Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
-` 1~8~S;~;
This invention rQlates to derivatives of 4'-
deoxy VLB "A" and 4'-deoxy VLB "B" produced by oxidation
thereof. :-
The invention provides a process for the prep-
aration of l-_ormyl dimeric indole-dihydroindole of the
formula
7' ~'/\ ,~ `.
- - - R4
( ~ Formula II
H 1 0
CHO C~CH3
`~ : 1~ :. .;,,`' :'
~,~ o ~::
wherein Rl is OH or O-C-CH ;
" 3
O .. ,
~-~ one of R3 and R4 is H and the other C2H5;
and pharmaceutically acceptable salts thereof;
whlch comprises reacting l-methyl dimeric indole-dihydro- ~-
indole of the formula
~',`," .
i~ X-4730A -2- -~
, ~
.:. , - . .. . . . . .
~O~
For~ula III
H ~ 0 ~ ,
CI18/CH~
CH30-- 7~51~
CH3 C~ 3 -~.
, ~ o
wherein Rl, R3 or R4 are as described above with a chromic `~ -,
acid oxidizing agent in a reaction mixture at low temperature, _
;~ and recovering the free base or a pharmaceutically acceptable
~ salt thereof.
., ~ , .
Several naturally-occurring alkaloids obtainable
from V1nca rosea have been found active in the treatment of
20 experimental malisnancies in animals. Among the e~are ~-
leurosine ~U.S. Patent No. 3,370,057), vincaleukoblastine
v m blastlne) to be referred to hereinafter as VLB ~U.S. ~ I ;
Patent No. 3,097,137), leurosidine (vinrosidine) and leuro-
crist1ne lVCR or vincristine) (both in U.S. Patent No. r -.
; 3,2~05,~2201, 4'-deoxy VLB "A" and "B", Tetrahedron Letters,
783 (1968) (desacetyl leurQSine hydrazide is also dl-closed
therein): 4-desacatoxy vinblastine ~V.S. Patent No. 3,95q,7731;
4-desacetoxy-3'-hydroxyvinblastine ~U.S. Patent No. 3,944,554)~
urocolomb1ne (U.s. Patent NO. 3,890,325~, leurofor~1
X-4730A -3-
~ ,
,
1~8S~;
~N-formylleurosine, see Belgian Patent No. 811,110) and
vincadioline ~U.S- Patent No- 3,887,565)- TWO of these
alkaloids, VLB and leurocristine, are now marketed as drug~
for the treatment of malignancies in humans, particularly
the leukemias and related diseases.
The dimeric indole-dihydroindole alkaloids ob-
tainable from Vinca rosea can be represented by formula I.
F - - R-
Formula I
H I 0
~<2
O ,~
.
,
~ .
' ' ""` `~
'` ' ~ '`
.
.
X-~730A _4_
:
~ . .
In fonmula I where Rl is acetoxy, R2 i~ methyl, R3 i8
hydroxyl, R4 is ethyl and RS is H, VLB is represented; where
R is acetoxy, R2 is formyl, R3 is hydroxyl, R4 i8 ethyl and
R5 is H, vincristine is representeds where Rl i8 acetoxy, R2
is methyl, R3 is ethyl, R4 i8 hydroxyl, and R5 i8 H, leuro-
sidine is represented; where Rl is acetoxy, R2 i8 methyl, R3
and RS are H and R4 is ethyl, 4'-deoxy VLB "A" is repre-
sented; where Rl, R2 and R5 are the same as in 4'-deoxy VLB
~A" but R3 is ethyl and R4 is hydrogen, 4'-deoxy VLB ~B~ i8
represented; and where Rl is acetoxy, R2 is methyl, R3 i8
ethyl and R4 and R5 taken together form an a-epoxide ring,
leurosine is represented.
Of the above alkaloids, vincristine is the most
useful, and the least available, from vinca. Recently,
Jovanovics et al., U.S. Patent 3,899,493, have developed an
oxidative method for converting the relatively more abundant
VLB into vincristine by chromic acid oxidation at low
~-60C.) temperatures. There are other relatively abundant
alkaloids such as leurosine in the dimeric indole-dihydro-
ndole fraction from vinca and it would be desirable to
convert these directly or indirectly to vincristine or to a
druq of comparable oncolytic activity. It is known that
'' .
~ X-4730A - 4a -
,~ ' ' .
' ~
1(}~8S;~
leurosine can be converted to 4'-deoxy VLB "B" (along with
varying amounts of 4'-deoxy VLB "A") by treatment with Raney
nickel in refluxing absolute ethanol--see Neuss, Gorman,
Cone and Huckstep, Tetrahedron Letters 783-7 (1968). While
leurosine demonstrated oncolytic activity in experimental
tumors in mice, clinical response was limited. 4'-Deoxy VLB
"A" and 4'-deoxy VLB "B" were reported to lack reproducible
activity in experimental tumors in mice.
It is an object of this invention to convert
leurosine via 4'-deoxy VLB "A" and "B" to oncolytically
active derivatives of 4'-deoxy VLB "A" and "B", thereby
converting indirectly the relatively abundant alkaloid
leurosine into a drug of greater potential clinical utility. -~
A compound of formula I in which R4 is ethyl, Rl
- is acetoxy, R2 is CHO, and R3 and R5 are hydrogen is named
4'-deoxyvincristine; a compound where Rl is hydroxy but the
other groups are the same is named 4'-deoxy-4-desacetyl-
vincristine. Since the companion alkaloid to vincristine
having a reverse configuration of hydrogen and ethyl at 4'
from that found in vincristine is not known, those compounds
in which R3 is ethyl, and R4 is hydrogen will be referred
`~ back to leurosidine which has the same configuration at 4'
as 4'-deoxy VL8 "B" and will be referred to as derivatives
~ ~ .
of l-formylleurosidine; i.e., 4'-deoxy-1-formylleurosidine
(or 4'-deoxyepivincristine) and 4'-deoxy-4-desacetyl-1-
- formyileurosidine where R is acetoxy or hydroxy, respectively.
In each of the above names, it will be under stood that the
l-methyl group of leurosidine has been replaced by a formyl
group and that the "l-desmethyl" term has been omitted to
simplify the nomenclature.
X-4730A -5-
1~8~
Non-toxic acids useful for forming pharmaceu-
tically-acceptable acid addition salts of the compounds of
this invention include salts derived from inorganic acids
such as: hydrochloric acid, nitric acid, phosphoric acid,
sulfuric acid, hydrobromic acid, hydriodic acid, nitrous
acid, phosphorus acid and the like, as well as salts of
non-toxic organic acids including aliphatic mono and di-
carboxylic acids, phenyl-substituted alkanoic acids hydroxy
alkanoic and alkandioic acids, aromatic acids, aliphatic and
aromatic sulfonic acids, etc. Such pharmaceutically-
acceptable salts thus include the sulfate pyrosulfate,
bisulfate, sulfite, bisulfite, nitrate, phosphate, mono-
hydrogenphosphate, dihydrogenphosphate, metaphosphate,
pyrophosphate, chloride, bromide, iodide, acetate, pro-
pionate, decanoate, caprylate, acrylate, formate, iso-
butyrate, caprate, heptanoate, propiolate, oxalate, malonate,
succinate, suberate, sebacate, fumarate, maleate, benzoate,
chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxy-
benzoate, methoxybenzoate, phthalate, terephthalate,
benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate,
xylenesulfonate, phenylacetate, phenylpropionate, phenyl- `:
butyrate, citrate, lactate, 2-hydroxybutyrate, glycollate,
malate, tartrate, methanesul~onate, propanesulfonate,
naphthalene-l-sulfonate, naphthalene-2-sulfonate and the
like salts. `
The compounds of this invention according to
Formula I above wherein R2 is formyl are prepared by low
temperature chromic acid oxidation of either 4'-deoxy VLB
"A" or 4'-deoxy VLB "B" under acidic condition, e.g. chromium
trioxide and acetic acid.
X-4730A -6-
10~8S~6
4'-Deoxy VLB "A" and HB" or their l-formyl deriva-
tives can be hydrolyzed to the corresponding 4-desacetyl
derivatives under acidic or basic conditions. These 4-
desacetyl derivatives of 4'-deoxy VLB "An and "B" can then
be oxidized at low temperature (-60C) with chromium tri-
oxide without converting the 4-hydroxy group to a ketone to
yield 4'-deoxy-4-desacetylvincristine and 4'-deoxy-4-
desacetyl-l-formylleurosidine.
The preferred method of carrying out the above
hydrolysis reaction, uses sodium carbonate in methanol
at reflux temperature. Hydrazine hydrate may be used.
Other bases which can be employed include potassium t-
butoxide, sodium or potassium methoxide or ethoxide,~
pyridine, triethylamine (or other tertiary amine), urea and -
the like in polar organic solvents such as the lower
alkanols. Dilute sodium and potassium hydroxide can also be
employed, in methanol for example, but precautions must be --
taken not to operate with base concentrations or reaction
temperatures at which other hydrolysable groups in 4'-deoxy- -
vincristine or 4'-deoxy-1-formylluerosidine are affected.
Bases which operate only in non-polar solvents can also be `-
usedi i.e., sodium or lithium hydride in benzene, ether,
THF, etc. or the sodium salt of dimethyl-sulfoxide in DMSO.
`~ Temperatures varying from ambient temperature (25C.) to
boiling point of the particular solvent may be used. On the
other hand hydrolysis can be carried out under acidic
conditions, e.g., absolute methanol saturated with anhydrous
hydrogen chloride at 0C.
X-4730A -7-
1~8S;2t;
More specifically, the compounds of thi8 invention
can be prepared according to the following examples.
Example 1
Preparation of 4'-Deoxyvincristine
582 mg. of chromium trioxide are dissolved in
5.8 ml. of acetic acid and 0.6 ml. of water. This oxidizing
solution i~ added in dropwiæe fa~hion over a five-minute
period to a stirred solution of 462 mg. of 4'-deoxy VLB ~An
in 58 ml. of acetone and 2.9 ml. of glacial acetic acid at a
temperature of about -50C. The reaction mixture i8 stirred
at this temperature for about 30 minutes and then cooled
to -65C. at which temperature the reaction mixture is
quenched with 12 ml. of 14 N aqueous ammonium hydroxide.
The alkalinized reaction mixture is then poured onto 40Q ml.
of an ice-water mixture and the aqueous layer extracted with
~;~ 150 ml. of ether followed by three extractions with 150 ml.
-~ of chloroform each. The organic layers are combined and the -
combined layers washed with dilute aqueous sodium bisulfite, ~ ;
separated and dried. Evaporation of the organic solvents ^~
leaves, as a residue, 4'-deoxyvincristine. Chromatography
of the residue over 50 gm. of activity I silica is employed
to further purify the desired compound. The chromatogram iæ
developed as follows: 300 ml. of 3:1 ethyl acetate-methanol
followed by 300 ml. of 1:1 ethyl acetate-methanol. A~ter an
initial 100 ml. ~raction, 20 ml. fractions are collected.
Fractions 8-20 are combined. Evaporation of the solvents
from the combined fraction~ yield~ 279 mg. of a light tan ``~
solid which is substantially one spot (pure) material by
thin-layer chromatography.
` X-4730A -8-
-. .
~08~5;~
4'-Deoxyvincristine free base thus prepared has
the following physical characteristics:
Mass spectrum: m/e 808 (M ), 806,707
Infrared spectrum: 3465, 1745, 1687, 1220 cm 1
Ultraviolet spectrum: 210, 222, 255, 290, 298 nm
100 MHz NMR spectrum: methyl singlets at 3.88,
3.67 and 2.07 ~.
4'-Deoxyvincristine, as a tan solid is dissolved
in acetone and the acetone solution treated with 0.96 ml. of
0.36 M (2 percent volume/volume) sulfuric acid in absolute
ethanol. A green solution results which is maintained at
about 0C. overnight. crystallization is induced by
scratching or seeding, and the solid crystalline 4'-deoxy-
vincristine sulfate is separated by filtration. The filter ~ ~
cake is washed with cold acetone. The sulfate salt is ~- -
somewhat soluble in acetone so the filtrate is evaporated to ~-
dryness and the resulting residue recrystallized from
ethanol. Crystalline 4'-deoxyvincristine sulfate thus
- obtained from ethanol was filtered and the filter cake
washed with ethanol. Total yield of 4'-deoxyvincristine
~`~ sulfate is 266 mg.
In similar fashion, 794 mg. of 4'-deoxy VLB nBn
can be oxidized with 900 mg. of chromium trioxide in 10 ml.
of glacial acetic acid and 1 ml. of water to yield 4'-deoxy-
formylleurosidine. Thin-layer chromatography of the
residue obtained directly from the oxidation mixture prior
to purification indicates the presence of a major and a
minor spot plus traces of other components. Recrystal-
` ~ lization of the residue from anhydrous ethanol yields
.~ :
--~ 30 `
X-4730A -9-
.
108~
qubstantially one ~pot crystalline material which is isolated
by filtration and the crystals washed with cold ethanol.
Chromatography of the crystalline free base thus
obtained over 50 g. of silica using a 1:1 methylenedi-
chloride-ethyl acetate solvent system containinq 20, 30, 45
and 60 percent by volume of methanol as the eluant as follow~:
System Quantity
1:1 20% 200
1:1 30% 100
1:1 45% 100
1:1 60% 400
yields the following fractions:
FractionVolume of Eluate
1 160 ml.
2 100 ml.
3 50 ml.
4 50 ml.
50 ml.
6 120 ml.
7 120 ml.
Fractions 4-7 are combined to yield 597 mg. of a
tan residue which in turn yields 435 mg. of white crys-
talline 4'-deoxy-1-formylleurosidine (from ethanol). The
compound has the following physical characteristics:
Mass spectrum: m/e 808 ~M ), 806, 777, 775,
336, 138, 136.
~-~ Infrared spectrum: v (CHC13) 3470, 1743, 1690,
; 1222 cm 1.
X-4730A -10-
1~8S;~
Ultraviolet spectrum: (C2H5OH) 210, 222, 254,
290, 298.
100 MHzNMR spectrum: methyl singlets 3.87,
3.65 and 2.07 ~.
PKa = 9.0 and 4.9 (in 66% DMF).
The sulfate salt is prepared by dissolving 435 mg. of the
free base in 10 ml. of hot ethanol and adding 1.5 ml. of 2
percent sulfuric acid in ethanol thereto. Crystalline
4'-deoxy-1-formylleurosidine sulfate deposits on cooling.
EXAMPLE 2
Preparation of 4'-Deoxy-4-desacetyl-1-formylleurosidine
About 744 mg. of 4'-deoxy-1-formylleurosidine are
mixed with 10 ml. of anhydrous methanol and the mixture
heated to refluxing temperature, at which temperature the
compound dissolves to give a clear solution. 200 mg. of
; solid sodium carbonate are added and the reaction mixture is
stirred for about 7.2 hrs. at which time TLC of the crude
reaction components shows that virtually all starting 4'-
deoxy-l-formylleurosidine has disappeared. The solvent is
removed by evaporation and the residue containing 4'-
deoxy-4-desacetyl-1-formylleurosidine formed in the above
reaction is partitioned between water and methylene di- -
chloride. The organic layer is separated and dried and the
solvent is removed by evaporation yielding 506 mg. of a
white solid which is substantially pure 4'-deoxy-4-desacetyl-
l-formylleurosidine,
The compound had the following physical char-
acteristics: `
mass spectrum: m~e 766(M ), 764, 735, 254, 252,
205, 138
X-4730A -11- ` -
~ 8S;~
infrared spectrum: ~ (CHC13) 3450, 1734, 1680,
1596, 1495, 1456, 1434 cm 1.
100 MHz pmr spectrum: ~CDC13) include~ N-formyl
at ~8.80, methyl singlets at 3.89 (C16-OCH3) and 3.66
(C18-C02CH3), broadened multiplet at 3.82 (C3-C02CH3), and
no N-CH3 around 2.75 (or OCOCH3 around 2.06).
The corresponding sulfate salt is formed as in the
previous examples using acetone as a solvent and 0.26 ml. of
2% sulfuric acid in ethanol. Other solvents can be used and
i~ is preferred to use a solvent in which the base is
readily soluble but the sulfate salt substantially insoluble.
4'-Deoxy-4-desacetylvincristine and its sulfate
salt are prepared in entirely analogous fashion from 4'-
deoxyvincristine.
EXAMPLE 3
Alternate preparation of 4'-deoxy-4-desacetyl-1-formyl-
leurosidine
A reaction mixture was prepared containing 1.48 g.
of 4'-deoxy VLB "Bn, 1 g. of sodium carbonate and 100 ml. of
methanol and was heated to reflux under a nitrogen atmosphere.
Thin-layer chromatography of an aliquot taken at two hours
~ indicated that the hydrolysis reaction to remove the 4-
,~ acetyl group was about half completed. The reaction mixture,
'~ after standing overnight at room temperature, was heated to `-
. ~
-~ reflux again for eight and one-half hours. Thin-layer
chromatography of an aliquot using a 20:1:1:1 ether/di-
ethylamine/toluene/methanol solvent indicated that the
reaction had gone to completion. The solvent was removed
from the reaction mixture by evaporation and the resulting
X-4730A -12-
^` 101~8S;~f~
residue was dissolved in a mixture of methylene dichloride
and water. The methylene dichloride phase was separated ana
dried. Evaporation of the methylene dichloride yielded a
residue comprising by TLC a very polar substance pluq the
expected 4'-deoxy-4-desacetylleurosidine. The re~idue which
weighed 1.33 g. was dissolved in benzene. The highly polar
material was substantially insoluble in benzene and WAS
separated by filtration. The filtrate was evaporated to
dryness and the residue weighing 500 mg. was chromatographed
on Woelm silica gel using a 20:1:1 ether/diethylamine/toluene
solvent system (with increasing quantities of methanol) as
the eluant. The progress of the chromatography was followed
by thin-layer chromatography and fractions shown to contain
4'-deoxy-4-desacetylleurosidine were combined and yielded
348 mg. of base on evaporation of the solvent. The residue
was treated with 1.28 ml. of 2 percent sulfuric acid in `
methanol (0.36M) and the resulting solution was filtered to
; yield 315 mg. of 4'-deoxy-4-desacetylleurosidin~ sulfate.
4'-Deoxy-4-desacetylleurosidine had the following
physical characteristics:
~; Mass spectrum: m/e 752 (M~), 750, 693, 691, 555,
338, 240, 138 `-
Infrared spectrum: v (CHC13) 3455, 1724, 1610,
1497, 1457, 1431 cm~l
100 MHz pmr spectrum: ~ TDsC13 9 43 (br s, 1,
.
C3-OH), 7.92 (brs, 1, indole N-H), 7.47-7.63 (m, 1, Cll, -
~ ~ C12,_14,-H), 6-58 ~s, 1, C14-H), 6.10
(s, 1, C17-H), 5.78-5.87 (m, 2, C6 7-H), 4.10 (m, 1, C4-H),
3.83 (s, 3, C16-OCH3), 3.78 (s, 3, C3-CO2CH3), 3.70 ~s, 1,
X-4730A -13-
`` 11~8S;~;
C2-H), 3.58 (s, 3, C18,-C02CH3), 2.75 (s, 3, N-CH3), 0.76-1.06
~m~ 6~ C21,21' H)-
4'-Deoxy-4-desacetylleurosidine (834 mg.) obtained
from filtrates and including solid filtered material was
combined. The combined material probably contained 30-40
percent of the highly polar material referred to above. The
combined material was dissolved in 100 ml. of acetone con-
taining 7 ml. of acetic acid. The solution was stirred for
15 minutes at room temperature and then cooled to -65C. in
a dry-ice acetone bath under a nitrogen atmosphere. 1110 mg.
of chromium trioxide were dissolved in 13 ml. of glacial
acetic acid and 2 ml. of water. This solution was added in
dropwise fashion to the solution of 4'-deoxy-4-desacetylleuro-
sidine. The reaction mixture was stirred in the temperature
range -60 to -65C. for one hour and then quenched by the
addition of 35 ml. of 14 M aqueous ammonium hydroxide. The
reaction mixture was next poured onto ice and the resulting
aqueous suspension extracted several times with chloroform.
The chloroform extracts were combined, washed with water~
and dried. Removal of the chloroform in vacuo yielded
794 mg. of a residue shown by thin-layer chromatography to
contain essentially one-spot material besides the original
very low Rf impurity. This residue was chromatographed over
Woelm silica gel using an initial eluant of 20:1:1 ethyl -
ether/diethylamine/toluene solvent mixture containing
0.9 percent methanol. The eluant was employed in 150 ml.
portions. The percent of methanol was increased for each
successive 150 ml. eluant portion up to 15 percent. Frac-
tions shown to contain 4'-deoxy-4-desacetyl-1-formylleuro-
X-4730A -14-
lO~S;~
sidine by thin-layer chromatography were combined and
yielded 293 mg. of pure 4'-deoxy-4-desacetyl-1-formyl-
leurosidine after evaporation of the solvent. The sulfate
salt was prepared as before.
4'-Deoxy-4-desacetylvincristine can be prepared as
above by hydrolysing 4'-deoxy VLB "A" to yield 4'-deoxy-
4-desacetyl VLB and then oxidizing this compound with CrO3
in acetic acid at -60C.
The compounds of this invention, as represented by
Formula I above, particularly those in which Rl is acetoxy,
are powerful anti-tumor agents. The compound 4'-deoxy-
4-desacetylleurosidine is also an oncolytic agent. In
demonstrating the activity of these drugs against trans-
planted tumors in mice, a protocol was used which involved
the administration of the drug by the intraperitoneal route
at a given dose level for 7-10 days after innoculation with - -
the tumor or alternatively, on the first, fifth, and ninth ~ `
days after innoculation. -~
Table I gives the results of several experiments -`
in which transplanted tumors in mice were treated successfully ~
with a compound of this invention. -"
In the table, column 1 gives the name of the -~
compound; column 2, the transplanted tumor; column 3, the
dose level or dose level range and the number of days the
dosage was administered; column 4, the route of admini- `"
stration, and column 5, the percent inhibition of tumor `
growth or percent prolongation of survival time, e.g., B16.
(ROS is an abbreviation of Ridgeway osteogenic sarcoma; GLS
, ~
for Gardner lymphosarcoma; P1534(J) and L1210 are leukemias;
30 CA755 is an adenocarcinoma; and B16 is a melanoma.).
X-4730A -15-
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X-4730A -17-
` 10~8S;~i
In utilizing the novel compounds of this invention
as anti-tumor agents, either the parenteral or oral route of
administration may be employed. For oral dosage, a suitable
quantity of a pharmaceutically-acceptable salt of a base
according to Formula II formed with a non-toxic acid, such
as the sulfate salt, is mixed with starch or other excipient
and the mixture placed in telescoping gelatin capsule~ each
containing from 7.5 to 50 mg. of active ingredients.
Similarly, the anti-neoplastically active salt can be mixed
with starch, a binder and a lubricant and the mixture
compressed into tablets each containing from the 7.5-50 mgs.
of salt. The tablets may be scored if lower or divided
dosages are to be used. Parenteral administration is
preferred however. For this purpose, isotonic solutions are
employed containing 1-10 mg./ml. of a salt of an indole-
dihydroindole of Formula II such as the sulfate salt. The
compounds are administered at the rate of from 0.01 to
1 mg/kg. and preferably from 0.1 to 1 mg./kg. of mammalian
body weight once or twice a week or every two weeks de-
pending on both the activity and the toxicity of the drug.An alternative method of arriving at a therapeutic dose is
based on body-surface area with a dose in the range 0.1 to
10 mg./meter squared of mammalian body surface every 7 or 14
days being admini~tered.
In utilizing a compound of this invention clin-
ically, the clinical physician would administer the compound
initially by the same route and in the same vehicle and
probably against the same types of tumors as are indicated
for vincristine or VLB. The dose levels employed would
X-4730A -18-
S;~Ji
reflect the difference in dose levels found in the treatment
of experimental tumors in mice, the dose levels of the
compounds of this invention being less than those used with
vincristine and VLB. In clinical tests, as with other
anti-tumor agents, particular attention would be paid to the
effect of the oncolytic compounds of this invention against
the ten nsignal" tumors set forth at page 266 of "The Design
of Clinical Trials in Cancer Therapy" edited by Staquet
(Futura Publishing Company, 1973).
X-4730A -19-
