Organic compounds -- part of the class 532-570 series – Organic compounds – Heterocyclic carbon compounds containing a hetero ring...
Reexamination Certificate
2000-03-28
2001-04-10
Higel, Floyd D. (Department: 1626)
Organic compounds -- part of the class 532-570 series
Organic compounds
Heterocyclic carbon compounds containing a hetero ring...
C435S123000, C549S510000
Reexamination Certificate
active
06215000
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention is directed to novel crystalline complexes of baccatin III with imidazole, 2-methylimidazole or isopropanol which are useful for isolating and purifying baccatin III from a crude plant cell culture solution. The complex is also useful for isolation and purification of baccatin III from crude extracts of bark and leaves of the plant, such as
Taxus bevifolia
(Western yew)
Taxus baccata
etc, which contain baccatin III with other impurities or from plant tissue culture (PTC) solutions.
The taxane family of terpenes, of which baccatin III is a member, is of considerable importance, since the latter is a starting material for the semi-synthesis of paclitaxel (Taxol®). Paclitaxel (Taxol®) is now considered one of the most promising treatments for breast and ovarian cancer. Its effectiveness is also demonstrated against lung cancer and melanoma.
The isolation of baccatin III from the Yew needles has been described in U.S. Pat. Nos. 9,330,696, 930,312. According to the method of these U.S. Pat. Nos. 9,330,696, 930,312, Yew needles are dewaxed with a critical or nearly critical fluid, followed by extraction of the taxoids with a mixture of critical or nearly critical fluid and a polar solvent and the resulting extract is chromatographed. The chromatography is carried out using two columns. The first column, a normal-phase silica absorption column, retains the taxoids and the second column, a reverse-phase absorption column, separates the taxoids. The taxoids, such as taxol, baccatin III, 10-deacetylbaccatin III and cephalomannine, are eluted with a mixture of critical or nearly-critical fluid and a polar solvent. Patents describing the production of baccatin III and paclitaxel PTC solutions are U.S. Pat. No. 5,407,816 and U.S. 5,637,484.
Another method for the isolation of baccatin III has been described in J. Liq, chromatogr., 12 (11), 2117-32 (1989). In this method, large quantities of methanol extracts derived from the bark of the Western yew,
Taxus brevifolia
and containing a mixture of five taxanes was resolved on adsorbent types (cyano and Ph bonded silica gel phases) using isocratic and gradient elution. The five taxanes obtained were, namely, Taxol, cephalomannine, 10-deacetylcephalomannine, baccatin III and 10-deacetylbaccatin III.
In addition, an improved method for isolating Taxol and certain clinically important analogs of Taxol including baccatin III has been described in U.S. Pat. No. 5,475,120. The improved method which comprises treating the crude extract of a naturally occurring Taxus species by reverse phase liquid chromatography on an adsorbent, causing the taxol and taxol analogs to be adsorbed on the adsorbent and recovering the taxol and its analogs from the adsorbent by elution with an eluant. The compounds isolated were taxol, 10-deacetylbaccatin III, baccatin III etc.
Thus, all of the above processes require an alcoholic extract or a polar solvent extract obtained from the bark or needles of the Taxus species and then chromatographing the crude extract on a column of silica gel.
SUMMARY OF THE INVENTION
An object of the present invention is to provide a novel crystalline complex of baccatin III and imidazole, 2-methylimidazole or isopropanol, when baccatin III is mixed together in solution with either imidazole, 2-methylimidazole or isopropanol. Another object of the present invention is to provide a method of isolating baccatin III from plant cell culture extracts containing a mixture of several taxanes including baccatin III by treating the cell culture with imidazole, 2-methylimidazole or isopropanol and collecting the precipitated complex of baccatin III and imidazole, 2-methylimidazole or isopropanol. This precipitated complex of baccatin III and imidazole, 2-methylimidazole or isopropanol is then broken down to give baccatin III.
Another additional object of the present invention is to provide a method of isolating baccatin III from plant extracts of bark of
Taxus brevifolia
or needle (leaves) or any plant extract containing baccatin III in admixture with several other taxanes. The extract of bark or needles is treated with imidazole, 2-methylimidazole or isopropanol and the precipitated complex of baccatin III and imidazole, 2-methylimidazole or isopropanol is collected and broken down to give baccatin III.
Another object of the present invention is to purify crude baccatin III solid containing other taxanes by heating it in isopropanol to form a solution and then allowing it to cool to form a complex of baccatin III with isopropanol.
DETAILED DESCRIPTION OF THE INVENTION
Briefly, therefore, the present invention is directed to a novel crystalline complex of baccatin III and imidazole (I), 2-methylimidazole (II) or isopropanol (III) having the structural formulas:
The x-ray structure of the three complexes (IA, IIB, IIIC) are determined by a single crystal x-ray analysis and are depicted herein below.
The x-ray analysis shows the above chemical structure of baccatin III and imidazole with a molecular formula of C
37
H
38
O
11
.C
3
H
4
N
2
.0.25H
2
O. In the crystal, two hydrogen bonds are formed between baccatin III and imidazole, O7-H—N=1.743 Å) and N1-H—O (the carbonyl oxygen of the C-10 acetyl group) (154.0°, N—O=2.898 Å, H—O=2.000 Å). The water molecule also forms two hydrogen bonds with baccatin III, Owater-H—O1 (O—O=2.750 Å) and Owater-H—O13 (O—O=3.060 Å).
The x-ray analysis shows the chemical structure of baccatin III and 2-methylimidazole with a molecular formula of C
31
H
38
O
11
.C
4
H
6
N
2
. Similar to imidazole, the 2-methylimidazole complex links molecules of baccatin III through two hydrogen bonds, O7-H—N3 (171.4°, O—N=2.671 Å, H—N=1.680 Å) and W1-H—O (the carbonyl oxygen of the C-10 acetyl group) (148.5°, N—O=2.960 Å, H—O=2.155 Å). The 2-methylimidazole also interacts with baccatin III via three C—H—O hydrogen bonds, two of them involving the methyl group.
The x-ray analysis shows the chemical structure of baccatin III and isopropanol complex with a molecular formula of C
31
H
38
O
11
.C
3
H
3
O. There are two hydrogen bonds formed between baccatin III and isopropanol.
The single crystal of the complexes of imidazole and 2-methylimidazole are grown in the solvent acetonitrile. The single crystal of the complex of isopropanol is grown in methylene chloride and isopropanol.
The x-ray powder diffraction pattern of the three complexes, namely baccatin III-imidazole, baccatin III-2-methylimidazole or baccatin III-isopropanol were obtained using a Debye-Scherrer powder camera irradiated by copper targer x-ray tube for 2 hours at 40 kv, 20 ma through a nickel filter.
The d-spacings and the relative intensities of the three complexes are tabulated below.
TABLE 1
Baccatin III-imidazole complex
Relative
Line
Spacings D(Å)
Intensity I/Io
1
10.52
30
2
9.57
30
3
8.95
100
4
6.95
20
5
6.60
10
6
6.24
20
7
5.61
10
8
5.20
40
9
4.82
40
10
4.13
50
11
3.90
20
12
3.75
10
TABLE 2
Baccatin III-2-methylimidazole complex
Relative
Line
Spacings D(Å)
Intensity I/Io
1
10.52
30
2
9.41
30
3
8.70
100
4
6.72
20
5
6.13
20
6
5.50
10
7
5.09
30
8
4.69
30
9
4.28
10
10
4.14
40
11
3.89
10
12
3.75
20
13
3.73
10
TABLE 3
Baccatin III-isopropanol complex
Relative
Line
Spacings D(Å)
Intensity I/Io
1
10.52
30
2
9.24
20
3
8.52
100
4
7.19
20
5
6.60
20
6
6.13
20
7
5.35
10
8
5.03
30
9
4.70
25
10
4.09
50
11
3.78
20
12
3.72
20
13
3.56
20
14
3.24
10
15
3.12
10
16
2.95
10
17
2.83
20
The complex is formed by dissolving baccatin III having the structural formula
in dichloromethane or another appropriate organic solvent such as n-butyl acetate, ethyl acetate, or dichloroethane, at room temperature and then adding to the solution, with stirring, imidazole or 2-methylimidazole as a solid or in a solution of organic solvent, such as dichloromethane. In the case of isopropanol, the baccatin III is dissolved directly in the solvent by heating and then allowed to cool to crystallize the complex. The solution
Dillon, Jr. John L.
Gibson Francis S.
Vemishetti Purushotham
Wei Jianmei
Bristol--Myers Squibb Company
Higel Floyd D.
Lopez Gabriel
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