Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
~0~179
4-Desacetylcarbox~de derivatives of vincaleukoblastine (VLB) leuro-
sidine, leurocristine and l-desrnethyl-l-formyl leurosidine
are useful as anti-neoplastic agents.
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. 3,370,057), vincaleukoblastine
(vinblastine or VLB) (U.S. Patent No. 3,097,137), leuro-
sidine (vinrosidine) and leurocristine (VCR or vincristine)
(U.S. Patent No. 3,205,220), deoxy VLB "A" and "B" and
4-deascetyl leurosine hydrazide, Tetrahedron Letters, 783
(1958); 4-desacetoxy vinblastine (U.S. Patent No. 3,954,773;
4-desacetoxy-3'-hydroxyvinblastine (U.S. Patent No. 3,944,554;
leurocolombine (U.S. Patent No. 3,890,325) and vincadioline
(U.S. Patent No. 3,887,565). Two of these alkaloids, VLB
and leurocristine, are now marketed as drugs for the treat-
ment of malignancies, particularly the leukemias and related
diseases in humans. Of these marketed compounds, leuro-
cristine 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 chemo-therapeutic properties have been recovered
from Vinca rosea fractions, and a determination of their
X-3754S- -2-
.
.
:, :
~823l~79
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-active alkaloids has been the preparation of
dihydro VLB (U. S. Patent No. 3,352,868) and the replacement
of the acetyl group at C-4 (carbon no. 4 of the VLB ring
system-see the numbered structure below) with higher al-
kanoyl group or with unrelated acyl groups (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 VLB was also a useful intermediate
for the preparation of structurally modified VLB compounds
in which an N,N-dialkylglycl group replaced the C-4 acetyl
group of VLB (U. S. Patent No. 3,387,001). An intermediate
compound, namely 4-desacetyl VLB, was produced during the
chemical reactions leading to these latter derivatives.
ThiR C-4 hydroxy intermediate has been reported to be a
toxic material having little in vivo chemotherapeutic
activity against the P1534 murine leukemia system by
Hargrove, Lloydia, 27, 340 (1964).
A series of C-3 carboxamide derivatives of the
indole-dihydroindole has been prepared and found to have
significant in vivo activity against transplanted tumors
in mice (Belgium Patent 813,168).
.. . .
X-3754S -3-
'.
_ . _ _ _ . . . . . .. . . .
1~8~79
A number of compounds an presently available which
show activity ayainst one or more neoplastic diseases. They
show considerable specificity in their activity. Neoplastic
diseases often develop resistance to an active agent.
These new compounds provide the clinician additional weapons
to use against the spectrum of neoplastic diseases. They
also offer alternatives to ayents to which resistance has
developed.
The present invention provides a dimeric indole-
dihydroindole carboxamide of the formula
H J
j~ - - - CH~-CHD
R
C-N/R4
Il R5
6
wherein R4 is -(CH )m~C\ where m is 1, 2 or 3 and R6 is -CHO,
-O-C-Cl-C17 alkyl, -0-C-C2-C7 alkenyl, -0-Cl-C
O O -~
alkyl, -NH-C-Cl-C3 alkyl, or -S-Y when Y is
X-3754S -4-
1082179
H, Cl-C3 alkyl or a bond, said bond joininq :~
the sulfur atoms in two moieties of Formula I
wherein Y is a bond, and R7 is H; or R6 and ~ -
R each are -O-Cl-C3 alkyl; and R5 is H;
one of R and R is H or -OH and the other is
-C2H5; Rl is -CH3 or -CHO; and its pharma-
ceutically acceptable salts.
The present invention provides a pharmaceutical
composition for inhibiting a tumor or prolonging the life of
a host mammal comprising an inert carrier and as active
ingredient a dimeric indoledihydroindole carboxamide of ;
Formula I or a pharmaceutically acceptable salt thereof.
The present invention also provldes a prooess of ..
preparing a dimeric indole-dihydroindolecarboxamide of the
formula
-R`
H I O
CH:3-CH~
6 1 ""
O , -, .
:
X-3754S -5-
,~' ~,''
' ~'"",
_
: . , 1 . `
. ~ ' : , :
Zl~g
wherein R4 is -(CH ) -HC\ wherein m is 1, 2 or 3 and R6 is -CHO,
-O-C-Cl-C17 alkyl, -O-C-C2-C7 alkenyl, -O-Cl-C3
O . O
alkyl, -NH-C-Cl-C3 alkyl, or -S-Y wherein Y is
H, Cl-C3 alkyl or a bond, said bond joininq
the sulfur atoms in two moieties of Formula I
- wherein Y is a bond, and R7 is H; or R6 and
R7 each are -O-Cl-C3 alkyl; and RS is H;
one of R2 and R3 is H or -OH and the other is
-C2H5; R is -CEI3 or -CHO; and its pharma-
ceutically acceptable salts
comprising reactinq a dimeric indole-dihydroindole car-
boxazide of the formula
I t - C-O-CH3 Formula II
H I O
1 8 .
CH~-CHo
- CH3-O-- /Q\ ~ OH
R1 1 ~
C-N3
X-3754S -6-
_ _ . . . . . . . . . _: _ .
- . . : ,: ~ - :
~8~179
with an amine R4R5NH wherein R through R7 are as defined
above with the proviso that Y in R6 is other than a bond,
and recovering the product of formula I in the form of the
free amine or a pharmaceutically acceptable salt.
The carboxamides of this invention are limited to
the carboxamides of vincaleukoblastine, leurocristine,
leurosidine and l-desmethyl-l-formyl-leurosidine and their
deoxy "A" and "B" analogs and the pharmaceutically-acceptable
salts of the above bases.
Vincaleukoblastine, leurocristine and leurosidine
are found naturally. The l-desmethyl-l-formyl-leurosidine
and some of the deoxy "A" and "B" although not found in
compounds have been synthesized. Vincaleukoblastine and
leurocristine are clinically used for the treatment of
neoplastic diseases in humans.
Compounds can be described generically as deriva-
tives of vincaleukoblastine (vinblastine or VLB) when Rl is
CH3, R2 is OH and R3 is -CH2CH3. In derivatives of leuro-
cristine (vincristine or VCR) Rl is CHO, R2 is OH and R3
is -C2H5. In derivatives of leurosidine Rl is CH3, R2 is
CH2CH3 and R3 is OH. In derivatives of l-desmethyl l-formyl
leurosidine Rl is CHO, R2 is CH2CH3 and R3 is OH. The deoxy
analogs of the above compounds in which one of R2 or R3 is H
and the other is -C2H5 are described as "A" when R2 is H and
"B" when R3 is H.
The term "Cl-C3 alkyl" as employed hereinabove
includes the methyl, ethyl, n-propyl and iso-propyl groups.
X-3754S -7-
108Z~79
The term "Cl-C17-alkyl-CO" means an alkanoyl group derived
from alkanoic acids having from 2-18 carbon atoms; i.e.,
- acetyl, propionyl, isobutyryl, stearyl, palmitoyl, lauryl,
myristoyl, caproyl (C6), iso-valeroyl, capryloyl (C8),
capryl (C10) and the like. The term "C2-C7-alkenyl-CO"
means an unsaturated acid group having from 3 to 8 carbons;
i.e., acrylyl, crotonyl, methacrylyl, allylacetyl, vinyl-
acetyl, tigIyl, 2-methyl-2-hexenoyl, 2-octenoyl and the
like.
1~ Illustrative groups which are the nitrogen con-
taining moiety of the C3-carboxamido group in the various
modified dimeric indole-dihydroindole alkaloids represented
by the above formula include:
acetaldehydeamide, 2-methoxypropylamide, 2-
acetyloxyethylamide, 2-butyryloxyethylamide, 2-ethoxy-
ethylamide, 2-dimethoxyethylamide, 2-acrylyloxyethyl,
pyrrolidinylamide, 2-mercaptoethylamide, 3-methylmercapto-
propylamide, N-2-n-propylmercaptopropylamide, 4-acetyl-
amino-n-propylamide, and the like.
Non-toxic acids useful for forminy pharmaceutically-
acceptable acid addition salts of the amine bases include
salts derived from inorganic acids such as: hydrochloric
acid, nitric acid, phosphoric acid, sulfuric acid, hydro-
bromic acid, hydriodic acid, nitrous acid, pho~phor~us acid
and the like, as well as salts of non-toxic orsanic acids
including aliphatic mono and dicarboxylates, phenyl-sub-
stituted 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,
X-3754S -8-
_
.
7~
phosphate, monohydrogenphosphate, dihydrogenphosphate,metaphosphate, pyrophosphate, chloride, bromide, iodide,
acetate, propionate, decanoate, caprylate, acrylate, formate,
isobutyrate, caprate, heptoanate, propiolate, oxalate,
malonate, succinate, suberate, sebacate, fumarate, maleate,
butyne-1,4-dioate, hexyne-1,6-dioate, benzoate, chlorobenzoate,
methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxy-
benzoate, 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.
Illustrative compounds coming within the scope of
this invention include:
4-desacetyl deoxy VLB "A" C-3 N-2-mercaptoethyl-
carboxamide
4-desacetyl deoxy VLB "B" C-3 N-2-mercaptoethyl-
carboxamide :
4-desacetyl deoxy VCR "A" C-3 N-2-mercaptoethyl-
carboxamide
4-desacetyl deoxy VCR "B" C-3 N-2-mercaptoethyl-
carboxamide
4-desacetyl deoxy VLB "A" C-3 N-2-methylmercap-
toethylcarboxamide
4-desacetyl deoxy VLB "B" C-3 N-2-methylmercap-
toethylcarboxamide
4-desacetyl deoxy VCR "A" C-3 N-2-methylmercap-
toethylcarboxamide
X-3754S _g_
~,
~1~8~
4-desacetyl deoxy VCR "B" C-3 N-2-methylmercap-
toethylcarboxamide
4-desacetyl deoxy VLB "A" C-3 N-2-methoxyethyl-
carboxamide
4-desacetyl deoxy VLB "B" C-3 N-2-methoxyethyl-
carboxamide
4-desacetyl deoxy VCR "A" C-3 N-2-methoxyethyl-
carboxamide
4-desacetyl deoxy VCR "B" C-3 N-2-methoxyethyl-
carboxamide
4-desacetyl VCR C-3 N-2-mercaptoethylcarboxamide
4-desacetyl VCR C-3 N-2-methylmercaptoethylcar-
boxamide
4-desacetyl leurosidine C-3 N-2-mercaptoethyl-
carboxamide
4-desacetyl leurosidine C-3 N-2-methylmercapto-
ethylcarboxamide
4-desacetyl-1-desmethyl-1-formyl leurosidine C-3
N-2-mercaptethylcarboxamide
4-desacetyl-1-desmethyl-1-formyl leurosidine C-3
N-2-methylmercaptethylcarboxamide
4-desacetyl VCR C-3 N-2-methoxyethylcarboxamide
4-desacetyl leurosidine C-3 N-2-methoxyethylcar-
boxamide.
The particular derivatives which are the subject
of this invention are those in which the carbomethoxyl group
at C-3 of certain known indole-dihydroindole alkaloids
obtained either from plants or by partial synthesis is
transformed to a derivative of a carboxamide. Not all of
these derivatives are ordinarily prepared by a single
X-3754S -10-
_ . . . . .. . . . . . .
7~
process. For example, the compounds of this invention of
formula I can be prepared as follows: Treatment of VLB,
leurocristine, leurosidine, l-desmethyl-l-formyl-leurosidine
or their deoxy analogs with hydrazine yields the corre-
sponding hydrazide. The product of this reaction with
starting materials having an intact 4-acetyl group is
usually a mixture of compounds in which the carbomethoxy
group at C-3 is transformed to a 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.
Generally, the reaction would be carried out starting with
the 4-desacetyl derivative of VLB, leurocristine, leuro-
sidine or the 1-desmethyl-1-formyl leurosidine.
The C-4-desacetyl C-3 carboxhydrazide derivatives
are transformed into the corresponding azides 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 a suitable amine. The above azide-amine transformation
follows the procedure originated by Stoll and Huffman,
Helv. Chim. A~ta., 26, 944 (1943) -- see also U. S. Patents
2,090,429 and 2,090,430. In the examples of this invention
the reaction was carried out in methylene dichloride. Other
suitable solvents which do not react with the azide are
chloroform, acetonitrile, acetone, benzene and toluene.
Compounds in which an aldehyde amide group,
NH-CH2-CHO, is present are preferably prepared from the
corresponding acetal amide NH-CH2-(O-C1-C3 alkyl)2 by acidic
hydrolysis. Compounds in which the amide group contains an
X-3754S -11-
1~2i79
ester function such as in the group NH-(CH2)n-OAc, wherein n
and Ac are as defined above, are preferably prepared by
esterifying an hydroxy amide containing the group NH(CH2)nOH
with a suitable acid anhydride, Ac2O, wherein Ac is Cl-C17-
alkyl-CO or C2-C7-alkenyl-CO. Similarly, compounds in which
R6 is alk-X wherein X is NH-CO-Cl-C3-alkyl are prepared by
acylating, with an acid anhydride, an aminoalkylamide group
of the structure NH-alk-NH2. The azide reacts with NH2(CH)nSH
in the presence of a base, preferably, a pyridine, to produce
a mixture of the N-2-mercaptoalkyl carboxamide and the bis
N-2-alkylcarboxamide disulfide.
The hydrazides can be used to prepare the corre-
sponding azides which are in turn used to prepare other
amides directly. Similarly, the hydroxyalkylamides and
aminoalkylamides can be acylated (with care) to form the
corresponding carboalkoxy or acylamidoalkyl amides. The
acetalamides are, of course, hydrolyzed with acid to yield
the corresponding acetaldehydeamides.
An alternative and presently preferred method of
preparing a primary amide is from the hydrazide 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.
The novel derivatives of this invention will be
named with reference only to the new group formed at a given
carbon atom. For example, the compound produced by replacinq
the methyl ester function in VLB at C-3 with an amide
function will be called simply VLB C-3 carboxamide, and not
VLB C-3 descarbomethoxy C-3 carboxamide.
X-3754S -12-
10~z;~ 79
The compounds of this invention, in the form of
their free bases, including both carboxamides, are white or
tan-colored amorphous solids. It is preferable, however,
where possible, to isolate and crystallize the carboxamides
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 of this invention
is more fully illustrated in the following specific examples:
Example 1
4-Desacetyl VLB C-3 carboxhydrazide
4-Desacetyl VLB was heated in anhydrous ethanol
with an excess of anhydrous hydrazine 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 VLB C-3 carbox-
hydrazide, was taken up in methylenechloride, the methylene-
- chloride solution washed with water, separated and dried,
and the methylenechloride removed by evaporation in vacuo.
The resulting residue was dissolved in a 1:1 chloroform:benzene
solvent mixture and chromatographed over silica gel. A
benzene-chloroform-triethylamine solution (100:50:7.5) was
employed to develop the chromatogram. The initial chroma-
tographic fractions contained unreacted 4-desacetyl VLB.
Further fractions were found to contain 4-desacetyl 18'-
descarbomethoxy VLB C-3 carboxhydrazide previously described
by Neuss et al., Tetrahedron Letters, 1968, 783. The next
fractions, found to contain 4-desacetyl VLB C-3 carbox-
hydrazide by thin layer chromatography, were combined, and
X-3754S -13-
..
1~82~79
the solvents evaporated therefrom in vacuo. The resulting
solid melted at about 219-220C. with decomposition. 4-Des-
acetyl VLB C-3 carboxyhydrazide thus prepared had a carbo-
methoxy absorption band in the IR at 1725-1735 cm 1 thereby
differentiating it from the 18'-descarbomethoxy compound of
Neuss et al. supra, and a 1690 cm 1 band in the IR attrib-
utable to the hydrazide function. Molecular weight by mass
spectrography was 768 in agreement with the theoretical
value calculated for C43H56N6O7. The nmr spectrum contained
the prominent resonance at ~ 3.6 representing the methyl
group of the C-18 carbomethoxy function.
Example 2
4 Desacetyl VLB C-3 carboxazide
A solution of 678 mg. of 4-desacetyl VLB C-3 car-
boxhydrazide (from Example 1) was prepared in lS ml. of
anhydrous methanol. About 50 ml. of lN aqueous hydrochloric
acid were added, and the resulting solution cooled to about
0C. Approximately 140 mg. of sodium nitrite were then
added, and the resulting reaction mixture stirred for 10
minutes while maintaining the temperature at about 0C. The
solution turned dark red-brown upon the addition of the
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. 4-Desacetyl VLB C-3 carboxazide
formed in the above reaction passed into the methylene
dichloride.
While ordinarily the methylene dichloride solution
of 4-desacetyl vinblastine C-3 carboxazide is used without
further purification, an aliquot was treated as follows in
X-3754S -14-
:
.. . ..
- .- ,' : . ,: ,. ~ ' '
: .- . ~ , ' : ' ''
order to characterize the azide: Evaporation of the methyl-
ene dichloride left the azide in an amorphous state. The
azide residue was washed with ether, and the resulting
suspension filtered. The residual tan powder had the fol-
lowing distinguishing physical characteristics: ultraviolet
spectrum lambda maX=269 mu. (epsilon = 16,700); shoulder at
about 290 mu; 309 mu. (epsilon = 7,100); infrared absorption
maximum at 1690 cm. 1 (carboxhydrazide) was absent, while
the maximum at 1730 cm. 1 was not affected. Furthermore, a
sharply defined maximum at 2135 cm. 1 was noted characteris-
tic of the carboxazide function. The mass spectrogram re-
vealed a molecular ion m/e = 708 showing a loss of 71 mass
units (H, CON3) from the molecular weight calculated for
C43 53 7 7
Example 3
4-Desacetyl VLB C-3 N-ethylcarboxamide
A solution of 4-desacetyl VLB C-3 carboxazide was
prepared in methylene dichloride solution according to the
procedure of Example 2 from 900 mg. of 4-desacetyl VLB C-3
carboxhydrazide. The methylene dichloride solution was
dried, and the volume reduced to about 20 ml.
The solution of the azide in methylene dichloride
was then placed in a flask fitted with a drying tube and
stirrer. 50 ml. of anhydrous ethylamine were added thereto,
and the reaction mixture was stirred at room temperature for
about two hours. Evaporation of the volatile constituents
in vacuo yielded a tan amorphous powder which was chromato-
graphed over silica gel. The chromatogram was developed
with an ethyl acetate-anhydrous ethanol (3:1) solvent
mixture. Fractions containing 4-desacetyl VLB C-3 N-ethyl-
X-3754S -15-
'79
carboxamide as determined by thin-layer chromatoqraphy were
combined, and the solvent was removed from the combined
fractions in vacuo. 450 mg. of a tan amorphous powder were
obtained with the following distinctive physical charac-
teristics: molecular ion spectrum, m/e = 781 (corresponding
to C45H59N507); infrared spectrum; absorption maxima at 1730
cm. 1 (ester), 1670 cm. 1 (amide), 3420 cm. 1 ~N-H amide),
nmr. ~1.18 (triplet-~-methyl of ethyl amide group), ~3.28
(quartet-a-methylene of ethyl amide group), ~3.59 (singlet-
methyl ester),
4-desacetyl VLB C-3 N-ethylcarboxamide sulfate was
prepared by dissolving the above amorphous powder in an-
hydrous ethanol and adjusting the pH to about 4.0 with 2
percent sulfuric acid in anhydrous ethanol. Evaporation of
the solvent _ vacuo yielded a water-soluble tan powder com-
prising 4-desacetyl VLB C-3 N-ethylcarboxamide sulfate.
.;
X-3754S -16-
108~:~79
Example 4
4-Desacetyl VLB C-3 N-~-butyryloxyethylcarboxamide
Following the procedure of Example 3, 4-desacetyl
VLB C-3 N-~-butyryloxyethylcarboxamide was prepared with the
following physical characteristics: infrared spectrum;
peaks at 3420 cm. 1, 1735 cm. 1, and 1680 cm. 1; molecular
spectrum; molecular ion, M+ = 867 consistent with empirical
formula C49H6 N O
Example 5
4-Desacetyl VLB C-3 N-(2-hydroxyethyl)carboxamide
Following the procedure of Example 3, 4-desacetyl
VLB C-3 N-(2-hydroxyethyl)carboxamide was prepared by
reacting 4-desacetyl VLB C-3 carboxazide with ethanol amine.
It was a tan amorphous solid with the following physical
characteristics: infrared spectrum; peaks at 3420 cm. 1
(NH), 1732 cm. 1 (COO), 1670 cm. 1 (CON). Molecular ion M+
= 797 consistent with empirical formula C45H59N5O8. The
corresponding sulfate salt was prepared by the above procedure
and was a water soluble tan amorphous powder.
Example 6
4-Desacetyl VLB C-3 N-(2-acetoxyethyl)carboxamide
4-Desacetyl VLB C-3 N-(2-acetoxyethyl)carboxamide
was prepared from the N-hydroxyethylcarboxamide by acetylation.
It was a tan amorphous powder with the following physical
characteristics: infrared spectrum, peaks at 3420 cm. 1
(NH), 1740 cm. 1 (COO), and 1670 cm. 1 (CON). Molecular
spectrum, molecular ion M+ = 839 consistent with empirical
formula C47H61N5Og; nmr spectrum consistent with structure,
particularly with added peak at ~1.91 (acetylmethyl).
.
X-3754S -17-
,
lOB~79
,:
Example 7
4-Desacetyl vLs C-3 N-(2-aminoethyl)carboxamide
4-Desacetyl VLB C-3 N-(2-aminoethyl) carboxamide
was prepared by the above procedure and was a tan amorphous
powder with the following physical characteristics: pka=
6.8, 9.0, 4.6. Infrared spectrum; peaks at 3420 cm. 1
(NH), 1730 cm. 1 (COO), 1670 cm. 1 (CON; molecular spectrum,
molecular ion, M+ = 796 consistent with empirical formula
C45H60N6O7. A sulfate salt was also prepared as a tan
amorphous powder.
Example 8
4-Desacetyl VLB C-3 N-2-dimethoxyethylcarboxamide
Following the procedure of Example 3, 4-desacetyl
VLB C-3 N-2-dimethoxyethylcarboxamide was prepared by
reacting 4-desacetyl VLB C-3 carboxazide with 2-dimethoxy-
ethylamine. The amide thus produced had the following
physical characteristics: infrared absorption maxima at
1665 cm 1 (amide) and at 1730 1 (carboxyl); molecular ion ~ -
spectrums (m/e), molecular ion at 841 other peaks at 782,
20 651, 500, 355, and 154; NMR ~4.42 (triplet) ~3.41 (doublet
Cl-2H), ~3.36-3.45 (6 methyl ether hydrogens).
The sulfate salt was prepared by dissolving the
free base prepared as above in methanol and adding a solution
of 2 percent sulfuric acid also in methanol thereto.
Evaporation of the resulting solution to dryness yielded a
tan amorphous water soluble powder.
X-3754S -18-
. ,
, ' ' ~ '. ' . ' , ~ :
108217t~
Example 9
4-Desacetyl VLB C-3 N-(2-methylmercaptoethyl)carboxamide
1.8 g. of 4-Desacetyl VLB C-3 carboxhydrazide was
converted to the azide with the procedure of Example 2 using
100 ml. lN hydrochloric acid and 180 mg. sodium nitrite in
anhydrous methanol. A solution of the azide in methylene
dichloride was reacted with 4 g. methylmercaptoethylamine
after the procedure of Example 3. The reaction mixture was
stirred overnight of room temperature. The solution was
then washed once with water, dried over sodium sulfate,
filtered and the solvent evaporated in vacuo. The residue
was applied to a silica column and eluted with an ethyl
acetate-methanol (3:1) eluant. Fractions containing the
N-(2-methylmercaptoethyl)carboxamide were combined and the
solvent evaporated in vacuo. The yield was 540 mg. The
carboxamide had the following distinctive physical charac-
teristics: elemental analysis S found 3.47~ (3.87% cal-
culated); infrared absorption maxima peaks at 1740 and 1675
cm ; nmr ~2.12 (-SCH3) ~2.80 (-NCH3) ~3.58 (-COOCH3) ~3.78
20 (ArOCH3); mass spectrum in 827 m/e 841 (transmethylation)
486 (vindoline half) no peak at 813.
The sulfate salt was prepared in the usual manner.
Example 10
4-Desacetyl VLB C-3 N-(3-methylmercaptopropyl)carboxamide
4.0 g. 4-Desacetyl VLB hydrazide was connected
to the azide with 200 ml. lN hydrochloric acid and 400 mg.
sodium nitrite with the procedure of Example 2. 5 g. Methylmer-
captopropylamine was added to a solution of the azide in
methylenedichloride and the solution stirred overnight at
X-3754S -19-
, .
1~8~17~
room temperature under the conclitions of Example 3. The
solution was washed once with water, dried over sodium
sulfate filtered and evaporated. The residue was applied to
a silica column and eluted with a methylenedichloride:ethyl
acetate-methanol (1:1:1) eluant. The appropriate fractions
were combined and the solvent removed in vacuo. Yield was
1.86 g. The carboxamide had the following distinguishing
physical characteristics: elemental analysis 53.71% (cal-
culated 3.80~); infrared absorption maxima peaks at 1720 and
10 1660 cm ; nmr, ~2.08 (-SCH3) ~2.79 (1-NCH3) ~3.58 (-COOCH3)
~3.76 (-~rOCH3); mass spectrum ion 841 m/e 855 (trans-
methylation) 500 (vindoline half).
The sulfate salt was prepared in the usual manner.
- Example 11
4-Desacetyl VLB C-3 ~N-2-mercaptoethyl)carboxamide and bis-
[4-desacetyl VLB C-3 (N-2-ethylcarboxamide)]disulfide
12 Gms of 4-desacetyl VLB C-3 carboxhydrazide were
converted to the azide by the procedure of Example 2. Next
68.2 g. of 2-mercaptoethylamine hydrochloxide were dissolved
in a minimum amount of water and the resulting acidic solution
made alkaline with concentrated aqueous sodium hydroxide.
2-Mercaptoethylamine free base thus formed, being insoluble
in the alkaline layer, separated and was extracted with
ethyl acetate. The aqueous layer was further extracted with
ether and with methylene dichloride. The organic extracts
were combined and the solvents removed therefrom by evap-
oration. The residual amine was dissolved in a minimal
quantity of methylene dichloride and added to a solution of
the azide, prepared as above, in 500 ml. of methylene
X-3754S -20-
_ . . , _, . ,,, _ . , _ . . , _
: - .
. . , ,
. .
- , .
108~179
dichloride. The reaction mixture was heated at 100C. for
five minutes and then cooled. 20 ml. of pyridine were added
and the mixture stirred overnight at room temperature.
Next, an excess of five percent aqueous ~odium bicarbonate
was added and the organic and aqueous layers separated. The
organic layer was washed three times with water and then
dried. The solvent as removed by evaporation in vacuo. The
residue, comprising a mixture of 4-desacetyl VLB C-3 (N-
2-mercaptoethyl)carboxamide and bis-[4-desacetyl VLB C-3
(N-2-ethylcarboxamide)]disulfide formed in the above reaction
was separated by chromatography over silica using a 1:1:1
methylene dichloride/ethyl acetate/methanol solvent mixture
containing 2 percent triethylamine. Two fractions were
obtained, one with a Rf=0.5 and second with Rf=0.25. Both
fractions had several virtually identical physical chemical
properties as follows: Molecular spectrum: m/e=827
~molecular ion + transmethylation), 486; Infrared spectrum:
peaks at 1730 and 1670 cm 1 (in chloroform); NMR virtually
superinposable.
The materials were differentiated as to structure
by the following criteria: The faster moving material
referred to as Rf=0.5 fraction had three titratable groups
(in 66 percent aqueous dimethylformamide) at 5.3, 7.38, and
11.8. The slower moving fraction, Rf=.25, had only two
titratable groups, these occurring at pK 5.2 and 7.5. The
Rf=.5 thus had an extra titratable group which would be the
sulfhydryl group of the C-3 amide- The sulfhydryl group is,
of cour~e, missing in the disulfide which was the Rf=.25
fraction. In addition, 13C NMR analysis indicated that both
X-3754S -21-
~. , .
. .
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108~179
fractions had peaks in the 173.6-173.8 region consistent
with a secondary amide carbon (vindesine--a C-3 carboxamide
and also a primary amide--has a peak at 176.7). Both samples
had many identical peaks and only two extraneous peaks the
Rf=.5 fraction at 42.3 and 24.2 and the Rf=.25 fraction at
38.0 and 37.6. An interpretation of these 13C NMR spectra
indicates that the former peaks are consistent with the
carbons in the unsubstituted side chain (mercaptoethyl
amide) and the latter is consistent with the same inter-
pretation except that the sulfur is substituted (as in adisulfide). Molecular weight by osmotic determination for
the Rf=.25 fraction was 1770 (calculated = 1624) again
consistent with a disulfide structure. Sulfide analysis for
the Rf=.5 fraction was 0.8 and for the Rf=.25 fraction O.
Example 12
4-Desacetyl VLB C-3 N-Acetaldehydecarboxamide
4-Desacetyl VLB C-3 N-2-dimethoxyethylcarboxamide
prepared by the procedure of Example 8 was dissolved in lN -
aqueous hydrochloric acid. The reaction mixture was allowed
to stand at room temperature for 4 hours and was then made
basic with 14N aqueous ammonium hydroxide. The amide, being
insoluble in the alkaline solution, separated and was ex-
tracted into methylene dichloride. The methylene dichloride
layer was separated, dried, and the solvent removed by
evaporation. Chromatography of the residual powder over
silica gel using a 3:1 ethyl acetate-ethanol solvent mixture
as the eluant yielded purified 4-desacetyl VLB C-3 N-acet-
aldehydecarboxamide having the following physical character-
istics: Rf=.43 (compared with Rf=1.50 for dimethylacetal).
X-3754S -22-
.,
.
- , : . . .
79
Infrared spectrum:peaks at 3420 cm 1 (N-H), 1735 cm 1
(carboxyl), 1675 cm 1 (carboxamide); nmr ~7.78 (triplet-
amide H) ~9.67 (aldehyde H).
The sulfatP salt was prepared by dissolving the
above amide in absolute ethanol and adjusting the pH of the
resulting solution to 5.0 with 2 percent sulfuric acid in
absolute ethanol. Evaporation of the solvent to dryness
yielded the sulfate salt as a tan amorphous powder.
Example 13
4-Desacetyl VLB C-3 N-2-acetylaminoethylcarboxamide
A solution was prepared with 1600 mg. of 4-des-
acetyl VLB C-3 N-2-aminoethylcarboxamide as provided by
Example 8 in 30 ml. of methylenedichloride to which was
added 5 ml. of pyridine. 200 mg. of acetic anhydride were
next added. The reaction vessel was sealed and the reaction
mixture stirred at ambient temperature for 24 hours. Methanol
was then added to react with excess anhydride. The volatile
constituents were removed by evaporation, and the residue,
comprising 4-desacetyl VLB C-3 N-2-acetylaminoethylcarbox-
amide, was dissolved in methylene dichloride. The methylene
dichloride layer was washed several times with dilute
aqueous ammonium hydroxide followed by a water wash. The
methylene dichloride layer was dried, and the methylene
dichloride evaporated therefrom. Chromatography of the
resulting residue on silica gel using a 1:1 ethyl acetate-
methanol solvent mixture yielded purified 4-desacetyl VLB
C-3 N-2-acetylaminoethylcarboxamide having the following
physical characteristics: Molecular spectrum (m/e) molecular
ion = 838 consistent for C47H62N6O8. Infrared spectrum;
30 peaks at 3429 cm 1 (N-H), 1735 cm 1 (carboxyl), 1670 cm
X-3754S -23-
_ _ . . . . . . . . .
Z17'~
(amide). nmr showed peaks at ~4.17 and 1.965 (acetyl hydrogenson ~-amino group).
Example 14
4-Desacetyl VLB C-3 N-2-Acrylyloxyethylcarboxamide
A solution was prepared containing 1100 mg. of
4-desacetyl VLB C-3 N-2-hydroxyethylcarboxamide from Example
6 in 50 ml. of benzene. 150 mg. of acrylyl chloride were
added. The reaction vessel was sealed and the reaction kept
at ambient temperature for 18 hours. The reaction vessel
was then opened and 200 mg. of acrylyl chloride were added.
The reaction vessel was again sealed and maintained at
ambient temperature for 10 additional hours. The reaction
vessel was then opened and the reaction mixture worked up by
contacting the organic solution with dilute ammonium hydroxide
to remove any excess acid chloride. The organic layer was
then dried and the solvents evaporated therefrom. Chroma-
tography of the residue comprising 4-desacetyl VLB N-2-
acrylyloxyethylcarboxamide with 3:1 ethyl acetate-ethanol
solvent mixture over silica gel yielded purified amide (27
mg.) as a tan amorphous powder with the following physical
characteristics: Molecular spectrum (m/e) molecular ion =
851 consistent with C48H51N5Og. Infrared spectrum peaks as
follows: 3427 cm 1, (NH), 1730 cm 1 (carboxyl), 1675 cm 1
(amide).
Example 15
4-Desacetyl VLB C-3 N-2-stearoyloxyethylcarboxamide
Using 2 g. of 4-desacetyl VLB C-3 N-2-hydroxy-
ethylcarboxamide following the procedure of Example 14 but
substituting stearic anhydride for acrylyl chloride, 4-desacetyl
VLB C-3 N-2-stearoyloxyethylcarboxamide was prepared having
X-3754S -24-
. ' ,
, , .
a molecular ion at 1063 consistent with C63H93N5O9 andothers peaks at 1004, 651, 355 and 154. The sulfate was
prepared in the usual manner using anhydrous ethanol. The
resulting sulfate salt (151 mg.) was a tan amorphous powder
insoluble in water.
Example 16
Preparation of 4-Desacetyl VLB N-~-methoxyethylcarboxamide
5.0 g. of 4-desacetyl VLB hydrazide was converted
to the azide as in Example 2. 10 ml. of ~-methoxyethylamine
was added to a methylene dichloride solution of the azide
and the reaction solution was stirred overnight at room
temperature under the conditions of Example 3. The solution
was washed once with water, dried over sodium sulfate,
filtered and evaporated. The residue was applied to a
silica column and eluted with methylene dichloride-methyl-
ethyl acetate (1:1:1) eluant. The appropriate fractions
were combined and the solvent removed in vacuo. Physical
characterictics were determined on 50 mg. material: infrared
maxima peaks at 3670, 3550, 3470, 1730, 1670, cm 1; nmr in
20 (CDC13) ~2.80, 3.34, 3.58, 3.77; mass spectrum in 811, m/e
825 (transmethylation), 780, 752, 571, 470, 353, 154, 124,
122; titer (66% DMF) pKa 5.35 and 7.38. The remaining
material was converted to the sulfate salt in the usual
manner. Yield was 1.8 g.
Example 17
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
X-3754S -25-
'
'79
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 2 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 compounds of this invention requires
10 extra care in the preparation of salts so as to avoid .
hydrolysis, transesterification and other reactions which
take place at high temperatures, at extremely acid pH's etc.
The compounds of this invention have been shown to
be active against transplanted mouse tumors in vivo. For
example, 4-desacetyl VLB C-3 N-2-butyroxyethylcarboxamide
sulfate, 4-desacetyl VLB C-3 N-2-mercaptoethyl)carboxamide
sulfate, 4-desacetyl VLB C-3 N-2-dimethyloxyethylcarboxamide
sulfate, 4-desacetyl VLB C-3 N-2-acetaldehydecarboxamide
sulfate and 4-desacetyl VLB C-3 N-2-acryloxyethylcarbox-
amide, as well as other compounds coming within the scope ofthe above formula, demonstrate such activity. In demon-
strating activity of the drugs of this invention against
these tumors, a protocol was used which involved the admin- ~ -
istration of the drug, usually by the intraperitoneal .
route, at a given dose level for 7-10 days after innoculation
with the tumor.
X-3754S -26-
- 10~3~179
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 inhibition
of tumor growth or percent prolongation of survival time.
ROS is an abbreviation for Ridgeway osteogenic sarcoma; GLS
for Gardner lymphosarcoma; B16 for melanoma; P388 for
lymphocytic leukemia; and P1533 leukemia. :
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Indefinite survivors were found with 4-desacetyl
VLB C-3 N-2-methoxyethylcarboxamide sulfate.
The compounds of this invention, as with the
marketed drugs leurocristine and VLB, 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 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.
As would be expected, the novel carboxamides
of this invention differ in their anti-tumor spectrum from
VLB, leurocristine and leurosine, as well as from the C-4
N,N-dialkylglycyl esters of VLB in the same way that the
anti-tumor spectra of those compounds differ among them-
selves, some being more effective against certain tumors or
classes of tumors and less effective against others.
However, in utilizing a compound of this invention clinically,
the clinical physician would administer them initially by
the same route in the same vehicle and against the same
types of tumors as for clinical use of leurocristine and
VLB. Differences in dosage level would, of course, be based
on relative activity between leurocristine and the new drug
in the same experimental tumor in mice. The amides of this
invention apparently show decreased neurotoxicity compared
with leurocristine.
X-3754S -31-
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1082179
In utilizing the novel carboxamides of this
invention 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 formed with a non-
toxic acid is mixed with starch or other excipient and the
mixture placed in telescoping gelatin capsules each containing
from 7.5-50 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. For this purpose, isotonic solutions are employed
containing 1-10 mg./ml. of a salt of an indole-dihydroindole-
carboxamide of formula I. 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 depending 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.
X-3754S -32-
. . _ , _ .. , . , _
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