" means that a moiety has one or more units of unsaturation.

[0051] As used herein, the term "divalent hydrocarbon chain" refers to straight or branched divalent alkylene, alkenylene, and alkynylene chains, as defined herein.

[0052] The term "alkylene" refers to a divalent alkyl group. An "alkylene chain" is a polymethylene group, i.e., -(CH2) n - (where n is a positive integer, preferably 1 to 6, 1 to 4, 1 to 3, 1 to 2, or 2 to 3). The substituted alkylene chain is a polymethylene group in which one or more methylene hydrogen atoms are replaced with a substituent. Suitable substituents include those described below for substituted aliphatic groups.

[0053] The term "alkenylene" refers to a divalent alkenyl group. A substituted alkenylene chain is a polymethylene group containing at least one double bond in which one or more hydrogen atoms are replaced with a substituent. Suitable substituents include those described below for substituted aliphatic groups.

[0054] The term "alkynylene" refers to a divalent alkynyl group. A substituted alkynylene chain is a polymethylene group containing at least one triple bond in which one or more hydrogen atoms are replaced by a substituent. Suitable substituents include those described below for substituted aliphatic groups.

[0055] As used herein, the term "cyclopropylenyl" refers to a divalent cyclopropyl group of the following structure: [ka]

[0056] The term "halogen" means F, Cl, Br, or I.

[0057] The term "aryl" used alone or as part of a larger moiety such as "aralkyl", "aralkoxy" or "aryloxyalkyl" refers to a monocyclic or bicyclic ring system having a total of 5 to 14 ring members, where at least one ring in the system is aromatic and each ring in the system contains 3 to 7 ring members. The term "aryl" may be used interchangeably with the term "aryl ring". In certain embodiments of the invention, "aryl" refers to an aromatic ring system which may bear one or more substituents, including, but not limited to, phenyl, biphenyl, naphthyl, anthracyl, and the like. Also included within the scope of the term "aryl" as used herein are groups in which an aromatic ring is fused to one or more non-aromatic rings, such as, for example, indanyl, phthalimidyl, naphthimidyl, phenanthridinyl, or tetrahydronaphthyl.

[0058] The terms "heteroaryl" and "heteroar-" used alone or as part of a larger moiety, such as "heteroaralkyl" or "heteroaralkoxy", refer to a group having 5 to 10 ring atoms, preferably 5, 6, or 9 ring atoms; having 6, 10, or 14 pi electrons shared in a cyclic array; and, in addition to the carbon atoms, having 1 to 5 heteroatoms. The term "heteroatom" means nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur and any quaternized form of a basic nitrogen. Heteroaryl groups include, but are not limited to, 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, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyrido[2,3-b]-1,4-oxazin-3(4H)-one. Heteroaryl groups are monocyclic or bicyclic. The term "heteroaryl" may be used interchangeably with the term "heteroaryl ring," "heteroaryl group," or "heteroaromatic," all of which include rings that are optionally substituted. The term "heteroaralkyl" refers to an alkyl group substituted by a heteroaryl, where the alkyl and heteroaryl portions independently are optionally substituted.

[0059] As used herein, the terms "heterocycle", "heterocyclyl", "heterocyclic radical" and "heterocyclic ring" are used interchangeably and refer to a stable 5-7 membered monocyclic or 7-10 membered bicyclic heterocyclic moiety that is saturated or partially unsaturated and has, in addition to carbon atoms, one or more, preferably 1-4 heteroatoms as defined above. When used in reference to a ring atom of a heterocycle, the term "nitrogen" includes substituted nitrogen. By way of 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 . + It may also be NR (as in the case of N-substituted pyrrolidinyl).

[0060] Heterocyclic ring can be bonded to its pendant group at any heteroatom or carbon atom to provide a stable structure, and any of the ring atoms can be optionally substituted. Examples of such saturated or partially unsaturated heterocyclic radicals include, but are not limited to, tetrahydrofuranyl, tetrahydrothiophenylpyrrolidinyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, triazepinyl, 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 a heterocyclyl ring is fused to one or more aryl, heteroaryl or cycloaliphatic rings, such as indolinyl, 3H-indolyl, chromanyl, phenanthridinyl, or tetrahydroquinolinyl. A heterocyclyl group may be monocyclic or bicyclic. The term "heterocyclylalkyl" means an alkyl group substituted by a heterocyclyl, where the alkyl and heterocyclyl portions each independently may be optionally substituted.

[0061] As used herein, the term "partially unsaturated" means a ring moiety that contains at least one double or triple bond. The term "partially unsaturated" is intended to include rings having multiple sites of unsaturation, but is not intended to include aryl or heteroaryl moieties, as defined herein.

[0062] As described herein, the compounds of the present invention can include "optionally substituted" moieties. In general, the term "substituted", whether preceded by the term "optionally" or not, means that one or more hydrogens of the specified moiety are replaced by suitable substituents. Unless otherwise specified, an "optionally substituted" group can have a suitable substituent at each substitutable position of the group, and when multiple positions in any given structure can be substituted by multiple substituents selected from a specified group, the substituents can be the same or different at all positions. The combinations of substituents envisioned by the present invention are preferably those that result in the formation of stable or chemically feasible compounds. As used herein, the term "stable" refers to compounds that are not substantially altered when subjected to conditions that allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the compounds disclosed herein.

[0063] Suitable monovalent substituents on a substitutable carbon atom of an "optionally substituted" group are independently halogen, -(CH2), 0-4 R°;-(CH2) 0-4 OR°;-O(CH2) 0-4 R°, -O-(CH2) 0-4 C(O)OR°;-(CH2) 0-4 CH(OR°)2;-(CH2) 0-4 SR°; may be substituted by R° -(CH2) 0-4 Ph; optionally substituted by R° -(CH2) 0-4 O(CH2)0-1 Ph; optionally substituted by R° -CH=CHPh; optionally substituted by R° -(CH2) 0-4 O(CH2) 0-1 -Pyridyl; -NO2; -CN; -N3; ​​-(CH2) 0-4 N(R°)2;-(CH2) 0-4 N(R°)C(O)R°;-N(R°)C(S)R°;-N(R°)C(NR°)N(R°)2;-(CH2) 0-4 N(R°)C(O)NR°2;-N(R°)C(S)NR°2;-(CH2) 0-4 N(R°)C(O)OR°;-N(R°)N(R°)C(O)R°;-N(R°)N(R°)C(O)NR°2;-N(R°)N(R°)C(O)OR°;-(CH2) 0-4 C(O)R°;-C(S)R°;-(CH2) 0-4 C(O)OR°;-(CH2) 0-4 C(O)SR°;-(CH2) 0-4 C(O)OSiR°3;-(CH2) 0-4 OC(O)R°;-OC(O)(CH2) 0-4 SR-, -SC(S)SR°;-(CH2) 0-4 SC(O)R°;-(CH2) 0-4 C(O)NR°2;-C(S)NR°2;-C(S)SR°;-(CH2) 0-4 OC(O)NR°2;-C(O)N(OR°)R°;-C(O)C(O)R°;-C(O)CH2C(O)R°;-C(NOR°)R°;-(CH2) 0-4 SSR°;-(CH2) 0-4 S(O)2R°;-(CH2) 0-4 S(O)2OR°;-(CH2) 0-4 OS(O)2R°;-S(O)2NR°2;-(CH2) 0-4 S(O)R°;-N(R°)S(O)2NR°2;-N(R°)S(O)2R°;-N(OR°)R°;-C(NH)NR°2;-P(O)2R°;-P(O)R°2;-OP(O)R°2;-OP(O)(OR°)2;-SiR°3;-(C 1-4 Linear or branched alkylene)ON(R°)2; or -(C 1-4linear or branched alkylene)C(O)ON(R°)2, where each R° is optionally substituted as defined below and is independently hydrogen, C 1-6 Aliphatic, -CH2Ph, -O(CH2) 0-1 Ph, -CH2- (a 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° taken together with their intervening atoms form a 3-12 membered saturated, partially unsaturated, or aryl monocyclic or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur, which may be optionally substituted as defined below.

[0064] Suitable monovalent substituents on R° (or the ring formed by taking two independent occurrences of R° together with their intervening atoms) are independently halogen, halogen, -(CH2), 0-2 R ● , -(Halo R ● ), -(CH2) 0-2 OH, -(CH2) 0-2 OR ● , -(CH2) 0-2 CH(OR ● )2;-O(HaloR ● ), -CN, -N3, -(CH2) 0-2 C(O)R ● , -(CH2) 0-2 C(O)OH, -(CH2) 0-2 C(O)OR ● , -(CH2) 0-2 S.R. ● , -(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, if preceded by "halo", is substituted only with one or more halogens; C 1-4 Aliphatic, -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. Suitable divalent substituents on a saturated carbon atom of R° include =O and =S.

[0065] Suitable divalent substituents on a saturated carbon atom of an "optionally substituted" group include: =O, =S, =NNR * 2, =NNHC(O)R * , =NNHC(O)OR * , =NNHS(O)2R * , =NR * , =NOR * , -O(C(R * 2)) 2-3 O-, or -S(C(R * 2)) 2-3 S-, where R *の Each independent occurrence is hydrogen, optionally substituted as defined below. 1-6 Aliphatic or unsubstituted 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents attached to adjacent substitutable carbons of the "optionally substituted" group include: -O(CR * 2) 2-3 O-(where R * Each independent occurrence of is hydrogen, optionally substituted as defined below C 1-6 aliphatic, or unsubstituted 5-6 membered saturated, partially unsaturated or aryl rings having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur.

[0066] R *Suitable substituents on the aliphatic group include halogen, -R ● , -(Halo R ● ), -OH, -OR ● , -O(HaloR ● ), -CN, -C(O)OH, -C(O)OR ● , -NH2, -NHR ● , -NR ● 2, or -NO2, where each R ● is unsubstituted or, if preceded by "halo", is substituted only with one or more halogens, and independently represents C 1-4 Aliphatic, -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.

[0067] Suitable substituents on a substitutable nitrogen of an "optionally substituted" group include -R † , -NR † 2. -C(O)R † , -C(O)OR † , -C(O)C(O)R † , -C(O)CH2C(O)R † , -S(O)2R † , -S(O)NR † 2. -C(S)NR † 2. -C(NH)NR † 2, or -N(R † )S(O)2R † where each R †は , independently hydrogen, C optionally substituted as defined below 1-6 aliphatic, 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, R † two independent occurrences of together with their intervening atoms form an unsubstituted 3-12 membered saturated, partially unsaturated, or aryl monocyclic or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0068] R † Suitable substituents on the aliphatic group are independently halogen, -R ● , -(Halo R ● ), -OH, -OR ● , -O(HaloR ● ), -CN, -C(O)OH, -C(O)OR ● , -NH2, -NHR ● , -NR ● 2, or -NO2, where each R ● is unsubstituted or, if preceded by "halo", is substituted only with one or more halogens, and independently represents C 1-4 Aliphatic, -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.

[0069] As used herein, the term "pharmaceutical acceptable salt" refers to a salt that is suitable for use in contact with human and lower animal tissues without excessive toxicity, irritation, allergic reaction, etc., within the scope of sound medical judgment, and is commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al. describe pharmaceutical acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19 (incorporated herein by reference). In addition, pharmaceutical acceptable salts are described in detail in Pharmaceutical Salts: Properties, Selection, and Use, 2nd Revised Edition, (2011), P. Heinrich Stahl (Editor), Camille G. Wermuth (Editor), (ISBN: 978-3-906-39051-2) (incorporated herein by reference in its entirety). Pharmaceutically acceptable salts of the compounds of the present invention include those derived from suitable inorganic and organic acids and bases. Examples of pharma-ceutically acceptable non-toxic acid addition salts are salts of amino groups formed with inorganic acids such as hydrochloric, hydrobromic, phosphoric, sulfuric and perchloric acids, or organic acids such as acetic, oxalic, maleic, tartaric, citric, succinic or malonic acids, or by other methods used in the art, such as ion exchange.Other pharma- ceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, besylate, benzoate, hydrogensulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, malate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxybutanol ... Salts of hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, mesylate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, and valerate.

[0070] Salts derived from appropriate bases include alkali metal salts, alkaline earth metal salts, ammonium salts and N + (C 1-4 Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharma- ceutically acceptable salts include, where appropriate, halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, (C 1-6 Examples of the counter ions include non-toxic ammonium salts, quaternary ammonium salts, and amine cation salts formed using counter ions such as alkyl)sulfonates, arylsulfonates, and the like.

[0071] Unless otherwise specified, structures depicted herein are 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 compounds of the invention are within the scope of the invention. Unless otherwise specified, all tautomeric forms of the compounds of the invention are within the scope of the invention.

[0072] As used herein, "therapeutically effective amount" refers to 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 to treat, diagnose, prevent, and / or delay the onset of a disease, condition, or disorder when administered as part of a dosing regimen to a subject suffering from or susceptible to a disease, condition, or disorder. As will be understood by those skilled in the art, the effective amount of a substance can vary depending on factors such as the desired biological endpoint, the substance delivered, the target cell or tissue, etc. For example, an effective amount of a compound in a formulation for treating a disease, condition, or disorder is an amount that relieves, improves, alleviates, inhibits, prevents, delays the onset, reduces the severity, and / or reduces the incidence of one or more symptoms or characteristics of a disease, condition, or disorder.

[0073] The term "treat" or "treating" as used herein means to partially or completely alleviate, inhibit, delay onset, prevent, improve and / or alleviate a disease or disorder, or one or more symptoms of a disease or disorder. As used herein, the terms "treatment", "treat" and "treating" mean to partially or completely alleviate, inhibit, delay onset, prevent, improve and / or alleviate a disease or disorder, or one or more symptoms of a disease or disorder, as described herein. In some embodiments, treatment may be administered after one or more symptoms have developed. In some embodiments, the term "treating" includes preventing or arresting the progression of a disease or disorder. In other embodiments, treatment may be administered in the absence of symptoms. For example, treatment may 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 may also be continued after symptoms have resolved, for example to prevent or delay recurrence. Thus, in some embodiments, the term "treating" includes preventing the relapse or recurrence of a disease or disorder.

[0074] The expression "unit dosage form" as used herein means a physically discrete unit of therapeutic preparation suitable for the subject to be treated. However, it will be understood that the total daily usage of the composition of the present invention will be determined by the attending physician within the scope of sound medical judgment. The specific effective dose level for any particular subject or organism will depend on various factors, including the disorder to be treated and the severity of the disorder; the activity of the specific active agent used; the specific composition used; the age, weight, general health, sex and diet of the subject; the administration time and excretion rate of the specific active agent used; the duration of treatment; the drugs and / or additional therapies used in combination or simultaneously with the specific compound(s) used, and factors as are well known in the medical art.

[0075] Bicyclic toxin conjugate BT8009 has the structure shown below, and the preparation of BT8009 (BCY8245) is described in WO 2019 / 243832, the entirety of which is incorporated herein by reference. [ka]

[0076] 3. Description of the synthesis of bicyclic toxin conjugates of formula I and related intermediates In some embodiments, the invention is a method for preparing a bicyclic toxin conjugate of formula I according to Scheme I, wherein each of the variables, reagents, intermediates, and reaction steps are as defined below and described in the embodiments (both alone and in combination) herein.

[0077] The compounds of formula I in Scheme I comprise constrained bicyclic peptides that bind 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 / GB 2019 / 051740 (International Publication No. WO 2019 / 243832), the entire contents of which are incorporated herein by reference. In some embodiments, the bicyclic peptide is a peptide covalently attached to a molecular scaffold. In some embodiments, the bicyclic peptide comprises three cysteine ​​residues (C in the sequence below) that are capable of forming covalent bonds to the molecular scaffold. i , C ii and C iii In some embodiments, the bicyclic peptide comprises a peptide having Peptide C i -P / A / Hyp-F / YG / AC ii -X1-X2-X3-W / 1-Nal / 2-Nal-S / A-X4-PI / D / AW / 1-Nal / 2-Nal-C iii (SEQ ID NO:1); C i -W / APLD / SS / DYWC ii -X5-RIC iii (SEQ ID NO:2); C i -VTTSYDC ii -F / WL / VH / R / TLL / GG / Q / HC iii (SEQ ID NO:3); C i -X6-X7-X8-C ii -X9-X 10 -X 11 -X 12 -X 13 -X 14 -X 15 -X 16 -X 17 -C iii (SEQ ID NO:4); and C i -W / A / YP / ALD / S / AS / D / P / AYW / 1-Nal-C ii -X5-R / HArg / AIC iii(SEQ ID NO:5); X1-X5 represent any amino acid residue, including modified and unnatural amino acids; X6 represents Gly; Pro or an unnatural derivative of Pro selected from azetidine (Aze), hydroxyproline (HyP), 4-amino-proline (Pro(4NH)), oxazolidine-4-carboxylic acid (Oxa), octahydroindoline carboxylic acid (Oic) or 4,4-difluoroproline (4,4-DFP); Ala or an unnatural derivative of Ala selected from aminoisobutyric acid (Aib); or sarcosine (Sar); X7 represents Phe or an unnatural derivative of Phe selected from 3-methyl-phenylalanine (3MePhe), 4-methyl-phenylalanine (4MePhe), homophenylalanine (HPhe), 4,4-biphenylalanine (4,4-BPA) or 3,4-dihydroxyphenylalanine (DOPA); Tyr; or Ala or an unnatural derivative of Ala selected from 1-naphthylalanine (1-Nal), 2-naphthylalanine (2-Nal) or 2-pyridylalanine (2Pal); X8 represents Gly; Ala; Asp; Lys or a non-natural derivative of Lys selected from acetyl-lysine (KAc or Lys(Ac)); Phe; Glu; Gln; Leu; Ser; Arg; or cysteic acid (Cya); X9 represents absent or a non-natural derivative of Met selected from Met or a non-natural derivative of Met(O2); Gln or a non-natural derivative of Gln selected from homoglutamine (HGln); Leu or a non-natural derivative of Leu selected from homoleucine (HLeu) or norleucine (Nle); Lys; Ile; t-butyl-alanine (tBuAla); or homoserine-methyl (HSe(Me)); 10denotes Pro; Lys or a non-natural derivative of Lys selected from acetyl-lysine (KAc or Lys(Ac)); Arg or a non-natural derivative of Arg selected from 2-amino-4-guanidinobutyric acid (Agb), homoarginine (HArg) or N-methyl-homoarginine; Glu; Ser; Asp; Gln; Ala; hydroxyproline (HyP); or cysteic acid (Cya); X 11 denotes Asn or a non-natural derivative of Asn selected from N-methyl-asparagine; Thr; Asp; Gly; Ser; His; Ala or a non-natural derivative of Ala selected from thienyl-alanine (Thi), 2-(1,2,4-triazol-1-yl)-alanine (1,2,4-TriAz) or beta-(4-thiazolyl)-alanine (4ThiAz); Lys; or cysteic acid (Cya); X 12 denotes Trp or an unnatural derivative of Trp selected from azatryptophan (AzaTrp), 5-fluoro-L-tryptophan (5FTrp) or methyl-tryptophan (TrpMe); or Ala or an unnatural derivative of Ala selected from 1-naphthylalanine (1-Nal) or 2-naphthylalanine (2-Nal); X 13 represents Ser or a non-natural derivative of Ser selected from homoserine (HSer); Ala; Asp; or Thr; X 14denotes Trp or an unnatural derivative of Trp selected from azatryptophan (AzaTrp); Ser; Ala or an unnatural derivative of Ala selected from 2-(1,2,4-triazol-1-yl)-alanine (1,2,4-TriAz), 1-naphthylalanine (1-Nal) or 2-naphthylalanine (2-Nal); Asp; Phe or an unnatural derivative of Phe selected from 3,4-dihydroxyphenylalanine (DOPA); Tyr; Thr or an unnatural derivative of Thr selected from N-methyl-threonine; tetrahydropyran-4-propanoic acid (THP(O)); or dioxo-4-tetrahydrothiopyranylacetic acid (THP(SO2)); X 15 represents Pro or a non-naturally occurring derivative of Pro selected from azetidine (Aze), pipecolic acid (Pip) or oxazolidine-4-carboxylic acid (Oxa); X 16 denotes Ile or an unnatural derivative of Ile selected from N-methyl-isoleucine (NMeIle); Ala or an unnatural derivative of Ala selected from 3-cyclohexyl-alanine (Cha) or cyclopropyl-alanine (Cpa); Pro or an unnatural derivative of Pro selected from hydroxyproline (HyP); Asp; Lys; cyclopentyl-glycine (C5A); tetrahydropyran-4-propanoic acid (THP(O)); or dioxo-4-tetrahydrothiopyranylacetic acid (THP(SO2)); X 17 denotes Trp or a non-natural derivative of Trp selected from azatryptophan (AzaTrp) or 5-fluoro-L-tryptophan (5FTrp); Phe; Tyr; 1-naphthylalanine (1-Nal); or 2-naphthylalanine (2-Nal); Hyp indicates hydroxyproline, 1-Nal indicates 1-naphthylalanine, 2-Nal indicates 2-naphthylalanine, HArg indicates homoarginine, and C i , C ii and C iiirepresent a first cysteine ​​residue, a second cysteine ​​residue and a third cysteine ​​residue, respectively, or a pharma- ceutically acceptable salt thereof.

[0078] In some embodiments, the bicyclic peptide comprises a peptide selected from the following: CPFGCMETWSWPIWC (SEQ ID NO:6); CPFGCMRGWSWPIWC(SEQ ID NO:7); CPFGCMSGWSWPIWC(SEQ ID NO:8); CPFGCMEGWSWPIWC(SEQ ID NO:9); CPFGCMEDWSWPIWC(SEQ ID NO:10); CPFGCMPGWSWPIWC (SEQ ID NO:11); CPFGCMKSWSWPIWC (SEQ ID NO:12); CPFGCMKTWSWPIWC (SEQ ID NO:13); CPFGCMKGWSWPIWC (SEQ ID NO:14); CPFGCQEHWSWPIWC (SEQ ID NO:15); CPFGCIKSWSWPIWC (SEQ ID NO: 16); CPFGCQEDWSWPIWC (SEQ ID NO: 17); CPFGCMSDWSWPIWC (SEQ ID NO: 18); CPFGCM[HArg]NWSWPIWC (SEQ ID NO: 19); CPFGCM[K(Ac)]NWSWPIWC(SEQ ID NO:20); CPFGCM[K(Ac)]SWSWPIWC (SEQ ID NO: 21); CPFGC[Nle]KSWSWPIWC (SEQ ID NO:22); CPFGCM[HArg]SWSWPIWC (SEQ ID NO:23); CPFGCM[dK]SWSWPIWC (SEQ ID NO:24); CP[dA]GCMKNWSWPIWC (SEQ ID NO:25); CPF[dA]CMKNWSWPIWC (SEQ ID NO:26); CPFGCM[dA]NWSWPIWC (SEQ ID NO:27); CPFGCMK[dA]WSWPIWC (SEQ ID NO:28); CPFGCMKN[dA]SWPIWC (SEQ ID NO:29); CPFGCMKNWSWP[dA]WC (SEQ ID NO:30); C[dA]FGCMKNWSWPIWC (SEQ ID NO:31); CPFGC[tBuAla]KNWSWPIWC (SEQ ID NO:32); CPFGC[HLeu]KNWSWPIWC (SEQ ID NO:33); CPFGCMKNWSWPI[1Nal]C (SEQ ID NO:34); CPF[dD]CM[HArg]NWSWPIWC (SEQ ID NO: 35); CPF[dA]CM[HArg]NWSWPIWC (SEQ ID NO: 36); CP[3MePhe]GCMKNWSWPIWC (SEQ ID NO: 37); CP[4MePhe]GCMKNWSWPIWC (SEQ ID NO: 38); CP[HPhe]GCMKNWSWPIWC (SEQ ID NO:39); CPF[dD]CMKNWSWPIWC (SEQ ID NO: 40); CPFGC[Hse(Me)]KNWSWPIWC (SEQ ID NO: 41); CPFGCMKN[AzaTrp]SWPIWC (SEQ ID NO:42); CPFGCMKNWSFPIWC (SEQ ID NO:43); CPFGCMKNWSYPIWC (SEQ ID NO:44); CPFGCMKNWS[1Nal]PIWC (SEQ ID NO: 45); CPFGCMKNWS[2Nal]PIWC (SEQ ID NO: 46); CPFGCMKNWS[AzaTrp]PIWC (SEQ ID NO: 47); CPFGCMKNWSW[Aze]IWC (SEQ ID NO: 48); CPFGCMKNWSW[Pip]IWC (SEQ ID NO: 49); CPFGCMKNWSWPIFC (SEQ ID NO:50); CPFGCMKNWSWPIYC (SEQ ID NO:51); CPFGCMKNWSWPI[AzaTrp]C (SEQ ID NO:52); CGFGCMKNWSWPIWC (SEQ ID NO:53); C[Aze]FGCMKNWSWPIWC (SEQ ID NO:54); CPF[K(Ac)]CMKNWSWPIWC (SEQ ID NO:55); CPFGCLKNWSWPIWC(SEQ ID NO:56); CPFGC[MetO2]KNWSWPIWC (SEQ ID NO:57); CPFGCMPNWSWPIWC (SEQ ID NO:58); CPFGCMQNWSWPIWC(SEQ ID NO:59); CPFGCMKNWSWPPWC (SEQ ID NO:60); CP[2Pal]GCMKNWSWPIWC (SEQ ID NO:61); CPFGCMKN[1Nal]SWPIWC (SEQ ID NO:62); CPFGCMKN[2Nal]SWPIWC (SEQ ID NO:63); CPFGCMKNWSWPI[2Nal]C (SEQ ID NO: 64); C[HyP]FGCMKNWSWPIWC (SEQ ID NO:65); CPF[dD]CM[HArg]NWSTPIWC (SEQ ID NO: 66); CPF[dD]CM[HArg][dK]WSTPIWC (SEQ ID NO: 67); CPF[dD]CM[HArg]NWSTPKWC (SEQ ID NO: 68); C[Pro(4NH)]F[dD]CM[HArg]NWSTPIWC (SEQ ID NO: 69); CPF[dD]CMKNWSTPIWC (SEQ ID NO:70); CPF[dK]CM[HArg]NWSTPIWC (SEQ ID NO:71); CPF[dD]CK[HArg]NWSTPIWC (SEQ ID NO:72); CPF[dD]CM[HArg]KWSTPIWC (SEQ ID NO:73); C[Oxa]F[dD]CM[HArg]NWSTPIWC (SEQ ID NO:74); CPF[dD]CM[HArg][Thi]WSTPIWC (SEQ ID NO: 75); CPF[dD]CM[HArg][4ThiAz]WSTPIWC (SEQ ID NO: 76); CPF[dD]CM[HArg][124TriAz]WSTPIWC (SEQ ID NO: 77); CPF[dD]CM[HArg]NWS[124TriAz]PIWC (SEQ ID NO: 78); CPF[dD]CM[HArg]NWST[Oxa]IWC (SEQ ID NO: 79); CP[DOPA][dD]CM[HArg]NWSTPIWC (SEQ ID NO: 80); CPF[dD]CM[HArg]NWS[DOPA]PIWC (SEQ ID NO: 81); CPF[dD]CM[HArg]NWS[THP(SO2)]PIWC (SEQ ID NO: 82); CPF[dD]CM[HArg]NWSTP[THP(SO2)]WC (SEQ ID NO: 83); CPF[dD]CM[HArg]N[5FTrp]STPIWC (SEQ ID NO: 84); CPF[dD]CM[HArg]NWSTPI[5FTrp]C (SEQ ID NO: 85); CPF[dD]CM[HArg]NWS[THP(O)]PIWC (SEQ ID NO: 86); CPF[dD]CM[HArg]NWSTP[THP(O)]WC (SEQ ID NO: 87); C[44DFP]F[dD]CM[HArg]NWSTPIWC (SEQ ID NO: 88); C[Oic]F[dD]CM[HArg]NWSTPIWC (SEQ ID NO: 89); CPF[dF]CM[HArg]NWSTPIWC (SEQ ID NO: 90); CPF[dE]CM[HArg]NWSTPIWC (SEQ ID NO:91); CPF[dQ]CM[HArg]NWSTPIWC (SEQ ID NO:92); CPF[dL]CM[HArg]NWSTPIWC (SEQ ID NO: 93); CPF[dS]CM[HArg]NWSTPIWC (SEQ ID NO:94); CPF[dD]CM[HArg]NW[HSer]TPIWC (SEQ ID NO: 95); CPF[dD]CM[HArg]NWSTP[C5A]WC (SEQ ID NO: 96); CPF[dD]CM[HArg]NWSTP[Cpa]WC (SEQ ID NO: 97); CPF[dD]CM[HArg]NWSTP[Cha]WC (SEQ ID NO: 98); CPF[dD]C[HGln][HArg]NWSTPIWC (SEQ ID NO: 99); CPF[dD]C[C5A][HArg]NWSTPIWC (SEQ ID NO: 100); CPF[dD]CM[HArg]N[Trp(Me)]STPIWC (SEQ ID NO: 101); CPF[dD][NMeCys]M[HArg]NWSTPIWC (SEQ ID NO: 102); CPF[dD]C[HArg]NWS[NMeThr]PIWC (SEQ ID NO: 103); CP[1Nal][dD]CM[HArg]NWSTPIWC (SEQ ID NO: 104); CP[2Nal][dD]CM[HArg]NWSTPIWC (SEQ ID NO: 105); CP[44BPA][dD]CM[HArg]NWSTPIWC (SEQ ID NO: 106); CPF[dD]CM[HArg]NWSTPPWC (SEQ ID NO: 107); CPF[dD]CM[HArg]NWSTP[HyP]WC (SEQ ID NO: 108); CPF[dD]CL[HArg]NWSTPPWC (SEQ ID NO: 109); CPF[dD]CL[HArg]NWSTPIWC (SEQ ID NO: 110); CPY[dD]CM[HArg]NWSTPIWC (SEQ ID NO: 111); C[Aib]F[dD]CM[HArg]NWSTPIWC (SEQ ID NO: 112); C[Sar]F[dD]CM[HArg]NWSTPIWC (SEQ ID NO: 113); CPF[dR]CM[HArg]NWSTPIWC (SEQ ID NO: 114); CPF[dD]CM[HArg]NWSTPKWC (SEQ ID NO: 115); CP[1Nal][dD]CM[HArg]NWSTP[HyP]WC (SEQ ID NO: 116); CP[1Nal][dD]CM[HArg]HWSTP[HyP]WC (SEQ ID NO: 117); CP[1Nal][dD]CM[HArg]DWSTP[HyP]WC (SEQ ID NO: 118); CP[1Nal][dD]CM[HArg]DWSTPIWC (SEQ ID NO: 119); CP[1Nal][dR]CM[HArg]NWSTP[HyP]WC (SEQ ID NO: 120); CP[1Nal][dR]CM[HArg]HWSTP[HyP]WC (SEQ ID NO: 121); CPF[dD]CM[NMeHArg]NWSTPIWC (SEQ ID NO: 122); CPF[dD]CM[HArg][NMeAsn]WSTPIWC (SEQ ID NO: 123); CPF[dD]CM[HArg]NWS[NMeThr]PIWC (SEQ ID NO: 124); CPF[dD]CM[HArg]NWSTP[NMeIle]WC (SEQ ID NO: 125); CP[1Nal][dD]CM[HArg][Cya]WSTP[HyP]WC (SEQ ID NO: 126); CP[1Nal][dD]CM[Cya]DWSTP[HyP]WC (SEQ ID NO: 127); CP[1Nal][DCya]CM[HArg]DWSTP[HyP]WC (SEQ ID NO: 128); CP[1Nal][dD]CM[HArg]DWDTP[HyP]WC (SEQ ID NO: 129); CP[2Nal][dD]CM[HArg]DWSTP[HyP]WC (SEQ ID NO: 130); CP[1Nal][dD]CM[HArg]DWTTP[HyP]WC (SEQ ID NO: 131); CP[1Nal][dD]CM[HArg]DW[HSer]TP[HyP]WC (SEQ ID NO: 132); CP[1Nal][dD]CM[HArg]DW[dS]TP[HyP]WC (SEQ ID NO: 133); CP[1Nal][dD]CM[HArg]DWSSP[HyP]WC (SEQ ID NO: 134); CP[1Nal][dD]CM[Agb]DWSTP[HyP]WC (SEQ ID NO: 135); CP[1Nal][dD]CMPDWSTP[HyP]WC (SEQ ID NO: 136); CP[1Nal][dD]CM[HyP]DWSTP[HyP]WC (SEQ ID NO: 137); CP[1Nal][dR]CM[HArg]DWSTP[HyP]WC (SEQ ID NO: 138); CP[1Nal][dR]CM[HArg]DWDTP[HyP]WC (SEQ ID NO: 139); CP[2Nal][dR]CM[HArg]DWSTP[HyP]WC (SEQ ID NO: 140); CP[1Nal][dR]CM[HArg]DWTTP[HyP]WC (SEQ ID NO: 141); CP[1Nal][dR]CM[HArg]DW[HSer]TP[HyP]WC (SEQ ID NO: 142); CP[1Nal][dR]CM[HArg]DW[dS]TP[HyP]WC (SEQ ID NO: 143); CP[1Nal][dR]CM[HArg]DWSSP[HyP]WC (SEQ ID NO: 144); CP[1Nal][dR]CM[Agb]DWSTP[HyP]WC (SEQ ID NO: 145); CP[1Nal][dR]CMPDWSTP[HyP]WC (SEQ ID NO: 146); CP[1Nal][dR]CM[HyP]DWSTP[HyP]WC (SEQ ID NO: 147); CP[1Nal][dD]CL[HArg]DWSTPIWC (SEQ ID NO: 148); CP[1Nal][dD]CL[HArg]DWSTP[HyP]WC (SEQ ID NO: 149); CP[1Nal][dR]CL[HArg]DWSTP[HyP]WC (SEQ ID NO: 150); CP[1Nal][dR]CL[HArg]HWSTP[HyP]WC (SEQ ID NO: 151); CP[1Nal][dR]CM[HArg]DWSTPIWC (SEQ ID NO: 152); CP[1Nal][DCya]CM[Cya]DWSTP[HyP]WC (SEQ ID NO: 153); CP[1Nal][DCya]CM[HArg][Cya]WSTP[HyP]WC (SEQ ID NO: 154); CP[1Nal][dD]CM[Cya][Cya]WSTP[HyP]WC (SEQ ID NO: 155); CP[1Nal][dK]CM[HArg]DWSTP[HyP]WC (SEQ ID NO: 156); CP[1Nal][dD]CMKDWSTP[HyP]WC (SEQ ID NO: 157); CP[1Nal][dD]CM[HArg]D[dW]STP[HyP][dW]C (SEQ ID NO: 158); and CPFGCM[HArg]DWSTP[HyP]WC (sequence number: 159).

[0079] In some embodiments, the bicyclic peptide is [ka] [In the formula, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , and R 9 are each independently defined below and as described in the embodiments (both alone and in combination) herein.

[0080] In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , and R 9 are each independently hydrogen or C 1-6 an optionally substituted group selected from an aliphatic, a 3-8 membered saturated or partially unsaturated monocyclic carbocyclic ring, a phenyl, an 8-10 membered bicyclic aromatic carbocyclic ring, a 4-8 membered saturated or partially unsaturated monocyclic heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen or sulfur, a 5-6 membered monocyclic heteroaromatic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen or sulfur, an 8-10 membered bicyclic heteroaromatic ring having 1-5 heteroatoms independently selected from nitrogen, oxygen or sulfur.

[0081] In certain embodiments, R 1 is hydrogen or optionally substituted C 1-6 In certain embodiments, R 1 teeth, [ka] It is.

[0082] In certain embodiments, R 2 is hydrogen or optionally substituted C 1-6 In certain embodiments, R 2 teeth, [ka] It is.

[0083] In certain embodiments, R 3 is hydrogen or optionally substituted C 1-6 In certain embodiments, R3 teeth, [ka] It is.

[0084] In certain embodiments, R 4 is hydrogen or optionally substituted C 1-6 In certain embodiments, R 4 teeth, [ka] It is.

[0085] In certain embodiments, R 5 is hydrogen or optionally substituted C 1-6 In certain embodiments, R 5 teeth, [ka] It is.

[0086] In certain embodiments, R 6 is hydrogen or optionally substituted C 1-6 In certain embodiments, R 6 teeth, [ka] It is.

[0087] In certain embodiments, R 7 is hydrogen or optionally substituted C 1-6 In certain embodiments, R 7 teeth, [ka] It is.

[0088] In certain embodiments, R 8 is hydrogen or optionally substituted C1-6 In certain embodiments, R 8 teeth, [ka] It is.

[0089] In certain embodiments, R 9 is hydrogen or optionally substituted C 1-6 In certain embodiments, R 9 teeth, [ka] It is.

[0090] In certain embodiments, R 10 is hydrogen or optionally substituted C 1-6 In certain embodiments, R 10 teeth, [ka] It is.

[0091] In certain embodiments, R 11 is hydrogen or optionally substituted C 1-6 In certain embodiments, R 11 teeth, [ka] It is.

[0092] In some embodiments, the bicyclic toxin conjugate of formula I is [ka] [In the formula, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R8 , R 9 , R 10 , and R 11 are each independently hydrogen or C 1-6 an optionally substituted group selected from an aliphatic, a 3-8 membered saturated or partially unsaturated monocyclic carbocyclic ring, a phenyl, an 8-10 membered bicyclic aromatic carbocyclic ring, a 4-8 membered saturated or partially unsaturated monocyclic heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur, a 5-6 membered monocyclic heteroaromatic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an 8-10 membered bicyclic heteroaromatic ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur; m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15; and n is 0, 1 or 2] and pharma- ceutically acceptable salts thereof.

[0093] In certain embodiments, R 1 is hydrogen or optionally substituted C 1-6 In certain embodiments, R 1 teeth, [ka] It is.

[0094] In certain embodiments, R 2 is hydrogen or optionally substituted C 1-6 In certain embodiments, R 2 teeth, [ka] It is.

[0095] In certain embodiments, R 3 is hydrogen or optionally substituted C 1-6 In certain embodiments, R 3 teeth, [ka] It is.

[0096] In certain embodiments, R 4 is hydrogen or optionally substituted C 1-6 In certain embodiments, R 4 teeth, [ka] It is.

[0097] In certain embodiments, R 5 is hydrogen or optionally substituted C 1-6 In certain embodiments, R 5 teeth, [ka] It is.

[0098] In certain embodiments, R 6 is hydrogen or optionally substituted C 1-6 In certain embodiments, R 6 teeth, [ka] It is.

[0099] In certain embodiments, R 7 is hydrogen or optionally substituted C 1-6 In certain embodiments, R 7 teeth, [ka] It is.

[0100] In certain embodiments, R 8 is hydrogen or optionally substituted C 1-6In certain embodiments, R 8 teeth, [ka] It is.

[0101] In certain embodiments, R 9 is hydrogen or optionally substituted C 1-6 In certain embodiments, R 9 teeth, [ka] It is.

[0102] In certain embodiments, R 10 is hydrogen or optionally substituted C 1-6 In certain embodiments, R 10 teeth, [ka] It is.

[0103] In certain embodiments, R 11 is hydrogen or optionally substituted C 1-6 In certain embodiments, R 11 teeth, [ka] It is.

[0104] In certain embodiments, m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

[0105] In certain embodiments, m is 0. In certain embodiments, m is 1. In certain embodiments, m is 2. In certain embodiments, m is 3. In certain embodiments, m is 4. In certain embodiments, m is 5. In certain embodiments, m is 6. In certain embodiments, m is 7. In certain embodiments, m is 8. In certain embodiments, m is 9. In certain embodiments, m is 10. In certain embodiments, m is 11. In certain embodiments, m is 12. In certain embodiments, m is 13. In certain embodiments, m is 14. In certain embodiments, m is 15.

[0106] In certain embodiments, n is 0, 1 or 2.

[0107] In certain embodiments, n is 0. In certain embodiments, n is 1. In certain embodiments, n is 2.

[0108] R in Scheme I 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 and R 11 each independently is hydrogen or C 1-6 an optionally substituted group selected from an aliphatic, a 3-8 membered saturated or partially unsaturated monocyclic carbocyclic ring, a phenyl, an 8-10 membered bicyclic aromatic carbocyclic ring, a 4-8 membered saturated or partially unsaturated monocyclic heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur, a 5-6 membered monocyclic heteroaromatic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an 8-10 membered bicyclic heteroaromatic ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0109] In certain embodiments, R 1 is hydrogen or optionally substituted C 1-6 In certain embodiments, R 1 teeth, [ka] It is.

[0110] In certain embodiments, R 2 is hydrogen or optionally substituted C 1-6 In certain embodiments, R 2 teeth, [ka] It is.

[0111] In certain embodiments, R 3 is hydrogen or optionally substituted C 1-6 In certain embodiments, R 3 teeth, [ka] It is.

[0112] In certain embodiments, R 4 is hydrogen or optionally substituted C 1-6 In certain embodiments, R 4 teeth, [ka] It is.

[0113] In certain embodiments, R 5 is hydrogen or optionally substituted C 1-6 In certain embodiments, R 5 teeth, [ka] It is.

[0114] In certain embodiments, R 6 is hydrogen or optionally substituted C 1-6 In certain embodiments, R 6 teeth, [ka] It is.

[0115] In certain embodiments, R 7 is hydrogen or optionally substituted C 1-6 In certain embodiments, R 7 teeth, [ka] It is.

[0116] In certain embodiments, R 8 is hydrogen or optionally substituted C 1-6 In certain embodiments, R 8 teeth, [ka] It is.

[0117] In certain embodiments, R 9 is hydrogen or optionally substituted C 1-6 In certain embodiments, R 9 teeth, [ka] It is.

[0118] In certain embodiments, R 10 is hydrogen or optionally substituted C 1-6 In certain embodiments, R 10 teeth, [ka] It is.

[0119] In certain embodiments, R 11 is hydrogen or optionally substituted C 1-6 In certain embodiments, R 11 teeth, [ka] It is.

[0120] In Scheme I, m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

[0121] In certain embodiments, m is 0. In certain embodiments, m is 1. In certain embodiments, m is 2. In certain embodiments, m is 3, and in certain embodiments, m is 4. In certain embodiments, m is 5. In certain embodiments, m is 6. In certain embodiments, m is 7. In certain embodiments, m is 8. In certain embodiments, m is 9. In certain embodiments, m is 10. In certain embodiments, m is 11. In certain embodiments, m is 12. In certain embodiments, m is 13. In certain embodiments, m is 14. In certain embodiments, m is 15.

[0122] In Scheme I, n is 0, 1 or 2.

[0123] In certain embodiments, n is 0. In certain embodiments, n is 1. In certain embodiments, n is 2.

[0124] R in Scheme II 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R8 and R 9 are each independently hydrogen or C 1-6 an optionally substituted group selected from an aliphatic, a 3-8 membered saturated or partially unsaturated monocyclic carbocyclic ring, a phenyl, an 8-10 membered bicyclic aromatic carbocyclic ring, a 4-8 membered saturated or partially unsaturated monocyclic heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur, a 5-6 membered monocyclic heteroaromatic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an 8-10 membered bicyclic heteroaromatic ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0125] In certain embodiments, R 1 is hydrogen or optionally substituted C 1-6 In certain embodiments, R 1 teeth, [ka] It is.

[0126] In certain embodiments, R 2 is hydrogen or optionally substituted C 1-6 In certain embodiments, R 2 teeth, [ka] It is.

[0127] In certain embodiments, R 3 is hydrogen or optionally substituted C 1-6 In certain embodiments, R 3 teeth, [ka] It is.

[0128] In certain embodiments, R 4is hydrogen or optionally substituted C 1-6 In certain embodiments, R 4 teeth, [ka] It is.

[0129] In certain embodiments, R 5 is hydrogen or optionally substituted C 1-6 In certain embodiments, R 5 teeth, [ka] It is.

[0130] In certain embodiments, R 6 is hydrogen or optionally substituted C 1-6 In certain embodiments, R 6 teeth, [ka] It is.

[0131] In certain embodiments, R 7 is hydrogen or optionally substituted C 1-6 In certain embodiments, R 7 teeth, [ka] It is.

[0132] In certain embodiments, R 8 is hydrogen or optionally substituted C 1-6 In certain embodiments, R 8 teeth, [ka] It is.

[0133] In certain embodiments, R 9 is hydrogen or optionally substituted C 1-6 In certain embodiments, R 9 teeth, [ka] It is.

[0134] In Scheme II, m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

[0135] In certain embodiments, m is 0. In certain embodiments, m is 1. In certain embodiments, m is 2. In certain embodiments, m is 3. In certain embodiments, m is 4. In certain embodiments, m is 5. In certain embodiments, m is 6. In certain embodiments, m is 7. In certain embodiments, m is 8. In certain embodiments, m is 9. In certain embodiments, m is 10. In certain embodiments, m is 11. In certain embodiments, m is 12. In certain embodiments, m is 13. In certain embodiments, m is 14. In certain embodiments, m is 15.

[0136] Fragment F-3 can generally be prepared or isolated by synthetic and / or semi-synthetic methods known to those of skill in the art for similar compounds (e.g., as described in WO 2019 / 243832, the entire contents of which are incorporated herein by reference), and as described in detail in the Examples herein.

[0137] In some embodiments, fragment F-3 in Scheme I is [ka] [In the formula, R 1 , R 2 , R 3 , R4 , R 5 , R 6 , R 7 , R 8 , R 9 and m are each defined below and as described in the embodiments (both alone and in combination) herein, or a salt thereof.

[0138] In some embodiments, fragment F-3 in Scheme I is [ka] or a salt thereof.

[0139] In some embodiments, fragment F-2 in Scheme I is [ka] [In the formula, R 10 , R 11 and n are each defined below and as described in the embodiments (both alone and in combination) herein, or a salt thereof.

[0140] In some embodiments, fragment F-2 in Scheme I is [ka] or a salt thereof.

[0141] In some embodiments, fragment F-3 in Scheme I is [ka] [In the formula, R 10 and R 11 are each defined below and as described in the embodiments (both alone and in combination) herein, or a salt thereof.

[0142] In some embodiments, fragment F-3 in Scheme I is [ka] or a salt thereof.

[0143] In step S-1 (amide formation via ring opening of anhydride), fragment F-1 or a salt thereof is coupled to compound A or a salt thereof to form fragment F-2 or a salt thereof. Suitable coupling reactions are well known to those skilled in the art and typically involve activated ester derivatives (e.g., anhydrides) whose treatment with an amine moiety results in the formation of an amide bond. The coupling reaction is typically carried out in the presence of an excess of base. In some embodiments, the base is a tertiary amine base. In some embodiments, the tertiary amine base is triethylamine. In some embodiments, the base is a tertiary amine base. In some embodiments, the tertiary amine base is N,N-diisopropylethylamine (DIPEA). The coupling reaction can be carried out in a suitable solvent that solubilizes all the reagents. In some embodiments, the solvent is a dipolar aprotic solvent. In some embodiments, the dipolar aprotic solvent is N,N-dimethylacetamide (DMA). In some embodiments, the dipolar aprotic solvent is dimethylsulfoxide (DMSO), N,N-dimethylformamide (DMF), acetone, ethyl acetate, hexamethylphosphoramide (HMPA) or N,N'-dimethylpropyleneurea (DMPU). In some embodiments, the reaction mixture is mixed with an acidic aqueous solution to precipitate fragment F-2 or a salt thereof. In some embodiments, the reaction mixture is mixed with an acidic brine solution to precipitate fragment F-2 or a salt thereof. In some embodiments, the brine solution is a 13% brine solution. In some embodiments, the brine solution is a saturated brine solution. In some embodiments, the fragment F-2 or a salt thereof obtained by precipitation and filtration has a purity of about 80% or more. In some embodiments, the fragment F-2 or a salt thereof obtained by precipitation and filtration has a purity of about 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96% or 98%. In some embodiments, the fragment F-2 or a salt thereof obtained by precipitation and filtration is further purified by column chromatography.

[0144] In step S-2 (amide formation), fragment F-2 or a salt thereof and fragment F-3 or a salt thereof participate in an amide formation reaction to form a compound of formula I or a salt thereof. Suitable amide formation reactions are well known to those skilled in the art and typically involve an activated ester moiety, such that treatment with an amine moiety results in the formation of an amide bond. The coupling reaction is typically carried out in the presence of an excess of base. In some embodiments, the base is a tertiary amine base. In some embodiments, the tertiary amine base is triethylamine. In some embodiments, the base is a tertiary amine base. In some embodiments, the tertiary amine base is DIPEA. The coupling reaction can be carried out in a suitable solvent that solubilizes all the reagents. In some embodiments, the solvent is a dipolar aprotic solvent. In some embodiments, the dipolar aprotic solvent is DMA. In some embodiments, the dipolar aprotic solvent is DMSO, DMF, acetone, ethyl acetate, HMPA or DMPU. In some embodiments, the reaction mixture is mixed with a non-polar solvent to precipitate the compound of formula I or a salt thereof. In some embodiments, the reaction mixture is mixed with a non-polar solvent at room temperature or low temperature to form a suspension or slurry. In some embodiments, the suspension or slurry is further stored at room temperature or lower temperature for a period of time, with or without mixing, before filtering the compound of formula I or its salt. In some embodiments, the low temperature is about 15°C, 10°C, 5°C, 0°C, -5°C, -10°C, -15°C or -20°C. In some embodiments, the low temperature is less than -20°C. In some embodiments, the non-polar solvent is ether. In some embodiments, the non-polar solvent is diethyl ether. In some embodiments, the non-polar solvent is methyl tert-butyl ether (MTBE). In some embodiments, the compound of formula I or its salt obtained by precipitation and filtration has a purity of about 70% or more.In some embodiments, the compound of formula I or its salt obtained by precipitation and filtration has a purity of about 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96% or 98%.In some embodiments, the compound of formula I or its salt obtained by precipitation and filtration is further purified by column chromatography.

[0145] In some embodiments, the present invention provides a method for preparing fragment F-2 or a salt thereof, comprising the steps of: 1) providing fragment F-1 or a salt thereof; 2) reacting fragment F-1 or a salt thereof with compound A or a salt thereof to form fragment F-2 or a salt thereof; and 3) isolating fragment F-2 or a salt thereof from the reaction mixture by precipitation, where compound A and fragments F-1 and F-2 are as described above, respectively. In some embodiments, the method further comprises purifying fragment F-2 or a salt thereof by column chromatography. In some embodiments, the solvent and conditions of the method are as described in step S-1 above.

[0146] In some embodiments, the present invention provides a method for preparing a compound of formula I or a salt thereof, comprising the steps of: 1) providing a fragment F-2 or a salt thereof; 2) reacting fragment F-2 or a salt thereof with fragment F-3 or a salt thereof to form a compound of formula I or a salt thereof; and 3) isolating the compound of formula I or a salt thereof from the reaction mixture by precipitation, where fragments F-2 and F-3 and the compound of formula I are as described above. In some embodiments, the method further comprises purifying the compound of formula I or a salt thereof by column chromatography. In some embodiments, the solvent and conditions of the method are as described in step S-2 above.

[0147] In some embodiments, the present invention provides a method for preparing a compound of formula I or a salt thereof, comprising: 1) providing a fragment F-1 or a salt thereof; 2) reacting the fragment F-1 or a salt thereof with compound A or a salt thereof to form a fragment F-2 or a salt thereof; 3) isolating the fragment F-2 or a salt thereof from the reaction mixture by precipitation; 4) reacting the fragment F-2 or a salt thereof with fragment F-3 or a salt thereof to form a compound of formula I or a salt thereof; and 5) isolating the compound of formula I or a salt thereof from the reaction mixture by precipitation. In some embodiments, the method further comprises purifying the compound of formula I or a salt thereof by column chromatography. In some embodiments, the fragment F-2 or a salt thereof obtained from step 3) is not further purified by column chromatography before being used in step 4). In some embodiments, the solvent and conditions of the method are as described for steps S-1 and S-2 above.

[0148] In some embodiments, the present invention provides a heterogeneous mixture comprising fragment F-2 or a salt thereof and a non-polar solvent. In some embodiments, the heterogeneous mixture is a suspension. In some embodiments, the heterogeneous mixture is a slurry. In some embodiments, the present invention provides a solid composition comprising fragment F-2 or a salt thereof and a small amount of a non-polar solvent. In some embodiments, the heterogeneous mixture and / or the solid composition further comprises TBTU. In some embodiments, the non-polar solvent in the heterogeneous mixture and / or the solid composition is as described in step S-1 above. In some embodiments, the temperature of the heterogeneous mixture and / or the solid composition is as described in step S-1 above. In some embodiments, the purity of fragment F-2 or a salt thereof after filtration from the heterogeneous mixture is as described in step S-1 above. In some embodiments, the purity of fragment F-2 or a salt thereof in the solid composition is as described in step S-1 above.

[0149] In some embodiments, the present invention provides a heterogeneous mixture comprising a compound of formula I or a salt thereof and a non-polar solvent. In some embodiments, the heterogeneous mixture is a suspension. In some embodiments, the heterogeneous mixture is a slurry. In some embodiments, the present invention provides a solid composition comprising a compound of formula I or a salt thereof and a small amount of a non-polar solvent. In some embodiments, the non-polar solvent in the heterogeneous mixture and / or solid composition is as described in step S-2 above. In some embodiments, the temperature of the heterogeneous mixture and / or solid composition is as described in step S-2 above. In some embodiments, the purity of the compound of formula I or a salt thereof after filtering from the heterogeneous mixture is as described in step S-2 above. In some embodiments, the purity of the compound of formula I or a salt thereof in the solid composition is as described in step S-2 above.

[0150] 4. Description of Exemplary Bicyclic Toxin Complexes In some embodiments, the bicyclic toxin conjugate of formula I is [ka] [In the formula, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , m and n are each defined below and as described in the embodiments (both alone and in combination) herein, or a pharma- ceutically acceptable salt thereof.

[0151] In some embodiments, the bicyclic toxin conjugate of formula I is [ka] or a pharma- ceutically acceptable salt thereof.

[0152] In some embodiments, the bicyclic toxin conjugate of formula I is BT8009 or a pharma- ceutically acceptable salt thereof.

[0153] 5. Use, Formulation and Administration Pharmaceutically acceptable compositions According to another embodiment, the present invention provides a composition comprising a bicyclic toxin conjugate of the present invention, or a pharma- ceutically acceptable derivative thereof, and a pharma- ceutically acceptable carrier, adjuvant, or vehicle.

[0154] The term "patient", as used herein, means an animal, preferably a mammal, and most preferably a human.

[0155] The term "pharmaceutically acceptable carrier, adjuvant or vehicle" refers to a non-toxic carrier, adjuvant or vehicle that does not destroy the pharmacological activity of the compound in which it is incorporated.The pharmaceutically acceptable carrier, adjuvant or vehicle that can be used in the composition of the present invention includes, but is not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol and wool fat.

[0156] "Pharmaceutically acceptable derivatives" means non-toxic salts, esters, salts of esters, or other derivatives of a compound of the invention which, upon administration to a recipient, are capable of directly or indirectly providing a compound of the invention, or an inhibitory active metabolite or residue thereof.

[0157] The compositions of the present invention may be administered parenterally, by inhalation spray, topically, rectally, nasally, bucally, vaginally, or via an implanted reservoir. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injection or infusion techniques. Preferably, the compositions are administered intraperitoneally or intravenously. Sterile injectable forms of the compositions of the present invention may be aqueous or oleaginous suspensions. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. Sterile injectable preparations may also be sterile injectable solutions or suspensions in non-toxic parenterally acceptable diluents or solvents, such as solutions in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and physiological saline solution.

[0158] These solutions or suspensions may also contain long chain alcohol diluents or dispersants, such as carboxymethylcellulose, or similar dispersants commonly used in formulating pharma- ceutically acceptable dosage forms, including emulsions and suspensions. For formulation purposes, other commonly used surfactants, such as Tweens, Spans, and other emulsifiers, or bioavailability enhancers commonly used in the manufacture of pharma- ceutically acceptable solid, liquid, or other dosage forms, may also be used.

[0159] In some embodiments, formulations suitable for lyophilization and reconstitution for use in parenteral administration, for example by dilution into isotonic saline or dextrose-containing infusion fluids, may include one or more of the following excipients: acid buffering components, such as citric acid, succinic acid, acetic acid, or amino acids, such as glycine or histidine; bases, such as sodium hydroxide or potassium hydroxide, or organic bases, such as tris(hydroxymethyl)aminomethane; · Mineral acids, such as HCl, to adjust the pH to the desired range, usually pH 3-9; a dispersing agent or surfactant, such as polysorbate 20 or polysorbate 80; and / or · Sugars, e.g. sucrose, lactose, dextrose, trehalose or mannitol, to provide stability and control moisture content of the lyophilized product.

[0160] The mixture is typically lyophilized from an aqueous solution and reconstituted with purified water before dilution into the desired infusion solution.

[0161] Alternatively, the pharma- ceutically acceptable compositions of the present invention can be administered in the form of suppositories for rectal administration. These can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature, and therefore melts in the rectum to release the drug. Such materials include cocoa butter, beeswax, and polyethylene glycols.

[0162] The pharma- ceutically acceptable compositions of the present invention may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye, the skin, or the lower intestinal tract. Suitable topical formulations are readily prepared for each of these areas or organs.

[0163] Topical application for the lower intestinal tract can be effected in a rectal suppository formulation (see above) or in a suitable enema formulation. Topical-transdermal patches may also be used.

[0164] For topical application, the provided pharma- ceutically acceptable composition can be formulated into a suitable gel, ointment, lotion or cream containing the active ingredient suspended or dissolved in one or more carriers.Carriers for topical administration of the compounds of the present invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compounds, emulsifying wax and water.Alternatively, the provided pharma-ceutically acceptable composition can be formulated into a suitable lotion or cream containing the active ingredient suspended or dissolved in one or more pharma-ceutically acceptable carriers.Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

[0165] For ophthalmic use, the provided pharma- ceutically acceptable compositions can be formulated as a micronized suspension in isotonic pH-adjusted sterile saline, with or without a preservative such as benzylalkonium chloride, or preferably as a solution in isotonic pH-adjusted sterile saline. Alternatively, for ophthalmic use, the pharma-ceutically acceptable compositions can be formulated in an ointment such as petrolatum.

[0166] The pharma- ceutically acceptable compositions of the present invention may also be administered by nasal aerosol or inhalation.Such compositions are prepared according to techniques well known in the art of pharmaceutical formulation and may be prepared as a solution in saline using benzyl alcohol or other suitable preservatives, absorption enhancers to enhance bioavailability, fluorocarbons, and / or other conventional solubilizing or dispersing agents.

[0167] The amount of the compounds of the present invention that can be combined with the carrier materials to produce a composition in a single dosage form will vary depending on the host treated, the particular mode of administration. Preferably, the provided compositions should be formulated so that a dosage of 0.01-100 mg / kg body weight / day of the inhibitor can be administered to a patient receiving these compositions.

[0168] It should also be understood that the specific dosage and treatment regimen for a particular patient will depend on a variety of factors, including the activity of the particular compound employed, age, body weight, general health, sex, diet, time of administration, excretion rate, drug combination, and the judgment of the treating physician, and the severity of the particular disease being treated. The amount of a compound of the invention in a composition will also depend on the particular compound in the composition.

[0169] Uses of the Compounds and Pharmaceutically Acceptable Compositions In some embodiments, the present invention provides a method of preventing and / or treating a cancer described herein, comprising administering to a patient a bicyclic toxin conjugate of the present invention.

[0170] As used herein, the terms "treatment", "treat" and "treating" refer to reversing, alleviating, delaying the onset, or inhibiting the progression of a disease or disorder, or one or more symptoms thereof, as described herein. In some embodiments, treatment may be administered after one or more symptoms have developed. In other embodiments, treatment may be administered in the absence of symptoms. For example, treatment may 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 may also be continued after symptoms have resolved, for example to prevent or delay recurrence.

[0171] Cancer, in one embodiment, is selected from the group consisting of leukemia (e.g., acute leukemia, acute lymphocytic leukemia, acute myeloid leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma (e.g., Hodgkin's disease or non-Hodgkin's disease), Waldenstrom's macroglobulinemia, multiple myeloma, heavy chain disease, and solid tumors, such as sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovial tumors, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, and the like). pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary carcinoma adenocarcinomas), cystadenocarcinomas, medullary carcinomas, bronchogenic carcinomas, renal cell carcinomas, hepatomas, bile duct carcinomas, choriocarcinomas, seminomas, embryonal carcinomas, Wilms tumors, cervical cancers, uterine cancers, testicular cancers, lung cancers carcinoma), small cell lung carcinoma, bladder carcinoma, epithelial carcinomacarcinoma), glioma, astrocytoma, glioblastoma multiforme (GBM, also known as glioblastoma), medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, neurofibrosarcoma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0172] In some embodiments, the cancer is a glioma, astrocytoma, glioblastoma multiforme (GBM, also known as glioblastoma), medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, neurofibrosarcoma, meningioma, melanoma, neuroblastoma, or retinoblastoma.

[0173] In some embodiments, the cancer is an acoustic neuroma, an astrocytoma (e.g., grade I-pilocytic astrocytoma, grade II-low grade astrocytoma, grade III-malignant astrocytoma, or grade IV-glioblastoma (GBM)), chordoma, CNS lymphoma, craniopharyngioma, brainstem glioma, ependymoma, mixed glioma, optic nerve glioma, subependymoma, medulloblastoma, meningioma, metastatic brain tumor, oligodendroglioma, pituitary tumor, primitive neuroectodermal tumor (PNET) tumor, or schwannoma. In some embodiments, the cancer is a type that is more commonly seen in children than adults, such as brainstem glioma, craniopharyngioma, ependymoma, juvenile pilocytic astrocytoma (JPA), medulloblastoma, optic nerve glioma, pineal tumor, primitive neuroectodermal tumor (PNET), or rhabdoid tumor. In some embodiments, the patient is an adult human. In some embodiments, the patient is a child or a pediatric patient.

[0174] In some embodiments, the cancer is mesothelioma, hepatobiliary (liver and bile duct), bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular melanoma, ovarian cancer, colon cancer, rectal cancer, anal cancer, gastric cancer, gastrointestinal (stomach, colorectum and duodenum), uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, testicular cancer, chronic or acute leukemia, chronic myeloid leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal cell carcinoma, The cancers include, but are not limited to, renal cell carcinoma, carcinoma of the renal pelvis, non-Hodgkin's lymphoma, spinal axis tumor, brain stem glioma, pituitary adenoma, adrenal cortical carcinoma, gallbladder carcinoma, multiple myeloma, cholangiocarcinoma, fibrosarcoma, neuroblastoma, retinoblastoma, or a combination of one or more of the foregoing cancers.

[0175] In some embodiments, the cancer is hepatocellular carcinoma, ovarian cancer, ovarian epithelial cancer, or fallopian tube cancer; papillary serous cystadenocarcinoma or uterine papillary serous carcinoma (UPSC); prostate cancer; testicular cancer; gallbladder cancer; hepatocholangiocarcinoma; soft tissue and bone synovial sarcoma; rhabdomyosarcoma; osteosarcoma; chondrosarcoma; Ewing's sarcoma; angiosarcoma-associated thyroid cancer; adrenal cortical adenoma; pancreatic cancer; pancreatic ductal carcinoma or pancreatic adenocarcinoma; gastrointestinal / gastric (GIST) cancer; lymphoma; squamous cell carcinoma of the head and neck, SCCHN); salivary gland cancer; glioma, or brain cancer; neurofibromatosis type 1-associated malignant peripheral nerve sheath tumor (MPNST); Waldenstrom's macroglobulinemia; or medulloblastoma.

[0176] In some embodiments, the cancer is selected from hepatocellular carcinoma (HCC), hepatoblastoma, colon cancer, rectal cancer, ovarian cancer, ovarian epithelial cancer, fallopian tube cancer, papillary serous adenocarcinoma, uterine papillary serous carcinoma (UPSC), hepatocholangiocarcinoma, soft tissue and bone synovial sarcoma, rhabdomyosarcoma, osteosarcoma, anaplastic thyroid carcinoma, adrenal cortical adenoma, pancreatic cancer, pancreatic ductal carcinoma, pancreatic adenocarcinoma, glioma, neurofibromatosis-1 associated malignant peripheral nerve sheath tumor (MPNST), Waldenstrom's macroglobulinemia, or medulloblastoma.

[0177] In some embodiments, the cancer is a solid tumor, such as a sarcoma, carcinoma, or lymphoma. A solid tumor generally comprises an abnormal mass of tissue that usually does not contain cysts or liquid areas. In some embodiments, the cancer is renal cell carcinoma, or kidney cancer; hepatocellular carcinoma (HCC) or hepatoblastoma, or liver cancer; melanoma; breast cancer; colorectal carcinoma, or colorectal cancer; colon cancer; rectal cancer; anal cancer; lung cancer, such as non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC); ovarian cancer, ovarian epithelial cancer, ovarian carcinoma, or fallopian tube cancer; papillary serous cystadenocarcinoma or uterine papillary serous carcinoma (UPSC); prostate cancer; testicular cancer; gallbladder cancer; hepatocholangiocarcinoma; soft tissue and bone synovial sarcoma. sarcoma); rhabdomyosarcoma; osteosarcoma; chondrosarcoma; Ewing's sarcoma; anaplastic thyroid carcinoma; adrenal cortical carcinoma; pancreatic cancer; pancreatic ductal carcinoma or pancreatic adenocarcinoma; gastrointestinal / gastric (GIST) cancer; lymphoma; squamous cell carcinoma of the head and neck (SCCHN); salivary gland cancer; glioma, or brain cancer; neurofibromatosis type 1-associated malignant peripheral nerve sheath tumor (MPNST); Waldenstrom's macroglobulinemia; or medulloblastoma.

[0178] In some embodiments, the cancer is selected from the group consisting of renal cell carcinoma, hepatocellular carcinoma (HCC), hepatoblastoma, colorectal carcinoma, colorectal cancer, colon cancer, rectal cancer, anal cancer, ovarian cancer, ovarian epithelial cancer, ovarian cancer, fallopian tube cancer, papillary serous cystadenocarcinoma, uterine papillary serous carcinoma (UPSC), hepatocholangiocarcinoma, soft tissue and bone synovial sarcoma, rhabdomyosarcoma, osteosarcoma, chondrosarcoma, anaplastic thyroid cancer, adrenal cortical carcinoma, pancreatic cancer, pancreatic ductal carcinoma, pancreatic adenocarcinoma, pancreatic ductal car ... adenocarcinoma, glioma, brain cancer, neurofibromatosis type 1-associated malignant peripheral nerve sheath tumor (MPNST), Waldenstrom's macroglobulinemia, or medulloblastoma.

[0179] In some embodiments, the cancer is selected from the group consisting of hepatocellular carcinoma (HCC), hepatoblastoma, colon cancer, rectal cancer, ovarian cancer, ovarian epithelial cancer, ovarian carcinoma, fallopian tube cancer, papillary serous adenocarcinoma, uterine papillary serous carcinoma (UPSC), hepatocholangiocarcinoma, soft tissue and bone synovial sarcoma, rhabdomyosarcoma, osteosarcoma, anaplastic thyroid cancer, adrenal cortical carcinoma, pancreatic cancer, pancreatic ductal carcinoma, pancreatic adenocarcinoma, pancreatic ductal car ... adenocarcinoma, glioma, neurofibromatosis type 1-associated malignant peripheral nerve sheath tumor (MPNST), Waldenstrom's macroglobulinemia, or medulloblastoma.

[0180] In some embodiments, the cancer is hepatocellular carcinoma (HCC). In some embodiments, the cancer is hepatoblastoma. In some embodiments, the cancer is colon cancer. In some embodiments, the cancer is rectal cancer. In some embodiments, the cancer is ovarian cancer or ovarian carcinoma. In some embodiments, the cancer is ovarian epithelial cancer. In some embodiments, the cancer is fallopian tube cancer. In some embodiments, the cancer is papillary serous cystadenocarcinoma. In some embodiments, the cancer is uterine papillary serous carcinoma (UPSC). In some embodiments, the cancer is hepatocholangiocarcinoma. In some embodiments, the cancer is soft tissue and bone synovial sarcoma. In some embodiments, the cancer is rhabdomyosarcoma. In some embodiments, the cancer is osteosarcoma. In some embodiments, the cancer is histoplastic thyroid cancer. In some embodiments, the cancer is an adrenocortical carcinoma. In some embodiments, the cancer is pancreatic cancer or pancreatic ductal carcinoma. In some embodiments, the cancer is pancreatic adenocarcinoma. In some embodiments, the cancer is a glioma. In some embodiments, the cancer is a malignant peripheral nerve sheath tumor (MPNST). In some embodiments, the cancer is a neurofibromatosis-1 associated MPNST. In some embodiments, the cancer is Waldenstrom's macroglobulinemia. In some embodiments, the cancer is a medulloblastoma.

[0181] In some embodiments, the cancer is a virus-associated cancer, including human immunodeficiency virus (HIV)-associated solid tumor, human papillomavirus (HPV)-16 positive refractory solid tumor, adult T-cell leukemia, a highly aggressive form of CD4+ T-cell leukemia caused by human T-cell leukemia virus type I (HTLV-I) and characterized by clonal integration of HTLV-I in leukemia cells (see https: / / clinicaltrials.gov / ct2 / show / study / NCT02631746); and virus-associated tumors in gastric cancer, nasopharyngeal carcinoma, cervical cancer, vaginal cancer, vulvar cancer, squamous cell carcinoma of the head and neck, Merkel cell carcinoma. (https: / / clinicaltrials.gov / ct2 / show / study / NCT02488759; see also https: / / clinicaltrials.gov / ct2 / show / study / NCT0240886; https: / / clinicaltrials.gov / ct2 / show / NCT02426892)

[0182] In some embodiments, the cancer is melanoma cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is small cell lung cancer (SCLC). In some embodiments, the cancer is non-small cell lung cancer (NSCLC).

[0183] In some embodiments, cancer is treated by preventing further growth of tumor.In some embodiments, cancer is treated by reducing tumor size (e.g., volume or mass) by at least 5%, 10%, 25%, 50%, 75%, 90% or 99% compared to the tumor size before treatment.In some embodiments, cancer is treated by reducing the tumor volume of the patient by at least 5%, 10%, 25%, 50%, 75%, 90% or 99% compared to the tumor volume before treatment.

[0184] The compounds and compositions according to the method of the present invention may be administered using any amount and any route of administration effective for treating or reducing the severity of cancer. The exact amount required will vary from subject to subject, depending on the species, age, general condition, severity of the disease or condition, the particular agent, its method of administration, and the like. The compounds of the present invention are preferably formulated in dosage unit form for ease of administration and uniformity of dosage. As used herein, the expression "unit dosage form" means a physically discrete unit of agent appropriate for the patient being treated. However, it will be understood that the total daily usage of the compounds and compositions of the present invention will be determined by the attending physician within the scope of sound medical judgment. The specific effective dosage level for a particular patient or organism will depend on a variety of factors, including the disorder or severity of the disorder being treated; the activity of the particular compound used; the particular composition used; the age, weight, general health, sex, and diet of the patient; the time of administration, route of administration, and excretion rate of the particular compound used; the duration of treatment; drugs used in combination or simultaneously with the particular compound used, and similar factors well known in the medical arts. As used herein, the term "patient" refers to an animal, preferably a mammal, and most preferably a human.

[0185] The pharma- ceutically acceptable compositions of the present invention can be administered to humans and other animals rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (by powders, ointments or drops), buccally, orally or as a nasal spray, etc., depending on the severity of the disease or disorder being treated. In certain embodiments, the compounds of the present invention can be administered parenterally at dosage levels of about 0.01 mg / kg to about 50 mg / kg, preferably about 1 mg / kg to 25 mg / kg of subject body weight per day.

[0186] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions, can be formulated according to known techniques using suitable dispersing or wetting agents and suspending agents. Sterile injectable preparations can be sterile injectable solutions, suspensions or emulsions in non-toxic parenterally acceptable diluents or solvents, for example, solutions in 1,3-butanediol. Among the acceptable vehicles and solvents that can be used are water, Ringer's solution, USP and physiological saline solution. In addition, sterile fixed oils are conventionally used as solvents or suspending media. For this purpose, any bland fixed oil can be used, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

[0187] Injectable preparations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0188] In order to prolong the effect of the compounds of the present invention, it is often desirable to delay the absorption of the compound from subcutaneous or intramuscular injection. This can be accomplished by using a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the compound then depends on its rate of dissolution, which in turn may depend on the crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered compound form is accomplished by dissolving or suspending the compound in an oil vehicle. Injectable depot forms are prepared by forming microencapsule matrices of the compound in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of the compound to the polymer and the nature of the particular polymer used, the release rate of the compound can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the compound in liposomes or microemulsions that are compatible with body tissues.

[0189] Compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing a compound of the present invention with a suitable non-irritating excipient or carrier, such as cocoa butter, polyethylene glycol or a suppository wax, which are solid at ambient temperature but liquid at body temperature and will melt in the rectal or vaginal cavity and release the active compound.

[0190] Dosage forms for topical or transdermal administration of the compounds of the present invention include ointments, pastes, creams, lotions, gels, foams, powders, solutions, sprays, inhalants or patches. The active ingredient is mixed under sterile conditions with a pharma- ceutically acceptable carrier and any necessary preservatives or buffers as required. Ophthalmic formulations, ear drops, and eye drops are also considered to be within the scope of the present invention. In addition, the present invention contemplates the use of transdermal patches, which have the added advantage of providing controlled delivery of the compound to the body. Such dosage forms can be prepared by dissolving or dispensing the compound in a suitable medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by providing a rate-controlling membrane or by dispersing the compound in a polymer matrix or gel.

[0191] Example The following examples are illustrative of the invention described above, but 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 invention can also be determined by other test models known as such to those skilled in the relevant art.

[0192] A list of common abbreviations used in the experimental section. AA(s): Amino acid(s) ACN: Acetonitrile Ac2O: acetic anhydride AcOH: acetic acid API: API Aq.: Water-based A%: Peak area percent 1,4-BDMT: 1,4-benzenedimethanethiol Boc: t-butyloxycarbonyl BV: Bed volume ℃: Celsius C of A: Certificate of Analysis Cat: Category CPP: Current Preferred Procedure CV: column volume DIC: Diisopropylcarbodiimide DIPEA: Diisopropylethylamine DITU: Diisopropylthiourea DMA: N,N-Dimethylacetamide DMF: Dimethylformamide DM1: Mertansine / emtansine DTT: 1,4-dithiothreitol eq.: Molar equivalent Eq.:Equivalent Expt: Experiment h: Hours H:Hour HPLC: High-performance liquid chromatography Imp: Impurity Info: IPA: Isopropyl alcohol IPC: In-Process Control Lab: Testing room LC: Liquid chromatography Lyo:Lyophilization MBHA: 4-Methylbenzhydrylamine Fmoc: Fluorenylmethyloxycarbonyl MeOH: Methanol Min: Minutes mL: milliliter Mol: Mol Mol. Wt.:Molecular weight MTBE: Methyl tert-butyl ether non-GMP: Non-Good Manufacturing Practices NMT: Less than Oxyma: Ethyl cyano(hydroxyimino)acetate PD: Process Development Pdt:Product RO / DI: Reverse Osmosis RP-HPLC: Reversed-phase high-performance liquid chromatography RP-18: Reversed phase C18 bonded silica RRT: Relative retention time Rt: room temperature SAFC:Sigma-Aldrich Fine Chemicals SM: starting material SPP: N-Succinimidyl 2-pyridyldithio-carboxylate SPPS: Solid Phase Peptide Synthesis TATA: 1,3,5-triacryloylhexahydro-1,3,5-triazine TFA: Trifluoroacetic acid TIPS: Triisopropylsilane TLC: Thin Layer Chromatography USP: United States Pharmacopoeia v / v: volume / volume vol: volume wt%: weight percent Wt: Weight EXAMPLES

[0193] Example 1: Preparation of BicycleBCY8234 The synthesis of BCY8234 was revisited with the aim of reducing the high levels of aspartimide-related impurities previously found. A series of experiments were performed using different deblocking cocktails, and a cocktail using 3% oxymer in 10% piperidine / DMF was selected for further processing.

[0194] Cleavage from the resin and global deprotection of the peptide was performed in a one-step process using a TFA cocktail containing 90% TFA, 15% DTT, and 5% TIPS, 0.25% NHI, and 5% water. 150 g of peptide-resin was cleaved to yield, after precipitation and drying, 142 g of crude linear peptide with spent resin.

[0195] The crude cyclic product was generated by cyclization of the linear peptide with TATA under basic conditions. Two sets of cyclization experiments were performed (each with crude linear peptide from a different cleavage method) using a total of 121 g of crude linear peptide with spent resin. The quality of the cyclic crude solution was similar.

[0196] Initial purification was achieved by reversed-phase HPLC using a C18 column media (Daiso gel, 120 Å, 10μ) with 0.1 M NH4OAc in water / ACN buffer system. Subsequent purification was performed with 0.1% TFA in water / ACN on the same reversed-phase C18 column. The TFA main pool was desalted with water / ACN and lyophilized to give approximately 24 g of final product with a purity of >95%.

[0197] The sequence is β-Ala 1 -Sar 2 -Sar 3 -Sar 4 -Sar 5 -Sar 6 -Sar 7 -Sar 8 -Sar 9 -Sar 10 -Sar 11 -*Cys 12 - Pro 13 -1Nal 14 -d-Asp 15 -*Cys 16 -Met 17 -hArg 18 -Asp 19 -Trp 20 -Ser 21 -Thr 22 -Pro 23 -Hyp 24 -Trp 25 -*Cys-NH2.

[0198] Here, * denotes a cysteine ​​residue that forms a bicyclic thioether with 1,1,1″-(1,3,5-triazinane-1,3,5-triyl)tris(propan-1-one) as follows: [ka]

[0199] Solid-phase synthesis of BCY8234 The synthesis protocol for BCY8234 used in the last GMP batch had to be optimized due to the high presence of aspartimide-related impurities. The aspartimide impurity is believed to form during the deblocking process with 20% piperidine in DMF. The amount of aspartimide impurity was reduced from 20.4% to 18.6% by adding 0.1 M oxymer to the deblocking solution.

[0200] Previous attempts have suppressed these impurities by using 0.15 M Oxima in 3% piperidine / DMF. A drawback of this cocktail was the presence of incomplete deblocking of the Fmoc protecting group as evidenced by the deletion sequence impurity present in the crude linear peptide.

[0201] Based on data from previous work and previous knowledge, optimization experiments aimed at mitigating the aspartimide problem were designed.

[0202] In addition to aspartimide reduction, new improved techniques were tested. These techniques included a 60 min preactivation, oxidation inhibition with DITU, and one post-coupling wash to reduce the amount of DMF. Additionally, experiments were performed with a fully loaded resin (degree of substitution >0.8 mmoles / g) to see if there was an advantage to the partial loading used in previous work.

[0203] Synthesis Optimization Synthesis optimization experiments were performed on a Symphony XTM Synthesizer. Three factors were screened: piperidine, oxymer, and formic acid concentrations in the deblocking cocktail. Minitab was used to design the screening experiments. The first four experiments were designed to explore trends and relationships. The experiments are described below: · Deblocking reactions are 5' and 20'. 2 eq. of amino acids, 2 eq. of oxyma, and 2.1 eq. of DIC Fmoc-Sar-Sar-OH was used instead of Fmoc-Sar-OH. 60 min activation time (except for Cys and homoArg residues) · 0.2eq of DITU was added to all coupling solutions. The coupling time was 3 hours. No acetylation Wash once after coupling

[0204] The experimental results are summarized in Table 1. [Table 1]

[0205] Consideration The results of these experiments confirmed that the addition of organic acids reduced aspartimide formation. Of the two acids tested, the less reactive oxyma shows a positive impact on the synthesis yield. 3% formic acid in 5% piperidine / DMF (Run No. 3) reduced the synthesis yield to less than 50%, as observed by analytical HPLC analysis of the crude sample. The oxyma was investigated further in the next set of experiments.

[0206] Additional Oxyma and Piperidine Concentration Experiments In this section, the effect of increasing the oxymer concentration to 5% in a 10% piperidine solution was investigated. The effect of removing the final deblocking by using Boc-β-Ala-OH in the final coupling was tested in experiment #6 compared to Fmoc-β-Ala-OH #5. Additional deblocking conditions suggested by BicycleTX were also evaluated in experiment #7. The results are shown in Table 2. [Table 2]

[0207] The first deblocking condition had the poorest crude purity and the lowest yield. There is no significant difference in yield and purity when Fmoc-β-Ala-OH is replaced with Boc-β-Ala-OH. Aspartimide-related impurities are unusually high in run #5 and appear to be out of trend compared to the other conditions.

[0208] The quality of the crude peptide from the synthesis can be improved by using methods that limit aspartimide formation without causing sequence loss or truncation. The use of formic acid caused cleavage likely due to formylation of the free amine. This was common in Exp. Std order #3, with a yield of 45.6%. Furthermore, due to the acidity of formic acid (pKa=3.75), it may have caused some of the identified loss of sequence or Des impurities by reducing the effectiveness of the piperidine solution. For these reasons, formic acid was deemed an unsuitable additive to prevent aspartimide formation.

[0209] The use of oxyma (pKa=4.60) to buffer the basicity of the piperidine solution may be more effective than formic acid. This may be because oxyma is less reactive and does not cause any cleavage of the sequence. Designed experiments gave similar results to the use of oxyma additive, but replication of the original protocol was poor. The conditions used in experiment std. order #2 were selected for the GMP manufacturing process.

[0210] Cleavage and global deprotection Cutting optimization experiments:

[0211] A series of cleavage experiments were performed to find the best conditions for cleaving the peptide from the resin. First, different TFA cocktails were tested. Then, the cocktail-to-resin ratio was evaluated to find the best reaction concentration for cleavage. After the cocktail and reaction concentration, the operating temperature was tested. The cleavage reaction was performed with 10 g peptide-resin for 3 h, and the peptide with the spent resin was precipitated using MTBE (4x) at -40 °C.

[0212] TFA cocktail selection experiment Comparison of 1,4-BDMT and DTT as thiol scavengers

[0213] 1,4-Benzenedimethanethiol (1,4-BDMT) has been reported by PPL to be a better scavenger than DTT (Pawlas and Rasmussen, Green Chemistry 2019 (21) 5990-5998). This reagent was tested in the cleavage optimization. The experiments performed are summarized below. Cleavage concentration of 10mL / g Cocktail: 85% TFA, 5% water, 5% TIPS, 0.2% NH4I and 5% DTT or 1,4-BDMT · Cool cocktails to 10±2℃ After 1 h, TIPS was added 3h reaction time at RT

[0214] The results are summarized in Table 3. [Table 3]

[0215] Cleavage of 1,4-BDMT reduced t-butylation by 3.5%, but the overall purity was similar, albeit slightly lower at 2.5% for 1,4-BDMT. This difference was not significant enough to justify the introduction of a new chemical into the process, so DTT was used to further optimize the cleavage process.

[0216] Cocktail screening experiments Use Minilab to plan your experiments Cleavage concentration of 10mL / g Cocktail: 85% TFA, 5% water, 5% TIPS, 0.2% NH4I and 5% DTT or 1,4-BDMT · Cool cocktails to 10±2℃ After 1 h, TIPS was added 3h reaction time at RT [Table 4]

[0217] The results of the cocktail screening experiments were analyzed using Minitab and are described below.

[0218] Factor regression: +56(%) versus TFA(%), DTT(%), CenterPt for +56 impurity is shown in Figure 1.

[0219] A Pareto plot of the effect for the +56 impurity is shown in Figure 2. Note that it was not possible to graph the specified residual type due to MSE=0 or degrees of freedom for error=0.

[0220] Factorial regression: +163 (%) versus TFA (%), DTT (%), CenterPt is shown in Figure 3, which shows a normal plot of the effect on the +163 impurity.

[0221] A Pareto plot of the effect for the +163 impurity is shown in Figure 4. Note that it was not possible to graph the specified residual type due to MSE=0 or degrees of freedom for error=0.

[0222] Response optimization: +163(%), +56(%), purity(%) is shown in Table 5 below. [Table 5]

[0223] FIG. 5 shows reaction optimization of the cleavage cocktail.

[0224] TFA and DTT content showed no significant effect on both target impurities. Minitab response optimizer selected standard sequence #4, but the team selected standard sequence #3 as the better result. The 10% DTT cocktail was superior to 5% DTT. Therefore, the liquid mixture (i.e., TFA, water, and TIPS) was tested in the 15% DTT experiment.

[0225] % DTT experiment Initial screening experiments showed that increasing the amount of DTT by 10% improved crude quality. Two 10% DTT cocktails will be increased to 15% DTT and compared to the current BPR cocktail. All experiments used 15% DTT Solid scavengers (DTT and NH4I) are excluded from the total cocktail volume NH4I retains 0.25% of the total volume · Amount of water in cocktail = amount of TIPS Total volume = TFA + water + TIPS The cocktail was cooled to 10±2°C. Add TIPS after 1 hour 3h reaction time at RT The experiments are summarized in Table 6 below. [Table 6]

[0226] Although there were no significant differences between the three runs, some improvement in overall purity was observed from the 10% DTT results (Table 4). Run 7 was slightly better and was selected for use in the concentration and temperature experiments.

[0227] Cocktail to resin ratio (cutting concentration) experiment After selection of the cocktail composition, the cleavage concentration, i.e. the cocktail to resin ratio (mL / g), was evaluated. Experiments were performed with cocktail #7 (Table 6) using 10 g of peptide-resin each. The results of this experiment are reported in Table 7 below. [Table 7]

[0228] No improvement in purity or crude recovery was observed at any ratio tested, so the 10 mL / g ratio was maintained for large scale cleavage.

[0229] Cutting Temperature Experiment With the cocktail and concentration selected, the next step was to check whether temperature had a significant effect on purity or yield. Low (15°C) and high (30°C) temperatures were tested and the results of these experiments are reported below. [Table 8]

[0230] The results of Exp.11 (15℃) showed the highest crude purity, but the recovery rate from this cleavage was very low, 67% lower than the other cleavage. The cleavage at 30℃ showed low crude purity. Therefore, the cleavage condition considered optimal was to keep at room temperature.

[0231] Summary of cutting conditions and conclusions A clear trend observed in the optimization experiments is that the crude purity improves as the amount of DTT increases from 5% to 15%. The optimal conditions for cleavage are a cocktail of 90% TFA, 5% water, 15% DTT, 0.25% ammonium iodide and 5% TIPS (added after 1 h). 90% TFA + 5% water + 5% TIPS = 100% (10 mL / g). 15% DTT and 0.25% NHI are added on top. The cocktail was cooled to 10 ± 2 °C before adding the resin. The total reaction time is 3 h at room temperature. The crude with spent resin is precipitated with 4 times the cocktail volume of cold MTBE (≦-30 °C) and the precipitate is washed 3 times with MTBE.

[0232] Determination of crude yield without spent resin Using the optimized conditions above, two cuts of 10 g each were performed. In one cut, crude without resin was isolated, and in the other, crude with spent resin (control) was isolated. The isolated spent resin was washed with methanol, dried, and weighed. The experimental results are summarized below. [Table 9]

[0233] From the above results it can be estimated that 67% crude is present in the crude isolated from cleavage plus spent resin.

[0234] Displaying large-scale cuts To test the scalability of the optimized cleavage conditions, cleavage was performed at 150 g. The cleavage and results are summarized below. · 150g of peptide resin was used. Cocktail: 90% TFA (1350 mL), 5% (75 mL) water, 15% (225 g) DTT, 0.25% (3.75 g) NHI, and 5% (75 mL) TIPS · Cool cocktails to 10±2℃ Add TIPS after 1h 3h reaction time at RT Precipitate 4 times with MTBE (6 L) at -40 °C and wash 3 times with 150 mL MTBE After drying, 142g was recovered, purity 74.04%

[0235] cyclization Cyclization optimization experiment A series of experiments were performed to find the best method for the cyclization reaction. Two different setups were investigated: the current method of BPR and the setup provided by Bicycle. Four experiments, shown in Table 10, were performed with 2.5 g of crude linear peptide (purity about 71.6%). Reactant concentrations and addition times were tested. 2 eq. were used for the current PPL protocol (3 pot) and 1.3 eq. for the Bicycle method (2 pot). After 24 h, the reaction was quenched by the addition of 6.5 eq. (43 mg) AC-Cys-OH and stirred for 1 h. The pH of the solution was then adjusted to pH=4 with acetic acid. [Table 10]

[0236] Consideration Results show that increasing the final crude concentration from 510 g / L to 10 g / L did not result in a trend in purity. The use of 50% ACN is not beneficial since a 3-fold dilution is required before loading the crude peptide onto the column. The TATA equivalents can be reduced to 1.3 eq. without any loss in purity. The experiments and results are shown in Table 10.

[0237] Comparison of 2-pot and 3-pot setups The original 3-pot setup was compared to the 2-pot setup proposed by Bicycle. Both reactions were carried out at 5g / L in 30% ACN / 0.1M NH4HCO3. 1.3eq of TATA was added over 2 hours and the reaction was quenched after 24 hours. [Table 11]

[0238] Consideration Both methods yield similar results. Although the 2-pot setup appears more attractive as it requires less equipment, the 3-pot setup will remain the setup for standardized TATA safety and GMP manufacturing.

[0239] Cyclization experiment (decrease rate of acetonitrile) Using 30% acetonitrile in water for cyclization means that the solution has to be diluted 2x with water before loading onto the purification column. This means a higher volume and a longer packing time. Therefore, to avoid this issue, it was necessary to reduce the acetonitrile concentration in the cyclization solution. The following experiment was performed to see if the acetonitrile content could be reduced during cyclization. 3.7g of linear crude + spent resin (purity about 70.86%). The results are summarized in Table 12 below. [Table 12]

[0240] Decreasing the percentage of acetonitrile appears to increase the purity slightly. No turbidity or precipitation was observed at 20% ACN, but some precipitation was observed at 15% ACN. Therefore, for large scale cyclizations, a 20% ACN final solution is used.

[0241] Large-scale cyclization Large scale cyclization was carried out in a 22 L three-neck flask with constant nitrogen bubbling. Two reactions were carried out using 121 g of linear with spent resin. The first reaction was carried out with 150 g of linear from the cleavage + spent resin, and the second cyclization was carried out with the mixed residual crude from the cleavage optimization process + spent resin. The cyclization reaction protocol is shown below.

[0242] procedure A 0.15M solution of NH4HCO3 (130g, 1.65mol) in 10.75L of 16.3% ACN / water (1.75L ACN & 9L H2O) was prepared under N2. 121 g (27.7 mmol) of linear peptide was dissolved in 20% ACN / water (3.75 L). Filter the linear solution and wash the spent resin with 3x250mL of 20% ACN / water. 9.6 g (38.6 mmol) of TATA was dissolved in 1 L of 50% ACN. ·TATA and crude linear peptide were added to the stirred NH4HCO3 solution under nitrogen over a period of 2 h. The final reaction volume was 16.25L. The reaction completion was monitored by HPLC. 41 g of Ac-Cys-OH (250 mmol) was dissolved in 500 mL of water. Once the reaction was complete, the Ac-Cys-OH solution was added to the reaction flask to quench the reaction. The reaction was stirred for an additional hour. 800 mL of 50% AcOH in water was added to the reaction mixture to adjust the pH of the first sublot to 4.5. The second sublot was adjusted to pH=6.8. Proceeded to refinement. [Table 13]

[0243] purification Purification Optimization The current purification method was first tested to see if it was sufficient to purify high quality crude product from the optimized upstream process. The loading amounts at each step are tested. RPC3 was added for TFA desalting.

[0244] RPC1 purification test The crude conjugate obtained from the cyclization was purified using conventional purification methods as follows: Column medium: Daiso gel C18 120Å, 10μm ·Buffer A=0.1M NH4OAc, Buffer B=ACN Gradient: 20-35% B, 105 min The crude cyclics were diluted in 15% ACN (pH=4.5) prior to loading. Three replicates were performed with different column loadings. The results are as follows: [Table 14]

[0245] conclusion The current RPC1 method was used for purification of the crude from the non-optimized cyclization experiment. The optimized synthesis and cyclization steps allowed the crude load to be doubled (2.6-fold) without affecting purity and recovery. The column was overloaded when loaded three-fold.

[0246] Purification test of RPC2 The current RPC2 method was used for testing. Fractions with purity greater than 85% from 0.1M NH4OAc(aq.) were combined. Lower sample purity (side cuts) were used to test the purification power of the method. Column medium: Daiso gel C18 120Å, 10μm Column diameter: 2.5cm ·Filling amount: approx. 1.56g, purity=88.04, SLI=3.76% Samples were diluted with an equal volume of water before loading. Buffer A was 0.1% TFA (aq.). Buffer B was acetonitrile. The gradient used was 15% B to 35% B in 100 min. The product was eluted with 29% B. -15mL fractions were collected.

[0247] Results and Discussion The main pool purity = 95.64, SLI = 1.43%, amount = 827.2 mg, and recovery of 53% were obtained. This result indicates that the current RPC2 method can be used to purify products with purity less than 90% (RPC1 main pool), but with a negative impact on recovery. Therefore, a criterion of RPC1 main pool purity ≥ 90% was selected for RPC2 purification.

[0248] Development of RPC3 This step (RPC3) was added to reduce TFA in the final lyophilized product, since high TFA content has a negative effect on the stability of the lyophilized product. First, a salt selection exercise was performed.

[0249] Salt selection experiment Two runs were performed for the salt selection work and the resulting main pool was lyophilized and tested for stability. Runs were as follows: (a) TFA salt loaded, washed, and eluted with 30% ACN / water, and (b) TFA salt loaded, salt exchanged with 0.1 M NH4Cl, pH=4.5, and eluted with 30% ACN / water.

[0250] TFA salt column cleaning and elution Column: Daiso Gel C18, 120Å, 15μm, 0.46X1cm The column was conditioned with 2BV of 5% ACN / 0.1% TFA. 113 mg of lyophilized product was dissolved in 10% ACN / 0.1% TFA and packed. Passed 2BV of 5% ACN in water Gradient: 5%B to 10%B, 10 min, then 10%B to 30%B, 10 min ·Mobile phase A: water; mobile phase B: ACN Product elutes after 30% B in 2BV Main pool pH = 6.5, lyophilized to recover 76.6 mg; purity = 94.81% (TFA method)

[0251] Ammonium chloride to chloride experiment Column: Daiso Gel C18 120Å 15μm, 0.46X1cm The column was conditioned with 2BV of 5% ACN / 0.1% TFA. 116 mg of lyophilized product was dissolved 10 times with 5% ACN / 0.1% TFA and filled. ·3BV of 0.1M ammonium chloride / 5% ACN was passed through. Passed 1BV of 5% ACN in water. Gradient: 5%B-10%B in 10 min, then 10%B-30%B in 10 min ·Mobile phase A: water, mobile phase B: ACN The product eluted after 2BV of 30% B. Main pool pH = 6.81, lyophilization yielded 81 mg; purity = 94.99% (TFA method)

[0252] conclusion A sample of each run was given to Analytical Development for analysis. Salt content and stability were tested. Both samples were confirmed to be counterion free, meaning the product was in the free base form. Stability was similar for both samples and was found to be superior to the original TFA salt. A TFA column wash was selected for further development.

[0253] Purification and development of RPC3 The RPC2 main pool was desalted at this stage and further purified. A purification method was developed using the same media and flow rates as used for RPC1 and RPC2. The experiment is shown below: Column medium: Daiso gel C18 120Å, 10μm Column diameter: 2.5cm ·Buffer A=water, Buffer B=ACN The column was conditioned with 2BV of 5% ACN / 0.1% TFA. RPC2 main pool purity = 95.64, SLI = 1.43%, amount = 827.2mg was diluted with an equal volume of water and loaded. TFA was removed by passing through 2BV of 10% ACN / water. Gradient: 20-35%B, 60 min Eluted with 32.5% B A fraction of 20 mL was collected. Results: Main pool purity = 95.91, SLI = 1.21%, amount = 800mg Recovery rate: 96.7%

[0254] This method was chosen for large-scale demonstration.

[0255] Large-scale purification and lyophilization Purification of 0.1M NH4OAc (RPC1) The crude cyclic solution was filtered through a 2.4 μm filter and loaded onto a preparative reversed phase column. The purification method used is as follows:

[0256] 0.1M NH4OAc purification condition (RPC1) Column diameter: 10cm Column medium: DaisogelTM C18, 120 Å, 10 μm Amount of filled medium: 1.2kg Buffer A: 0.1M NH4OAc / H2O, Buffer B: 100% ACN Gradient: 10-20% buffer B, 10 min, then 20-35% buffer B, 105 min Flow rate: 175mL / min Wavelength: 230nm procedure · Pass 2 bed volumes of 5% ACN in 0.1% TFA (aq.). Filter the resulting solution through a 2.4 μm filter. -Pack the sample into the column. · Pass one bed volume of 10% buffer B through. Start the gradient as above. When the product begins to elute, collect the fraction (approximately 250 mL). Backwash the column with 3BV of 80% MeOH in water.

[0257] Results and Discussion The resulting cyclic crude was purified. The pH of crude sample #1 was pH=4.5, while the second crude sample was pH=6.8. The results of the runs are summarized below. [Table 15]

[0258] The recovery from this purification step was 83%. The retention time of the main pool is reported in Section 11.

[0259] TFA purification (RPC2) The main pool from the 0.1M NH4OAc (aq.) purification was diluted with an equal volume of water and loaded onto the same column. 5% ACN in 0.1% TFA (aq.) was then passed through the column to facilitate salt exchange. Purification and elution with 0.1% TFA (aq.) were performed under the conditions shown below.

[0260] 0.1% TFA condition (RPC2) Column diameter: 10cm Column medium: DaisogelTM C18, 120 Å, 10 μm Amount of filled medium: 1.2kg Buffer A: 0.1% TFA in water, Buffer B: 100% ACN Gradient: 5-15% B, 10 min, then 15-35% buffer B, 100 min. Wavelength: 230nm Flow rate: 175mL / min The product was eluted at approximately 29% buffer B. procedure · Pass 2 bed volumes of 5% acetonitrile in 0.1% TFA in water. ·Dilute the NH4OAc main pool with an equal volume of water. Load the diluted main pool onto the column. Pass 2 bed volumes of 10% buffer B through. Run the gradient specified by the RPC2 conditions. When the product begins to elute, collect the fraction (approximately 300 mL). Backwash the column with 3BV of 80% MeOH in water.

[0261] The column load was approximately 30 g (25 g / kg column media). Approximately 23 g (77%) of the estimated loaded product (RPC1 main pool by peak area) loaded onto the column for RPC2 purification was recovered with an HPLC purity of 95.22% and a single maximum impurity of 1.43% (see Figure 18 below). No side cuts were performed at this stage. The TFA main pool was stable for 28 days at 5°C (Section 11).

[0262] TFA desalination (RPC3) The main pool from the 0.1% TFA (aq.) purification was diluted with an equal volume of water and loaded onto the same column. Then, 10% ACN in purified water was passed through the column to desalt the TFA salt. Purification and elution with purified water and ACN were performed under the conditions shown below.

[0263] Desalination conditions (RPC3) Column diameter: 10cm Column medium: DaisogelTM C18, 120 Å, 10 μm Amount of filled medium: 1.2kg Buffer A: Water, Buffer B: 100% ACN Gradient: 10-20% Buffer B, 10 min, then 20-35% Buffer B, 60 min Wavelength: 230nm Flow rate: 175mL / min The product was eluted at approximately 32% buffer B. procedure · Pass 2 bed volumes of 5% acetonitrile in 0.1% TFA in water. · Dilute the TFA main pool with an equal volume of water. Load the diluted main pool onto the column. Pass 2 bed volumes of 10% buffer B through. Run the gradient specified in the RPC3 condition. When the product begins to elute, collect the fraction (approximately 500 mL). Backwash the column with 3BV of 80% MeOH in water.

[0264] An estimated 23 g of HPLC purity 95.22% and single largest impurity 1.43% was loaded onto the column, and approximately 10 g of purity = 95.91 and SLI = 1.49% was in the main pool. This means that no purification was observed and recovery was only 43%. This indicates that the results observed with the 2.5 cm column purification could not be reproduced. To see if this was due to scalability or simply poor performance of the column, this experiment was repeated using a 5 cm column.

[0265] RPC3 side cut handling The side cut solution of RPC3 (approximately 11 g) was diluted with an equal volume of water and loaded onto the column.

[0266] Desalting conditions (RPC3) Column diameter: 5cm Column medium: DaisogelTM C18, 120Å, 10μm Filled media amount: 300g ·Buffer A: Water, Buffer B: 100% ACN Gradient: 10-35% B, 10 min, then 20-40% Buffer B, 50 min ·Wavelength: 230nm ·Flow rate: 43.7 mL / min The product was eluted with approximately 32% buffer B.

[0267] procedure Pass 2 bed volumes of 5% acetonitrile in 0.1% TFA in water. · Dilute side cut with an equal volume of water. Load the diluted main pool onto the column. Pass 2 bed volumes of 10% buffer B through. Run the gradient specified in the RPC3 conditions. Once the organisms begin to elute, collect a fraction (approximately 50 mL). Backwash the column with 3BV of 80% MeOH in water.

[0268] Only 6 g was recovered from a possible 11 g (54% recovery). This confirms that the results from the 2.5 cm column desalting run are not feasible on a large scale. Fast gradient kick-out experiments are required.

[0269] Desalination and kickout All fractions from Section 9.2.3.1 were combined, diluted with an equal volume of water and reloaded onto the column to perform this experiment.

[0270] Desalination conditions (RPC3) Column diameter: 5cm Column medium: DaisogelTM C18, 120Å, 10μm Filled media amount: 300g ·Buffer A: Water, Buffer B: 100% ACN Gradient: 10-35% B, 10 min, then held at 35% buffer B until all product was eluted. ·Wavelength: 230nm ·Flow rate: 43.7 mL / min The product was eluted with approximately 32% buffer B.

[0271] procedure Pass 2 bed volumes of 5% acetonitrile in 0.1% TFA in water. · Dilute side cut with an equal volume of water. Load the column with the diluted main pool. Pass 2 bed volumes of 10% buffer B through. Run the gradient specified in the RPC3 conditions. When the product begins to elute, collect the fraction (approximately 50 mL). Backwash the column with 3BV of 80% MeOH in water.

[0272] Approximately 10.8 g was recovered from the 11 g loaded (column medium 36.7 g / kg), so it can be considered that all the loaded product was recovered. The concentration of the final main pool was 25 g / L. This method is recommended for large-scale desalting.

[0273] Freeze drying The main pools from the desalting run were combined and freeze-dried in a bottle. After freeze-drying, 24 g of the final freeze-dried product was recovered. The purity of the final freeze-dried product was 95.77%, with the maximum impurity being 1.49%, and the overall yield was about 10.6%.

[0274] Retention Time Test The retention time test was performed on the final solution of each step starting from cyclization at room temperature (samples were left in the room) and 2 to 8°C (stored in the refrigerator).

[0275] Coarse goods Retention time studies were performed on the crude at pH=4.5 and pH=6.8 and a summary of these studies is shown in the table below. [Table 16] [Table 17]

[0276] The crude product (pH=4.5) is more stable under both conditions and can be left at room temperature for 3 weeks and refrigerated for 1 month. The crude product (pH=6.8) can be stored at room temperature for 1 week and refrigerated for 2 weeks.

[0277] RPC1 main pool The ammonium acetate main pool was stored at room temperature and monitored weekly for purity, and the results are summarized below. [Table 18]

[0278] The main pool at this stage can be stored at room temperature or refrigerated for up to one month.

[0279] RPC2 main pool The TFA main pool was stored at room temperature and monitored weekly for purity, and the results are summarized below. [Table 19]

[0280] The TFA main pool should be stored refrigerated for up to one month. Storage at room temperature is not recommended.

[0281] RPC3 Main Pool The stability of the final desalted solution before lyophilization was investigated and the results are reported in the table below. [Table 20]

[0282] Both room temperature and refrigerated solutions are stable for one month.

[0283] Conclusion: synthesis The SPPS of BCY8234 was optimized to minimize the formation of aspartimide impurities.

[0284] A variety of deblocking cocktails were tested using DOE-style screening experiments.

[0285] A deblocking cocktail containing 3% Oxyma in 10% piperidine / DMF performed slightly better than 3% Oxyma in 5% piperidine / DMF and 5% Oxyma in 10% piperidine.

[0286] For GMP manufacture, 3% oxymer in 10% piperidine / DMF was chosen.

[0287] DITU was added to the coupling solution to suppress cysteine ​​oxidation.

[0288] For sarcosine coupling, a sarcosine dipeptide derivative was used.

[0289] High loading (>0.8 mmol / g) resin was successfully used in this optimization work.

[0290] Disconnect A series of cleavage experiments was carried out.

[0291] When 1,4-BDMT was compared with DTT, no significant differences were observed between the two.

[0292] A screening experiment with DTT was designed using Minitab.

[0293] The screening experiment showed two possible cocktail choices: selection by Minitab's response optimizer and team selection.

[0294] Further optimization work revealed that the optimal method was to cool 90% TFA, 15% DTT, 5% water to 10°C before adding the resin, followed by addition of 5% TIPS after 1 hour.

[0295] The cocktail to resin ratio was 10 mL / g and the reaction was carried out at RT for 3 h.

[0296] Precipitation was performed at -40°C (4x TFA cocktail), filtered and washed 3 times with MTBE.

[0297] 150 g was cut and 142 g was recovered with a purity of 74.04%.

[0298] cyclization A series of optimization experiments were carried out for the cyclization. The main findings were: TATA can be reduced to 1.3 eq. The reaction time can be reduced to 4 hours. The ACN content in the reaction solution can be reduced to 20%. Two cyclizations of 121 g were carried out to yield an estimated 18 g of product. The crude cyclic solution is loaded onto the column at pH=6.8. A pH of 4.5 is required for long-term storage of the crude.

[0299] purification The crude cyclic peptide was purified using 0.1M NH4OAc (aq.).

[0300] It was then further purified using 0.1% TFA (aq.) to produce the final TFA salt.

[0301] The purified TFA salt was desalted by washing with water and eluted with 35% ACN / water.

[0302] Without wishing to be bound by any particular theory, it is believed that the primary advantage of desalting is the long-term stability of the solid peptide intermediate which does not contain acidic or basic counterions.

[0303] Freeze drying The best pool of desalted TFA was bottle lyophilized to give 24 g of product with a purity of 95.77% and a single maximum impurity of 1.49%.

[0304] GMP Manufacturing Recommendations synthesis Rink Amide MBHA Resin 3000g (2.4 moles) The reaction vessel must be under inert conditions (N2 or argon) at all times.

[0305] After coupling of Asp19, all deblocking should use 3% oxymer in 10% piperidine / DMF. All deblocking times must be between 5 and 20 minutes.

[0306] Avoid leaving the piperidine solution in the container for long periods of time (ideally, total drain time should be less than 5 minutes).

[0307] 0.2 eq. of DITU should be added to the coupling solution.

[0308] All sarcosine couplings must be performed with Fmoc-Sar-Sar-OH.

[0309] Without wishing to be bound by any particular theory, it is believed that the primary advantage of using Fmoc-Sar-Sar-OH for all sarcosine couplings is that it reduces the number of peptide synthesis and deprotection cycles while maintaining overall coupling efficiency on the solid phase, minimizing the chance of asparitimid formation. Disconnect

[0310] A range of 1000-2000 g of peptide-resin is recommended.

[0311] Cleavage and global deprotection of the peptide performed by treating the resin for 3 h with a cocktail (10 mL / g) of 90% TFA, 5% water, 0.25% NHI, 5% DTT and 5% TIS (added after 1 h).

[0312] The reaction mixture with the spent resin is precipitated with cold MTBE and the resulting precipitate is isolated and dried.

[0313] cyclization 500g linear crude + spent resin is recommended. 40 g of TATA was pre-weighed into a 5 L screw-cap flask. The weighing and dispensing of TATA must be done in a segregated system. Exposure to TATA should be minimized. The reaction solution is 66 L of 0.1 M NH4HCO3 in 20% ACN (aq.). The reaction should take between 4 and 20 hours. Holding time at 2-8°C = 5 days (after acetic acid treatment, pH = 4.28)

[0314] purification 20cm column packed with Daisogel C18 120Å Buffer B=ACN Hold the current BPR in RPC1 and RPC2. For RPC3, 3BV of 10% ACN / water was used to wash residual TFA and the product was eluted with 35% ACN / water.

[0315] HPLC conditions and test report [Table 21] [Table 22]

[0316] Test report BCY8234 Item number: 512175 Lot number: P200462 Molecular weight: 2954.3 Appearance: White powder Peptide purity: ≥95% Peptide content: 92.8% (based on nitrogen content) Moisture (KF): 7.1% TFA: Not detected Mass balance: 99.9% Identity: Mass spectrometry (ESI) shows the correct molecular ion (2953.3) Storage: Keep dry and store below -20°C.

[0317] References Acid-Mediated Prevention of Aspartimide. Michels, Tillmann, et al. 2012, ORGANIC LETTERS, Vol. 14, No. 20, pp. 5218-5221. ReGreen SPPS: enabling circular chemistry in. Pawlas, Jan: Rasmussen Jon H. 2019, Green Chemistry (21), pp. 5990-5998.

[0318] Example 2: Preparation of BT8009 Introduction Objective: Develop a new process for kilolab-scale production of BT8009. This example describes the process development activities that were undertaken to address issues identified during process development. [ka]

[0319] Results and Discussion Step 1: Formation of gvcMMAE [ka]

[0320] Five experiments were performed on the 1-3 g scale (Table 23). Entry 1 was performed to simplify the work-up procedure and improve yield. The brine solution was extracted with 1:1 EtOAc / THF. Both the organic and aqueous layers were rich in gvcMMAE. Extraction of the aqueous reaction with EtOAc / THF was unsuccessful. Therefore, the reaction was poured into an acidic brine solution. A filterable suspension was obtained. The product was obtained with 94.3% LC purity and 88% yield. The product contained only 0.07% w / w sodium chloride.

[0321] To reduce the workup volume, the experiment in entry 2 was performed. The workup volume was reduced from 70 vol to 50 vol. The reaction was poured into an acidic aqueous HCl solution. The product precipitated out of solution. The product was obtained in 93.5% LC purity and 79% yield. The aqueous solution dissolved more gvcMMAE than saline, resulting in a lower yield.

[0322] The experiment in entry 3 was conducted to investigate why the entry 2 experiment showed a lower yield than entry 1. In the experiment in entry 3, the experimental procedure in entry 1 was repeated, except that the brine was replaced with water in the workup. The product was obtained with an LC purity of 94.6% and a yield of 72%. The results showed that the brine was important for achieving a high yield.

[0323] The experiment in entry 4 was conducted to continue to investigate why the yield in the experiment in entry 2 was lower than that in entry 1. In the experiment in entry 4, the procedure in the experiment in entry 1 was repeated, except that the reaction mixture was distilled to remove DIPEA. The product was obtained in 74% yield with 94.6% LC purity. This result indicates that distillation is not important to achieve high yields.

[0324] The results of entries 1-4 demonstrated that salt water is important to achieve high yields. To confirm this hypothesis, the experiment of entry 5 was carried out. The reaction solution was poured into acidic saturated salt water. The suspension was filtered. The product was obtained in 91% yield with 95.4% LC purity. The sodium chloride in the product was only 0.45% w / w. These conditions are used as the preferred procedure (see attached document). [Table 23]

[0325] Step 2: Formation of BT8009 [ka]

[0326] Eleven experiments were performed at the 0.708-2.832 g scale (Table 24). Two lots of BCY8234 were used in these experiments, Lot C was prepared by the earlier route and Lot P was prepared by the later route. The experiment in entry 1 was performed to produce enough crude BT8009 to study the work-up procedure and optimize the column purification conditions. The starting material for BCY8234 was from Lot C, which had 7.62% w / w TFA. This BCY8234 was readily soluble in DMA. After stirring the reaction for 1 hour, the IPC showed 1.73% BCY8234, 0.75% gvcMMAE and 0.07% RRT0.93 impurity. The reaction was poured into MTBE solution. The product precipitated as a filterable suspension. The suspension was filtered through a class D funnel. Assay analysis showed no product present in the filtrate. The solvent was kept on top of the cake during filtration to avoid the formation of sticky solids. The vacuum was turned off as soon as the rinse was completed and the solvent stopped dripping. The crude product was obtained with an estimated yield of 100% and an LC purity of 86.1%.

[0327] To investigate the column purification conditions, experiments in entries 2 to 4 were carried out. In each experiment, 1 g of theoretical BT8009 was extracted from the crude product in entry 1 and purified on a 60 g Ultra C18 column.

[0328] In the experiment of entry 2, 10-40% ACN / HO+0.1% AcOH was used for gradient elution on an ultra C18 column. After lyophilization, BT8009 was obtained in 89.8% yield with 96.2% LC purity and no impurities at RRT0.93.

[0329] In the experiment of entry 3, 10-40% ACN / HO+0.05% AcOH was used for gradient elution on an ultra C18 column. After lyophilization, BT8009 was obtained in 61.1% yield with 95.6% LC purity and no impurities at RRT0.93.

[0330] In the experiment of entry 4, 10-35% ACN / H2O + 0.1% AcOH was used for gradient elution on an ultra C18 column. After lyophilization, BT8009 was obtained with 96.9% LC purity, 0.1% RRT0.93 impurity, and 62.7% yield. The results showed that the purification of entry 2 was the best condition, but optimization is required.

[0331] To determine whether lot P of BCY8234 would provide an acceptable final product, the experiment in entry 5 was performed. In this experiment, 5 equiv. DIPEA and 1 equiv. gvcMMAE / TBTU were used. This BCY8234 did not contain TFA and was not soluble in DMA. After stirring the suspension for 1 hour, the IPC showed 36.14% BCY8234 with 2.78% gvcMMAE and 2.81% RRT0.93 impurity. After an additional charge of gvcMMAE / TBTU (2x0.1 equiv.), the IPC showed 3.32% BCY8234 with 3.55% gvcMMAE and 3.88% RRT0.93 impurity.

[0332] In the experiment for entry 6, the experiment for entry 5 was repeated except that 11 equivalents of DIPEA were used to see if the reaction would improve. The IPC was similar to that of entry 5. After stirring the suspension for 1 hour and 13 minutes, the IPC showed 27.33% BCY8234 and 3.76% gvcMMAE and 1.19% RRT0.93 impurity. After an additional charge of gvcMMAE / TBTU (3x0.1 equivalents), the IPC showed 0.16% BCY8234 and 4.37% gvcMMAE and 1.66% RRT0.93 impurity.

[0333] For the entry 7 experiment, BCY8234 from lot P was dissolved in DMA and 4 equiv. of TFA before mixing with gvcMMAE / TBTU. After stirring for 17 h, the IPC showed 4.62% BCY8234 and 0.99% gvcMMAE and 0.38% RRT0.93 impurity. After an additional charge of gvcMMAE / TBTU (0.1 equiv.), the IPC showed 0.26% BCY8234 and 1.53% gvcMMAE and 0.79% RRT0.93 impurity. In this experiment, 10-40% ACN / H2O + 0.1% AcOH was used for gradient elution on an Ultra C18 column. After lyophilization, BT8009 was obtained with 94.7% LC purity, 0.99% RRT0.93 impurity, and 59.1% yield.

[0334] In the experiment for entry 8, BCY8234 from lot P was dissolved in DMA, 3 equivalents of TFA, and 12 equivalents of water before mixing with gvcMMAE / TBTU. After stirring for 1 hour, the IPC showed 3.82% BCY8234 and 1.14% gvcMMAE and 0.38% RRT0.93 impurity. After an additional charge of gvcMMAE / TBTU (0.1 equivalents), the IPC showed 0.21% BCY8234 and 1.98% gvcMMAE and 1.69% RRT0.93 impurity. In this experiment, a gradient elution was used on the ultra C18 column from 10 to 38% ACN / H2O + 0.1% AcOH, followed by 45% ACN / H2O + 0.1% AcOH to ensure all product was eluted from the C18 column. A catch-release column was performed. After lyophilization, BT8009 was obtained with an LC purity of 95.5%, RRT 0.93 impurity % of 1.36%, and a yield of 68.5%.

[0335] To determine whether the direct addition of 1.1 equivalents of gvcMMAE / TBTU would minimize the RRT0.93 impurity, experiments 9-10 were performed. In the experiment for entry 9, lot P BCY8234 was used in the reaction. After 1 hour of stirring, the IPC showed 0.55% BCY8234, 1.72% gvcMMAE, and 1.04% RRT0.93 impurity. In the experiment for entry 10, lot C BCY8234 was used in the reaction. After 1 hour of stirring, the IPC showed 0.23% BCY8234, 1.68% gvcMMAE, and 0.87% RRT0.93 impurity. This result indicated that an excess of gvcMMAE / TBTU would result in the RRT0.93 impurity, and that 1 equivalent of gvcMMAE / TBTU should be used for the reaction in step 2. This impurity occurred from both lot P and lot C BCY8234.

[0336] For the entry 11 experiment, BCY8234 from lot P was dissolved in DMA and 4 equivalents of TFA before mixing with gvcMMAE / TBTU. 1 equivalent of gvcMMAE / TBTU was used for this reaction. After stirring for 1 hour, IPC showed 3.45% BCY8234, 2.00% gvcMMAE and 0.01% RRT0.93 impurity. After lyophilization, BT8009 was obtained in 64.7% yield with 96.9% LC purity and no RRT0.93 impurity. This experiment will be used as the preferred procedure.

[0337] [Table 24]

[0338] Step 2: Identification of impurities RRT0.97 impurity: The chromatogram of crude BT8009 contains 4.5% RRT0.97 impurity. This impurity is also present in the IPC chromatogram. This impurity was identified by LC-MS of BT8009 as impurity BT8009+OH.

[0339] RRT0.93 impurity: In the final BT8009 chromatogram, there is 1.4% of the RRT0.93 impurity. This impurity is also present in the IPC chromatogram when excess gvcMMAE / TBTU is used. This impurity was identified by LC-MS of BT8009 as impurity BT8009-H2O. LC-MS analysis of BCY8234 lots C and P confirmed the presence of this impurity BCY8234-H2O, which partially co-dissolves with the main peak. BCY8234 lot C appears to contain more of this impurity.

[0340] conclusion A two-step process for producing BT8009 was developed with a yield of 44% and LC purity of 96.9%. The process of step 1 was simplified to improve the yield. One equivalent of gvcMMAE / TBTU was used in the reaction of step 2 to minimize the RRT0.93 impurity. The filtration and column purification of step 2 were optimized.

[0341] Having described a number of embodiments of the invention, it will be apparent that the basic examples may be modified to provide other embodiments that utilize the compounds and methods of the invention. It will therefore be appreciated that the scope of this invention is to be defined by the appended claims rather than by the specific embodiments that have been represented by way of example.

Claims

1. A process for preparing a compound of formula I or a salt thereof, comprising: 1) Fragment F-2: 【Chemical 1】 or a salt thereof; and 2) Fragment F-2 is coupled to fragment F-3 using a coupling agent N,N,N',N'-tetramethyl-O-(benzotriazol-1-yl)uronium tetrafluoroborate (TBTU): 【Chemistry 2】 or a salt thereof to form a compound of formula I: 【Chemistry 3】 or a salt thereof; Including, where: R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 and R 11 are each independently hydrogen or C 1-6 an optionally substituted group selected from an aliphatic, a 3-8 membered saturated or partially unsaturated monocyclic carbocyclic ring, a phenyl, an 8-10 membered bicyclic aromatic carbocyclic ring, a 4-8 membered saturated or partially unsaturated monocyclic heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur, a 5-6 membered monocyclic heteroaromatic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an 8-10 membered bicyclic heteroaromatic ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur; m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15; and The method wherein n is 0, 1, or 2.

2. The method of claim 1, wherein the method uses about 1 equivalent of fragment F-2.

3. The method of claim 1, comprising reacting fragment F-3 with about 1 equivalent of fragment F-2 and TBTU.

4. 2. The method of claim 1, wherein the reaction of step 2) is carried out in a dipolar aprotic solvent.

5. 5. The method of claim 4, wherein the dipolar aprotic solvent is N,N-dimethylacetamide (DMA).

6. The method of claim 1, wherein step 2) is carried out in the presence of an excess of base.

7. The method of claim 6, wherein the base is N,N-diisopropylethylamine (DIPEA).

8. 10. The method of claim 1, further comprising purifying the compound of formula I or a salt thereof by column chromatography.

9. 2. The method of claim 1, wherein the RRT0.93 impurity is formed at a relative area of ​​less than about 5% relative to the compound of formula I.

10. 10. The method of claim 9, wherein the impurities are less than about 2.5%.

11. 11. The method of claim 10, wherein the impurities are less than about 1%.

12. 12. The method of claim 11, wherein the impurities are less than about 0.5%.

13. 13. The method of claim 12, wherein the impurities are less than about 0.05%.

14. R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 and R 11 are each independently, C 1-6 2. The method of claim 1, wherein the optionally substituted group is selected from an aliphatic, 3-8 membered saturated or partially unsaturated monocyclic carbocyclic ring, a phenyl, an 8-10 membered bicyclic aromatic carbocyclic ring, a 4-8 membered saturated or partially unsaturated monocyclic heterocyclic ring having 1 to 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur, a 5-6 membered monocyclic heteroaromatic ring having 1 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an 8-10 membered bicyclic heteroaromatic ring having 1 to 5 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

15. R 1 but, 【Chemistry 7】 15. The method of claim 14, wherein:

16. R 2 but, 【Chemistry 8】 15. The method of claim 14, wherein:

17. R 3 but, 【Chemistry 6】 15. The method of claim 14, wherein:

18. R 4 but, 【Chemistry 7】 15. The method of claim 14, wherein:

19. R 5 but, 【Chemistry 8】 15. The method of claim 14, wherein:

20. R 6 but, 【Chemistry 9】 15. The method of claim 14, wherein:

21. R 7 but, 【Chemistry 10】 15. The method of claim 14, wherein:

22. R 8 but, 【Chemistry 11】 15. The method of claim 14, wherein

23. R 9 but, 【Chemistry 12】 15. The method of claim 14, wherein

24. R 10 but, 【Chemistry 13】 15. The method of claim 14, wherein

25. R 11 but, 【Chemistry 14】 15. The method of claim 14, wherein

26. The compound of formula I is 【Chemistry 15】 The method of claim 1, wherein

27. Fragment F-3, 【Chemistry 16】 or a salt thereof.

28. Fragment F-2, 【Chemistry 17】 or a salt thereof.

29. Fragment F-3, 【Chemistry 18】 or a salt thereof.

30. Fragment F-2, 【Chemistry 19】 or a salt thereof.

31. 2. The method of claim 1, wherein the compound of formula I is BT8009 or a salt thereof.

32. A method for preparing a composition comprising a compound of formula I or a salt thereof and a pharmaceutically acceptable carrier, adjuvant, or vehicle, comprising: (a) carrying out the method of any one of claims 1 to 31 to produce a compound of formula I or a salt thereof; and (b) formulating a compound of formula I or a salt thereof into a composition together with a pharmaceutically acceptable carrier, adjuvant or vehicle; A method comprising:

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