Process for the preparation and purification of thiol-containing maytansinoids

Abstract

The present invention provides a process for the preparation and purification of thiol-containing maytansinoids comprising the steps of: (1) reductive hydrolysis of a maytansinoid C-3 ester with a reducing agent selected from the group consisting lithium trimethoxyaluminum hydride (LiAl (OMe)3H), lithium triethoxyaluminum hydride (LiAl(OEt)3H), lithium tripropoxyaluminum hydride (LiAl (OPr)3H), sodium trimethoxyaluminum hydride (NaAl (OMe)3H), sodium triethoxyaluminum hydride (NaAl(OEt)3H) and sodium tripropoxyaluminum hydride (NaAl(OPr)3H) to yield a maytansinol; (2) purifying the maytansinol to remove side products when present; (3) esterifying the purified maytansinol with a carboxylic acid to yield a mixture of an L- and a D-aminoacyl ester of maytansinol; (4) separating the L-aminoacyl ester of maytansinol from the reaction mixture in (3); (5) reducing the L-aminoacyl ester of maytansinol to yield a thiol-containing maytansinoid; and (5) purifying the thiol-containing maytansinoid.

Description

[0001]U.S. application Ser. No. 10 / 758,264, filed Jan. 16, 2004 (abandoned), is a divisional application of this application.<?insert-end id="INS-S-00001" ?>FIELD OF THE INVENTION[0002]The present invention relates to a process for preparing and purifying cytotoxic agents. More specifically, the invention relates a process for preparing and purifying cytotoxic agents comprising thiol-containing maytansinoids. These cytotoxic agents can be used as therapeutic agents by linking them to a cell binding agent, through the thiol group, and then delivering them to a specific cell population in a targeted fashion.BACKGROUND OF THE INVENTION[0003]In recent years, a myriad of reports have appeared on the attempted specific targeting of tumor cells with monoclonal antibody-drug conjugates (R. V. J. Chari., 31 Adv. Drug Deliv. Res., 89-104 (1998); G. A. Pietersz and K. Krauer, 2 J. Drug Targeting 183-215 (1994); Sela et al., in Immunoconjugates 189-216 (C. Vogel, ed. 1987); Ghose et al., ...

AI Technical Summary

Benefits of technology

[0026]Thus, the object of the present invention is to provide an improved process for the preparation and purification of thiol-containing maytansinoids that reduces the complexity of the process, allows scalability and improves the yield.

Problems solved by technology

In vitro cytotoxicity tests, however, have revealed that antibody-drug conjugates rarely achieved the same cytotoxic potency as the free unconjugated drugs.

This suggested that mechanisms by which drug molecules are released from the antibodies are very inefficient.

One reason for the lack of disulfide linked antibody-drug conjugates is the unavailability of cytotoxic drugs possessing a sulfur atom containing moiety that can be readily used to link the drug to an antibody via a disulfide bridge.

Furthermore, chemical modification of existing drugs is difficult without diminishing their cytotoxic potential.

Another major drawback with existing antibody-drug conjugates is their inability to deliver a sufficient concentration of drug to the target site because of the limited number of targeted antigens and the relatively moderate cytotoxicity of cancerostatic drugs like methotrexate, daunorubicin and vincristine.

However, such heavily modified antibodies often display impaired binding to the target antigen and fast in vivo clearance from the blood stream.

A further drawback to the therapeutic use of maytansinoids, of interest here, is that the process for the preparation and purification of thiol-containing maytansinoids involves several inefficient chromatographic steps that are cumbersome, not easily scalable and result in only moderate yields.

However, this process involves several inefficient steps that are cumbersome and result in moderate yields.

However, the reaction with lithium aluminum hydride results in the formation of several side products which can be removed only by careful preparative thin layer chromatography on silica gel.

Thus, this process is not amenable for industrial scale use.

While the unreacted maytansinol is readily separated from its esters by column chromatography on silica gel, the diastereomeric maytansinoid esters are barely separable.

Thus, this process is also not amenable for industrial scale use.

However, this step results in low yields and is also not amenable for industrial scale use.

Thiol-containing maytansinoids are not very soluble in the ethanol / water solvent mixture used for the reaction.

Furthermore, purification by HPLC on a reverse phase C-18 column using acetonitrile / water as the mobile phase results in poor recovery and can result in the dimerization of some product.

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