US20140249315A1

US20140249315A1 - Processes for preparing tubulysins

Info

Publication number: US20140249315A1
Application number: US13/841,078
Authority: US
Inventors: Iontcho R. Vlahov; Christopher P. Leamon
Original assignee: Endocyte Inc
Current assignee: Endocyte Inc
Priority date: 2013-03-01
Filing date: 2013-03-15
Publication date: 2014-09-04
Also published as: EP2961764A1; EP2961764A4; CA2901337A1; US9771391B2; US20160016993A1; WO2014134543A1

Abstract

Processes for preparing tubulysins and derivatives thereof are described. In addition, processes for preparing unnatural tubulysins are described.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit, under 35 U.S.C. §119(e), of U.S. Provisional Application No. 61/771,429, filed Mar. 1, 2013, and U.S. Provisional Application 61/793,082, filed Mar. 15, 2013, the entirety of each of the disclosures of which are hereby incorporated herein by reference.

TECHNICAL FIELD

The invention described herein pertains to processes for preparing tubulysins and derivatives thereof. In particular, the processes pertain to the preparation of unnatural tubulysins.

BACKGROUND AND SUMMARY OF THE INVENTION

The tubulysins are members of a new class of natural products isolated from myxobacterial species (F. Sasse, et al., J. Antibiot. 2000, 53, 879-885). As cytoskeleton interacting agents, the tubulysins are mitotic poisons that inhibit tubulin polymerization and lead to cell cycle arrest and apoptosis (H. Steinmetz, et al., Chem. Int. Ed. 2004, 43, 4888-4892; M. Khalil, et al., ChemBioChem. 2006, 7, 678-683; G. Kaur, et al., Biochem. J. 2006, 396, 235-242). Tubulysins are extremely potent cytotoxic molecules, exceeding the cell growth inhibition of any clinically relevant traditional chemotherapeutic e.g. epothilones, paclitaxel, and vinblastine. Furthermore, they are potent against multidrug resistant cell lines (A. Domling, et al., Mol. Diversity. 2005, 9, 141-147). These compounds show high cytotoxicity tested against a panel of cancer cell lines with IC₅₀values in the low picomolar range; thus, they are of interest as potential anticancer therapeutics. Accordingly, processes for preparing tubulysins, including non-naturally occurring tubulysins are needed.
Tubulysins are described herein. Structurally, tubulysins often include linear tetrapeptoid backbones, including illustrative compounds having formula T or AT
and pharmaceutically acceptable salts thereof; wherein
Ar₁is optionally substituted aryl;
R₁is hydrogen, alkyl, arylalkyl or a pro-drug forming group;
R₂is selected from the group consisting of optionally substituted alkyl and optionally substituted cycloalkyl;
R₁₂is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl or heteroarylalkyl, each of which is optionally substituted;
R₄is optionally substituted alkyl or optionally substituted cycloalkyl;
R₃is optionally substituted alkyl;
R₅and R₆are each independently selected from the group consisting of optionally substituted alkyl and optionally substituted cycloalkyl;
R₇is optionally substituted alkyl; and
n is 1, 2, 3, or 4.
Another illustrative group of tubulysins described herein are more particularly comprised of one or more non-naturally occurring or hydrophobic amino acid segments, such as N-methyl pipecolic acid (Mep), isoleucine (Ile),
and analogs and derivatives of each of the foregoing. A common feature in the molecular architecture of the more potent natural occurring tubulysins is the acid and/or base sensitive N-acyloxymethyl substituent (or a N,O-acetal of formaldehyde) represented by R2-C(O) in the formula (T).
Another illustrative group of tubulysins described herein are those having formula 1.
Structures of Several Natural Tubulysins


Tubulysin	R_A	R₂

A	OH	CH₂CH(CH₃)₂
B	OH	CH₂CH₂CH₃
C	OH	CH₂CH₃
D	H	CH₂CH(CH₃)₂
E	H	CH₂CH₂CH₃
F	H	CH₂CH₃
G	OH	CH═C(CH₃)₂
H	H	CH₃
I	OH	CH₃

A total synthesis of tubulysin D possessing C-terminal tubuphenylalanine (R_A=H) (H. Peltier, et al., J. Am. Chem. Soc. 2006, 128, 16018-16019) has been reported. Recently, a modified synthetic protocol toward the synthesis of tubulysin B (R_A=OH) (O. Pando, et at., Org. Lett. 2009, 11, 5567-5569) has been reported. However, attempts to follow the published procedures to provide larger quantities of tubulysins were unsuccessful, being hampered in part by low yields, difficult to remove impurities, the need for expensive chromatographic steps, and/or the lack of reproducibility of several steps. The interest in using tubulysins for anticancer therapeutics accents the need for reliable and efficient processes for preparing tubulysins, and analogs and derivatives thereof. Described herein are improved processes for making tubulysins, or analogs or derivatives thereof, including compounds of formula (AT).
In one illustrative embodiment of the invention, processes for preparing tubulysins, or analogs or derivatives thereof, including compounds of formula (AT). The processes include one or more steps described herein. In another embodiment, a process is described for preparing a compound of formula B, wherein R₅and R₆are as described in the various embodiments herein, such as each being 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 R₆may each include conventional protection groups on the optional substituents.
In another embodiment, a process is described for preparing a compound of formula C, wherein R₅and R₆are as described in the various embodiments herein, such as each being independently selected from optionally substituted alkyl or optionally substituted cycloalkyl; R₈is C1-C6 n-alkyl; and R₂is as described in the various embodiments herein, such as being selected from optionally substituted alkyl or optionally substituted cycloalkyl; wherein the process comprises the step of treating a compound of formula B with a base and a compound of the formula ClCH₂OC(O)R₂in an aprotic solvent at a temperature below ambient temperature, such as in the range from about −78° C. to about 0° C.; wherein the molar ratio of the compound of the formula ClCH₂OC(O)R₂to the compound of formula B from about 1 to about 1.5.
It is to be understood that R₂, R₅and R₆may each include conventional protection groups on the optional substituents.
In another embodiment, a process is described for preparing a compound of formula D, wherein R₅and R₆are as described in the various embodiments herein, such as being selected from optionally substituted alkyl or optionally substituted cycloalkyl; R₈is C1-C6 n-alkyl; R₂is as described in the various embodiments herein, such as being selected from optionally substituted alkyl or optionally substituted cycloalkyl; and R₇is optionally substituted alkyl; wherein the process comprises the steps of
a) preparing a compound of formula (E1) where X₁is a leaving group from a compound of formula E; and
b) treating a compound of formula C under reducing conditions in the presence of the compound of formula E1.
It is to be understood that R₂, R₅, R₆, and R₇may each include conventional protection groups on the optional substituents.
In another embodiment, a process is described for preparing a compound of formula AF, wherein R₅and R₆are as described in the various embodiments herein, such as being selected from optionally substituted alkyl or optionally substituted cycloalkyl; R₂is as described in the various embodiments herein, such as being selected from optionally substituted alkyl or optionally substituted cycloalkyl; and R₇is optionally substituted alkyl; wherein the process comprises the step of contacting compound D with an alcohol, R₁₂OH, where R₁₂is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl or heteroarylalkyl, each of which is optionally substituted; and a transesterification catalyst. In another embodiment, the transesterification catalyst is selected from the group consisting of (R₁₃)₈Sn₄O₂(NCS)₄, (R₁₃)₂Sn(OAc)₂, (R₁₃)₂SnO, (R₁₃)₂SnCl₂, (R₁₃)₂SnS, (R₁₃)₃SnOH, and (R₁₃)₃SnOSn(R₁₃)₃, where R₁₃is independently selected from alkyl, arylalkyl, aryl, or cycloalkyl, each of which is optionally substituted. In another embodiment, the transesterification catalyst is (R₁₃)₂SnO. Illustrative examples of R₁₃are methyl, n-butyl. n-octyl, phenyl, o-MeO-phenyl, p-MeO phenyl, phenethyl, and benzyl.
It is to be understood that R₅, R₆, R₁₂, and R₇may each include conventional protection groups on the optional substituents.
In another embodiment, a process is described for preparing a compound of formula AG, wherein R₅and R₆are as described in the various embodiments herein, such as being selected from optionally substituted alkyl or optionally substituted cycloalkyl; R₂is as described in the various embodiments herein, such as being selected from optionally substituted alkyl or optionally substituted cycloalkyl; and R₇is optionally substituted alkyl; wherein the process comprises the step of contacting compound F with an alcohol, R₁₂OH, where R₁₂is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl or heteroarylalkyl, each of which is optionally substituted; and a transesterification catalyst. In another embodiment, the transesterification catalyst is selected from the group consisting of (R₁₃)₈Sn₄O₂(NCS)₄, (R₁₃)₂Sn(OAc)₂, (R₁₃)₂SnO, (R₁₃)₂SnCl₂, (R₁₃)₂SnS, (R₁₃)₃SnOH, and (R₁₃)₃SnOSn(R₁₃)₃, where R₁₃is independently selected from alkyl, arylalkyl, aryl, or cycloalkyl, each of which is optionally substituted. In another embodiment, the transesterification catalyst is (R₁₃)₂SnO. Illustrative examples of R₁₃are methyl, n-butyl. n-octyl, phenyl, o-MeO-phenyl, p-MeO phenyl, phenethyl, and benzyl.
It is to be understood that R₂, R₅, R₆, R₇, and R₁₂may each include conventional protection groups on the optional substituents.
In another embodiment, a process is described for preparing a compound of formula BG, wherein R₅and R₆are as described in the various embodiments herein, such as being selected from optionally substituted alkyl or optionally substituted cycloalkyl; R₂is as described in the various embodiments herein, such as being selected from optionally substituted alkyl or optionally substituted cycloalkyl; R₁₂is as described in the various embodiments herein, such as being selected from alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl or heteroarylalkyl, each of which is optionally substituted; and R₇is optionally substituted alkyl; wherein the process comprises the step of contacting compound AF with a metal hydroxide or carbonate. Illustrative examples of a metal hydroxide or carbonate include LiOH, Li₂CO₃, NaOH, Na₂CO₃, KOH, K₂CO₃, Ca(OH)₂, CaCO₃, Mg(OH)₂, MgCO₃, and the like.
It is to be understood that R₅, R₆, R₇, and R₁₂may each include conventional protection groups on the optional substituents.
In another embodiment, a process is described for preparing a compound of formula AH, wherein R₅and R₆are as described in the various embodiments herein, such as being selected from optionally substituted alkyl or optionally substituted cycloalkyl; R₂and R₄are as described in the various embodiments herein, such as being selected from optionally substituted alkyl or optionally substituted cycloalkyl; R₁₂is as described in the various embodiments herein, such as being selected from alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl or heteroarylalkyl, each of which is optionally substituted; and R₇is optionally substituted alkyl; wherein the process comprises the step of treating a compound of formula BG with an acylating agent of formula R₄C(O)X₂, where X₂is a leaving group.
It is to be understood that R₄, R₅, R₆, and R₇may each include conventional protection groups on the optional substituents.
In another embodiment, a process is described for preparing a tubulysin of formula (AT), wherein Ar₁is aryl or heteroaryl each of which is optionally substituted; R₁is hydrogen, optionally substituted alkyl, optionally substituted arylalkyl or a pro-drug forming group; R₅and R₆are as described in the various embodiments herein, such as being selected from optionally substituted alkyl or optionally substituted cycloalkyl; R₃is optionally substituted alkyl; R₂and R₄are as described in the various embodiments herein, such as being selected from optionally substituted alkyl or optionally substituted cycloalkyl; R₁₂is as described in the various embodiments herein, such as being selected from alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl or heteroarylalkyl, each of which is optionally substituted; and R₇is optionally substituted alkyl; wherein the process comprises the step of forming an active ester intermediate from a compound of formula AH; and reacting the active ester intermediate with a compound of the formula I to give a compound of the formula AT.
It is to be understood that Ar₁, R₁, R₂, R₄, R₅, R₆, R₇, and R₁₂may each include conventional protection groups on the optional substituents.
In another embodiment, a process is described for preparing a tubulysin linker derivative of formula (TL-2), wherein Ar₁is optionally substituted aryl or optionally substituted heteroaryl; Ar₂is optionally substituted aryl or optionally substituted heteroaryl; L is selected from the group consisting of
where p is an integer from about 1 to about 3, m is an integer from about 1 to about 4, and * indicates the points of attachment;
R^a, R^b, and R are each independently selected in each instance from the group consisting of hydrogen and alkyl; or at least two of R^a, R^b, or R are taken together with the attached carbon atoms to form a carbocyclic ring;
R_Arrepresents 0 to 4 substituents selected from the group consisting of amino, or derivatives thereof, hydroxy or derivatives thereof, halo, thio or derivatives thereof, alkyl, haloalkyl, heteroalkyl, aryl, arylalkyl, arylheteroalkyl, heteroaryl, heteroarylalkyl, heteroarylheteroalkyl, nitro, sulfonic acids and derivatives thereof, carboxylic acids and derivatives thereof; R₁is hydrogen, optionally substituted alkyl, optionally substituted arylalkyl or a pro-drug forming group; R₅and R₆are as described in the various embodiments herein, such as being selected from optionally substituted alkyl or optionally substituted cycloalkyl; R₃is optionally substituted alkyl; R₂and R₄are as described in the various embodiments herein, such as being selected from optionally substituted alkyl or optionally substituted cycloalkyl; and R₇is optionally substituted alkyl; wherein the process comprises the step of contacting compound TL, with an alcohol, R₁₂OH, where R₁₂is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl or heteroarylalkyl, each of which is optionally substituted; and a transesterification catalyst. In one embodiment the transesterification catalyst is TFA. In another embodiment, the transesterification catalyst is selected from the group consisting of (R₁₃)₈Sn₄O₂(NCS)₄, (R₁₃)₂Sn(OAc)₂, (R₁₃)₂SnO, (R₁₃)₂SnCl₂, (R₁₃)₂SnS, (R₁₃)₃SnOH, and (R₁₃)₃SnOSn(R₁₃)₃, where R₁₃is independently selected from alkyl, arylalkyl, aryl, or cycloalkyl, each of which is optionally substituted. In another embodiment, the transesterification catalyst is (R₁₃)₂SnO. Illustrative examples of R₁₃are methyl, n-butyl. n-octyl, phenyl, o-MeO-phenyl, p-MeO phenyl, phenethyl, and benzyl. It is to be understood that Ar₁, Ar₂, R₁, R₂, R₄, R₅, R₆, R₇, and R₁₂may each include conventional protection groups on the optional substituents.
In another embodiment, a process is described for preparing a tubulysin linker derivative of formula (TL-2), wherein Ar₁is optionally substituted aryl or optionally substituted heteroaryl; Ar_eis optionally substituted aryl or optionally substituted heteroaryl; L is selected from the group consisting of
wherein
p is an integer from about 1 to about 3, m is an integer from about 1 to about 4, and * indicates the points of attachment;
R^a, R^b, and R are each independently selected in each instance from the group consisting of hydrogen and alkyl; or at least two of R^a, R^b, or R are taken together with the attached carbon atoms to form a carbocyclic ring;
R_Arrepresents 0 to 4 substituents selected from the group consisting of amino, or derivatives thereof, hydroxy or derivatives thereof, halo, thio or derivatives thereof, alkyl, haloalkyl, heteroalkyl, aryl, arylalkyl, arylheteroalkyl, heteroaryl, heteroarylalkyl, heteroarylheteroalkyl, nitro, sulfonic acids and derivatives thereof, carboxylic acids and derivatives thereof;
R₁is hydrogen, optionally substituted alkyl, optionally substituted arylalkyl or a pro-drug forming group; R₅and R₆are as described in the various embodiments herein, such as being selected from optionally substituted alkyl or optionally substituted cycloalkyl; R₃is optionally substituted alkyl; R₂and R₄are as described in the various embodiments herein, such as being selected from optionally substituted alkyl or optionally substituted cycloalkyl; and R₇is optionally substituted alkyl;
wherein the process comprises the step of forming an active ester intermediate from a compound of formula AH; and reacting the active ester intermediate with a compound of the formula IL to give a compound of the formula TL-2.
In another embodiment, the process described in any of the embodiments described herein wherein Ar₁is optionally substituted aryl is described.
In another embodiment, the process described in any of the embodiments described herein wherein Ar₁is optionally substituted heteroaryl is described.
It is to be understood that Ar₁, Ar₂, R₁, R₁₂, R₃, R₄, R₅, R₆, and R₇may each include conventional protection groups on the optional substituents in any of the embodiments described herein.

DETAILED DESCRIPTION

In one embodiment, a process is described for preparing a compound of formula B, wherein R₅and R₆are as described in the various embodiments herein, such as being 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 preparations of the intermediate silyl ether of formula 2, use of a large excess of triethylsilyl trifluoromethylsulfonate (TESOTf) and lutidine is described (see, for example, Peltier, et al., 2006). It was found that the reported process makes it necessary to submit the product of the reaction to a chromatographic purification step. Contrary to that reported, it has been surprisingly discovered herein that the less reactive reagent TESCl may be used. It has also been surprisingly discovered herein that although TESCl is a less reactive reagent, it may nonetheless be used in nearly stoichiometric amounts in the processes described herein. It is appreciated herein that the use of the less reactive TESCl may also be advantageous when the process is performed on larger scales, where higher reactivity reagents may represent a safety issue. It has also been discovered that the use of TESCl in nearly stoichiometric amounts renders the chromatographic purification step unnecessary. In an alternative of the embodiment, the process is performed without subsequent purification. In another alternative of the foregoing embodiments, and each additional embodiment described herein, R₅is isopropyl. In another alternative of the foregoing embodiments, and each additional embodiment described herein, R₆is sec-butyl. In another alternative of the foregoing embodiments, and each additional embodiment described herein, R₈is methyl. In another alternative of the foregoing embodiments, and each additional embodiment described herein, the silyl ether is TES.
In an illustrative example of the processes described herein, a process for preparing the silyl ether 2 in high yield is described wherein compound 1 is treated with 1.05 equivalent of TESCl and 1.1 equivalent of imidazole.
In one alternative of the foregoing example, the compound 2 is not purified by chromatography.
In another embodiment, a process is described for preparing a compound of formula C, wherein R₅and R₆are each independently selected from the group consisting of optionally substituted alkyl and optionally substituted cycloalkyl; R₈is C1-C6 n-alkyl; and R₂is selected from the group consisting of optionally substituted alkyl and optionally substituted cycloalkyl; wherein the process comprises the step of treating a compound of formula B with from about 1 equivalent to about 1.5 equivalent of base and from about 1 equivalent to about 1.5 equivalent of a compound of the formula ClCH₂OC(O)R₂in an aprotic solvent at a temperature from about −78° C. to about 0° C.
In another embodiment, the process of the preceding embodiment is described wherein the compounds of formulae B and C have the stereochemistry shown in the following scheme for B′ and C′.
In another illustrative embodiment, the process of any one of the preceding embodiments is described wherein about 1 equivalent to about 1.3 equivalent of a compound of the formula ClCH₂OC(O)R₂is used. In another illustrative example, the process of any one of the preceding embodiments is described, wherein about 1.2 equivalent of a compound of the formula ClCH₂OC(O)R₂is used. In another illustrative example, the process of any one of the preceding embodiments is described wherein R₂is n-propyl. In another alternative of the foregoing embodiments, and each additional embodiment described herein, R₂is CH₂CH(CH₃)₂, CH₂CH₂CH₃, CH₂CH₃, CH═C(CH₃)₂, or CH₃.
In an illustrative example of the processes described herein, a process for preparing the N,O-acetal 3 is described. In another illustrative example, compound 2 is treated with 1.1 equivalent of potassium hexamethyldisilazane (KHMDS) and 1.2 equivalent of chloromethyl butanoate in a nonprotic solvent at about −45° C. In another illustrative example, the product formed by any of the preceding examples may be used without chromatographic purification.
In another embodiment, a process is described for preparing a compound of formula D, wherein R₅and R₆are each independently selected from the group consisting of optionally substituted alkyl and cycloalkyl; R₈is C1-C6 n-alkyl; R₂is selected from the group consisting of optionally substituted alkyl and cycloalkyl; and R₇is optionally substituted alkyl; wherein the process comprises the steps of
a) preparing a compound of formula (E1) where X₁is a leaving group from a compound of formula E; and
b) treating a compound of formula C under reducing conditions with the compound of formula E1.
In one illustrative example, a mixture of compound 3 and the pentafluorophenyl ester of D-N-methyl-pipecolic acid is reduced using H₂and a palladium-on-charcoal catalyst (Pd/C) to yield compound 4. It has been discovered herein that epimerization of the active ester of pipecolic acid can occur during reaction or during its preparation or during the reduction under the previously reported reaction conditions. For example, contrary to prior reports indicating that epimerization does not occur (see, for example, Peltier, 2006), upon repeating those reported processes on a larger scale it was found here that substantial amounts of epimerized compounds were formed. In addition, it was discovered herein that substantial amounts of rearrangement products formed by the rearrangement of the butyryl group to compound 8 were formed using the reported processes. Finally, it was discovered herein that the typical yields of the desired products using the previously reported processes were only about half of that reported. It has been discovered herein that using diisopropylcarbodiimide (DIC) and short reaction times lessens that amount of both the unwanted by-product resulting from the epimerization reaction and the by-product resulting from the rearrangement reaction. In another alternative of the foregoing embodiments, and each additional embodiment described herein, n is 3. In another alternative of the foregoing embodiments, and each additional embodiment described herein, R₇is methyl.
In one illustrative example, it was found that limiting the reaction time for the preparation of pentafluorophenyl D-N-methyl-pipecolate to about 1 hour lessened the formation of the diastereomeric tripeptide 9. It has also been discovered that using dry 10% Pd/C as catalyst, rather than a more typically used wet or moist catalyst, lessens the amount of epimer 9 formed during the reduction. It has also been discovered that using dry 10% P/C and/or shorter reaction times also lessens the formation of rearranged amide 8.
It has been previously reported that removal of the protecting group from the secondary hydroxyl group leads to an inseparable mixture of the desired product 5 and a cyclic O,N-acetal side-product as shown in the following scheme.
Further, upon repeating the reported process, it has been discovered herein that removal of the methyl ester using basic conditions, followed by acetylation of the hydroxyl group leads to an additional previously unreported side-product, iso-7. That additional side-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 the N-hydroxymethyl group to the secondary hydroxyl group, as shown below.
In another embodiment, a process is described for preparing a compound of formula AF, wherein R₅and R₆are as described in the various embodiments herein, such as being selected from optionally substituted alkyl or optionally substituted cycloalkyl; R₂is as described in the various embodiments herein, such as being selected from optionally substituted alkyl or optionally substituted cycloalkyl; and R₇is optionally substituted alkyl; wherein the process comprises the step of contacting compound D with an alcohol, R₁₂OH, where R₁₂is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl or heteroarylalkyl, each of which is optionally substituted; and a transesterification catalyst. In another embodiment the transesterification catalyst is trifluoroacetic acid (TFA). In another embodiment, the transesterification catalyst is selected from the group consisting of (R₁₃)₈Sn₄O₂(NCS)₄, (R₁₃)₂Sn(OAc)₂, (R₁₃)₂SnO, (R₁₃)₂SnCl₂, (R₁₃)₂SnS, (R₁₃)₃SnOH, and (R₁₃)₃SnOSn(R₁₃)₃, where R₁₃is independently selected from alkyl, arylalkyl, aryl, or cycloalkyl, each of which is optionally substituted. In another embodiment, the transesterification catalyst is (R₁₃)₂SnO. Illustrative examples of R₁₃are methyl, n-butyl. n-octyl, phenyl, o-MeO-phenyl, p-MeO phenyl, phenethyl, and benzyl.
It is to be understood that R₅, R₆, R₁₂, and R₇may each include conventional protection groups on the optional substituents.
In an illustrative example, compound 4 is heated with an alcohol and di-n-butyltin oxide at about 100° C. to yield ether 10. It is appreciated that a co-solvent may be present. In one embodiment, the molar ratio (tin oxide)/(compound 10) is about 0.01 to about 0.30, or about 0.02 to about 0.20, or about 0.05 to about 0.15, or about 0.05 to about 0.10
In another embodiment, a process is described for preparing a compound of formula BG, wherein R₅and R₆are as described in the various embodiments herein, such as being selected from optionally substituted alkyl or optionally substituted cycloalkyl; R₂is as described in the various embodiments herein, such as being selected from optionally substituted alkyl or optionally substituted cycloalkyl; R₁₂is as described in the various embodiments herein, such as being selected from alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl or heteroarylalkyl, each of which is optionally substituted; and R₇is optionally substituted alkyl; wherein the process comprises the step of contacting compound AF with a metal hydroxide or carbonate. Illustrative examples of a metal hydroxide or carbonate include LiOH, Li₂CO₃, NaOH, Na₂CO₃, KOH, K₂CO₃, Ca(OH)₂, CaCO₃, Mg(OH)₂, MgCO₃, and the like.
It is to be understood that R₅, R₆, R₇, and R₁₂may each include conventional protection groups on the optional substituents.
In an illustrative example, compound 10 is treated with LiOH.H₂O in a mixture of THF and water at about room temperature to yield compound 11. It is appreciated that the THF may be replaced with other solvents.
In another embodiment, a process is described for preparing a compound of formula AH, wherein R₅and R₆are each independently selected from the group consisting of optionally substituted alkyl and optionally substituted cycloalkyl; R₂and R₄are independently selected from the group consisting of optionally substituted alkyl and optionally substituted cycloalkyl; and R₇is optionally substituted alkyl; wherein the process comprises the step of treating a compound of formula BG with an acylating agent of formula R₄C(O)X₂, where X₂is a leaving group. It is appreciated that the resulting product may contain varying amounts of the mixed anhydride of compound AH and R₄CO₂H. In another embodiment, the process described in the preceding embodiment further comprises the step of treating the reaction product with water to prepare AH, free of or substantially free of anhydride. In another embodiment, the process of the preceding embodiments wherein X₂is R₄CO₂, is described. In another embodiment, the process of any one of the preceding embodiments wherein R₄is C1-C4 alkyl is described. In another alternative of the foregoing embodiments, and each additional embodiment described herein, R₄is methyl. In another embodiment, the process of any one of the preceding embodiments wherein R₆is sec-butyl is described. In another embodiment, the process of any one of the preceding embodiments wherein R₇is methyl is described. In another embodiment, the process of any one of the preceding embodiments wherein R₅is iso-propyl is described.
In an illustrative example, compound 11 is treated with acetic anhydride in pyridine. It is appreciated that the resulting product may contain varying amounts of the mixed anhydride of 12 and acetic acid. In another embodiment, treatment of the reaction product resulting from the preceding step with water in dioxane yields compound 12, free of or substantially free of anhydride. It is to be understood that other solvents can be substituted for dioxane in the hydrolysis of the intermediate mixed anhydride. Alternatively, the step may be performed without solvent.
In another embodiment, a process is described for preparing a tubulysin of formula (AT), wherein Ar₁is optionally substituted aryl; R₁is hydrogen, optionally substituted alkyl, optionally substituted arylalkyl or a pro-drug forming group; R₅and R₆are as described in the various embodiments herein, such as being selected from optionally substituted alkyl or optionally substituted cycloalkyl; R₃is optionally substituted alkyl; R₂and R₄are as described in the various embodiments herein, such as being selected from optionally substituted alkyl or optionally substituted cycloalkyl; R₁₂is as described in the various embodiments herein, such as being selected from alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl or heteroarylalkyl, each of which is optionally substituted; and R₇is optionally substituted alkyl; wherein the process comprises the step of forming an active ester intermediate from a compound of formula AH; and reacting the active ester intermediate with a compound of the formula I to give a compound of the formula AT.
It is to be understood that Ar₁, R₁, R₂, R₄, R₅, R₆, R₇, and R₁₂may each include conventional protection groups on the optional substituents.
In one embodiment, compound AH is treated with an excess amount of active ester forming agent and pentafluorophenol to form the pentafluorophenol ester of compound AH, followed by removal of the excess active ester forming agent prior to the addition of compound I. In another alternative of the foregoing embodiments, and each additional embodiment described herein, Ar₁is phenyl. In another alternative of the foregoing embodiments, and each additional embodiment described herein, Ar₁is substituted phenyl. In another alternative of the foregoing embodiments, and each additional embodiment described herein, Ar₁is 4-substituted phenyl. In another alternative of the foregoing embodiments, and each additional embodiment described herein, Ar₁is R_A-substituted phenyl. In another alternative of the foregoing embodiments, and each additional embodiment described herein, Ar₁is 4-hydroxyphenyl, or a hydroxyl protected form thereof. In another alternative of the foregoing embodiments, and each additional embodiment described herein, R₃is methyl. In another alternative of the foregoing embodiments, and each additional embodiment described herein, R₁is hydrogen.
In an illustrative example, compound 12 is treated with an excess amount of a polymeric version of a carbodiimide and pentafluorophenol to form the pentafluorophenyl ester of 12, the polymeric carbodiimide is removed by filtration; and amino acid (S)-tubutyrosine is added to the solution to yield the tubulysin, compound 13. In another embodiment, the process of any one of the preceding embodiments wherein the polymeric carbodiimide is polystyrene-CH₂—N═C═N-cyclohexane (PS-DCC) is described.
In another embodiment, a compound AF is described wherein R₁₂, R₅, R₆, and R₇are as described in the any of the embodiments described herein.
In another embodiment, the following compound is described wherein R₁₂, R₅, R₆, R₇and R₈are as described in the any of the embodiments described herein.
In another embodiment, the compound having formula 10 is described.
In another embodiment a compound BG, is described, wherein R₁₂, R₅, R₆, and R₇are as described in any of the embodiments described herein.
In another embodiment, compound 11 is described.
In another embodiment, compound 7 is described.
In another embodiment, a compound AH is described wherein R₄is Me and R₁₂, R₅, R₆, and R₇are as described in any of the embodiments described herein; and the compound H is free of or substantially free of the compound H wherein R₄and R₂are both Me.
In another alternative of the foregoing embodiments, and each additional embodiment described herein, R₅is isopropyl.
In another alternative of the foregoing embodiments, and each additional embodiment described herein, R₆is sec-butyl.
In another alternative of the foregoing embodiments, and each additional embodiment described herein, R₈is methyl.
In another alternative of the foregoing embodiments, and each additional embodiment described herein, R₂is CH₂CH(CH₃)₂, CH₂CH₂CH₃, CH₂CH₃, CH═C(CH₃)₂, or CH₃.
In another alternative of the foregoing embodiments, and each additional embodiment described herein, R₁₂is CH₂CH═CH₂, or CH₂(CH₂)nCH₃, where n is 1, 2, 3, 4, 5, or 6.
In another alternative of the foregoing embodiments, and each additional embodiment described herein, R₁₂is CH₂CH═CH₂, CH₂CH₂CH₂CH₃, or CH₂CH₂CH₂CH₂CH₃.
In another alternative of the foregoing embodiments, and each additional embodiment described herein, n is 3.
In another alternative of the foregoing embodiments, and each additional embodiment described herein, R₇is methyl.
In another alternative of the foregoing embodiments, and each additional embodiment described herein, R₄is methyl.
In another alternative of the foregoing embodiments, and each additional embodiment described herein, Ar₁is phenyl. In another alternative of the foregoing embodiments, and each additional embodiment described herein, Ar₁is substituted phenyl. In another alternative of the foregoing embodiments, and each additional embodiment described herein, Ar₁is 4-substituted phenyl. In another alternative of the foregoing embodiments, and each additional embodiment described herein, Ar₁is R_A-substituted phenyl. In another alternative of the foregoing embodiments, and each additional embodiment described herein, Ar₁is 4-hydroxyphenyl, or a hydroxyl protected form thereof.
In another alternative of the foregoing embodiments, and each additional embodiment described herein, R₃is methyl.
In another alternative of the foregoing embodiments, and each additional embodiment described herein, R₁is hydrogen.
Illustrative embodiments of the invention are further described by the following enumerated clauses:
1. A process for preparing a compound of the formula
or a pharmaceutically acceptable salt thereof; wherein Ar₁is optionally substituted aryl or optionally substituted heteroaryl;
R₁is hydrogen, alkyl, arylalkyl or a pro-drug forming group;
R₂is selected from the group consisting of optionally substituted alkyl and optionally substituted cycloalkyl;
R₁₂is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl or heteroarylalkyl, each of which is optionally substituted;
R₃is optionally substituted alkyl;
R₄is optionally substituted alkyl or optionally substituted cycloalkyl;
R₅and R₆are each independently selected from the group consisting of optionally substituted alkyl and optionally substituted cycloalkyl; R₇is optionally substituted alkyl; and n is 1, 2, 3, or 4;
wherein the process comprises the step of treating a compound of formula A with triethylsilyl chloride and imidazole in an aprotic solvent, where R₈is C1-C6 unbranched alkyl
or
the step of treating a compound of formula B with a base and a compound of the formula ClCH₂OC(O)R₂in an aprotic solvent at a temperature from about −78° C. to about 0° C.; wherein the molar ratio of the compound of the formula ClCH₂OC(O)R₂to the compound of formula B from about 1 to about 1.5, where R₈is C1-C6 unbranched alkyl
or
the steps of a) preparing a compound of formula (E1), where X₁is a leaving group, from a compound of formula E
and
b) treating a compound of formula C under reducing conditions in the presence of the compound of formula E1, where R₈is C1-C6 unbranched alkyl
or
the step of contacting compound D with an alcohol, R₁₂OH, where R₁₂is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl or heteroarylalkyl, each of which is optionally substituted; and a transesterification catalyst selected from TFA or the group consisting of (R₁₃)₈Sn₄O₂(NCS)₄, (R₁₃)₂Sn(OAc)₂, (R₁₃)₂SnO, (R₁₃)₂SnCl₂, (R₁₃)₂SnS, (R₁₃)₃SnOH, and (R₁₃)₃SnOSn(R₁₃)₃, where R₁₃is independently selected from alkyl, arylalkyl, aryl, or cycloalkyl, each of which is optionally substituted;
or
the step of treating the compound AF with a metal hydroxide or a metal carbonate;
or
the step of treating a compound of formula BG with an acylating agent of formula R₄C(O)X₂, where X₂is a leaving group
or
the steps of c) forming an active ester intermediate from a compound of formula AH
and
d) reacting the active ester intermediate with a compound of the formula I
or
one or more combinations thereof.
2. The process of clause 1 wherein Ar₁is optionally substituted aryl.
3. The process of clause 1 wherein Ar₁is optionally substituted heteroaryl.
4. A process for preparing a compound having formula (TL-2)
wherein
L is selected from the group consisting of
where p is an integer from about 1 to about 3, m is an integer from about 1 to about 4, and * indicates the points of attachment;
R^a, R^b, and R are each independently selected in each instance from the group consisting of hydrogen and alkyl; or at least two of R^a, R^b, or R are taken together with the attached carbon atoms to form a carbocyclic ring;
R_Arrepresents 0 to 4 substituents selected from the group consisting of amino, or derivatives thereof, hydroxy or derivatives thereof, halo, thio or derivatives thereof, alkyl, haloalkyl, heteroalkyl, aryl, arylalkyl, arylheteroalkyl, heteroaryl, heteroarylalkyl, heteroarylheteroalkyl, nitro, sulfonic acids and derivatives thereof, carboxylic acids and derivatives thereof;
wherein the process comprises the step of contacting a compound having formula (TL)
with R₁₂OH, where R₁₂is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl or heteroarylalkyl, each of which is optionally substituted; and a transesterification catalyst selected from TFA or the group consisting of (R₁₃)₈Sn₄O₂(NCS)₄, (R₁₃)₂Sn(OAc)₂, (R₁₃)₂SnO, (R₁₃)₂SnCl₂, (R₁₃)₂SnS, (R₁₃)₃SnOH, and (R₁₃)₃SnOSn(R₁₃)₃, where R₁₃is independently selected from alkyl, arylalkyl, aryl, or cycloalkyl, each of which is optionally substituted.
5. The process of any one of the preceding clauses wherein R₄is optionally substituted alkyl.
6. The process of any one of the preceding clauses comprising the step of treating a compound of formula A with triethylsilyl chloride and imidazole in an aprotic solvent, where R₈is C1-C6 unbranched alkyl
7. The process of any one of the preceding clauses comprising the step of treating a compound of formula B with a base and a compound of the formula ClCH₂OC(O)R₂in an aprotic solvent at a temperature from about −78° C. to about 0° C.; wherein the molar ratio of the compound of the formula ClCH₂OC(O)R₂to the compound of formula B from about 1 to about 1.5, where R₈is C1-C6 unbranched alkyl
8. The process of any one of the preceding clauses comprising the steps of
a) preparing a compound of formula (E1), where X₁is a leaving group, from a compound of formula E
and
b) treating a compound of formula C under reducing conditions in the presence of the compound of formula E1, where R₈is C1-C6 unbranched alkyl
9. The process of any one of the preceding clauses comprising the step of treating compound D with an alcohol, R₁₂OH, where R₁₂is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl or heteroarylalkyl, each of which is optionally substituted; and a transesterification catalyst selected from TFA or the group consisting of (R₁₃)₈Sn₄O₂(NCS)₄, (R₁₃)₂Sn(OAc)₂, (R₁₃)₂SnO, (R₁₃)₂SnCl₂, (R₁₃)₂SnS, (R₁₃)₃SnOH, and (R₁₃)₃SnOSn(R₁₃)₃, where R₁₃is independently selected from alkyl, arylalkyl, aryl, or cycloalkyl, each of which is optionally substituted;
10. The process of any one of the preceding clauses comprising the step of treating the compound AF with a metal hydroxide or a metal carbonate;
11. The process of any one of the preceding clauses comprising the step of treating a compound of formula BG with an acylating agent of formula R₄C(O)X₂, where X₂is a leaving group
12. The process of any one of the preceding clauses comprising the steps of
c) forming an active ester intermediate from a compound of formula AH
and
d) reacting the active ester intermediate with a compound of the formula I
13. A process for preparing a compound of the following formula
the process comprising the step of contacting a compound of the formula
with an acid and R₁₂OH, wherein R₂, R₅, R₆, R₈, and R₁₂are as described in any of the embodiments described herein.
14. A process for preparing a compound of the following formula
the process comprising the step of contacting a compound of the formula
with a transesterification catalyst selected from the group consisting of (R₁₃)₈Sn₄O₂(NCS)₄, (R₁₃)₂Sn(OAc)₂, (R₁₃)₂SnO, (R₁₃)₂SnCl₂, (R₁₃)₂SnS, (R₁₃)₃SnOH, and (R₁₃)₃SnOSn(R₁₃)₃, where R₁₃and R₁₂OH, wherein R₂, R₅, R₆, R₈, R₁₂, and R₁₃are as described in any of the embodiments described herein.
15. A process for preparing a compound of the following formula
the process comprising the step of contacting a compound of the formula
with a base and R₁₂OCH₂X, where X is Cl or Br; and wherein R₂, R₅, R₆, R₈, R₁₂, and R₁₃are as described in any of the embodiments described herein. In another embodiment, R₁₂OCH₂X is n-C₅H₁₁OCH₂Br.
16. A process for preparing a compound of the following formula
the process comprising the step of contacting a compound of the formula
with an acid and R₁₂OH, wherein R₁₄is Et₃Si or R₄C(O), and R₂, R₄, R₅, R₆, R₈, and R₁₂are as described in any of the embodiments described herein.
17. A process for preparing a compound of the following formula
the process comprising the step of contacting a compound of the formula
with a transesterification catalyst selected from the group consisting of (R₁₃)₈Sn₄O₂(NCS)₄, (R₁₃)₂Sn(OAc)₂, (R₁₃)₂SnO, (R₁₃)₂SnCl₂, (R₁₃)₂SnS, (R₁₃)₃SnOH, and (R₁₃)₃SnOSn(R₁₃)₃, where R₁₃; and R₁₂OH, wherein R₂, R₅, R₆, R₈, R₁₂, and R₁₃are as described in any of the embodiments described herein.
18. A process for preparing a compound of the following formula
the process comprising the step of contacting a compound of the formula
with an acid and R₁₂OH, wherein n, R₂, R₃, R₄, R₅, R₆, R₇, R₈, Ar₁, Ar₂, L and R₁₂are as described in any of the embodiments described herein.
19. The process of any one of the preceding clauses wherein R₁is hydrogen, benzyl, or C1-C4 alkyl.
19A. The process of any one of the preceding clauses wherein R₁is hydrogen.
20. The process of any one of the preceding clauses wherein R₂is C1-C8 alkyl or C3-C8 cycloalkyl.
20A. The process of any one of the preceding clauses wherein R₂is n-butyl.
20B. The process of any one of the preceding clauses wherein R₂is CH₂CH(CH₃)₂, CH₂CH₂CH₃, CH₂CH₃, CH═C(CH₃)₂, or CH₃.
21. The process of any one of the preceding clauses wherein R₃is C1-C4 alkyl.
21A. The process of any one of the preceding clauses wherein R₃is methyl.
22. The process of any one of the preceding clauses wherein Ar₁is phenyl or hydroxyphenyl.
22A. The process of any one of the preceding clauses wherein Ar₁is 4-hydroxyphenyl.
23. The process of any one of the preceding clauses wherein R₄is C1-C8 alkyl or C3-C8 cycloalkyl.
23A. The process of any one of the preceding clauses wherein R₄is methyl.
24. The process of any one of the preceding clauses wherein R₅is branched C3-C6 or C3-C8 cycloalkyl.
24A. The process of any one of the preceding clauses wherein R₅is iso-propyl.
25B. The process of any one of the preceding clauses wherein R₅is sec-butyl.
26. The process of any one of the preceding clauses wherein R₆is branched C3-C6 or C3-C8 cycloalkyl.
27. The process of any one of the preceding clauses wherein R₇is C1-C6 alkyl.
27A. The process of any one of the preceding clauses wherein R₇is methyl.
28. The process of any one of the preceding clauses wherein R₁₂is CH₂CH═CH₂, or CH₂(CH₂)nCH₃, where n=1, 2, 3, 4, 5, or 6.
28A. The process of any one of the preceding clauses wherein R₁₂is CH₂CH═CH₂, CH₂CH₂CH₂CH₃, or CH₂CH₂CH₂CH₂CH₃.
29. The process of any one of the preceding clauses wherein Ar₁is substituted phenyl.
29A. The process of any one of the preceding clauses wherein Ar₁is 4-substituted phenyl.
29B. The process of any one of the preceding clauses wherein Ar₁is R_A-substituted phenyl.
29C. The process of any one of the preceding clauses wherein Ar₁is 4-hydroxyphenyl, or a hydroxyl protected form thereof.
30. The process of any one of the preceding clauses wherein R₁₃is CH₂CH₂CH₂CH₃.
31. The process of any one of the preceding clauses wherein the metal hydroxide is LiOH.
32. A compound of the formula
or a pharmaceutically acceptable salt thereof; wherein Ar₁is optionally substituted aryl;
R₁is hydrogen, alkyl, arylalkyl or a pro-drug forming group;
R₂is selected from the group consisting of optionally substituted alkyl and optionally substituted cycloalkyl;
R₁₂is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl or heteroarylalkyl, each of which is optionally substituted;
R₃is optionally substituted alkyl;
R₄is optionally substituted alkyl or optionally substituted cycloalkyl;
R₅and R₆are each independently selected from the group consisting of optionally substituted alkyl and optionally substituted cycloalkyl; R₇is optionally substituted alkyl; and n is 1, 2, 3, or 4.
33. A compound of formula
35. A compound of formula
35. A compound of formula
36. A compound of formula
37. The compound of any one of the preceding clauses wherein R₁is hydrogen, benzyl, or C1-C4 alkyl.
37A. The compound of any one of the preceding clauses wherein R₁is hydrogen.
38. The compound of any one of the preceding clauses wherein R₂is C1-C8 alkyl or C3-C8 cycloalkyl.
38A. The compound of any one of the preceding clauses wherein R₂is n-butyl.
38B. The compound of any one of the preceding clauses wherein R₂is CH₂CH(CH₃)₂, CH₂CH₂CH₃, CH₂CH₃, CH═C(CH₃)₂, or CH₃.
39. The compound of any one of the preceding clauses wherein R₃is C1-C4 alkyl.
39A. The compound of any one of the preceding clauses wherein R₃is methyl.
40. The compound of any one of the preceding clauses wherein Ar₁is phenyl or hydroxyphenyl.
40A. The compound of any one of the preceding clauses wherein Ar₁is 4-hydroxyphenyl.
40B. The compound of any one of the preceding clauses wherein Ar₁is substituted phenyl.
40C. The compound of any one of the preceding clauses wherein Ar₁is 4-substituted phenyl.
40D. The compound of any one of the preceding clauses wherein Ar₁is R_A-substituted phenyl.
40E. The compound of any one of the preceding clauses wherein Ar₁is 4-hydroxyphenyl, or a hydroxyl protected form thereof.
41. The compound of any one of the preceding clauses wherein R₄is C1-C8 alkyl or C3-C8 cycloalkyl.
41A. The compound of any one of the preceding clauses wherein R₄is methyl.
42. The compound of any one of the preceding clauses wherein R₅is branched C3-C6 or C3-C8 cycloalkyl.
42A. The compound of any one of the preceding clauses wherein R₅is iso-propyl.
42B. The compound of any one of the preceding clauses wherein R₅is sec-butyl.
43. The compound of any one of the preceding clauses wherein R₆is branched C3-C6 or C3-C8 cycloalkyl.
44. The compound of any one of the preceding clauses wherein R₇is C1-C6 alkyl.
44A. The compound of any one of the preceding clauses wherein R₇is methyl.
45. The compound of any one of the preceding clauses wherein R₁₂is CH₂CH═CH₂, or CH₂(CH₂)nCH₃, where n=1, 2, 3, 4, 5, or 6.
45A. The compound of any one of the preceding clauses wherein R₁₂is CH₂CH═CH₂, CH₂CH₂CH₂CH₃, or CH₂CH₂CH₂CH₂CH₃.
46. The compound selected from the group consisting of
where n=1, 2, 3, 4, 5, or 6.
In any of the embodiments described herein, the acid selected for the conversion of the NCH₂OC(O)R₂moiety to the NCH₂OR₁₂moiety is TFA.
In any of the embodiments described herein, the catalyst selected for the conversion of the NCH₂OC(O)R₂moiety to the NCH₂OR₁₂moiety is (n-Bu)₂SnO.
It is to be understood that as used herein, the term tubulysin refers both collectively and individually to the naturally occurring tubulysins, and the analogs and derivatives of tubulysins. Illustrative examples of a tubulysin are shown in Table 1.
As used herein, the term tubulysin 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 illustrative embodiments described herein.
It is to be understood that such derivatives may include prodrugs of the compounds described herein, compounds described herein that include one or more protection or protecting groups, including compounds that are used in the preparation of other compounds described herein.
In addition, as used herein the term tubulysin also refers to prodrug derivatives of the compounds described herein, and including prodrugs of the various analogs and derivatives thereof. In addition, as used herein, the term tubulysin refers to both the amorphous as well as any and all morphological forms of each of the compounds described herein. In addition, as used herein, the term tubulysin refers to any and all hydrates, or other solvates, of the compounds described herein.
It is to be understood that each of the foregoing embodiments may be combined in chemically relevant ways to generate subsets of the embodiments described herein. Accordingly, it is to be further understood that all such subsets are also illustrative embodiments of the invention described herein.
The compounds described herein may contain one or more chiral centers, or may otherwise be capable of existing as 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 that include them may be optically pure, or may be any of a variety of stereoisomeric mixtures, including racemic and other mixtures of enantiomers, other mixtures of diastereomers, and the like. It is also to be understood that such mixtures of stereoisomers may include a single stereochemical configuration at one or more chiral centers, while including mixtures of stereochemical configuration 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 is to be understood 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 that include them may be pure, or may be any of a variety of geometric isomer mixtures. It is also to be understood that such mixtures of geometric isomers may include a single configuration at one or more double bonds, while including mixtures of geometry at one or more other double bonds.
As used herein, the term aprotic solvent refers to a solvent which does not yield a proton to the solute(s) under reaction conditions. Illustrative examples of nonprotic solvents are tetrahydrofuran (THF), 2,5-dimethyl-tetrahydrofuran, 2-methyl-tetrahydrofuran, tetrahydropyran, diethyl ether, t-butyl methyl ether, dimethyl formamide, N-methylpyrrolidinone (NMP), and the like. It is appreciated that mixtures of these solvents may also be used in the processes described herein.
As used herein, an equivalent amount of a reagent refers to the theoretical amount of the reagent necessary to transform a starting material into a desired product, i.e. if 1 mole of reagent is theoretically required to transform 1 mole of the starting material into 1 mole of product, then 1 equivalent of the reagent represents 1 mole of the reagent; if X moles of reagent are theoretically required to convert 1 mole of the starting material into 1 mole of product, then 1 equivalent of reagent represents X moles of reagent.
As used herein, the term active ester forming agent generally refers to any reagent or combinations of reagents that may be used to convert a carboxylic acid into an active ester.
As used herein, the term active ester generally refers to a carboxylic acid ester compound wherein the divalent oxygen portion of the ester is a leaving group resulting in an ester that is activated for reacting with compounds containing functional groups, such as amines, alcohols or sulfhydryl groups. 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,N′-disubstituted isoureas, substituted hydroxyheteroaryls, such as but not limited to 2-pyridinols, 1-hydroxybenzotriazoles, 1-hydroxy-7-aza-benzotriazoles, cyanomethanol, and the like. Illustratively, the reaction conditions for displacing the active ester with a compound having an amino, hydroxy or thiol group are mild. Illustratively, the reaction conditions for displacing the active ester with a compound having an amino, hydroxy or thiol group are performed at ambient or below ambient temperatures. Illustratively, the reaction conditions for displacing the active ester with a compound having an amino, hydroxy or thiol group are performed without the addition of a strong base. Illustratively, the reaction conditions for displacing the active ester with a compound having an amino, hydroxy or thiol group are performed with the addition of a tertiary amine base, such as a tertiary amine base having a conjugate acid pKa of about 11 or less, about 10.5 or less, and the like.
As used herein, the term “alkyl” includes a chain of carbon atoms, which is optionally branched. As used herein, the term “alkenyl” and “alkynyl” includes a chain of carbon atoms, which is optionally branched, and includes at least one double bond or triple bond, respectively. It is to be understood that alkynyl may also include one or more double bonds. It is to be further understood that in certain embodiments, alkyl is advantageously of limited length, including C₁-C₂₄, C₁-C₁₂, C₁-C₈, C₁-C₆, and C₁-C₄. Illustratively, such particularly limited length alkyl groups, including C₁-C₈, C₁-C₆, and C₁-C₄may be referred to as lower alkyl. It is to be further understood that in certain embodiments alkenyl and/or alkynyl may each be advantageously of limited length, including C₂-C₂₄, C₂-C₁₂, C₂-C₈, C₂-C₆, and C₂-C₄. Illustratively, such particularly limited length alkenyl and/or alkynyl groups, including C₂-C₈, C₂-C₆, and C₂-C₄may be referred to as lower alkenyl and/or alkynyl. It is appreciated herein that shorter alkyl, alkenyl, and/or alkynyl groups may add less lipophilicity to the compound and accordingly will have different pharmacokinetic behavior. In embodiments of the invention described herein, it is to be understood, in each case, that the recitation of alkyl refers to alkyl as defined herein, and optionally lower alkyl. In embodiments of the invention described herein, it is to be understood, in each case, that the recitation of alkenyl refers to alkenyl as defined herein, and optionally lower alkenyl. In embodiments of the invention described herein, it is to be understood, in each case, that the recitation of alkynyl refers to alkynyl as defined herein, and optionally lower alkynyl. Illustrative alkyl, alkenyl, and alkynyl 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, and the corresponding groups containing one or more double and/or triple bonds, or a combination thereof.
As used herein, the term “cycloalkyl” includes a chain of carbon atoms, which is optionally branched, where at least a portion of the chain in cyclic. It is to be understood that cycloalkylalkyl is a subset of cycloalkyl. It is to be understood that cycloalkyl may be polycyclic. Illustrative cycloalkyl include, but are not limited to, cyclopropyl, cyclopentyl, cyclohexyl, 2-methylcyclopropyl, cyclopentyleth-2-yl, adamantyl, and the like. As used herein, the term “cycloalkenyl” includes a chain of carbon atoms, which is optionally branched, and includes at least one double bond, where at least a portion of the chain in cyclic. It is to be understood that the one or more double bonds may be in the cyclic portion of cycloalkenyl and/or the non-cyclic portion of cycloalkenyl. It is to be understood that cycloalkenylalkyl and cycloalkylalkenyl are each subsets of cycloalkenyl. It is to be understood that cycloalkyl may be polycyclic. Illustrative cycloalkenyl include, but are not limited to, cyclopentenyl, cyclohexylethen-2-yl, cycloheptenylpropenyl, and the like. It is to be further understood that chain forming cycloalkyl and/or cycloalkenyl is advantageously of limited length, including C₃-C₂₄, C₃-C₁₂, C₃-C₈, C₃-C₆, and C₅-C₆. It is appreciated herein that shorter alkyl and/or alkenyl chains forming cycloalkyl and/or cycloalkenyl, respectively, may add less lipophilicity to the compound and accordingly will have different pharmacokinetic behavior.
As used herein, the term “heteroalkyl” includes a chain of atoms that includes both carbon and at least one heteroatom, and is optionally branched. Illustrative heteroatoms include nitrogen, oxygen, and sulfur. In certain variations, illustrative heteroatoms also include phosphorus, and selenium. As used herein, the term “cycloheteroalkyl” including heterocyclyl and heterocycle, includes a chain of atoms that includes both carbon and at least one heteroatom, such as heteroalkyl, and is optionally branched, where at least a portion of the chain is cyclic. Illustrative heteroatoms include nitrogen, oxygen, and sulfur. In certain variations, illustrative heteroatoms also include phosphorus, and selenium. Illustrative cycloheteroalkyl include, but are not limited to, tetrahydrofuryl, pyrrolidinyl, tetrahydropyranyl, piperidinyl, morpholinyl, piperazinyl, homopiperazinyl, quinuclidinyl, and the like.
As used herein, the term “aryl” includes monocyclic and polycyclic aromatic carbocyclic groups, each of which may be optionally substituted. Illustrative aromatic carbocyclic groups described herein include, but are not limited to, phenyl, naphthyl, and the like. As used herein, the term “heteroaryl” includes aromatic heterocyclic groups, each of which may be optionally substituted. Illustrative aromatic heterocyclic groups include, but are not limited to, pyridinyl, pyrimidinyl, pyrazinyl, triazinyl, tetrazinyl, quinolinyl, quinazolinyl, quinoxalinyl, thienyl, pyrazolyl, imidazolyl, oxazolyl, thiazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, triazolyl, benzimidazolyl, benzoxazolyl, benzthiazolyl, benzisoxazolyl, benzisothiazolyl, and the like.
As used herein, the term “amino” includes the group NH₂, alkylamino, and dialkylamino, where the two alkyl groups in dialkylamino may be the same or different, i.e. alkylalkylamino. Illustratively, amino includes methylamino, ethylamino, dimethylamino, methylethylamino, and the like. In addition, it is to be understood that when amino modifies or is modified by another term, such as aminoalkyl, or acylamino, the above variations of the term amino are included therein. Illustratively, aminoalkyl includes H₂N-alkyl, methylaminoalkyl, ethylaminoalkyl, dimethylaminoalkyl, methylethylaminoalkyl, and the like. Illustratively, acylamino includes acylmethylamino, acylethylamino, and the like.
As used herein, the term “amino and derivatives thereof” includes amino as described herein, and alkylamino, alkenylamino, alkynylamino, heteroalkylamino, heteroalkenylamino, heteroalkynylamino, cycloalkylamino, cycloalkenylamino, cycloheteroalkylamino, cycloheteroalkenylamino, arylamino, arylalkylamino, arylalkenylamino, arylalkynylamino, heteroarylamino, heteroarylalkylamino, heteroarylalkenylamino, heteroarylalkynylamino, acylamino, and the like, each of which is optionally substituted. The term “amino derivative” also includes urea, carbamate, and the like.
As used herein, the term “hydroxy and derivatives thereof” includes OH, and alkyloxy, alkenyloxy, alkynyloxy, heteroalkyloxy, heteroalkenyloxy, heteroalkynyloxy, cycloalkyloxy, cycloalkenyloxy, cycloheteroalkyloxy, cycloheteroalkenyloxy, aryloxy, arylalkyloxy, arylalkenyloxy, arylalkynyloxy, heteroaryloxy, heteroarylalkyloxy, heteroarylalkenyloxy, heteroarylalkynyloxy, acyloxy, and the like, each of which is optionally substituted. The term “hydroxy derivative” also includes carbamate, and the like.
As used herein, the term “thio and derivatives thereof” includes SH, and alkylthio, alkenylthio, alkynylthio, heteroalkylthio, heteroalkenylthio, heteroalkynylthio, cycloalkylthio, cycloalkenylthio, cycloheteroalkylthio, cycloheteroalkenylthio, arylthio, arylalkylthio, arylalkenylthio, arylalkynylthio, heteroarylthio, heteroarylalkylthio, heteroarylalkenylthio, heteroarylalkynylthio, acylthio, and the like, each of which is optionally substituted. The term “thio derivative” also includes thiocarbamate, and the like.
As used herein, the term “acyl” includes formyl, and alkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, heteroalkylcarbonyl, heteroalkenylcarbonyl, heteroalkynylcarbonyl, cycloalkylcarbonyl, cycloalkenylcarbonyl, cycloheteroalkylcarbonyl, cycloheteroalkenylcarbonyl, arylcarbonyl, arylalkylcarbonyl, arylalkenylcarbonyl, arylalkynylcarbonyl, heteroarylcarbonyl, heteroarylalkylcarbonyl, heteroarylalkenylcarbonyl, heteroarylalkynylcarbonyl, acylcarbonyl, and the like, each of which is optionally substituted.
As used herein, the term “carboxylic acid and derivatives thereof” includes the group CO₂H and salts thereof, and esters and amides thereof, and CN.
The term “optionally substituted” as used herein includes the replacement of hydrogen atoms with other functional groups on the radical that is optionally substituted. Such other functional groups illustratively include, but are not limited to, amino, hydroxyl, halo, thiol, alkyl, haloalkyl, heteroalkyl, aryl, arylalkyl, arylheteroalkyl, heteroaryl, heteroarylalkyl, heteroarylheteroalkyl, nitro, sulfonic acids and derivatives thereof, carboxylic acids and derivatives thereof, and the like. Illustratively, any of amino, hydroxyl, thiol, alkyl, haloalkyl, heteroalkyl, aryl, arylalkyl, arylheteroalkyl, heteroaryl, heteroarylalkyl, heteroarylheteroalkyl, and/or sulfonic acid is optionally substituted.
As used herein, the terms “optionally substituted aryl” and “optionally substituted heteroaryl” include the replacement of hydrogen atoms with other functional groups on the aryl or heteroaryl that is optionally substituted. Such other functional groups illustratively include, but are not limited to, amino, hydroxy, halo, thio, alkyl, haloalkyl, heteroalkyl, aryl, arylalkyl, arylheteroalkyl, heteroaryl, heteroarylalkyl, heteroarylheteroalkyl, nitro, sulfonic acids and derivatives thereof, carboxylic acids and derivatives thereof, and the like. Illustratively, any of amino, hydroxy, thio, alkyl, haloalkyl, heteroalkyl, aryl, arylalkyl, arylheteroalkyl, heteroaryl, heteroarylalkyl, heteroarylheteroalkyl, and/or sulfonic acid is optionally substituted.
Illustrative substituents include, but are not limited to, a radical —(CH₂)_xZ^x, where x is an integer from 0-6 and Z^xis selected from halogen, hydroxy, alkanoyloxy, including C₁-C₆alkanoyloxy, optionally substituted aroyloxy, alkyl, including C₁-C₆alkyl, alkoxy, including C₁-C₆alkoxy, cycloalkyl, including C₃-C₈cycloalkyl, cycloalkoxy, including C₃-C₈cycloalkoxy, alkenyl, including C₂-C₆alkenyl, alkynyl, including C₂-C₆alkynyl, haloalkyl, including C₁-C₆haloalkyl, haloalkoxy, including C₁-C₆haloalkoxy, halocycloalkyl, including C₃-C₈halocycloalkyl, halocycloalkoxy, including C₃-C₈halocycloalkoxy, amino, C₁-C₆alkylamino, (C₁-C₆alkyl)(C₁-C₆alkyl)amino, alkylcarbonylamino, N—(C₁-C₆alkyl)alkylcarbonylamino, aminoalkyl, C₁-C₆alkylaminoalkyl, (C₁-C₆alkyl)(C₁-C₆alkyl)aminoalkyl, alkylcarbonylaminoalkyl, N—(C₁-C₆alkyl)alkylcarbonylaminoalkyl, cyano, and nitro; or Z^xis selected from —CO₂R⁴and —CONR⁵R⁶, where R⁴, R⁵, and R⁶are each independently selected in each occurrence from hydrogen, C₁-C₆alkyl, aryl-C₁-C₆alkyl, and heteroaryl-C₁-C₆alkyl.
The term “prodrug” as used herein generally refers to any compound that when administered to a biological system generates a biologically active compound as a result of one or more spontaneous chemical reaction(s), enzyme-catalyzed chemical reaction(s), and/or metabolic chemical reaction(s), or a combination thereof. In vivo, the prodrug is typically acted upon by an enzyme (such as esterases, amidases, phosphatases, and the like), simple biological chemistry, or other process in vivo to liberate or regenerate the more pharmacologically active drug. This activation may occur through the action of an endogenous host enzyme or a non-endogenous enzyme that is administered to the host preceding, following, or during administration of the prodrug. Additional details of prodrug use are described in U.S. Pat. No. 5,627,165; and Pathalk et al., Enzymic protecting group techniques in organic synthesis, Stereosel. Biocatal. 775-797 (2000). It is appreciated that the prodrug is advantageously converted to the original drug as soon as the goal, such as targeted delivery, safety, stability, and the like is achieved, followed by the subsequent rapid elimination of the released remains of the group forming the prodrug.
Prodrugs may be prepared from the compounds described herein by attaching groups that ultimately cleave in vivo to one or more functional groups present on the compound, such as —OH—, —SH, —CO₂H, —NR₂. Illustrative prodrugs include but are not limited to carboxylate esters where the group is alkyl, aryl, aralkyl, acyloxyalkyl, alkoxycarbonyloxyalkyl as well as esters of hydroxyl, thiol and amines where the group attached is an acyl group, an alkoxycarbonyl, aminocarbonyl, phosphate or sulfate. Illustrative esters, also referred to as active esters, include but are not limited to 1-indanyl, N-oxysuccinimide; acyloxyalkyl groups such as acetoxymethyl, pivaloyloxymethyl, β-acetoxyethyl, β-pivaloyloxyethyl, 1-(cyclohexylcarbonyloxy)prop-1-yl, (1-aminoethyl)carbonyloxymethyl, and the like; alkoxycarbonyloxyalkyl groups, such as ethoxycarbonyloxymethyl, α-ethoxycarbonyloxyethyl, β-ethoxycarbonyloxyethyl, and the like; dialkylaminoalkyl groups, including di-lower alkylamino alkyl groups, such as dimethylaminomethyl, dimethylaminoethyl, diethylaminomethyl, diethylaminoethyl, and the like; 2-(alkoxycarbonyl)-2-alkenyl groups such as 2-(isobutoxycarbonyl)pent-2-enyl, 2-(ethoxycarbonyl)but-2-enyl, and the like; and lactone groups such as phthalidyl, dimethoxyphthalidyl, and the like.
Further illustrative prodrugs contain a chemical moiety, such as an amide or phosphorus group functioning to increase solubility and/or stability of the compounds described herein. Further illustrative prodrugs for amino groups include, but are not limited to, (C₃-C₂₀)alkanoyl; halo-(C₃-C₂₀)alkanoyl; (C₃-C₂₀)alkenoyl; (C₄-C₇)cycloalkanoyl; (C₃-C₆)-cycloalkyl(C₂-C₁₆)alkanoyl; optionally substituted aroyl, such as unsubstituted aroyl or aroyl substituted by 1 to 3 substituents selected from the group consisting of halogen, cyano, trifluoromethanesulphonyloxy, (C₁-C₃)alkyl and (C₁-C₃)alkoxy, each of which is optionally further substituted with one or more of 1 to 3 halogen atoms; optionally substituted aryl(C₂-C₁₆)alkanoyl, such as the aryl radical being unsubstituted or substituted by 1 to 3 substituents selected from the group consisting of halogen, (C₁-C₃)alkyl and (C₁-C₃)alkoxy, each of which is optionally further substituted with 1 to 3 halogen atoms; and optionally substituted heteroarylalkanoyl having one to three heteroatoms selected from O, S and N in the heteroaryl moiety and 2 to 10 carbon atoms in the alkanoyl moiety, such as the heteroaryl radical being unsubstituted or substituted by 1 to 3 substituents selected from the group consisting of halogen, cyano, trifluoromethanesulphonyloxy, (C₁-C₃)alkyl, and (C₁-C₃)alkoxy, each of which is optionally further substituted with 1 to 3 halogen atoms. The groups illustrated are exemplary, not exhaustive, and may be prepared by conventional processes.
It is understood that the prodrugs themselves may not possess significant biological activity, but instead undergo one or more spontaneous chemical reaction(s), enzyme-catalyzed chemical reaction(s), and/or metabolic chemical reaction(s), or a combination thereof after administration in vivo to produce the compound described herein that is biologically active or is a precursor of the biologically active compound. However, it is appreciated that in some cases, the prodrug is biologically active. It is also appreciated that prodrugs may often serves to improve drug efficacy or safety through improved oral bioavailability, pharmacodynamic half-life, and the like. Prodrugs also refer to derivatives of the compounds described herein that include groups that simply mask undesirable drug properties or improve drug delivery. For example, one or more compounds described herein may exhibit an undesirable property that is advantageously blocked or minimized may become pharmacological, pharmaceutical, or pharmacokinetic barriers in clinical drug application, such as low oral drug absorption, lack of site specificity, chemical instability, toxicity, and poor patient acceptance (bad taste, odor, pain at injection site, and the like), and others. It is appreciated herein that a prodrug, or other strategy using reversible derivatives, can be useful in the optimization of the clinical application of a drug.
As used herein, the term “treating”, “contacting” or “reacting” when referring to a chemical reaction means to add or mix two or more reagents under appropriate conditions to produce the indicated and/or the desired product. It should be appreciated that the reaction which produces the indicated and/or the desired product may not necessarily result directly from the combination of two reagents which were initially added, i.e., there may be one or more intermediates which are produced in the mixture which ultimately leads to the formation of the indicated and/or the desired product.
As used herein, the term “composition” generally refers to any product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combinations of the specified ingredients in the specified amounts. It is to be understood that the compositions described herein may be prepared from isolated compounds described herein or from salts, solutions, hydrates, solvates, and other forms of the compounds described herein. It is also to be understood that the compositions may be prepared from various amorphous, non-amorphous, partially crystalline, crystalline, and/or other morphological forms of the compounds described herein. It is also to be understood that the compositions may be prepared from various hydrates and/or solvates of the compounds described herein. Accordingly, such pharmaceutical compositions that recite compounds described herein are to be understood to include each of, or any combination of, the various morphological forms and/or solvate or hydrate forms of the compounds described herein. Illustratively, compositions may include one or more carriers, diluents, and/or excipients. The compounds described herein, or compositions containing them, may be formulated in a therapeutically effective amount in any conventional dosage forms appropriate for the methods described herein. The compounds described herein, or compositions containing them, including such formulations, may be administered by a wide variety of conventional routes for the methods described herein, and in a wide variety of dosage formats, utilizing known procedures (see generally, Remington: The Science and Practice of Pharmacy, (21^sted., 2005)).

EXAMPLES

Example

Synthesis of Dipeptide 3

4.9 g of dipeptide 1 (11.6 mmol) was dissolved in 60 mL dichloromethane, imidazole (0.87 g, 12.7 mmol) was added to the resulting solution at 0° C. The reaction mixture was warmed slightly to dissolve all solids and re-cooled to 0° C. TESCl (2.02 mL, 12.1 mmol) was added drop-wise at 0° C., the reaction mixture was stirred under argon and warmed to room temperature over 2 h. TLC (3:1 hexanes/EtOAc) showed complete conversion. The reaction was filtered to remove the imidazole HCl salt, extracted with de-ionized water, and the aqueous phase was back-washed with dichloromethane, the combined organic phase was washed with brine, dried over Na₂SO₄, filtered to remove the Na₂SO₄, concentrated under reduced pressure, co-evaporated with toluene and dried under high-vacuum overnight to give 6.4 g of crude product 2 (vs 5.9 g of theoretical yield).
The crude product 2 was co-evaporated with toluene again and used without further purification. TES protected dipeptide was dissolved in 38 mL THF (anhydrous, inhibitor-free) and cooled to −45° C. and stirred for 15 minutes before adding KHMDS (0.5 M in toluene, 25.5 mL, 12.8 mmol, 1.1 equiv) drop-wise. After the addition of KHMDS was complete, the reaction mixture was stirred at −45° C. for 15 minutes, and chloromethyl butyrate (1.8 mL, 1.2 equiv, 14 mmol) was added. The reaction mixture changed from light yellow to a blueish color. TLC (20% EtOAc/petroleum ether) showed the majority of starting material was converted. LC-MS showed about 7% starting material left. The reaction was quenched by adding 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 passed through short silica plug to remove the potassium salt. The plug was washed with 13% EtOAc/petroleum ether, and the collected eluates were combined and concentrated under reduced pressure. The crude alkylated product was passed through an additional silica plug (product/silica=1:50) and eluted with 13% EtOAc/petroleum ether to remove residual starting material to give 5.7 g of product 3 (two steps, yield 76%)

Example

Synthesis of Tripeptide 4

Alkylated dipeptide 3 (4.3 g, 7.0 mmol), N-methyl pipecolinate (MEP) (4.0 g, 28.0 mmol, 4 equiv) and pentafluorophenol (5.7 g, 30.8 mmol. 4.4 equiv) were added to a flask. N-methylpyrrolidone (NMP, 86 mL) was added to the mixture. To the mixture was added diisopropylcarbodiimide (DIC, 4.77 mL, 30.8 mmol, 4.4 equiv) was added to the mixture. The mixture was stirred at room temperature for 1 h. Pd/C (10%, dry, 1.7 g) 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 less than 3%. The mixture was filtered through diatomaceous earth. The diatomaceous earth was extracted with 200 mL ethyl acetate. The filtrate and the ethyl acetate extract were combined and transferred to a separatory funnel and washed with 1% NaHCO₃/10% NaCl solution (200 mL×4). The organic layer was isolated and evaporated on a rotary evaporator under reduced pressure. The crude product was dissolved in 40 mL of MeOH/H₂O (3:1). The crude product solution was loaded onto a Biotage C18 column (Flash 65i, 350 g, 450 mL, 65×200 mm) and eluted with buffer A [10 mM NH₄OAc/ACN (1:1)] and B (ACN, acetonitrile). The fractions were collected and organic solvent was removed by evaporating on a rotary evaporator. 100 mL of 10% NaCl solution and 100 mL of methyl tert-butyl ether (MTBE) were added to the flask and the mixture was transferred to a separatory funnel. The organic layer was isolated and dried over anhydrous Na₂SO₄, filtered and evaporated on a rotary evaporator to dryness. 2.5 g of tripeptide intermediate 4 was obtained (yield 50%).

Example

Large Scale Synthesis of Dipeptide 3

10.2 g of dipeptide 1 (25.6 mmol) was dissolved in 130 mL dichloromethane, imidazole (1.9 g, 28.1 mmol) was added to the resulting solution at 0° C. The reaction mixture was warmed slightly to dissolve all solids and re-cooled to 0° C. TESCl (4.5 mL, 26.8 mmol) was added drop-wise at 0° C., the reaction mixture was stirred under argon and warmed to room temperature over 2 h. TLC (3:1 hexanes/EtOAc) showed complete conversion. The reaction was filtered to remove the imidazole HCl salt, extracted with de-ionized water, and the aqueous phase was back-washed with dichloromethane, the combined organic phase was washed with brine, dried over Na₂SO₄, filtered to remove the Na₂SO₄, concentrated under reduced pressure, co-evaporated with toluene and dried under high-vacuum overnight to give 12.2 g of product 2.
The crude product 2 was co-evaporated with toluene again and used without further purification. TES protected dipeptide was dissolved in 80 mL THF (anhydrous, inhibitor-free) and cooled to −45° C. and stirred for 15 minutes before adding KHMDS (0.5 M in toluene, 50 mL, 25.0 mmol, 1.05 equiv) drop-wise. After the addition of KHMDS was complete, the reaction mixture was stirred at −45° C. for 15 minutes, and chloromethyl butyrate (3.6 mL, 1.2 equiv, 28.3 mmol) was added. The reaction mixture changed from light yellow to a blueish color. TLC (20% EtOAc/petroleum ether) showed the reaction was complete. The reaction was quenched by adding 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 passed through short silica plug to remove the potassium salt. The plug was washed with 13% EtOAc/petroleum ether, and the collected eluents were combined and concentrated under reduced pressure to give 12.1 g of product 3 (two steps, yield 76%)

Example

Large Scale Synthesis of Tripeptide 4

Alkylated dipeptide 3 (7.6 g, 12.4 mmol), N-methyl pipecolinate (MEP) (7.0 g, 48.9 mmol, 4 equiv) and pentafluorophenol (10.0 g, 54.3 mmol. 4.4 equiv) were added to a 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) was added to the mixture. The mixture was stirred at room temperature for 1 h. Pd/C (10%, dry, 3.0 g) 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 and washed with 1% NaHCO₃/10% NaCl solution (250 mL×4). The organic layer was isolated and evaporated on a rotary evaporator under reduced pressure. The crude product was dissolved in dichloromethane and the urea was filtered. The crude product solution was loaded onto a Teledyne Redisep Silica Column (330 g) and purified with EtOAc/petroleum ether on CombiFlash flash chromatography system. The fractions were collected and organic solvent was removed by evaporating to give 5.0 g of the tripeptide (61%). NMR and mass spectral data were consistent with those measured for the Example
Also described herein, is the conversion of 4 to 10 (R remains Me) by contacting 4 with TFA and an alcohol. In some illustrative examples of compound 10, R is allyl, or CH₂(CH₂)nCH₃, where n is 1, 2, 3, 4, 5, or 6.

Example

Compound 4 (50 mg, 0.07 mmol) in allyl alcohol (5 mL) was treated with di-n-butyltin oxide (1.75 mg, 0.007 mmol, 10% mol). The reaction mixture was heated to reflux for 22 hrs till the reaction was complete. The reaction was concentrated and purified with HPLC in 10-100% ACN/NH₃HCO₃buffer (pH7.0) to give the title compound (32.4 mg, yield 65%). LCMS: [M+H]⁺ m/z=707.73. ¹H NMR (CD₃OD, δ in ppm): 8.35 (s, 1H), 6.01 (m, 2H); 5.2-5.5 (m, 3H), 5.14 (d, J=10.26 Hz, 1H), 5.04 (d, J=5.87 Hz, 1H), 4.88 (s, 3H), 4.82 (d, J=5.5 Hz, 2H), 4.70 (d, J=8.79 Hz, 1H), 4.50 (d, J=10.26 Hz, 1H), 4.42 (b, 1H), 4.06 (s, 2H), 2.92 (d, J=11.36 Hz, 1H), 2.55 (d, J=9.17 Hz, 1H), 1.95-2.20 (m, 7H), 1.45-1.82 (m, 7H), 1.22 (m, 2H), 0.82-1.00 (m, 17H), 0.77 (d, J=6.23 Hz, 3H), 0.59-0.70 (m, 6H); ¹³C NMR (CD₃OD, δ in ppm): 176.97, 175.08, 174.09, 160.95, 146.02, 134.13, 132.05, 127.94, 117.38, 116.37, 73.85, 70.32, 69.14, 68.40, 65.34, 56.89, 55.20, 53.55, 43.35, 40.37, 36.38, 31.59, 30.15, 24.80, 24.27, 22.93, 19.09, 18.71, 15.31, 9.52, 5.77, 4.41.

Example

Compound 10a (15.3 mg, 0.02 mmol) was subjected to hydrolysis with LiOH.H₂O (0.99 mg, 0.024 mmol) in 4:1 THF/H₂O (2.5 mL) for 19 hrs at room temperature (rt). The reaction was purified with HPLC in 10-100% ACN/NH₃HCO₃buffer (pH7.0) to provide compound 11a (9.2 mkg, yield 83%). LCMS: [M+H]⁺ m/z=553.55. ¹H NMR (CD₃OD, δ in ppm): 7.94 (s, 1H), 6.00 (m, 1H), 5.1-5.4 (m, 3H), 4.68 (d, J=9.09 Hz, 2H), 4.10 (d, J=3.81 Hz, 2H), 2.80 (b, 1H), 2.56 (s, 2H), 1.4-2.2 (m, 11H), 1.20 (m, 1H), 0.80-0.99 (m, 13H); ¹³C NMR (CD₃OD, δ in ppm): 17.90, 167.53, 153.18, 134.05, 123.09, 116.53, 68.63, 67.25, 54.85, 54.44, 42.10, 37.75, 36.53, 30.60, 29.13, 24.26, 23.25, 21.37, 20.32, 19.53, 14.72, 9.51.

Example

To compound 11a (9.2 mg, 0.017 mmol) in pyridine (1 mL) was added acetic anhydride (15.7 μL, 0.165 mmol) and a catalytic amount of 4-dimethylamino pyridine (0.053 M in pyridine, 5 μL) at rt under argon. The reaction was stirred for 24 hrs. To the reaction mixture was added 0.4 mL of dioxane/water (1:1) and stirred for 10 min, and then the solvent was removed in vacuo. The residue was purified with HPLC in 10-100% ACN/NH₃HCO₃buffer (pH7.0) to provide the product 12a 10.4 mg (quantitative yield). LCMS: [M+H]⁺ m/z=595.59. ¹H NMR (CD₃OD, δ in ppm): 7.96 (s, 1H), 5.8-6.0 (m, 2H), 5.33 (d, J=17.59 Hz, 1H), 5.19 (d, J=10.56 Hz, 1H), 4.71 (d, J=9.23 Hz, 2H), 4.05 (d, J=5.71 Hz, 2H), 3.30 (m, 6H), 2.50 (b, 4H), 2.10 (s, 3H), 1.40-2.00 (m, 7H), 1.20 (m, 1H), 0.80-1.02 (m, 11H); ¹³C NMR (CD₃OD, δ in ppm): 175.11, 170.44, 167.29, 153.45, 133.92, 123.40, 116.79, 116.55, 68.62, 67.82, 67.11, 54.75, 54.16, 42.39, 36.31, 36.12, 34.91, 30.55, 29.26, 24.09, 23.26, 21.25, 20.24, 19.48, 19.20, 14.78, 9.56.

Example

Compound 12a (10.4 mg, 0.017 mmol) was dissolved in anhydrous methylene chloride (4 mL) and to this solution was added DCC-resin (2.3 mmol/g, 0.038 g, 0.087 mmol) and followed by pentafluorophenol (PFP, 6.26 mg, 0.034 mmol) at rt under argon. The reaction was stirred for 19 hrs at rt. The reaction mixture was filtered and the solution was concentrated. The residue was redissolved in dry DMF (4 mL). Then, (2S,4R)-4-amino-5-(4-hydroxyphenyl)-2-methylpentanoic acid (Tut acid) was added into the solution, followed by DIPEA (8.9 μL, 0.051 mmol). When completed, the reaction was concentrated in vacuo and the residue was purified with HPLC. Product 13a was obtained (13.1 mg, 96% yield). LCMS: [M+H]⁺ m/z=800.88. ¹H NMR (CD₃OD, δ in ppm): 8.08 (s, 1H), 7.02 (d, J=8.43 Hz, 2H), 6.68 (d, J=8.06 Hz, 2H), 5.99 (d, J=10.99 Hz, 1H), 5.80 (m, 1H), 5.38 (d, J=9.53 Hz, 1H), 5.31 (d, J=17.23 Hz, 1H), 5.13 (d, J=10.63 Hz, 1H), 4.66 (d, J=8.79 Hz, 1H), 4.55 (d, J=10.28 Hz, 1H), 4.30 (b, 2H), 4.00 (b, 2H), 3.16 (b, 2H), 2.80 (d, J=5.86 Hz, 2H), 2.40 (b, 4H), 2.10-2.30 (b, 2H), 1.40-1.90 (b, 6H), 1.23 (s, 3H), 1.17 (d, J=6.96 Hz, 3H), 1.05 (d, J=6.23 Hz, 2H), 0.94 (d, J=6.97 Hz, 2H), 0.90 (d, J=7.70 Hz, 2H), 0.79 (d, J=6.6 Hz, 3H); ¹³C NMR (CD₃OD, δ in ppm): 179.24, 174.88, 170.97, 170.43, 170.20, 161.29, 155.62, 149.30, 133.70, 130.23, 128.44, 123.54, 116.41, 114.72, 69.92, 68.15, 67.87, 54.96, 53.92, 49.27, 42.40, 39.62, 37.72, 36.91, 36.08, 35.29, 31.01, 29.51, 29.33, 24.08, 23.72, 21.93, 19.40, 19.34, 18.89, 17.24, 15.00, 9.34.

Example

Compound 12a (26.4 mg, 0.044 mmol) was dissolved in anhydrous methylene chloride (5 mL) and to this solution was added DCC-resin (2.3 mmol/g, 0.096 g, 0.22 mmol), followed by pentafluorophenol (PFP, 16.4 mg, 0.089 mmol) at rt under argon. The reaction was stirred for 19 hrs at rt. The reaction was filtered and concentrated and the residue was redissolved in dry DMF (5 mL). 2-((3-nitropyridin-2-yl)disulfanyl)ethyl 2-((2S,4R)-4-((tert-butoxycarbonyl)amino)-5-(4-hydroxyphenyl)-2-methylpentanoyl)hydrazinecarboxylate (40.0 mg, 0.067 mmol) was deprotected with TFA/DCM (1:1, 5 mL, 1 drop of TIPS as scavenger) at rt for 1 hr. The solvent was removed under reduced pressure, 5 mL more of DCM was added, and then the solvent was co-evaporated to dryness. The residue was dissolved in dry DMF (2 mL) and was added to the solution of PFP ester intermediate in DMF made above after the addition of DIPEA (23.2 μL, 0.13 mmol) at rt under argon. The reaction was stirred for 19 hrs and diluted with EtOAc (20 mL). The organic phase was washed with water (5 mL×3) and brine. The organic layer was dried over anhydrous Na₂SO₄and concentrated after filtration to give the crude product 15a (52.8 mg), which could be used for conjugation with folate. LCMS: [M+H]⁺ m/z=1072.92.

Example

Compound 4 (75.9 mg, 0.11 mmol) in n-butanol (4 mL) was treated with n-Bu₂SnO (2.12 mg, 0.0085 mmol, 8.0 mol %) at rt and the reaction was heated to 100° C. for 2 days. The solvent was reduced to a minimum and the product was purified with CombiFlash (Teledyne Redisep Silica column, eluted with 0 to 15% of MeOH/DCM) to give 44.0 mg (56%) of intermediate 10b. LCMS: [M+H]⁺ m/z=739.61. ¹H NMR (CDCl₃, δ in ppm): 8.07 (s, 1H), 7.02 (d, J=9.68 Hz, 1H), 5.27 (d, J=9.67 Hz, 1H), 5.02 (dd, J=8.36, 2.64 Hz, 1H), 4.69 (t, J=9.23 Hz, 4.20-4.40 (m, 4H), 3.47 (td, J=6.6, 1.76 Hz, 2H), 2.88 (d, J=11.44 Hz, 1H), 2.46 (dd, J=10.55, 3.08 Hz, 2H), 1.90-2.24 (m, 8H), 1.10-1.79 (m, 18H), 0.80-1.00 (m, 19H), 0.58-0.78 (m, 6H).

Example

The same procedure as compound 11a was followed. 11b (11.7 mg, 35%) was obtained from intermediate 10b (44.0 mg). LCMS: [M+H]⁺ m/z=569.51. ¹H NMR (CDCl₃drops of CD₃OD, δ in ppm) 8.00 (s, 1H), 5.23 (b, 1H), 4.80 (b, 1H), 4.58 (d, J=8.80 Hz, 1H), 4.42 (b, 1H), 3.45 (t, J=6.38 Hz, 1H), 3.33 (b, 3H), 2.15-2.40 (m, 3H), 1.80-2.10 (m, 2H), 1.40-1.79 (m, 4H), 1.04-1.38 (m, 3H), 0.60-1.02 (m, 9H).

Example

In a 10 mL round bottom flask, 11b (11.7 mg, 0.021 mmol) and acetic anhydride (20 μL, 0.212 mmol) were dissolved in pyridine (1 mL). To this solution was added a catalytic amount of dimethylaminopyridine (1 mg, 0.008 mmol). This solution was stirred at room temperature for 16 h under Argon. LCMS (10-100% ACN, 50 mM NH₄HCO₃pH7) indicated all of the starting material had been consumed and product had been formed. To the flask was added a 1:1 mixture of 1,4-dioxane and water (0.4 mL) and the solution was stirred for 10 min to hydrolyze any potential diacetate side product. The reaction mixture was concentrated under reduced pressure, then purified by preparative HPLC (10-100% ACN, 50 mM NH₄HCO₃pH7) to yield 12b (9.6 mg, 76%). LCMS: [M+H]⁺=611.53. ¹H NMR (CDCl₃w/2 drops CD₃OD): 7.97 (s, 1H) 5.83 (d, J=9.9 Hz, 1H) 5.28 (s, 1H) 4.58 (d, J=9.0 Hz, 1H) 4.24 (d, J=9.3 Hz, 2H) 3.42 (m, 3H) 2.60-2.95 (br, 7H) 2.20-2.58 (br, 6H) 1.76-2.20 (br, ¹H) 1.40-1.56 (br, 12H) 1.02-1.20 (br, 12H) 0.40-1.10 (br, 27H) 0.04 (s, 8H). ¹³C NMR: 175.04, 170.53, 67.78, 53.74, 44.33, 36.79, 35.64, 31.69, 29.89, 24.86, 20.96, 20.49, 19.52, 15.95, 13.99, 10.65, 1.21

Example

In a 25 mL round bottom flask, 12b (9.6 mg, 0.016 mmol) and pentafluorophenol (28.2 mg, 0.153 mmol) were dissolved in dry dichloromethane (5 mL). N-cyclohexylcarbodiimide, N′-methyl polystyrene (33.4 mg, 2.3 mmol/g, 0.077 mmol) was added and the reaction mixture was stirred at room temperature for 16 h under Argon. LCMS (10-100% ACN, 50 mM NH₄HCO₃pH7) indicated all of the starting material had been consumed and activated intermediate had been formed. The reaction mixture was filtered and concentrated under reduced pressure, and the residue was dissolved in a solution of N,N-dimethylformamide (2 mL) and N,N-diisopropylethylamine (8 μL, 0.046 mmol). PFP ester intermediate (6.0 mg, 0.023 mmol) was added and the reaction mixture was stirred at room temperature for 2 h under argon. LCMS (10-100% ACN, 50 mM NH₄HCO₃pH7) indicated all of the activated intermediate had been consumed and product had been formed. The reaction mixture was purified by preparative HPLC (10-100% ACN, 50 mM NH₄HCO₃pH7) to yield 13b (4.7 mg, 37%). LCMS: [M+H]⁺ m/z=816.71. ¹H NMR (CDCl₃, δ in ppm): 8.04 (s, 1H) 7.05 (d, J=8.4 Hz, 2H) 6.80 (d, J=8.4 Hz, 2H) 5.90 (m, 1H) 5.38 (d, J=10.2 Hz, 1H) 4.63 (t, J=9.3 Hz, 1H) 4.38 (br, 1H) 4.27 (d, J=9.9 Hz, 1H) 3.48 (m, 1H) 3.34 (m, 2H) 2.86 (m, 6H) 2.56 (m, 3H) 2.23 (s, 3H) 2.16 (s, 3H) 1.22-2.10 (br, 16H) 1.12 (d, J=6.9 Hz, 3H) 1.03 (d, J=6.6 Hz, 3H) 0.88 (m, 14H). ¹³C NMR: 174.90, 170.44, 161.73, 155.52, 149.37, 130.77, 128.56, 124.33, 115.91, 70.40, 69.69, 67.62, 55.45, 53.70, 49.25, 44.61, 40.40, 36.94, 36.69, 35.93, 31.77, 31.16, 30.06, 24.94, 23.14, 21.08, 20.74, 20.20, 19.55, 17.78, 16.08, 14.05, 10.70

Example

Compound 4 (73.9 mg, 0.10 mmol) in n-pentanol (4 mL) was treated with n-Bu₂SnO (2.10 mg, 0.0083 mmol, 8.0 mol %) at rt and the reaction was heated to 100° C. for 2 days. The solvent was reduced to a minimum and the product was purified with CombiFlash (Teledyne Redisep Silica column, eluted with 0 to 15% of MeOH/DCM) to give 51.2 mg (64%) of intermediate 10b. LCMS: [M+H]⁺ m/z=767.64. ¹H NMR (CDCl₃, δ in ppm): 8.07 (m, 1H), 7.06 (t, J=9.23 Hz, 1H), 5.95 (d, J=12.3 Hz, 1H), 5.43 (d, J=12.32 Hz, 1H), 5.26 (d, J=9.68 Hz, 1H), 5.03 (dd, J=8.36, 2.64 Hz, 1H), 4.93 (dd, J=8.36, 6.24 Hz, 1H), 4.71 (dd, J=15.83, 8.80 Hz, 1H), 4.20-4.33 (m, 3H), 3.46 (m, 1H), 2.88 (d, J=11.43 Hz, 1H), 2.30-2.60 (m, 2H), 2.20 (s, 2H), 1.95-2.18 (m, 3H), 1.50-1.80 (m, 6H), 1.10-1.44 (m, 6H), 0.80-1.04 (m, 13H), 0.50-0.77 (m, 6H).

Example

The same procedure as for compound 11a was followed, intermediate 11c (14.9 mg, 38%) was obtained from 10c (51.2 mg). LCMS: [M+H]⁺ m/z=583.56. ¹H NMR (CD₃OD, δ in ppm): 7.97 (s, 1H), 5.27 (d, J=9.67 Hz, 1H), 4.67 (d, J=9.23 Hz, 1H), 4.58 (d, J=9.68 Hz, 1H), 3.53 (m, 3H), 2.80 (b, 1H), 2.58 (b, 4H), 1.48-2.18 (m, 13H), 1.10-1.42 (m, 6H), 0.70-1.08 (m, 18H).

Example

In a 10 mL round bottom flask, 11c (14.9 mg, 0.026 mmol) and acetic anhydride (20 μL, 0.212 mmol) were dissolved in pyridine (1 mL). This solution was added a catalytic amount of dimethylaminopyridine (1 mg, 0.008 mmol). This solution was stirred at room temperature for 16 h under argon. LCMS (10-100% ACN, 50 mM NH₄HCO₃pH7) indicated all of the starting material had been consumed and product had been formed. To the flask was added a 1:1 mixture of 1,4-dioxane and water (0.4 mL) and the solution was stirred for 10 min to hydrolyze any potential diacetate side product. The reaction mixture was concentrated under reduced pressure, then purified by preparative HPLC (10-100% ACN, 50 mM NH₄HCO₃pH7) to yield 12c (4.8 mg, 30%). LCMS: [M+H]⁺ m/z=625.58. ¹H NMR (CDCl₃w/2 drops CD₃OD) 7.98 (s, 1H) 5.82 (d, J=10.8 Hz, 1H) 5.26 (s, 1H) 4.57 (d, J=8.4 Hz, 1H) 4.23 (d, J=8.4 Hz, 2H) 3.42 (m, 3H) 2.60-2.92 (br, 8H) 2.15-2.40 (br, 4H) 1.90-2.12 (m, 7H) 1.38-1.90 (br, 14H) 1.00-1.38 (br, 13H) 0.50-1.00 (br, 22H), 0.03 (s, 13H). ¹³C NMR: 175.15, 150.56, 125.47, 69.55, 68.09, 55.33, 53.71, 44.59, 36.77, 35.74, 31.34, 30.19, 29.86, 29.32, 28.51, 24.84, 22.85, 22.55, 20.86, 20.40, 19.91, 15.94, 14.10, 10.63, 1.17

Example

In a 25 mL round bottom flask, 12c (4.8 mg, 0.008 mmol) and pentafluorophenol (14.1 mg, 0.077 mmol) were dissolved in dry dichloromethane (5 mL). N-cyclohexylcarbodiimide, N-methyl polystyrene (16.7 mg, 2.3 mmol/g, 0.038 mmol) was added and the reaction mixture was stirred at room temperature for 16 h under Argon. LC-MS (10-100% ACN, 50 mM NH₄HCO₃pH7) indicated all of the starting material had been consumed and activated intermediate had been formed. The reaction mixture was filtered and concentrated under reduced pressure, and the residue was dissolved in a solution of N,N-dimethylformamide (2 mL) and N,N-diisopropylethylamine (4 μL, 0.023 mmol). PFP ester intermediate (3.0 mg, 0.012 mmol) was added and the reaction mixture was stirred at room temperature for 2 h under Argon. LC-MS (10-100% ACN, 50 mM NH₄HCO₃pH7) indicated all of the activated intermediate had been consumed and product had been formed. The reaction mixture was purified by preparative HPLC (10-100% ACN, 50 mM NH₄HCO₃pH7) to yield 13c (1.1 mg, 17%). LCMS: [M+H]⁺ m/z=830.76. ¹H NMR (CDCl₃w/2 drops CD₃OD): 8.00 (s, 1H) 7.01 (d, J=8.7 Hz, 2H) 6.74 (d, J=8.4 Hz, 2H) 5.89 (d, J=12.6 Hz, 1H) 5.25 (d, J=9.0 Hz, 1H) 4.55 (d, J=8.7 Hz, 1H) 4.30 (m, 3H) 3.39 (m, 3H) 3.21 (m, 2H) 2.81 (m, 3H) 2.04-2.60 (br, 45H) 1.76-2.04 (m, 5H) 1.34-1.76 (br, 9H) 1.20 (m, 6H) 1.12 (d, J=7.2 Hz, 4H) 1.01 (d, J=6.3 Hz, 3H) 0.89 (t, J=7.1 Hz, 6H) 0.78 (m, 6H)

Example

In a 5 mL round bottom flask, 14 (10.0 mg, 0.009 mmol) was dissolved in a solution of trifluoroacetic acid (125 μL, 1.632 mmol) and dichloromethane (0.5 mL) and stirred at room temperature for 1 hr under argon, then 1-butanol (200 μL, 2.186 mmol) added and reaction mixture stirred at room temperature for 30 min under argon. LCMS (10-100% ACN, 50 mM NH₄HCO₃pH7) indicated all of the starting material had been consumed and product had been formed. The reaction mixture was purified by preparative HPLC (10-100% ACN, 50 mM NH₄HCO₃pH7) to yield 15b (3.2 mg, 32%). LCMS: [M+H]⁺ m/z=1088.79. ¹H NMR (CDCl₃w/2 drops CD₃OD): 8.86 (s, 1H) 8.47 (d, J=8.0 Hz, 1H) 7.99 (s, 1H) 7.31 (d, J=9.5 Hz, 2H) 7.01 (d, J=7.5 Hz, 2H) 6.73 (d, J=8.5 Hz, 2H) 5.94 (d, J=10.5 Hz, 1H) 5.34 (d, J=10.0 Hz, 1H) 4.58 (m, 3H) 4.38 (t, J=6.0 Hz, 4H), 4.27 (d, J=10.0 Hz, 2H) 3.37 (m, 2H) 3.18 (m, 2H) 3.09 (t, J=6.3 Hz, 3H) 2.70-2.90 (br, 6H) 2.43 (dd, J=11.0 Hz, 3.0 Hz, 2H) 2.26-2.36 (br, 4H) 2.12-2.22 (br, 10H) 2.02-2.12 (br, 2H) 1.86-2.02 (br, 11H) 1.69-1.80 (br, 6H) 1.54-1.69 (br, 10H) 1.34-1.52 (br, 12H) 1.09-1.34 (br, 16H) 1.047 (dd, J=15.0 Hz, 6.5 Hz, 19H) 0.88 (m, 19H) 0.75 (m, 17H). ¹³C NMR: 174.95, 174.59, 170.64, 170.23, 161.92, 156.91, 156.06, 153.88, 149.00, 133.77, 130.79, 123.92, 120.98, 115.53, 69.95, 69.61, 67.03, 63.82, 55.32, 53.21, 44.78, 41.42, 40.40, 36.84, 36.38, 35.62, 35.22, 31.54, 31.40, 30.37, 24.99, 24.66, 23.20, 20.68, 20.24, 19.56, 19.27, 17.69, 15.71, 13.72, 10.35

Example

In a 5 mL round bottom flask, 14 (10.0 mg, 0.009 mmol) was dissolved in a solution of trifluoroacetic acid (125 μL, 1.632 mmol) and dichloromethane (0.5 mL) and stirred at room temperature for 1 hr under argon, then 1-pentanol (200 μL, 1.840 mmol) added and reaction mixture stirred at room temperature for 30 min under argon. LC-MS (10-100% ACN, 50 mM NH₄HCO₃pH7) indicated all of the starting material had been consumed and product had been formed. The reaction mixture was purified by preparative HPLC (10-100% ACN, 50 mM NH₄HCO₃pH7) to yield 15c (3.6 mg, 36%). LCMS: [M+H]⁺ m/z=1102.77.

Example

In a 25 mL round bottom flask, 15b (3.2 mg, 0.003 mmol) was dissolved in dimethylsulfoxide (2 mL). A solution of 16 (4.9 mg, 0.003 mmol) in 20 mM, pH7, sodium phosphate buffer (2 mL) was added dropwise, stirring at room temperature with argon bubbling for 30 min. LCMS (10-100% ACN, 50 mM NH₄HCO₃pH7) indicated all of the starting material had been consumed and product had been formed. The reaction mixture was purified by preparative HPLC (10-100% ACN, 50 mM NH₄HCO₃pH7) to yield 17b (4.3 mg, 56%). LCMS: [M+H]⁺ m/z=1306.82. ¹H NMR (9:1 DMSO-d6:D₂O): 8.60 (s, 1H) 8.14 (s, 1H) 7.59 (d, J=8.5 Hz, 2H) 6.94 (d, J=7.5 Hz, 2H) 6.60 (dd, J=13.3 Hz, 8.8 Hz, 3H) 5.77 (d, J=11.5 Hz, 1H) 5.20 (d, J=9.5 Hz, 1H) 4.46 (m, 3H) 4.00-4.40 (br, 12H) 3.48-3.62 (br, 11H) 3.28-3.48 (br, 12H) 3.10-3.28 (br, 4H) 2.80-3.08 (br, 7H) 2.60-3.80 (br, 3H) 2.48 (s, 1H) 2.26-2.40 (br, 2H) 2.00-2.26 (br, 19H) 1.58-2.00 (br, 20H) 1.28-1.58 (br, 8H) 1.18 (q, J=7.5 Hz, 3H) 0.84-1.10 (br, 8H) 0.75 (m, 9H) 0.60 (d, J=6.5 Hz, 3H). ¹³C NMR: 175.25, 174.93, 174.36, 173.59, 173.22, 172.76, 172.70, 172.02, 171.85, 171.67, 170.85, 170.34, 169.68, 166.48, 161.94, 160.67, 156.42, 155.80, 154.22, 150.98, 149.56, 149.21, 149.08, 130.64, 129.17, 128.69, 128.08, 124.89, 122.00, 115.31, 111.84, 72.31, 72.23, 71.82, 71.69, 69.84, 69.74, 68.21, 66.59, 63.52, 63.09, 55.04, 53.74, 53.56, 53.23, 52.96, 52.48, 46.11, 43.63, 42.39, 37.43, 35.69, 35.41, 35.19, 32.17,

Example

1.1 g of dipeptide 1 (2.77 mmole), was mixed with 53 mg (0.21 mmole, 0.08 eq) of n-Bu₂SnO in 15 mL of benzyl alcohol and heated to 130° C. for 2½ hours, then 100° C. overnight. LC/MS showed no starting material left. The reaction mixture was loaded onto a 330 g of Combiflash column, purified with petroleum ether/EtOAc to give some clean fractions. Mixed fractions were repurified to give a combined yield of 0.67 g (51%) of pure benzyl ester 18. LCMS: [M+H]⁺ m/z=474.46. ¹H NMR (CDCl₃, 6 in ppm): 8.12 (s, 1H), 7.46-7.43 (m, 2H), 7.40-7.32 (m, 3H), 6.68 (d, J=9.6 Hz, 1H), 5.41 (d, J=12.3 Hz, 1H), 5.36 (d, J=12.3 Hz, 1H), 5.24 (d, J=4.5 Hz, 1H), 4.87 (m, 1H), 4.02-3.90 (m, 2H), 2.24-2.13 (m, 2H), 1.88-1.78 (m, 2H), 1.42-1.30 (m, 2H), 1.07 (d, J=6.9 Hz, 3H), 0.97-0.90 (m, 9H). ¹³C NMR (CDCl₃, 6 in ppm): 176.1, 170.2, 161.3, 146.5, 135.7, 128.6, 128.5, 128.4, 127.8, 69.6, 68.8, 66.9, 51.6, 41.1, 38.6, 31.8, 24.1, 19.7, 18.3, 16.0, 11.7.

Example

0.67 g (1.42 mmole) of dipeptide benzyl ester 18 was dissolved in 5 mL dichloromethane. To this solution was added 263 μL of TESCl (236 mg, 1.56 mmole, 1.1 eq), and 117 mg (1.72 mmole, 1.2 eq) of imidazole. The reaction was stirred at 0° C. and solid formed. After 2 hours, the solid was filtered away and the filtrate was concentrated. The residue was on the Combiflash (24 g of silica column) with petroleum ether/EtOAC. After concentration, 763 mg (92%) of the desired product 19 was recovered. ¹H NMR (CDCl₃, δ in ppm): 8.12 (s, 1H), 7.46-7.43 (m, 2H), 7.40-7.32 (m, 3H), 6.68 (d, J=8.4 Hz, 1H), 5.41 (d, J=12.3 Hz, 1H), 5.36 (d, J=12.3 Hz, 1H), 5.13 (t, J=5.7 Hz, 1H), 4.03-3.95 (m, 1H), 3.83 (d, 1H), 2.20-2.05 (m, 1H), 1.95-1.86 (m, 2H), 1.48-1.38 (m, 1H), 1.30-1.20 (m, 2H), 1.03 (d, 3H), 0.96-0.82 (m, 18H), 0.65 (t, 6H). ¹³C NMR (CDCl₃, δ in ppm): 178.2, 168.4, 161.1, 146.5, 135.7, 128.6, 128.5, 128.4, 127.7, 70.7, 70.1, 66.9, 51.3, 39.9, 38.3, 31.6, 24.2, 18.3, 17.6, 16.0, 11.5, 6.8, 4.6.

Example

746 mg (1.27 mmole) of TES protected dipeptide benzyl ester 19 was dissolved in 8 mL of THF (anhydrous, inhibitor-free) and cooled to −45° C. After 15 minutes of cooling, 2.8 mL of 0.5 M KHMDS (1.1 eq., 1.4 mmole) in toluene solution was added dropwise. After an additional 15 mins, 175 μL of chloromethyl butyrate (1.1 eq., 1.4 mmole) was added dropwise. After 30 mins, TLC showed only a trace amount of starting material left. After 2 hours, the reaction mixture was quenched 1 mL MeOH, and allowed to warm to room temperature. The reaction was extracted with EtOAc/brine. The organic layer was washed with brine and then concentrated to give 759 mg (87%) of crude product 20. LCMS: [M+Na]⁺ m/z=710.57. ¹H NMR (CDCl₃, δ in ppm) 8.10 (s, 1H), 7.44-7.40 (m, 2H), 7.39-7.30 (m, 3H), 5.43 (d, J=12.3 Hz, 1H), 5.37 (d, J=12.3 Hz, 1H), 5.35 (s, 2H), 4.98 (t, J=5.1 Hz, 1H), 4.40-4.20 (br, 1H), 3.52 (d, J=16.0 Hz, 1H), 2.42-2.38 (t, J=6.7 Hz, 2H), 2.25-2.05 (m, 2H), 1.78-1.72 (m, 2H), 1.68-1.55 (m, 3H), 1.30-1.20 (m, 1H), 1.00-0.85 (m, 24H), 0.65 (t, 6H). ¹³C NMR (CDCl₃, δ in ppm) 177.6, 173.0, 171.0, 161.1, 146.6, 135.7, 128.6, 128.42, 128.36, 127.6, 77.2, 70.8, 66.8, 63.5, 40.9, 35.9, 34.9, 31.1, 25.0, 20.1, 19.5, 18.1, 15.7, 13.6, 10.5, 6.8, 4.7.

Example

239 mg of MEP (1.67 mmole, 1.5 eq), 316 mg of EDC (1.65 mmole, 1.5 eq), and 300 mg of pentafluorophenol (1.63 mmole, 1.5 eq) were dissolved in 8 mL of N-methyl-2-pyrrolidone. The reaction was stirred overnight. 759 mg (1.1 mmole) of the alkylated dipeptide 20 in 1 mL NMP was then added. An additional 0.8 mL of NMP was used to rinse the flask/syringe to transfer residue to the hydrogenation flask. 60 mg (0.05 eq) of 10% Pd/C was then added and the reaction mixture was hydrogenated at 35 PSI, overnight. LC/MS showed the major product is the benzyl ester, along with 10% free acid. The reaction was filtered through celite, and the filter cake was washed with EtOAc. The filtrate was extracted with brine, washed with brine, and concentrated. Combiflash purification with petroleum ether/EtOAc resulted in the recovery of 215 mg (25%) of benzyl ester 21. LCMS: [M+H]⁺ m/z=787.66. ¹H NMR (CDCl₃, δ in ppm): 8.09 (s, 1H), 7.44-7.40 (m, 2H), 7.39-7.30 (m, 3H), 7.07 (d, J=15.5 Hz, 1H), 5.93 (d, J=12.3 Hz, 1H), 5.42 (d, J=12.3 Hz, 1H), 5.34 (s, 2H), 4.93 (dd, J=8.4, 2.7 Hz, 1H), 4.70-4.60 (m, 1H), 4.50-4.30 (br, 1H), 2.88 (m, 1H), 2.60-2.28 (m, 4H), 2.21 (s, 3H), 2.08-1.89 (m, 4H), 1.80-1.40 (m, 8H), 1.36-1.1.07 (m, 3H), 1.00-0.80 (m, 21H), 0.77 (d, 3H), 0.65 (t, 6H). ¹³C NMR (CDCl₃, 6 in ppm): 177.5, 175.1, 174.1, 173.0, 161.1, 146.5, 135.8, 128.6, 128.4, 128.3, 127.6, 77.2, 70.7, 69.5, 69.2, 66.7, 57.3, 55.4, 53.5, 53.4, 44.8, 41.3, 36.8, 35.9, 31.4, 30.3, 25.0, 24.7, 23.2, 20.2, 19.4, 18.1, 16.2, 13.6, 10.6, 6.8, 5.1, 4.7.

Example

Synthesis of EC1759

Paraformaldehyde (1.5 g, 1.25 eq) was added to 16 mL of TMSBr. The resulted suspension was cooled to 0° C., and 1-pentanol (4.36 mL, 40 mmole, 1 equiv.) was added dropwise. The reaction was stirred at 0° C. and warmed up to room temperature. After overnight, TMSBr was evaporated under reduced pressure. Vacuum distillation of the residue was carried out at 7 mm Hg pressure, the fraction came out at 56° C. was the desired product EC1759 (4.3 g, 59%).

Example

Synthesis of EC1760

1.58 g (3.09 mmole) TES-dipeptide EC0997 was dissolved in 12 mL THF (anhydrous, inhibitor-free). The resulted solution was cooled to −45° C. To the solution, 6.5 mL of 0.5 M KHMDS in toulene (3.25 mmole, 1.05 equiv.) was added dropwise. After finishing the addition, the reaction mixture was stirred at −45° C. for 15 minutes. 600 μL of bromomethyl pentyl ether EC1759 (4.1 mmole, 1.33 equiv.) was added dropwise. The reaction mixture was warmed from −45° C. to −10° C. in 90 minutes, then quenched with 10% NaCl/1% NaHCO₃aqueous solution, extracted with EtOAc. The organic phase was washed with 10% NaCl/1% NaHCO₃aqueous solution three times, then brine. The separated organic phase was dried over Na₂SO₄. Na₂SO₄was filtered off and the solvent was evaporated under vacuum to give 2.4 g of crude product. The crude product was purified with EtOAc/petroleum ether to give 1.47 g of product EC1760 (78%)

Example

Synthesis of EC1761

0.38 g of MEP (2.65 mmole, 1.4 equiv.) was suspended in 1.2 mL NMP, 0.53 g of PFP (2.88 mmole, 1.5 equiv.) and 0.55 g of EDC (2.87 mmole, 1.5 equiv.) were added. The reaction mixture was stirred overnight in a hydrogenation vessel. 1.17 g (1.91 mmole) of alkylated dipeptide EC1760 was dissolved in 0.3 mL NMP and transferred to the above hydrogenation vessel, and the residue of the dipeptide was rinsed with 0.3 mL NMP and transferred to the hydrogenation vessel. 154 mg of 10% Pd/C (dry, 0.05 equiv.) was added to the solution. The hydrogenation was carried out at 35 PSI. After 5 hrs, LC/MS showed there was no starting material. The reaction mixture was filtered through celite pad and the reaction vessel was washed with EtOAc and filtered through celite pad. The combined solution was washed with 10% NaCl/1% Na₂CO₃solution to remove PFP, then with brine. The organic phase was dried over Na₂SO₄. Na₂SO₄was filtered off and the solvent was evaporated under vacuum to give 1.20 g (88%) of crude product EC1761.

Example

Synthesis of EC1602

1.17 g (1.65 mmole) of tripeptide ester EC1761 was dissolved in 15 mL MeOH, the solution was cooled to 0° C. 300 mg of LiOH hydrate (7.15 mmole, 4.3 equiv.) dissolved in 5 mL H₂O was added to the ester solution, the resulted reaction mixture was stirred and warmed up to room temperature in 2 hours. LC/MS showed no starting material left. MeOH was removed using rotary evaporator, and the residual was worked up by extraction between EtOAc/brine. The organic phase was dried over Na₂SO₄. Na₂SO₄was filtered off and the solvent was evaporated under vacuum to give 0.80 g (83%) of crude product EC1602.

Example

Synthesis of EC1633

0.80 g (1.37 mmole) of tripeptide acid EC1602 was dissolved in 6.4 mL of pyridine, the solution was cooled to 0° C. 6.0 mg (0.049 mmole, 0.035 equiv) DMAP was added and then 2 mL of acetic anhydride (21.2 mmole, 15.5 equiv) was added, the reaction mixture was warmed up to room temperature in 5 hours and stored in −20 0° C. for 2 days. 20 mL dioxane/20 mL H2O was added to the reaction mixture at 0° C. and stirred for 1 hour. The solvent was evaporated under reduced pressure. 20 mL of phosphate buffer (20 mM) and 5 mL acetonitrile were added to the residue, the pH of the resulted solution was adjusted to 5.4 using saturated NaHCO₃solution. The solution was loaded on Biotage 120 g C18 column. The flask containing the crude product was rinsed with 1 mL acetonitrile/5 mL phosphate buffer and loaded on the column. The purification was done using a gradient from 20% ACN/80% water to 70% ACN/30%. The fractions containing the desired product were combined and ACN was evaporated under reduced pressure. There were white precipitate coming out from solution, brine was added to the suspension and EtOAc was used to extract the desired product. The organic phase was dried over Na₂SO₄. Na₂SO₄was filtered off and the solvent was evaporated under vacuum to give 0.49 g (57%) of product EC1633.

Example

General Procedures

Synthesis of EC1623 (Scheme 2)

EC1008 (I: R₁=n-propyl. 103 mg) was dissolved in anhydrous dichloromethane (DCM, 2.0 mL) and to this solution was added trifluoroacetic acid (TFA, 0.50 mL). The resulting solution was stirred at ambient temperature under argon for 20 minutes, and to which was added 1-pentanol (0.72 mL). The reaction mixture was stirred at ambient temperature for 3 minutes, concentrated on a Buchi Rotavapor at 30° C. for 10 minutes, residue stirred at ambient temperature under high vacuum for 75 minutes, and to which was added saturated NaHCO₃solution (10 mL) with vigorous stirring, followed by addition of acetonitrile (ACN, 3.0 mL). The resulting white suspension was stirred at ambient temperature for 3 minutes and let stand to settle. The top clear solution was loaded onto a Biotage SNAP 12 g KP-C18-HS column on a Biotage system. The white solid left in the reaction flask was dissolved in water (5.0 mL) and the solution was also loaded onto the Biotage column. The remaining solid stuck on the glass wall of the reaction flask was dissolved in ACN (2.0 mL). To this solution was added water (6.0 mL) and the resulting cloudy solution was loaded onto the same Biotage column. The reaction mixture was eluted following these parameters: Flow rate: 15 mL/min. A: water; B: CAN. Method: 25% B 2 CV (column volume), 25-50% B 3 CV, and 50% B 5 CV (1 CV=15 mL). Fractions containing the desired product was collected and freeze-dried to afford EC1623 (II: R=n-pentyl. 95.9 mg) as a white powder.

Example

Synthesis of EC1662 (Scheme 3)

Step 1: Anhydrous DCM (5.0 mL) was added to a mixture of EC1623 (II: R=n-pentyl. 114 mg), pentafluorophenol (PFP, 67.3 mg), and DCC-resin (2.3 mmol/g, 396 mg) and the suspension was stirred at ambient temperature under argon for 23 hours. The resin was filtered off and washed with anhydrous DCM (3.0 mL) and the combined filtrates were concentrated under reduced pressure to give a residue, which was vacuumed at ambient temperature for 1 hour prior to use in Step 3.
Step 2: EC1426 (114 mg) was dissolved in anhydrous DCM (1.5 mL) and to which was added TFA (0.50 mL). The resulting solution was stirred at ambient temperature under argon for 70 minutes and concentrated under reduced pressure to give a residue, which was co-evaporated with anhydrous DCM (2.0 mL×3) and vacuumed at ambient temperature for 9 hours prior to use in Step 3.
Step 3: The residue from Step 1 was dissolved in anhydrous DCM (1.5 mL) and to this solution was added DIPEA (0.50 mL) followed by a solution of the residue from Step 2 dissolved in anhydrous dimethylformamide (DMF, 1.5 mL). The resulting solution was stirred at ambient temperature under argon for 1 hour, diluted with ethyl acetate (EtOAc, 60 mL), and washed with brine (20 mL×3). The organic layer was separated, dried (Na₂SO₄), and concentrated under reduced pressure to give a residue, which was vacuumed at ambient temperature for 2 hours, dissolved in DCM (3.5 mL), and loaded onto a 24 g silica gel column on a CombiFlash system for purification. The materials were eluted with 0-5% MeOH in DCM to afford EC1662 (III: R=n-pentyl. 171 mg) as a white solid.

Synthesis of EC1664 (Scheme 3)

A solution of EC1454 (SPACER-SH; See FIG. 1 for structure. 44.1 mg.) in 20 mM phosphate buffer (pH 7.0, 4.0 mL) was added to a solution of EC1662 (24.1 mg) in MeOH (4.8 mL), followed by addition of saturated Na₂SO₄(0.30 mL). The reaction mixture was stirred at ambient temperature under argon for 30 minutes and the solution was injected onto a preparative HPLC (A: 50 M NH₄HCO₃buffer, pH 7.0; B: CAN. Method: 10-80% B in 20 minutes.) for purification. Fractions containing the desired product were collected and freeze-dried to afford EC1664 (IV: R=n-pentyl. 42.8 mg) as a fluffy yellow solid.

Claims

1. A process for preparing a compound of the formula

or a pharmaceutically acceptable salt thereof; wherein

Ar₁is optionally substituted aryl or optionally substituted heteroaryl;

R₁is hydrogen, alkyl, arylalkyl or a pro-drug forming group;

R₂is selected from the group consisting of optionally substituted alkyl and optionally substituted cycloalkyl;

R₁₂is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl or heteroarylalkyl, each of which is optionally substituted;

R₃is optionally substituted alkyl;

R₄is optionally substituted alkyl or optionally substituted cycloalkyl;

R₅and R₆are each independently selected from the group consisting of optionally substituted alkyl and optionally substituted cycloalkyl; R₇is optionally substituted alkyl; and n is 1, 2, 3, or 4;

wherein the process comprises the step of treating a compound of formula A with triethylsilyl chloride and imidazole in an aprotic solvent, where R₈is C1-C6 unbranched alkyl

or

the step of treating a compound of formula B with a base and a compound of the formula ClCH₂OC(O)R₂in an aprotic solvent at a temperature from about −78° C. to about 0° C.; wherein the molar ratio of the compound of the formula ClCH₂OC(O)R₂to the compound of formula B from about 1 to about 1.5, where R₈is C1-C6 unbranched alkyl

or

the steps of a) preparing a compound of formula (E1), where X₁is a leaving group, from a compound of formula E

and b) treating a compound of formula C under reducing conditions in the presence of the compound of formula E1, where R₈is C1-C6 unbranched alkyl

or

the step of contacting compound D with an alcohol, R₁₂OH, where R₁₂is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl or heteroarylalkyl, each of which is optionally substituted; and a transesterification catalyst selected from the group consisting of (R₁₃)₈Sn₄O₂(NCS)₄, (R₁₃)₂Sn(OAc)₂, (R₁₃)₂SnO, (R₁₃)₂SnCl₂, (R₁₃)₂SnS, (R₁₃)₃SnOH, and (R₁₃)₃SnOSn(R₁₃)₃, where R₁₃is independently selected from alkyl, arylalkyl, aryl, or cycloalkyl, each of which is optionally substituted;

or

the step of treating the compound AF with a metal hydroxide or a metal carbonate;

or

the step of treating a compound of formula BG with an acylating agent of formula R₄C(O)X₂, where X₂is a leaving group

or

the steps of c) forming an active ester intermediate from a compound of formula AH

and d) reacting the active ester intermediate with a compound of the formula I;

or

one or more combinations thereof.

2. The process of claim 1 wherein R₄is optionally substituted alkyl.

3. The process of claim 1 er-2 comprising the step of treating a compound of formula A with triethylsilyl chloride and imidazole in an aprotic solvent, where R₈is C1-C6 unbranched alkyl

4. The process of claim 1 comprising the step of treating a compound of formula B with a base and a compound of the formula ClCH₂OC(O)R₂in an aprotic solvent at a temperature from about −78° C. to about 0° C.; wherein the molar ratio of the compound of the formula ClCH₂OC(O)R₂to the compound of formula B from about 1 to about 1.5, where R₈is C1-C6 unbranched alkyl

5. The process of claim 1 comprising the steps of a) preparing a compound of formula (E1), where X₁is a leaving group, from a compound of formula E

6. The process of claim 1 comprising the step of treating compound D with an alcohol, R₁₂OH, where R₁₂is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl or heteroarylalkyl, each of which is optionally substituted; and a transesterification catalyst selected from the group consisting of (R₁₃)₈Sn₄O₂(NCS)₄, (R₁₃)₂Sn(OAc)₂, (R₁₃)₂SnO, (R₁₃)₂SnCl₂, (R₁₃)₂SnS, (R₁₃)₃SnOH, and (R₁₃)₃SnOSn(R₁₃)₃, where R₁₃is independently selected from alkyl, arylalkyl, aryl, or cycloalkyl, each of which is optionally substituted;

7. The process of claim 1 comprising the step of treating the compound AF with a metal hydroxide or a metal carbonate;

8. The process of claim 1 comprising the step of treating a compound of formula BG with an acylating agent of formula R₄C(O)X₂, where X₂is a leaving group

9. The process of claim 1 comprising the steps of c) forming an active ester intermediate from a compound of formula AH

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

10. The process of claim 1 wherein R₁is hydrogen, benzyl, or C1-C4 alkyl.

11. The process of claim 1 wherein R₂is C1-C8 alkyl or C3-C8 cycloalkyl.

12. The process of claim 1 wherein R₂is CH₂CH(CH₃)₂, CH₂CH₂CH₃, CH₂CH₃, CH═C(CH₃)₂, or CH₃.

13. The process of claim 1 wherein R₃is C1-C4 alkyl.

14. The process of claim 1 wherein Ar₁is phenyl or hydroxyphenyl.

15. The process of claim 1 wherein R₄is C1-C8 alkyl or C3-C8 cycloalkyl.

16. The process of claim 1 wherein R₅is branched C3-C6 or C3-C8 cycloalkyl.

17. The process of claim 1 wherein R₆is branched C3-C6 or C3-C8 cycloalkyl.

18. The process of claim 1 wherein R₇is C1-C6 alkyl.

19. The process of claim 1 wherein R₁₂is CH₂CH═CH₂, or CH₂(CH₂)nCH₃, where n=1, 2, 3, 4, 5, or 6.

20. The process of claim 1 wherein the metal hydroxide is LiOH.