METHODS FOR PREPARING MAYTANSINOID DERIVATIVES WITH SELF-IMMOLATIVE PEPTIDE LINKERS

RELATED APPLICATION

This application claims priority to U.S. Provisional Application No. 63/406,938, filed on September 15, 2022, the entire content of which is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to novel methods for preparing maytansinoid derivatives with self-immolative peptide linkers as well as their synthetic precursors.

BACKGROUND OF THE INVENTION

Cell binding agent-drug conjugates, including antibody-drug conjugates (ADC) are emerging as a powerful class of agents with efficacy across a range of abnormal cell growth or proliferative diseases (e.g., cancers). Cell binding agent-drug conjugates (such as ADCs) are commonly composed of three distinct elements: a cell-binding agent (e.g., an antibody); a linker; and a cytotoxic moiety. The linker component of ADC is an important element in developing targeted anti-cancer agents that possess an optimal therapeutic window, i.e., high activity at a low, non-toxic dose.

Maytansinoids are highly cytotoxic compounds, including maytansinol and C-3 esters of maytansinol, e.g., C-3 esters with N-methyl-L-alanine (MayNMA). It has been found that conjugates of maytansinoid derivatives with self-immolative peptide linkers and cell binding agents are highly potent as anti-proliferative agents, in particularly, as anticancer agents.

There exists a need to develop new processes for producing maytansinoid derivatives with these self-immolative peptide linkers.

SUMMARY OF THE INVENTION

The present invention provides new methods for preparing maytansinoid derivatives with self-immolative peptide linkers as well as their synthetic precursors. The methods disclosed herein can eliminate the cumbersome chromatography purifications, which are more efficient and suitable for large scale manufacturing process. In addition, the present methods only introduce highly toxic maytansinoid moiety in the last reaction step, thereby reducing worker exposure to highly toxic material.

In one aspect, the present invention relates to a method of preparing a compound represented by Formula (I): or a salt thereof, comprising the steps of:

(a) reacting a compound of Formula (II): or a salt thereof, with a deprotecting agent to form a compound of Formula (III): or a salt thereof; and

(b) reacting the compound of Formula (III) or a salt thereof with a compound of

Formula (A): to form the compound of Formula (I) or a salt thereof.

In another aspect, the present invention relates to a method of preparing a compound of Formula (IV): or a salt thereof, comprising the step of reacting a compound of Formula (I): or a salt thereof (e.g., a TEA salt), with a compound of Formula (V): (V), to form the compound of Formula (IV) or a salt thereof, wherein DM is represented by the following formula:

In another aspect, the present invention provides a method of preparing a compound of Formula (II): or a salt thereof, comprising the step of reacting a compound of Formula (VI): with a compound of Formula (B) to form the compound of Formula (II) or a salt thereof, wherein the compound of Formula (II) is purified by precipitation. DETAILED DESCRIPTION OF THE INVENTION

Reference will now be made in detail to certain embodiments of the invention, examples of which are illustrated in the accompanying structures and formulas. While the invention will be described in conjunction with the enumerated embodiments, it will be understood that they are not intended to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents which may be included within the scope of the present invention as defined by the claims. One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention.

It should be understood that any of the embodiments described herein can be combined with one or more other embodiments of the invention, unless explicitly disclaimed or improper. Combination of embodiments are not limited to those specific combinations claimed via the multiple dependent claims.

DEFINITIONS

Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings that are commonly understood by those of ordinary skill in the art to which this invention belongs. Generally, nomenclature used in connection with the compounds, composition and methods described herein, are those well-known and commonly used in the art.

Chemistry terms used herein are used according to conventional usage in the art, as exemplified by “The McGraw-Hill Dictionary of Chemical Terms”, Parker S., Ed., McGraw- Hill, San Francisco, C.A. (1985).

The term “herein” means the entire application.

Throughout this specification, the word “comprise” or variations such as “comprises” or “comprising” will be understood to imply the inclusion of a stated integer (or components) or group of integers (or components), but not the exclusion of any other integer (or components) or group of integers (or components).

Throughout the specification, where compositions are described as having, including, or comprising (or variations thereof), specific components, it is contemplated that compositions also may consist essentially of, or consist of, the recited components.

Similarly, where methods or processes are described as having, including, or comprising specific process steps, the processes also may consist essentially of, or consist of, the recited processing steps. Further, it should be understood that the order of steps or order for performing certain actions is immaterial so long as the compositions and methods described herein remains operable. Moreover, two or more steps or actions can be conducted simultaneously.

The term “including” is used to mean “including but not limited to.” “Including” and “including but not limited to” are used interchangeably.

As used herein, “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system.

The use of the terms “a” and “an” and “the” and similar referents in the context of describing the elements (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

The term “or” as used herein should be understood to mean “and/or,” unless the context clearly indicates otherwise.

Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the embodiments and does not pose a limitation on the scope of the claims unless otherwise stated. No language in the specification should be construed as indicating any non-claimed element as essential.

The term “compound” is intended to include compounds for which a structure or formula or any derivative thereof has been disclosed in the present invention or a structure or formula or any derivative thereof that has been incorporated by reference. The term also includes, stereoisomers, geometric isomers, tautomers, solvates, and salts (e.g., pharmaceutically acceptable salts) of a compound of all the formulae disclosed in the present invention. The term also includes any solvates, hydrates, and polymorphs of any of the foregoing. The specific recitation of “stereoisomers,” “geometric isomers,” “tautomers,” “solvates,” “salt” “hydrate,” or “polymorph” in certain aspects of the invention described in this application shall not be interpreted as an intended omission of these forms in other aspects of the invention where the term “compound” is used without recitation of these other forms.

The term “chiral” refers to molecules that have the property of non-superimposability of the mirror image partner, while the term “achiral” refers to molecules that are superimposable on their mirror image partner.

The term “stereoisomer” refers to compounds that have identical chemical constitution and connectivity, but different orientations of their atoms in space that cannot be interconverted by rotation about single bonds.

The term “diastereomer” refers to a stereoisomer with two or more centers of chirality and whose molecules are not mirror images of one another. Diastereomers have different physical properties, e.g. melting points, boiling points, spectral properties, and reactivities. Mixtures of diastereomers can separate under high resolution analytical procedures such as crystallization, electrophoresis and chromatography.

The term “enantiomers” refer to two stereoisomers of a compound that are non- superimposable mirror images of one another.Stereochemical definitions and conventions used herein generally follow S. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., “Stereochemistry of Organic Compounds,” John Wiley & Sons, Inc., New York, 1994. The compounds of the invention can contain asymmetric or chiral centers, and therefore exist in different stereoisomeric forms. It is intended that all stereoisomeric forms of the compounds of the invention, including but not limited to, diastereomers, enantiomers and atropisomers, as well as mixtures thereof such as racemic mixtures, form part of the present invention. Many organic compounds exist in optically active forms, i.e., they have the ability to rotate the plane of plane-polarized light. In describing an optically active compound, the prefixes D and L, or R and S, are used to denote the absolute configuration of the molecule about its chiral center(s). The prefixes d and 1 or (+) and (-) are employed to designate the sign of rotation of plane-polarized light by the compound, with (-) or 1 meaning that the compound is levorotatory. A compound prefixed with (+) or d is dextrorotatory. For a given chemical structure, these stereoisomers are identical except that they are mirror images of one another. A specific stereoisomer can also be referred to as an enantiomer, and a mixture of such isomers is often called an enantiomeric mixture. A 50:50 mixture of enantiomers is referred to as a racemic mixture or a racemate, which can occur where there has been no stereoselection or stereospecificity in a chemical reaction or process. The terms “racemic mixture” and “racemate” refer to an equimolar mixture of two enantiomeric species, devoid of optical activity.

The term “tautomer” or “tautomeric form” refers to structural isomers of different energies that are interconvertible via a low energy barrier. For example, proton tautomers (also known as prototropic tautomers) include interconversions via migration of a proton, such as keto-enol and imine-enamine isomerizations. Valence tautomers include interconversions by reorganization of some of the bonding electrons.

The term “salt” as used herein, refers to an organic or inorganic salts of a compound of the invention. Exemplary salts include, but are not limited, to sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate “mesylate,” ethanesulfonate, benzenesulfonate, p- toluenesulfonate, pamoate (/'.<?., l,l’-methylene-bis-(2-hydroxy-3 -naphthoate)) salts, alkali metal (e.g., sodium and potassium) salts, alkaline earth metal (e.g., magnesium) salts, and ammonium salts. A salt can involve the inclusion of another molecule such as an acetate ion, a succinate ion or other counter ion. The counter ion can be any organic or inorganic moiety that stabilizes the charge on the parent compound. Furthermore, a salt can have more than one charged atom in its structure. Instances where multiple charged atoms are part of the salt can have multiple counter ions. Hence, a salt can have one or more charged atoms and/or one or more counter ion.

If the compound of the invention is a base, the desired salt can be prepared by any suitable method available in the art, for example, treatment of the free base with an inorganic acid, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, methanesulfonic acid, phosphoric acid and the like, or with an organic acid, such as acetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, a pyranosidyl acid, such as glucuronic acid or galacturonic acid, an alpha hydroxy acid, such as citric acid or tartaric acid, an amino acid, such as aspartic acid or glutamic acid, an aromatic acid, such as benzoic acid or cinnamic acid, a sulfonic acid, such as p-toluenesulfonic acid or ethanesulfonic acid, or the like.

If the compound of the invention is an acid, the desired salt can be prepared by any suitable method, for example, treatment of the free acid with an inorganic or organic base, such as an amine (primary, secondary or tertiary), an alkali metal hydroxide or alkaline earth metal hydroxide, or the like. Illustrative examples of suitable salts include, but are not limited to, organic salts derived from amino acids, such as glycine and arginine, ammonia, primary, secondary, and tertiary amines, and cyclic amines, such as triethylamine, piperidine, morpholine and piperazine, and inorganic salts derived from sodium, calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminum and lithium.

In certain embodiments, the salt is a pharmaceutically acceptable salt. The phrase “pharmaceutically acceptable” indicates that the substance or composition must be compatible chemically and/or toxicologically, with the other ingredients comprising a formulation, and/or the mammal being treated therewith.

The term “solvate” means a compound that further includes a stoichiometric or non- stoichiometric amount of solvent such as water, isopropanol, acetone, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine dichloromethane, 2-propanol, or the like, bound by non-covalent intermolecular forces. Solvates or hydrates of the compounds are readily prepared by addition of at least one molar equivalent of a hydroxylic solvent such as methanol, ethanol, 1 -propanol, 2-propanol or water to the compound to result in solvation or hydration of the imine moiety.

The term “precursor” of a given group refers to any group which may lead to that group by any deprotection, a chemical modification, or a coupling reaction.

The term “amino acid” refers to naturally occurring amino acids or non-naturally occurring amino acid. In some embodiments, the amino acid is represented by NH2- C(R ^aa R ^aa )-C(=O)OH, wherein R ^aa and R ^aa are each independently H, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having 1 to 10 carbon atoms, aryl, heteroaryl or heterocyclyl or R ^aa and the N-terminal nitrogen atom can together form a heterocyclic ring (e.g., as in proline). The term “amino acid residue” refers to the corresponding residue when one hydrogen atom is removed from the amine and/or carboxy end of the amino acid, such as -NH-C(R ^aa R ^aa )-C(=O)-.

The term “peptide” refers to short chains of amino acid monomers linked by peptide (amide) bonds. In some embodiments, the peptides contain 2 to 20 amino acid residues. In other embodiments, the peptides contain 2 to 10 or 2 to 8 amino acid residues. In yet other embodiments, the peptides contain 2 to 5 amino acid residues. As used herein, when a peptide is a portion of a cytotoxic agent or a linker described herein represented by a specific sequence of amino acids, the peptide can be connected to the rest of the cytotoxic agent or the linker in both directions.

The term “immunoconjugate,” “conjugate,” or “ADC” as used herein refers to a compound or a derivative thereof that is linked to a cell binding agent (e.g., an antibody or antigen -binding fragment thereof).

The term “cell-binding agent” in the immunoconjugates of the present invention can be of any kind presently known, or that become known, including peptides and non-peptides that binds to a cell or cell component (e.g., receptor, protein, DNA, RNA, etc.). Generally, these can be antibodies (such as polyclonal antibodies and monoclonal antibodies, especially monoclonal antibodies) or fragments thereof, lymphokines, hormones, growth factors, vitamins (such as folate etc., which can bind to a cell surface receptor thereof, e.g., a folate receptor), nutrient-transport molecules (such as transferrin), probodies, nanobodies, or any other cell-binding molecule or substance.

The term “cation” refers to an ion with positive charge. The cation can be monovalent (e.g., Na ⁺ , K ⁺ , etc.), bi-valent (e.g., Ca ²⁺ , Mg ²⁺ , etc.) or multi-valent (e.g., Al ³⁺ etc.). Preferably, the cation is monovalent.

The term “acid” refers to any substance that in water solution tastes sour, changes the color of certain indicators (e.g., reddens blue litmus paper), reacts with some metals (e.g., iron) to liberate hydrogen, reacts with bases to form salts, and promotes certain chemical reactions (acid catalysis). Examples of acids include the inorganic substances known as the mineral acids — sulfuric, nitric, hydrochloric, and phosphoric acids — and the organic compounds belonging to the carboxylic acid, sulfonic acid, and phenol groups. Such substances contain one or more hydrogen atoms that, in solution, are released as positively charged hydrogen ions. Examples of acid includes formic acid, acetic acid, trifluoroacetic acid (TFA), pyridinium p-toluenesulfonate (PPTS), p-toluenesulfonic acid, methanesulfonic acid, camphorsulfonic acid, phosphoric acid, sulfuric acid, hydrochloric acid (HC1), and trichloroacetic acid.

The term “base” refers to a substance that can accept hydrogen ions (protons) or donate a pair of valence electrons. Examples of the suitable bases include imidazole, piperidine, 4-methylpiperidine, tetramethylpiperidine, morpholine, N-methylmorpholine, pyridine, 2,6-lutidine, dimethylformamide, piperazine, pyrrolidine, 1-methylpyrrolidine, 1,8- diazabicyclo[5.4.0]undec-7-ene (DBU), diethylamine (DEA), a trialkylamine (e.g., N,N- diisopropylethylamine (DIPEA), triethylamine (TEA), and l,8-Diazabicycloundec-7-ene), a metal alkoxide (e.g., sodium tert-butoxide and potassium tert-butoxide), an alkyl metal (e.g., tert-butyllithium, methyl lithium, n-butyl lithium, tert-butyl lithium, lithium diisopropylamide, pentyl sodium, and 2-phenyl isopropyl-potassium), an aryl metal (e.g., phenyl lithium), a metal hydride (e.g., sodium hydride), a metal amide (e.g., sodium amide, potassium amide, lithium diisopropylamide and lithium tetramethylpiperidide), and a silicon- based amide (e.g., sodium bis(trimethylsilyl)amide and potassium bis(trimethylsilyl)amide).

The term “Lewis acid” refers to an acid substance which can employ an electron lone pair from another molecule in completing the stable group of one of its own atoms. Exemplary Lewis acids for use in the disclosed methods include boron trifluoride etherate (BF3*OEt2), zinc triflate, zinc chloride, magnesium bromide, magnesium triflate, copper triflate, copper (II) bromide, copper (II) chloride, magnesium chloride, and aluminum chloride (AICI3).

The term “amorphous solid” refers to any non-crystalline solid in which the atoms and molecules are not organized in a definite lattice pattern. Such solids include glass, plastic, and gel.

The “semi-amorphous solid” refers to semi-crystalline materials that display crystalline regions, called crystallites, within an amorphous matrix.

The term “crystallization” refers to the process by which a solid forms, where the atoms or molecules are highly organized into a structure known as a crystal. Some of the ways by which crystals form are precipitating from a solution, freezing, or more rarely deposition directly from a gas. Attributes of the resulting crystal depend largely on factors such as temperature, air pressure, and in the case of liquid crystals, time of fluid evaporation.

The term “precipitation” refers to the process of transforming a dissolved substance into an insoluble solid from a solution comprising the substance (e.g., saturated solution of the substance). The solid formed is called the precipitate. The clear liquid remaining above the precipitated or the centrifuged solid phase is also called the 'supernate' or 'supernatant'. In some embodiments, precipitation can occur by adding a co-solvent, in which the substance has low or no solubility, to a solution of the substance. In some embodiments, cooling a solution comprising the substance can result in precipitation.

The term “organic solvent” refers to carbon-based substances capable of dissolving or dispersing one or more other substances. Many classes of chemicals are used as organic solvents, including aliphatic hydrocarbons, aromatic hydrocarbons, amines, esters, ethers, ketones, and nitrated or chlorinated hydrocarbons. Exemplary organic solvents include dichloromethane (CH2CI2 or DCM), dichloroethane (DCE), acetonitrile (ACN or MeCN), ethyl acetate, methanol (MeOH), ethanol, tetrahydrofuran (THF), toluene, N- methylmorpholine (NMM), dimethylformamide (DMF), dimethyl sulfoxide (DMSO), dimethylacetamide (DMA or DM Ac), or any combination thereof. METHODS OF THE PRESENT INVENTION

The present invention provides methods of preparing maytansinoid derivatives with self-immolative peptide linkers, which can be linked to cell binding agents (e.g., antibodies) to form immunoconjugates that are useful as medicaments, in particular as anti-proliferative agents (anticancer agents). The present invention also provides methods of preparing the related synthetic precursors.

In a first embodiment, the present invention provides a method of preparing a compound represented by Formula (I): or a salt thereof, comprising the steps of:

(a) reacting a compound of Formula (II): or a salt thereof, with a deprotecting agent to form a compound of Formula (III): or a salt thereof; and

(b) reacting the compound of Formula (III) or a salt thereof with a compound of

Formula (A): to form the compound of Formula (I) or a salt thereof.

In a 1 ^st specific embodiment, the compound of Formula (I) is represented by a compound of Formula (la): or a salt thereof, and the method comprises the steps of:

(a) reacting a compound of Formula (Ila): with a deprotecting agent to form a compound of Formula (Illa): or a salt thereof; and

(b) reacting the compound of Formula (Illa) or a salt thereof with the compound of Formula (A): to form the compound of Formula (la) or a salt thereof.

In some embodiments, the deprotecting agent in the reaction of step (a) is a base. In some embodiments, the base is piperidine, 4-methylpiperidien, piperazine, 1,8- Diazabicyclo[5.4.0]undec-7-ene (DBU), or morpholine. In a specific embodiment, the base is morpholine.

Any suitable amount of deprotecting agent (e.g., morpholine) can be used. In some embodiments, the molar ratio of the deprotecting agent to the compound of Formula (II) or (Ila) is in the range of 1:1 to 20:1, 1:1 to 10:1, 1:1 to 5:1, or 1.5:1 to 2.5:1. In a specific embodiment, the molar ratio of the deprotecting agent to the compound of Formula (II) or (Ila) is 2:1.

Any suitable solvents can be used for the reaction of step (a), which include but are not limited to DMF, DMSO, THF, CH2CI2, acetonitrile, dichloroethane, dimethylacetamide, methanol, ethanol, and toluene. In a specific embodiment, the solvent is DMF.

The reaction of step (a) can be carried out at a suitable temperature. In some embodiments, the reaction is carried out at a temperature between -50°C and 50°C, between 0°C and 40°C, between 5°C and 35°C, between 10°C and 30°C, or between 15°C and 25°C. In a specific embodiment, the reaction is carried out at a temperature between 15 °C and 25°C.

In some embodiments, the compound of Formula (III) or (Illa) is purified by precipitation. Any suitable solvents can used for the precipitation, which include DMF, DMSO, THF, CH2CI2, acetonitrile, dichloroethane, dimethylacetamide, methanol, ethanol, toluene, and tert-butyl methyl ether. In a specific embodiment, the precipitation is carried out in DMF and CH2CI2. In some embodiments, the compound of Formula (III) or (Illa) can be precipitated by mixing CH2CI2 with a DMF solution comprising the compound. The volume ratio of DMF to CH2CI2 can be in the range of 1:100 to 100:1, 1:80 to 40:1, 1:60 to 1:1, 1:60 to 1:30, or 1:50 to 1:40. In a specific embodiment, the volume ratio of DMF to CH2CI2 is 1:48.

In some embodiments, the compound of Formula (III) or (Illa) is obtained as an amorphous or semi-amorphous solid. In a specific embodiment, the compound of Formula (III) or (Illa) is obtained as an amorphous solid.

In some embodiments, the compound of Formula (III) or (Illa) is further purified by crystallization. Any suitable solvents can used for the crystallization. In some embodiments, the crystallization is carried out in one or more solvents including DMF, DMSO, THF, CH2CI2, acetonitrile, dichloroethane, dimethylacetamide, methanol, ethanol, toluene, or tertbutyl methyl ether. In a specific embodiment, the crystallization is carried out in acetonitrile.

The crystallization can be carried out at a suitable temperature. In some embodiments, the crystallization is carried out at a temperature between 0°C and 100°C, between 5°C and 80°C, between 10°C and 70°C, between 15 °C and 55 °C, between 17 °C and 23 °C, or between 47 °C and 53 °C. In a specific embodiment, the crystallization is carried out at a temperature between 17 °C and 53 °C.

In some embodiments, the compound of Formula (III) or (Illa) is further purified by precipitation after crystallization. Any suitable solvents can used for the precipitation. In some embodiments, the precipitation is carried out in one or more solvents including DMF, DMSO, THF, CH2CI2, acetonitrile, dichloroethane, dimethylacetamide, methanol, ethanol, toluene, or tert-butyl methyl ether. In a specific embodiment, the precipitation is carried out in DMF and CH2CI2. In some embodiments, the compound of Formula (III) or (Illa) can be precipitated by mixing CH2CI2 with a DMF solution comprising the compound.

The precipitation can be carried out at a suitable temperature. In some embodiments, the precipitation is carried out at a temperature between 0°C and 100°C, between 5°C and 80°C, between 10°C and 70°C, between 15 °C and 55 °C, between 17 °C and 23 °C, or between 47 °C and 53 °C. In a specific embodiment, the precipitation is carried out at a temperature between 17 °C and 53 °C.

In some embodiments, the compound of Formula (III) or (Illa) is obtained as an amorphous solid after the second precipitation.

In some embodiments, the reaction in step (b) between the compound of Formula (III) or (Illa) or a salt thereof and the compound of Formula (A) is carried out in the presence of a base. In some embodiments, the base is N-methylmorpholine, triethylamine (TEA), 1,8- diazabicyclo[5.4.0]undec-7-ene (DBU), N-methylpiperidine, 1 -methyip yrrolidine, or diisopropylethyalamine (DIPEA). In a specific embodiment, the base is triethylamine (TEA).

Any suitable amount of the compound of Formula (A) can be used. In some embodiments, the molar ratio of the compound of Formula (A) to the compound of Formula (III) or (Illa) is in the range of 1:1 to 5:1, 1:1 to 3:1, 1:1 to 2:1, 1:1 to 1.5:1, 1:1 to 1.2:1 or 1:1 to 1.1 : 1. In a specific embodiment, the molar ratio of the compound of Formula (A) to the compound of Formula (III) or (Illa) is 1.05:1.

Any suitable amount of the base can be used the reaction of step (b). In some embodiments, the molar ratio of base to the compound of Formula (III) or (Illa) is in the range of 1:1 to 20:1, 1:1 to 10:1, 1:1 to 5:1, 2:1 to 5:1 , 3:1 to 5:1 or 4.2:1 to 4.6:1. In a specific embodiment, the molar ratio of the base to the compound of Formula (III) or (Illa) is 4.4:1.

Any suitable solvents can be used for the reaction of step (b). The suitable solvents include DMF, DMSO, THF, CH2CI2, acetonitrile, dichloroethane, dimethylacetamide, methanol, ethanol, and toluene. In a specific embodiment, the reaction in step (b) is carried out in acetonitrile.

The reaction of step (b) can be carried out at a suitable temperature. In some embodiments, the reaction is carried out at a temperature between -50°C and 50°C, between 0°C and 40°C, between 5°C and 35°C, between 10°C and 30°C, or between 15°C and 25°C. In a specific embodiment, the reaction is carried out at a temperature between 15 °C and 25°C.

In some embodiments, the compound of Formula (I) or (la) is purified by precipitation. Any suitable solvents can used for the precipitation, which include DMF, DMSO, THF, CH2CI2, acetonitrile, dichloroethane, dimethylacetamide, methanol, ethanol, toluene, and tert-butyl methyl ether. In a specific embodiment, the precipitation is carried out in tert-butyl methyl ether. In some embodiments, the precipitation is carried out by adding tert-butyl methyl ether to an acetonitrile solution comprising the compound of Formula (I) or (la). The precipitation can be carried out at a suitable temperature. In some embodiments, the precipitation is carried out at a temperature between -100°C and 100°C, between -50°C and 80°C, between -40°C and 60°C, between -30 °C and 40 °C, between -30 °C to 25 °C, or between -20 °C to -16 °C. In a specific embodiment, the precipitation is carried out at a temperature of -30 °C.

In some embodiments, the compound of Formula (I) or (la) is obtained as a salt. In a specific embodiment, the compound of Formula (I) or (la) is obtained as a triethylamine (TEA) salt. The ratio of the compound of Formula (I) or (la) to TEA is in the range of 1:1.2 to 1:0.1, 1:1 to 1:0.8, or 1:0.8 to 1:0.5. In a specific embodiment, the compound of Formula (I) or (la) is obtained as triethylamine (TEA) salt, wherein the molar ratio of the compound and TEA is 1:1.

In a second embodiment, the present invention provides a method of preparing a compound of Formula (IV): or a salt thereof, comprising the step of reacting a compound of Formula (I): or a salt thereof (e.g., a TEA salt), with a compound of Formula (V): to form the compound of Formula (IV) or a salt thereof, wherein DM is represented by the following formula:

In a 2 ^nd specific embodiment, the compound of Formula (IV) is represented by a compound of Formula (IVa): or a salt thereof, and the method comprises the step of reacting a compound of Formula (la): or a salt thereof (e.g., a TEA salt), with a compound of Formula (Va):

(Va), to form the compound of Formula (IVa) or a salt thereof, wherein DM _a is represented by the following formula: In some embodiments, the reaction between the compound of Formula (I) or(Ia) or a salt thereof (e.g., a TEA salt) and the compound of Formula (V) or (Va) is carried out in the presence of an amide coupling reagent. As used herein, the “amide coupling reagent” is a reagent that activates a carboxylic group (e.g., the carboxylic group in the compound of Formula (I), (la) or a salt thereof (e.g., a TEA salt)) to facilitate the coupling reaction with an amine group (e.g., the amine group in the compound of Formula (V) or (Va)) to form an amide group (e.g., the amide group in the compound of Formula (IV) or (IVa) or a salt thereof).

In some embodiments, the amide coupling reagent is a 2,4,6-trialkyl-l,3,5,2,4,6- trioxatriphosphorinane 2,4,6-trioxide, carbodiimide (e.g., A,A’-dicyclohexylcarbodiimide (DCC) or l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)), 1,1 ’-carbonyldiimidazole (CDI), a uronium, an activated ester, a phosphonium, 2-alkyl-l-alkylcarbonyl-l,2- dihydroquinoline, 2-alkoxy-l-alkoxycarbonyl-l,2-dihydroquinoline, or alkylchloroformate. In a specific embodiment, the amide coupling reagent is 2,4,6-trialkyl-l,3,5,2,4,6- trioxatriphosphorinane 2,4,6-trioxide. In a more specific embodiment, the amide coupling reagent is 2,4,6-tripropyl-l,3,5,2,4,6-trioxatriphosphorinane 2,4,6-trioxide (T3P).

The amide coupling reagent can be used in any forms including powder, crystals, liquid or solution. In a specific embodiment, the amide coupling reagent is a solution in a solvent. Any suitable solvents can be used to make a solution of an amide coupling reagent. In a more specific embodiment, the amide coupling reagent is a solution of T3P in ethyl acetate (EtOAc). The concentration of an amide coupling reagent in a solvent is in the range of 0-100 wt%, 20-100 wt%, 30-100 wt%, 50-100 wt%, or 60-100 wt%,. In a specific embodiment, the concentration of T3P in EtOAc is in the range of or 40-60 wt%.

Any suitable amount of the amide coupling reagent can be used in the reaction between the compound of Formula (I), (la) or a salt thereof (e.g., a TEA salt) and the compound of Formula (V) or (Va). In some embodiments, between 1.0 and 10.0 molar equivalents of the amide coupling reagent (e..g., T3P) relative to the amount of the compound of formula (V) or (Va) is used in the reaction. In a specific embodiment, 1.5-2.5, 1.0-3.0, 1.0-5.0, 1.0-6.0, 2.0-3.0, 2.0-4.0, 3.0-5.0, or 4.0-6.0 molar equivalent of T3P relative to the amount of the compound of formula (V) or (Va) is used. In a more specific embodiment, 5.0 equivalent of T3P is used.

In some embodiments, the reaction between the compound of Formula (I), (la) or a salt thereof (e.g., a TEA salt) and the compound of Formula (V) or (Va) is carried out in the presence of a base. Suitable bases include, but are not limited to, triethylamine, imidazole, A(.V-diisopropylethylamine, pyridine, 2,6-lutidine, dimethylformamide, 1,8- diazabicyclo[5.4.0]undec-7-ene (DBU), and tetramethylpiperidine. In a specific embodiment, the base is pyridine.

In a specific embodiment, the reaction between the compound of Formula (I), (la) or a salt thereof (e.g., a TEA salt) and the compound of Formula (V) or (Va) is carried out in the presence of 2,4,6-tripropyl-l,3,5,2,4,6-trioxatriphosphorinane 2,4,6-trioxide (T3P) as the amide coupling reagent and pyridine as the base.

The reaction between the compound of Formula (I), (la) or a salt thereof (e.g., a TEA salt) and the compound of Formula (V) or (Va) can be carried out in any suitable organic solvent(s). The suitable solvents include DMF, DMSO, THF, CH2CI2, acetonitrile, ethyl acetate, dichloroethane, dimethylacetamide, methanol, ethanol, and toluene. In a specific embodiment, the solvent is acetonitrile.

The reaction between the compound of Formula (I), (la) or a salt thereof (e.g., a TEA salt) and the compound of Formula (V) or (Va) can be carried out at a suitable temperature. In some embodiments, the reaction is carried out at a temperature between -50°C and 50°C, between -30°C and 30°C, between -25°C and 25°C, or between -20°C and 20°C. In a specific embodiment, the reaction is carried out between -25°C and 25°C.

In some embodiments, the reaction between the compound of Formula (I), (la) or a salt thereof (e.g., a TEA salt) and the compound of Formula (V) or (Va) is quenched with a mixture of an organic solvent and water. In other embodiments, said reaction is quenched with water. The organic solvent can be DMF, DMSO, THF, CH2CI2, acetonitrile, ethyl acetate, dichloroethane, dimethylacetamide, methanol, ethanol, or toluene. In a specific embodiment, the reaction is quenched with a mixture of acetonitrile and water. A volume ratio between 1:5 and 5:1, between 1:4 and 4:1, between 1:3 and 3:1, between 1:2 and 2:1, or a volume ratio of 1:1 of an organic solvent to water can be used for the quench. In a specific embodiment, the reaction is quenched with a mixture of acetonitrile and water at a volume ratio of 1:1. Said reaction is quenched at a temperature between -78 °C and 0 °C, between - 78 °C and -15 °C, between -78 °C and -20 °C, between -20 °C and -15 °C, between -20 °C and -10 °C, or at a temperature of -15 °C .

In some embodiments, the reaction between the compound of Formula (I), (la) or a salt thereof (e.g., a TEA salt) and the compound of Formula (V) or (Va) is carried out in the presence of N-hydroxy succinimide (NHS).In some embodiments, the present invention provides a method of preparing a compound of Formula (IV): or a salt thereof, with a deprotecting agent to form a compound of Formula (III): or a salt thereof; and

(b) reacting the compound of Formula (III) or a salt thereof with a compound of

Formula (A): to form a compound of Formula (I): or a salt thereof (e.g., a TEA salt); and

(c) reacting the compound of Formula (I) or a salt thereof (e.g., a TEA salt), with a compound of Formula (V): (V), to form the compound of Formula (IV) or a salt thereof, wherein DM is represented by the following formula:

In a specific embodiment, the present invention provides a method of preparing a compound of Formula (IVa): or a salt thereof, comprising the steps of:

(a) reacting a compound of Formula (Ila): or a salt thereof, with a deprotecting agent to form a compound of Formula (Illa): (Illa), or a salt thereof; and

(b) reacting the compound of Formula (Illa) or a salt thereof with the compound of Formula (A): to form a compound of Formula (la): or a salt thereof (e.g., a TEA salt); and

(c) reacting the compound of Formula (la) or a salt thereof (e.g., a TEA salt), with a compound of Formula (Va):

(Va), to form the compound of Formula (IVa) or a salt thereof, wherein DM _a is represented by the following formula:

In some embodiments, the reactions of steps (a)-(c) are carried out as the corresponding reactions described in the first and second embodiments and any additional embodiments described therein.

In a third embodiment, the present invention provides a method of preparing a compound of Formula (II): or a salt thereof, comprising the step of reacting a compound of Formula (VI): with a compound of Formula (B) to form the compound of Formula (II) or a salt thereof, wherein the compound of Formula (II) is purified by precipitation.

In a 3 ^rd specific embodiment, the compound of Formula (II) is represented by a compound of Formula (Ila): or a salt thereof, and the method comprises the step of reacting a compound of Formula (Via): with the compound of Formula (B) to form the compound of Formula (Ila) or a salt thereof, wherein the compound of Formula (Ila) is purified by precipitation.

Any suitable solvents can be used for said precipitation. The suitable solvents include DMF, DMSO, THF, CH2CI2, acetonitrile, dichloroethane, dimethylacetamide, methanol, ethanol, toluene, and tert-butyl methyl ether. In a specific embodiment, the precipitation is carried out in CH2CI2 and tert-butyl methyl ether. In some embodiments, the precipitation is carried out by mixing tert-butyl methyl ether with a solution of the compound of Formula (II) or (Ila) or a salt thereof in CH2CI2.

In some embodiments, the reaction between the compound of Formula (VI) or (IV a) and the compound of Formula (B) is carried out in the presence of an acid. Any suitable acids can be used for said reaction, which include but are not limited to, trifluoroacetic acid (TFA), formic acid, acetic acid, hydrochloric acid, phosphoric acid, pyridinium p- toluenesulfonate (PPTS), p-toluenesulfonic acid, methanesulfonic acid, camphorsulfonic acid, and sulfuric acid. In a specific embodiment, the acid is trifluoroacetic acid (TFA).

In a fourth embodiment, the method of the present invention comprises the steps of: (a) reacting the compound of Formula (II) prepared by the method of the third embodiment with a compound of Formula (V): (V), to form a compound of Formula (VII): or a salt thereof;

(b) reacting the compound of Formula (VII) or a salt thereof with a deprotecting agent to form a compound of Formula (VIII): or a salt thereof; and

(c) reacting the compound of Formula (VIII) or a salt thereof with a compound of

Formula (A): to form a compound of Formula (IV): or a salt thereof, wherein DM is represented by the following formula:

In a 4 ^th specific embodiment, the method of present invention comprises the steps of:

(a) reacting the compound of Formula (Ila) prepared by the method of the 3 ^rd specific embodiment with a compound of Formula (Va):

(Va), to form a compound of Formula (Vila): (Vila), or a salt thereof;

(b) reacting the compound of Formula (Vila) or a salt thereof with a deprotecting agent to form a compound of Formula (Villa): (Villa), or a salt thereof; and

(c) reacting the compound of Formula (Villa) or a salt thereof with a compound of Formula (A): to form a compound of Formula (IVa): or a salt thereof, wherein DM _a is represented by the following formula:

In some embodiments, the reaction between the compound of Formula (II) or (Ila) and the compound of Formula (V) or (Va) in step (a) is carried out in the presence of an amide coupling reagent.

In some embodiments, the amide coupling reagent is a 2,4,6-trialkyl-l,3,5,2,4,6- trioxatriphosphorinane 2,4,6-trioxide, carbodiimide (e.g., VV-dicyclohcxylcarbodiimidc (DCC) or l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)), 1,1 ’-carbonyldiimidazole (CDI), a uronium, an activated ester, a phosphonium, 2-alkyl-l-alkylcarbonyl-l,2- dihydroquinoline, 2-alkoxy-l-alkoxycarbonyl-l,2-dihydroquinoline, or alkylchloroformate. In a specific embodiment, the amide coupling reagent is l-ethyl-3-(3- dimethylaminopropyl)c arbodiimide (EDC ) . In some embodiments, the reaction between the compound of Formula (II) or (Ila) and the compound of Formula (V) or (Va) in step (a) is carried out in the presence of a base.

In some embodiments, the base is triethylamine, imidazole, N,N- diisopropylethylamine, pyridine, 2,6-lutidine, dimethylformamide, 1,8- diazabicyclo[5.4.0]undec-7-ene (DBU), or tetramethylpiperidine. In a specific embodiment, the base is V A-diisopropylcthylaminc.

In some embodiments, the compound of Formula (VII), (Vila) or a salt thereof in step (b) is reacted with a deprotecting agent that is a base. In some embodiments, the base is piperidine, 4-methylpiperidien, piperazine, l,8-Diazabicyclo[5.4.0]undec-7-ene (DBU), or morpholine. In a specific embodiment, the base is morpholine.

The reaction between the compound of Formula (VII), (Vila) or a salt thereof and the deprotecing agent in step (b) is carried out in a suitable solvent including DMF, DMSO, THF, CH2C12, acetonitrile, dichloroethane, dimethylacetamide, methanol, ethanol, or toluene. In a specific embodiment, the solvent is DMF.

In some embodiments, the reaction between the compound of Formula (VIII), (Villa) or a salt thereof and the compound of Formula (A) in step (c) is carried out in a solvent selected from DMF, DMSO, THF, CH2CI2, acetonitrile, dichloroethane, dimethylacetamide, methanol, ethanol, and toluene. In a specific embodiment, the solvent is DMF.

In a fifth embodiment, the present invention provides a method of preparing a compound of Formula (I): or a salt thereof, comprising the steps of:

(a) reacting the compound of Formula (II) prepared by the method of the third embodiment with a deprotecting agent to form a compound of Formula (III): or a salt thereof; and

(b) reacting the compound of Formula (III) or a salt thereof with a compound of Formula (A): to form the compound of Formula (I) or a salt thereof.

In a 5 ^th specific embodiment, the present invention provides a method of preparing a compound of Formula (la): or a salt thereof, comprising the steps of:

(a) reacting the compound of Formula (Ila) prepared by the method of the 3 ^rd specific embodiment with a deprotecting agent to form a compound of Formula (Illa): or a salt thereof; and

(b) reacting the compound of Formula (Illa) or a salt thereof with the compound of Formula (A): to form the compound of Formula (la) or a salt thereof.

In a sixth embodiment, the present invention provides a method of preparing a compound of Formula (IV): or a salt thereof, comprising the steps of:

(a) reacting the compound of Formula (II) prepared by the method of the third embodiment with a deprotecting agent to form a compound of Formula (III): or a salt thereof; and (b) reacting the compound of Formula (III) or a salt thereof with a compound of Formula (A): to form a compound of Formula (I): or a salt thereof (e.g., a TEA salt); and

(c) reacting the compound of Formula (I) or a salt thereof (e.g., a TEA salt), with a compound of Formula (V): (V), to form the compound of Formula (IV) or a salt thereof, wherein DM is represented by the following formula:

In a 6 ^th specific embodiment, the present invention provides a method of preparing a compound of Formula (IVa): or a salt thereof, comprising the steps of: (a) reacting the compound of Formula (Ila) prepared by the method of the 3 ^rd specific embodiment with a deprotecting agent to form a compound of Formula (Illa): or a salt thereof; and

(b) reacting the compound of Formula (Illa) or a salt thereof with the compound of Formula (A): to form a compound of Formula (la): or a salt thereof (e.g., a TEA salt); and

(c) reacting the compound of Formula (la) or a salt thereof (e.g., a TEA salt), with a compound of Formula (Va):

(Va), to form the compound of Formula (IVa) or a salt thereof, wherein DM _a is represented by the following formula:

All references cited herein and in the examples that follow are expressly incorporated by reference in their entireties.

EXAMPLES

The following solvents, reagents, protecting groups, moieties and other designations may be referred to by their abbreviations in parenthesis:

DCM dichloromethane

DMF N, A-di methyl formamide

EtOAc ethyl acetate eq molar equivalents

HPLC high performance liquid chromatography

IP Ac isopropyl acetate

IPC in-process control h Hour

MCNE or= EMCS 6-Maleimidocaproic acid N-succinimidyl ester

MeCN acetonitrile

MP mobile phase

MTBE tert-butyl methyl ether

NMT not more than

LDPE low density polyethylene

NaHCCE sodium bicarbonate

Na2SO4 sodium sulfate r.t. Room temperature

TFA trifluoroacetic acid

THF tetrahydrofuran

UPLC ultra performance liquid chromatography vol volumes wt weight

°C degree Celsius Example 1. Synthesis of (5S,8R,llS)-l-(9H-fhioren-9-yl)-5,8,ll-trimethyl-3,6,9,12- tetraoxo-2-oxa-15-thia-4,7,10,13-tetraazahenicosan-21-oic add (Compound Ila)

To a reaction vessel containing (5S,8R,l lS)-l-(9H-fluoren-9-yl)-5,8,l l-trimethyl- 3,6,9,12-tetraoxo-2-oxa-4,7,10,13-tetraazatetradecan-14-yl acetate (Compound Via, 1.0 eq, 1 wt) under nitrogen was added a solution of 6-mercaptohexanoic acid (Compound B, 1.25 eq) and trifluoroacetic acid (1 vol) in CH2CI2 (8.5 vol) at a temperature of 20 ± 5 °C. The reaction continued to stir for 1-3 hours and was concentrated under vacuum to remove the solvent. CH2CI2 (12 vol) was added and it was concentrated again to give a golden yellow residue that was re-dissolved in CH2CI2 (3.0 vol). The resulting solution was added dropwise to tert-butyl methyl ether (MTBE, 16 vol) at 20 °C ± 5 °C under nitrogen and then cooled to 5 °C ± 3 °C. Let it stir for 60 minutes at the same temperature and then stored for 18 hours at 20 °C ± 5 °C under nitrogen. The solid was filtered, washed with MTBE (2 x 1.5 vol) and dried in vacuum oven for more than 12 hours at 25 ± 5 °C to afford Compound Ila as a white to off-white solid (yield, quantitative). The largest scale run using this process was 50 g with a second run using 43 g Compound Via. Compound Ila was taken directly into the next step without further purification. ¹ H NMR (400 MHz, d6-DMSO) 6 8.37 (t, J = 6.3 Hz, 1H), 8.12 (d, J = 7.2 Hz, 1H), 7.95 (d, J = 7.6 Hz, 1H), 7.88 (d, J = 7.6 Hz, 2H), 7.70 (t, J = 8.4 Hz, 2H), 7.55 (d, J = 7.0 Hz, 1H), 7.40 (t, J = 7.4 Hz, 2H), 7.32 (t, J = 7.5 Hz, 2H), 4.40 - 4.24 (m, 3H), 4.23 - 4.11 (m, 5H), 4.04 (p, J = 7.2 Hz, 2H), 2.47 (s, 1H), 2.16 (t, J = 7.3 Hz, 2H), 1.48 (ddt, J = 15.0, 12.1, 7.4 Hz, 5H), 1.28 (qd, J = 7.5, 6.6, 4.4 Hz, 3H), 1.22 - 1.14 (m, 10H). ¹³ C NMR (101 MHz, d6-DMSO) 8 174.88, 172.90, 172.51, 172.24, 156.28, 144.32, 141.20, 128.12, 127.56, 125.72, 120.58, 66.14, 50.68, 48.81, 47.13, 34.07, 30.26, 29.37, 28.31, 24.57, 18.47, 18.39. MS (ESI+): m/z = 613.83. Example 2. Synthesis of (2S,5R,8S)-2-amino-5,8-dimethyl-3,6,9-trioxo-12-thia-4,7,10- triazaoctadecan- 18-oic acid (Compound Illa)

A solution of Compound Ila (1 eq. 1 wt) and morpholine (2.0 eq) in DMF (5 vol) stirred for 20 h at 20 ± 5 °C under nitrogen. The reaction mixture was filtered to remove the solid and the filter cake was washed with DMF (0.5 vol). The combined filtrates were added slowly over 5 h to a vessel containing CH2CI2 (24 vol) and seed crystals (0.02 wt%) under nitrogen. The resulting slurry stirred for another 1 h at 20 ± 5 °C, filtered under vacuum and washed with CH2CI2 (3 vol). The product was transferred to an oven equipped with slow nitrogen bleed and dried at NMT than 28 °C until a constant weight is maintained to afford Compound Illa as amorphous or semi-amorphous material. Amorphous Compound Illa is preferred for the next step of reaction because of higher solubility in MeCN. The largest scale run using this process was 30 g with a second run using another 30 g Compound Ila.

Crystallization of Compound Illa

Amorphous Compound Illa (1.0 eq, 1 wt) was dissolved in MeCN (30 vol) and heated to 50 ± 3 °C under nitrogen. The suspension was cooled to 20 ± 3 °C over 20 minutes and continued to stir for 1 h. The solid was filtered and washed with MeCN (2 x 5 vol) and dried to provide Compound Illa (yield, 80.7%) as a highly crystalline white solid. The largest scale run using this crystallization method is 30 g amorphous of Compound Illa.

Produce Amorphous Compound Illa from Crystalline

Crystalline Compound Illa (1.0 eq, 1 wt) was dissolved in DMF (8 vol) and heated to 50 ± 3 °C until all solids dissolved. The solution was cooled to 20 ± 3 °C and added dropwise to CH2Ch (40 vol) over 15 minutes. The resulting white suspension is stirred overnight at 20 ± 3 °C and filtered. The solid was washed with CH2CI2 (3 vol) and dried to provide compound Illa (yield, 97.7%) as an amorphous solid. The largest scale run using this method is 5 g amorphous of Compound Illa. ’ H NMR (400 MHz, d6-DMSO) 6 8.48 (t, J = 6.3 Hz, 1H), 8.30 (d, J = 7.8 Hz, 1H), 8.22 (d, J = 4.0 Hz, 1H), 4.58 (s, 4H), 4.28 - 4.16 (m, 4H), 3.35 (t, J = 6.9 Hz, 1H), 2.52 (d, J = 7.3 Hz, 3H), 2.14 (t, J = 7.2 Hz, 2H), 1.57 - 1.41 (m, 4H), 1.36 - 1.25 (m, 4H), 1.23 (d, J = 2.7 Hz, 6H), 1.19 (d, J = 7.1 Hz, 7H), 1.13 (d, J = 7.0 Hz, 3H), 0.89 - 0.80 (m, 5H). ¹³ C NMR (101 MHz, d6-DMSO) 8 175.40, 175.22, 172.56, 172.40, 50.30, 48.72, 48.62, 34.66, 31.73, 30.33, 29.41, 28.84, 28.36, 24.66, 22.57, 21.32, 18.90, 18.48, 14.42. MS (ESI+): m/z = 390.99.

Example 3. Synthesis of (HS,14R,l’7S)-24-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)- 11,14,17-trimethyl- 10,13,16,19-tetraoxo-7-thia-9,12,15,18-tetraazatetracosanoic add (Compound la)

To a solution of Compound Illa (1.0 eq) in MeCN (40 vol) was added TEA (3.0 eq) and 6-maleimidocaproic acid N-succinimidyl ester (MCNE or Compound A, 1.05 eq) at 20 ± 5 °C. The reaction continued to stir at this temperature until IPC showed complete. MTBE (40 vol) was added and the resulting slurry stirred for 1 h before cooled via ramp to -18 ± 2 °C over 1 h. Let the slurry hold for an additional 1-16 and continue to cool to -30 °C over 1 h and hold for 1 h. The solid was filtered, washed with MTBE (10 vol) and dried to afford Compound la (yield, 79.5%) as the triethylamine salt (1:1). The largest scale run using this method is 4 g Compound Illa. ¹ H NMR (400 MHz, d6-DMSO) 8 8.35 (t, J = 6.3 Hz, 1H), 8.14 (d, J = 7.3 Hz, 1H), 8.01 (dd, J = 13.1, 7.0 Hz, 2H), 6.98 (s, 2H), 4.27 - 4.11 (m, 6H), 3.36 (t, J = 7.1 Hz, 2H), 2.61 - 2.53 (m, 2H), 2.53 - 2.46 (m, 4H), 2.17 (t, J = 7.3 Hz, 2H), 2.08 (t, J = 7.2 Hz, 2H), 1.57 - 1.40 (m, 9H), 1.36 - 1.22 (m, 3H), 1.22 - 1.09 (m, 12H), 0.97 (t, J = 7.2 Hz, 2H). ¹³ C NMR (101 MHz, d6-DMSO) 8 174.55, 172.42, 172.19, 172.09, 171.79, 171.04, 134.43, 48.61, 48.40, 48.26, 45.58, 36.96, 34.73, 33.77, 29.76, 28.90, 27.86, 27.76, 25.75, 25.21, 24.61, 24.13, 17.84, 17.65, 10.95. MS (ESI+): m/z = 584.3. Example 4. Synthesis of maytansinoid derivative with self-immolative peptide linkers (Compound IVa)

To a solution of L-MayNMA (Compound Va, 1.0 eq, 1 wt) in MeCN (5 vol) was added Compound la (1.5 eq based on non-TEA salt MW 567.70) and pyridine (5.0 eq). The reaction was cooled to -20 ± 5 °C and added T3P in ethyl acetate (5.0 eq). After warmed up to 20 °C ± 5 °C, the reaction continued to stir until all the solids were dissolved and solution formed and then was quenched with 1:1 MeCN/thO (2 vol) after IPC showed complete. Once the quench was over, the crude mixture was loaded onto Teledyne Isco Combiflash companion with C18 30 micron cartridge (450 g) for purification. Elution was conducted with CO2 saturated HPLC grade water with a linear gradient of HPLC grade MeCN from 5% to 35%. The combined fractions were extracted twice with IP Ac (200 vol and 50 vol) to afford Compound IVa (yield, 79%). The largest scale run using this method is 1 g Compound la. ’H NMR (400 MHz, d6-DMSO) 8 8.11 (t, J = 6.3 Hz, 1H), 7.92 (d, J = 7.3 Hz, 1H), 7.81 (d, 7 = 6.5 Hz, 1H), 7.76 (d, 7 = 7.5 Hz, 1H), 6.97 (d, 7 = 1.6 Hz, 1H), 6.78 (s, 3H), 6.68 (s, 1H), 6.45 - 6.29 (m, 3H), 5.71 (s, 1H), 5.36 (dd, 7 = 14.7, 9.0 Hz, 1H), 5.13 (q, 7 = 6.8 Hz, 1H), 4.31 (dd, 7 = 12.0, 3.0 Hz, 1H), 4.04 - 3.81 (m, 5H), 3.72 (s, 3H), 3.25 (dd, 7 = 21.5, 10.6 Hz, 2H), 3.19 - 3.03 (m, 7H), 3.00 (d, 7 = 12.5 Hz, 1H), 2.88 (s, 3H), 2.59 (d, 7 = 9.7 Hz, 1H), 2.49 (s, 5H), 2.19 (dq, 7 = 13.7, 7.2 Hz, 2H), 2.01 - 1.90 (m, 2H), 1.93 - 1.79 (m, 3H), 1.73 (d, 7 = 16.5 Hz, 1H), 1.69 (d, 7 = 7.9 Hz, 1H), 1.39 (s, 3H), 1.24 (dtd, 7 = 11.2, 7.2, 3.2 Hz, 5H), 1.06 (q, 7 = 8.2, 7.7 Hz, 2H), 0.98 (d, 7 = 7.0 Hz, 9H), 0.97 - 0.88 (m, 5H), 0.57 (s, 3H). ¹³ C NMR (101 MHz, d6-DMSO) 8 172.91, 172.67, 172.53, 172.32, 172.25, 171.52, 171.23, 168.65, 155.77, 151.71, 141.82, 141.62, 138.74, 134.91, 133.07, 129.01, 125.73, 122.15, 117.65, 114.40, 88.67, 80.47, 78.12, 73.65, 67.30, 60.51, 57.03, 56.59, 51.94, 49.09, 48.96, 48.86, 48.72, 45.85, 38.21, 37.44, 36.82, 35.74, 35.20, 32.99, 32.43, 30.58, 30.20, 30.12, 29.48, 29.38, 28.34, 28.25, 26.23, 25.08, 24.45, 18.29, 18.12, 17.70, 15.49, 14.90, 13.54, 11.83. MS (ESI+): m/z = 1215.40.