Organic compounds -- part of the class 532-570 series – Organic compounds – Heterocyclic carbon compounds containing a hetero ring...
Reexamination Certificate
2000-07-13
2002-08-13
Owens, Amelia (Department: 1625)
Organic compounds -- part of the class 532-570 series
Organic compounds
Heterocyclic carbon compounds containing a hetero ring...
C549S511000
Reexamination Certificate
active
06433198
ABSTRACT:
FIELD OF THE INVENTION
The present invention is directed to an enzymatic hydroxylation method for the preparation of 6-&agr;-hydroxy-7-deoxytaxanes, useful as intermediates in the preparation of taxanes, and particularly in the preparation of paclitaxel, paclitaxel analogues, and second and third generation paclitaxel-like compounds.
BACKGROUND OF THE INVENTION
Taxanes are diterpene compounds which find utility in the pharmaceutical field. For example, paclitaxel (Taxol®), a taxane having the structure:
has been found to be an effective anticancer agent.
Naturally occurring taxanes such as paclitaxel, 10-deacetylpaclitaxel and baccatin III can be extracted with some difficulty from the trunk bark of different species of Taxus (yew). Paclitaxel, in particular, may be extracted from the inner bark of
Taxus brecifolia.
Although
T. brevifolia
is a relatively common tree in the Pacific Northwest, it is a slow growing plant and is indigenous to the ecologically threatened old-growth forests of this area, and harvesting is thus increasingly restricted because of environmental concerns.
As yields of paclitaxel extracted from
T. brevifolia
are generally low, of the order of 100 mg/kg, semisynthetic methods of producing paclitaxel from baccatin III and 10-deacetylbaccatin have proven successful and are routinely practiced. Baccatin III, 10-deacetylbaccatin, as well as other paclitaxel precursors may be isolated from the needles of the European yew,
Taxus baccata
in relatively larger quantities, e.g. approximately 300 mg/kg of 10-deacetylbaccatin may be obtained from yew leaves. Although yew needles generally provide an adequate supply of the necessary starting materials for synthesizing paclitaxel, the supply is not endless and other methods easing the supply dilemma and producing adequate amounts of paclitaxel have become a priority. The art has thus continued to search for synthetic, including semisynthetic routes for the preparation of naturally occurring taxanes such as paclitaxel, as well as the preparation of paclitaxel analogues and second and third generation paclitaxel-like compounds thereof.
Recently, endophytic microbes associated with
T. brevifolia
were examined as potential alternative sources of paclitaxel. Stierle et al., in “Bioactive Metabolites of the Endophytic Fungi of Pacific Yew,
Taxus brevifolia
”, ACS 1995, have confirmed that the fungus
Taxomyces andreanae,
isolated from the inner bark of a yew tree in Montana, has demonstrated the ability to produce paclitaxel.
Paclitaxel is converted to 6-&agr;-hydroxy paclitaxel in human liver by cyp2C8, and loses most of its cytotoxicity as a result of this hydroxylation Such activity is evidenced by studies performed by Kumar et al, “Comparative in vitro Cytotoxic Effects of Paclitaxel and Its Major Human Metabolite 6&agr;-hydroxypaclitaxel”,
Cancer Chemother. Pharmacol.
36: 129-135 (1995), and by Rahman et al., “Selective Biotransformation of Paclitaxel to 6-&agr;-hydroxypaclitaxel by Human Cytochrome P450 2C8”,
Cancer Research,
54: 5543-5546 (1994). To avoid this problem, second generation analogs are being developed. Compounds such as 6-&agr;-hydroxy-7-deoxytaxanes or compounds derived therefrom may be useful as second generation drugs. The chemical preparation of these compounds requires a long sequence of reactions to incorporate oxygen at C6. An enzyme able to hydroxylate the 6-position of a 7-deoxytaxane would afford a much simpler route. Although human cyp2C8 converts paclitaxel to 6-&agr;-hydroxypaclitaxel, it has not been shown to be effective with 7-deoxypaclitaxel or 7-deoxybaccatin. We have, therefore, derived another enzyme source for this transformation.
SUMMARY OF THE INVENTION
The present invention provides for a process of preparing 6-&agr;-hydroxy-7-deoxytaxanes using microorganisms or enzymes derived therefrom for hydroxylation of 7-deoxytaxanes at C-6 to give 6-&agr;-hydroxy-7-deoxytaxanes from which taxanes having a desired sidechain at C-13 or containing another modification, may subsequently be synthesized.
In particular, the present invention provides a method for the preparation of at least one taxane having a hydroxyl group directly bonded at C-6, comprising the steps of contacting at least one taxane having a deoxygenated C-7 with an enzyme or microorganism capable of effecting said hydroxylation at C-6.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides an efficient method for the preparation of 6-&agr;-hydroxy-7-deoxytaxanes from 7-deoxytaxanes. The present invention is described further as follows.
In a preferred embodiment, the present invention provides a method for the preparation of at least one 6-&agr;-hydroxy-7-deoxytaxane of the following formula I:
where
R
1
is hydrogen, hydroxyl, R
4
—O—, or R
5
—C(O)—O—;
R
2
and R
3
are independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, or heterocyclo;
R
4
is a hydroxyl protecting group;
R
5
is a hydrogen, alkyl, alkenyl, alkynyl, cyclocalkyl, cycloalkenyl, aryl or heterocyclo; and
R
6
is hydrogen, a hydroxyl protecting group such as triethylsilyl, or an acyl group such as a paclitaxel side chain, cephalomannine side chain, or taxol analog side chain;
or salts, thereof, comprising the steps of contacting at least one 7-deoxytaxane of the following formula II:
where R
1
, R
2
, R
3
and R
6
are as defined above,
or salts thereof, with an enzyme or microorganism capable of effecting the hydroxylation at C-6 on said taxane. All stereoconfigurations of the unspecified chiral centers of the compounds of the formulae I and II are contemplated in the method of the present invention, either alone (that is, substantially free of other stereoisomers) or in admixture with other stereoisomeric forms. Furthermore, this method may also be utilized in the preparation of the 6-&bgr;-hydroxy-deoxytaxane isomer.
In another embodiment, the present invention provides a method for the preparation of at least one first taxane, having a desired 6-&agr;-hydroxy-7-deoxytaxane, from at least one second taxane having a C-10 acetyl, and having no hydroxyl group directly bonded at C-7, by enzymatic hydrolysis of the C-10 acetyl to provide at least one 10-deacetyl-6,7-dideoxy analogue by the method described herein, followed by attachment of the desired hydroxyl group at C-6 to provide the former. In this embodiment, the present invention provides, for example, a method for the preparation of a desired taxane having a hydroxyl group directly bonded at C-6, from a starting material of 7-deoxy-10-acetyltaxane by simultaneous or sequential hydrolysis of the 7-deoxy-10-acetyltaxane to provide a C-10 deacetyltaxane, followed by the coupling of the desired hydroxyl group at C-6. The 6-&agr;-hydroxy-7-deoxytaxane product of the enzymatic process of the present invention includes a hydroxyl group directly bonded at C-13 and which may undergo later subsequent reaction as part of the semisynthesis of paclitaxel from the 7-deoxytaxane starting material.
The terms “enzymatic process” or “enzymatic method”, as used herein, denote a process or method of the present invention employing an enzyme or microorganism. The term “hydroxylation”, as used herein, denotes the formation of a hydroxyl group, and may be achieved, for example, by contact with oxygen and a suitable reductant according to the method of the present invention. Use of “an enzyme or microorganism” in the present method includes use of a single, as well as two or more, enzymes or microorganisms.
The terms “alkyl” or “alk”, as used herein alone or as part of another group, denote optionally substituted, straight and branched chain saturated hydrocarbon groups, preferably having 1 to 12 carbons in the normal chain. Exemplary unsubstituted such groups include methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, pentyl, hexyl, isohexyl, heptyl, 4,4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl, nonyl, decyl, undecyl, dodecyl and the like. Exemplary substituents may include one or more of the following groups: halo, alkoxy, alkylthio, alkenyl, alkynyl, aryl, cycl
Hanson Ronald L.
Patel Ramesh N.
Bristol--Myers Squibb Company
Owens Amelia
Peist Kenneth W.
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