CA1082179A - Amide derivatives of vlb, leurosidine, leurocristine and related dimeric alkaloids - Google Patents

Abstract

Abstract This invention relates to novel C-3 carboxamide derivatives of 4-desacetyl dimeric indole-dihydroindole compounds. They inhibit the growth of tumors and pro-long the life of their host animals.

Description

~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 .

.
:, :

~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).

.. . .

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_ . _ _ _ . . . . . .. . . .

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

Il R5 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 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 , -, .
:

,~' ~,'' ' ~'"", _ : . , 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 ~

_ _ . . . . . . . . . _: _ .

- . . : ,: ~ - :

~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.

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, _ .

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 _ . . . . .. . . . . . .

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 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.

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 ..

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 :

.. . ..

- .- ,' : . ,: ,. ~ ' ' : .- . ~ , ' : ' '' 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 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-'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.
.;

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).
.

, 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.

. , , ' ' ~ '. ' . ' , ~ :

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 , .

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 _ . . , _, . ,,, _ . , _ . . , _ : - .
. . , , . .
- , .

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 ~. , .

. .
_.
- : . , . , .. . . . .. :
- ''' '. ' . . .. .' . ' , ., : ~ . , .. ': . .. - , :

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).

., .
- , : . . .

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 _ _ . . . . . . . . .

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 . ' , , , .

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 ' '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.

- 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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108Z1~9 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.

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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.

. . _ , _ .. , . , _ .

Claims (21)

The embodiment of the invention for which an exclusive property or privilege is claimed are as follows:

1. A process of preparing a dimeric indole-dihydroindolecarboxamide of the formula Formula I

wherein R4 is wherein m is 1, 2 or 3 and R6 is -CHO, alkyl, alkenyl, -O-C1-C3 alkyl, alkyl, or -S-Y wherein Y is H, C1-C3 alkyl or a bond, said bond joining 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-C1-C3 alkyl; and R5 is H;
one of R2 and R3 is H or -OH and the other is -C2H5; R1 is -CH3 or -CHO; and its pharma-ceutically acceptable salts comprising reacting a dimeric indole-dihydroindole car-boxazide of the formula Formula II

with an amine R4R5NH wherein R1 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.

2. The process of claim 1 for preparing a dimeric indole-dihydroindole carboxamide of Formula I as defined in claim 1 wherein the reaction takes place in methylene dichloride.

3. A compound of formula I as defined in claim 1 or a phar-maceutically acceptable salt thereof, when prepared by the process of claim 1 or 2 or by an obvious chemical equivalent thereof.

4. A process according to claim 1 for preparing a dimeric indole-dihydroindole carboxamide of Formula I as defined in claim 1 wherein the reaction takes place at room temperature.

5. A compound of Formula I as defined in claim 1 or a phar-maceutically acceptable salt thereof, when prepared by the process of claim 4 or by an obvious chemical equivalent thereof.

6. A process for preparing a dimeric indole-dihydro-indolecarboxamide of Formula I as defined in claim 1 wherein R4 is wherein m is 1, 2 or 3, R6 is -S-Y wherein Y

is a bond, R7 is H and R5 is H, which comprises reacting a dimeric indoledihydroindolecarboxazide of Formula II as defined in claim 1 with an amine of the formula H2N-C1-C3 alkayl-S-H
in the presence of a base.

7. A dimeric indole-dihydroindolecarboxamide of Formula I

as defined in claim 1 wherein R4 is wherein m is 1, 2 or 3, R is -S-Y wherein Y is a bond, R7 is H
and R5 is H, when prepared by the process of claim 6 or by an obvious chemical equivalent thereof.

8. A process for preparing bis-[4-desacetyl VLB
(vincaleukoblastine) C-3 (N-2-ethylcarboxamide)]disulfide which comprises reacting 4-desacetyl VLB C-3 carboxazide with 2-aminoethylmercaptan in the presence of a base.

9. Bis-[(4-desacetyl VLB C-3 (N 2-ethylcarboxamide)]
disulfide, when prepared by the process of claim 8 or by an obvious chemical equivalent thereof.

10. A process for preparing 4-desacetyl VLB C-3 N-.beta.-butyryloxyethyl carboxamide which comprises reacting 4-desacetyl VLB C-3 carboxazide with butyryloxyethylamine.

11. 4-Desacetyl VLB C-3 N-.beta.-butyryloxyethyl carboxazide, when prepared by the process of claim 10 or by an obvious chemical equivalent thereof.

12. A process for preparing 4-desacetyl VLB C-3 N-2-dimethoxyethylcarboxamide which comprises reacting 4-desacetyl VLB C-3 carboxazide with 2-dimethoxyethylamine.

13. 4-Desacetyl VLB C-3 N-2-dimethoxyethylcar-boxamide, when prepared by the process of claim 12 or by an obvious chemical equivalent thereof.

14. A process for preparing 4-desacetyl VLB C-3 N-2-methylmercaptoethylcarboxamide which comprises reacting 4-desacetyl VLB C-3 carboxazide with 2-methylmercaptoethyl-amine.

15. 4-Desacetyl VLB C-3 N-2-methylmercaptoethyl-carboxamide, when prepared by the process of claim 14 or by an obvious chemical equivalent thereof.

16. A process for preparing 4-desacetyl VLB C-3 N-(3-methylmercaptopropyl)carboxamide which comprises reacting 4-desacetyl VLB C-3 carboxazide with 3-methylmercaptopropyl-amine.

17. 4-Desacetyl VLB C-3 N-(3-methylmercapto-propyl)carboxamide, when prepared by the process of claim 16 or by an obvious chemical equivalent thereof.

18. A process for preparing 4-desacetyl VLB C-3 N-2-mercaptoethylcarboxamide which comprises reacting 4-desacetyl VLB C-3 carboxazide with 2-aminoethylmercaptan.

19. 4-Desacetyl VLB C-3 N-2-mercaptoethylcarbox-amide, when prepared by the process of claim 18 or by an obvious chemical equivalent thereof.

20. A process for preparing 4-desacetyl VLB C-3 N-.beta.-methoxyethylcarboxamide which comprises reacting 4-desacetyl VLB C-3 carboxazide with .beta.-methoxyethylamine.

21. 4-Desacetyl VLB C-3 N-.alpha.-methoxyethylcarbox-amide, when prepared by the process of claim 20 or by an obvious chemical equivalent thereof.