Taxane derivatives, their preparation and use in oncology

The preparation of new 3,11-cyclotaxanes having the general formula 1. Cyclotaxane 1a (1, R'.dbd.R".dbd.H) may be isolated from plants of the genus Taxus. New cyclotaxanes 1 have antiblastic activity.

The present invention pertains to new 3,11-cyclotaxane derivatives, to 
their preparation and to their use in oncology. 
The new compounds have the general formula 1 
##STR1## 
wherein each of R' and R", independently of the other, is hydrogen, 
alkanoyl of 2 to 7 carbon atoms, --CO--CHOHCH(C.sub.6 H.sub.5) NHCOC.sub.6 
H.sub.5, or --COCHOHCH(C.sub.6 H.sub.5)NHCOOC(CH.sub.3).sub.3. 
The cyclotaxane la (1, R'.dbd.R".dbd.H) is a new natural compound 
obtainable substantially free of other taxicins and taxines by extraction 
of needles of plants of the Taxus genus, e.g. Taxus baccata L.. Its 
structure was determined by spectroscopical analysis, namely lH-NMR and 
13C-NMR. The structure 5-O-cinnamoyl10-acetyl-phototaxicine was in fact 
assigned to la. The new cyclotaxane la can be isolated by extraction at 
room temperature of the vegetable materials with alcohols, e.g. methanol 
or ethanol, or aliphatic ketones, e.g. acetone, or with their mixtures 
with water. The extracts, after concentration under vacuum till the 
removal of the organic solvent, are filtered from any precipitated 
insoluble material and treated with a water immiscible aprotic solvent 
such as methylene chloride, chloroform or ethylacetate. The organic 
extract containing 1a is evaporated to dryness and purified by column 
chromatography, using silica gel as stationary phase and solvent mixtures 
such as nhexane, ethyl acetate, methylene chloride/methanol, or 
acetone/toluene as eluents. The fractions containing 1a are concentrated 
to dryness under vacuum and the residue crystallized from ethyl acetate. 
The compound 1a also can be obtained starting from the natural compound 2, 
which also can be isolated from Taxus plants, by irradiation at 240 nm in 
ethanol solution with a mercury lamp. 
Under these conditions, the formation of the bond between the carbon atoms 
in the position 3 and 11 and the migration of the proton from the position 
3 to the position 12 take place. 
##STR2## 
The invention concerns therefore also compound 2 as an intermediate for the 
synthesis of la. The compound 2 is also per se active as an anti-tumor 
agent, as hereinbelow specified. 
The photochemical rearrangement of 2, having configuration E on the 
cinnamoyl residue, is accompanied by partial isomerization of the olefinic 
double bond. The material from the photocyclization consists in fact of a 
5:1 mixture of the E and Z isomers. The natural product la has however a 
unitary stereoisomery, as does compound 2, having E configuration. 
Re-isomerization of the portion of the Z isomer obtained from the 
photosynthesis to the E isomer can be achieved by refluxing the mixture 
from the photocyclization in tetrahydrofuran in the presence of 
diphenylsulfide. 
The complete conversion of 2 to la occurs. 
The photocyclization reaction of taxicins was described by K. Nakanishi (J. 
Chem. Soc. Chem. Commun. 1201, 1967) and it presumably occurs through a 
diradical intermediate in positions 3 and 11, formed by hydrogen transfer 
from the carbon in 3 to the carbon in 12. However the Nakanishi method 
relying on dioxane as a solvent gives low yields (about 50%) and, when 
applied to taxicins containing the cinnamoyl residues, yields 
stereoisomeric mixtures having high content in the Z isomer. 
We have found that it is possible to obtain 1a containing at least 85% of 
the E isomer by carrying out the photocyclization in ethanol solution and 
with a low pressure mercury lamp. We also have found that it is possible 
to achieve the complete transformation of the mixture into the E isomer by 
refluxing in tetrahydrofuran in the presence of diphenylsulfide in the 
amount of 0.1-0.4 mol/mole of 2. 
The stereochemically pure product is isolated after removal of the 
diphenylsulfide by silica gel chromatography, eluting with solvent 
mixtures such as toluene/acetone or n-hexane/ethyl acetate. 
The compound 2 is extracted from needles of T. baccata L. similarly to the 
method described for compound la, for instance by extraction of the 
vegetable material with alcohols at room temperature, then with water 
immiscible solvents, silica gel chromatography and preparative HPLC. 
The derivatives 1 wherein R' and/or R" represent COR"' groups can be 
obtained by reacting 1a (1, R'.dbd.R".dbd.H) with a suitably activated 
derivative of the acid R"'COOH, e.g., the acid chloride, anhydride, or in 
the presence of a condensing agent, such as dicyclohexylcarbodiimide. In 
the first instance, the esterification is preferably carried out in a 
basic solvent, e.g. pyridine, with a stoichiometric amount of the 
acylating reagent. The reaction mixture is diluted with water and 
extracted with chlorinated solvent (e.g. methylene chloride or chloroform) 
or with an ether (e.g. ethyl ether). The organic phase is separated, 
washed with water and evaporated to dryness under vacuum. 
The residue is then chromatographed on silica gel to give the desired 
ester. When dicyclohexylcarbodiimide is used, the esterification is 
carried out in aprotic solvent, such as dichloromethane, chloroform or 
dioxane. 
After removal of the formed dicyclohexylurea by filtration, the reaction 
mixture is evaporated to dryness under vacuum and the residue is 
chromatographed on silica gel using as eluent solvent mixtures such as 
n-hexane/ethyl acetate or toluene/acetone. 
It is possible to obtain a selective acylation since the hydroxy groups in 
position 2 and 9 of la have different reactivity. 
In particular, the hydroxy in the 9-position can be acylated under mild 
conditions, at temperatures ranging from -50.degree. C to the room 
temperature. For acylation in the 2-position, it is necessary to use 
stronger conditions, for instance by heating the mixture to 30-80.degree. 
C, using suitable catalysts such as 4-dimethylaminopyridine, or prolonging 
the reaction times. 
For instance compound 1b (1, R'.dbd.R".dbd.COCH.sub.3) is obtained by 
reacting 1a (R'.dbd.R".dbd.H) with acetic anhydride in dry pyridine 
solution. 
After standing overnight at room temperature, the reaction mixture is 
diluted with water and extracted with chloroform. The organic phase is 
washed with a sodium bicarbonate aqueous solution, then with water and 
evaporated to dryness. The residue, after silica gel column chromatography 
eluting with 7:3 ethyl acetate/hexane mixture, yields 1b. 
The compounds of formula 1 and 2 have antimitotic activity comparable to 
that of known taxanes such as taxol or derivatives thereof, and in vivo, 
antitumor activity. 
In vitro, they exhibited activity on the brain tubuline (Shelanski, Proc. 
Natl. Acad. Sci. USA, 70, 765, 1973) and on human cultured leucocytes. The 
compounds of the invention have an activity on tubuline which is twice 
that of the corresponding derivatives of baccatine III. The compounds can 
be administered orally or parenterally, alone or in combination with other 
therapeutic agents including anti-neoplastic agents, steroids, etc., to a 
mammal in need of such treatment. Parenteral routes of administration 
include intramuscular, intrathecal, intravenous and intraarterial. As with 
any drug of this type, dosage regimens must be titrated to the particular 
neoplasm, the condition of the patient, and the response observed but 
generally doses will be from about 10 to about 30 mg/m.sup.2 per day for 5 
days or 150 to 250 mg/m.sup.2 given once every three weeks. While having a 
low toxicity as compared to other agents now in use, a toxic response 
often can be eliminated by either or both of reducing the daily dosage or 
administering the compound on alternative days or at longer intervals such 
as every three to five days. Oral dosage forms include tablets and 
capsules containing from 1-10 mg of drug per unit dosage. Isotonic saline 
solutions containing 20-100 mg/ml can be used for parenteral 
administration. 
The following examples will clarify the main aspects of the invention.

EXAMPLE 1 
Isolation of 5-O-cinnamoyl-10-acetylcyclotaxane 1a (1, R'.dbd.R".dbd.H) 
from Taxus baccata leaves 
500 kg of Taxus baccata leaves were extracted with 10 portions, 1500 1 each 
of ethanol at room temperature. The collected extracts were concentrated 
till a 900 1 volume and allowed to stand for 24 hours at room temperature. 
The undissolved material was separated by centrifugation and the solution 
was extracted with 5 portions, 300 1 each, of methylene chloride. The 
organic phases were collected and the solvent was distilled off under 
reduced pressure. The obtained residue, 3.5 kg, was dissolved in a mixture 
containing chloroform and methanol (98:2) and was passed through a 
chromatography column containing 70 kg of silica gel, using the same 
solvent mixture as eluent. 
The fractions containing pure la were collected, the eluent was distilled 
off under reduced pressure and the residue was crystallized from ethyl 
acetate. The compound 1a was obtained in the form of a microcrystalline 
white powder; m.p. 126.degree. C., [.alpha.].sub.D.sup.20 +7.5 (CH.sub.2 
Cl.sub.2 c.dbd.0.77) UV.lambda..sub.max (EtOH) 279, 217, 201 nm. 
IR.nu..sub.max (KBr): 3475, 1700, 1630, 1450, 1370, 1245, 1040, 990, 900, 
770, 710 cm.sup.-1. CI-MS (NH.sub.3) 556 (C.sub.31 H.sub.38 
O+NH.sub.4).sup.+. 
EXAMPLE 2 
Isolation of 5-0-cinnamoyl-10-acetyltaxicine I (2) 
500 kg of dried needles and small branches of T. baccata leaves were 
extracted with ethanol at room temperature. The residue was suspended in 
water and extracted with hexane and then with CHC13 After evaporating the 
chloroform phase, the residue (3.5 kg) was subjected to silica gel column 
chromatography, using methylene chloride containing increasing amounts of 
methanol as eluent. The CH.sub.2 Cl.sub.2 -MeOH (98:2) fractions gave 12 g 
of a yellowish powder, which, after HPLC chromatography (hexane - ethyl 
acetate 1:1) yielded 1.5 g of 2 as a white powder, m.p. 145.degree. C., 
[.alpha.].sub.D.sup.15 +185 (CHCl.sub.3, c.dbd.0.61); UV.lambda..sub.max 
EtOH nm: 280, 223, 217; IR.nu..sub.max KBr cm.sup.-1 : 3450, 1720, 1670, 
1645, 1320, 1230, 1180, 1010, 990; CI-MS (NH.sub.3) 140 eV, m/z (rel. 
int.): 556. 
EXAMPLE 3 
Preparation of 5-O-cinnamoyl-10-acetylcyclotaxane 1a (1, R".dbd.R".dbd.H) 
by photocyclization 
1.05 g of 5-O-cinnamoyl-10-acetyltaxicine I (2) were dissolved in 500 ml of 
ethanol. The solution was put into a quartz tube and air was completely 
removed by bubbling nitrogen. The tube was placed into a Rayonet 
photochemical reactor and the solution was irradiated at 240 nm for 5 
hours. The reaction mixture was then vacuum distilled to dryness and the 
residue was dissolved in 200 ml of tetrahydrofurane. 100 mg of 
diphenylsulfide were added and the mixture was refluxed for 5 hours. 
The reaction mixture was then distilled to dryness and the residue was 
purified with a chromatography column containing 250 g of silica gel, 
using a mixture of n-hexane-ethyl acetate 9:1 as eluent. The 
chromatography was continued till complete elimination of the disulphide. 
Subsequent elution with n-hexaneethyl acetate 1:1 gave 1 g of la, which 
was crystallized from ethyl acetate. The obtained product had the same 
physico-chemical characteristics as the product obtained in Example 1. 
EXAMPLE 4 
Preparation of 5-O-cinnamoyl-2,9,10-triacetylcyclotaxane 1b (1, R'.dbd.H 
R".dbd.COCH.sub.3) 
200 mg of la were dissolved in 2 ml of anhydrous pyridine and treated with 
2 ml of acetic anhydride. The reaction mixture was allowed to stand for 12 
hours at room temperature, than was diluted with 15 ml of water and 
extracted with two portions, 5 ml each, of methylene chloride. The 
collected organic phases were washed with a saturated NaHCO.sub.3 aqueous 
solution then with water and finally was evaporated to dryness. The 
residue was submitted to column chromatography, through 15 g of silica 
gel, using a n-hexane-ethyl acetate 3:7 mixture as eluent. The eluates 
containing 1b were vacuum distilled to dryness and gave 170 mg of white 
product, m.p. p.f. 90.degree. C., CI-MS (NH.sub.3) m/z 598 (C.sub.31 
H.sub.38 O.sub.8 +NH.sub.4).sup.+.