Description:FIELD OF THE INVENTION
[0001] The present invention generally relates to organic compounds. Specifically, the present invention relates to a process for the preparation of tubulysins and its derivatives of formula (I) or its stereoisomer or a pharmaceutically acceptable salt thereof and pharmaceutical composition containing them. More particularly, the present invention relates to a new, rapid, gram scale, cost effective synthesis of tubulysins and its derivatives of formula (I) or a pharmaceutically acceptable salt thereof and use of the tubulysins and its derivatives of formula (I) or a pharmaceutically acceptable salt thereof as anticancer agents.

BACKGROUND OF THE INVENTION
[0002] Background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
[0003] Tubulysins are naturally occurring antimitotic tetra peptides with potent anticancer activity against multidrug-resistant (MDR) cancer cells, acting by inhibition of tubulin polymerization. They were first reported in 2000, when they were identified through a screen of the culture broths from myxobacteria Archangium gephyra and Angiococcus disciformis against mammalian cell lines. Structurally, tubulysins are tetra peptides, composed of D-methylpipecolate (D-Mep), L-isoleucine (L-Ile), L-tubuvaline (L-Tuv), and L-tubuphenylalanine (L-Tup) residues. Later on, additional members of the family were described (Angew Chem, 2004, 4888). Most naturally occurring tubulysins showed up to pM cytotoxic activity against cancer cell lines that correlated with tubulin polymerization inhibition. The mechanism of action was described by Sasse (Chem bioChem 2006, 678). Furthermore, Tubulysin A, B, D and E processed potent antiproliferative activity against L929 mouse fibroblasts, ptK2 kidney cancer cells, and KN-3-1 cervix cancer cells (IC50 20 pg/mL to 1 ng/mL) (Sasse et al. J. Antibiot. 2000, 53, 879-885; WO 98/13375). Additionally, all retained their high potency against the MDR cervical cancer cell line KB-VI (IC50 80 pg/mL to 1 ng/mL).

Tubulysin A: R′=CH2CH(CH3)2; R″=OH
Tubulysin B: R′=CH2CH2CH3; R″=OH
Tubulysin C: R′=CH2CH3; R″=OH
Tubulysin D: R′=CH2CH(CH3)2; R″=H
Tubulysin E: R′=CH2CH2CH3; R″=H
Tubulysin F: R′=CH2CH3; R″=H
[0004] Strikingly, tubulysins are 20-fold to 1000-fold more potent than the epothilones, vinblastine, and taxol as cell growth inhibitors and thus they were promising lead compounds for the development of new anticancer drugs (Chem Biol Drug Des., 2007, 70, 75). Recently, tubulysins as payloads, folic acid conjugates, or antibody drug conjugates (ADCs) showed high clinical promise (Sci. Rep., 2018, 8, 8943, Can Res., 2014, 74, 5700).
[0005] To develop new ADCs, tubulysins have been emerged as a promising class of molecules for use as payloads. The variability in structure of the tubulysins is mostly observed in the Tuv portion of the molecule. The first series (A-I) contains an unusual and synthetically challenging N,O-acetal group, as well as an acetylated hydroxyl group. A second series (U-Z) is missing the N,O-acetal group and/or the acetyl group, and is less active. On the basis of their complex structure, limited availability and potential as anticancer agents, significant efforts have been undertaken on the synthesis of tubulysins, including total syntheses of Tubulysin B17, D18, U19 and V20. Work has also been completed on evaluating the key structural features that impact the tubulysins with their activity, looking to deliver structurally simplified versions of these molecules. Of particular interest in simplified analogues have been pretubulysins 22 and N 14 - Desacetoxytubulysin H23 (1), both of which replace the Tuv N,O-acetal group with a simple N-methyl group without significant loss of activity.
[0006] The Multi Component Reactions (MCRs) combine at least three reactants in one pot to generate a product containing most (preferably all) atoms of the starting materials. Intrinsically, MCRs feature high convergence and step economy which has a great positive impact on the resource side (namely the starting materials utilization and time) of a chemical synthesis route. MCRs bring additional advantages in terms of mildness of reaction conditions and compatibility with green chemistry principles (e.g. atom economy, waste prevention, benign solvents, less hazardous synthesis) which improve the sustainability side of the process. All these justify a central position of multicomponent reactions in the toolbox of modern synthetic organic methodologies in contrast with conventional multi step sequential synthesis.
[0007] The large-scale fermentation of tubulysins is still a poorly solved challenge which is overcome by chemical synthesis. Several laboratories around the world have disclosed the total synthesis of tubulysins. All the classical total synthesis of the non-ribosomal peptide structure of Tubulysins involves multistep approach and proceeds through sequential coupling of four amino acid fragments such as D-N-methyl pipecolic acid (Mep), isoleucine (Ile), tubuvaline (Tuv) and the tubuphenylalanine (Tup) as logical precursors. The common problem of classical tubulysins synthesis is the high step count which obviously gives low yields of the product. Firstly, the syntheses of Tuv and Tup were only accomplished through extensive functional group manipulations and chiral separations. Secondly, the sequential difficult coupling of sterically hindered Mep, Ile and Tuv amino acids was challenging. Even though many short syntheses have been developed for the synthesis of Tuv and Tup, the concept of sustainable diversity-oriented synthesis is still limited.
[0008] Therefore, there is an unmet need to provide an efficient synthesis of tubulsyins, to develop tubulysins which can be used as anticancer agents that disrupts microtubule dynamics.

OBJECTIVES OF THE INVENTION
[0009] An object of the present invention is to provide a process for the preparation of tubulysins and its derivatives of formula (I) or its stereoisomer or a pharmaceutically acceptable salt thereof and pharmaceutical composition containing them.
[0010] Another object of the present invention is to provide a new, rapid, gram scale, cost effective synthesis of tubulysins and its derivatives of formula (I) or a pharmaceutically acceptable salt thereof.
[0011] Another object of the present invention is to provide a tubulysins and its derivatives of formula (I) or a pharmaceutically acceptable salt thereof as anticancer agents.

SUMMARY OF THE INVENTION
[0012] The present invention generally relates to organic compounds. Specifically, the present invention relates to a process for the preparation of tubulysins and its derivatives of formula (I) or its stereoisomer or a pharmaceutically acceptable salt thereof and pharmaceutical composition containing them. More particularly, the present invention relates to a new, rapid, gram scale, cost effective synthesis of tubulysins and its derivatives of formula (I) or a pharmaceutically acceptable salt thereof and use of the tubulysins and its derivatives of formula (I) or a pharmaceutically acceptable salt thereof as anticancer agents.
[0013] In one aspect, the present invention relates to a tubulysins compound and its derivatives of formula (I) or a stereoisomer, a tautomer, a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof.

(I)
wherein
n is 0, 1, 2 or 3;
R1 is CH3 or H; R2 is CH3 or H; R3 is CH3, CH2CH3, alkyl, aryl or optionally substituted bifunctional alkyl groups; R4 is esters, ethers, or amides; X is H, OH or NH2, NH-Y (Y = any alkyl or acetyl)
[0014] In another aspect, the present invention relates to a process for preparation of a tubulysins compound and its derivatives of formula (I) or a stereoisomer, a tautomer, a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof, wherein said process comprises the steps of:
a) reacting a compound A, a compound B and a compound C in a solvent in a Passerini reaction to obtain a compound D;

b) subjecting the compound D to cyclodehydration reaction by incubating compound in the presence of TiCl4, followed by oxidation in the presence of MnO2 to obtain a compound F;

c) subjecting the compound F to acyl migration reaction by exposure to diethylamine (DEA), followed by exposure to trimethylamine (TEA) to obtain a compound G;

d) oxidizing a hydroxyl group of the compound G using Dess-Martin Periodinane at room temperature to obtain compound H;

e) alkylating secondary amide of the compound H using alkyl halides in presence of strong base at low temperature to obtain a compound I;

f) reducing ketone of the compound I diastereoselectively using chiral catalyst to obtain a compound J;

g) hydrolyzing the ester of the compound J to obtain a carboxylic acid compound K;

h) reacting carboxylic acid of the compound K with a tubuphenylalanine compound to obtain a compound M;

i) coupling of the compound M and a compound N, followed by protecting group cleavage, reductive amination and further hydrolysis to obtain a compound P; and

j) acylating hydroxyl group of the compound P to obtain a compound of Formula (I).

(I)
wherein
R10 is acetyl, acyl (substituted) alkyl, acyl cycloalkyl, or acyl benzyl, preferably acetyl or acyl derivative of methyl, ethyl, tert-butyl or benzyl.
[0015] In another aspect of the present invention, the compound A is a carboxylic acid having formula:

A
wherein,
R1 is a substituted or unsubstituted alkyl; a substituted or unsubstituted cycloalkyl; or a substituted or unsubstituted benzyl,
R2 is H, a substituted or unsubstituted alkyl, or a substituted or unsubstituted cycloalkyl.
[0016] In another aspect of the present invention, the compound B is an aldehyde having formula:

B
wherein,
R3, R4 each independently is H, F, a substituted or unsubstituted alkyl, a substituted or unsubstituted cycloalkyl or a substituted or unsubstituted benzyl;
Pg1 is an amine protecting group, a carbamate, a substituted or an unsubstituted benzyl.
[0017] In another aspect of the present invention, compound C is an isocyanide having formula:

C
wherein,
R5 is a substituted or unsubstituted alkyl, a substituted or unsubstituted cycloalkyl, or a substituted or unsubstituted benzyl;
X is O, S, Se, or -NH-; and
Pg2 is an X- protecting group selected from trityl, tert-butyl, adamantyl and substituted benzyl, more preferably trityl or tert-butyl.
[0018] In another aspect of the present invention, the solvent in step (a) is selected from a group consisting of CH2Cl2, CHCl3, CCl4, benzene, THF, CH3CN, 1,4-dioxane, or 1,2-dichloroethane, or a combination thereof.
[0019] In another aspect of the present invention, the compound A, compound B and compound C are present in a ratio in a range of 0.8-1.2: 0.8-1.2: 0.8-1.2.
[0020] In another aspect of the present invention, the activated MnO2 in step (b) has a pore size of ≤ 5 microns.
[0021] In another aspect of the present invention, the compound I obtained in step (e), wherein R6 is optionally, substituted alkyl, aryl or optionally substituted bifunctional alkyl groups.
[0022] In another aspect of the present invention, the strong base in step (e) is selected from KHMDS, NAHMDS and NaH.
[0023] In another aspect of the present invention, the temperature in step (e) is in the range of -70℃ to -80℃.
[0024] In another aspect of the present invention, the chiral catalyst in step (f) is selected from (S)-CBS catalyst and BH3.DMS.
[0025] In another aspect of the present invention, the compound N having formula:

wherein R8 and R9 is isopropyl, tert-butyl, iso-butyl, sec-butyl, cyclopropylmethyl or cyclobutyl methyl; R9 is H;
n is 0, 1, 2, 3, 4 or 5; and
Pg moiety is an amine protecting group, a carbamate, a substituted or an unsubstituted benzyl.
[0026] In another aspect, the present invention relates to a pharmaceutical composition comprising of a tubulysins compound and its derivatives of formula (I) or a stereoisomer, a tautomer, a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof.
[0027] In an aspect of the present invention, the pharmaceutical composition of a tubulysins compound and its derivatives of formula (I) is in the form of a tablet, a capsule, a solution, a gel, a suspension or a powder.
[0028] In another aspect, the present invention relates to a tubulysins compound and its derivatives of formula (I) or a stereoisomer, a tautomer, a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof for use in the treatment of cancer.
[0029] Various objects, features, aspects and advantages of the inventive subject matter will become more apparent from the following detailed description of preferred embodiments.

DETAILED DESCRIPTION OF THE INVENTION
[0030] The following is a detailed description of embodiments of the disclosure. The embodiments are in such detail as to clearly communicate the disclosure. However, the amount of detail offered is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure as defined by the appended claims.
[0031] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.
[0032] Reference throughout this specification to “one embodiment” or “an embodiment” or “another embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.
[0033] In some embodiments, the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term “about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.
[0034] As used in the description herein and throughout the claims that follow, the meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise.
[0035] Unless the context requires otherwise, throughout the specification which follow, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open, inclusive sense that is as “including, but not limited to.”
[0036] Also, use of "(s)" as part of a term, includes reference to the term singly or in plurality, for example, the term pharmaceutically acceptable salt(s) indicates a single salt or more than one salt of the compound of formula (I).
[0037] The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual 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 with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.
[0038] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description that follows, and the embodiments described herein, is provided by way of illustration of an example, or examples, of particular embodiments of the principles and aspects of the present disclosure. These examples are provided for the purposes of explanation, and not of limitation, of those principles and of the disclosure.
[0039] It should also be appreciated that the present disclosure can be implementedin numerous ways, including as a system, a method or a device. In this specification, these implementations, or any other form that the invention may take, may be referred to as processes. In general, the order of the steps of the disclosed processes may be altered within the scope of the invention.
[0040] The headings and abstract of the invention provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.
[0041] The following discussion provides many example embodiments of the inventive subject matter. Although each embodiment represents a single combination of inventive elements, the inventive subject matter is considered to include all possible combinations of the disclosed elements. Thus if one embodiment comprises elements A, B, and C, and a second embodiment comprises elements B and D, then the inventive subject matter is also considered to include other remaining combinations of A, B, C, or D, even if not explicitly disclosed.
[0042] Unless otherwise indicated, the following definitions are set forth to illustrate and define the meaning and scope of the various terms used to describe the invention herein and the appended claims. These definitions should not be interpreted in the literal sense as they are not intended to be general definitions and are relevant only for this application.
[0043] The term "or", as used herein, is generally employed in its sense including "and/or" unless the content clearly dictates otherwise.
[0044] The term, "therapeutically effective amount" as used herein refers to an amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof or a composition comprising a compound of formula (I) or a salt thereof, effective in producing the desired therapeutic response in a particular patient (subject) suffering from a disease or disorder.
[0045] The term "pharmaceutically acceptable excipient(s)" as used herein refers to a diluent, binder, disintegrant, glidant, lubricant, coating material or the like, which is non-toxic, and inert, which does not have undesirable effects on a subject to whom it is administered and is suitable for delivering a therapeutically active agent to the target site without affecting the therapeutic activity of the said agent.
[0046] The term, "subject" as used herein refers to an animal, preferably a mammal, and most preferably a human. The term "mammal" used herein refers to warm-blooded vertebrate animals of the class 'mammalia' , including humans, characterized by a covering of hair on the skin and, in the female, milk-producing mammary glands for nourishing the young, the term mammal includes animals such as cat, dog, rabbit, bear, fox, wolf, monkey, deer, mouse, pig and human.
[0047] The terms, “treatment", "treat" and "therapy" and the like as used herein refer to alleviate, slow the progression, attenuation, prophylaxis or as such treat the existing diseases or condition (e.g. bacterial infection or fungal infection). Treatment also includes treating, preventing development of, or alleviating to some extent, one or more of the symptoms of the diseases or condition.
[0048] In a general embodiment, the present invention relates to a process for the preparation of tubulysins and its derivatives of formula (I) or its stereoisomer or a pharmaceutically acceptable salt thereof and pharmaceutical composition containing them. Also, discloses the intermediates useful for preparing tubulysins and its derivatives of formula (I). More particularly, the present invention relates to a new, rapid, gram scale, cost effective synthesis of tubulysins and its derivatives of formula (I) or a pharmaceutically acceptable salt thereof and use of the tubulysins and its derivatives of formula (I) or a pharmaceutically acceptable salt thereof as anticancer agents.
[0049] In one embodiment, the present invention relates to a tubulysins compound and its derivatives of formula (I) or a stereoisomer, a tautomer, a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof.

(I)
wherein
n is 0, 1, 2 or 3;
R1 is CH3 or H; R2 is CH3 or H; R3 is CH3, CH2CH3, alkyl, aryl or optionally substituted bifunctional alkyl groups; R4 is esters, ethers, or amides; X is H, OH or NH2, NH-Y (Y = any alkyl or acetyl).
[0050] In another embodiment, the present invention relates to a tubulysins compound and its derivatives of formula (I) or a stereoisomer, a tautomer, a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof.

(I)
wherein
n is 3, and R1, R2 is H, R3 is methyl, R4 is acetyl, and X is H.
[0051] In an embodiment of the present invention, the present inventors develop a new synthetic approach towards potent novel tubulysin derivatives which is short (in numbers of steps), cheap, allows for full stereoisomer control and has a high yield, preferably at a gram scale. The present invention provides a highly efficient synthesis route (overall yield nearly 28%) based on only four building blocks, three of which (compounds A, B and C) are reacted in a multicomponent Passerini reaction. The tubulysin derivatives thus obtained are super potent and can be attached towards biological matter (e.g. mAbs) through at least 3 different linker positions. Furthermore, the synthetic method offers full stereo-control. Thus, the process helps to fine-tune the properties of tubulysin ADCs in a much better way than offered by current synthetic procedures.
[0052] In another embodiment, the present invention provides a method for preparing a tubulysins compound and its derivatives of formula (I), comprising reacting compounds A, B and C in a 3-component Passerini reaction,
wherein compound A is a carboxylic acid according to the general formula:

wherein
R1 is a substituted or unsubstituted alkyl; a substituted or unsubstituted cycloalkyl; or a substituted or unsubstituted benzyl,
R2 is H, a substituted or unsubstituted alkyl, or a substituted or unsubstituted cycloalkyl;
wherein compound B is an aldehyde according to the general formula:

wherein
R3 and R4, each independently represent H, F, a substituted or unsubstituted alkyl, a substituted or unsubstituted cycloalkyl or a substituted or unsubstituted benzyl;
Pg1 is an amine protecting group, preferably a carbamate, a substituted or an unsubstituted benzyl; and
wherein compound C is an isocyanide according to the general formula:

wherein
R5 represents a substituted or unsubstituted alkyl, a substituted or unsubstituted cycloalkyl, or a substituted or unsubstituted benzyl;
X represents O, S, Se, or -NH-, preferably S; and
Pg2 represents an X- protecting group, preferably selected from trityl, tert-butyl, adamantyl and substituted benzyl, more preferably trityl or tert-butyl.
[0053] In another embodiment of the present invention, R1 is isopropyl, tert-butyl, iso-butyl, sec-butyl, cyclopropylmethyl or cyclobutyl methyl; R2 is H; R3 is isopropyl, tert-butyl, iso-butyl, sec-butyl, cyclopropylmethyl or cyclobutyl methyl; R4 is H; R5 is selected from the group consisting of methyl, ethyl, propyl, butyl, isopropyl, cyclopropyl and cyclopropylmethyl.
[0054] In another embodiment, a moiety may be substituted, such as by use of “unsubstituted or substituted” or “optionally substituted” phrasing as in “unsubstituted or substituted alkyl” or “optionally substituted benzyl,” such moiety may have one or more independently selected substituents, preferably one to five in number, more preferably one or two in number. Substituents and substitution patterns can be selected by one of ordinary skill in the art, having regard for the moiety to which the substituent is attached, to provide compounds that are chemically stable and that can be synthesized by techniques known in the art as well as the methods set forth herein.
[0055] In another embodiment, the present invention relates to a process for preparation of a tubulysins compound and its derivatives of formula (I) or a stereoisomer, a tautomer, a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof, wherein said process comprises the steps of:
a) Compound A, compound B and compound C can be reacted in any suitable solvent or solvent mixture. The reaction is performed at room temperature. The Passerini reaction is preferably carried out from 1 mmol to 20 mmol scale. After completion of the Passerini reaction, the solvent(s) may evaporate under reduced pressure. The reaction products are suitably purified through flash chromatography (e.g. cyclohexane/EtOAc 20:80 v/v). The by-product can also be recovered and coupled with dipeptide acid to form an ester or compound D can be used for the next step without further purification. Finally, isolating the 3-component α-acyloxy amide compound D.

b) Subjecting the Passerini reaction product of compound D to cyclodehydration of Cys-amide to afford compound E. Compound D (wherein X = S) is treated with 1M TiCl4 in CH2Cl2 at 0◦C in CH2Cl2 solvent. The reaction time ranges from about 6 to 12 h. After completion of the reaction, the reaction mixture is quenched with Sat NaHCO3 under 0◦C. The organic solvent is separated, dried and purified through flash chromatography (e.g. cyclohexane/EtOAc 20:80 v/v). The compound E could be further oxidized to thiazole using activated MnO2 (activated MnO2 having a pore size of ≤ 5 microns is preferred). Compound E is reacted with 20 eq. of activated MnO2 in CHCl3 at reflux conditions (70 ◦C). The reaction time can range from 6 h to 12 h. The reaction steps of producing compounds E and F can also be carried out from 1 mmol to 20 mmol scale.

c) The next step is the acyl migration reaction, deprotection of an amine protecting group followed by base treatment yields hydroxyl moiety of compound G in excellent yield. The acyl migration reaction is suitably performed in a one-pot-two-step process involving exposure to amine deprotection (Fmoc is used diethylamine (DEA) is treated to remove Fmoc group, followed by exposure to trimethylamine (TEA)) disclosing conditions for Amine Deprotection−Acyl Migration.

d) As a next step, oxidizing the hydroxyl group to afford ketone compound H as an advanced intermediate. The great advantage of this intermediate is the wide diversity at tubuvaline (L-Tuv) building block could be placed. Specifically, hydroxyl group is oxidized using Dess-Martin periodinane in CH2Cl2, the reaction finishes in excellent yield under mild conditions (at room temperature for 1 hr).

e) Preparing compound I, where in R6 is independently selected from optionally, substituted alkyl, aryl or optionally substituted bifunctional alkyl groups; wherein the process comprises the step of treating compound H with a strong base such as KHMDS, NAHMDS or NaH and a respective leaving groups such as alkyl/aryl halides or corresponding tosylate or mesylates or bifunctional alkyl halide. Preferably, NaHMDS at -78℃ is reacted with compound H and the alkyl halides could afford desired compound I. The reaction time can range from 1-5 hr. The reaction is preferably carried out from 1 mmol to 20 mmol scale. The reaction could be easily monitored by TLC and quenched by MeOH and extracted with EtOAc and purified by column chromatography.

f) Stereoselective reduction of ketone is carried out using (S)-CBS catalyst and BH3.DMS for the synthesis of compound J. After completion of the reduction, the reaction mixture was quenched with MeOH extracted with EtOAc. The desired diastereomer of the compound J could be isolated by column chromatography in quantitative yield.

g) Treating a compound J with a metal hydroxide or carbonate selected from the group consisting of LiOH, Li2CO3, NaOH, Na2CO3, KOH, K2CO3, Ca(OH)2, CaCO3, Mg(OH)2 and MgCO3 or a combination thereof. Additionally, acid-cleavable protecting groups could be removed by the treatment of CF3COOH or HCl. The compound K could be isolated as pure by simple acidification and/or evaporation of the solvent.

h) The carboxylic acid compound of the compound K is reacted with tubuphenylalanine compound L or a salt thereof, such as the Na, K, Li or Ca salt,

A tubuphenylalanine compound L using Boc-Phe-OH (or phenyl substitution thereof), method of synthesis comprises the steps of: (i) conversion of Boc-Phe-OH (or phenyl substitution thereof) to corresponding alcohol followed by oxidation to aldehyde. The desired aldehyde could also be obtained by reduction of esters or Weinreb amide. (ii) Next, aldehyde is reacted with commercially available Wittig ylide or readily preparable to give corresponding alkene derivatives, which upon hydrogenation using palladium gives a mixture of diastereomers. The desired diastereomer could be separated by column chromatography or recrystallization techniques. Further, deprotecting of the protecting groups gives compound L. Alternatively, carboxylic acid of the compound L could be converted into corresponding methyl ester or salts thereof.

i) The carboxylic acid of the compound K is activated by coupling reagent followed by treatment of compound L or ester thereof to give compound M. The coupling could be performed by using onium salts such as HATU, HTBU in the presence of HOBt as an additive. Alternatively, an active ester of the compound M could be prepared by using DIC/pentafluoro phenol or p-nitrophenyl ester followed by the addition of compound L to give compound M. The desired peptide adduct is purified by column chromatography.

j) A carboxylic acid compound N, wherein R1 and R2 represents isopropyl, tert-butyl, iso-butyl, sec-butyl, cyclopropylmethyl or cyclobutyl methyl; R2 is H; wherein n is 0, 1, 2, 3, 4 or 5. The Pg moiety in compound N is an amine protecting group, preferably a carbamate, a substituted or an unsubstituted benzyl. Preferably, Pg is selected from the group consisting of benzyloxycarbonyl, 4-azidobenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, 4,5-dimethoxy-2-nitrobenzyloxycarbonyl, 3,5-dimethoxybenzyloxycarbonyl, 1-naphthylmethoxycarbonyl, 4-acetyloxybenzyloxycarbonyl, fluorenyloxycarbonyl, tert-butyloxycarbonyl, allyloxycarbonyl, methyl carbamate, ethyl carbamate, benzyl, 4-methoxybenzyl and 3,4-dimethoxybenzyl.

k) The Azide group of the compound M is reduced to amine using palladium catalyzed hydrogenation. The palladium is filtered off and the compound N is treated using suitable coupling reagents. Preferably, coupling reagents are HBTU, HATU using HOBt as an additive in the presence of a base. Removal of the carboxyl protecting group (Methyl ester) gives a compound O. Hydrolysis is carried out using metal hydroxide or carbonate include LiOH, Li2CO3, NaOH, Na2CO3, KOH, K2CO3, Ca(OH)2, CaCO3, Mg(OH)2, MgCO3, and the like. Additionally, a transesterification catalyst is also selected from the group consisting of Bu3SnOH, depending on the other functional group sensitivity in the molecule.

l) The acylation of the hydroxyl group of compound P to obtain an ac(et)ylated tubulysin derivative compound (1), wherein R10 represent acetyl, acyl (substituted) alkyl, acyl cycloalkyl, or acyl benzyl, preferably acetyl or acyl derivative of methyl, ethyl, tert-butyl or benzyl.

[0056] In another embodiment, in step (a), a non-coordinating solvent or solvent mixture is used selected from a group consisting of CH2Cl2, CHCl3, CCl4, benzene, THF, CH3CN, 1,4-dioxane, and 1,2-dichloroethane or a combination thereof. Preferably, compound A, compound B and compound C reacted in a mixture of CH2Cl2: THF (1:1 v/v).
[0057] In another embodiment, in step (a), the reaction time can range from a few hours up to a few days. Preferably, the reaction time ranges from16 to 60 h. More preferably, the reaction time ranges from 24 to 48 h. The compound A, compound B and compound C may be present in the starting reaction mixture in about equimolar amounts, wherein A: B: C is 0.8-1.2: 0.8-1.2: 0.8-1.2. Preferably the ratio is 0.9-1.1:0.9-1.1:09-1.1.
[0058] In another embodiment, the order in which the acyl migration reaction and the cyclodehydration reaction are performed are not critical. In one embodiment, acyl migration precedes cyclodehydration reaction. In another, cyclodehydration reaction precedes acyl migration. Preferably, the method comprises cyclodehydration reaction, then oxidation followed by Fmoc deprotection and acyl migration.
[0059] In another embodiment, the method of the present invention allows for the introduction of a variety of acid groups at R10 position which can be used in the conjugation of payloads to small molecules, polymers, peptides, proteins, antibodies, antibody fragments etc. can be adopted and thereby, many different conjugation methods can be applied. Spacer systems at different positions can be used either directly for conjugation by using different conjugation technologies such as chemical conjugation methods known in the art, or enzymatic conjugations using transglutaminases, sortases or other enzymes or which can be used in combination with commonly described linker systems known in the art. More in particular, a method as herein disclosed provides tubulysin derivatives that can be attached toward biological matter, e.g. monoclonal antibodies, through at least 3 different linker positions. Herewith, the properties of, for example, ADC’s can be fine-tuned in a better way as compared to existing products. Therefore, the present invention particularly finds its use in the manufacture of a cytotoxic tubulysin derivative or tubulysin prodrug.
[0060] In another embodiment of the present invention, the process for the preparation of tubulysins compound and its derivatives of formula (I) is depicted below in general scheme-1:

Scheme-1
[0061] In another embodiment, the present invention provides a cost-effective synthesis of tubulysins compound and its derivatives of formula (I) by using the suitable designed and bench-stable building blocks. Also, discloses that strategy can be applicable to completely different tubulysins molecular scaffolds targeting the wide modifications at N14- tubuvaline (Tuv) position, which could be the most demanding anticancer agents. The present invention may also bring a practical solution to the process development for kilogram scale synthesis. The green synthesis of tubulysins may pave the way to produce novel tubulysins based ADCs having improved biophysical properties in the future. Further, the process of the present invention affords multigram scale synthesis, better purity, involves less number of steps and affords a diversity of products. Thus, provides a greener, rapid and low cost production of Tubulysins.
[0062] In another embodiment, the tubulysins compound and its derivatives of formula (I) of present invention can be converted into a pharmaceutically acceptable salt. The pharmaceutical acceptable salts of the tubulysins compound and its derivatives of formula (I) of present invention according to the invention are prepared in a manner known to one skilled in the art. Pharmaceutically acceptable salts of the tubulysins compound and its derivatives of formula (I) of the present invention include but not limited to, an acid salt of a compound of the present invention containing an amine or other basic group can be obtained by reacting the compound with a suitable organic or inorganic acid, resulting in pharmaceutically acceptable anionic salt forms. Examples of anionic salts include the acetate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, calcium edetate, camsylate, carbonate, chloride, citrate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, glyceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, malate, maleate, mandelate, mesylate, methylsulfate, mucate, napsylate, nitrate, pamoate, pantothenate, phosphate/diphosphate, polygalacturonate, salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate, teoclate, tosylate, and triethiodide salts.
[0063] In yet another embodiment, the pharmaceutically acceptable salts of the tubulysins compound and its derivatives of formula (I) of the present invention containing acidic functional groups can be prepared by reacting with a suitable base. Such a pharmaceutically acceptable salt may be made with a base which affords a pharmaceutically acceptable cation, which includes alkali metal salts (especially sodium and potassium), alkaline earth metal salts (especially calcium and magnesium), aluminum salts and ammonium salts, as well as salts made from physiologically acceptable organic bases such as trimethylamine, triethylamine, morpholine, pyridine, piperidine, picoline, dicyclohexylamine, N,N'-dibenzylethylenediamine, 2-hydroxyethylamine, bis-(2-hydroxyethyl)amine, tri-(2-hydroxyethyl)amine, procaine, dibenzylpiperidine, dehydroabietylamine, N,N'-bisdehydroabietylamine, glucamine, N-methylglucamine, collidine, quinine, quinoline, and basic amino acids such as lysine and arginine.
[0064] In another aspect, the present invention relates to pharmaceutical compositions that contain a therapeutically effective amount of a tubulysins compound and its derivatives of formula (I) of present invention or its pharmaceutically acceptable salt in addition to customary pharmaceutically acceptable excipients. The present invention also relates to a process for the production of the pharmaceutical composition, which includes bringing the compound of present invention, into a suitable administration form using a pharmaceutically acceptable excipient or a carrier and, if appropriate, further suitable a pharmaceutically acceptable carriers, additives or auxiliaries. The pharmaceutical compositions containing the compound of present invention according to the invention are prepared in a manner known to one skilled in the art.
[0065] In an embodiment, the pharmaceutical compositions can be administered orally, for example in the form of pills, tablets, coated tablets, capsules, granules or elixirs. Administration, however, can also be carried out rectally, for example in the form of suppositories, or parenterally, for example intravenously, intramuscularly or subcutaneously, in the form of injectable sterile solutions or suspensions, or topically, for example in the form of ointments or creams or transdermally, in the form of patches, or in other ways, for example in the form of aerosols or nasal sprays.
[0066] For the production of oral dosages form of the tubulysins compound and its derivatives of formula (I) of the present invention such as the pills, tablets, coated tablets and hard gelatin capsules, it is possible to use, for example, lactose, corn starch or compounds thereof, gum arabica, magnesia or glucose, etc. Pharmaceutically acceptable excipients that can be used for soft gelatin capsules and suppositories are, for example, fats, waxes, natural or hardened oils, etc. Suitable pharmaceutically acceptable excipients for the production of solutions, for example injection solutions, or of emulsions or syrups are, for example, water, physiological sodium chloride solution or alcohols, for example, ethanol, propanol or glycerol, sugar solutions, such as glucose solutions or mannitol solutions, or a mixture of the said solvents.
[0067] In another embodiment, the pharmaceutical compositions normally contain about 1% to 99%, for example, about 5% to 70%, or from about 10% to about 30% by weight of the compound of formula (I) or its pharmaceutically acceptable salt. The amount of the compound of formula (I) or its pharmaceutically acceptable salt in the pharmaceutical compositions normally is from about 5 to 500 mg or may be lower than or higher than the lower and the upper limit respectively. The dose of the compound of the present invention, which is to be administered, can cover a wide range depending on the type of disease or disorder to be treated. The dose to be administered daily is to be selected to suit the desired effect. A suitable dosage is about 0.01 to 100 mg/kg of the compound of present invention or its pharmaceutically acceptable salt depending on the body weight of the recipient (subject) per day, for example, about 0.1 to 50 mg/kg/day of a compound of the present invention or a pharmaceutically acceptable salt of the compound. If required, higher or lower daily doses can also be administered.
[0068] The selected dosage level will depend upon a variety of factors including the activity of a tubulysins compound and its derivatives of formula (I) of the present invention, or its salt employed, the route of administration, the time of administration, the rate of excretion of the particular compound being administered, the duration of the treatment, other concurrently administered drugs, compounds and/or materials, the age, sex, weight, condition, general health and prior medical history of the patient (subject) being treated, and like factors well known in the medical arts.
[0069] In addition to the tubulysins compound and its derivatives of formula (I) of present invention or its pharmaceutically acceptable salt and the pharmaceutically acceptable carrier substances, the pharmaceutical compositions of the present invention can contain additives such as, for example, fillers, antioxidants, dispersants, emulsifiers, defoamers, flavors, preservatives, solubilizers or colorants. Furthermore, in addition to a compound of present invention or its pharmaceutically acceptable salt, the pharmaceutical compositions can also contain one or more other therapeutically or prophylactically active agents.
[0070] The present invention also encompasses within its scope the use of a tubulysins compound and its derivatives of formula (I) of present invention or its pharmaceutically acceptable salt in combination with other therapeutically active agents.
[0071] In an embodiment, the combination of tubulysins compound and its derivatives of formula (I) of present invention with another therapeutic agent or treatment includes co-administration of a compound of pres with the other therapeutic agent or treatment as either a single combination dosage form or as multiple, separate dosage forms, administration of the compound of the present invention first, followed by the other therapeutic agent or treatment and administration of the other therapeutic agent or treatment first, followed by the compound of present invention. Further therapeutic agents are administered either simultaneously or sequentially.
[0072] In another embodiment of the present invention, the other therapeutic agent may be any agent that is known in the art to treat, prevent, or reduce the symptoms of a disease or disorder. The selection of other therapeutic agent(s) is based upon the particular disease or disorder being treated. Such choice is within the knowledge of a treating physician. Furthermore, the additional therapeutic agent may be any agent when administered in combination with the administration of a compound of the present invention provides benefit to the subject in need thereof.
[0073] In another aspect, the present invention relates to a tubulysins compound and its derivatives of formula (I) or a stereoisomer, a tautomer, a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof as an anticancer agent for use in the treatment of cancer.
[0074] While the foregoing describes various embodiments of the disclosure, other and further embodiments of the disclosure may be devised without departing from the basic scope thereof. The scope of the invention is determined by the claims that follow. The invention is not limited to the described embodiments, versions or examples, which are included to enable a person having ordinary skill in the art to make and use the invention when combined with information and knowledge available to the person having ordinary skill in the art.
EXAMPLES
[0075] The present invention is further explained in the form of the following examples. However, it is to be understood that the following examples are merely illustrative and are not to be taken as limitations upon the scope of the invention.
[0076] Example 1:

In 200 mL of methanol at O °C was added thionyl chloride (20.96 mL, 288.88 mmol) in a drop wise fashion followed by addition of L-Cysteine Hydrochloride (10.0 g, 82.54 mmol) at same maintained temperature. The solution was allowed to warm up to room temperature and then refluxed at 80 °C for 5 h using an oil bath. The solvent was removed under reduced pressure added DCM and subjected to rotavapor for solvent removal 3-4 times until white solid crude product obtained (14.0 g 81.56 mmol). Washed with diethyl ether to get pure product a1 which is used as the next step.

To a solution of triphenylmethanol (42.27 g, 163.13 mmol) in 100.0 mL of trifluoroacetic acid L-cysteine methyl ester hydrochloride a1 (14.0 g, 81.56 mmol). The resulting pale yellow solution was left to stir at room temperature for 2 h after it was concentrated using 3 times 50 mL DCM to obtain yellow oil. To this yellow oil added 100.0 mL DCM followed by saturated aqueous NaHCO3 solution until solution became basic in nature (confirmed with pH paper). DCM layer collected and water layer washed with 2 x 50 mL DCM. Organic layer (DCM) concentrated under reduced vacuum to get light yellow gum of a2 (28.0 g).

Crude (R)-methyl 2-amino-3-(tritylthio)propanoate a2 (28.0 g, 74.17 mmol) was dissolved in methyl formate (80.0 mL, solvent) and some small amount of DCM and the assembly was allowed to reflux in an oil bath at 60 °C until TLC showed complete consumption of starting material (time taken 5 h). The solvent was evaporated under reduced pressure. Purified by Flash prep using Hexane/EtOAc as eluent to get pure product a3 (24.0 g).
NMR (300 MHz, CDCl3): 7.95 (s, 1H), 7.41-7.35 (m, 5H), 7.29-7.16 (m, 10H), 5.82 (d, 1H, J= 6.0 Hz), 4.66-4.60 (m, 1H), 3.69 (s, 1H), 2.79-2.58 (m, 2H).
[0077] Example 2:

A solution of N-formyl Cys(Trt)-methyl ester (24.0 g, 59.18 mmol) in CH2Cl2 (150.0 mL), was cooled to -78 °C. N-methylmorpholine (13.0 mL, 118.37 mmol, 2.0 eq.) was added. After 5 min triphosgene (7.0 g, 23.67 mmol, in CH2Cl2 (50 mL) was added drop wise over 30 min. After addition was complete the reaction mixture was stirred for 7h at - 78 oC (TLC analysis). Saturated NaHCO3 solution (100 mL) was added at the same temperature and then allowed to warm to room temperature. The organic layer was separated and the aqueous phase was washed CH2Cl2 (3 X 50 mL).The combined organic extracts were separated, dried over anhydrous MgSO4, filtered, and evaporated under reduced pressure. Added 5 mL DCM and 30 mL Diethyl Ether kept in refrigerator for 4 hr and scratch with spatula to get solid product which was filtered using funnel followed by 2 time washing with Diethyl Ether to get crude (R)-methyl 2-isocyano-3-(tritylthio)propanoate a4 (16.0 g) was used further without purification.
NMR (300 MHz, CDCl3, PPM): 7.48-7.23 (m, 15H), 3.74 (s, 3H), 3.39-3.35 (m, 1H), 2.84-2.76 (m, 2H).
[0078] Example 3:

Sodium Azide (25.0 g, 381 mmol) was dissolved in distilled H2O (61.0 mL) with CH2Cl2 (102.0 mL) and cooled in an ice bath. Triflyl anhydride (12.80 mL, 76.24 mmol) was added slowly over 5 min with stirring continued for 2 h. The mixture was placed in a separatory funnel and the CH2Cl2 phase removed. The aqueous portion was extracted with DCM (2 × 50 mL). The organic fractions, containing the triflyl azide, were pooled, and washed once with saturated NaHCO3 and added Na2SO4 to remove any water content to be used without further purification. L-Ile (5.0 g, 38.12 mmol) was combined with K2CO3 (7.90 g, 57.18 mmol) and Cu(II)SO4 pentahydrate (0.095.0 g, 0.01 eq, 0.0762 mmol) distilled H2O (123.0 mL) and CH3OH (246.0 mL). The triflyl azide in DCM (already prepared) was added and the mixture was stirred at ambient temperature and pressure overnight. Subsequently, the organic solvents were removed under reduced pressure and the aqueous slurry was diluted with H2O (100 mL). This was acidified to pH 6 with conc. HCl and extracted with EtOAc (2 × 100 mL) to remove sulphonamide by-product. The aqueous phase was again acidified to pH 2 with conc. HCl and extracted with EtOAc (2 x 50 mL). These EtOAc extracts were combined, dried (Na2SO4) and purified by flash to get the pale oil of a5 (4.62 g).
NMR (300 MHz, CDCl3): 8.13 (brs, 1H), 3.82 (d, 1H, J= 5.7 Hz), 2.05-1.96 (m, 1H), 1.61-1.49 (m, 1H), 1.39-1.24 (m, 1H), 1.04 (d, 3H, J= 6.9 Hz), 0.94 (t, 3H, J= 6.0 Hz).
[0079] Example 4:
Under an argon atmosphere triethylamine (25.0 mL, 176 mmol, 2.0 eq.) and ethyl chloroformate (10.1 mL, 106 mmol, 1.2 eq.) were carefully added at -20 °C to the Fmoc-Val-OH (30.0 g, 88.0 mmol, 1.0 eq.) dissolved in dry THF (300 mL). The reaction mixture was stirred at -20 °C for 30 min. In a separate conical flask containing KOH (60 g) was dissolved in 120.0 mL water and was cooled to 0 ◦C. To this cooled solution of KOH, diethyl ether (100 mL) was added followed by Nitroso-N-methyl urea (3.0 eq. with respect to Fmoc-Val-OH, 28.0 g) was added slowly in portion wise, the diazomethane generated in situ was collected in diethyl ether, which was decanted to another conical flask containing KOH, this dried ethereal solution of diazomethane was transferred to the round bottomed flask containing Fmoc-Val-mixed anhydride solution. This procedure was repeated three more times until all the diazomethane was transferred. The reaction mixture was stirred overnight. Afterwards, the excess of diazomethane was quenched by addition of glacial acetic acid (5 mL) and the mixture was washed with sat NaHCO3 (3 X 200 mL) and extracted with EtOAc (3 X 300 mL). The combined organic phases were washed with saturated NaCl, dried over MgSO4 and the solvent was removed under reduced pressure. The product was recrystallized from diethyl ether to get 25.0 g as solid of a6.

The diazo ketone a6 (25.0 g, 68 mmol) was dissolved in Dioxane/H2O (9:1, 500 mL) and under exclusion of light silver benzoate (3.15 g, 13.76 mmol, 0.2 eq.) was added at 0 ˚C and the reaction mixture was stirred at same temperature for 30 min. Then the reaction mixture was refluxed in an oil bath at 70 ˚C for 5 h. After completion of the reaction (TLC analysis), the mixture was cooled down to room temperature and diluted with H2O (110.0 mL), adjusted to pH = 2-3 by the addition of 1.0 M HCl and extracted with EtOAc (3 X 100 mL). The combined organic phases were dried over MgSO4 and the solvent was removed under reduced pressure. The crude product was taken up in DCM (40.0 mL) and was cooled to -22 °C, cold pentane (26.0 mL) was added dropwise. The obtained precipitate was filtered off, washed with cold pentane (2 X 20 mL) and dried overnight under reduced pressure to yield desired product as white solid of a7 (22.0 g).

A solution of (R)-3-((((9H-fluoren-9-yl) methoxy) carbonyl)amino)-4-methylpentanoic acid a7 (20.0 g, 56.6 mmol, 1.0 eq.) in THF (150.0 mL) was cooled to -15 °C (ice/ salt bath) under a nitrogen atmosphere. NMM (18.2 mL, 169.7 mmol, 3.0 eq.) and ethyl chloroformate (8.08 mL, 84.8 mmol, 1.5 eq.) were added successively in a dropwise manner. After 3 h, the reaction mixture was filtered. The filtrate was cooled to -15 °C (ice/ salt bath), and a solution of NaBH4 (8.56 g, 226 mmol, 4.0 eq.) in H2O (5.0 mL) was added in one portion. After complete reduction (TLC analysis), the suspension was diluted with EtOAc and H2O (1:1). The organic layer was separated, and the aqueous layer was extracted with EtOAc (3 X 80 mL) and the combined organic layer was dried over MgSO4 and evaporated under vacuum. The crude product was purified through flash purification to get pure (18.0) product as solid of a8.

IBX (44.5 g, 159 mmol, 3.0 eq.) was added to a solution of the compound a8 (18.0 g, 53 mmol, 1.0 eq.) in EtOAc (300.0 mL) and the mixture was refluxed (70 ◦C) for 3.0 h opened to the atmosphere. The mixture was cooled to r.t., using ice cold water bath filtered and the solvent was removed under reduced pressure and subjected to flash purification to afford (R)-(9H-fluoren-9-yl)methyl (4-methyl-1-oxopentan-3-yl)carbamate of a9 as (15.0 g) solid.
To a solution of menthol (10.0 g, 64 mmol, 1.0 eq.) in dry THF (60.0 mL) Et3N (8.9 mL, 64 mmol, 1.0 eq.) was added. After cooling at 0 °C, bromoacetyl bromide (5.6 mL, 64.0 mmol, 1.0 eq.) was added dropwise. The temperature was allowed to warm to rt and the reaction mixture was stirred for 2.0 h. After cooling at 0 °C, the reaction was quenched with a 1 N HCl aqueous solution (10.0 mL) and AcOEt was added (60.0 mL). The layers were separated, and the organic phase was dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The crude was purified by FC to give (1S,2S,5R)-2-isopropyl-5-methylcyclohexyl 2-bromoacetate (8.0 g) as a colorless oil of a10.

To a solution of a10 (8 g, 29 mmol, 1.0 eq.) in dry THF (60.0 mL), under a nitrogen atmosphere, PPh3 (7.57 g, 29.0 mmol, 1.0 eq.) was added. After refluxing for 2.5 h, the reaction mixture was concentrated in vacuo. The resulting solid was washed with a 7 : 3 mixture of hexane–Et2O and filtered to give (11.0 g) phosphonium salt of a11 as a white solid, which was used in the next step without further purification.

To a suspension of a11 (8.0 g, 229.0 mmol) in toluene (150.0 mL) a 0.38 N NaOH aqueous solution (25.0 mL) was added dropwise over a period of 5 min. The reaction mixture was stirred for 3.0 h and the layers were separated. The organic phase was dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The resulting white phosphonium salt was in CH2Cl2 (60.0 mL), cooled at 0 °C, MeI (2.7 mL, 44 mmol, 1.5 eq.) was added dropwise. After stirring for 10 min the temperature was allowed to warm to rt. The reaction mixture was stirred overnight, and the solvent was evaporated. The crude was dissolved in toluene (100.0 mL) and a 0.38 N NaOH aqueous solution (25.0 mL) was added. The mixture was stirred for 2.0 h and the layers were separated. The organic phase was dried over anhydrous Na2SO4, filtered and concentrated in vacuo to give crude which was purified by FC to give pure(1S,2S,5R)-2-isopropyl-5-methylcyclohexyl 2-(triphenylphosphoranylidene)propanoate a12 (6.0 g).

A solution of (S)-2-((tert-butoxycarbonyl)amino)-3-phenylpropanoic acid (10.0 g, 37.69 mmol) in THF (120 mL) was cooled to -15 °C (ice/ salt bath) under a nitrogen atmosphere. NMM (12.43 mL, 113.08 mmol, 3 eq.) and ethyl chloroformate (6.08 mL, 56.54 mmol, 1.5 eq.) were added successively in a dropwise manner. After 3 h, the reaction mixture was filtered. The filtrate was cooled to -15 °C (ice/ salt bath), and a solution of NaBH4 (5.70 g, 150.77 mmol, 4.0 eq.) in H2O (7.7 mL) was added in one portion. After complete reduction (TLC analysis), the suspension was diluted with EtOAc and H2O (1:1). The organic layer was separated, and the aqueous layer was extracted with EtOAc (3 X 80 mL) and the combined organic layer was dried over Na2SO4 and evaporated under vacuum. The crude product was purified on silica gel flash column chromatography to afford a13.

To a solution of 13 (4.53 g, 9.6 mmol) in DCM (80 mL), cooled at 0 °C, aldehyde 95 (1.6 g, 6.4 mmol) was added. After stirring for 15 min at 0 °C, the temperature was allowed to warm to rt and the reaction mixture was stirred for 8 h. The reaction was quenched with a 1 N NaHSO4 aqueous solution (50 mL) and extracted with DCM (2 × 50 mL). The organic layer was washed with brine (1 × 50 mL), dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The crude was purified by FC (2 : 8 IBX (26.6 g, 95.5 mmol, 3.0 eq.) was added to a solution of the a13 (8.0 g, 31.83 mmol, 1 eq.) in EtOAc (150 mL) with ACN (50 mL) and the mixture was refluxed for 2 h opened to the atmosphere. The mixture was cooled to r.t., using ice cold water bath filtered and the solvent was removed under reduced pressure and subjected to flash purification to afford a14.

To a solution of a14 (4.53 g, 9.6 mmol) in DCM (80 mL), cooled at 0 °C, a12 (1.6 g, 6.4 mmol) was added. After stirring for 15 min at 0 °C, the temperature was allowed to warm to rt and the reaction mixture was stirred for 8 h. The reaction was quenched with a 1 N NaHSO4 aqueous solution (50 mL) and extracted with DCM (2 × 50 mL). The organic layer was washed with brine (1 × 50 mL), dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The crude was purified by FC to yield a15.

A solution of a15 (10 g, 22.54 mmol, 1.0 eq.) in EtOAc (200 mL) was treated with Pd/C (2.4 g, 2.25 mmol, 0.1 eq.), stirred under an atmosphere of H2 overnight at rt, filtered on Celite, and concentrated. The crude residue was purified by chromatography on SiO2 (hexanes/EtOAc 85:15) to afford a16 (a16a and a16b) a mixture of diastereomers. Desired diastereomer a16b was isolated as 5.0 g (68% yield).

A suspension of a16b (500 mg, 1.12 mmol, 1.0 eq.) in 6 M HCl (15.0 mL) and AcOH (5.0 mL) was heated at reflux (110 °C) for 1.5 h, diluted with H2O, washed with Et2O, and concentrated to afford (2R,4S)-4-carboxy-1-phenylpentan-2-aminium chloride (0.72 g) as a colorless crystalline solid of a17.
To the stirred solution of NaHCO3 (2.60 g, 30.97 mmol) in H2O:Dioxane (1:1, 70 mL) at 0 °C added (R)-piperidine-2-carboxylic acid (2.0 g, 15.49 mmol) followed by dropwise addition of Benzyl chloroformate (2.42 mL, 17.03 mmol) and stirred the reaction mixture at 0 °C to room temperature for overnight. Progresses of the reaction monitored by TLC, and then quench the Sodium bicarbonate with 1N HCl (used 80 mL) until pH 2 of the solution. This solution was extracted with EtOAc (3 X 40 mL) treated with brine and dried over Na2SO4. The solvent was evaporated under reduced pressure to get the colourless liquid compound which required further purification to get pure (R)-1-((benzyloxy)carbonyl)piperidine-2-carboxylic acid (3.5 g) as solid id a18.

Under nitrogen atmosphere, to a stirred solution of a9 (7.0 g, 20.75, 1.0 eq.) compound a5 (3.36 g, 20.75 mmol, 1.0 eq.) was added and the reaction mixture was stirred for 5 min. at room temperature, then compound a4 (8.04 g, 20.75 mmol, 1.0 eq.), was added at room temperature. The resulting solution was stirred for 18 h at room temperature. TLC analysis showed complete consumption of the starting materials. Removed the solvent under reduced pressure to get crude a19 (15.0 g) as oil which was used as it is for the next reaction.

A solution of a19 (15.0 g, 17.1 mmol) in dry CH2Cl2 (200.0 mL) was treated with TiCl4 (1.0 M solution in CH2Cl2, 51.02 mmol) and stirred at 0 °C for 18 h. The reaction mixture was stirred vigorously with cold saturated aqueous NaHCO3 (80.0 mL) for 30.0 min. The aqueous layer was extracted with CH2Cl2 (2 X 100 mL), and the combined organic layers were dried over MgSO4, filtered, and concentrated under reduced pressure to get compound a20 (9.0 g) as oil. The resultant crude product was used for the next reaction.
A solution of a20 (8.0 g, 13.0 mmol, 1.0 eq.) in CHCl3 (200.0 mL) was treated with activated MnO2 (12.0 g, 128.67 mmol, 10.0 equiv.) and the reaction mixture was refluxed (nearly 3 h) and monitored by TLC. The reaction mixtures were cooled to rt, then the solution was filtered through celite and the solvent was evaporated and purified by flash column to get compound a21 as mixture of diastereomers (7.5 g) isolated as oil.
To a solution of a21 (7.0 g, 11.30 mmol, 1.0 eq.) in CH3CN (16.0 mL) at 0 °C under Ar was added Et2NH (23 mL, 225.90 mmol, 20.0 eq.). The solution was stirred at 0 °C for 1 h (TLC analysis). After complete deprotection of Fmoc group (TLC analysis) the mixture was evaporated and co-evaporated with CH2Cl2 to remove DEA. The crude product which was dissolved in CH2Cl2 (12 mL) and triethylamine (3.15 mL, 22.59 mmol, 2.0 eq.) was added and the resulting mixture was stirred at room temperature 16 h. The crude hydroxyl derivative a22 was purified by column chromatography (4.0 g).
To a solution of a22 (4.0 g, 10.0 mmol, 1.0 eq.) in CH2Cl2 (30.0 mL) at 0 °C under, Dess-Martin Periodinane (6.4 g, 15.1 mmol, 1.5 eq.) was added. The solution was stirred at rt for 2.0 h (TLC analysis). The reaction mixture was then quenched with aqueous 1M sodium metabisulfite and sat NaHCO3 solution. The organic layer was separated, and the aqueous phase was extracted two times with CH2Cl2 (30.0 mL). The collected organic phase was dried over Na2SO4 and evaporated under reduced pressure and purified by column chromatography to get pure product of a23 as white solid.

A 0.30 M solution of a23 (2.0 g, 5.0 mmol, 1.0 eq.) in THF (17.0 mL) was cooled to -45 °C and KHMDS (18.0 mL, 8.60 mmol, 0.50 M in toluene) was added. The resulting mixture was stirred for 20 minutes at -45 °C. Methyl iodide (1.26 ml, 20.23 mmol, 4.0 eq.) was added, and the reaction mixture was allowed to warm to rt over 4.5 h at which time the reaction was quenched with MeOH (15.0 mL). The crude product was diluted with EtOAc (500.0 mL) and washed with brine (300.0 mL). The aqueous layer was extracted with EtOAc (2 x 100 mL). The organic portions were dried, filtered, and concentrated followed by flash purification giving compound a24 (1.50 g) as oil.

To an ice-cooled stirred solution of (S)-CBS catalyst (1.1mL, 1.0 M in THF, 1.1 mmol, 0.3 eq.) in THF (13.5 mL) was added BH3•SMe2 (2.0 mL, 2.0 M in THF, 3.66 mmol, 1.0 eq.) and stirring was continued for 10 min at 0 °C. Then, a solution of ketone a24 (1.5 g, 3.66 mmol) in THF (5.6 mL) was added dropwise to the reaction mixture and stirring was continued for 18 h while the temperature gradually increased to 25 °C. The reaction was quenched with MeOH (10.0 mL) and the solvent was removed under reduced pressure. The resulting residue was purified using flash column chromatography (silica gel, 10→30% EtOAc in hexanes) to furnish alcohol methyl 2-((1R,3R)-3-((2S,3R)-2-azido-N,3-dimethylpentanamido)-1-hydroxy-4-methylpentyl)thiazole-4-carboxylate (0.58 g, 82% yield) of a25 as a colourless oil.
LiOH.H2O (82.0 mg, 1.94 mmol, 1.6 eq.) was added to a solution of a26 (500 mg, 1.22 mmol, 1.0 eq.) in a THF/H2O 4:1 mixture (10.0 mL). The reaction was stirred for 12 h, then H2O (10.0 mL) and AcOEt (10.0 mL) were added. The layers were separated and a 1 M aqueous solution of HCl was added to the aqueous phase until pH 1-2 was reached. The resulting mixture was extracted with AcOEt. The organic phase was dried over anhydrous Na2SO4, filtered and the solvent was removed in vacuo to give pure compound a26 (480 mg) which was used in the next step without further purification.
To a solution of a26 (150.0 mg, 0.37 mmol, 1.0 eq.) was dissolved in CH2Cl2 (10.0 mL) and was cooled to 0 ◦C. To this solution DIPEA (0.2 mL, 1.12 mmol, 3.0 eq.), PYBOP (235.65 mg, 0.5 mmol, 1.2 eq.) was added followed by compound a17a (97 mg, .004 mmol, 1 eq.) was added. The reaction mixture was stirred for 12.0 h. The crude reaction was directly purified through flash column purification to give pure compound a27 (170.0 mg).

A catalytic amount of Pd/C 10% was added to a solution of a27 (0.13 mg, .25 mmol, 1.eq.) in MeOH (10 mL). The reaction mixture was stirred under a hydrogen atmosphere for 5 h and the palladium was filtered through celite. The filtrate was concentrated under reduced pressure to give pure corresponding free amine which was used in the next step without further purification. The resulting free amine (135.0 mg, 0.22 mmol, 1.1 eq.) was dissolved in CH2Cl2 (10 mL) and was cooled to 0 ◦C. To this solution DIPEA (0.07 mL, 1.12 mmol, 3.0 eq.), PYBOP (176.0 mg, 0.3 mmol, 1.5 eq.) was added followed by compound a18 (60.0 mg, 0.22 mmol, 1 eq.) was added. The reaction mixture was stirred at rt for 12.0 h. The crude reaction was directly purified through flash column purification to get compound a28 (135.0 mg) as foamy solid.
LiOH.H2O (11 mg, 0.26 mmol, 1.6 eq.) was added to a solution of a28 (135 mg, 0.164 mmol, 1.0 eq.) in a THF/H2O 4:1 mixture (5.0 mL). The reaction was stirred for 12 h, then H2O (5.0 mL) and AcOEt (5.0 mL) were added. The layers were separated and a 1 M aqueous solution of HCl was added to the aqueous phase until pH 1-2 was reached. The resulting mixture was extracted with AcOEt. The organic phase was dried over anhydrous Na2SO4, filtered and the solvent was removed in vacuo to give pure a29 (125.0 mg) which was used in the next step without further purification.
Compound a29 (120.0 mg, 0.15 mmol) was dissolved in MeOH (15 mL) added 40% aqueous solution of paraformaldehyde (200 µL) to it followed by Pd/C (100.0 mg, 0.099 mmol) and stirred the reaction mixture for 16 h under Hydrogen atmosphere. Remove the solvent under reduced pressure and purify the reaction mixture using 0-10% MeOH in DCM as eluent to get white solid of compound a30 (90.0 mg).

To a solution of a30 (80 mg, 0.15 mmol, 1.0 eq.) in pyridine (4 mL), acetic anhydride (2 mL) was added, and the reaction mixture was stirred for 24 h at room temperature. The excess pyridine and acetic anhydride were removed under vacuum and the crude product was purified by column chromatography. The crude was further purified through the RP column using 30-70% H2O:ACN in 40 mins to afford a31 (55 mg).
[0080] The foregoing examples are merely illustrative and are not to be taken as limitations upon the scope of the invention. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications may be made without departing from the scope of the invention.

ADVANTAGES
1. The present invention provides a cost-effective synthesis of tubulysins compound and its derivatives of formula (I) by using the suitable designed and bench-stable building blocks.
2. The present invention provides a process for the synthesis of tubulysins compound and its derivatives of formula (I) that affords multigram scale synthesis, better purity, involves less number of steps and affords a diversity of products.
3. The present invention provides a greener, rapid and low cost production of Tubulysins compound and its derivatives of formula (I).
, Claims:1. A process for preparation of a tubulysins compound and its derivatives of formula (I) or a stereoisomer, a tautomer, a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof,

(I)
wherein,
n is 0, 1, 2 or 3;
R1 is CH3 or H; R2 is CH3 or H; R3 is CH3, CH2CH3, alkyl, aryl or optionally substituted bifunctional alkyl groups; R4 is esters, ethers, or amides; X is H, OH or NH2, NH-Y (Y = any alkyl or acetyl);
wherein said process comprises the steps of:
a) reacting a compound A, a compound B and a compound C in a solvent in a Passerini reaction to obtain a compound D;

b) subjecting the compound D to cyclodehydration reaction by incubating compound in the presence of TiCl4, followed by oxidation in the presence of MnO2 to obtain a compound F;

c) subjecting the compound F to acyl migration reaction by exposure to diethylamine (DEA), followed by exposure to trimethylamine (TEA) to obtain a compound G;

d) oxidizing a hydroxyl group of the compound G using Dess-Martin Periodinane at room temperature to obtain compound H;

e) alkylating secondary amide of the compound H using alkyl halides in presence of strong base at low temperature to obtain a compound I;

f) reducing ketone of the compound I diastereoselectively using chiral catalyst to obtain a compound J;

g) hydrolyzing the ester of the compound J to obtain a carboxylic acid compound K;

h) reacting carboxylic acid of the compound K with a tubuphenylalanine compound to obtain a compound M;

i) coupling of the compound M and a compound N, followed by protecting group cleavage, reductive amination and further hydrolysis to obtain a compound P; and

j) acylating hydroxyl group of the compound P to obtain a compound of Formula (I).

(I)
wherein,
R10 is acetyl, acyl (substituted) alkyl, acyl cycloalkyl, or acyl benzyl, preferably acetyl or acyl derivative of methyl, ethyl, tert-butyl or benzyl.
2. The process as claimed in claim 1, wherein the compound A is a carboxylic acid having formula:

A
wherein,
R1 is a substituted or unsubstituted alkyl; a substituted or unsubstituted cycloalkyl; or a substituted or unsubstituted benzyl,
R2 is H, a substituted or unsubstituted alkyl, or a substituted or unsubstituted cycloalkyl.
3. The process as claimed in claim 1, wherein the compound B is an aldehyde having formula:

B
wherein,
R3, R4 each independently is H, F, a substituted or unsubstituted alkyl, a substituted or unsubstituted cycloalkyl or a substituted or unsubstituted benzyl;
Pg1 is an amine protecting group, a carbamate, a substituted or an unsubstituted benzyl.
4. The process as claimed in claim 1, wherein the compound C is an isocyanide having formula:

C
wherein,
R5 is a substituted or unsubstituted alkyl, a substituted or unsubstituted cycloalkyl, or a substituted or unsubstituted benzyl;
X is O, S, Se, or -NH-; and
Pg2 is an X- protecting group selected from trityl, tert-butyl, adamantyl and substituted benzyl, more preferably trityl or tert-butyl.
5. The process as claimed in claim 1, wherein the compound A, compound B and compound C are present in a ratio in a range of 0.8-1.2: 0.8-1.2: 0.8-1.2.
6. The process as claimed in claim 1, wherein the solvent in step (a) is selected from a group consisting of CH2Cl2, CHCl3, CCl4, benzene, THF, CH3CN, 1,4-dioxane and 1,2-dichloroethane, or a combination thereof.
7. The process as claimed in claim 1, wherein the activated MnO2 in step (b) have a pore size of ≤ 5 microns.
8. The process as claimed in claim 1, wherein the compound I obtained in step (e), R6 is optionally, substituted alkyl, aryl or optionally substituted bifunctional alkyl groups.
9. The process as claimed in claim 1, wherein the strong base in step (e) is selected from the group consisting of KHMDS, NAHMDS and NaH.
10. The process as claimed in claim 1, wherein the temperature in step (e) is in the range of -70℃ to -80℃.
11. The process as claimed in claim 1, wherein the chiral catalyst in step (f) is selected from (S)-CBS catalyst and BH3.DMS.
12. The process as claimed in claim 1, wherein the compound N having formula:

wherein,
R8 and R9 is isopropyl, tert-butyl, iso-butyl, sec-butyl, cyclopropylmethyl or cyclobutylmethyl; R9 is H;
n is 0, 1, 2, 3, 4 or 5; and
Pg moiety is an amine protecting group, a carbamate, a substituted or an unsubstituted benzyl.
13. A pharmaceutical composition comprising of a tubulysins compound and its derivatives of formula (I) or a stereoisomer, a tautomer, a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate thereof as claimed in claim 1 and one or more pharmaceutically acceptable excipients.
14. The pharmaceutical composition comprising a tubulysins compound and its derivatives of formula (I) as claimed in claim 13, wherein the pharmaceutical composition is in the form of a tablet, a capsule, a solution, a gel, a suspension or a powder.