Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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This invention relates to a novel alkaloid deriva-
tive, its acid addition salts and pharmaceutical compositions
containing the same, furthermore to a process for the prepara-
tion thereof. More particularly, the invention relates to
the novel diindole alkaloid 4-deacetoxy-vincristine, its acid
addition salts, pharmaceutical compositions containing the
same, as well as to a process for their preparation.
As known, the minor alkaloids of Vinca roses L. with
diindole skeleton possess valuable cytostatic effects (U.S.
patents Nos. 3,097,137, 3,205,220, 3,887,565 and 3,890,325;
Hungarian patents Nos. 153,200, 154,715 and 160,967). Phar-
macologically effective semisynthetic alkaloid derivatives,
such as N-formyl-leurosine (Hungarian patent No. 165,986),
various carboxamide and deacetyl derivatives ~published
German patent applications Nos. 1,795,763 and 2,415,890) and
4-deacetoxy-vinblastine (N. Neuss, A.J. Barnes and L.L.
Huckstep: Experimentia 31/1, 18-19 /1975/) have also been
prepared.
Now it has bean found that 4-deacetoxy-vinblastine
can be converted by oxidative formylation into 4-deacetoxy-
vincristine, a novel compound possessing excellent cytostatic
effects and being less toxic than vincristine.
The cytostatic effects of 4-deacetoxy-vincristine
were examined on subcutaneously transplantable tumors of rats
and mice. m e effects on L 1210/VS (vinca-sensitive) mouse
lymphoid leukemia and L 1210/Ref (vinca-refractive) mouse
lymphoid leukemia were tested on BDFl mice, the effects on
Erlich ascites sarcoma were examined on Swiss mice, whereas
the effects on Yoshida sarcoma and ~ovikoff hepatoma were
examined on Wistar rats. 4-Deacetoxy-vincristine sulfate was
admixed with physiological saline and the mixture was adminis-
tered intraperitoneally once a day into the animals to be
- 1 - ~a~
5~C~.
tested. In the tests the average prolongation of survival
period and the tumor-inhibiting effect were examined in re-
lation to the untreated controls. The results are listed in
Table 1. In the Table the symbol T/c represents the ratio of
treated animals to the controls.
:J~O'~ ~SSl
s~ ~ ~ ;^~ ~ ~
~ ~ ~ o~ ~ ~
1` 0 0 ~ ~1 0 0 ~ CS~ 1~ t`~ 0 d' ~ O
X ~ O ~ t` d~ ~ Ln t` ~ 0 ~D 0 ~ d'
~ ~I N ~ 1 r~ 1 r I r~ 1 ~I t~ 3 N (~)
o ~ o ~ n Ln Ln ~ ~ ~ o~ co 0 co 0
0 0 0 ~ ~
.~q ~ ~ ~
-'I -
3 ~ ~ ~ 0 ~ 0 o r~ 1~ ~ Ln o o
~3a) O ~ ~ d' d~ ~P Ln O ~ ~1 0 ~ ~ c5~ Ln
i~ f~ E I ~ -1 ~1 ~1 ~1 ~1 ~1 ~ t~l t~ ~ ~ d' .
Lo~
~ ~ ~ I` t` l` l` ~ ~ ~ ~ 0 0
~ l u ~ ~
X ~1~ d' Ln ~ d~ 0 ~ 9 . .. . U) n
3?~ ~
~ ~ U ~ O
o ~ ~ u o ~o
~ ~l ~l ~l v ~
E~ ~ ~ ~ ~
-- 3 --
109'15S1.
It appears from the table that when administering
the compound into rats or mice in daily intraperitoneal dos-
ages of 0.1 to 1.0 mg/kg, the survival period increases by
about 70 to 190 per cents in relation to the untreated con-
trols. The activity of the compound is dosage-dependent.
The novel compound is particularly effective against Ehrlich
ascites sarcoma, Yoshida sarcoma and Novikoff hepatoma.
The approximative acute toxicity data of 4-deace-
toxy-vincristine, compared to those of other known vinca al-
0 kaloids, are summarized in Table 2.TABLE 2
C o m p o u n d LD50 mg/kg
4-Deacetoxy-vincristine (mouse, i.p.) 5
4-Deacetoxy-vincristine (mouse, i.v.) 6
4-Deacetoxy-vincristine (rat, i.p.) 1.3
Vincristine (mouse, i.p.) 4.2
Vincristine (rat, i.p~) 1.3
The data of Table 2 show that the novel compound
is by 25% less toxic than vincristine, a compound effective
in the same dosage interval. It is a particularly important
advantage that neurotoxicity accompanied with paralytic symp-
toms, a characteristic adverse side effect of vincristine,
does not appear upon administering the 4-deacetoxy compound
even in lethal dosages.
In the human therapy the intravenous or infusion
methods of administration are recommended. The compound can
be administered in daily amounts of 0.01 to 0.3 mg/kg, pre-
ferably 0.05 to 0.2 mg/kg, either as a single dosage or in
subdivided forms.
4-Deacetoxy-vincristine can be prepared, in accord-
lO9 ~S~l
ance with the invention, by subjecting 4-deacetoxy-vinblastine
to oxidative formylation. If desired, the resulting product
can be purified and/or converted into its acid addition salts.
Thus, in accordance with the invention, 4-deacetoxy-
vinblastine containing an N-methyl group is converted into
the respective ~-formyl derivative by oxidative formylation.
The term "oxidative formylation" refers to oxidation and
formylation performed either simultaneously or in two subse-
quent steps.
When oxidation and formylation are performed in two
subsequent steps, a mixture consisting of 4-deacetoxy-vin-
cristine and ~-demethyl-4-deacetoxy-vinblastine is obtained
in the first oxidation step. If desired, the components of
this mixture can be separated from each other and then the
N-demethyl compound is formylated, one can also proceed, how-
ever, by formylating the mixture as such.
When the reaction is performed in the simultaneous
presence of oxidizing and formylating agents, the required 4-
deacetoxy-vincristine is obtained directly.
Oxidation can be performed in the presence of an
oxidizing acid or a salt or anhydride thereof, one can also
use, however, oxygen or an oxygen-containing gas, such as air,
and an oxidizing catalyst for the same purpose.
As oxidizing agent e.g. nitric acid, a nitrate salt,
or, preferably, chromium trioxide or an alkali dichromate
(such as potassium or sodium dichromate) can be applied.
As oxidizing catalyst a metal of the platinum group,
such as platinum, palladium or rhodium can be applied, pref-
erably in supported form.
As formylating agent e.g. formic acid can be used.
The process according to the invention can be per-
formed e.g. as follows:
1()~4.S~.
4-Deacetoxy-vinblastine, optionally liberated from
its acid addition salt prior to the reaction, is dissolved in
an inert organic solvent and/or in the formylating agent it-
self. As solvent the formylating agent, preferably formic
acid, furthermore optionally a benzene-type hydrocarbon, such
as benzene, toluene or xylene can be applied. senzene proved
to be a particularly advantageous inert solvent.
Thereafter an oxidizing catalyst, preferably a
supported noble metal of the platinum group, particularly
platinum black or palladium-on-carbon, is introduced into the
reaction mixture. Oxidation is perforrned at a temperature of
O to 30C,preferably at room temperature, in oxygen atmosphere
~preferably in an atmosphere of oxygen with 1 to 3 atm. pres-
sure). When the reaction is over the catalyst is removed
from the mixture, the pH of the resulting mixture is adjusted
to a value of 7 to 10, preferably 8.5 to 9, with an inorganic
base (preferably ammonium hydroxide), and then the alkaline or
neutral solution is extracted with an inert organic solvent,
such as e.g. benzene, toluene, xylene or a chlorinated hydro-
carbon, preferab:Ly methylene chloride or chloroform. The ex-
tract is evaporated, and the resulting product is purified by
recrystallization, if desired. As recrystallization solvent
preferably an aqueous lower alcohol, such as aqueous methanol,
ethanol or propanol can be applied. If desired, the obtained
product is converted into its acid addition salt, preferably
into the sulfate.
According to a preferred method of the invention one
proceeds as follows:
4-Deacetoxy-vinblastine is dissolved in a lower ali-
phatic ketone, such as acetone, methyl ethyl ketone or methylisobutyl ketone, and an oxidizing agent, preferably chromium
trioxide or an alkali dichromate, is added to the reaction
~0~ 15'j~.
mixture. Oxidation is performed in the presence of acetic
anhydride and glaci~ll acetic acid at a temperature below 0C,
preferably at -90 to -30C. When the reaction is over the pH
of the mixture is adjusted to a value of 5 to 10. This opera-
tion is performed preferably in two steps by adjusting first
the pH to a value of 5 to 7, and then further increasing the pH
to a value of 8 to 9. Thereafter the products of oxidation
(i.e. the aimed compound and the N-demethyl derivative) are
separated from each other e.g. by column chromatography, and
the N-demethyl component is formylated. It is more approp-
riate, however, to formylate the mixture as such~
Formylation can be performed in a solvent inert
towards the reaction, such as in a benzene-type hydrocarbon,
it is more preferable, however, to apply the formylating
mixture itself (a mixture of formic acid and acetic anhydride)
as reaction medium. The temperature of formylation amounts
from 0C to 15Ct preferably 0C to 5C. Thereafter the pH of
the mixture is adjusted to a value of 7 to 10, preferably 8
to 9.5. The resulting alkaline or neutral solution is ex-
tracted with an inert organic solvent, preferably with a ben-
zene-type hydrocarbon, such as benzene, xylene or toluene, or
a chlorinated hydrocarbon, particularly methylene chloride or
chloroform. The crude product obtained after evaporating
the extracts is purified e.g. by column chromatography and/or
recrystallization.
As chromatographic adsorbent partially deactivated
alumina (preferably alumina with an activity grade of III),
whereas as eluting agent an inert organic solvent, such as
benzene, toluene, xylene and/or a chlorinated hydrocarbon,
preferably chloroform or methylene chloride, can be applied,
In oxder to separate the aimed product the fractions contain-
ing the major part of 4-deacetoxy-vincristine are evaporated,
whereas the previous and subsequent cuts are evaporated and
the product is subjected to a second chromatographical treat-
ment. The two product fractions can be recrystallized, if
desired, either separately or after combination. If desired,
the resulting product can be converted into its acid addition
salt, preferably into the sulfate.
As already mentioned above, 4-deacetoxy-vincristine
or its acid addition salts are applied in the therapy pre-
ferably as parenterally administerable compositions. In
order to prepare a parenterally administerable composition
the active agent is dissolved in distilled water or physio-
logical saline and/or optionally in a lower aliphatic alcohol
or glycol ether. Preferably a freeze-dried form of the
active agent is dissolved in the respective solvent. The
solution may also contain various additives, such as preserva-
tives (e.g. benzyl alcohol, p-oxybenzoic acid esters, etc.),
antioxidants (e.g. ascorbic acid, tocopherol, etc.), lactose,
buffers, and related substances. The compositions may also
contain other pharmaceutically active agents, such as local
anaesthetics. The solution is filled into vials, and steril-
ized either before or after filling.
In accordance with a broad aspect of the invention,
there is provided a process for the preparation of 4-deace-
toxy-vincristine or its acid addition salts, in which 4-
deacetoxy-vinblastine is subjected to oxidative formylation
in the presence of an organic solvent, chromium oxide or an
alkali metal dichromate being used as oxidizing agent, the
resulting product is purified, if desired, and optionally
converted into its pharmaceutically acceptable acid addition
salt.
The invention is elucidated in detail by the aid of
the following non-limiting Examples.
~0~55~
The physical constants of 4-deacetoxy-vincristine
were determined as follows: The optical rotation power was
measured on an Opton type polarimeter, whereas the PMS spec-
tra were recorded on a Varian EM 360 type spectrophotometer.
EXAM2LE 1
12 g. (0.0159 moles~ of 4-deacetoxy-vinblastine are
dissolved in 7640 ml. of acetone, and 600 ml. of glacial
acetic acid, distilled from chromium trioxide, are added to
the solution. The solution is cooled to -55C, and a solu-
tion of 5.94 g. (0.135 moles) of chromium trioxide in 2000
ml. of acetic anhydride, cooled to -55 to -60C, is added
under stirring. When the reaction terminates the pH of the
mixture is adjusted to 6 with cc. aqueous ammonia pre-cooled
to -35 to -40C. During this operation the mixture should
be cooled so that its temperature does not raise a~oye 50C.
The nearly neutral reaction mixture is diluted with 9 1. of
distilled water, and the pH of the resulting mixture is ad-
justed to 8.5 with 25% aqueous ammonia. The reaction mixture
is extracted with 4 x 1500 ml. of methylene chloride. The
methylene chloride solutions are combined and washed with
3 x 1000 ml. of distilled water in order to remove the am~on-
ium acetate by-product.
The phases are allowed to separate from each other.
The methylene chloride solution is dried over sodium sulfate,
filtered, and the filtrate is evaporated to dryness under
reduced pressure. The resulting dry residue, consisting of
crude 4-deacetoxy-vincristine and ~-demethyl-4-deacetoxy-
vinblastine, is dissolved in a mixture of 60 mL of cc. formic
acid and 10 ml. of acetic anhydride, the solution is poured
into 300 ml. of water pre-cooled to 0 to 5C, and the pH of
the resulting solution is ad~usted, under stirring, to 9 with
cc. aqueous ammonia pre-cooled to 0 to 5C. The alkaline solu-
tion is extracted with 3 x 100 ml. of methylene chloride. The
g _
5'~i1.
methylene chloride solutions are combined, dried, and evaporat-
ed to dryness under reduced pressure. The resulting 9.8 g. of
4-deacetoxy-vincristine is purified by column chromatograph~.
9.~ g. of deacetoxy-vincristine are dissolved in 60 ml. of
benzene, and the solution is poured onto a chromatographic
column, 45 mm. in diameter, filled with 500 g, of alumina
(activity grade: III) in benzene. Elution is started with
1200 ml. of benzene, and then continued with 5000 ml. of a
2:1 mixture of benzene and chloroform, 3000 ml. of a 1:1 mix-
ture of benzene and chloroform, and 800 ml. of chloroform.The effluent is collected into fractions of 400 ml. Frac-
tions Nos. 15 to 20, containing the major part of the pro-
duct, are combined and evaporated to dryness under reduced
pressure. 6.5 g. of 4-deacetoxy-vincristine (first product
fraction) are obtained.
Fractions Nos. 11 to 14 and 21 to 26 are combined,
evaporated under reduced pressure, the obtained residue,
weighing 1.75 g., is dissolved in 15 mlO of benzene, and the
solution is poured onto a chromatographic column filled with
90 g. of alumina (activity grade: III) in benzene. The
column is eluted with 1200 ml. of a 2:1 mixture of benzene
and chloroform, and the effluent is evaporated under reduced
pressure. 1.05 g. of 4-deacetoxy-vincristine (second product
fraction) are obtained.
The first and second product fractions, obtained
as described above, are combined, dissolved in 60 ml. of a 3:2
mixture of methanol and water, and allowed to stand at 5~C for
16 hours. The separated crystals are filtered of, washed
with 10 ml. of a 3:2 mixture of methanol and water pre-cooled
to 5C, and dried in vacuo.
7.0 g. of 4-deacetoxy-vincristine are obtained; m.p.:
205-209~C, ~ D = +105.7 (c = 1, in chloroform).
-- 10 --
'10~ ~5~1
The IR absorption spectrum of the obtained compound
is similar to that of 4-deacetoxy-vinblastine, there appears,
however, an absorption band characteristic of the N-formyl
radical at v = 1672 cm 1,
In the mass spectrum of the obtained com~?ound a
molecular ion peak appears at a mass number of m/e = 766,
which corresponds to the empirical formula of C44H54N3O8.
PMR spectrum: When comparing the PMR spectrum oE
the obtained compound with those of vinblastine and vincris-
10 tine it can be stated that the peak characteristic of the 4-
acetoxy group, appearing at ct'= 2.09 ppm in the spectra of
vincristine and vinblastine, fails to appear in the spectrum
of the new compound. Characteristic PMR peaks: J~= 3.65 ppm
(carbomethoxy protons), 3.81 ppm (aromatic OCH3 protons),
3.85 ppm (carbomethoxy protons) and 8.30 ppm (~-formyl
proton).
EXAMPEE 2
7.0 g. of 4-deacetoxy-vincristine, obtained as des-
cribed in Example 1, are dissolved in 20 ml. of methylene
20 chloride, and the solution is evaporated to dryness under
reduced pressure. The dry residue is dissolved in 30 ml. of
dry ethanol, and the pH of the solution is adjusted to 4 with
dry ethanol containing 1% of sulfuric acid. The sulfate
starts to crystallize immediately. The crystal suspension is
allowed to stand at room temperature, thereafter the separat-
ed product is filtered off, washed with 30 ml. of dry ethanol,
and dried.
7.1 g. (63.7%) of 4-deacetoxy-vincristine monosulfate
are obtained: r~_7D = +58.3 (c = 1, in water) and +42.0
30 ~c = 1, in methanol).
EXAMPLE 3
0.50 g. (0.000655 moles) of 4-deacetoxy-vinblastine
~09 ~
sulfate are dissolved in 15 ml. of distilled water. The pH
of the solution is adjusted to 8.5 to 9.0 with cc. aqueous
ammonia, and the liberated 4-deacetoxy-vinblastine base is
extracted with 3 x 10 ml. of chloroform. The chloroform
solutions are combined, dried over sodium sulfate, filtered,
and the filtrate is evaporated under reduced pressure.
The 0.4 g. of 4-deacetoxy-vinblastine base, ob-
tained as residue, are dissolved in 30 ml. of 98% formic
acid. me solution is stirred at room temperature, the air
space above the solution is flushed with oxygen, and then
0.5 g. of platinum black are added to the solution. An
oxygen atmosphere is maintained above the solution during
the oxidation process, requiring about 18 to 20 hours, and
the run of the reaction is monitored by thin layer chroma-
tography. At the end of the reaction the platinum black is
removed by filtration, the filtrate is diluted with fivefold
amount of 5C distilled water, and the pH of the resulting
mixture is adjusted to 8.5 to 9.0 with cc. aqueous ammonia.
The alkaline solution is extracted with 3 x 10 ml. of chloro-
form. The chloroform solutions are combined, dried oversodium sulfate, :~iltered, and the filtrate is evaporated to
dryness under reduced pressure. 0.38 g. of crude 4-deacetoxy-
vincristine are obtained as a residue. If desired, this crude
product can be recrystallized as follows:
0.38 g. of crude 4-deacetoxy-vincristine are dis-
solved in 3 ml. of a 3:2 mixture of methanol and water, and
the solution is allowed to stand at 5aC for 16 hours. The
separated crystals are filtered off, washed with 1 ml. of a
3:2 mixture of methanol and water, and dried. 0.33 g. of
crude 4-deacetoxy-vincristine are obtained. mis product
is recrystallized as described in Example 1 to obtain 0.3 g.
of 4-deacetoxy-vincristine (recovery: 75%).
- 12 -
~10~4.~S~.
The physical constants of the obtained product are
identical with those indicated in Example l.
EXAMæLE 4
50 mg. of 4-deacetoxy-vinblastine sulfate are
treated as described in Example 3 to liberate the base, and
the base is isolated by chloroform extraction and evapora-
tion. The resulting 4-deacetoxy-vinblastine base is dis-
solved in 3 ml. of 98% formic acid, and a suspension of 200
mg. of 5% palladium-on-carbon catalyst in 2 ml. of 98 to 100%
formic acid is added to the solution with stirring. The
addition flask containing the suspension is flushed with
2x0.5 ml. of formic acid in order to introduce the total
amount of the suspension into the reaction mixture. The air
above the mixture is displaced with oxygen, and the reaction
mixture is stirred at room temperature for 18 to 20 hours
under oxygen atmosphere. The run of the reaction is moni-
tored by thin layer chromatography. The reaction mixture is
processed as described in Example 3.
25 mg. (62%, calculated for the 4-deacetoxy-vin-
0 blastine base) of 4-deacetoxy-vincristine are obtained.
EXAMPLE 5
One proceeds as described in Example 4 with the
difference that a 6:1 mixture of benzene and formic acid is
applied instead of formic acid to dissolve the 4-deacetoxy-
vinblastine base and to suspend the catalyst.
20 mg. (50/O~ calculated for the 4-deacetoxy-vin-
blastine base) of 4-deacetoxy-vincristine are obtained.
EXAMPLE 6
An injectable composition comprising a dry ampoule
containing the active agent and a solvent ampoule is prepared
as follows:
a) 5 g. of benzyl alcohol and 5 g. of lactose are
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~0~155~
dissolved in 500 ml. of saline, the solution is sterilized by
filtration, and filled into ampoules 5 ml. in volume.
b) Sterile solutions of 0.05 mg., 0.10 mg., 0.20
mg., 0.50 mg. or 1.00 mg. of 4-deacetoxy-vincristine sulfate
are filled into ampoules under sterile conditions, and the
contents of the ampoules are freeze-dried.
Prior to administration the contents of the dry
ampoule and of the solvent ampoule are admixed with each
other.
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