Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
11746~7Z
The invention relates to a process for the preparation of new cyto-
static compounds, and pharmaceutical compositions containing them.
More particularly, the invention concerns the preparation of new com-
pounds of the general formula
N ~ 3
~;~H
H ~I COOCH3 ~I)
~72~1 :
C 3 ~0- R
CH2HO
OR'
in which
R4 is ~-hydroxyl and
R is ~-ethyl; or
R4 is hydrogen and
R is ~-ethyl;
R" is a straight chained alkyl having from 1 to 10 carbon atoms except ethyl,
or a branched chained alkyl having from 3 to 10 carbon atoms, in which the
carbon atom attached to the ~Na-CH2-O- group has a primary or secondary con-
figuration or an aralkyl having from 1 to 3 carbon atoms in the alkyl moiety;
Rl is methoxy and
R is acetyl.
The new compounds of the general formula (I), in which Rl, R , R3, R
and R" are as defined above, show cytostatic activity.
-- 1 --
r~
. . , -
. . . .
,. . .
, , , :
, , , . :
- :
. .,
117~67~
Compounds of the general formula (I) can be prepared from the corres-
ponding compounds of the general formula ~II)
3~ R
~H
COOCH3
/ ~ ~12 ~ (II)
CH3O
or
the acid addition salts thereof by oxidation in the presence of compounds of the
general formula
R" - 0~l ~III)
In the above formulae Rl, R, R, R4 and R" have the same meaning as hereinbefore
defined. The reaction essentially involves the alteration of the structure of
the group ~Na-CH3 in the compounds of the general formula (II). This alteration
can be performed in any bis-indol alkaloid containing a group `N -CH3.
Starting compounds of the general formula ~II) are known in the art.
Vinblastine ~Rl = methoxy, R2 = acetyl, R3 = ~-ethyl and R4 = ~-hydroxyl) is for
example disclosed in the U.S. Patent Specification No. 3,097,137 and 20'-desoxy-
-leurosidine ~Rl = methoxy, R = acetyl, R3 = ~-ethyl and R4 = hydrogen) and its
isolation are described by N. Neuss et al. [Tetrahedron Letters 1968, 783] while
its synthesis is reported by Potier et al. [J. Am. Chem. Soc. 98, 7017 ~1976)].
-- 2
. .. ..
.
,
-
;72
The starting compounds possess cytostatic activity but are more toxic than the
compounds of the general formula ~I) prepared therefrom.
The acute toxicity of the compounds according to the invention was
tested on male Swiss mice weighing 27 to 31 g. The tests were carried out on
groups of six mice. Tes~ compounds were administered intraperitoneally, as
injectable solutions prepared by physiological saline solution and optionally
one drop of Tween* 80 polysorbate. Doses were successively increased from a
dose causing no mortality up to the lethal dose. The results were evaluated by
the method of Lichfield and Wilcoxon and are listed in the following Table 1, in
comparison with vinblastine, vindesine and vincristine.
Table 1
LD50 Paralytic
C o m p o u n d (mg /kg., effect
mice)
~ . .
N-desmethyl-N-(methoxymethyl)- ~100 0
vinblastine
N-desmethyl-N-(propoxymethyl)- ~ 80 0
vinblastine
N-desmethyl-N-(isobutoxymethyl)- ~ 60 0
vinblastine
N-desmethyl-N-(heptoxymethyl)- ~ 60 0
~ . .
vinblastine
N-desmethyl-N-(benzyloxymethyl)- ~ 70 0
vinblastine
vincristine 4.2
vinblastine 7.6 0
vindesine 4.0 +
i
*Trade Mark - 3 ~
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1~7~672
According to the results set forth in Table 1 the new compounds are 15-
to 25-times less toxic than vincristine and vindesine and 8- to 13-times less
toxic than vinblastine. In contrary to vincristine and vindesine no paralytic
side-effects were observed at doses up to LD50.
The cytostatic activity of the new compounds according to the inven-
tion was tested also in tissue cultures and various transplanted tumour strains.
The test compounds were dissolved in the tissue culture (HeLa cell
culture) in concentrations ranging from a threshold dose of lxlO 3 ~g./ml. up to
100 ~g./ml. In 24-hour cultures, by in vivo microscopic evaluation of the meta-
phase-arresting effect the following results were obtained.
Table 2
Dose inducing a
C o m p o u n d strong block
(~g-/ml-)
N-desmethyl-N-(methoxymethyl)- 0.001
vinblastine
N-desmethyl-N-(propoxymethyl)- 0.001
vinblastine
N-desmethyl-N-(isobutoxymethyl)- 0.001
vinblastine
N-desmethyl-N-(heptoxymethyl)- 0.001
vinblastine
N-desmethyl-N-(benzyloxymethyl)- 0.01
vinblastine
Under similar conditions but using stained tissue cultures also
slighter differences could be observed which can be classified as follows:
li74~7~
The first stage ~minimal effective dose) is characterized by an in-
creased ratio of blocked mitoses. A part of these is abnormal mitoses, for
example three-group mitosis or polar chromosomes also occur. Generally the
anaphascs have already disappeared.
In the second stage a strong metaphase block is observed and practic-
ally there is no regular mitosis present. Chromosomas are in the form of a
loose glomus. The proportion of interphase cells is low.
In the third stage the chromosomas are aggregated in a mass in the
middle of the cell. This state is called pycnomitosis of "Ball-Metaphase".
In the fourth stage already the interphase cells are influenced. More
particularly, the number of the blocked cells is decreased since they are not
able to take part in mitosis. The citoplasm of the cells ~in interphase) is
! extended, its border is irregular, "fringy", the cells frequently have an elon-
gated, fibroplast-like shape.
Finally, in the fifth stage the citoplasm is filled in by a fine,
reticular structure and the interphase cell has unambiguously been killed by thetreatment.
The above stages could be well distinguished when testing the com-
pounds according to the invention.
The following observations were made: The most effective compound was
the heptoxy-derivative, which induced a strong metephasis block already in a
dose of 0.001 ~g./ml. Pycnomitosis was observed at a dose of 0.1 ~g./ml. while
the interphase cells were influenced at doses between 1 and 10 ~g./ml. The iso-
butoxy derivative had an order of a magnitude lower effect, namely at a dose of
0.001 ~g./ml. still regular mitoses could be observed. The benzyloxy derivative
had the weakest effect. At the lowest dose applied this compound induced a
; minimum block with several deformed mitoses but without anaphases. At 0.01
-- 5 --
' C
~ ~ ~ ._c
. . . ' ' .,
:
67~
~g./ml. the block was moderate and a strong block and pycnomitosis were induced
only by a dose of 1 ~g./ml.
The effect of the new compounds on intraperitoneally transplantable
tumours (P 388 mouse leukaemia and NK/Ly ascites lymphoma) is described herein-
below. P 388 leukaemia test was performed on BDF hybride mice. The tests were
carried out on groups of six mice and 106 tumour cells/animal were transplanted
intraperitoneally. Administration of the test compounds was started in the 24th
hour after transplantation. Treatment was performed intraperitoneally and the
body weight and state of animals was controlled every day. The effect obtained
on the treated animals is expressed in % of the mean length of life of the con-
trol group, given in days.
The results set forth in the following table show that the life span
of the mice having a P 388 leukaemia is considerably increased by the test com-
pounds.
~1 7467Z
Table 3
DoseMean length Treated/ Toxicity
of life Control
Compound (days) (%)
Treated Control
_
N-dGsmethyl-N- 8x0.4 14.310.3 139
heptoxy- 8x4.0 18.710.3 181
methyl-VLB 8x8.0 21.3 9.9 217
N-desmethyl-N- 8x0.4 13.010.3 126
(benzyloxymethyl)- 8x4.018.3 10.3 178
vinblastine 5x8.0 19.2 9.9 195
N-desmethyl-N- 8x0.4 13.710.3 133
~isobutoxymethyl)- 8x4.020.0 10.3 194
vinblastine 8x8.0 20.5 9.9 208
N-desmethyl-N- 8xl.0 12.710.3 122
~methoxymethyl)- 8x2.0 18.7;10.5 178;155
vinblastine 16.8
8x4.0 19.0;11.1 171;163
17.7
8x6.0 19.210.5 182
8x8.0 18.7,10.8 172;205
20.2
N-desmethyl-N- 8xl.0 15.010.3 145
~propoxymethyl)- 8x2.0 20.810.5 198
vinblastine 8x4.0 19.011.1 171
8x8.0 20.2 9.9 205
8xlO.0 14.710.3 142 toxic~
The animals died tumour-free
Into groups of ten Swiss-H/Riop outbred mice from our own breed 5X106
ascites tumour cells were transplanted intraperitoneally. In the 24th hour
after transplantation treatment was started and the compounds were administered
daily, altogether five times. The mean length of life of the control group
amounted to 15.7 days.
Similar tests were performed with N-desmethyl-N-~methoxymethyl)-20'-
-desoxy-leurosidine. In these tests the doses and the number of treatments were
.
'
s
il74~72
varied. The following results were obtained:
Table 4
Dose Mean length Treated/
~mg./kg.) of life Control
Compoundi.p. (days) (%)
Treated Control
N-desmethyl- 4x8 21.8 12.1 180
N-(methoxymethyl)- 8x4 20.8 11.6 180
-20'-desoxy- 4x7 17.1 12.1 142
leurosidine 8xl 21.7 12.2 178
8x0.5 15.3 12.2 126
The compounds result in a considerable increase of life span as shown
in the following table.
Table 5
Dose on the 25th day on the 30th
Compound~mg./kg.)alive tumour-free day
i.p. tumour-free
N-desmethyl-
N-(heptoxy- 5x6.0 10/107/10 3/10
methyl)-VLB
- N-desmethyl-
N-~isobut- Sx4.0 10/1010/10 7/10
, oxymethyl)-
` VLB
, .,
,~ ,
The tumour inhibitory effect of the new compounds on P 388 and NK/Ly
strains is significant in a 4 to 8 mg./kg./day dose and is equal to the effect of
- the known diindole alkaloids. In the same time, the instant compounds are far
less toxic than the known compounds having an analogous structure.
10For human treatment the compounds can best be employed intravenously
or as infusions.
According to a further aspect the present invention ~elates to a pro-
cess for the preparation of compounds of the general formula
- 8 -
,j
'
.
117~67Z
~Nl~< R
I~ 11
H I C00CH 3
~--'2 ( I )
CH30 I H ~ 1
OR"
wherein R3, R4, R", Rl and R are as hereinbefore defined. According to the
invention compounds of the general formula (I) are prepared by reacting a com-
pound of the general formula
R3
H I C00CH3
CH3 ~ (I I)
CH3HO
wherein Rl, R2, R and R4 are as hereinbefore defined, or an acid addition salt
thereof, with a large excess of an alcohol of the general formula
s~ ~
.
', ~,' ~. .
J
, ' . - '
,
' '' ~ ': ' .
: ~ .
117~672
R" - OH (III)
wherein R" has the same meaning as defined above, in the presence of chromium
trioxide, a suitable organic solvent and acetic acid anhydride and an acid, at a
temperature between -60 C and -30 C, preferably -50 C and -60 C, adjusting
the pH of the reaction mixture to 8 to 10 and optionally purifying the product
of the general formula ~I) obtained followed by isolation.
According to the above process a compound of the general foTmula ~II)
or an acid addition salt thereof is used as a starting material.
The starting compound is dissolved in a suitable organic solvent, pre-
ferably acetone or a chlorinated hydrocarbon, e.g. in alcohol-free dichloro-
methane, chloroform or dichloroethane. The dissolution and the reaction are sub-
stantially promoted by the presence of an acid. It is therefore advisable to
perform the reaction in a slightly acidic medium. Preferably an organic acid,
e.g. glacial acetic acid, adipinic acid, pelargonic acid and optionally a small
amount of an inorganic acid are added to the reaction mixture. As an inorganic
acid inorganic acids forming an acid addition salt with the compounds of the
general formula ~II) can be used, which may be employed in an amount from traces
to approximately equimolar amount.
The reactant of the general formula (III) is used in a large molar
excess. As an alcohol for example straight chained alkanols having from 1 to 10
carbon atoms can be employed. The reaction with ethanol is disclosed in the co-
-pending European Patent Appln. No. 0018231. First of all methanol, propanol,
butanol, amyl alcohol, heptanol can advantageously be employed. Branched
chained alkanols with from 3 to 10 carbon atoms in which the OH group is attached
to primary or secondary carbon atom can also be used. With tertiary alkanols the
desired reaction cannot be performed. Preferably isobutanol, isopropanol, etc.
are used. Finally, aryl-alkyl alcohols, having from 1 to 3 carbon atoms in the
- 10-
,~
-
:1~74672
alkyl moiety are also suitable alcohol reactants. Of these compounds for
example benzylalcohol or phenylethyl alcohol can be employed.
Alcohols of the general formula (III) are used in a large molar excess.
The molar ratio of the alcohol and the compounds of the general formula ~II) can
be varied depending on the alcohol, but preferably 50 to 150 molar equivalents
of the alcohols of the general formula (III) are used.
Compounds of the general formula ~II) and ~III) are reacted in the pre-
sence of chromium trioxide. Chromium trioxide preferably is added to the reac-
tion mixture as a solution, preferably in acetic acid anhydride.
The reaction is preferably performed in an inert gas atmosphere, at a
temperature between -60 C and -30 C, preferably -50 C and -60 C. The pro-
gress of the reaction is monitored by an analytical method, for example by thin
layer chromatography. Depending on the alcohol of the general formula ~III)
employed the reaction terminates in about 20 minutes to 1 hour and 40 minutes.
When the reaction is complete, the pH of the reaction mixture is ad-
justed to 8 to 10 to eliminate the acetic anhydride completely. The adjustment
of the pH value preferably is started at a temperature of -50 C and generally
is ended at a temperature between 0 C and ~30 C. The reaction mixture prefer-
ably is stirred at room temperature for 5 to 10 minutes.
The crude product obtained is then isolated by extraction and evapora-
tion. The crude product may contain a small amount of N-desmethyl- and N-des-
methyl-N-formyl derivatives, respectively, which preferably are separated from
the desired product of chromatography. Chromatographic purification is prefer-
ably carried out on a partially deactivated alumina or grainy silica gel adsor-
bent, and the isolated product, if desired, is further purified by recrystalliza-
tion.
Further details of the invention are illustrated by the following
examples, which are not intended to limit the scope of the invention in any way.
~, - 11 -
.
.
7467~
N-Desmethyl-N-(methoxymethyl?-vinblastine
1.0 g. (1.1 moles) of vinblastine sulfate are dissolved in a mixture
of 240 ml. alcohol-free absolute dichloromethane, 8.0 ml. of methanol and 25 ml.
of glacial acetic acid. The solution is cooled to -55 C. A solution of 0.5 g.
(5.0 mmoles) of chromium trioxide in 40 ml. of acetic anhydride is cooled to -55
C and is then added dropwise to the vinblastine solution under dry nitrogen
atmosphere, with vigorous stirring in 5 minutes. During the addition the
temperature of the reaction mixture must not exceed -50 C. The progress of the
oxidation reaction is monitored by thin layer chromatography (a silica gel DC-
-Alufolien* Kieselgel* 60 F254; 10:0.5:0.5:0.5 mixture of diethyl ether, ethanol,
benzene and diethyl amine). Generally the oxidation terminates in 20 to 30
minutes. Thereafter a mixture of 190 ml. of concentrated aqueous ammonium
hydroxide solution and 200 g. of ice is added to the reaction mixture under cool-
ing. The temperature raises from -55 C to a temperature between 0 C and +10
C. When this temperature is reached outer cooling is terminated and the solu-
tion having a pH of 8.5 to 9 is vigorously stirred for 10 minutes. The phases
are separated, the aqueous phase is extracted with three 30-ml. portions of
dichloromethane, and the extracts are washed with four 25-ml. portions of a 1:1
mixture of water and ammonium hydroxide and then with two 30-ml. portions of
water. The organic phase is dried with anhydrous magnesium sulfate~ filtered
off and the filtrate is evaporated in vacuo. 0.35 g. of a crude product are ob-
tained. The product is purified by chromatography on a column filled with 0.3 g.
of alumina having an activity grade II-III in a dichloromethane solution. 6-ml.
dichloromethane fractions are collected, which are analysed by thin layer
chromatography (DC-Alufolien* Kieselgel* 60 F254, Art. 5554; solvent: 5:0.4 mix-
ture of dichloromethane and methanol; detection: iodine vapour or ultraviolet
light of 254 nm).
*Trade Mark - 12 -
.,
i72
Fractions containing the same alkaloid are combined and evaporatedseparately. The desired end product is contained in the 6th to 25th fractions.
Yield: 64 % title compound, which is chromatographically uniform.
M.p.: 205 to 210 C (ethanol).
[a]D = +23 (c = 1, chloroform).
H-NMR (CDC13, 100 MHz): ~ 9.05/s, lH, Cl6-OH/, 8.05/s, lH, NaH/, 7.35/s, lH,
C12,-H/, 7.13-7.26/m, 31-1, Cg,-Cll,-H/, 6.68/s, lH, Cg-H/, 6.33/s, lH, C12-H/,
5.84/d, lH, C14-H/, 5.37/s, lH, C17-H/, 5.28/d, lH, C15-H/, 4.42/2H, ,Na-CH2-O-/,
4.18/s, lH,C2-H/, 3.97/s, 3H, C16-CO2CH3/, 3.77/s, 3H, Cll-OCH3/, 3.62/s, 3H,
C16,-CO2CH3/, 3.26/s, 3H, -OCH3/, 2.73/s, lH, C21-H/, 2.00/s, 3H, OCOCH3/,
o.g o.s6/2t, 6H, C18~H3~ C18' 3/
MS m/e: 840 (M 100 %), 810, 809, 781, 751, 681, 651, 650, 601, 499, 355, 282,
243, 241, 154, 149.
xample 2
N-Desmethyl-N-(isobutoxymethyl)-vinblastine
0.5 g. (0.55 mmoles) of vinblastine sulfate are dissolved in a mixture
of 120 ml. of absolute dichloromethane, 3.7 ml. of isobutanol and 12.5 ml. of
glacial acetic acid and the solution is cooled to -55 C. 0.25 g. (2.5 mmoles)
of chromium trioxide in 40 ml. of glacial acetic acid cooled to -55 C are then
added and the progress of the reaction is monitored by thin layer chromatography
as described in Example 1 ~DC-Plastikfolien* Kieselgel* 60 F254, Art. 5735;
10:0.5:0.5:0.5 mixture of ether, ethanol, benzene and diethyl amine). The reac-
tion terminates in 140 minutes. Furtheron treating the reaction mixture as des-
cribed in Example 1 0.65 g. of crude product are obtained. The crude product is
purified by column chromatography on a Kieselgel 60 (Art. 9385) adsorbent, in
dichloromethane solvent. The column is washed and eluted with dichloromethane.
10-ml. fractions are collected. The first 330 ml. of eluate do not contain any
*Trade Mark - 13 -
~'~
.....
,
`` :
,
'
~174~7Z
alkaloid. Elution is then continued with dichloromethane containing 3 %
methanol. The first 9O ml. eluate contains the desired title compound, which is
isolated by evaporation.
Yield: 169 mg. (35 %) of the title compound.
Melting point: 215 to 218 C (acetone and ether).
IR spectrum (KBr): 3400 ~NH,OH), 1730 (CO2CH3), 1605, 1220(cm/OAc). H-NMR/CDC13,
100 MHZ/: ~ 8.1/s, lH, Na,H/, 7.5/m, lH, C12,-H/, 7.05-7.2/m, 3H, Cg~-Cll~-H/~
6-70/s, lH, Cg-H/, 6.36/s, lH, C12-H/, 5.87/d, lH, C14-H/, 5.37/s, lH, C17-H/,
5-30/d, lH, C15-H/, 4-75, 4.15/2H, JAB10 Hz, ~ Na-CH2-O-j, 4.0/s, lH, C2-H/, 3.78,
3.75/2s, 6H, CO2CH3/, 3.63/s, 3H, Cll-OCH3/, 2.75/s, lH, C21H/, 2-1/s, 3H,
OCOCH3/, 0.95-0.70/12H, CH3-groups/.
MS m/e: 882 (M , 100 %), 864, 851, 823, 810, 779, 751, 723, 651, 650, 514, 355,
346, 329, 154.
Example 3
N-Desmeth~l-N-~heptoxymethyl)-~inblastine
The procedure described in Example 2 is followed except that 3.7 ml.
of isobutanol and replaced by 3.7 ml. of l-heptanol. Reaction time: about 90
minutes. 4 g. of crude product are obtained from the reaction mixture. Puri-
fication is carried out on a Kieselgel* 60 (Art. 9385) chromatographic column in
30 ml. of dichloromethane. The column is washed and eluted with dichloromethane.
10-ml. fractions are collected. The first 300 ml. do not contain any alkaloid.
Elution is continued with dichloromethane containing 3 % of methanol. The first
120 ml. eluate contains the desired title compound.
Yield: 21 % of title compound.
Melting point: 200 to 205 C (Acetone/ether).
IR spectrum (KBr): 3450 /OH,NH/, 1740 /CO2CH3/, 1610, 1220/cm/OAc/. H-NMR
(CDC13, 100 MHz): ~ 8.07/s, lH, N ,H/, 7.52/m, lH, C12,-H/, 7.2-7.02/m, 3H,
*Trade Mark - 14 -
~;'`'
`` 117'~72
Cg,-Cll,-H/, 6.70/s, lH, Cg-H/, 6.35/s, lH, C12-H/, 5.85/dd, lH, C14-H/, 5.39/s,
lH, C17-H/, 5-30/d, lH, C15-H/, 4.75, 4-15/2H, JAB 10Hz, ~Na-CH2-O-/, 4.0/s, lH,
, 2CH3/, 3.65/s, 3H, Cll-OCH3/, 2.75/s, lH C -H/
2.10/s, lH, OCOCH3/, 0.7-1.0/3t, 9H, CH3-groups/.
MS m/e: 924 ~M , 100 %), 906, 893, 865, 822, 810, 765, 751, 737, 651, 650, 649,
469, 455, 355, 282, 154, 135, 122.
Example 4
N-Desmethyl-4-(benzyloxymethyl)-vinblastine
The procedure described in Example 2 is followed but 3.7 ml. of iso-
butanol are replaced by 4.3 ml. of benzyl alcohol. After 45 minutes 4 ml. of an
oily product are obtained, which is then isolated by preparative thin layer
chromatography ~Kieselgel* PF254+366; a mixture of lOO ml. of dichloromethane
and 8 ml. of methanol).
Yield: 75 mg. (15 %) of the title compound.
Melting point: 215 to 218 C (methylene chloride/ether).
IR spectrum (KBr): 3400 /OH, NH/, 1740 /CO2CH3/, 1610, 1230/cm/OAc/. lH-NMR
/CDC13, 100 M~lz/: ~ 8.07/s, lH, Na,H/, 7.52/m, lH, C12,-H/, 7.2-7.08/m, 3H,
Cg,-Cll,-H, 3H, aromatic/, 6.75/s, lH, Cg-H/, 6.20/s, lH, C12-H/, 5.85/dd, lH,
C14-H/, 5-41/s, lH, C17-H/, 5.30/d, lH, C15-H/, 4.80, 4.20/2H, JAB lOHz,
,Na-CH2-O-/, 4.40/s, 2H, benzyl-CH2/, 4.00/s, lH, C2-H/, 3.70, 3.68/2s, 6H,
CO2CH3/, 3.63/s, 3H, Cll-OCH3/, 2.75/s, lH, C21-H/, 1.0-0.70/2t, 6H, CH3-groups/-
MS m/e: 916 (M , 100 %), 885, 857, 822, 810, 796, 779, 757, 751, 737, 651, 650,
649, 514, 355, 346, 329, 282, 154, 135.
Example 5
N-Desmethyl-N-(methoxymethyl)-vinblastine
5 g. (5.5 mmoles) of vinblastine sulfate are dissolved in 150 ml. of
water. The vinblastine base in set free by adding a concentrated aqueous
*Trade Mark - 15 -
.
.
~174~
ammonium hydroxide solution (pH = 8) and is treated with four 50-ml. fractions
of dichloromethane. The dichloromethane solution is dried over sodium sulfate,
filtered and the filtrate is evaporated to dryness in vacuo. 4 g. of vinblas-
tine base are obtained, which are then dissolved in a mixture of 1000 ml. of
alcohol-free dichloromethane, 120 ml. of acetic acid and 6 ml. of methanol. The
solution is cooled to -55 C. To the solution a solution of 2~5 g. (25 mmoles)
chromium trioxide in 470 ml. of acetic anhydride cooled to -55 C is added, in
5 minutes. The reaction mixture is stirred at -55 C for 45 minutes, whereupon
it is added to a mixture of 940 ml. of concentrated aqueous ammonium hydroxide
solution and 940 ml. of ice water. The temperature must not exceed 300 C. The
phases are separated and the dichloromethane phase is admixed with 1000 ml. of a1 % aqueous ammonium hydroxide solution. The organic phase is separated, dried
; over anhydrous sodium sulfate, filtered and the filtrate is evaporated to dry-
ness _ vacuo 3.8 g. of a crude product are obtained. Purification s carried
out on a chromatographic column prepared from 230 g. of alumina having an
activity grade of II-III (Brockmann) and benzene. l'he crude product is dis-
solved in 20 ml. of a 8:2 mixture of benzene and chloroform and is layered on
the column. Elution is performed with about 8 litres of a mixture containing
benzene and chloroform in the above proportion. 100-ml. fractions are collected,which are analysed by thin layer chromatography (silicagel; 5:5:5:100 mixture ofbenzene, ethanol, diethyl amine and diethyl ether). Fractions containing the
same substance are combined and evaporated. 2.1 g. of the title compound are ob-tained.
Yield (after recrystallization from 6 ml. of ethanol): 1.5 g.
The physical characteristics of the product are the same as in Example
1 .
.
"~,~, . .~
i~7~67;~
Example 6
N-Desmethyl-N-(propoxymethyl)-vinblastine
Following the procedure described in Example 5 but using 4.5 ml. of
propanol instead of methanol as a reactant, 0.6 g. of the title compound are ob-
tained.
Melting point: 200 to 210 C (ethanol).
[~]20 = +31.2 (c = 1, chloroform)
H-NMR /CDC13, 100 MHz/: ~ 9.05/s, lH, C16-OH/, 8.06/s, lH, N ,H/, 7.34/m, lH,
C12,-H/, 7.34-7.12/m, 3H, Cg,-Cll,-H/, 6.68/s, lH, Cg-H/, 6.35/s, lH, C12-H/,
5-84/dd, lH, C14-H/, 5.36/s, lH, C17-H/, 5.28/d, lH, C15-H/, 4.75, 4-15/2H,
JAB lOHz, ,Na-CH2-0-/, 4.18/s, lH, C2-H/, 3.78/s, 3H, C02CH3/, 3.75/s, 3H,
Cll-OCH3/, 3.62/s, 3H, C02CH3/, 2.73/s, lH, C21-H/, 2.09/s, 3H, OCOCH3/,
0.65-0.94/m, gH, CH3-groups/.
Example 7
-Desmethyl-N-(methoxymethyl)-vinblastine
Following the procedure described in Example l but starting from
vinblastine dihydrochloride instead of vinblastine sulfate, the title compound
is obtained.
Yield: 28 %.
The physical characteristics of the product are identical with those
given in Example 1.
Example 8
N-Desmethyl-N-(methoxymethyl)-vinblastine
Following the procedure described in Example 1 but using alcohol-free
chloroform, dichloroethane or acetone as a solvent instead of dichloromethane,
the title compound is obtained.
Yield: 40 to 50 % depending on the solvent employed.
The physical characteristics are the same as given in Example 1.
! ~ 17 -
li7~f~72
Example 9
N-Desmethyl-N-(methoxymethyl)-20'-desoxyleurosidine
60 mg. ~0.067 mmoles) of 20'-desoxyleurosidine sulfate prepared by
catalytic hydrogenation (Pd/C, methanol) of 3',4'-anhydrovinblastine and a sub-
sequent conversion into the corresponding sulfate salt, are dissolved in a mix-
ture of 20 ml. of absolute dichloromethane, 1.5 ml. of glacial acetic acid and
0.5 ml. of methanol. The solution is cooled to -55 C and a solution of 30 mg.
(0.3 mmoles) of chromium trioxide in 3.5 ml. of acetic anhydride, cooled to -55
C is added. The reaction terminates in 60 minutes. To the reaction mixture a
mixture of 12 ml. of concentrated aqueous ammonium hydroxide solution and 12 g.
of ice is added and the phases are allowed to separate. The aqueous phase is
shaken with three 10-ml. portions of dichloromethane. The combined organic
phase is washed with two 10-ml. portions of a 1:1 mixture of ammonium hydroxide
and water and subsequently with two 5-ml. portions of water. Drying and evapora-
tion of the organic phase affords 48 mg. of a crude product. Purification is
carried out by column chromatography (silica gel 0.040 to 0.063 mm. Merck, Art.
9385; solvent: CH2C12 followed by a mixture of 3 % methanol and CH2C12). 18 mg.
(32 %) of the title compound are obtained.
Melting point: 185 to l9O C (decomp.).
[~]D = +50 (c = 1, chloroform).
IR spectrum (KBr): 3400 /NH/, 1720/CO2CH3/, 1610, 1230/cm/OAc/. H-NMR/CDC13/:
7.95/s, lH, Na,-H/, 7.54/m, lH, C12,-H/, 7.2-7.1/m, 3H, Cg,-Cll,-H/, 6.56/s,
lH, Cg-H/, 6.11/s, lH, C12-H/, 5.87/dd, lH, C14-H/, 5.46/s, lH, C17-H/, 5-37/d,
lH, C15-H/, 4.72, 4.15/2H, ~Na-CH2-O-/, 3.80, 3.75, 3.61 /9H, CO2CH3/, 2.11/s,
1ll, OCOCH3/, 1.0-0.6/6H, CH3-groups/.
By the above chromatographic purification step 46 mg. (22 %) of
N-desmethyl-N-formyl-20'-desoxyleurosidine can be isolated as a by-product.
Melting point: 205 to 210 C (decomp.).
[~]D = +54 (c = 1, chloroform).
- 18 -
V
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