IE45558B1 - C3-carboxamido derivatives of vinblastine and related alkaloids - Google Patents

Abstract

A dimeric indole-dihydroindolecarboxamide of the formula in which the substituents are defined in Claim 1, is prepared. This compound is obtained by reacting a corresponding dimeric indole-dihydroindolecarboxazide with an amine in order to introduce the group -NR<4>R<5>. The resulting product is isolated as a free amine or as a pharmaceutically tolerated salt. The resulting compounds can be used as antineoplastic active compounds.

Description

This invention relates to 4,- Desacetyl - C3 carboxamide derivatives of VLB, leurosidine, leurocristine and 1 cdaanethyl-l-formyl leurosidine which are useful as anti-neoplastic agents.

Several naturally-occurring alkaloids obtainable 5 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), leurosidine (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 treatment of malignancies, particularly the leukemias and related diseases in humans. Of these marketed compounds, leurocristine is a most active and useful agent in the treatment of leukemias but is also the least abundant of the antineoplastic alkaloids of Vinca roBea.

Chemical modification of the Vinca alkaloids has been.rather limited. Zn the first place, the molecular structures involved are extremely complex end ehemioal reactions which affect a specific function of the moleoule are difficult to develop. Secondly, alkaloids lacking desirable chemo-therapeutic properties have been recovered from Vinoa rosea fractions, and a determination of their *2™ 455sg structures has led to the conclusion that these compounds are closely related to the active alkaloids. Thus, antineoplastic 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 correspondingly 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 alkanoyl 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 Ν,Ν-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.

This 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).

This invention provides a number of compounds which show activity against 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 agents to which resistance has developed.

The present invention provides a dimeric indol 10 dihydroindole carboxamide of the formula 12' 13' ll H CH® I wherein R4 is -(CH ) where m is 1, 2 or 3 and either R6 is -CHO, -O-C-^1-Cl7) alkyl, -O-C-^-C.) alkenyl, -O^-C^ 0 0 alkyl, -NH-C-^-Cg') alkyl, or -S-Y wherein Y is 0 4SBS8 H, - C3 alkyl or a bond, said bond joining the sulfur atoms in two moieties of Formula I wherein Y is a bond, 6 7 and R is H; or R and R each are - O-iC^-Cj) alkyl; and r5 is H; one of R2 and R2 is H or -OH and the other is -CjHg,· R^ is -CH3 or -CHO; and its pharmaceutically acceptable acid addition salts. 7 Preferred Compounds are those wherein m is 1 and R and R are each -O-(Cj-C3)alkyl; m is 1 or 2, R6 is -OCH3, -O-C-(C1-C17)alkyl or -0~C~ (Cj-C·?) alkenyl and R is H; or m is 1, 2 or 3, Rg is -NH-C-(Cj-C3)alkyl or -S-Y il wherein Y is H or C^-Cj alkyl, and R is H.

The present Invention also provides a pharmaceutical composition for inhibiting a tumor or prolonging life of a host mammal comprising an inert carrier associated with a dimeric indoledihydroindole carboxamide of Formula 1 or a pharmaceutically acceptable salt thereof.

The present invention also provides a method of inhibiting a tumor or prolonging the life of a non-hu»an host mammal by the administration to a mammal host of a tumor of an anti20 tumar effective dose of a dimeric indoledihydroindole carboxamide compound of Formula I as defined above.

The present invention further provides a process of preparing a dimeric indole-dihydroindolecarboxamide of the invention and its p^hrmaceutically acceptable acid addition salts comprising reacting a dimeric indole-dihydroindolecarboxazide of the formula 43533 13*1 ν'/’Χ r3 Ρ 8·Γ 3'3 , ι! 16·11 I ι ±Ζ «--ς _ θ _ α Formula IX 9/\ -----Νλ \7 14 IT ι iifi /\3 \12/ν/ 5γ—-03^ CH, - Ο _ _Γ16 I 1 12 4 I-ΟΗ ί CH, ι C - Ν, with an amine R4R5NH wherein R1 to are as defined above with the proviso that θ Y in R is other than a bond, and recovering the product of formula I in the form of the free amine or a pharmaceutically acceptable salt.

In the case when R is -^’mHC K 7 Rz vherein m is 1, 2 or 3, R is -S-Y, Y being a bond joining the S atoms of tv» moieties of formula I, and R and R are both H, a dimeric indole dihydro indole carbcxazide of formula II may be reacted with an amine of formula HjNfCg - Cj)alkyl -S - H in the presence of a base.

The carboxamides of this invention comprise the carboxamides of vincaleukoblastine, leurocristine, leurosidine and 1-desmsthyl-l-fornylleurosidins and their deoxy A and B analogs and the pharmaceuticallyacceptable salts o£ the above bases.

Vincaleukoblastine, leurocristine and leurosidine are found naturally. The 1-desmethyl-l-formyl-leurosidine and seme of the deoxy Ά and B although not found in nature have been synthesized. Vincaleukoblastine and leurocristine are clinically used for the treatment of neoplastic diseases in humans. 4S5S8 Compounds can be described generically as derivatives of vincaleukoblastine (vinblastine or VLB) when R^ is CH3, 3 R is OH and R is -CH2CH3· In derivatives of leurocristine (vincristine or VCR) R1 is CHO, R2 is OH and R3 is -C2Hg. 2 In derivatives of leurosidine R is CHj, R is CH^CHj and R3 is OH. In derivatives of 1 - desmethyl 1 - formyl leurosidine R1 is CHO, R2 is CH2CH3 and R3 is OH. The deoxy analogs of the above compounds in which R 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 C^ - C3 alkyl as employed hereinabove means the methyl, ethyl, n-propyl and iso-propyl groups. - 7 4 5 55 0 The term C^-C^-alkyl-CO means an alkanoyl group derived from alkanoic acids having from 2-18 carbon atoms; e.g. acetyl, propionyl, isobutyryl, stearyl, palmitoyl, lauryl, myristoyl, caproyl (cg), iso-valeroyl, oapryloyl (Cg) and capryl (C10). The term C2 - C? -alkenyl -CO means an olefinieally unsaturated acid group having from 3 to 8 carbons; i.e., acrylyl, crotonyl, methacrylyl, allylacetyl, vinylacetyl, tiglyl, 2-methyl-2-hexenoyl and 2-octenoyl.

Illustrative groups which are the nitrogen con10 taining moiety of the c3~carboxamido group in the various modified dimeric indole-dihydroindole alkaloids represented by the above formula include: acetaldehydeamide, 2-methoxypropylamide, 2acetyloxyethylamide, 2-butyryloxyethylamide, 2-ethoxy15 efchylamide,2.,2-dimethoxyethylamide, 2-acrylyloxyethyl, 2-mercaptoethylamide·, 3-methylmercaptopropylamide, 2-n-propylmercaptopropylamide, and 4-acetylamino-n-butylamide.

Non-toxic acids useful for forming pharmaoeutically20 acceptable acid addition salts of the amine bases include salts derived from inorganic acids such as: hydrochloric acid, nitric aeid, phosphoric acid, sulfuric acid, hydrobromic acid, hydriodic acid, nitrous acid > and phosphorus acid, as well as salts of hon-toxic organic acids including aliphatic mono and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and alkandioates, aromatic acids, aliphatic and aromatic eulfonic acids.

Such pharmaceutically-acceptable salts thus include the sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, -8V 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-l,4-dioate, hexyne-l,6-dioate, benzoate, chlorobenzoate, 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 and naphthalene-2-sulfonate salts. illustrative compounds coming within the scope of this invention include: 4-desacetyl deoxy VLB A C-3 N-2-mercaptoethylcarboxamide 4-desacetyl deoxy VLB 8 C-3 N-2-mercaptoethylcarboxamide 4-desacetyl deoxy VCR A C-3 N-2-mercaptoethylcarboxaraide 4-desacetyl deoxy VCR B C-3 N-2-meroaptoethylcarboxamide 4-desacetyl deoxy VLB A C-3 N-2-methylmeroap25 toethylcarboxamide 4-desaoetyl deoxy VLB B C-3 N-2-methylmercaptoethylcarboxamide 4-desacetyl deoxy VCR A C-3 N-2-methylmercaptoethylcarboxamide 958 4-desacetyl deoxy VCR B toethyloarboxamide 4-desacetyl deoxy VLB A carboxamide 4-desacetyl deoxy VLB B carboxamide C-3 N-2-methylmercapC-3 N-2-methoxyethylC-3 N-2-methoxyethyl4-desacetyl carboxamide' 4-desacetyl carboxamide deoxy VCR A deoxy VCR B C-3 N-2-methoxyethylC-3 N-2-methoxyethyl4-desacetyl VCR C-3 N-2-mercaptoethylcarboxamide 4-desacetyl VCR C-3 N-2-methylmercaptoethylcarboxamide 4-desacetyl leurosidine C-3 N-2-mercaptoethylcarboxamide 4-desacetyl leurosidine C-3 N-2-methylmercaptoethylcarboxamide 4-desacetyl-l-desmethyl-l-formyl leurosidine C-3 N-2-mercaptoethylcarboxamide 4-desacetyl-1-desmethyl-l-formyl leurosidine C-3 N-2-methylmercaptoethylearboxamide 4-desacetyl VCR C-3 N-2-methoxyethyloarboxamide 4-desacetyl leurosidine C-3 N-2-methoxyethylcarboxamide.

Compounds of this invention may be prepared by transforming the carbomethoxyl group at C-3 of certain known indole-dihydroindole alkaloids obtained either from plants or by partial synthesis into a derivative of a carboxamide. Not all of these derivatives are ordinarily prepared by a single -1045558 process. For example, the compounds of this invention of formula I can be prepared as follows: Treatment of VLB, leurocristine, leurosidine, 1-desmethyl-l-formyl-leurosidine or their deoxy analogs with hydrazine yields the corre5 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 earboxhydrazide 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 may be separated by chromatography. Generally, the reaction would be carried out starting with the 4-desacetyl derivative of VLB, leurocristine, leurosidine or the 1-desmethyl-l-formyl leurosidine.

The C-4-desacetyl C-3 earboxhydrazide derivatives may be 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 a2ide-araine transformation follows the procedure originated by Stoll and Huffman, Helv. Chim. Acta., 26, 944 (1943) -- see also ϋ. S. Patents 2,090,429 and 2,090,430. In the examples of this invention the reaction was carried out at 'rem temperature 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-C^-Cj alkyl)2 by acidic hydrolysis. Compounds in which the amide group contains an -1145358 ester function such as in the group NH-(CHj) -OAc, wherein n and Ac are as defined above, are preferably prepared by esterifying an hydroxy amide containing the group NH(CH2)n0H with a suitable acid anhydride, Ac20, wherein Ac is alkyl-CO or C^-C^-alkenyl-CO. Similarly, compounds in which g R is alk-X wherein X is NH-CO-C^-Calkyl may be prepared by acylating, with an acid anhydride, an aminoalkylamide group of the structure NH-alk-NH.. The azide reacts with NH.(CH) SH & 2 n 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 corresponding 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 involving 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 replacing 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 desoarbomethoxy C-3 carboxamide. -124S558 The compounds of this invention, in the form of their free bases, including both carboxamides, are white or tan-colored amorphous solids. It i3 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.

Bie preparation, of' intemediate conpounds and conpounds of this invention is nore fully illustrated in the following specific examples: Example 1 4-Desacetyl VLB C-3 carboxhydrazide 4-Desacatyl VLB was heated in anhydrous ethanol with an excess of anhydrous hydrazine in a sealed reaction vessel at about 60°C. 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 carboxhydrazide, was taken up in methylenechloride, the methylenechloride 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 chromatographic fractions contained unreacted 4-desacetyl VLB.

Further fractions were found to contain 4-desacetyl 18’descarbomethoxy VLB C-3 carboxhydrazide previously described by Neuse et al., Tetrahedron Letters, 1968, 783. The next fractions, found to contain 4-desacetyl VLB C-3 carboxhydrazide by thin layer chromatography, were combined, and -134-5558 the solvents evaporated therefrom in vacuo. The resulting solid melted at 219-220°C. with decomposition. 4-Desacetyl VLB C-3 carboxyhydrazide thus prepared had a carbomethoxy 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 attributable to the hydrazide function. Molecular weight by mass spectrography was 768 in agreement with the theoretical value calculated for C^HggNgO?. The nmr spectrum contained the prominent resonance at δ 3.6 representing the methyl group of the C-18 carbomethoxy function.

Example 2 (Intermediate) 4rDesacetyl VLB C-3 carboxazide A solution of 678 mg. of 4-desacetyl VLB C-3 carboxhydrazide (from Example 1) was prepared in 15 ml. of anhydrous methanol. About 50 ml. of IN aqueous hydrochloric acid were added, and the resulting solution cooled to about 0°C. Approximately 140 mg. of sodium nitrite were then added, and the resulting reaotion mixture stirred for 10 minutes while maintaining the temperature at about 0°C. 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 diehloride. 4-Desacetyl VLB C-3 carboxazide formed in the above reaction passed into the methylene diehloride.

While ordinarily the methylene diehloride solution of 4-desacetyl vinblastine C-3 carboxazide is used without further purification, an aliquot was treated as follows in -14ίο order to characterize the azide: Evaporation of the methylene 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 following distinguishing physical characteristics: ultraviolet spectrum lambda =269 mu. (epsilon 16,700); shoulder at max about 290 mu; 309 mu. (epsilon = 7,100); infrared absorption -1 (earboxhydrazide) was absent, while -1 maximum at 1690 cm. the maximum at 1730 cm.’2 was not affected. Furthermore, a sharply defined maximum at 2135 cm.-·1· was noted characteristic of the carboxazide function. The mass spectrogram revealed a molecular ion m/e = 708 showing a loss of 71 mass units (H, CONg) from the molecular weight calculated for G43H53N7O7 779.

* Preparative 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 earboxhydrazide. 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 chromatographed 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-ethyl30 1545558 carboxamide as determined by thin-layer chromatography 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 charac5 teristios: molecular ion spectrum, m/e = 781 (corresponding to C45H59N5O7); infrared spectrum; absorption maxima at 1730 cm.-}· (ester), 1670 cm.-1 (amide), 4320 cm.-1 (N-H amide), nmr. 61.18 (triplet-|3-niethyl of ethyl amide group), 63.28 (quartet-a-methylene of ethyl amide group), 63.59 (singlet10 methyl ester), 4-desacetyl VLB C-3 N-ethylcarboxamide sulfate was prepared by dissolving the above amorphous powder in anhydrous ethanol and adjusting the pH to about 4.0 with 2 percent sulfuric acid in anhydrous ethanol. Evaporation of the solvent in vacuo yielded a water-soluble tan powder comprising 4-desacetyl VLB C-3 N-ethylcarboxamide sulfate. -1645558 Example 4 4-Desacetyl VLB C-3 N-p-butyryloxyethylcarboxamida Following the procedure of Example 3, 4-desacetyl VLB C-3 Ν-β-butyryloxyethylcarboxamide was prepared with the following physical characteristics: infrared spectrum; -l -1 -1 peaks at 3420 cm. , 1735 cm. , and 1680 cm. ; molecular spectrum; molecular ion, M+ = 867 consistent with empirical formula C4'gHg5N5og.

Example 5’(Intermediate) IO1 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-desaeetyl 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 (COM). Molecular ion M+ =» 797 consistent with empirical formula C45H5gN5Og. 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 C47Hg^NgOg; nmr spectrum consistent with structure, particularly with added peak at 51.91 (acetyImethyl). -1745 5 58 Example 7(Intermediate) 4-Desacetyl VLB C-3 N-(2-aminoethyl)carboxamide 4-Desacetyl VLB C-3 N-(2-aminoethyl) carboxamide was prepared by the procedure of preparative Example 3 and was a tan amcaphous powder with the following physical characteristics: pk = a 6.8, 9.0, 4.6. Infrared spectrum; peaks at 3420 cm.-'*' (NH), 1730 cm.-1 (COO), 1670 cm.-1 (CON; molecular spectrum, molecular ion, M+ = 796 consistent with empirical formula C45H60N6°7 A su^fate salt was also prepared as a tan amorphous powder.

Example 8 4-Desa’cetyl VLB C-3 N-2-dimethoxyethylcarboxamide Following the procedure of Example 3, 4-desacetyl VLB C-3 N-2,2-dimethoxyethylcarboxamide was prepared by reacting 4-desa.cetyl VLB C-3 carboxazide with 2,2-dimethoxyethylamine. The amide thus produced had the following physical characteristics: infrared absorption maxima at 1665 cm 1 (amide) and at 1730-^ (carboxyl); molecular ion spectrums (m/e), molecular ion at 841 other peaks at 782, 651, 500, 355, and 154; NMR 64.42 (triplet) 63.41 (doublet C^-2H), 63.36-3.45 (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. -184555 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. IN hydrochloric acid and 180 mg. sodium nitrite in anhydrous methanol. A solution of the azide in methylene diehloride was reacted with 4 g. methylmercaptoethylamine after the procedure of Example 3. The reaction mixture was stirred overnight at 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-methyimercaptoethyl)carboxamide were combined and the solvent evaporated in vacuo. The yield was 540 mg. The carboxamide had the following distinctive physical characteristics: elemental analysis S found 3.47% (3.87% calculated) ; infrared absorption maxima peaks at 1740 and 1675 cm”1,· nmr 62.12 (-SCHg) 62.80 (-NCH3) 63.58 (~COOCH3) 63.78 (ArOCHj); 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 converted to the azide with 200 ml. IN hydrochloric acid and 400 mg. sodium nitrite with the procedure of Example 2. 5 g. 3-Methylmer captopropylamine was added to a solution of the azide in methylenediohloride and the solution stirred overnight at 19' 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 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% (calculated 3.80%); infrared absorption maxima peaks at 1720 and 1660 cm-1; nmr, 62.08 (-SCH3) 62.79 (1-NCH3) 63.58 (-COOCH3) 63.76 (-ArOCH3); mass spectrum ion 841 m/e 855 (transmethylation) 500 (vindoline half).

The sulfate salt was prepared in the usual manner. Example 11 4-Desacetyl VLB C-3 iH^t-mercaptoethyl)carboxamide and bis[4-desacetyl VLB C-3 N-fZ-ethylcarboxamide)]disulfide 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 hydrochloride 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 evaporation. 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 -2045558 dichloride. The reaction mixture was heated at 100°C. 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 sodium bicarbonate was added and the organic and aqueous layers separated. The organic layer was washed three times with water and then dried. The solvent was removed by evaporation in vacuo. The residue, comprising a mixture of 4-desacetyl VLB C-3 (N2-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 lsl:1 methylene dichloride/ethyl acetate/methanol solvent mixture containing 2 percent triethylamine. Two fractions were obtained, one with Rf=0.5 and second with R^=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 (in chloroform); NMR virtually super imposable.

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, R£=.25, had only two titratabie groups, these occurring at pK 5.2 and 7.5, The Rjj=.5 thus had an extra titratable group which would be the sulfhydryl group of the C-3 amide. The sulhydryl group is, of course, missing in the disulfide which was the Rf=.25 13 fraction. In addition, C NMR analysis indicated that both 21fractions 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 R^=.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 interpretation except that the sulfur is substituted (as in a disulfide). Molecular weight by osmotic determination for the Rf=.25 fraction was 1770 (calculated = 1624) again consistent with a disulfide structure. Sulfide analysis for Rj=.5 fraction was 0.8 and for the Rf=.25 fraction 0. 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 IN 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 extracted 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-acetaldehydecarboxamide having the following physical characteristics: Rf=.43 (compared with Rj=1.50 for dimethylacetal). 224SSS3 Infrared spectrum speaks at 3420 cm”1· (N-H) , 1735 cm 1 (carboxyl), 1675 cm-1 (carboxamide); nmr 67.78 (tripletamide H) 69.67 (aldehyde H).

The sulfate salt was prepared by dissolving the 5 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 sulfata salt as a tan amorphous powder.

Example 13 4-Desacetyl VLB C-3 N-^-aoetylaminoethy^carboxamide A solution was prepared with 1600 mg. of 4-desaoetyl VLB C-3 N^S-aminoethy^arboxamide as provided by Example 7 in 30 ml. of methylenediohloride to which was added 5 ml. of pyridine. 200 mg. of acetic anhydride were next added. The reaction vessel was sealed and the reaotion 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-aoetylaminoethylcarbox20 . 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 lsl ethyl acetatemethanol solvent mixture yielded purified 4-desacetyl VLB C-3 N-2-acetylaminoethylcarboxamide having the following physical characteristics: Molecular spectrum (m/e) molecular ion a 838 consistent for C47H62NgO8. Infrared spectrum; peaks at 3429 cm1 (N-H), 1735 cm1 (carboxyl), 1670 cm^· -234SS5S (amide), nmr showed peaks at 54.17 and 1.965 (acetyl hydrogens on β-amino group).

Example 14 4-Desacetyl VLB C-3 N-2-Acrylyloxyethylcarboxamide 5 A solution was prepared containing 1100 mg. of 4-desacetyl VLB C-3 N-^2-hydroxyethy^oarboxamide from Example 5 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. Chromatography of the residue comprising 4-desacetyl VLB C-3 N-2acrylyloxyethylcarboxamide 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 C4gH5jN5Og. Infrared spectrum peaks as follows: 3427 cm-1, (NH), 1730 cm”1 (carboxyl), 1675 cm1 (amide).

Example 15 4-Desaoetyl VLB C-3 N-^-stearoyloxyethy^carboxamide Using 2 g. of 4-desacetyl VLB C-3 N-2-hydroxyethylcarboxamide following the procedure of Example 14 but substituting stearic anhydride for acrylyl chloride, 4-desaoetyl VLB C-3 N-2-stearoyloxyethylcarboxamide was prepared having -2445558 a molecular ion at 1063 consistent with C63H93N5°9 and others 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^&-mathoxyethy3)3arboxaraide .0 g. of 4-desacetyl VLB hydrazide was converted to the azide as in Example 2. 10 ml. of B-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-methylethyl acetate (ltltl) 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 (CDClj) 62.80, 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 ..and nitrate as well as salts with organic anions such as acetate, chloroacefcate, trichloroacetate, benzoate, alkyl or aryl -254SS5Q sulfonates 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 extra care in the preparation of salts so as to avoid hydrolysis, transesterification and other reactions which take place for exanple at high temperatures and at extremely acid pH's.

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 03 N-2,2-dimethyloxyethylcarbaxamide sulfate, 4-desacetyl VLB C-3 N-2-acetaldehydecarboxamide sulfate and 4-desacetyl VLB C-3 N-2-acryloxyethylcarbox20 amide, as well as other compounds coming within the scope of the above formula, demonstrate such activity. In demonstrating activity of the drugs of this invention 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 innoculation with the tumor. -2645558 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. Xn 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. -27- fi ο μ·μ fi μ Φ ·Η οχι Μ·Η ΟΛ A fi Η Ο «τ ο ω Η ι I m γ* CM Μ* ο ο ο ο > ΗΗΓΟ I I I σνσκ ιο ρ- Μ* co ο ι Ο CM Η Γο σ «-ι Ο I Ο ΡΗ ω ο ο r-l tn OOO O o « rd rd rd o rd d r-H Ό X XXX X x X Xo X co o in o O CM ΙΛ · • · CM rd CM rd · CM • • o rd Ο · a O • X bi O 1 1 o X O X 1 o Ai ι m in tn ι 1 in in X rd O rd rd CM CM rd CM O rd O Cn El O O OOO O O O o o o Table στ CO co CO w w tn cn ω cn cn co oo O Hl nJ O rd O J Q 1-4 tJ os υ 0 tf 04 « o Pi o 0 04 Φ μ Η » tn Φ *α φ +> Φ μ rd η CQ Φ gs >,1 »0 μ Φ •rl 3 μ g Λ « ι μ I ft CM Η CS Φ 0 1 fi 110 Λ 22 tn13 E μ <ΰ CO 0 <η φ cn ib 1 »0 1 X 1 rdu e U 0 0 S>t 4} afi 03 § CQ W CQ μ •rl μ 0 'ϋ £ ΦΛ g cn Β □ μ 3 1 X φ φ 0 O 0 in ο Q- Λ φ S Λ Μ - Ω co 0 J ιΒ 1 (J > 0 «Μ* Ο go ^•s £3 Φ H Ο >1 (Β Λ in +> ω u ?5? o g ϋ ι—I iM >< >ιΛ +J L 0 Φ 0 >t Φ IB 5?+) ω ο Φ φ +> Ή β «rl ι ο o v ib tn 28' β P-H δϋ ϋ Λ P-H Φ «fi fa fi H © © H I CO *3* «*· ω © © in m* h vo m i oti co rc o vo cm *4» in H rd CM o o r*i I Φ «Ρ •H fi H cn tw Φ Ό c •H H > β to cm «Φ Table 1 (Continued) cn ti Ό & s E o OY 1 I r* cn 1 r- i> cn X cn X X X „ © © 5j* X i-d «* rd · u* m O · cn X © X 1 o H · I tn 1 1 ! 51* H -M* © CM CO VO • · a · • · · © © o o © © © σ> r* c o I CM © O rd X in r* « © co X σ» eo ¢0 ω »cn co cn cn w eo co □ Π tfi tfi r-ί CO fa U fa 0 ϋ fa fa © cn VD 00 tfi H cn ϋ fa fa Φ •ri s s 1*1 f—I I N ΟΛ 4J Μ Φ £ Η H >t 5h dJrd Φ >1 Ο Μ Φ ti □ 4J CO ti ti φ I CM I “ a 4} —r p •H cn ιμ •Q 1 rd μ U fi ti co o fa Ή rd tfi Ό φ N cn |5 1 «fi 1 <·» a +> u rt Φ φ N3 +i ·Η 0 § O 3 4J ffl XrH cn Q nJ oa in Q H „ t ra a> » oa ·—' r-i flj I NW tl) Xi H •rl JJ 3 te φ w 294555s C Si 0 ίβ 4M Q fi -P »*-* Φ·Η OA o to*H o ΙΛ DA H Γ- Oi fi I I H f'4 cn to cn cn co m (Continued) © • 0) pH Γ- 5h a «0 X OJ to if) © Φ κ • O • o Λ O pH pH pH •P ά ( in X ι X OH J4 Γ4 0 pH in 04 © ta • · fi β © o O pH 0 •to 4-) rt tft ω φ υ to Φ 4) rH ΟΗ rt Φ to pH Q η .fi ω ω ω ω >1 <8 3 u ο Ω (ΰ Eh u Pi ϋ « Ό ώ ο Ό Ό '3 ta (3 C X •Η 0 > Λ •rl Μ rH υ to Η to m >, Φ fQ 01 -fi I 4-) ► CJ 4J •pi u rt •rl ffl c 04 > ffl 0 ?3 0i pH to g c X U fi fi 0 > ω ω Λ pH (3 to pH QJ SnH rt >ιΌ 4J 0 Sn 0 4-i *rH •H Ό ω -u Φ S fi C 0 (U (U fQ φ υ rt rH 3 nJ ο -μ I 4) rt X OH 0 tn io nj □ rt W 0 0) η 0 1 Ή 04 Φ Λ Π3 S D N H « pH ο to fi 0 IIS U S ι rt H ο B W > » xf o * •3045558 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 typioal 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 Ν,Ν-dialkylglycyl esters of VLB in the same way that the anti-tumor spectra of those compounds differ among themselves, 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. 3l· 4S558 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 nontoxic 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-dihydroindole15 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 (7)

CLAIMS:

1. A compound of the formula Formula - CHs-CHa . / , wherein R s is -(CH ) —HGf „ wherein m is 1, 2 or 3 and R° is -CHO, 2. ID \^7 -O-C-CCj-C,^) alkyl, -O-C-^-C^ alkenyl, -O-^-Cg) 0 0 alkyl, -NH-C-fc^-C^ alkyl, or -S-Y wherein Y is 0 H, C^-Cg alkyl or a bond, said bond joining the sulfur atoms in two moieties of Formula I ~1 fi wherein Y is a bond, and R is H; or R and *7 fi r' each are -O-iC^-Cg) alkyl; R is H; -3343553 2 3 one of R and R is H or -OH and the other is ~C 2 Hg; and R 1 is -CH^ or -CHO; and its pharmaceutically acceptable acid addition salts.

2. A compound according to claim 1 wherein R 1 is 2 3 5 CH 3 , R is -OH, R is -C 2 H 5 and lts pharmaceutically acceptable acid addition salts.

3. A compound according to claim 1 wherein R·*· . 2 3 is -CH 3 , R is “CjHg and R is -OH and its pharmaceutically acceptable acid addition salt. 10 4. A compound according to claim 1 wherein S'*· 2 3 is -CHO, R is -OH and R is -C 2 Hg an< ^ it Pharmaceutically acceptable acid addition salts. 5. A compound according to claim 1 wherein R 1 2 3 is -CHO, R is and R is -OH and its pharmaceutically 15 acceptable acid:addition salts. 6. A compound according to claim 1 wherein R 1 is 2 . 3 CH 3 , R is H and R is -CgHg and lts pharmaceutically acceptable acid addition salts. 7. A compound according to claim 1 wherein R 1 2 3 20 is -CHg, R is “CjHg and R is H and its pharmaceutically acceptable acid addition salts. 8. A compound according to claim 1 wherein R 1 is 2 . 3 CHO, R is H and R is -C 2 H 5 and lts pharmaceutically acceptable acid addition salts. 25 9. A compound according to claim 1 wherein R is 2 · 3 CHO, R is “ C 2 H 5 and R is H and its pharmaceutically acceptable acid addition salts. 344S558 10. 4-desacetyl VLB C-3 N-B-butyryloxyethylcarboxamide. 11. 4-desacetyl VLB C-3 N-(2-acetoxyethyl)carboxamide. 12. 4-desacetyl VLB C-3 N-G-dimethoxyethyScarboxamide. 13. 4-desacetyl VLB C-3 N-G-methylmercaptoethyDcarboxamide. 14. 4-desacetyl VLB C-3 N-(3-methylmercaptopropyl)carboxamide. 15. 4-desacetyl VLB C-3 N-G-mercaptoethy])carboxamide. 16. 4-desacetyl VLB C-3 N-acetaldehydecarboxamide. 17. 4-desacetyl VLB C-3 N-G-acetylaminoethyDcarboxamide. 18. 4-desacetyl VLB C-3 N-(2-acrylyloxyethy])carboxamide. 19. 4-desacetyl VLB C-3 N-G-stearoyloxyethyDcarboxamide. 20. 4-desacetyl VLB C-3 N-0-methoxyethylcarboxamide. 21. Bis-[4-desacetyl VLB C-3 N-G-ethylcarboxamide)[disulfide. 22. The pharmaceutically acceptable acid addition salt of a compound according to any of claims 1 to 21. 23. The sulfate salt of a compound according to any of claims 1 to 22. 351S5G8 24. A pharmaceutical composition comprising an inert carrier associated with a compound or a pharmaceutically acceptable acid addition salt thereof according to any of claims 1 to 22. 5 25. A process of preparing a compound according to claim 1; and its pharmaceutically acceptable acid addition salts comprising reacting dimeric indole-dihydroindole oarboxazide of the formula

4. 5 17 10 with an amine R R NH wherein R to R are as defined in olaim g 1 with the proviso that Y in R is other than a bond, and recovering the product of formula I in the form of the free amine or a pharmaceutically acceptable salt. 26. A prooess of claim 25 for preparing a 15 dimeric indole-dihydroindole carboxamide of Formula I wherein the reaction takes place in methylene dichloride. <15 5 s 8 27. A process of claim 25 or 26 for preparing a dimeric indole-dihydroindole carboxamide of Formula I wherein the reaction takes place at room temperature. 28. The process of claim 25 for preparing a dimeric . 4 indoledihydro-indolecarboxamide of Formula I wherexn R xs -(CH ) -Hcf wherein m is 1, 2 or 3, R 6 is -S-Y wherexn Y is a bond a m \„7 * joining the sulphur atoms of two moieties of Formula X, 7 6 R is H and R is H which comprises reactxng a dimeric indoledihydroindolecarboxazide of Formula XX with an amine of the formula “ C 4) alkyl-S-H in the presence of a base. 29. The process of any of claims 25 to 28 for preparing bis[4-desacetyl VLB C-3 (N-2-ethylcarboxamxde)]disulfide which comprises reacting 4-desacetyl VLB C-3 carboxhydrazide with 2-aminoethylmercaptan in the presence of a base. 30. A compound of formula I according to Claim 1 whenever prepared by a method according to any one of Claims 25 to 29. 31. A compound according to Claim 1 or Claim 30 6 7 wherein m is 1 and R and R are each -0-(^- Cg)alkyl; π is 1 or 2, E 6 is - OCH3, - 0 -C -(C.-C,.,) alkyl or - 0 - c - (C--C-)alkenyl and R 7 is H; II II 0 0 or m is 1, 2 or 3, R 6 is - NH - C -(C,-C,)alkyl or - S - Y wherein ϊ is H or - Cg alkyl, and R is H. - 37 32. A method of inhibiting a tumor or prolonging the life of a non-human host mammal by the administration to a mammal host of a tumor of an anti-tumor effective dose of a dimeric indole-dihydroindole carboxamide

5. Compound of Formula I as defined in any one of claims 1 to 23, 30 and 31. 33. A compound of Formula I as defined in claim 1 substantially as hereinbefore described in any one of Examples 4, 6 and 8 to 16.

6. 10 34. A pharmaceutical composition as defined in claim 24 substantially as hereinbefore described. 35. A process for preparing a compound of formula I defined in claim 1 substantially as hereinbefore described in any of Examples 4, 8, 9, 10, 11 and 16.

7. 15 36. A method according to claim 32 substantially as hereinbefore described.