Note: Descriptions are shown in the official language in which they were submitted.
~V6q~73
The invention relates to novel amide derivatives
of dimeric alkaloids and to a process for the preparation
thereof. The compounds are useful as anti-viral and
anti-neoplastic agents or as intermediates in the prepa-
ration of such agents.
The invention provides a compound of the formula
, ~ R~
~ CH2-CH3
~ ,~o
14 't--- y 117
~ 5 ~ CH2-CH3
CH3 o-l~ 3~I_Rl
H ' Formula I
wherein R is NH2, NH-NH2, N(CH3)2, pyrrolidinyl, NH-alk-X,
N~-(C3-C8)-cyclo-alk, NH-alk-(oH)l 3, or N3;
_ . . . .
20 wherein alk is (Cl-C6) aLkyl, _ __
and X is hydrogen;
_ . . . . . .
Rl is hydrogen, hydroxyl, 0-(Cl-C3)-alkanoyl
or O-chloro-(Cl-C3)-alkanoyl;
R2, R3 and R4 are hydrogen or hydroxyl with the
provisos that when R2 is hydrogen, R3 and R4 are hydrogen;
when R2 is hydroxyl at least one of R3 or R4 is hydroxyl.
The invention also provides a process for preparing
a compound of Formula I by (a) reacting a compound having
X-3754M -2-
~o67~73
a structure of Formula I wherein R is O-CH3, R ls hydrogen,
hydroxyl or acetyloxy, and R , R and R are as defined in
Formula I with a c~mpound of the formula
NH2R5 Formula II
where R5 is hydrogen, methyl or amino and/or
(b) when R is NH-NH2 reacting the compound so
obtained with a nitrosating agent and with a compound of the
formula
0 ~R7
" wherein R6 is hydrogen or methyl and R7 is methyl, -alk-X,
(C3-C8)-cycloalk, or -alk-(OH)l 3 wherein alk is
(Cl-C6) alkyl and X is as defined in Formula I, and/or (c)
acylating the compound obtained in (a) or Ib)
above wherein Rl is hydroxyl to provide a compound wherein
Rl is other than hydroxyl and, if desired, reacting any of
the products obtained above with a non-toxic inorganic acid
- or organic acid to provide the pharmaceutically acceptable
acid addition salt of the product.
2u
Several naturally-occurring alkaloids obtainable
from Vinca rosea have been found active in the treatment of
experimental malignancies in animals. Among these are
leurosine (U. S. Patent No. 3,370,û57), vinblastine
(vincaleukoblastine) (U.S. Patent No. 3,097,137), leuro-
sidine (vinrosidine) and leurocristine (vincristine) (both
in U. S. Patent No. 3,2û5,220). Two of these alkaloids,
vinblastine and leurocristine, are now marketed as drugs for
the treatment of malignancies, particularly the leukemias
and related diseases in humans. Of these marketed compounds,
X-3754M -3_
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r
1067073
leurocristine is a most active and useful agent in the
treatment of leukemias but is also the least abundant of the
anti-neoplastic alkaloids of Vinca rosea.
Chemical modification of the Vinca alkaloids has
been rather limited. In the first place, the molecular
structures involved are extremely complex and chemical
reactions which affect a specific function of the molecule
are difficult to develop. Secondly, alkaloids lacking
~ desirable chemotherapeutic properties have been recovered
; 10 from Vinca rosea fractions, and a determination of their
structures has led to the conclusion that these compounds
are closely related to the active alkaloids. Thus, anti-
neoplastic activity seems to be limited to very specific
structures, and the chances of obtaining more active drugs
by modification of these structures would seem to be cor-
respondingly slight. Among the successful modifications of
physiologically-actlve alkaloids has been the preparation of
dihydro vinblastine (U. S. Patent No. 3,352,868) and the
replacement of the acetyl group at C-4 (carbon no. 4 of
vinblastine ring system-see Formula I) with a higher al-
kanoyl group or with unrelated acyl groups. (See U. S.
Patent No. 3,392,173.) Several of these derivatives are
capable of prolonging the life of mice inoculated with P1534
leukemia. One of the derivatives in which a chloracetyl
group replaced the C-4 acetyl group of vinblastine was also
a useful intermediate for the preparation of structurally
modified vinblastine compounds in which an N,N-dialkylglycl
group replaced the C-4 acetyl group of vinblastine (see
U. S. Patent No. 3,387,001). An intermediate compound,
namely 4-desacetyl vinblastine, was produced during the
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chemical reactions leading to these latter derivatives.
This intermediate, in which the C-4 acyl group was lacking,
leaving an unesterified hydroxy group, has been reported to
be a toxic material having little in vivo chemotherapeutic
activity against the Pl534 murine leukemia system by
Hargrove, Lloydia, 27, 340 (1964).
A preferred group of compounds of Formula I
includes the amides of vincadioline, leurocolombine, 4-
desacetoxyvinblastine, 3'-hydroxy-4-desacetoxyvinblastine,
deoxyvinblastine and the 4-desacetyl derivative of any of
the above dimeric alkaloids having a C-4 acetoxy group, and
the pharmaceutically-acceptable salts of the above bases,
except those in which R is NH-NH2 and N3, formed with
nontoxic acids.
Illustrative of alk-(OH)l 3, alk-Am and alk-X in
the above formula are the following: methyl, 2-methyl-
pentyl, isohexyl, isopentyl, n-pentyl, n-hexyl, sec-hexyl,
ethyl, isopropyl, n-butyl, sec-butyl, cyanomethyl, cyano-
ethyl, 2-hydroxy-n-hexyl, 5-cyano-n-pentyl, 2-hydroxyethyl,
3-hydroxypropyl, 2-dimethylaminoethyl, 2-aminoethyl, 2-
methylaminoethyl, 2-hydroxypropyl, benzyl, phenethyl, 4-
phenylbutyl, 2-aminopropyl, 2-aminohexyl, 2-dimethylamino-
propyl, 2,2'-dihydroxyisopropyl, 2,2'-dihydroxy-t-butyl,
2,2',2''-trihydroxy-t-butyl, and the like.
In Formula I, the terms "(Cl-C3)-alkanoyl" and
"chloro-(Cl-C3)-alkanoyl" include groups such as acetyl,
chloroacetyl, propionyl, 2-chloropropionyl, 2-chlorobutyryl
and butyryl, these terms being represented by the formula
(Cl-C3)-alkyl-CO, an alkanoyl group, or by the formula
X-3754M -5_
1067073
(Cl-C3)-alkyl (Cl)-CO, a chloroalkanoyl group. The term
"NH-(C3-CB)-cycloalk" includes the radicals cyclopropyl-
amino, cyclobutylamino, cyclopentylamino, cyclohexylamino,
cycloheptylamino, and cyclooctylamino. The term "carbo-
(Cl-C3~-alkoxy" includes the radicals carbomethoxy, carbo-
ethoxy, carboisopropoxy and carbo-n-propoxy.
When "X" in the radical "alk-X" is phenyl, the
phenyl group can contain the standard aromatic substituents
including lower alkyl, lower alkoxy, hydroxy, halo, nitro
and the like and a given phenyl group can contain more than
one of the above substituents, either the same or different
from the original substituent; examples of such groups are
4-hydroxyphenyl, 2,4-dichlorophenyl, 2-methyl-4-chloro-
phenyl, 2,4-dinitrophenyl, 3,5-xylyl, 4-tolyl, 2-tolyl,
3-ethoxyphenyl and the like.
Non-toxic acids useful for forming pharmaceu-
tically-acceptable acid addition salts of the amine bases
include salts derived from inorganic acids such as: hydro-
chloric acid, nitric acid, phosphoric acid, sulfuric acid,
hydrobromic acid, hydroiodic acid, nitrous aci~, phosphorous
acid and the like, as well as salts of non-toxic organic
acids including aliphatic mono and dicarboxylates, phenyl-
substituted alkanoates, hydroxy alkanoates and alkandioates,
aromatic acids, aliphatic and aromatic sulfonic acids, etc.
Such pharmaceutically-acceptable salts thus include the
sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate,
phosphate, monohydrogenphosphate, dihydrogenphosphate,
metaphosphate, pyrophosphate, chloride, bromide, iodide,
acetate, propionate, decanoate, caprylate, acrylate, formate,
X-3754M -6-
1(~67073
isobutyrate, caprate, heptoanate, propiolate, oxalate,
malonate, succinate, suberate, sebacate, fumarate, maleate,
butyne-1,4-dioate, hexyne-1,6-dioate, benzoate, chloro-
benzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate,
methoxybenzoate, phthalate, terephthalate, benzenesulfonates,
toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate,
phenylacetate, phenylpropionate, phenylbutyrate, citrate,
lactate, 2-hydroxybutyrate, glycollate, malate, tartrate,
methanesulfonate, propanesulfonate, naphthalene-l-sulfonate,
naphthalene-2-sulfonate and the like salts.
A majority of the compounds of Formula I can be
described generally as derivatives of vinblastine. Vin-
blastine is a compound having the structure of Formula I
when R is CH30, Rl is acetoxy, R2 is ~-hydroxyl (the C-4'-
ethyl group is alpha) and R3 and R4 are hydrogen. Deoxy-
vinblastine occurs when in Formula I all of R2, R3 and R4
are hydrogen, R is CH30 and R is acetoxy. There are two
isomers of deoxyvinblastine identified as "A" and "B". In
the case of the "A" isomer, the R2 hydrogen is Beta(up) and
the C-4'ethyl group is Alpha(down). The "B" isomer has a
~-ethyl group and an a-hydrogen at C-4'.
Vincadioline is the 3'-hydroxy derivative (i.e.
R3 is hydroxyl) of vinblastine. Leurocolombine is the 2'-
hydroxy derivative (i.e. R is hydroxyl) of vinblastine.
Vincadioline has the following characteristics:
X-3754M _7_
1067073
Melting point = 218-220.5C. with decomposition;
X-ray powder diffraction pattern, using filtered
chromium radiation; ~ = 2.2896A.
d in A I/Il d in A I/I
9.55 100 -1 3.99 60
8.87 9o)-2 3.71 20
8.63 90) 3.64 15
7.78 05 3.44 10 B
7.57 60 3.19 20
7.21 50 3.05 05
6.00 40 2.85 20
5.88 40 2.78 10
5.58 70 -3 2.61 10
5.22 20 2.44 15 B
5.08 20 2.21 05 B
4.70 50 2.07 05
4.57 40 1.98 15
4.42 05 1.91 05
4.31 05
nmr spectrum, ~ at 7.13, 7.53, 8.04, 3.60, 6.61,
6.09, 3.79, 2.70, 9.77, 5.47, 2.09, 0.80, 5.85, 5.29, 5.63,
3.84, 0.91;
infra-red absorption maxima at 3480, 1745 and 1725
--1
cm
molecular weight, 826;
Empirical formula, C46H58N4O]o; and
Mass ions, m/e = 826, 170, 371.
Vincadioline is prepared according to the following pro-
cedure: Leaves of plants containing crude vinca alkaloids;
ie, Catharanthus roseus (Vinca rosea), are extracted with a
water-immiscible solvent such as benzene. The benzene is
distilled from the extract in the presence of aqueous
tartaric acid. The pH of the resulting aqueous acidic
extract is adjusted to pH=6 by the addition of base.
Alternatively, the leaves are contacted with an aqueous acid
at pH=3, and the resulting acidic layer extracted with
benzene. The benzene layer is separated and discarded, and
X-3754M -8-
lQ67073
the pH of the aqueous layer adjusted to pH=6 as before. The
dimeric alkaloids are then extracted from the aqueous layer
into an organic solvent, customarily benzene. An optional
` gel exclusion filtration step can be carried out on the
extracted alkaloids using a cross-linked dextran gel
("Sephadex G-25F"*), the mobile phase being a pH=3.0 ~.Ir
ammonium citrate buffer. A pressure of about 15 psi is
employed during gel-e~clusion chromatography. In this
process, the dimeric alkaloid fraction containing leluro-
cristine, vinblastine, des-N-methyl vinblastine, leuro-
formine, leurosine and leurosidine is eluted first. The
dimeric alkaloids are extracted from the pH = 3 buffer by
adjusting the pH to 7.0 with base and then contacting the
resulting aqueous solution with a water-immiscible solvent,
preferably again benzene. Evaporation of the benzene yields
a residue which can be dissolved in ethanol and leurosine
crystallized directly therefrom. The leurosine crystals are
separated by decantation, and the supernate thus obtained is
acidified to pH = 4.2 with 3 percent ethanolic sulfuric acid
to convert the remaining dimeric alkaloids to their sulfate
salts which precipitate. The precipitated salt~ are
collected and are converted to the corresponding free
alkaloidal bases by standard procedures as, for example, by
dissolving the salts in water, adjusting the pH to 8.0 with
ammonium hydroxide and extracting the dimeric alkaloids with
a water-immiscible organic solvent, preferably methylene-
dichloride. Evaporation of the methylenedichloride yields
the mixed dimeric alkaloids which are then chromatographed
at high pressure over alumina (Activity III) using an ethyl
acetate-methylenedichloride-water ~25:75:0.4) solvent system
X-3754M _g_
*Trademark
t~.. i
_. .
~ 067073
as the eluant.
Operating pressures employed have been in the
range 150-350 psi. As will be understood by those skilled
in the art of high-pressure chromatography, equipment is
available to carry out procedures at 4000-5000 psi and pres-
sures in the range 7500-8000 psi appear feasible. Alkaloidal
separation is in general more efficient at the higher pres-
sures. High-pressure chromatography procedures are carried
out in stainless steel equipment equipped with pressure-
resistant fittings.
The alkaloids are eluted in the following order in
this chromatographic procedure: residual leurosine, vin-
blastine, des-N-methyl vinblastine, leurocristine and
leurosidine. Identification of the dimeric alkaloid in the
eluant fraction is carried out by standard procedures known
to the art, as by thin layer chromatography~
After elution of the known alkaloids, there remain
on the column several more polar dimeric alkaloids. These
are eluted with methanol and rechromatographed until vinca-
dioline is obtained as a separate fraction substantially
` free from other dimeric alkaloids present in the polar
alkaloid fraction~
Leurocolombine has the following characteristics:
pKa's at 5~05, 6~3;
Infra-red absorption maxima at 2~80, 2~88, 3.35,
5.74, 6.18, 6.65, 6.83, 6.95, 7.25, 7.50, 8.11, 9.60, 9~90
and 10~75 microns;
Ultra-violet absorption maxima at 217 (am=51~091)
and 265 (am=15,666) millimicrons;
Molecular weight, 826;
X-3754M -10-
1067073
Empirical formula, C46H58N4Olo;
Ion fragments by mass spectroscopy, m/e 795, 767,
749, 667, 649, 282, 170, 156, 154, 152, 144, 143;
proton nmr spectrum, chemical shifts in ppm at
7.51, 7.13, 0.90, 3.60, 3.75, 7.01, 3.84, 6.15, 5.29, 5.85,
5.48, 0.78, 2.68, 3.79, 2.70, 2.10 and 4.16; and forming a
sulfate salt with the following x-ray powder diffraction
pattern using filtered chromium radiation at 2.2896A.
d in A I/ 2
17.00 30
12.50 100
9.45 50
7.70 10
7.20 60
6.20 20
5.70 30
4.95 05
4.65 20
Leurocolombine is prepared according to the
following procedure: Leaves of plants containing crude
vinca alkaloids; ie, Catharanthus roseus (Vinca rosea), are
extracted with a water-immiscible solvent such as benzene.
The benzene is distilled from the extract in the presence of
aqueous tartaric acid. The pH of the resulting aqueous
acidic extract is adjusted to pH=6 by the addition of base.
Alternatively, the leaves are contacted with an aqueous acid
at ph=3, and the resulting acidic layer extracted with
benzene. The benzene layer is separated and discarded, and
the pH of the aqueous layer adjusted to pH=6 as before. The
dimeric alkaloids are then extracted from the aqueous layer
X-3754M -11-
1067~)'73
into an organic solvent, customarily benzene. An optional
gel exclusion filtration step can be carried out on the ex-
tracted alkaloids using a cross-linked dextran gel ("Sepha~ex
G-25 F"*), the mobile phase being a pH=3.0, O.lM ammonium
citrate buffer. A pressure of about 15 psi is employed
during gel-exclusion chromatography. In this process, the
dimeric alkaloid fraction containing leurocristine, vin-
blastine, des-N-methyl vinblastine, leuroformine, leurosine
and leurosidine is eluted first. The dimeric alkaloids are
extracted from the pH=3 buffer by adjusting the pH to pH=7.0
with base and then extracting the resulting aqueous solution
with a water-immiscible solvent, preferably again benzene.
Evaporation of the benzene yields a residue which can be
dissolved in ethanol and leurosine crystallized directly
therefrom. The leurosine crystals are separated by decan-
tation, and the supernatant thus obtained is acidified to
pH=4.2 with 3 percent ethanolic sulfuric acid to convert the
remaining dimeric alkaloids to their sulfate ~alts which
precipitate. The precipitated salts are collected and are
converted to the corresponding free alkaloidal bases by
standard procedures as, for example, by dissolving the salts
in water, adjusting the pH ~ 8.0 with ammonium hydroxide and
extracting the dimeric alkaloids with a water-immiscible
organic solvent, preferably methylenedichloride. Evapo-
ration of the methylenedichloride yields the mixed dimeric
alkaloids which are then chromatographed at high pressure
over alumina (Activity III-IV) using a ethyl acetate-
methylenedichloride-water (25:75:0.4) solvent system as the
eluant.
X-3754M -12-
*Trademark
... .
1067073
Operating pressures employed have been in the
range 150-350 psi. As will be understood by those skilled
in the art of high-pressure chromatography, equipment is
available to carry out procedures at 4000-5000 psi and
pressures in the range 7500-8000 psi appear feasible.
Alkaloidal separation is in general more efficient at the
higher pressures. High-pressure chxomatography procedures
are carried out in stainless steel equipment equipped with
pressure-resistant fittings.
The alkaloids are eluted in the following order in
this chromatographic procedure: residual leurosine, vin-
blastine, leurocolombine, des-N-methyl vinblastine, leuro~
cristine and leurosidine. Identification of the dimeric
alkaloids in the eluant fraction is carried out by standard
procedures known to the art, as by thin layer chromatography.
4-DesacetoxyVinblastine has the following physical
and chemical characteristics: melting point = 183-190C.
with decomposition after recrystallization from methanol;
[]26= +95,3o (chloroform); molecular ion M+ = 752, corre-
sponding to an empirical formula C44H56N4O7.
Analysis Calcd. for: C44H56N4O7
Analysis Calc.: C, 70.19; H, 7.50; N, 7.44;
O, 14.87
Found: C, 69.71; H, 7.47; N, 7.08;
O, 15.00
4-Desacetoxyvinblastine is prepared according to
the following procedure: Leaves of plants containing crude
vinca alkaloids; i.e., Catharanthus roseus (Vinca rosea),
previously moistened with aqueous ammonia, are extracted
with a water-immisclble solvent such as benzene. The
X-3754M -13-
~067C~73
benzene is distilled from the extract in the presence of
aqueous tartaric acid. The tartaric acid layer is extracted
with a water-immiscible organic solvent and is then made
basic by the addition of ammonia. The dimeric alkaloids are
then extracted from the alkaline layer into an organic
solvent, customaril~ benzene. Evaporation of the benzene
yields a mixture of amorphous dimeric alkaloids which are
dissolved in benzene and chromatographed over alumina
(CAMAG - Activity III3.
The alkaloids are eluted in the following order:
leurosine, vinblastine, des-N-methyl vinblastine, leuro-
cristine and leurosidine. Identification of the dimeric
alkaloids in the eluant fraction is carried out by standard
procedures known to the art, as by thin layer chromatography.
Vinblastine is customarily eluted with a benzene-chloroform
(1:1) solvent mixture. The procedure for obtaining the V~B
fraction is more fully set forth in U. S. Patent 3,225,030.
Vinblastine fractions thus obtained were shown by
thin layer chromatography to contain small quantities of a
second alkaloid, identified as 4-desacetoxyvinblastine.
This second alkaloid is isolated as follows: The vinblastine
fraction is converted to the corresponding sulfate salts by
standard procedure and the sulfates subjected to a gradient
pH separation procedure in which the sulfates are dissolved
in 2 percent aqueous citric acid, and the citric acid
solution extracted twice with benzene. The pH is then
raised to pH = 5.5 by the addition of ammonia and two more
benzene extractions are carried out. The second fraction is
chromatographed over alumina (activity III). The chroma-
togram is developed with benzene. Fractions shown by thin
X-3754M -14-
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.. _ _ . . _ .. . . .
:1067073
layer chromatography to contain a secGnd alkaloid inaddition to vinblastine are combined and rechromatographed
over alumina and the chromatogram again developed with
benzene. This procedure is repeated. Fractions containing
substantially only the second alkaloid, 4-desacetoxy vin-
blastine with only minor amounts of vinblastine are combined
and recrystallized from methanol. 4-Desacetoxy vinblastine
thus recrystallized is then further purified by preparative
thin layer chromatography over silica using as eluant a
3:2;4 diethyla~ine-chloroform-benzene solvent mixture.
3'-Hydroxy-4-desacetoxyvinblastine has the
following physical characteristics:
proton nmr spectrum peaks at ~4.075~5), 5.85(15),
5.46-5.78 (broad m~ltiplet)
mass spectrum: ions at m/e 768, 411, 371, 224,
170, 102.
3'-Hydroxy-4-desacetoxyvinblastine is prepared
according to the following procedure: Defatted leaves of
plants containing crude vinca alkaloids; i.e., Catharanthus
roseus (Vinca rosea), previously moistened with aqueous
ammonia, are extracted with a water-immiscible solvent such
as benzene. The benzene is distilled from the extract in
the presence of aqueous tartaric acid. The tartaric acid
layer is then made basic by the addition of ammonia. The
dimeric alkaloids are extracted from the alkaline layer into
an organic solvent, customarily benzene. Evaporation of the
solvent yields a mixture of amorphous dimeric alkaloids.
The dimeric alkaloid fraction is dissolved in ethanol and
the corresponding sulfate salts formed by the addition of
ethanolic sulfuric acid. The crystalline mixed sulfate
salts are collected and then converted to the corresponding
X-3754M -lS-
, _
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1067073
free bases by solution in water, basifying the aqueous solu-
tion and extracting the alkaloids into a water-immiscible
organic solvent, customarily methylene dichloride. Evapora-
tion of the solvent yields a mixture of amorphous dimeric
alkaloids which are redissolved in methylene dichloride and
chromatographed over alumina (CAMAG - Activity III-IV).
The alkaloids are eluted in the following order:
leurosine, vinblastine, des-N-methyl vinblastine, leuro-
cristine and leurosidine. Identification of the dimeric
alkaloids in the eluant fraction is carried out by standard
procedures known to the art, as by thin layer chromatography.
Chromatography was carried out in a stainless steel column,
5 cm. by 730 cm., at a pressure of 200-400 psi. The
alumina-to-charge ratio was approximately 300 to 1. The
eluate was monitored at 280 m~, and fractions were separated
based upon the peaks observed in the ultraviolet profile.
Fractions were identified containing predominatntly leuro-
sine, vinblastine, des-N-methylvinblastine, and leuro-
cristine by thin layer chromatography. Post-des-N-methyl-
vinblastine, pre-leurocristine fractions were accumulated,
i.e., fractions containing more than one dimeric alkaloid
occurring after the peak des-N-methylvinblastine fraction
and prior to the peak leurocristine fraction, and were
converted to the corresponding sulfate salts by treatment
with an excess of 1 percent ethanolic sulfuric acid. The
sulfate salts were subjected to a gradient pH separation
procedure in which a solution of the sulfate salts in citric
acid buffer at pH = 3.4 was extracted with benzene. The pH
of the citric acid solution was raised in increments of
one-half pH unit, and the resulting aqueous layer extracted
X-3754M -16-
1067073
with benzene. 4-Desacetoxy-3'-hyaroxyvinblastine was found
to be present by thin layer chromatography in extracts at pH
= 5.4 and 5.9. Sulfates (VLB and leurocristine were shown
by TLC to be the chief dimeric alkaloid impurities present),
recovered from the pH = 5.4 extract, were dissolved in 5 ml.
of water and the acidity of the aqueous solution adjusted to
pH = 9 by the addition of ammonium acetate. The precip-
itated alkaloidal free bases were separated by centri-
fugation, dissolved in 3 ml. of methylenechloride and
chromatographed at high pressure in a stainless steel 5/16"
by 6 meter column packed with neutral alumina [Woelm N-18
(18-30 ~)~ using a linear gradient of 0-5 percent ethanol in
methylene chloride. The column was operated at about 1100
psi with a consequent flow rate of 180 ml/hr. Fractions
were collected every 3 minutes after material began to
appear in the column effluent as determined by ultra-violet
profile. Fractions 30-32 contained 4-desacetoxy-3'-
hydroxylvinblastine, as shown by TLC on silica gel using an
etherdiethylamine-toluenemethanol (100:5:5:5) solvent
system.
Illustrative compounds include:
3'-hydroxy-4-desacetoxyvinblastine C-3 N-methyl
carboxamide
3'-hydroxy-4-desacetoxyvinblastine C-3 N-cyclo-
propylmethyl carboxamide
2'-hydroxy-4-desacetoxyvinblastine C-3 N-cyano-
ethylamide
2'-hydroxy-4-desacetoxyvinblastine C-3 N-(2-
hydroxypropyl~amide
X-3754M -17-
1067073
deoxyvinblastine "A" C-3 carboxazide
deoxyvinblastine "A" C-3 N-~2-dimethylamino-
ethyl) carboxamide
deoxyvinblastine "B" C-3 N-(2,3-dihydroxypentyl)
carboxamide
4-desacetoxyvinblastine C-3 N-(3-hydroxypropyl)
carboxamide
4-desacetoxyvinblastine C-3 N-(2-aminoethylamino)
carboxamide
2'-hydroxyvinblastine C-3 N-(2-acetoxyethyl)
carboxamide
2'-hydroxyvinblastine C-3 N-(2-phenylethyl)
carboxamide
2'-hydroxyvinblastine C-3 N-(3-phenylpropyl)
carboxamide
3'-hydroxyvinblastine C-3 carboxhydrazide and
the like.
The present compounds are derivatives in which
the carbomethoxyl group at C-3 of certain known indole-
dihydroindole alkaloids is transformed to a carboxhydrazidegroup, a carboxazide group, a carboxamide group or a
derivative thereof. Not all of these derivatives are
ordinarily prepared by one process. The compounds in
which R in formula I above is NH2, NH-NH2 or NH-CH3 are
prepared as follows: Treatment of leurocolumbine, vin-
cadioline, their respective 4-desacetyl compounds or
deoxyvinblastine with either ammonia, methylamine or
hydrazine yields the corresponding amide, N-methylamide or
hydrazide. The product of this reaction with starting
materials having an intact 4-acetyl group is usually a
X-3754M -18-
1067073
mixture of compounds in which the carbomethoxy group at C-3
is transformed to a carboxamide, N-methylcarboxamide or
carboxhydrazide group, but also in which the acetyl group at
C-4 is completely or partially removed. For purification,
the C-4 desacetyl derivatives thus prepared are separated by
chromatography.
The compounds in which R is N(CH3~2, NH-alk-X
wherein X is H, CN or phenyl, NH-(C3-C8)-cycloalk, NH-
alk-Am or NH-alk-(OH)l 3 and alk and Am are as previously
defined are prepared by the following procedure: a
hydrazide, (Formula I wherein R is NH-NH2) prepared by
reaction of the C-3 carbomethoxyl compound with anhydrous
hydrazine, is transformed into the corresponding azide by
treatment with nitrous acid, nitrosyl chloride, nitrogen
tetroxide, amyl nitrite or a similar reagent according to
conventional procedures. The C-3 azide thus prepared is
then reacted with the primary of secondary amines HN (CH3)2,
NH2-alk-X, NH2-(C3-C8)-cycloalk, NH2-alk-(OH)1 3 or NH2-
alk-Am, to yield the desired C-3 amide. This C-3 azide-
amine reaction does not affect the C-4 acyl group which if
present remains intact during the reaction and workup. The
above azide-amine transformation follows the procedure
originated by Stoll and Huffman, Helv. Chim. Acta., 26, 944
(1943) -- see also U. S. Patents 2,090,429 and 2,090,430.
Compounds in which there is an acetyl group at
C-4 can be prepared, as has been stated above, by reaction
of vincadioline, leurocolumbine, or deoxyvinblastine
- directly with ammonia, methylamine or hydrazine followed by
separation of the 4-acetyl derivative from the 4-desacetyl
derivative, and, in the case of the hydrazide, conversion to
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the azide followed by reaction of the azide with an amine to
yield the amides. More generally, however, because of the
lability of the 4-acetyl group under basic reaction
conditions, the hydrazine-azide-amide reaction sequence is
carried out with a 4-desacetyl derivative. In general, the
4-desacetyl amides according to Formula I above can be
acylated with an aliphatic anhydride or acid chloride to
yield the corresponding C-4 acetate, propionate or butyrate
or a chloro derivative thereof. An acid chloride (Cl-
C3)-alkyl-COCl or chloro-(Cl-C3)-alkyl-CQ-Cl or an acid
anhydride l(Cl-C3)-alkyl-CO]=O or [chloro-tCl-C3)-alkyl-
CO]2=O can be used in the acylation reaction. The preferred
acylation procedure is that described in U. S. Patent
3,392,173 for vinblastine or leurocristine in which a diacyl
derivative is the first product of the reaction, and this
derivative is selectively hydrolysed to yield a 4-acyl
compound. Other procedures involving selective acylating or
multiple acylation followed by selective hydrolysis can be
employed to prepare the 4-acyl derivatives of this invention.
There are, however, certain provisos which must be
kept in mind when an acylation procedure is contemplated.
If the C-3 carboxamide group contains an acylable group;
i.e., hydroxy or amino, the C-4 acylation procedure must be
carried out prior to the azide-amine reaction which yields
the ultimate C-3 carboxamide group. The preferred procedure
here is to acylate, by the above procedures, the C-3
carboxhydrazide, first protecting the hydrazide group
itself, which would otherwise also be acylated. The pre-
ferred hydrazide protecting group is the propylidene group
formed by reaction of the NH2 portion of the hydrazide
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moiety with acetone. This group can be readily removed by
treatment with acid or, preferably, the propylidene deri-
vative itself can be reacted directly with nitrite to form
an azide group (see U. S. Patent 3,470,210, Example VII).
Other procedures involving selective acylation or
multiple acylation followed by selective hydrolysis or
selective protection of an acylable function followed by
acylation and subsequent removal of the protecting group
will be apparent to those skilled in the art.
Compounds according to Formula I above in which R
is NH-alk-X and X is carboxyl, carboxamido or carbo-(Cl-C3)-
alkoxy are prepared by reacting an amino acid, amino amide
preferably an amino ester of the structure NH2-C-COQ
wherein Z
Q is OH, NH2 or O-alk and Z is H or a Cl-C5 alkyl group,
with the chosen dimeric indole-dihydroindole azide. Amino
acids useful for this purpose, and coming within the scope
of the above formula, include leucine, isoleucine, valine,
glycine, alanine, norleucine and the like. As will be
apparent to those skilled in the art, other amino acids and
polypeptides can also be used to react with, for example,
4-desacetyl vinblastine C-3 carboxazide, to yield substi-
tuted C-3 carboxamides having anti-tumor properties.
An alternative method of preparing the primary
amide (R is NH2) from the hydrazide (R is NH-NH2) involves
the use of a procedure based on that of Ainsworth, U. S.
Patent 2,756,235, in which the hydrazide is hydrogenolyzed
with Raney nickel.
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. _ _, . _ _ . , _ _ . . . . . . . . _ .... _
1067~73
The novel derivatives will be named with referenceonly to the new group formed at a given carbon atom. For
example, the compound produced by replacing the methyl ester
function in vinblastine at C-3 with an amide function will
be called simply vinblastine C-3 carboxamide, and not vin-
blastine C-3 descarbomethoxy C-3 carboxamide.
The compounds in the form of their free bases,
including both carboxamides, carboxazides and carboxhydra-
zides are white or tan-colored amorphous solids. It is
preferable, however, where possible to isolate and crys-
tallize the amides in the form of their anionic salts formed
with non-toxic acids. Such salts are high-melting, white,
crystalline or amorphous, water-soluble solids.
The preparation of the compounds is more fully
illustrated in the following specific examples:
! Example 1
4-Desacetyl deoxyvinblastine "B" C-3 carboxhydrazide
Deoxyvinblastine "B" in the amount of 2.55 grams
was reacted with 30 ml. of anhydrous hydrazine in anhydrous
methanol in a sealed reaction vessel at about 60C for about
18 hours. The reaction vessel was cooled, and opened, the
contents removed, and the volatile constituents evaporated
therefrom in vacuo. The resulting residue, comprising
4-desacetyl deoxyvinblastine "s" C-3 carboxhydrazide, was
taken up in methylenechloride, the methylenechloride
solution washed with water, separated and dried, and the
methylenechloride removed in vacuo to yield an amorphous
powder with the following characteristics: infrared maxima;
3440 cm 1 (N-H), 1735 cm 1 (COO), 1675 cm (CON); molecular
ion spectrum; m/e = 752 (cc,nsistent with C43H56N6O6); nmr
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spectrum; ~3.78 (ArOCH3), ~3.58 (C18COOC_3), ~2.77 (N-CH3),
~4.15 (C4-H).
Example 2
4-Desacetoxyvinblastine C-3 N-(2-Hydroxyethyl)
Carboxamide
Following the procedure of Example 1, 4-des-
acetoxyvinblastine was reacted with anhydrous hydrazine in
methanol solution in a sealed tube at 46C. for three days.
4-Desacetoxyvinblastine C-3 carboxhydrazide thus prepared
was isolated and purified by the procedure of the same
example. The compound had the following physical char-
acteristics: PKa = 5.61 and 7.38; ultraviolet spectrum;
~max = 215 and 267 nm; infrared spectrum, peaks at 3450
cm 1 (N-H), 1725 cm 1 (COO), 1680 cm 1 (CON); molecular
spectrum m/e = 252, 411, 355, 244, 154. Molecular ion M+ =
752 consistent with empirical formula C43H56N6O6. The
compound was a tan amorphous powder.
The 4-desacetoxyvinblastine C-3 carboxhydrazide
was converted to the corresponding carboxazide in hydro-
chloric acid solution at 0C. with sodium nitrite. The
reaction mixture was next made basic by the addition of an
excess of cold 5 percent aqueous sodium bicarbonate. The
aqueous solution was extracted three times with methylene
dichloride.
Five ml. of ethanol amine were added to a solution
containing about 1.2 g. of 4-desacetoxyvinblastine C-3
carboxazide. The reaction mixture was sealed and protected
from light. After being allowed to stand for one day at
room temperature, the reaction vessel was opened and the
volatile constituents removed from the reaction mixture by
~-3754M -23-
1067073
evaporation in vacuo. The resulting residue containing
4-desacetoxyvinblastine C-3 N-(2-hydroxyethyl) carboxamide
formed in the above reaction was dissolved in methylene
dichloride and the methylene dichloride layer washed several
times with water. The methylene dichloride layer was
separated and dried and the solvent removed by evaporation
in vacuo. The resulting residue was chromatographed over
silica gel using a 3:1 ethyl acetate-ethanol solvent mixture
as the eluant. Fractions shown to contain the desired
product as determined by thin layer chromatography were
combined and evaporated to dryness in vacuo. 4-Desacetoxy-
vinblastine C-3 (N-(2-hydroxyethyl) carboxamide thus
prepared was a tan amorphous material with the following
physical characteristics: molecular ion; M+ = 781 con-
sistent with empirical formula C45H59N507; infrared spectrum
peaks at 3420 cm 1 (NH), 1735 cm 1 (C00), 1665 cm 1 (CON~.
The sulfate salt was prepared using ethanolic sulfuric acid
and adjusting the pH in the range 3.8-4.2. The sulfate salt
was recovered by evaporation of the volatile constituents in
vacuo.
Example 3
4-Desacetylvincadioline C-3 N-Methylamide
Following the procedure of Example 1, vincadioline
was reacted with hydrazine to form the corresponding C-3
carboxhydrazide. The hydrazide was in turn converted to the
corresponding carboxazide by the procedure of Example 8 and
the azide was reacted with methylamine according to the
procedure of the same example. The product of this re-
action, 4-desacetyl vincadioline C-3 N-methylamide, had the
following physical characteristics: infrared spectrum peaks
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1067~73
at 2.95 m~, 5.75 m~ and 5.97 m~; nmr spectrum consistent
with postulated structure with added doublet at ~3.82
(amidemethyl hydrogens); molecular spectrum, molecular ion
M+ = 783 consistent with C44H57N508
Following the above procedure 4-desacetylleuro-
colombine C-3 N-methylamide is prepared.
Example 4
Preparation of salts
Other salts, including salts with inorganic anions
such as chloride, bromide, phosphate, nitrate and the like,
as well as salts with organic anions such as acetate,
chloroacetate, trichloroacetate, benzoate, alkyl or aryl
sulfonates and the like, are prepared from the amide bases
of this invention by a procedure analogous to that set forth
in Example 1 above for the preparation of the sulfate salt
by substituting the appropriate acid in a suitable diluent
in place of the 1 percent aqueous sulfuric acid of that
example.
As will be apparent to those skilled in the art,
the presence of other ester and/or amide groups in the
indole-dihydroindole components requires extra care in the
preparation of salts so as to avoid hydrolysis, trans-
esterification and other reactions which take place at high
- temperatures, extremely acid pH's, etc.
The compounds have shown antiviral activity in
vitro against herpes virus employing a tissue culture system
in a plaque suppression test similar to that described by
Siminoff, Applied Microbiology~ 9, 66-72 (1961).
X-3754M -25-
,~
10671:~73
In addition, the compounds have been shown to be
active against transplanted mouse tumors in vivo. of
particular interest, however, is the activity of the
compounds against Ridgeway osteogenic sarcoma (ROS) and
Gardner lymphosarcoma (GLS). In demonstrating activity of
the drugs against these tumors, a protocol was used which
involved the administration of the drug, usually by the
intraperitoneal route, at a given dose level for 7-10 days
after inoculation with the tumor.
The following table - Table 1 - gives the results
of several experiments in which mice bearing transplanted
tumors 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; and column 4, the percent inhi-
bition of tumor growth. (ROS is an abbreviation for
Ridgeway osteogenic sarcoma; GLS for Gardner lymphosarcoma;
and CA 755 is an adenocarcinoma).
~o The compounds like leurocristine and vinblastine
become toxic to mice at doses above those at which they
produce 100 percent inhibition of the transplanted tumor.
In addition, for reasons that are not well understood, all
drugs in a given test including control drugs like vin-
blastine may show toxicity at dose levels where they
ordinarily give tumor inhibition without toxicity. Thus,
the results set forth in Table 1 are of typical experiments
where the control drugs give expected results and are not an
average of all runs.
X-3754M -26-
1~67073
o o
~ ~ o
a) ~1
C) Q
~1 ~
H
U~
X X
~ O Ln
O ~1
a ~ ,,
.Y X
10 ~s In r~
O O
E~ ~
~ o~
.. E~ ~; ~
2 0
~ .~
o
C)
~ ^
,
X
~a ~ o
o s~
~ d
o
I
o I ~:~
~ ~r ~ m
X-3754M -27-
1067073
The compounds are also active against other
transplanted tumors. For example, with Mecca lymphosarcoma,
parenteral injection of 0.25 mg./kg. for 9 days of vin-
blastine C-3 N-methylcarboxamide sulfate gave a 54 percent
inhibition of growth and of vinblastine C-3 amide, a 28
percent inhibition. At the same dose levels, vinblastine
itself was completely inactive.
In addition, in studies against CA 755 adeno-
carcinoma, 4-desacetyl vinblastine C-3 carboxamide sulfate
gave a 67 percent inhibition of tumor growth, 4-desacetyl
vinblastine C-3 N-methylcarboxamide sulfate 61 percent
inhibition and vinblastine C-3 carboxamide sulfate 49 per-
cent inhibition at a dose level of 0.25 mg./kg. for eight
days and 72 percent inhibition at 0.3 mg./kg. In a similar
experiment, vinblastine gave a 31 percent inhibition while
leurocristine at the somewhat lower dose level of 0.2 mg./kg.
gave 79 percent inhibition with an excellent effectiveness
rating. Against L5178Y lymphocytic leukemia, vinblastine
C-3 carboxamide sulfate at a dose level of 0.25 mg./kg. for
ten days in an experiment using five mice gave three
indefinite survivors; the life span of the two diseased mice
in this experiment was prolonged by 26 percent over that of
the control mice. In the same experiment, vinblastine gave a
36 percent prolongation but with no indefinite survivors and
rated only a minimal effectiveness rating.
In utilizing the novel amides and hydrazides as
anti-neoplastic 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 I except those in which R is
X-3754M -28-
~067073
NH-NH2 or N3, formed with a non-toxic acid is mixed with
starch or other excipient and the mixture placed in tele-
scoping gelatin capsules each containing from 7.5-50 mg. of
active ingredients. Similarly, the anti-neoplastic salt can
be mixed with starch, a binder, and a lubricant and the
mixture compressed into tablets each containing from the
7.5-50 mg. The tablets may be scored if lower or divided
dosages are to be used. With parenteral administration, the
intravenous route is preferred. For this purpose, isotonic
solutions are employed containing 1-10 mg./ml. of a salt of
an indole-dihydroindole amide of formula I except for the
hydrazides and azides. The compounds are administered at
the rate of from 0.1 to 1 mg./kg. of mammalian body weight
once a week, depending on both the activity and the toxicity
of the drug. Free bases of compounds according to formula I
in which R is NH-NH2 or N3 are compounded into suitable
dosage forms and administered in similar fashion at similar
dose levels.
While most of the compounds of this invention are
useful as antineoplastic or antiviral drugs, two types of
derivatives, the hydrazides and azides (compounds of formula
I wherein R is NH-NH2 or N3), are also useful as inter-
mediates as has been set forth above, in that the hydrazide
can be transformed to the azide by nitrosation, as by
nitrous acid treatment, or to the simple amide by hydro-
genolysis. The azide can in turn be made to react with
primary or secondary amines to yield the amides.
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