HK1185558B - Processes for preparing tubulysins - Google Patents

Description

Process for preparing tubulysins

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. provisional application serial No. 61/371,433 filed on 8/6/2010, pursuant to 35 u.s.c. § 119(e), the entire contents of which are incorporated herein by reference.

Technical Field

The invention described herein relates to a method for the preparation of tubulysins.

Background and summary of the invention

Tubulysins are members of a new class of natural products isolated from myxobacterial species (F. Sasse, et., J. Antibiot. 2000, 53, 879- -. As cytoskeletal influencing agents (interferongagents), tubulysins are mitotic toxicants which inhibit tubulin polymerization and lead to cell cycle arrest and apoptosis (h. steinmetz, et al, chem. int. ed. 2004, 43, 4888-. Tubulysins are very potent cytotoxic molecules, exceeding the cytostatic effects of any clinically relevant conventional chemotherapy, such as epothilones (epothilones), paclitaxel (paclitaxel) and vinblastine. In addition, they are effective against cell lines resistant to multiple drugs (A.D nanoting, et al, mol. university 2005, 9, 141-147). These compounds show high experimental cytotoxicity against a panel of cancer cell lines with IC50 values in the low picomolar range; therefore, they are of great interest as potential anticancer therapeutics.

Tubulysins are described herein. Structurally, tubulysins generally comprise a linear tetrapeptoid backbone, including exemplary compounds having the following formula T and pharmaceutically acceptable salts thereof;

(T)

wherein

Ar₁Is an optionally substituted aryl group;

R₁is hydrogen, alkyl, arylalkyl or a prodrug-forming group;

R₂selected from optionally substituted alkyl and optionally substituted cycloalkyl;

R₄is optionally substituted alkyl or optionally substituted cycloalkyl;

R₃is optionally substituted alkyl;

R₅and R₆Each independently selected from optionally substituted alkyl and optionally substituted cycloalkyl;

R₇is optionally substituted alkyl; and

n is 1, 2, 3 or 4.

Another group of exemplary tubulysins described herein more particularly consists of one or more non-naturally occurring or hydrophobic amino acid fragments, such as N-methylpipecacid (Mep), isoleucine (Ile),

Valine tubulin (Tubuvalin) (Tuv),

、

Tyrosine tubulin (Tut, an analogue of tyrosine)

、

Phenylalanine tubulin (tubuphenylalanine) (Tup, an analog of phenylalanine),

and analogs and derivatives of each of the foregoing compounds. One common feature in the molecular structure of the more potent naturally occurring tubulysins is the acid and/or base sensitive N-acyloxymethyl substituent (or N, O-acetal of formaldehyde), which is represented by R2-C (O) in formula (T).

Another exemplary set of tubulysins described herein are those compounds having formula 1.

In the formula 1, the compound is shown in the specification,

structure of several natural tubulysins

It has been reported that there is a C-terminal phenylalanine tubulin (R)_A= H) total synthesis of tubulysin D (H. Peltier, et al, j. Am. chem. soc. 2006, 128, 16018-. Recently, tubulysin B (R) has been reported_A= OH) synthesis (o. Pando, et at., org. lett. 2009, 11, 5567-. However, attempts to provide larger amounts of tubulysin following published procedures have not been successful, are partially hampered by low yields, are difficult to remove impurities, require costly chromatographic separation steps, and/or lack reproducibility of several steps. The key focus of the use of tubulysins as anticancer therapeutics is the need for reliable and efficient methods for preparing tubulysins and analogs and derivatives thereof. Described herein are improved methods for preparing native tubulysins or analogs or derivatives thereof, including compounds of formula (T) and formula (1).

In an exemplary embodiment of the invention, described herein is a method for the preparation of native tubulysin or analogues or derivatives thereof, including compounds of formula (T) and formula (1). The method comprises one or more steps described herein. In another embodiment, a process for preparing a compound of formula B is described, wherein R₅And R₆As described in various embodiments herein, for example, each is independently selected from optionally substituted alkyl or optionally substituted cycloalkyl; and R₈Is C1-C6 n-alkyl; wherein the process comprises the step of treating a compound of formula a with a silylating agent, such as triethylsilyl chloride, and a base, such as imidazole, in an aprotic solvent.

It is to be understood that R₅And R₆Each may include conventional protecting groups on the optional substituents.

In another embodiment, a process for preparing a compound of formula C is described, wherein R₅And R₆As described in various embodiments herein, for example, each is independently selected from optionally substituted alkyl or optionally substituted cycloalkyl; r₈Is C1-C6 n-alkyl; and R₂As described in various embodiments herein, such as selected from optionally substituted alkyl or optionally substituted cycloalkyl; wherein the process comprises contacting a compound of formula B with a compound of formula ClCH in an aprotic solvent at a temperature below room temperature, such as in the range of from about-78 ℃ to about 0 ℃₂OC(O)R₂A step of treating the compound; wherein the formula ClCH₂OC(O)R₂The molar ratio of compound to compound of formula B is from about 1 to about 1.5.

It is to be understood that R₂、R₅And R₆Each may include conventional protecting groups on the optional substituents.

In another embodiment, a process for preparing a compound of formula D is described wherein R₅And R₆As described in the various embodiments herein, for example, selected from optionally substituted alkyl or optionally substituted cycloalkyl; r₈Is C1-C6 n-alkyl; r₂As described in various embodiments herein, such as selected from optionally substituted alkyl or optionally substituted cycloalkyl; and R₇Is optionally substituted alkyl; wherein the method comprises the steps of:

a) preparation of a Compound of formula (E1) from a Compound of formula E, wherein X₁Is a leaving group; and

b) treating the compound of formula C under reducing conditions in the presence of a compound of formula E1.

It is to be understood that R₂、R₅、R₆And R₇Each may include conventional protecting groups on the optional substituents.

In another embodiment, a process for preparing a compound of formula F is described, wherein R is₅And R₆As described in the various embodiments herein, for example, selected from optionally substituted alkyl or optionally substituted cycloalkyl; r₂As described in various embodiments herein, such as selected from optionally substituted alkyl or optionally substituted cycloalkyl; and R₇Is optionally substituted alkyl; wherein the process comprises the step of treating the compound of formula D with a hydrolase.

It is to be understood that R₂、R₅、R₆And R₇Each may include conventional protecting groups on the optional substituents.

In another embodiment, a process for preparing a compound of formula G is described, wherein R₅And R₆As described in the various embodiments herein, for example, selected from optionally substituted alkyl or optionally substituted cycloalkyl; r₂As described in various embodiments herein, such as selected from optionally substituted alkyl or optionally substituted cycloalkyl; and R₇Is optionally substituted alkyl; wherein the process comprises the step of treating the silyl ether of compound F with a non-basic fluorine-containing reagent.

It is to be understood that R₂、R₅、R₆And R₇Each may include conventional protecting groups on the optional substituents.

In another embodiment, a process for preparing a compound of formula H is described, wherein R₅And R₆As described in the various embodiments herein, for example, selected from optionally substituted alkyl or optionally substituted cycloalkyl; r₂And R₄As described in various embodiments herein, such as selected from optionally substituted alkyl or optionally substituted cycloalkyl; and R₇Is optionally substituted alkyl; wherein the process comprises reacting a compound of formula G with a compound of formula R₄C(O)X₂Wherein X is₂Is a leaving group.

It is to be understood that R₂、R₄、R₅、R₆And R₇Each may include conventional protecting groups on the optional substituents.

In another embodiment, a process for preparing a compound of formula (T) is described, wherein Ar₁Is an optionally substituted aryl group; r₁Is hydrogen, optionally substituted alkyl, optionally substituted arylalkyl or a prodrug-forming group; r₅And R₆As described in the various embodiments herein, for example, selected from optionally substituted alkyl or optionally substituted cycloalkyl; r₃Is optionally substituted alkyl; r₂And R₄As described in the various embodiments herein, for example, selected from optionally substituted alkyl or optionally substituted cycloalkyl; and R₇Is optionally substituted alkyl; wherein the process comprises forming an active ester intermediate from a compound of formula H; and a step of reacting the active ester intermediate with a compound of formula I to obtain a compound of formula T.

It is understood that Ar₁、R₁、R₂、R₄、R₅、R₆And R₇Each may include conventional protecting groups on the optional substituents.

Detailed Description

In one embodiment, a process for preparing a compound of formula B is described, wherein R₅And R₆As described in the various embodiments herein, for example, selected from optionally substituted alkyl or optionally substituted cycloalkyl; and R₈Is C1-C6 n-alkyl; wherein the process comprises the step of treating a compound of formula a with triethylsilyl chloride and imidazole in an aprotic solvent.

In the previously reported preparation of the intermediate silyl ether of formula 2, the use of a large excess of triethylsilyl trifluoromethanesulfonate (TESOTf) and lutidine is described (see, e.g., Peltier, et al, 2006). It was found that the reported method necessitates a step of chromatographic purification of the product of the reaction. Contrary to what has been reported, it has been surprisingly found herein that the less active agent TESCl can be used. It has also been surprisingly found herein that TESCl, although a less active agent, can be used in near stoichiometric amounts in the methods described herein. It will be appreciated here that the use of less active TESCl is also advantageous when the process is carried out on a larger scale, whereas more active agents may represent a safety concern. It has also been found that the use of an approximately stoichiometric amount of TESCl makes a chromatographic purification step unnecessary. In an alternative embodiment, the process is carried out without subsequent purification. In another alternative to the foregoing embodiments and in various other embodiments described herein, R₅Is isopropyl. In another alternative to the foregoing embodiments and in various other embodiments described herein, R₆Is a middle schoolA butyl group. In another alternative to the foregoing embodiments and in various other embodiments described herein, R₈Is methyl. In another alternative to the preceding embodiments and in various other embodiments described herein, the silyl ether is TES.

In an illustrative example of the process described herein, a process is described for preparing silyl ether 2 in high yield, wherein compound 1 is treated with 1.05 equivalents of TESCl and 1.1 equivalents of imidazole.

In an alternative to the foregoing examples, compound 2 is not purified by chromatography.

In another embodiment, a process for preparing a compound of formula C is described, wherein R₅And R₆Each independently selected from optionally substituted alkyl or optionally substituted cycloalkyl; r₈Is C1-C6 n-alkyl; and R₂Selected from optionally substituted alkyl or optionally substituted cycloalkyl; wherein the process comprises contacting a compound of formula B with about 1 to about 1.5 equivalents of a base and about 1 to about 1.5 equivalents of a ClCH in an aprotic solvent at a temperature of about-78 ℃ to about 0 ℃₂OC(O)R₂And (3) treating the compound.

In another embodiment, the methods of the above embodiments are described wherein the compounds of formulae B and C have the stereochemistry shown in the following pairs of schemes B 'and C'.

In another exemplary embodiment, the above implementation is describedThe method of any of embodiments, wherein about 1 to about 1.3 equivalents of the formula ClCH are used₂OC(O)R₂The compound of (1). In another exemplary embodiment, a method is described in any of the above embodiments, wherein about 1.2 equivalents of the formula ClCH is used₂OC(O)R₂The compound of (1). In another exemplary embodiment, the method of any one of the above embodiments is described, wherein R is₂Is n-propyl. In another alternative to the preceding embodiments and various other embodiments described herein, R2 is CH₂CH(CH₃)₂、CH₂CH₂CH₃、CH₂CH₃、CH=C(CH₃)₂Or CH₃。

In an illustrative example of the process described herein, a process for preparing N, O-acetal 3 is described. In another illustrative example, compound 2 is treated with 1.1 equivalents of potassium hexamethyldisilazane (KHMDS) and 1.2 equivalents of chloromethyl butyrate in an aprotic solvent at about-45 ℃. In another illustrative example, the product formed from any of the preceding examples can be used without chromatographic purification.

In another embodiment, a process for preparing a compound of formula D is described wherein R₅And R₆Each independently selected from optionally substituted alkyl and cycloalkyl; r₈Is C1-C6 n-alkyl; r₂Selected from optionally substituted alkyl and cycloalkyl; and R₇Is optionally substituted alkyl; wherein the method comprises the steps of:

a) preparation of a Compound of formula (E1) from a Compound of formula E, wherein X₁Is a leaving group; and

b) treating the compound of formula C with a compound of formula E1 under reducing conditions.

In an exemplary embodiment, H is used₂And palladium on carbon catalyst (Pd/C), reducing a mixture of compound 3 and the pentafluorophenyl ester of D-N-methyl-pipecolic acid (pipecolic acid) to produce compound 4. It has been found herein that epimerization of the active ester of pipecolic acid can occur during the reaction or during its preparation or during the reduction under the reaction conditions reported above. For example, in a larger scale repetition of those reported methods, it is found herein that significant amounts of epimerized compounds are formed, as opposed to the previously reported absence of epimerization (see, e.g., Peltier, 2006). In addition, it is found herein that using the reported method, a significant amount of the rearrangement product of compound 8 formed via the butyryl rearrangement is formed. Finally, it is found herein that the typical yield of the desired product using the previously reported methods is only about half that reported. It has been found herein that the use of Diisopropylcarbodiimide (DIC) and short reaction times reduces the amount of both undesired by-products obtained from the epimerization reaction and by-products obtained from the rearrangement reaction. In another alternative to the foregoing embodiments, and in various other embodiments described herein, n is 3. In another alternative to the foregoing embodiments, and in various other embodiments described herein, R₇Is methyl.

In an exemplary embodiment, limiting the reaction time to produce D-N-methyl-pentafluorophenyl pipecolite to about 1 hour was found to reduce the formation of the stereoisomer tripeptide 9. It has also been found that the use of dry 10% Pd/C as the catalyst, rather than the more commonly used wet or moist catalyst, reduces the amount of epimer 9 formed during reduction. It has also been found that the use of dry 10% Pd/C and/or shorter reaction times can also reduce the formation of rearranged amide 8.

It has been previously reported that removal of the protecting group from the secondary hydroxyl group yields an inseparable mixture of the desired product 5 and cyclic O, N-acetal by-products, as shown in the scheme below.

In addition, in repeating the reported process, it was found herein that the removal of the methyl ester using basic conditions followed by acetylation of the hydroxyl group can form additional, previously unreported, byproduct iso-7. This additional by-product is difficult to detect and difficult to separate from the desired compound 7. Without being bound by theory, it is believed herein that iso-7 results from rearrangement of the butyrate group from a N-hydroxymethyl group to a secondary hydroxyl group, as shown below.

It has been found that the introduction of R at the secondary hydroxyl group₄After CO, readjusting (reordering) the sequence of the two deprotection steps and using different conditions for each deprotection reaction can improve the yield of a compound of formula H, e.g. compound 7, as detailed below.

In another embodiment, a process for preparing a compound of formula F is described, wherein R is₅And R₆Each independently selected from optionally substituted alkyl and optionally substituted cycloalkyl; r₂Selected from optionally substituted alkyl and optionally substituted cycloalkyl; and R₇Is optionally substituted alkyl; wherein the process comprises the step of treating the compound of formula D with a hydrolase.

In another embodiment, wherein the foregoing process of the treating step comprises adding a solution of compound D in a water miscible solvent to a buffered solution containing a hydrolase enzyme at a rate that minimizes precipitation of the ester. In another embodiment, the ester is added for a period of time from about 24 hours to about 100 hours. In another embodiment, the ester is added for a period of time from about 48 hours to about 100 hours. In another alternative to the foregoing embodiments, and in various other embodiments described herein, R₈Is methyl. In another embodiment, an embodiment of any of the preceding embodiments is described wherein the hydrolase is an esterase. In another embodiment, an embodiment of any of the preceding embodiments is described wherein the esterase is a porcine liver esterase.

In an exemplary embodiment, a solution of compound 4 in Dimethylsulfoxide (DMSO) is added to a buffered solution of pig liver esterase over a period of 90 hours. In another illustrative example, the buffer is a phosphate buffer. In another exemplary embodiment, the pH of the enzyme solution is 6.5-8.5. In another exemplary embodiment, an example of the enzyme solution has a pH of 7.4 to 7.8. It will be appreciated that the buffer substance used may be any buffer substance compatible with the hydrolase used to remove the ester.

In another embodiment, a process for preparing a compound of formula G is described, wherein R₅And R₆Each independently selected from optionally substituted alkyl and optionally substituted cycloalkyl; r₂Selected from optionally substituted alkyl and optionally substituted cycloalkyl; and R₇Is optionally substituted alkyl; wherein the process comprises the step of treating the silyl ether of the compound of formula F with a non-basic fluoride reagent. It has been found herein that the use of basic conditions can result in the formation of by-products resulting from the rearrangement of the ester group to form compound GAnd (4) generating.

In an exemplary embodiment, compound 6 is treated with Et in the preparation of the corresponding alcohol 6₃N.3HF treatment to crack TES-ether. It is understood that other non-basic fluoride reagents that cleave the silyl ether of compound F can be used in the methods and processes described herein, including but not limited to pyridine, HF, etc., to cleave TES-ethers.

In another embodiment, a process for preparing a compound of formula H is described, wherein R₅And R₆Each independently selected from optionally substituted alkyl and optionally substituted cycloalkyl; r₂And R₄Independently selected from optionally substituted alkyl and optionally substituted cycloalkyl; and R₇Is optionally substituted alkyl; wherein the process comprises reacting a compound of formula G with a compound of formula R₄C(O)X₂Wherein X is₂Is a leaving group. It will be appreciated that the resulting product may contain varying amounts of compounds H and R₄CO₂H, mixed anhydride. In another embodiment, the process described in the above embodiments further comprises the step of treating the reaction product with water to produce H free or substantially free of anhydride. In another embodiment, wherein X is described₂Is R₄CO₂The method of the above embodiment. In another embodiment, described are those in which R is₄A process according to any one of the above embodiments being a C1-C4 alkyl group. In another alternative to the foregoing embodiments, and in various other embodiments described herein, R₄Is methyl. In another embodiment, described are those in which R is₆The process of any one of the above embodiments being sec-butyl. In another embodiment, described are those in which R is₇Is methylThe method of any of the above embodiments. In another embodiment, described are those in which R is₅The method of any one of the above embodiments being isopropyl.

In an exemplary embodiment, compound 6' is treated with acetic anhydride in pyridine. It has been found herein that shortening the time of the steps of the process can improve the yield of compound H by limiting the amount of other acylated by-products (e.g., formula 7a) formed which have not been previously described. It is understood that the resulting product may contain varying amounts of the mixed anhydride of 7 and acetic acid. In another embodiment, the reaction product from the above step is treated with water in dioxane to produce compound 7, which is free or substantially free of anhydride. It will be clear that in the hydrolysis of the intermediate mixed anhydride, other solvents may be used instead of dioxane. Alternatively, this step may be carried out without a solvent.

In another embodiment, a method of preparing tubulin cytolysin T is described, wherein Ar₁Is an optionally substituted aryl group; r₁Is hydrogen, alkyl, arylalkyl or a prodrug-forming group; r₅And R₆Each independently selected from optionally substituted alkyl and optionally substituted cycloalkyl; r₃Is optionally substituted alkyl; r₂And R₄Independently selected from optionally substituted alkyl and optionally substituted cycloalkyl; and R₇Is optionally substituted alkyl; wherein the method comprises the following steps:

c) forming an active ester intermediate from the compound of formula H; and

d) reacting the activated ester intermediate with a compound of formula I.

It has been found herein that when the free acid of I (wherein R is₁Is hydrogen) when used in this step as previously reported, the desired product T can be reacted with additional amino acid I to form a polyamino acid by-product containing multiple copies of said amino acid I in side reactions not previously reported. It has also been found herein that removal of excess active ester former prior to addition of compound I can reduce or eliminate this side reaction to acceptable levels. In one embodiment, compound H is treated with an excess of active ester former and pentafluorophenol to form the pentafluorophenol ester of compound H, followed by removal of the excess active ester former prior to addition of compound I. In another alternative to the foregoing embodiments, and in various other embodiments described herein, Ar₁Is phenyl. In another alternative to the foregoing embodiments and in various other embodiments described herein, Ar₁Is a substituted phenyl group. In another alternative to the foregoing embodiments and in various other embodiments described herein, Ar₁Is a 4-substituted phenyl group. In another alternative to the foregoing embodiments and in various other embodiments described herein, Ar₁Is R_A-substituted phenyl. In another alternative to the foregoing embodiments and in various other embodiments described herein, Ar₁Is 4-hydroxyphenyl or a hydroxy protected form thereof. In another alternative to the foregoing embodiments and in various other embodiments described herein, R₃Is methyl. In another alternative to the foregoing embodiments and in various other embodiments described herein, R₁Is hydrogen.

In an exemplary embodiment, compound 7 is treated with an excess of carbodiimide in polymeric form and pentafluorophenol to form a pentafluorophenyl ester of 7, and the polymeric carbodiimide is removed by filtration; the amino acid (S) -tubulysine is then added to the solution to give tubulysin B. In another embodiment, a method is described in any of the above embodiments wherein the polymeric carbodiimide is polystyrene-CH 2-N = C = N-cyclohexane (PS-DCC).

In another embodiment, compounds having formula D are described wherein the compounds are free or substantially free of compounds having formula C-1 wherein R₂、R₅、R₆、R₇And R₈As described in any embodiment described herein. Without being bound by theory, it is believed herein that compound C-1 is formed from the corresponding compound C by acyl transfer.

In another embodiment, compound 4 is described which is free or substantially free of compound 8 and/or compound 9. In another embodiment, compound 4 is formed in optically pure form.

In another embodiment, compound H is described, wherein compound H is free or substantially free of a compound having the formula oxazin-2.

In another embodiment, compounds F are described wherein R is₂、R₅、R₆、R₇And R₈As described in any embodiment described herein.

In another embodiment, compounds having formula 6 are described.

In another embodiment, compounds G are described wherein the compounds are free or substantially free of compounds G', wherein R is₂、R₅、R₆And R₇As described in any embodiment described herein.

In another embodiment, compound 6 ' is described, wherein compound 6 ' is free or substantially free of the G ' isomer shown below.

In another embodiment, compound 7 is described, wherein compound 7 is free or substantially free of compound 7a described below.

In another embodiment, compounds H are described wherein R is₄Is Me, R₂、R₅、R₆And R₇As described in any of the embodiments described herein; and compound H is free or substantially free of compounds wherein R is₄And R₂All are Me compounds H.

In another alternative to the foregoing embodiments and in various other embodiments described herein, R₅Is isopropyl.

In another alternative to the foregoing embodiments and in various other embodiments described herein, R₆Is sec-butyl.

In another alternative to the foregoing embodiments and in various other embodiments described herein, R₈Is methyl.

In another alternative to the foregoing embodiments and in various other embodiments described herein, R₂Is CH₂CH(CH₃)₂、CH₂CH₂CH₃、CH₂CH₃、CH=C(CH₃)₂Or CH₃。

In another alternative to the foregoing embodiments and in various other embodiments described herein, n is 3.

In another alternative to the foregoing embodiments and in various other embodiments described herein, R₇Is methyl.

In another alternative to the foregoing embodiments and in various other embodiments described herein, R₈Is methyl.

In another alternative to the foregoing embodiments and in various other embodiments described herein, R₄Is methyl.

In another alternative to the preceding embodiment and hereinIn various other embodiments of the disclosure, Ar₁Is phenyl. In another alternative to the foregoing embodiments and in various other embodiments described herein, Ar₁Is a substituted phenyl group. In another alternative to the foregoing embodiments and in various other embodiments described herein, Ar₁Is a 4-substituted phenyl group. In another alternative to the foregoing embodiments and in various other embodiments described herein, Ar₁Is R_A-substituted phenyl. In another alternative to the foregoing embodiments and in various other embodiments described herein, Ar₁Is 4-hydroxyphenyl or a hydroxy protected form thereof.

In another alternative to the foregoing embodiments and in various other embodiments described herein, R₃Is methyl.

In another alternative to the foregoing embodiments and in various other embodiments described herein, R₁Is hydrogen.

Exemplary embodiments of the present invention are further illustrated by the following enumerated clauses:

1. a process for preparing a compound of the formula or a pharmaceutically acceptable salt thereof,

wherein Ar is₁Is an optionally substituted aryl group; r₁Is hydrogen, alkyl, arylalkyl or a prodrug-forming group; r₂Selected from optionally substituted alkyl and optionally substituted cycloalkyl; r₃Is optionally substituted alkyl; r₄Is optionally substituted alkyl or optionally substituted cycloalkyl; r₅And R₆Each independently selected from optionally substituted alkyl and optionally substituted cycloalkyl; r₇Is optionally substituted alkyl; and n is 1, 2, 3 or 4; wherein the method comprises the following steps: a step of treating the compound of formula A with triethylsilyl chloride and imidazole in an aprotic solvent, whichIn R₈Is C1-C6 unbranched alkyl

；

Or combining a compound of formula B with a base and a compound of formula ClCH in an aprotic solvent at a temperature of from about-78 ℃ to about 0 ℃₂OC(O)R₂A step of treating the compound; wherein the formula ClCH₂OC(O)R₂The molar ratio of compound to compound of formula B is from about 1 to about 1.5, wherein R₈Is C1-C6 unbranched alkyl

；

Or the following step a) preparing a compound of the formula (E1) from a compound of the formula E, wherein X₁Is a leaving group

；

And b) treating a compound of formula C, wherein R is R, under reducing conditions in the presence of a compound of formula E1₈Is C1-C6 unbranched alkyl

；

Or a step of treating the compound D with a hydrolase, wherein R₈Is C1-C6 unbranched alkyl

；

Or a step of treating the silyl ether of the compound F with a non-alkaline fluoride reagent

；

Or a compound of formula G with formula R₄C(O)X₂Wherein X is₂Is a leaving group

；

Or the following step c) formation of an active ester intermediate from the compound of formula H

；

And d) reacting the activated ester intermediate with a compound of formula I

；

Or a combination thereof. 1a. the method of clause 1, wherein R₄Is an optionally substituted alkyl group.

2. The process of clause 1 or 1a, which comprises the step of treating a compound of formula a with triethylsilyl chloride and imidazole in an aprotic solvent, wherein R₈Is C1-C6 unbranched alkyl

。

3. The method of clause 1 or 1a, which comprises contacting a compound of formula B with a base and a compound of formula ClCH in an aprotic solvent at a temperature of about-78 ℃ to about 0 ℃₂OC(O)R₂A step of treating the compound; wherein the formula ClCH₂OC(O)R₂The molar ratio of compound to compound of formula B is from about 1 to about 1.5, wherein R₈Is C1-C6 unbranched alkyl

。

4. The method of clause 1 or 1a, comprising the steps of: a) preparation of a Compound of formula (E1) from a Compound of formula E, wherein X₁Is a leaving group

；

And b) treating a compound of formula C, wherein R is R, under reducing conditions in the presence of a compound of formula E1₈Is C1-C6 unbranched alkyl

。

5. The method of clause 1 or 1a, comprising the step of treating compound D with a hydrolase, wherein R₈Is C1-C6 unbranched alkyl

。

6. The method of clause 1 or 1a, which comprises contacting a compound of formula G with a compound of formula R₄C(O)X₂Wherein X is₂Is a leaving group

。

7. The method of clause 1 or 1a, comprising the steps of: c) formation of an active ester intermediate from a compound of formula H

；

And d) reacting the activated ester intermediate with a compound of formula I

。

8. The method of any one of clauses 1-7 or 1a, wherein R₁Is hydrogen, benzyl or C1-C4 alkyl.

9. The method of any one of the preceding clauses wherein R₁Is hydrogen.

10. The method of any one of the preceding clauses wherein R₂Is C1-C8 alkyl or C3-C8 cycloalkyl.

11. The method of any one of the preceding clauses wherein R₂Is n-butyl.

12. The method of any one of the preceding clauses wherein R₃Is a C1-C4 alkyl group.

13. The method of any one of the preceding clauses wherein R₃Is methyl.

14. The method of any one of the preceding clauses wherein Ar is₁Is phenyl or hydroxyphenyl.

15. The method of any one of the preceding clauses wherein Ar is₁Is 4-hydroxyphenyl.

16. The method of any one of the preceding clauses wherein R₄Is C1-C8 alkyl or C3-C8 cycloalkyl.

17. The method of any one of the preceding clauses wherein R₄Is methyl.

18. The method of any one of the preceding clauses wherein R₅Is a branched C3-C6 or C3-C8 cycloalkyl.

19. The method of any one of the preceding clauses wherein R₅Is isopropyl.

20. The method of any of the preceding clausesWherein R is₆Is a branched C3-C6 or C3-C8 cycloalkyl.

21. The method of any one of the preceding clauses wherein R₅Is sec-butyl.

22. The method of any one of the preceding clauses wherein R₇Is a C1-C6 alkyl group.

23. The method of any one of the preceding clauses wherein R₇Is methyl.

24. The method of any one of the preceding clauses wherein R₂Is CH₂CH(CH₃)₂、CH₂CH₂CH₃、CH₂CH₃、CH=C(CH₃)₂Or CH₃。

25. The method of any one of the preceding clauses wherein Ar is₁Is a substituted phenyl group.

26. The method of any one of the preceding clauses wherein Ar is₁Is a 4-substituted phenyl group.

27. The method of any one of the preceding clauses wherein Ar is₁Is RA-substituted phenyl.

28. The method of any one of the preceding clauses wherein Ar is₁Is 4-hydroxyphenyl or a hydroxy protected form thereof.

It is to be understood that the term tubulysin as used herein refers to both the generic and individual designation of naturally occurring tubulysins, as well as analogs or derivatives of tubulysins. An illustrative example of tubulysins is given in table 1.

The term tubulysin as used herein generally refers to the compounds described herein and analogs and derivatives thereof. It is also to be understood that in each of the foregoing, any corresponding pharmaceutically acceptable salt is also included in the exemplary embodiments described herein.

It will be appreciated that such derivatives may include prodrugs of the compounds described herein, compounds described herein including one or more protecting groups, including compounds used in the preparation of other compounds described herein.

In addition, the term tubulysin as used herein also refers to prodrug derivatives of the compounds described herein, and includes prodrugs of various analogs and derivatives thereof. In addition, the term tubulysin as used herein refers to the amorphous form as well as any and all morphological forms of each of the compounds described herein. In addition, the term tubulysin, as used herein, refers to any and all hydrates or other solvates of the compounds described herein.

It is to be understood that the foregoing embodiments may be combined in a chemically related manner to produce subsets of the embodiments described herein. Thus, it is also to be understood that all such subgroups are also exemplary embodiments of the invention as described herein.

The compounds described herein may contain one or more chiral centers, or may otherwise have the ability to exist in multiple stereoisomers. It is to be understood that in one embodiment, the invention described herein is not limited to any particular stereochemical requirement, and that the compounds and compositions, methods, uses and medicaments containing them may be optically pure or may be in the form of any of a variety of stereoisomeric mixtures, including racemic and other mixtures of enantiomers, other mixtures of diastereomers, and the like. It is also understood that mixtures of these stereoisomers may include a single stereochemical configuration at one or more chiral centers, while including mixtures of stereochemical configurations at one or more other chiral centers.

Similarly, the compounds described herein may include geometric centers, such as cis, trans, (E) -and (Z) -double bonds. It will be appreciated that in another embodiment, the invention described herein is not limited to any particular geometric isomer requirement, and that the compounds and compositions, methods, uses and medicaments comprising them may be pure or may be mixtures of any of the various geometric isomers. It is also understood that such mixtures of geometric isomers may include a single configuration at one or more double bonds, while including mixtures of geometric isomers at one or more other double bonds.

The term aprotic solvent as used herein refers to a solvent that does not produce a solute under the reaction conditions. Illustrative examples of aprotic solvents are Tetrahydrofuran (THF), 2, 5-dimethyl-tetrahydrofuran, 2-methyl-tetrahydrofuran, tetrahydropyran, diethyl ether, t-butyl methyl ether, dimethylformamide, N-methylpyrrolidone (NMP), and the like. It is to be understood that mixtures of these solvents may also be used in the processes described herein.

As used herein, an equivalent of a reagent refers to the theoretical amount of reagent necessary to convert a starting material to the desired product, i.e., if 1 mole of reagent is theoretically needed to convert 1 mole of starting material to 1 mole of product, then 1 equivalent of reagent represents 1 mole of reagent; if X moles of reagent are theoretically required to convert 1 mole of starting material to 1 mole of product, then 1 equivalent of reagent represents X moles of reagent.

The term active ester former, as used herein, generally refers to an agent or combination of agents that can be used to convert a carboxylic acid to an active ester.

The term active ester as used herein generally refers to a carboxylate compound in which the divalent oxygen moiety of the ester is a leaving group, which results in the production of an ester that is activated to react with a compound containing a functional group (e.g., amine, alcohol, or thiol). Illustrative examples of active ester-forming compounds are N-hydroxysuccinimide, N-hydroxyphthalimide, phenols substituted with electron-withdrawing groups, such as but not limited to 4-nitrophenol, pentafluorophenol, N' -disubstituted isoureas, substituted hydroxyheteroaryl groups, such as but not limited to 2-pyridinol, 1-hydroxybenzotriazole, 1-hydroxy-7-aza-benzotriazole, cyanocarbinol, and the like. For example, the reaction conditions for replacing the active ester with a compound having an amino, hydroxyl or thiol group are mild. For example, the reaction conditions for replacing the active ester with a compound having an amino, hydroxyl or thiol group are conducted at room temperature or below. For example, the reaction conditions for replacing the active ester with a compound having an amino, hydroxyl or thiol group are carried out without adding a strong base. For example, the reaction conditions for replacing the active ester with a compound having an amino, hydroxyl or thiol group are carried out with the addition of a tertiary amine base, such as one having a conjugate acid pKa of about 11 or less, about 10.5 or less, and the like.

The term "alkyl" as used herein includes an optionally branched chain of carbon atoms. The terms "alkenyl" and "alkynyl" as used herein include a chain of carbon atoms, which is optionally branched and includes at least one double or triple bond, respectively. It is understood that alkynyl groups may also include one or more double bonds. It is also understood that in certain embodiments, alkyl groups advantageously have a limited length, including C₁-C₂₄、C₁-C₁₂、C₁-C₈、C₁-C₆And C₁-C₄. It is also understood that in certain embodiments, alkenyl and/or alkynyl groups may each advantageously have a finite length, including C₂-C₂₄、C₂-C₁₂、C₂-C₈、C₂-C₆And C₂-C₄. It is understood herein that shorter alkyl, alkenyl and/or alkynyl groups may increase the lipophilicity of the compound to a lesser extent and will therefore have different pharmacokinetic behavior. Exemplary alkyl groups are, but not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, 2-pentyl, 3-pentyl, neopentyl, hexyl, heptyl, octyl and the like.

The term "cycloalkyl" as used herein includes an optionally branched chain of carbon atoms, at least a portion of which is cyclic. It is understood that cycloalkylalkyl is a subset of cycloalkyl groups. It is understood that the cycloalkyl group may be polycyclic. Exemplary cycloalkyl groups include, but are not limited to, cyclopropyl, cyclopentyl, cyclohexyl, 2-methylcyclopropyl, cyclopentylethyl-2-yl, adamantyl, and the like. The term "cycloalkenyl" as used herein includes an optionally branched chain of carbon atoms,and comprises at least one double bond, wherein at least a portion of the chain is cyclic. It is understood that one or more double bonds may be in the cyclic portion of the cycloalkenyl group and/or the acyclic portion of the cycloalkenyl group. It is understood that cycloalkenylalkyl and cycloalkenylalkenyl are each a subset of cycloalkenyl. It is understood that the cycloalkyl group may be polycyclic. Exemplary cycloalkenyl groups include, but are not limited to, cyclopentenyl, cyclohexylethen-2-yl, cycloheptenylpropenyl, and the like. It will also be appreciated that the chains forming the cycloalkyl and/or cycloalkenyl groups advantageously have a defined length, including C₃-C₂₄、C₃-C₁₂、C₃-C₈、C₃-C₆And C₅-C₆. It is understood that shorter alkyl and/or alkenyl chains forming cycloalkyl and/or cycloalkenyl groups, respectively, may increase the lipophilicity to the compound to a lesser extent and will therefore have different pharmacokinetic behavior.

The term "heteroalkyl," as used herein, includes a chain of carbon atoms that includes carbon and at least one heteroatom and that is optionally branched. Exemplary heteroatoms include nitrogen, oxygen, and sulfur. In certain variations, exemplary heteroatoms also include phosphorus and selenium. The term "cycloheteroalkyl," as used herein, including heterocyclyl and heterocyclic rings, includes a chain of atoms including carbon and at least one heteroatom, such as heteroalkyl, and optionally branched, wherein at least a portion of the chain is cyclic. Exemplary heteroatoms include nitrogen, oxygen, and sulfur. In certain variations, exemplary heteroatoms also include phosphorus and selenium. Exemplary cycloheteroalkyl groups include, but are not limited to, tetrahydrofuranyl, pyrrolidinyl, tetrahydropyranyl, piperidinyl, morpholinyl, piperazinyl, homopiperazinyl, quinuclidinyl, and the like.

The term "aryl" as used herein includes monocyclic and polycyclic aryl groups, including aromatic carbocyclic and aromatic heterocyclic groups, each of which may be optionally substituted. The term "carboaryl" as used herein includes aromatic carbocyclic groups, each of which may be optionally substituted. Exemplary aromatic carbocyclic groups include, but are not limited to, phenyl, naphthyl, and the like. The term "heteroaryl" as used herein includes aromatic heterocyclic groups, each of which may be optionally substituted. Exemplary aromatic heterocyclic groups include, but are not limited to, pyridyl, pyrimidinyl, pyrazinyl, triazinyl, tetrazinyl, quinolinyl, quinazolinyl, quinoxalinyl, thienyl, pyrazolyl, imidazolyl, oxazolyl, thiazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, triazolyl, benzimidazolyl, benzoxazolyl, benzothiazolyl, benzisoxazolyl, benzisothiazolyl, and the like.

The term "amino" as used herein includes the group NH2, alkylamino and dialkylamino wherein the two alkyl groups in the dialkylamino group can be the same or different, i.e. alkylalkylamino. For example, alkyl groups include methylamino, ethylamino, dimethylamino, methylethylamino, and the like. In addition, it is to be understood that when an amino group is modified or modified by another term, such as aminoalkyl or acylamino, variations of the above-described term are included therein. For example, aminoalkyl includes H₂N-alkyl, methylaminoalkyl, ethylaminoalkyl, dimethylaminoalkyl, methylethylaminoalkyl and the like, for example, acylamino includes acylmethylamino, acylethylamino and the like.

The term "amino and derivatives thereof" as used herein includes amino and alkylamino, alkenylamino, alkynylamino, heteroalkylamino, heteroalkenylamino, heteroalkynylamino, cycloalkylamino, cycloalkenylamino, cycloheteroalkynylamino, cycloheteroalkenylamino, arylamino, arylalkylamino, arylalkenylamino, arylalkynylamino, acylamino and the like, each of which may be optionally substituted, as described herein. The term "amino derivative" also includes urea, carbamate, and the like.

The term "hydroxy and derivatives thereof" as used herein includes OH and alkoxy, alkenyloxy, alkynyloxy, heteroalkyloxy, heteroalkenyloxy, heteroalkynyloxy, cycloalkyloxy, cycloalkenyloxy, cycloheteroalkyloxy, cycloheteroalkenyloxy, aryloxy, arylalkyloxy, arylalkenyloxy, arylalkynyloxy, acyloxy and the like, each of which may be optionally substituted. The term "hydroxy derivative" also includes carbamates and the like.

The term "thiol (thio) and derivatives thereof" as used herein includes SH and alkylthio, alkenylthio, alkynylthio, heteroalkylthio, heteroalkenylthio, heteroalkynylthio, cycloalkylthio, cycloalkenylthio, cycloheteroalkenylthio, arylthio, arylalkylthio, arylalkenylthio, arylalkynylthio, acylthio and the like, each of which may be optionally substituted. The term "thiol derivative" also includes thiocarbamates and the like.

The term "acyl" as used herein includes formyl and alkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, heteroalkylcarbonyl, heteroalkenylcarbonyl, heteroalkynylcarbonyl, cycloalkylcarbonyl, cycloalkenylcarbonyl, cycloheteroalkylcarbonyl, cycloheteroalkenylcarbonyl, arylcarbonyl, arylalkylcarbonyl, arylalkenylcarbonyl, arylalkynylcarbonyl, acylcarbonyl, and the like, each of which may be optionally substituted.

As used herein, the term "carboxylate salt and derivatives thereof" includes the group CO₂H and its salts, and esters and amides thereof, and CN.

The term "optionally substituted" as used herein includes replacing a hydrogen atom with another functional group on the optionally substituted group. Such other functional groups illustratively include, but are not limited to, amino, hydroxyl, halo, thiol, alkyl, haloalkyl, heteroalkyl, aryl, arylalkyl, arylheteroalkyl, nitro, sulfonic acids and derivatives thereof, carboxylic acids and derivatives thereof, and the like. For example, any of the amino, hydroxyl, thiol, alkyl, haloalkyl, heteroalkyl, aryl, arylalkyl, arylheteroalkyl, and/or sulfonic acid may be optionally substituted.

The term "optionally substituted aryl" as used herein includes the replacement of a hydrogen atom with another functional group on the optionally substituted aryl. Such other functional groups illustratively include, but are not limited to, amino, hydroxyl, halo, thiol, alkyl, haloalkyl, heteroalkyl, aryl, arylalkyl, arylheteroalkyl, nitro, sulfonic acids and derivatives thereof, carboxylic acids and derivatives thereof, and the like. For example, any of the amino, hydroxyl, thiol, alkyl, haloalkyl, heteroalkyl, aryl, arylalkyl, arylheteroalkyl, and/or sulfonic acid may be optionally substituted.

Exemplary substituents include, but are not limited to, the group- (CH)₂)_xZ^XWherein x is an integer of 0 to 6 and Z^XSelected from halogen, hydroxy, alkanoyloxy, including C₁-C₆Alkanoyloxy, optionally substituted aroyloxy, alkyl, including C₁-C₆Alkyl, alkoxy, including C₁-C₆Alkoxy, cycloalkyl, including C₃-C₈Cycloalkyl, cycloalkoxy, including C_3-C₈Cycloalkoxy, alkenyl, including C₂-C₆Alkenyl, alkynyl, including C₂-C₆Alkynyl, haloalkyl, including C₁-C₆Haloalkyl, haloalkoxy, including C₁-C₆Haloalkoxy, halocycloalkyl, including C₃-C₈Halocycloalkyl, halocycloalkoxy, including C₃-C₈Halogenocycloalkoxy, amino, C₁-C₆Alkylamino radical, (C)₁-C₆Alkyl) (C₁-C₆Alkyl) amino, alkylcarbonylamino, N- (C)₁-C₆Alkyl) alkylcarbonylamino, aminoalkyl, C₁-C₆Alkylaminoalkyl, (C)₁-C₆Alkyl) (C₁-C₆Alkyl) aminoalkyl, alkylcarbonylaminoalkyl, N- (C)₁-C₆Alkyl) alkylcarbonylaminoalkyl, cyano and nitro; or Z^XIs selected from-CO₂R⁴and-CONR⁵R⁶Wherein R is⁴、R⁵And R⁶Each independently at each occurrence is selected from hydrogen, C₁-C₆Alkyl and aryl-C₁-C₆An alkyl group.

The term "prodrug" as used herein generally refers to any compound that, when administered to a biological system, produces a biologically active substance as a result of one or more spontaneous, enzyme-catalyzed, and/or metabolic chemical reactions or combinations thereof. In vivo, prodrugs generally act by releasing or regenerating a pharmacologically more active drug by enzymes (e.g., esterases, amidases, phosphatases, etc.), simple biochemical or other processes in the body. Such activation may occur by the action of endogenous host enzymes or non-endogenous enzymes before, after, or during administration of the prodrug to a host. Further details of prodrug use are found in u.s. Pat. number 5,627,165; and Pathalk et al, enzyme protecting group technology in organic synthesis (enzymic protecting groups in organic synthesis), Stereosel, biocat 775-797 (2000). It will be appreciated that once the goals of, for example, targeted delivery, safety, stability, etc., have been achieved, it is immediately advantageous to convert the prodrug to the initial drug, followed by a subsequent rapid elimination of the remaining prodrug-forming groups released.

Prodrugs can be prepared from the compounds described herein by linking a group that ultimately cleaves in vivo to one or more functional groups present on the compound (e.g., -OH-, -SH, -CO₂H、-NR₂) Illustrative prodrugs include, but are not limited to, carboxylic acid esters wherein the group is alkyl, aryl, aralkyl, acyloxyalkyl, alkoxycarbonyloxyalkyl, and esters of hydroxyl, thiol, and amine wherein the group attached is acyl, alkoxycarbonyl, aminocarbonyl, phosphate, or sulfate exemplary esters, also known as active esters, include, but are not limited to, 1-indanyl, N-oxysuccinimide, acyloxyalkyl such as acetoxymethyl, pivaloyloxymethyl, β -acetoxyethyl, β -pivaloyloxyethyl, 1- (cyclohexylcarbonyloxy) prop-1-yl, (1-aminoethyl) carbonyloxymethyl, and the like, alkoxycarbonyloxyalkyl such as ethoxycarbonyloxymethyl, α -ethoxycarbonyloxyethyl, β -ethoxycarbonyloxyethyl, and the like, dialkylaminoalkyl including di-lower alkylaminoalkyl such as dimethylaminomethyl, dimethylaminoethyl, diethylaminomethyl, diethylaminoethyl, and the like, 2- (alkoxycarbonyl) -2-alkenyl such as 2- (isobutoxycarbonyl) pent-2-enyl, 2- (ethoxycarbonyl) lactone, and the like, phthalidyl, and the likeAnd the like.

Other exemplary prodrugs comprise chemical moieties, such as amide or phosphorus groups, which function to increase the solubility and/or stability of the compounds described herein. Other exemplary prodrugs of amino groups include, but are not limited to (C)₃-C₂₀) An alkanoyl group; halo- (C)₃-C₂₀) An alkanoyl group; (C)₃-C₂₀) An alkenoyl group; (C)₄-C₇) A cycloalkanoyl group; (C)₃-C₆) -cycloalkyl (C)₂-C₁₆) An alkanoyl group; optionally substituted aroyl, such as unsubstituted aroyl or aroyl substituted with 1 to 3 substituents selected from: halogen, cyano, trifluoromethanesulfonyloxy, (C)₁-C₃) Alkyl and (C)₁-C₃) Alkoxy, each of which is optionally further mono-or polysubstituted with 1 to 3 halogen atoms; optionally substituted aryl (C)₂-C₁₆) Alkanoyl, such as aryl, unsubstituted or substituted with 1 to 3 substituents selected from: halogen, (C)₁-C₃) Alkyl and (C)₁-C₃) Alkoxy, each of which is optionally further substituted with 1 to 3 halogen atoms; and optionally substituted heteroarylalkanoyl having 1-3 heteroatoms selected from O, S and N on the heteroaryl group and 2-10 carbon atoms in the alkanoyl moiety, such as heteroaryl unsubstituted or substituted with 1-3 substituents selected from: halogen, cyano, trifluoromethanesulfonyloxy, (C)₁-C₃) Alkyl and (C)₁-C₃) Alkoxy, each of which is optionally further substituted with 1-3 halogen atoms. The exemplary groups are exemplary only, not exhaustive, and can be prepared by conventional methods.

It is understood that a prodrug may not possess significant biological activity itself, but upon administration undergoes one or more spontaneous, enzymatic, and/or metabolic chemical reactions or combinations thereof in vivo to yield a compound described herein that is biologically active or a precursor of the biologically active compound. However, it is understood that in some instances, a prodrug is biologically active. It is also understood that prodrugs are often used to improve drug efficacy or safety by improving oral bioavailability, pharmacodynamic half-life, and the like. Prodrugs also refer to derivatives of the compounds described herein that include groups that simply mask the undesirable properties of the drug or improve drug transport. For example, one or more of the compounds described herein may exhibit an undesirable property that is advantageously blocked or reduced, which may become a pharmacological, pharmaceutical or pharmacokinetic barrier in clinical drug applications, such as low oral drug absorption, lack of site specificity, chemical instability, toxicity and poor patient acceptance (poor taste, odor, injection site pain, etc.) and other properties. It is understood herein that prodrugs or other strategies using reversible derivatization may be used to optimize the clinical utility of the drug.

As used herein, the terms "treating," "contacting," or "reacting," when referring to a chemical reaction, refer to the addition or mixing of two or more reagents under appropriate conditions to produce the indicated and/or the desired product. It is to be understood that the reaction which produces the indicated and/or the desired product may not necessarily result directly from the combination of the two reagents initially charged, i.e., there may be one or more intermediates produced in the mixture which ultimately results in the formation of the indicated and/or the desired product.

The term "composition" as used herein generally refers to any product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts. It is to be understood that the compositions described herein can be prepared from each individual compound described herein or from salts, solutions, hydrates, solvates, or other forms of the compounds described herein. It is also understood that the compositions may be prepared from various amorphous, non-amorphous, partially crystalline, and/or other morphological forms of the compounds described herein. It is also understood that the compositions may be prepared from various hydrates and/or solvates of the compounds described herein. Thus, such pharmaceutical compositions listing the compounds described herein should be understood to include the various morphological forms and/or solvate or hydrate forms of each of the compounds described herein or any combination of these forms. For example, the composition may include one or more carriers, diluents, and/or excipients. The compounds described herein or compositions containing them can be formulated in therapeutically effective amounts into any of the usual dosage forms suitable for the methods described herein. The compounds described herein, or compositions containing them, including these formulations, can be administered by a wide variety of conventional routes for The methods described herein and in a wide variety of dosage formats using known procedures (see generally, Remington: The Science and Practice of Pharmacy, (21 step., 2005)).

Examples

Synthesis of dipeptide 3

4.9 g of dipeptide 1(11.6 mmol) was dissolved in 60 mL of dichloromethane, and imidazole (0.87g, 12.7 mmol) was added to the resulting solution at 0 ℃. The reaction mixture was warmed slightly to dissolve all solids and then cooled to 0 ℃. TESCl (2.02 mL,12.1 mmol) was added dropwise at 0 ℃ and the reaction mixture was stirred under argon and warmed to room temperature over 2 h. TLC (3:1 hexane/EtOAc) indicated complete conversion. The reaction was filtered to remove imidazole HCl salt, extracted with deionized water, the aqueous phase was washed back with dichloromethane, the combined organic phases were washed with brine, over Na₂SO₄Drying, filtering to remove Na₂SO₄Concentrated under reduced pressure, co-evaporated with toluene and then dried under high vacuum overnight to give 6.4 g of crude 2 (theoretical yield 5.9 g).

Crude product 2 was co-evaporated again with toluene and then used without further purification. The TES protected dipeptide was dissolved in 38 mL THF (anhydrous, no inhibitor) and cooled to-45 deg.C, stirred for 15 minutes, and then KHMDS (0.5M in toluene, 25.5mL, 12.8 mmol, 1.1 equiv) was added dropwise. After the addition of KHMDS was complete, the reaction mixture was stirred at-45 deg.C for 15 minutes and chloromethyl butyrate (1.8 mL, 1.2 equiv, 14 mmol) was added. The reaction mixture turned from light yellow to cyan. TLC (20% EtOAc/petroleum ether) indicated that most of the starting material had been converted. LC-MS indicated about 7% starting material remained. The reaction was quenched by addition of 3 mL MeOH, the mixture was warmed to room temperature and concentrated under reduced pressure to an oily residue. The residue was dissolved in petroleum ether and then passed through a short silica gel column to remove the potassium salt. The column was washed with 13% EtOAc/petroleum ether and the collected eluates were combined and concentrated under reduced pressure. The crude alkylation product was passed through an additional silica gel column (product/silica = 1:50) and eluted with 13% EtOAc/petroleum ether to remove residual starting material to give 5.7g of product 3 (2 steps, 76% yield).

Synthesis of tripeptide 4

Alkylated dipeptide 3 (4.3g, 7.0 mmol), N-methylpiperidine-2-carboxylate (MEP) (4.0g, 28.0 mmol, 4 equiv) and pentafluorophenol (5.7g, 30.8 mmol, 4.4 equiv) were added to the flask. N-methylpyrrolidone (NMP, 86mL) was added to the mixture. To the mixture was added diisopropylcarbodiimide (DIC, 4.77 mL, 30.8 mmol, 4.4 equiv). The mixture was stirred at room temperature for 1 h. Pd/C (10%, dry, 1.7g) was added. The flask was shaken under hydrogen (30-35 psi) for 5 hours. The reaction mixture was analyzed by HPLC. The starting material was found to be below 3%. The mixture was filtered through celite. The diatomaceous earth was extracted with 200mL ethyl acetate. The filtrate and ethyl acetate extract were combined and transferred to a separatory funnel with 1% NaHCO₃Wash with 10% NaCl solution (200 mL. times.4). The organic layer was separated and evaporated under reduced pressure on a rotary evaporator. The crude product was dissolved in 40 mL of MeOH/H₂O (3: 1). The crude solution was loaded onto a Biotage C18 column (Flash 65i, 350g, 450mL, 65X 200 mM) using buffer A [10mM NH ]₄OAc/ACN (1:1)]And B (CAN, acetonitrile). Fractions were collected and the organic solvent was evaporated on a rotary evaporator. 100 mL of 10% NaCl solutionAnd 100 mL of methyl tert-butyl ether (MTBE) were added to the flask, and the mixture was transferred to a separatory funnel. Separating the organic layer over anhydrous Na₂SO₄Dried, filtered and evaporated to dryness on a rotary evaporator. Yield was 2.5g tripeptide intermediate 4 (50% yield).

Synthesis of tripeptide acid 6

To 2L of 0.05M phosphate (pH =7.4) was added 3.6 g pig liver esterase (17 units/mg) at 30 ℃. 3.0g of methyl ester 4 were dissolved in 100 mL of DMSO. A first 50mL portion of this solution was added at a rate of 1.1 mL/h and a second 50mL portion was added via syringe at a rate of 1.2 mL/h. After the addition was complete, the reaction mixture was stirred at 30 ℃ for several hours. HPLC of EtOAc extracts of the reaction mixture indicated completion of the reaction. The reaction mixture was discharged from the reactor in 1L portions and extracted with EtOAc (3 × 1L). The combined organic extracts were washed with brine, over Mg₂SO₄Dried and concentrated under reduced pressure. 2.8 g of product 6(95%) are recovered. UPLC analysis indicated that the product was pure, removing pentafluorophenol remaining from the previous reaction.

Intermediate 6 spectroscopic data:

synthesis of tubulysin B

1.4 g (2.01 mmol) of tripeptide 6 are dissolved in 8.4 mL of THF, 327.4. mu.L (2.01 mmol) of 3HF NEt are added₃The reaction mixture was stirred for 30 minutes. LC-MS analysis (10% -100% acetonitrile, pH 7 buffer) confirmed TES group depletionAnd (5) finishing protection. THF was removed under reduced pressure. The residue was dried under high vacuum for 5 minutes. The crude product was dissolved in 8.4 mL of anhydrous pyridine. 2.85 mL (30.15 mmol, 15 equiv) of Ac was added at 0 deg.C₂And O. The resulting clear solution was stirred at room temperature for 3.5 hours. LC-MS analysis (10% -100% acetonitrile, pH 7.0) indicated conversion>98 percent. 56 mL of dioxane/H was added₂O, the resulting mixture was stirred at room temperature for 1 hour. The mixture was concentrated under reduced pressure. The residue was co-evaporated with toluene (3 ×) and dried under high vacuum overnight. The crude product 7 was used directly in the next reaction.

Intermediate 7 spectral data:

method A. the crude tripeptide acid 7 was dissolved in 28 mL EtOAc (anhydrous) and 740 mg (4.02 mmol, 2.0 equiv) pentafluorophenol was added followed by 1.04 g (5.03 mmol, 2.5 equiv) DCC. The resulting reaction mixture was stirred at room temperature for 1 hour. LC-MS (5% -80% acetonitrile, pH =2.0, formic acid) analysis indicated conversion>95 percent. The urea by-product was filtered off, the EtOAc was removed under reduced pressure and the residue was dried under high vacuum for 5 min. The residue was dissolved in 8.4 mL of DMF and tyrosine tubulin hydrochloride (Tut-HCl, 678.7 mg, 2.61 mmol, 1.3 equiv) was added followed by DIPEA (2.28 mL, 13.07 mmol, 6.5 equiv). The resulting clear solution was stirred at room temperature for 10 minutes. The reaction mixture was diluted with DMSO and purified by preparative-HPLC (X-bridge column, 10mM NH)₄OAc, pH =6.3, 25% -100% acetonitrile). The pure fractions were combined, acetonitrile removed under reduced pressure, extracted with EtOAc (3X), and dried Na₂SO₄. EtOAc was removed under reduced pressure and the residue was dried under high vacuum for 1 hour to give 513 mg of the desired product (combined yield from 6: 31%).

Method B. tripeptide 7 (229 mg, 0.367mmol) was dissolved in EtOAc (anhydrous), 134.9 mg (0.733 mmol, 2.0 equiv) pentafluorophenol was added, followed by 970 mg (1.84 mmol, 5.0 equiv) DCC resin. The resulting reaction mixture was stirred at room temperature for 16 hThen (c) is performed. LC-MS analysis shows conversion rate>96 percent. The reaction mixture was filtered and concentrated to dryness and dried under high vacuum for 5 minutes. The residue was dissolved in 3.5 mL of DMF and Tut-HCl (123.9 mg, 0.477 mmol, 1.3 equiv) was added followed by DIPEA (0.42 mL, 2.386 mmol, 6.5 equiv). The resulting clear solution was stirred at room temperature for 10 minutes. The reaction mixture was diluted with DMSO and purified on a prep-HPLC (X-bridge column, 10mM NH)₄OAc,25% -100% acetonitrile, two rounds). The pure fractions were combined, acetonitrile was removed under reduced pressure, the residue was extracted with EtOAc (2X), and the combined EtOAc extracts were extracted over Na₂SO₄And (5) drying. EtOAc was removed under reduced pressure. The residue was dried under high vacuum for 1 hour to give 175 mg of the desired product (58% combined yield from 6).

Large-Scale Synthesis of dipeptide 3

10.2 g of dipeptide 1(25.6 mmol) was dissolved in 130 mL of dichloromethane, and imidazole (1.9g, 28.1 mmol) was added to the resulting solution at 0 ℃. The reaction mixture was warmed slightly to dissolve all solids and then cooled to 0 ℃. TESCl (4.5 mL,26.8 mmol) was added dropwise at 0 ℃ and the reaction mixture was stirred under argon and warmed to room temperature over 2 h. TLC (3:1 hexane/EtOAc) indicated complete conversion. The reaction was filtered to remove imidazole HCl salt, extracted with deionized water, the aqueous phase was washed back with dichloromethane, the combined organic phases were washed with brine, over Na₂SO₄Drying and filtering to remove Na₂SO₄Concentrated under reduced pressure, co-evaporated with toluene and then dried under high vacuum overnight to give 12.2 g of product 2.

Crude product 2 was co-evaporated again with toluene and used without further purification. TES protected dipeptide was dissolved in 80 mL THF (anhydrous, no inhibitor) and cooled to-45 deg.C, stirred for 15 minutes, then KHMDS (0.5M in toluene, 50mL, 25.0 mmol, 1.05 equiv) was added dropwise. After the addition of KHMDS was complete, the reaction mixture was stirred at-45 deg.C for 15 minutes and chloromethyl butyrate (3.6 mL, 1.2 equiv, 28.3 mmol) was added. The reaction mixture turned from light yellow to cyan. TLC (20% EtOAc/petroleum ether) indicated completion of the reaction. The reaction was quenched by addition of 20 mL MeOH, the mixture was warmed to room temperature and concentrated under reduced pressure to an oily residue. The residue was dissolved in petroleum ether and then passed through a short silica gel column to remove the potassium salt. The column was washed with 13% EtOAc/petroleum ether and the collected eluates were combined and concentrated under reduced pressure to give 12.1 g of product 3 (2 steps, 76% yield).

Large-Scale Synthesis of tripeptide 4

Alkylated dipeptide 3 (7.6g, 12.4 mmol), N-methylpiperidine-2-carboxylate (MEP) (7.0g, 48.9 mmol, 4 equiv) and pentafluorophenol (10.0 g, 54.3 mmol, 4.4 equiv) were added to the flask. N-methylpyrrolidone (NMP, 152 mL) was added to the mixture. To the mixture was added diisopropylcarbodiimide (DIC, 8.43 mL, 54.4 mmol, 4.4 equiv). The mixture was stirred at room temperature for 1 h. Pd/C (10%, dry, 3.0g) was added. The flask was shaken under hydrogen (30-35 psi) for 5 hours. The reaction mixture was analyzed by HPLC. The reaction was complete. The mixture was filtered through celite. The celite was washed with 500 mL ethyl acetate. The solutions were combined and transferred to a separatory funnel, washed with 1% NaHCO 3/10% NaCl solution (250 mL. times.4). The organic layer was separated and evaporated under reduced pressure on a rotary evaporator. The crude product was dissolved in dichloromethane and the urea was filtered. The crude solution was loaded onto a Teledyne Redisep silica gel column (330g) and purified using EtOAc/petroleum ether on a Combiflash flash chromatography system. Fractions were collected and the organic solvent was removed by evaporation to yield 5.0g tripeptide (61% yield). NMR and mass spectral data were consistent with those determined in this example.

Large-Scale Synthesis of tripeptide acid 6

To 2L of 0.05M phosphate (pH =7.4) was added 3.6 g pig liver esterase (17 units/mg) at 30 ℃. 3.0g of methyl ester 4 were dissolved in 100 mL of DMSO. A first 50mL portion of this solution was added at a rate of 1.1 mL/h, followed by a second 50mL portion of this solution via syringe at a rate of 1.2 mL/h. After the addition was complete, the reaction mixture was stirred at 30 ℃ for several hours. HPLC of EtOAc extracts of the reaction mixture indicated completion of the reaction. The reaction mixture was discharged from the reactor in 1L portions and extracted with 94% EtOAc-6% MeOH (vol./vol.) solution (3 × 1L). The combined extracts were washed with brine, over Na₂SO₄Dried and concentrated under reduced pressure. 2.8 g of product 6(95%) are recovered. UPLC analysis indicated that the product was pure, removing pentafluorophenol remaining from the previous reaction.

Large-scale synthesis of tubulysin B

3.0g (4.30 mmol) of tripeptide 6 is dissolved in 18 mL of THF, 0.70 mL (4.30 mmol) of 3HF · NEt3 is added, and the reaction mixture is stirred for 30 minutes. LC-MS analysis (10% -100% acetonitrile, pH 7 buffer) confirmed the completion of the deprotection of the TES group. THF was removed under reduced pressure. The residue was dried under high vacuum for 5 minutes. The crude product was dissolved in 18 mL of anhydrous pyridine. 6.11 mL (64.50mmol, 15 equiv) of Ac were added at 0 deg.C₂And O. The resulting clear solution was stirred at room temperature for 5 hours. LC-MS analysis (10% -100% acetonitrile, pH 7.0) indicated conversion>98 percent. 117 mL of dioxane/H2O was added, and the resulting mixture was stirred at room temperature for 1 hour. The mixture was concentrated under reduced pressure. The residue was co-evaporated with toluene (3 ×) and dried under high vacuum overnight. The crude product 7 was used directly in the next reaction. LCMS (ESI) [ M + H ]]+ 625.2; the NMR spectroscopic data was consistent with structure 7.

Method B. crude tripeptide acid 7 (2.67 g, 4.30mmol) is dissolved in 43 mL DCM (anhydrous) and 1.59 g (8.6 mm) is addedol, 2.0 equiv) pentafluorophenol, followed by 9.33 g (21.5mmol, 5.0 equiv) of DCC on resin. The resulting reaction mixture was stirred at room temperature for 16 hours. LC-MS analysis shows conversion rate>96 percent. The reaction mixture was filtered and concentrated to dryness and dried under high vacuum for 5 minutes. The residue was dissolved in 16.5 mL of DMF and Tut-HCl (1.45 g, 5.59 mmol, 1.3 equiv) was added followed by DIPEA (4.88 mL,27.95 mmol, 6.5 equiv). The resulting clear solution was stirred at room temperature for 10 minutes. The reaction mixture was purified on preparative-HPLC (X-bridge column, 50mM NH)₄HCO₃25% -100% acetonitrile, 6 rounds). The pure fractions were combined, acetonitrile was removed under reduced pressure, the residue was extracted with EtOAc (2X), and the combined EtOAc extracts were extracted over Na₂SO₄And (5) drying. EtOAc was removed under reduced pressure. The residue is dried under high vacuum for 1 hour to yield 1.35 g of the desired product (combined yield from 4: 38%). The NMR spectroscopic data were consistent with tubulin cytolysin B.

Claims (29)

1. A process for preparing a compound of formula T or a pharmaceutically acceptable salt thereof,

wherein

Ar₁Is an optionally substituted aryl group;

R₁is hydrogen, alkyl, arylalkyl or a prodrug-forming group;

R₂selected from optionally substituted alkanesAnd optionally substituted cycloalkyl;

R₃is optionally substituted alkyl;

R₄is optionally substituted alkyl or optionally substituted cycloalkyl;

R₅and R₆Each independently selected from optionally substituted alkyl and optionally substituted cycloalkyl;

R₇is optionally substituted alkyl; and

n is 1, 2, 3 or 4;

wherein the method comprises the following steps:

a step of treating the compound D with a hydrolase, wherein R₈Is C1-C6 unbranched alkyl

Then the

Step of treating the silyl ether of Compound F with a non-basic fluoride reagent

2. The process of claim 1 further comprising the step of treating the compound of formula a, wherein R is triethylsilyl chloride and imidazole in an aprotic solvent₈Is C1-C6 unbranched alkyl

3. The process of claim 1 further comprising reacting the compound of formula ClCH with a base in an aprotic solvent at a temperature of from-78 ℃ to 0 ℃₂OC(O)R₂A step of treating the compound of formula B with a compound; wherein the formula ClCH₂OC(O)R₂The molar ratio of the compound to the compound of formula B is 1-1.5, wherein R₈Is C1-C6 unbranched alkyl

4. The method of claim 1, further comprising the steps of:

a) preparation of a Compound of formula (E1) from a Compound of formula E, wherein X₁Is a leaving group

And

b) treating a compound of formula C, wherein R is R, under reducing conditions in the presence of a compound of formula E1₈Is C1-C6 unbranched alkyl

5. The method of claim 1, further comprising treating the compound with a compound of formula R₄C(O)X₂A step of treating a compound of formula G wherein X₂Is a leaving group

6. The method of claim 1, further comprising the steps of:

c) formation of an active ester intermediate from a compound of formula H

And

d) reacting the activated ester intermediate with a compound of formula I

7. The method of any one of claims 1-6, wherein R₁Is hydrogen, benzyl or C1-C4 alkyl.

8. The method of any one of claims 1-6, wherein R₁Is hydrogen.

9. The method of any one of claims 1-6, wherein R₂Is C1-C8 alkyl or C3-C8 cycloalkyl.

10. The method of any one of claims 1-6, wherein R₂Is n-propyl.

11. The method of any one of claims 1-6, wherein R₃Is a C1-C4 alkyl group.

12. The method of any one of claims 1-6, wherein R₃Is methyl.

13. The method of any one of claims 1-6, wherein Ar₁Is phenyl or hydroxyphenyl.

14. The method of any one of claims 1-6, wherein Ar₁Is 4-hydroxyphenyl.

15. The method of any one of claims 1-6, wherein R₄Is C1-C8 alkyl or C3-C8 cycloalkyl.

16. The method of any one of claims 1-6, wherein R₄Is methyl.

17. The method of any one of claims 1-6, wherein R₅Is a branched C3-C6 or C3-C8 cycloalkyl.

18. The method of any one of claims 1-6, wherein R₅Is isopropyl.

19. The method of any one of claims 1-6, wherein R₆Is a branched C3-C6 or C3-C8 cycloalkyl.

20. The method of any one of claims 1-6, wherein R₅Is sec-butyl.

21. The method of any one of claims 1-6, wherein R₇Is a C1-C6 alkyl group.

22. The method of any one of claims 1-6, wherein R₇Is methyl.

23. The method of claim 1, wherein the method comprises:

a step of treating the compound D with a hydrolase, wherein R₈Is C1-C6 unbranched alkyl

Then the

Step of treating the silyl ether of Compound F with a non-basic fluoride reagent

Then the

With the formula R₄C(O)X₂A step of treating a compound of formula G wherein X₂Is a leaving group

Then the

The following steps

c) Formation of an active ester intermediate from a compound of formula H

And

d) reacting the activated ester intermediate with a compound of formula I

24. The method of claim 1, wherein the method comprises:

a step of treating a compound of formula A, wherein R is triethylsilyl chloride and imidazole, in an aprotic solvent₈Is a C1-C6 unbranched alkyl group,

then the

At a temperature of-78 ℃ to 0 ℃ in an aprotic solvent, with a base and a compound of formula ClCH₂OC(O)R₂A step of treating the compound of formula B with a compound; wherein the formula ClCH₂OC(O)R₂The molar ratio of the compound to the compound of formula B is 1-1.5, wherein R₈Is a C1-C6 unbranched alkyl group,

then the

The following steps

a) Preparation of a Compound of formula (E1) from a Compound of formula E, wherein X₁Is a leaving group

And

b) treating a compound of formula C, wherein R is R, under reducing conditions in the presence of a compound of formula E1₈Is C1-C6 unbranched alkyl

Then the

A step of treating the compound D with a hydrolase, wherein R₈Is C1-C6 unbranched alkyl

Then the

Step of treating the silyl ether of Compound F with a non-basic fluoride reagent

Then the

With the formula R₄C(O)X₂A step of treating a compound of formula G wherein X₂Is a leaving group

Then the

The following steps

c) Formation of an active ester intermediate from a compound of formula H

And

d) reacting the activated ester intermediate with a compound of formula I

25. The method of claim 1, wherein the method comprises:

a) a compound of formula (E1) is prepared from a compound of formula E by reacting the compound of formula E with pentafluorophenol in diisopropylcarbodiimide for 1 hour, wherein X₁Is a leaving group

And

b) treating a compound of formula C in the presence of a compound of formula E1 wherein R is₈Is C1-C6 unbranched alkyl

26. The method of any one of claims 23-25, wherein Ar₁Is 4-hydroxyphenyl.

27. The method of any one of claims 1-6 or 23-25, wherein R₁Is hydrogen, R₂Is C1-C8 alkyl or C3-C8 cycloalkyl, R₃Is C1-C4 alkyl, R₄Is C1-C8 alkyl or C3-C8 cycloalkyl, R₅Is a branched C3-C6 or C3-C8 cycloalkyl, R₆Is a branched C3-C6 or C3-C8 cycloalkyl, R₇Is C1-C6 alkyl, Ar₁Is benzeneA radical or a hydroxyphenyl radical.

28. The method of claim 27, wherein R₁Is hydrogen, R₂Is C1-C8 alkyl or C3-C8 cycloalkyl, R₃Is C1-C4 alkyl, R₄Is C1-C8 alkyl or C3-C8 cycloalkyl, R₅Is a branched C3-C6 or C3-C8 cycloalkyl, R₆Is a branched C3-C6 or C3-C8 cycloalkyl and R₇Is a C1-C6 alkyl group.

29. The method of any one of claims 1-6 or 23-25, wherein the compound of formula T is a compound of the formula or a salt thereof