Synthesis of epothilones

Details Synthesis of epothilones Synthesis of epothilones

2001-03-02

2002-10-01

Padmanabhan, Sreeni (Department: 1621)

Harvesters

Compressing and binding

Cord band and clip

C560S178000, C560S179000, C560S263000, C568S308000

Reexamination Certificate

active

06457303

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention is broadly concerned with methods for synthesizing various epothilone segments or precursors (either naturally occurring or analogs thereof) which can be used for the efficient synthesis of complete epothilones. More particularly, the invention pertains to such synthesis methods wherein, inter alia, the epothilone segment C is prepared using a unique Noyori reduction scheme, and epothilone segments B and C are connected via a novel aldol condensation reaction. These syntheses can be used to prepare the naturally occurring segments and a wide variety of corresponding analogs and homologs.
2. Description of the Prior Art
The epothilones (16-membered macrolides which were initially isolated from the myxobacterium
Sorangium cellulosum
) represent a class of promising anti-tumor agents, and have been found to be potent against various cancer lines, including breast cancer cell lines. These agents have the same biological mechanism of action as Taxol, an anti-cancer drug currently used as a primary therapy for the treatment of breast cancer. Other potential applications of the epothilones could be in the treatment of Alzheimer's disease, malaria and diseases caused by gram-negative organisms. Other cancers such as ovarian, stomach, colon, head and neck and leukemia could also, potentially be treated. The epothilones also may have application in the treatment of arthritis.
In comparison to Taxol®, the epothilones have the advantage of being active against drug-resistant cell lines. Drug resistance is a major problem in chemotherapy and agents such as the epothilones have overcome this problem and hold great promise as effective agents in the fight against cancer.
In addition, the poor water solubility of Taxol® has led to the formulation of this drug as a 1:1 ethanol-Cremophor concentrate. It has been determined that the various hypersensitive reactions in patients such as difficulty in breathing, itchiness of the skin and low blood-pressure are caused by the oil Cremophor used in the formulation. The epothilones are more water soluble than Taxol® which has positive implications in its formulation. Further advantages of the epothilones include easy access to multi-gram quantities through fermentation procedures. Also the epothilones are synthetically less complex, thus structural modifications for structure activity relationship studies are easily accessible.
The epothilones exhibit their activity by disrupting uncontrolled cell division (mitosis), a characteristic of cancer, by binding to organelles called microtubules that are essential for this process. Microtubules play an important role in cell replication and disturbing the dynamics of this component in the cell stops cell reproduction and the growth of the tumor. Antitumor agents that act on the microtubule cytoskeleton fall into two general groups: (1) a group that inhibits microtubule formation and depolymerizes microtubules and, (2) a group that promotes microtubule formation and stabilizes microtubules against depolymerization. The epothilones belong to the second group and have displayed cytotoxicity and antimitotic activity against various tumor cell lines.
It has been demonstrated on the basis of in vitro studies that the epothilones, especially epothilone B, are much more effective than Taxol® against multi-drug resistant cell line KBV-1. Preliminary in vivo comparisons with Taxol® in CB-17 SCID mice bearing drug-resistant human CCRF-CEM/VBL xenografts have shown that the reduction in tumor size was substantially greater with epothilone B in comparison to Taxol®.
In light of the great potential of the epothilones as chemotherapeutic agents, there is a need for techniques allowing the practical, large scale, economical synthesis thereof. Furthermore, there is a need for synthesis methods which facilitate the preparation of various homologs and analogs of the known epothilones, and those having affinity labels allowing study of the binding interactions of these molecules.
SUMMARY OF THE INVENTION
The present invention overcomes the problems outlined above, and provides various practical, commercially feasible synthesis routes for the production of important epothilone precursors or segments in high yield. The invention is particularly concerned with synthesis of the precursors or segments C, D (which is a combination of segments B and C) and vinyl halide epothilone precursors.
In a first aspect of the invention, an epothilone precursor of the formula
is synthesized using a Noyori reduction reaction. In the foregoing formula, n
1
is an integer from 0-4, R
4
is selected from the group consisting of H, C1-C10 straight and branched chain alkyl groups, substituted and unsubstituted benzyl groups, and C1-C10 alkoxy groups, R
5
and R
6
are each individually and respectively selected from the group consisting of H, substituted and unsubstituted aryl and heterocyclic groups, C1-C10 straight and branched chain alkyl groups, and substituted and unsubstituted benzyl groups, R
7
is H or straight or branched chain C1-C10 alkyl groups, and P′ is a protective group. The method comprises the steps of first providing a &bgr;-keto ester of the formula
where n
1
, R
5
, R
6
, R
7
and P′ are as defined above, and T is an alkyl group. This &bgr;-keto ester is then preferentially hydrogenated at the C3 keto group to form the corresponding hydroxyester. This is accomplished by reacting the &bgr;-keto ester with a hydrogenating agent in the presence of an asymmetric organometallic molecular catalyst comprising a metal atom or ion having one or more chiral ligands coupled thereto. The synthesis is completed by then converting the hydroxyester to the epothilone precursor.
More preferably, n
1
is an integer from 0-4, R
5
, R
6
and R
7
are each individually and respectively selected from the group consisting of H and the straight and branched chain C1-C4 lower alkyls, and the protective group is benzyl. In terms of preferred process parameters, the hydrogenating agent is preferably H
2
and the hydrogenating step is carried out at a pressure of from about 30-100 psi, more preferably 50-75 psi, and at a temperature of from about 40-100° C., more preferably from about 50-75° C. The reaction is normally allowed to proceed for a period of from about 12 hours to 5 days, and more usually for about 2-5 days. Typically, the reaction mixture is agitated during the hydrogenating step.
The catalyst used in the hydrogenation reaction is preferably one of the well-known Noyori catalysts such as RuBr
2
(S)-binap. However, a variety of other catalysts of this type can also be employed. The catalyst is generally used at a level of from about 1-25 mol % in the reaction mixture.
In order to complete the reaction sequence, the hydroxyester resulting from the Noyori reduction is converted to the epothilone precursor segment C. A number of routes can be used to effect this conversion. Preferably, however, the conversion involves: (1) removing the P′ protecting group from the hydroxyester to form a diol; (2) protecting the oxygen atoms of the diol, forming a protected diol; (3) reducing the ester function of the protected diol to a primary alcohol; (4) oxidizing the primary alcohol to the corresponding aldehyde; (5) reacting the aldehyde with a Grignard reagent having the R
4
group coupled thereto to form a secondary alcohol; and (6) oxidizing the secondary alcohol to form the final epothilone precursor.
Preferably, the P′ removal step involves reacting the hydroxyester with hydrogen in the presence of a catalyst (e.g., Pd(OH)
2
or Pd/C) at a pressure of from about 40-100 psi. The oxygen atom protecting step comprises reacting the diol with TBS chloride in a compatible solvent (i.e., one that will not interfere with the desired reaction) at a temperature of from about 40-100° C. for a period of from about 30-60 hours. The ester function reduction step is preferably carried out by reacting the protected diol with the reducing agent DIBAL-H at a temperatu

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