Improved methods for acylation of maytansinol

Abstract

To provide a method of preparing an amino acid ester of maytansinol.SOLUTION: Maytansinol is reacted with an N-carboxyanhydride (NCA) of an amino acid in the presence of a drying agent. An improved method is also disclosed in which a nucleophile is added to the reaction mixture after completion of the reaction between maytansinol and an N-carboxyanhydride of an amino acid.SELECTED DRAWING: None

Description

Technical Field

[0001] Reference to Related Cases This application claims the benefit of the filing date of U.S. Provisional Application No. 61 / 705,731, filed on September 26, 2012, under 35 U.S.C. § 119(e), and the entire content thereof is incorporated herein by reference.

[0002] The present invention is an improved method for preparing intermediates in the synthesis of maytansinoids and their antibody conjugates.

Background Art

[0003] Maytansinoids are highly cytotoxic compounds, such as maytansinol and C-3 esters of maytansinol as shown below (U.S. Patent No. 4,151,042).

[0004]

Chemical Formula

[0005] Natural and synthetic C-3 esters of maytansinol can be classified into two groups: (a) maytansine (2) and its analogs (e.g., DM1 and DM4) which are C-3 esters having N-methyl-L-alanine or a derivative of N-methyl-L-alanine (U.S. Patent Nos. 4,137,230; 4,260,608; 5,208,020; and Chem. Pharm. Bull. 12:3441 (1984)); (b) ansamitocin which is a C-3 ester having a simple carboxylic acid (U.S. Patent Nos. 4,248,870; 4,265,814; 4,308,268; 4,308,269; 4,309,428; 4,317,821; 4,322,348; and 4,331,598).

[0006] Mitansine (2), its analogs, and each ansamitocin species are C3 esters of mitansinol that can be prepared by esterification of mitansinol (1). U.S. Patent Nos. 7,301,019 and 7,598,375 describe a method of acylating mitansinol (1) with an N-carboxy anhydride of an amino acid (NCA, 5) in the presence of a base to form an amino acid ester of mitansinol (May-AA, 6) as shown below.

[0007]

Chemical formula

[0008] The amino acid esters of mitansinol are valuable intermediates that can be coupled to carboxylic acids to afford mitansinoids. For example, as shown below, the reaction of mitansinol with (4S)-3,4-dimethyl-2,5-oxazolidinedione (5a) forms N2'-deacetyl-mitansine (6a), which can then be coupled to 3-(methyldithio)propionic acid (7) to form DM1-SMe (8) using N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDAC).

[0009]

Chemical formula

Summary of the Invention

Problems to be Solved by the Invention

[0010] A major drawback of the acylation reaction to form amino acid esters of mitansinol is that it also forms a byproduct that contains an excess N-methyl-alanyl component (referred to as "excess NMA" (9)) in the C3 side chain. When N2'-deacetyl-mitansine is acylated, excess NMA (9) is also acylated to form excess NMA-DM1-SMe (9a). The structures of excess NMA (9) and excess NMA-DM1-SMe (9a) are shown below. When acylated, excess NMA (9) is also acylated to form excess NMA-DM1-SMe (9a). The structures of excess NMA (9) and excess NMA-DM1-SMe (9a) are shown below.

[0011]

Chem.

[0012] As shown below, DM1(3) can be prepared from DM1-SMe(8) by reduction, and in so doing, all of the excess NMA-DM1-SMe(9a) is also converted to excess NMA-DM1(10).

[0013]

Chem.

[0014] Removing the excess NMA-DM1(10) from DM1(3) is difficult because both compounds have similar polarities and show peak overlap in the HPLC trace of purified DM1(3). DM1(3) and DM4(4) are used to prepare antibody conjugates, some of which are currently in clinical trials.

[0015] Therefore, there is a need to improve the yield and robustness of methods for preparing such maytansinoids and to minimize the by-products formed during the reactions used in their preparation.

Means for Solving the Problems

[0016] As shown in Examples 1-4, it has now been found that adding a drying agent to the reaction of maytansinol with the N-carboxy anhydride of an amino acid substantially increases the yield of the amino acid ester of maytansinol. Also, as shown in Examples 6-8, it has been found that adding a preliminary quenching step using a nucleophile following the reaction of maytansinol with the N-carboxy anhydride of an amino acid substantially reduces the formation of unwanted by-products such as excess NMA. Based on these findings, an improved method for preparing the amino acid ester of maytansinol is disclosed herein.

[0017] The first embodiment of the present invention is a method for preparing an amino acid ester of maitansinol represented by formula (I):

[0018]

Chemical formula

[0019] This method involves reacting maitansinol with an N-carboxy anhydride in a reaction mixture containing a base and a desiccant added thereto. The N-carboxy anhydride has the following formula:

[0020]

Chemical formula

[0021] The second embodiment of the present invention is a method for preparing an amino acid ester of maitansinol represented by formula (I), comprising: a) reacting maitansinol with an N-carboxy anhydride represented by formula (II) in a reaction mixture containing a base added thereto; and b) reacting the unreacted N-carboxy anhydride in step a) with a nucleophile. All variables in formulas (I) and (II) are as defined in the first embodiment of the present invention.

Brief Description of the Drawings

[0022]

Figure 1

Figure 2

Figure 3

Figure 4

[0023] The present invention relates to a method for preparing an amino acid ester represented by formula (I) from mitansinol and an N-carboxy anhydride represented by formula (II). The amino acid ester can be further esterified to prepare mitansinoids such as DM1 and DM4, and then further converted into an antibody conjugate of the mitansinoid. Preferably, the amino acid ester is represented by formula (Ia) and the N-carboxy anhydride is represented by formula (IIa):

[0024] CHEMICAL FORMULA and

[0025] CHEMICAL FORMULA The variables in formulas (Ia) and (IIa) are as described for formulas (I) and (II).

[0026] In the case of formulas (I), (II), (Ia), and (IIa), preferably, R1 is the side chain of a natural amino acid, provided that if the side chain has a reactive functional group, the functional group may be protected; R2 is methyl. Alternatively, R1 is alkyl and R2 is methyl. More preferably, both R1 and R2 are methyl.

[0027] In a first embodiment of the present invention, the method comprises reacting mitansinol with an N-carboxy anhydride represented by formula (II) or (IIa) in a reaction mixture containing a base and a desiccant added thereto.

[0028] In one preferred embodiment, the reaction mixture further comprises a Lewis acid. Preferred Lewis acids contain metal cations.

[0029] In another preferred embodiment, first mitansinol is reacted with an N-carboxy anhydride, and then the reaction mixture is contacted with an aqueous solution containing hydrogen carbonate or carbonic acid, or the reaction mixture is contacted with a metal scavenger. As the metal scavenger, those known in the art can be used (see, for example, Chapter 9 of “The Power of Functional Resin in Organic Synthesis” by Aubrey Mendoca, Wiley-VCH Verlag GmbH & Co. KGaA, 2008). Examples of metal scavengers include, but are not limited to, polymer-based and silica-based metal scavengers (e.g., QuadraPure™ and QuadraSil™ from Sigma-Aldrich, SiliaMetS® from SiliCycle, Smopex® from Johnson Matthey, metal scavengers from Biotage), and carbon-based scavengers (e.g., QuadraPure™ C from Sigma-Aldrich).

[0030] In another preferred embodiment, first mitansinol and an N-carboxy anhydride are reacted, and then the metal cation of the Lewis acid is removed from the reaction mixture. For example, the metal cation of the Lewis acid is removed from the reaction mixture by contacting the reaction mixture with an aqueous solution containing hydrogen carbonate or carbonic acid, or by contacting the reaction mixture with a metal scavenger.

[0031] In a second embodiment, the method comprises: a) reacting mitansinol with an N-carboxy anhydride represented by formula (II) or (IIa) in a reaction mixture containing an added base; and b) reacting the unreacted N-carboxy anhydride in step a) with a nucleophile.

[0032] In one preferred embodiment, the reaction mixture of step a) further comprises a Lewis acid. Preferred Lewis acids contain metal cations.

[0033] In another preferred embodiment, the reaction mixture after step b) is contacted with an aqueous solution containing hydrogen carbonate or carbonic acid, or with a metal scavenger.

[0034] In another preferred embodiment, after carrying out step b), i.e., after reacting the nucleophile with the unreacted N-carboxy anhydride, the metal cation of the Lewis acid is removed from the reaction mixture. For example, the metal cation of the Lewis acid is removed from the reaction mixture by contacting the reaction mixture with an aqueous solution containing hydrogen carbonate or carbonic acid, or by contacting the reaction mixture with a metal scavenger.

[0035] In yet another preferred embodiment, the reaction mixture of step a) further contains a desiccant.

[0036] The term "base" refers to a substance that can accept a hydrogen ion (proton) or a substance that can donate a pair of valence electrons. Suitable examples of bases are non-nucleophilic and non-reactive with respect to the N-carboxy anhydride represented by formula (II). Examples of suitable bases include trialkylamines (e.g., diisopropylethylamine, triethylamine, and 1,8-diazabicycloundec-7-ene), metal alkoxides (e.g., sodium tert-butoxide and potassium tert-butoxide), alkyl metals (e.g., tert-butyllithium, methyllithium, n-butyllithium, tert-butyllithium, lithium di-isopropylamide, pentylsodium, and 2-phenylisopropyl-potassium), aryl metals (e.g., phenyllithium), metal hydrides (e.g., sodium hydride), metal amides (e.g., sodium amide, potassium amide, lithium diisopropylamide, and lithium tetramethylpiperidide), and silicon-based amides (e.g., sodium bis(trimethylsilyl)amide and potassium bis(trimethylsilyl)amide). Preferably, the base is a trialkylamine. More preferably, the base is diisopropylethylamine.

[0037] The term "desiccant" refers to an agent capable of removing moisture from a solution. Examples of suitable desiccants include, but are not limited to, molecular sieves, sodium sulfate, calcium sulfate, calcium chloride, and magnesium sulfate. Physical forms of the desiccant include, but are not limited to, granular beads or powder. Preferably, the desiccant is a molecular sieve. Alternatively, the desiccant is sodium sulfate.

[0038] The term "Lewis acid" refers to an acidic substance that can use the lone pair of electrons of another molecule in completing a stable group with one of its own atoms. Suitable examples of Lewis acids used in the methods of the present disclosure include zinc triflate, zinc chloride, magnesium bromide, magnesium triflate, copper triflate, copper(II) bromide, copper(II) chloride, and magnesium chloride. Preferably, the Lewis acid is zinc triflate.

[0039] The term "nucleophile" refers to a reactant that reacts with the positively charged center of the N-carboxy anhydride represented by formula (II) to decompose the N-carboxy anhydride. Examples of suitable nucleophiles include water, alcohols (methanol, ethanol, n-propanol, isopropanol, or tert-butanol), and primary or secondary amines (e.g., methylamine, ethylamine, dimethylamine, diethylamine, etc.). Preferably, the nucleophile is an alcohol. Alternatively, the nucleophile is water.

[0040] Suitable exemplary reaction conditions for preparing the amino acid ester of maitansinol represented by formula (I) are described below. Specific conditions are described in the examples.

[0041] An equimolar amount of mytansinol can be used for the N-carboxy anhydride, but more generally, the N-carboxy anhydride is used in excess. Suitable molar ratios of mytansinol to N-carboxy anhydride are in the range of 1:1 to 1:10, more generally 1:2 to 1:7 or 1:1 to 1:4. In one preferred embodiment, the molar ratio of mytansinol to N-carboxy anhydride is about 1:5.

[0042] In the method of the present disclosure, a Lewis acid may be used. In that case, typically, it is used in excess with respect to mytansinol, for example, up to 20 times in excess. More generally, the molar ratio of mytansinol to Lewis acid is in the range of 1:5 to 1:8, more preferably 1:7. A smaller amount of Lewis acid can also be used.

[0043] An amount of desiccant sufficient to remove the dissolved water from the reaction solvent is used. The amount of desiccant is not critically important, provided that the reaction solution is made substantially anhydrous. The desiccant can be used directly in the reaction vessel or placed in a semi-permeable barrier such as a sintered glass vessel in the vessel.

[0044] The time required for the reaction can be easily monitored by those skilled in the art using techniques such as high-performance liquid chromatography or thin-layer chromatography, but not limited thereto. A typical reaction is completed after stirring for 24 hours, but depending on various factors such as the reaction temperature and the concentration of the reactants, the reaction can be carried out slower or faster.

[0045] The reaction can be carried out between -20°C and 80°C, preferably between -10°C and 60°C, more preferably between -10°C and 40°C, and most preferably between 0°C and 35°C.

[0046] Suitable solvents can be easily determined by those skilled in the art and include polar aprotic solvents such as anhydrous dimethylformamide, dimethyl sulfoxide (DMSO) or dimethylacetamide (DMA), hexane, ethers (tetrahydrofuran, diethyl ether, dimethoxyethane, dioxane, etc.), dichloromethane, or mixtures thereof, but are not limited thereto.

[0047] When a Lewis acid is present in the reaction mixture, the reaction mixture after the reaction of mytansinol with the N-carboxy anhydride is preferably contacted with an aqueous solution containing hydrogen carbonate or carbonate or a metal scavenger. Preferably, the reaction mixture is reacted with a nucleophile to decompose excess N-carboxy anhydride before contacting with an aqueous solution containing hydrogen carbonate or carbonate or a metal scavenger.

[0048] When a Lewis acid containing a metal cation is present in the reaction mixture, as part of the work-up of the reaction, the metal cation is preferably removed from the reaction mixture. Removal of the metal cation can be achieved by contacting the reaction mixture with an aqueous solution containing hydrogen carbonate or carbonate or a metal scavenger. Preferably, the N-carboxy anhydride is reacted with a nucleophile before removal of the metal cation.

[0049] The amount of the nucleophile in step b) can be readily determined by those skilled in the art. Preferably, a sufficient amount of the nucleophile is used to decompose the unreacted N-carboxy anhydride. This is typically an equimolar amount of the nucleophile, but an excess amount of the nucleophile can also be used. A typical reaction is completed after stirring for 1 hour, but depending on various factors such as temperature, the reaction can be carried out slower or faster.

[0050] Also within the scope of the present invention is a method for acylating an amino acid ester of mytansinol. This method involves reacting an amino acid ester of mytansinol represented by formula (I) or formula (Ia), prepared as described above, with a carboxylic acid having the formula "R3COOH" in the presence of a condensing agent, or reacting it with an active carboxylic acid having the formula "R3COX" to form a compound represented by one of the following formulas:

[0051]

Chemical formula

[0052] [Chemical formula]

[0053] In formula (III) or (IIIa), R1 and R2 are as defined in formula (I), (II), (Ia), and (IIa); R3 is an alkyl group or a substituted alkyl group; X in R3COX is a leaving group. Preferably, X is a halide, an alkoxy group, an aryloxy group, imidazole, or -S-phenyl which may be substituted with nitro or chloride on the phenyl, or -OCOR where R is a straight-chain C1-C10 alkyl group, a branched C1-C10 alkyl group, a cyclic C3-C10 alkyl group, or a C1-C10 alkenyl group. In one embodiment, in the formula "R3COX" described above, -COX is a reactive ester, for example, an optionally substituted N-succinimidyl ester. Examples of reactive esters include, but are not limited to, N-succinimidyl, N-sulfosuccinimidyl, N-phthalimidyl, N-sulfophthalimidyl, 2-nitrophenyl, 4-nitrophenyl, 2,4-dinitrophenyl, 3-sulfonyl-4-nitrophenyl, and 3-carboxy-4-nitrophenyl esters.

[0054] Preferably, R3 is -Y-S-SR4, Y is C1-C10 alkylene, and R4 is C1-C10 alkyl, aryl, or heteroaryl. In another alternative form, Y is -CH2CH2- or -CH2CH2C(CH3)2-, and R4 is methyl.

[0055] In another embodiment, R3 is -L-E; L is

[0056] [Chemical formula] or -(CH2CH2O) m CH2CH2NHC(=O)CH2CH2- or

[0057] [Chemical formula] and E is

[0058] [Chemical formula] or

[0059] [Chemical formula] or

[0060] [Chemical formula] or

[0061] [Chemical formula] and X’ is a halide; n is 1, 2, 3, 4, 5, or 6; m is 0 or an integer from 1 to 20; q is 0 or 1. Alternatively, L is -(CH2) n - and n is as defined above or n is 5. In another alternative form, L is

[0062] [Chemical formula] and n and m are as described above; or n is 4 and m is 3.

[0063] In yet another alternative form, R3 is the following formula:

[0064]

[0065] [Chemical formula]

[0066] [Chemical formula]

[0067] [Chemistry]

[0068] [Chemistry]

[0069] [Chemistry]

[0070] [Chemistry]

[0071] [Chemistry] and

[0072] [Chemistry] is selected from.

[0073] The term "condensing agent" refers to a reagent that can react with the hydroxyl group of a carboxylic acid to convert it into a leaving group and replace it with an amine or a hydroxyl group. Examples of suitable condensing agents include carbodiimide (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride), uronium, active ester, phosphonium, 2-alkyl-1-alkylcarbonyl-1,2-dihydroquinoline (2-isobutoxy-1-isobutoxycarbonyl-1,2-dihydroquinoline), 2-alkoxy-1-alkoxycarbonyl-1,2-dihydroquinoline (2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline), or alkyl chloroformate (isobutyl chloroformate). Preferably, the condensing agent is carbodiimide. More preferably, it is N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride.

[0074] The term "leaving group" refers to a group, whether charged or uncharged, that can be readily replaced by a nucleophile such as an amine. Such leaving groups well-known in the art include, but are not limited to, halides, esters, alkoxy, hydroxyl, alkoxy, tosylate, triflate, me sylate, nitrile, azide, imidazole, carbamic acid, disulfide, thioester, thioether (i.e., -S-phenyl may be substituted), and diazonium compounds. Preferably, the leaving group is a halide, an alkoxy group, an aryloxy group, imidazole, or -S-phenyl optionally substituted with -NO2 or chloro, or -OCOR where R is a straight-chain C1-C10 alkyl group, a branched C1-C10 alkyl group, a cyclic C3-C10 alkyl group, or a C1-C10 alkenyl group. In another preferred embodiment, the leaving group is a substitutable component (e.g., -COX) in a reactive ester. Reactive esters include, but are not limited to, N-succinimidyl, N-sulfosuccinimidyl, N-phthalimidyl, N-sulfophthalimidyl, 2-nitrophenyl, 4-nitrophenyl, 2,4-dinitrophenyl, 3-sulfonyl-4-nitrophenyl, and 3-carboxy-4-nitrophenyl esters.

[0075] The present invention also includes a method for preparing derivatives using the C3 ester of mytansinol. The method includes reacting the C3 ester of mytansinol represented by formula (III) or (IIIa) prepared above with a reducing agent to form a compound represented by one of the following formulas:

[0076]

Chemical formula

[0077]

Chemical formula

[0078] In Formulas (IV) and (IVa), R1 and R2 are as defined in Formulas (I), (II), (Ia), and (IIa); Y is as defined in Formula (III) or (IIIa).

[0079] The term "reducing agent" refers to an element or compound of a redox reaction that converts a disulfide bond to a hydrogen sulfide group. Examples of suitable reducing agents include dithiothreitol (DTT), ( tris(2-carboxyethyl)phosphine) (TCEP), and NaBH4.

[0080] A compound of Formula (III) or (IIIa) where R3 is -LE, or a compound of Formula (IV) or (IVa), can react with an antibody or a modified antibody to form an antibody-mitansinoid complex. See, for example, U.S. Patent Nos. 7,521,541, 5,208,020, and 7,811,872. Alternatively, a compound of Formula (IV) or (IVa) can react with a bifunctional crosslinker to form a linker compound that retains a reactive group capable of reacting with an antibody to form an antibody-mitansinoid complex. See, for example, U.S. Patent No. 6,441,163, U.S. Patent Application Publication No. 2011 / 0003969 (A1), and U.S. Patent Application Publication No. 2008 / 0145374.

[0081] "Alkyl", as used herein, refers to straight-chain, branched, or cyclic alkyl.

[0082] "Straight-chain or branched alkyl", as used herein, refers to a saturated straight-chain or branched monovalent hydrocarbon radical having 1 to 20 carbon atoms. Examples of alkyl include, but are not limited to, methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 2-methyl-1-propyl, -CH2CH(CH3)2, 2-butyl, 2-methyl-2-propyl, 1-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, -CH2CH2CH(CH3)2, 1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl, 3,3-dimethyl-2-butyl, 1-heptyl, 1-octyl, etc. Preferably, the alkyl has 1 to 10 carbon atoms. More preferably, the alkyl has 1 to 4 carbon atoms.

[0083] "Alkylene", as used herein, refers to straight-chain, branched, or cyclic alkylene.

[0084] "Straight-chain or branched alkylene", as used herein, refers to a saturated straight-chain or branched-chain divalent hydrocarbon radical having 1 to 20 carbon atoms. Examples of alkyl include methylene, ethylene, 1-propylene, 2-propylene, 1-butylene, 2-methyl-1-propylene, -CH2CH(CH3)2-, 2-butylene, 2-methyl-2-propylene, 1-pentylene, 2-pentylene, 3-pentylene, 2-methyl-2-butylene, 3-methyl-2-butylene, 3-methyl-1-butylene, 2-methyl-1-butylene, -CH2CH2CH(CH3)2-, 1-hexylene, 2-hexylene, 3-hexylene, 2-methyl-2-pentylene, 3-methyl-2-pentylene, 4-methyl-2-pentylene, 3-methyl-3-pentylene, 2-methyl-3-pentylene, 2,3-dimethyl-2-butylene, 3,3-dimethyl-2-butylene, 1-heptylene, 1-octylene, etc., but are not limited thereto. Preferably, alkylene has 1 to 10 carbon atoms. More preferably, alkylene has 1 to 4 carbon atoms.

[0085] "Straight-chain or branched alkenyl" refers to a straight-chain or branched-chain monovalent hydrocarbon radical having 2 to 20 carbon atoms and having at least one unsaturated site, i.e., a carbon-carbon double bond. Alkenyl radicals include radicals having "cis" and "trans" configurations, or "E" and "Z" configurations. Examples include ethenyl or vinyl (-CH=CH2), allyl (-CH2CH=CH2), etc., but are not limited thereto. Preferably, alkenyl has 2 to 10 carbon atoms. More preferably, alkenyl has 2 to 4 carbon atoms to have.

[0086] "Cyclic alkyl" refers to a monovalent saturated carbocyclic radical. Preferably, cyclic alkyl is a three- to ten-membered monocyclic radical. More preferably, cyclic alkyl is cyclohexyl.

[0087] "Aryl" means a monovalent aromatic hydrocarbon radical having 6 to 18 carbon atoms, which is derived by removing one hydrogen atom from a single carbon atom of a parent aromatic ring system. Aryl includes an aromatic ring condensed with a saturated or partially unsaturated ring, or a bicyclic radical containing an aromatic carbocyclic or aromatic heterocyclic ring. Typical aryl groups include benzene (phenyl), substituted benzene (e.g., paranitrophenyl, orthonitrophenyl, and dinitrophenyl), naphthalene, anthracene, indenyl, indanyl, 1,2-dihydronaphthalene, 1,2,3,4-tetrahydronaphthyl, and radicals derived therefrom, but are not limited thereto. Preferably, aryl is phenyl which may be substituted (e.g., phenyl, phenol, or protected phenol).

[0088] "Heteroaryl" refers to a monovalent aromatic radical of a five-membered or six-membered ring, and includes a fused ring system of 5 to 18 atoms containing one or more heteroatoms independently selected from nitrogen, oxygen, and sulfur, at least one of which is aromatic. Examples of heteroaryl groups include pyridinyl (e.g., 2-hydroxypyridinyl), imidazolyl, imidazopyridinyl, pyrimidinyl (e.g., 4-hydroxypyrimidinyl), pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolinyl, isoquinolinyl, indolyl, benzimidazolyl, benzofuranyl, cinnolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl, triazolyl, thiadiazolyl, furazanyl, benzofurazanyl, benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, and furopyridinyl.

[0089] Suitable substituents for the alkyl group do not significantly interfere with the reactions of the present disclosure. Substituents that interfere with the reactions of the present disclosure can be protected according to methods well known in the art, for example, as described in T.W. Greene and P.G.M. Wuts “Protective Groups in Organic Synthesis” John Wiley & Sons, Inc., New York 1999. Exemplary substituents include aryl (e.g., phenyl, phenol, and protected phenol), heteroaryl (e.g., indolyl and imidazolyl), halogen, guanidium [-NH(C=NH)NH2], - OR 100 、NR 101 R 102 、-NO2、-NR 101 COR 102 、-SR 100 、-SOR 101 sulfoxide represented by -SO2R 101 sulfone represented by -SO3R, sulfuric acid -SO3R 100 sulfonic acid -OSO3R 100 、-SO2NR 101 R 102 sulfonamide represented by -COR 101 、-OCOR 101 、-OCONR 101 R 102 are mentioned; R 101 and R 102 are each independently selected from H, linear, branched, or cyclic alkyl, alkenyl or alkynyl having 1 to 10 carbon atoms.

[0090] The term “halide” refers to -F, -Cl, -Br, or -I.

[0091] The term “amino acid” refers to a natural or unnatural amino acid represented by NH2-C(R aa’ R aa )-C(=O)OH, where R aa and R aa’are each independently of the others, H, a linear, branched or cyclic alkyl which may be substituted, alkenyl or alkynyl having 1 to 10 carbon atoms, aryl, heteroaryl, or heterocy ryl. The term "amino acid" also refers to corresponding residues when one hydrogen atom is removed from the amine and / or carboxy terminus of the amino acid, such as -NH-C(R aa’ R aa )-C(=O)O-. The following specific examples are illustrative only and are not to be construed as limiting the other parts of the present disclosure in any way. Further, without further elaboration, it is believed that one of ordinary skill in the art can, based on the description herein, utilize the present invention to its fullest extent. All publications cited herein are hereby incorporated by reference in their entirety. Further, any techniques set forth below are not in any way limiting of the claimed invention.

Examples

[0092] Materials and Methods The process parameters given below can be adapted and employed by one of ordinary skill in the art to suit the particular needs of the one of ordinary skill in the art.

[0093] All reactions were carried out under argon with magnetic stirring. Tetrahydrofuran and dimethylformamide were purchased from Aldrich as anhydrous solvents. Myrtanol was generated as described in (Widdison et al., J. Med. Chem., 49:4392 - 4408 (2006)). N-Methyl-alanine, the N-carboxy anhydride of (4S)-3,4-dimethyl-2,5-oxazolidinedione was prepared as described in (Akssira, M. et al., J. Marocain de Chimie Heterocyclique, 1:44 - 47 (2002)). The nuclear magnetic resonance (NMR) spectrum ([[]] 1 H 400 MHz, 13C 100 MHz) was obtained using a Bruker ADVANCE (trademark) series NMR. HPLC / MS data was obtained using an Agilent 1100 series HPLC combined with a Bruker ESQUIRE (trademark) 3000 ion trap mass spectrometer. DM1 was analyzed using HPLC method 1. HPLC method 2 was used for all other analyses.

[0094] Analysis HPLC method 1: Water HPLC system equipped with a UV detector or equivalent Column: YMC-Pack ODS-AQ 250×4.6 mm; 5 μm (product number = AQ12S05-2546WT) Flow rate: 1 mL / min (gradient) Mobile phase: A = 1 ml of 85% H3PO4 in 1 liter of water; B = acetonitrile / tetrahydrofuran 30:70 (v / v) (Note: For LC / MS analysis, 0.1% TFA was used instead of H3PO4 in mobile phase A) Gradient table:

[0095]

Table 1

[0096] Analysis HPLC / MS method 2: Column: 150×4.6 mm C8, particle size 5 microns, Zorbax product number 993967-906 Solvent: A deionized water + 0.1% TFA Solvent B: acetonitrile Flow rate 1.0 mL / min Temperature: room temperature Injection volume: 15 μL Gradient

[0097]

Table 2

[0098] Sample preparation for Analytical HPLC method 2: An aliquot (20 μL) of the given mixture was added to acetonitrile (1.5 mL) in an autosampler vial. The vial was capped, shaken, and then placed in an autosampler at 15 °C. The injection volume (15 μL) was analyzed for each HPLC run.

[0099] Preparation of DM1-SMe with 4A molecular sieve as desiccant in Example 1 Mitansinol (50.1 mg, 0.0888 mmol), (4S)-3,4-dimethyl-2,5-oxazolidinedione (30.2 mg, 0.233 mmol, 2.6 equiv), zinc triflate (133 mg, 0.366 mol), and 4A molecular sieve (0.50 g) pre-dried under vacuum at 250 °C and then cooled to room temperature were added to a 10 ml flask. The contents were taken up in anhydrous dimethylformamide (0.75 mL), and diisopropylethylamine (62 μL, 0.357 mmol) was added. The mixture was stirred at room temperature for 24 hours. The crude mixture was analyzed by HPLC, and N 2’-Deacetyl-mytansine product accounted for 80% of the total HPLC area. The reaction mixture was diluted with 1:1 saturated NaHCO3:saturated NaCl (1.2 mL) and ethyl acetate (3 mL), mixed, then filtered through celite, and then washed with potassium phosphate buffer (1 mL, 400 mM, pH 7.5). The organic layer was dried over anhydrous magnesium sulfate, filtered, and then evaporated to form a yellow solid. To this solid, 3-methyldithiopropanoic acid (25 mg, 0.16 mmol), N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (30 mg, 0.16 mmol), and dichloromethane (3 mL) were added. After stirring for 2 hours, the mixture was diluted with ethyl acetate (8 mL), washed with 1.0 M potassium phosphate buffer, pH 6.5 (2 mL), and the aqueous solution was extracted with ethyl acetate (2 × 8 mL). The combined organic layers were dried over anhydrous magnesium sulfate, concentrated, and purified by silica chromatography, 95:5 dichloromethane:methanol to give 51 mg (70%) of DM1-SMe.

[0100] Example 2 Ten-fold scale-up of Example 1 The reaction of Example 1 was carried out on a 10-fold larger scale to give 490 mg (68%) of DM1-SMe.

[0101] Example 3 Preparation of DM1-SMe without addition of desiccant Mytanosinol (1.0 g, 1.77 mmol) was dissolved in anhydrous dimethylformamide (15 mL) in a 25 mL flask and cooled in an ice / water bath. After 2 minutes, diisopropylethylamine (DIPEA, 0.92 g, 7.07 mmol) and zinc triflate (3.8 g, 10.6 mmol) were added with magnetic stirring, then (4S)-3,4-dimethyl-2,5-oxazolidinedione (0.913 g, 7.07 mmol) was added quickly, and the mixture was stirred for 24 hours. Analysis of a sample of the crude mixture by HPLC showed that N 2’-Deacetyl-mytansine product accounted for 65% of the total HPLC area. The reaction mixture was diluted with 1:1 saturated NaHCO3:saturated NaCl (25 mL) and ethyl acetate (40 mL), mixed, then filtered through celite and washed with saturated NaCl. The organic layer was dried over anhydrous sodium sulfate, filtered, and then evaporated. The residue was taken up in dichloromethane (30 mL), and 3-methyldithiopropanoic acid (1.1 g, 7.0 mmol) and N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (1.34 g, 7.0 mmol) were quickly added, and the reaction was stirred at room temperature under argon for 2 h. The mixture was diluted with ethyl acetate (30 mL), washed with 1.0 M potassium phosphate buffer (30 mL), pH 6.5, and the aqueous solution was extracted with ethyl acetate (2×40 mL). The combined organic layers were dried over anhydrous sodium sulfate, concentrated, and purified by silica chromatography, 95:5 dichloromethane:methanol, to give 698 mg (50%) of DM1-SMe.

[0102] Example 4 Repetition of Example 3 The reaction of Example 3 was repeated on the same scale to give 735 mg (53%) of DM1-SMe.

[0103] Example 5 Crude N2 ’ -Deacetyl-mytansine stock solution Mytanosinol (0.5 g, 0.89 mmol) was dissolved in anhydrous dimethylformamide (7 mL) in a 25 mL flask and cooled in an ice / water bath. After 2 min, diisopropylethylamine (0.5 g, 3.5 mmol) and zinc triflate (1.9 g, 5.3 mmol) were added with magnetic stirring, then (4S)-3,4-dimethyl-2,5-oxazolidinedione (4.52 g, 3.5 mmol) was quickly added, and the mixture was stirred for 24 h. Aliquots (each 0.5 mL) of this stock solution were prepared from approximately 0.13 mmol of mytanosinol for use in the following experiments.

[0104] Example 6 N 2’ -Deacetyl-mytansine extraction and subsequent coupling to propionic acid (control)

[0105]

Chem.

[0106] As shown below, the following by-products were also formed from May-NMA2, a by-product of the aforementioned reaction.

[0107]

Chem.

[0108] The HPLC percent area ratio at 17:16 was 3.0:71.7. The MS (M+H+) of 16 was 706 (M+Na + ) 728; the MS (M+Na + ) of 17 was 813.

[0109] Example 7 The experiment of Example 6 was repeated At 17:16, the HPLC percent area ratio was 3.0:70.9.

[0110] Example 8 N 2’ -Deacetyl-mitansine extraction followed by methanol pre-quench to the next propionic acid binding (pre-quench to destroy excess 5a) N 2’-Add deacetyl-mitansine stock solution (0.50 mL) to a 6 mL volumetric vial, add methanol (75 μL, 1.8 mmol) thereto, cap the vial, and magnetically stir the contents for 1 hour. Then add ethyl acetate (1.5 mL) and 1:1 saturated NaCl:NaHCO3 (0.75 mL), cap the vial, and mix. Retain the organic layer and dry it over anhydrous Na2SO4 (120 mg). Take the organic layer (1.0 mL) and add propionic acid (20.0 μL, 0.27 mmol). Then transfer the solution to a vial containing N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (40 mg, 0.209 mmol). Allow the reaction to proceed for 2.5 hours, and then analyze it by HPLC. The HPLC peak for 17 was hardly detectable and could not be integrated. Repeating the reaction, again 17 was hardly detectable and could not be integrated. Thus, the prequenching method produces compounds 15 and 17 that are not more desirable.

[0111] Example 9 Synthesis of Excess NMA-DM1-SMe (9a)

[0112]

Chemical formula

[0113] Example 10 Synthesis of Excess NMA-DM1(10)

[0114]

Chem.

Claims

[Claim 1] The formula: 【Chemistry 1】 (In the formula, R １ is hydrogen, an optionally substituted C1-C10 alkyl group, or an amino acid side chain, provided that if the amino acid side chain has a reactive functional group, the reactive functional group may be protected; R ２ is hydrogen or an optionally substituted C1-C10 alkyl group, reacting maytansinol with an N-carboxyanhydride in a reaction mixture additionally comprising a base and a drying agent, thereby forming the compound of formula (I); The N-carboxyanhydride has the following formula: 【Chemistry 2】 The method of claim 1,

Images