GB1593231A - Vincristine derivatives - Google Patents

Abstract

Dimeric indolodihydroindoles have the following formula <IMAGE> In this formula: R<1> denotes the group OH or <IMAGE>, R<2> denotes the group CHO or, if R<1> denotes the group OH, the group CH3, one of the radicals R<3> and R<4> denotes hydrogen and the other of these radicals denotes the group C2H5 and R<5> denotes hydrogen. As active compound, antitumour medicaments contain a compound of the formula I or a pharmaceutically acceptable salt thereof. Two processes for the preparation of the novel compounds are described.

Description

(54) VINCRISTINE DERIVATIVES (71) We, ELI LILLY AND COMPANY, a Corporation of the State of Indiana, United States of America, having a principal place of business at 307 East McCarty Street, City of Indianapolis, State of Indiana, United States of America, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement:- This invention relates to vincristine derivatives, which are dimeric indoledihydroindoles.

The present invention provides a dimeric indole-dihydroindole of the formula

5/ I 6 1::8" I,, 2' 16r .17;1 1 5 ' U-0-CH3 Formula I Ir H O 'WY4 o1y;) y3 g \k 3 CH3 /;IOH .

R2 C+CH3 II 0 wherein Rl is OH or

R2 is CHO or when R' is OH R2 may be CH3; one of R3 and R4 is H and the other is C2Hs; RS is H; and pharmaceutically acceptable salts thereof.

The invention also provides an anti-tumor pharmaceutical composition comprising an inert carrier and as active ingredient a compound of formula I or its pharmaceutically acceptable salt.

The invention also provides a process for the preparation of l-formyl dimeric indole-dihydroindole of the formula

5, 7 / 111 - - 4 12' ' 13 I -C-O-CH Formula II 14 H O /1 CH3 AX CH / CH30-I r iRi 1 4 CHO : C-OCH3 II 0 wherein Rl is OH or

one of R3 and R4 is H and the other C2H5; and pharmaceutically acceptable salts thereof; which comprises reacting methyl dimeric indole-dihydroindole of the formula

s 7 8///\ 4 r t t .:; ~ - - R4 I, tolI: Y'' \/ 12/f'$U;: 14 X W CH3 Formula III I I 9 I !M '!.

CH30-- CH2/ai3 OH I : CH3 C+CH3 II 0 wherein Rl, R3 or R4 are as described above with a chromic acid oxidizing agent in a reaction mixture at low temperature, and recovering the free base or a pharmaceutically acceptable salt thereof.

The invention also provides a process for the preparation of a 4-desacetyl dimeric indole-dihydroindole of the formula

5, 7/ 8'R3 -d 4' u v H3 Formula IV I II H O I 9 I 10 A- CHz CH30~ / DX OH OH I : 2 R2 II 0 wherein R2 is CH3 or CHO; one of R3 and R4 is H and the other is C2H5; and pharmaceutically acceptable salts thereof which comprises hydrolysing a 4acetoxy dimeric indole, dihydroindole of the formula

5, 3 ,, ICHB Formula V 4 S 10' a, \9\/3I 13' t7I1,s1I21 t Formula V I II H O I S I es I 10N I S t CH2 CH3C U13 0H 8W 2 R2 C-0-CH3 II 0 wherein R2, R3 and R4 are as described above in a reaction mixture, and recovery of the 4-desacetyl free base or a pharmaceutically acceptable salt thereof The invention also provides a method of inhibiting a tumor in a non-human mammal host by the administration of an effective dose of a dimeric indoledihydroindole of Formula I.

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) to be referred to hereinafter as VLB (U.S. Patent No. 3,097,137), leurosidine (vinrosidine) and leurocristine (VCR or vincristine) (both in U.S. Patent No.

3,205,220), 4'-deoxy VLB "A" and "B", Tetrahedron Letters, 783 (1968) (desacetyl leurosine hydrazide is also disclosed therein); 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), leuroformine (Nformylleurosine, see Belgian Patent No. 811,110) and vincadioline (U.S. Patent No. 3,887,565). Two of these alkaloids, VLB and leurocristine, are now marketed as drugs for the treatment of malignancies in humans, particularly the leukemias and related diseases.

The dimeric indole-dihydroindole alkaloids obtainable from Vinca rosea can be represented by formula I. In compounds analogous to those of formula I where R' is acetoxy, R2 is methyl, R3 is hydroxyl, R4 is ethyl and R5 is H, VLB is represented; where R' is acetoxy, R2 is formyl, R3 is hydroxyl, R4 is ethyl and R5 is H, vincristine is represented; where Rl is acetoxy, R2 is methyl, R3 is ethyl, R4 is hydroxyl, and R5 is H, leurosidine is represented; where Rl is acetoxy, R2 is methyl, R3 and R5 are H and R4 is ethyl, 4'-deoxy VLB "A" is represented; where R', R2 and RS are the same as in 4'-deoxy VLB "A" but R3 is ethyl and R4 is hydrogen, 4'-deoxy VLB "B" is represented; and where R' is acetoxy, R2 is methyl, R3 is ethyl and R4 and R5 taken together form an a-epoxide ring, leurosine is represented.

Of the above alkaloids, vincristine is the most useful, and the least available, from vinca. Recently, Jovanovics et al., U.S. Patent 3,899,493, have developed an oxidative method for converting the relatively more abundant VLB into vincristine by chromic acidoxidation at-low (-60"C) temperatures. There are other relatively abundant alkaloids such as leurosine in the dimeric indoledihydroindole fraction from vinca and it would be desirable to convert these directly or indirectly to vincristine or to a drug of comparable oncolytic activity. It is known that leurosine can be converted to 4'-deoxy VLB "B" (along with varying amounts of 4'-deoxy VLB "A") by treatment with Raney nickel in refluxing absolute ethanol - see Neuss, Gorman, Cone and Huckstep.

Tetrahedron Letters 783-7 (1968). While leurosine demonstrated oncolytic activity in experimental tumors in mice, clinical response was limited. 4'-Deoxy VLB "A" and 4'-deoxy VLB "B" were reported to lack reproducible activity in experimental tumors in mice.

It is an object of this invention to convert leurosine via 4'-deoxy VLB "A" and "B" to oncolytically active derivatives of 4'-deoxy VLB "A" and "B", thereby converting indirectly the relatively abundant alkaloid leurosine into a drug of greater potential clinical utility.

A compound of formula I in which R4 is ethyl, Rl is acetoxy, R2 is CHO, and R3 and R5 are hydrogen is named 4'-deoxyvincristine; a compound where Rl is hydroxy but the other groups are the same is named 4' - deoxy - 4 desacetylvincristine. Since the companion alkaloid to vincristine having a reverse configuration of hydrogen and ethyl at 4' from that found in vincristine is not known, those compounds in which R3 is ethyl, and R4 is hydrogen will be referred back to leurosidine which has the same configuration at 4' as 4'-deoxy VLB "B" and will be referred to as derivatives of l-formylleurosidine; i.e., 4' - deoxy - 1 formylleurosidine (or 4'-deoxyepivincristine) and 4' - deoxy - 4 - desacetyl - 1 formylleurosidine where R' is acetoxy or hydroxy, respectively. In each of the above names, it will be understood that the methyl group of leurosidine has been replaced by a formyl group and that the "l-desmethyl" term has been omitted to simplify the nomenclature.

The pharmaceutically-acceptable salts of the compounds of this invention include acid addition salts. Non-toxic acids useful for forming these salts include inorganic acids such as: hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydriodic acid, nitrous acid and phosphorus acid, as well as non-toxic organic acids including aliphatic mono and dicarboxylic acids, phenyl-substituted alkanoic acids hydroxy alkanoic and alkandioic acids, aromatic acids, aliphatic and aromatic sulfonic acids. Such pharmaceuticallyacceptable salts thus include the sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, 2-hydroxybutyrate, glycollate, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-l-sulfonate and naphthalene2-sulfonate.

The compounds of this invention according to Formula I above wherein R2 is formyl may be prepared by low temperature chromic acid oxidation of either 4'deoxy VLB "A" or 4'-deoxy VLB "B" under acidic condition, e.g. chromium trioxide and acetic acid. The reaction temperature is preferably in the range -50 to -650C. The reaction mixture preferably comprises acetone and acetic acid.

4'-Deoxy VLB "A" and "B" or their l-formyl derivatives can be hydrolyzed to the corresponding 4-desacetyl derivatives under acidic or basic conditions.

These 4-desacetyl derivatives of 4'-deoxy VLB "A" and "B" can then be oxidized at low temperature (-60"C) with chromium trioxide without converting the 4hydroxy group to a ketone to yield 4' - deoxy - 4 - desacetylvincristine and 4' deoxy - 4 - desacetyl - 1 - formylleurosidine.

The preferred method of carrying out the above hydrolysis reaction, uses sodium carbonate in anhydrous methanol at reflux temperature. Hydrazine hydrate may be used. Other bases which can be employed include potassium tbutoxide, sodium or potassium methoxide or ethoxide, pyridine, triethylamine (or other tertiary amine) and urea in polar organic solvents such as the lower alkanols.

Dilute sodium and potassium hydroxide can also be employed, in anhydrous methanol for example, but precautions must be taken not to operate with base concentrations or reaction temperatures at which other hydrolysable groups in 4'deoxyvincristine or 4' - deoxy - 1 - formylleurosidine are affected. Bases which operate only in non-polar solvents can also be used; i.e., sodium or lithium hydride in benzene, ether or THF, or the sodium salt of dimethylsulfoxide in DMSO. Temperatures varying from ambient temperature (25"C) to boiling point of the particular solvent may be used. On the other hand hydrolysis can be carried out under acidic conditions, e.g., absolute methanol saturated with anhydrous hydrogen chloride at OOC.

The invention will be further understood from the following illustrative examples.

EXAMPLE 1 Preparation of 4'-Deoxyvincristine 582 mg of chromium trioxide are dissolved in 5.8 ml of acetic acid and 0.6 ml of water. This oxidizing solution is added in dropwise fashion over a five-minute period to a stirred solution of 462 mg of 4'-deoxy VLB "A" in 58 ml of acetone and 2.9 ml of glacial acetic acid at a temperature of about -50 C. The reaction mixture is stirred at this temperature for about 30 minutes and then cooled to -65"C at which temperature the reaction mixture is quenched with 12 ml of 14 N aqueous ammonium hydroxide. The alkalinized reaction mixture is then poured onto 400 ml of an ice-water mixture and the aqueous layer extracted with 150 ml of ether followed by three extractions with 150 ml of chloroform each. The organic layers are combined and the combined layers washed with dilute aqueous sodium bisulfite, separated and dried. Evaporation of the organic solvents leaves, as a residue, 4'-deoxyvincristine. Chromatography of the residue over 50 gm of activity I silica is employed to further purify the desired compound. The chromatogram is developed as follows: 300 ml of 3:1 ethyl acetate-methanol followed by 300 ml of 1:1 ethyl acetate-methanol. After an initial 100 ml fraction, 20 ml fractions are collected. Fractions 8-20 are combined. Evaporation of the solvents from the combined fractions yields 279 mg of a light tan solid which is substantially one spot (pure) material by thin-layer chromatography.

4'-Deoxyvincristine free base thus prepared has the following physical characteristics: Mass spectrum: m/e 808 (M+), 806, 707, Infrared spectrum: 3465, 1745, 1687, 1220 cm-1, Ultraviolet spectrum: 210, 222, 255, 290 298 nm, 100 MHz NMR spectrum: methyl singlets at 3.88, 3.67 and 2.07 .

4'-Deoxyvincristine, as a tan solid is dissolved in acetone and the acetone solution treated with 0.96 ml of 0.36 M (2 percent volume/volume) sulfuric acid in absolute ethanol. A green solution results which is maintained at about 0 C overnight. Crystallization is induced by scratching or seeding, and the solid crystalline 4'-deoxyvincristine sulfate is separated by filtration. The filter cake is washed with cold acetone. The sulfate salt is somewhat soluble in acetone so the filtrate is evaporated to dryness and the resulting residue recrystallized from ethanol. Crystalline 4'-deoxyvincristine sulfate thus obtained from ethanol was filtered and the filter cake washed with ethanol. Total yield of 4'-deoxyvincristine sulfate is 266 mg.

In similar fashion, 794 mg of 4'-deoxy VLB "B" can be oxidized with 900 mg of chromium trioxide in 10 ml of glacial acetic acid and 1 ml of water to yield 4' deoxy - 1 - formylleurosidine. Thin-layer chromatography of the residue obtained directly from the oxidation mixture prior to purification indicates the presence of a major and a minor spot plus traces of other components.

Recrystallization of the residue from anhydrous ethanol yields substantially one spot crystalline material which is isolated by filtration and the crystals washed with cold ethanol.

Chromatography of the crystalline free base thus obtained over 50 g of silica using a 1:1 methylenedichloride-ethyl acetate solvent system containing 20, 30, 45 and 60 percent by volume of methanol as the eluant as follows: System Quantity 1:120% 200 1:130% 100 1:145% 100 1:160% 400 vields the following fractions: Fraction Volume of Eluate 1 160 ml 2 100ml 3 50 ml 4 50 ml 5 50 ml 6 120 ml 7 120 ml Fractions 4--7 are combined to yield 597 mg of a tan residue which in turn yields 435 mg of white crystalline 4' - deoxy - 1 - formylleurosidine (from ethanol). The compound has the following physical characteristics: Mass spectrum: m/e 808 (M+), 806, 777, 775, 336, 138, 136.

Infrared spectrum: v (CHO3) 3470, 1743, 1690, 1222 cm-'.

Ultraviolet spectrum: (C2H5OH) 210, 222, 254, 290, 298.

100 MHz NMR spectrum: methyl singlets 3.87, 3.65 and 2.07 b. pKa'=9.0 and 4.9 (in 66% DMF).

The sulfate salt is prepared by dissolving 435 mg of the free base in 10 ml of hot ethanol and adding 1.5 ml of 2 percent sulfuric acid in ethanol thereto.

Crystalline 4' - deoxy - 1 - formylleurosidine sulfate deposits on cooling.

EXAMPLE 2 Preparation of 4'-Deoxy-4-desacetyl- 1 -formylleurosidine About 744 mg of 4' - deoxy - 1 - formylleurosidine are mixed with 10 ml of anhydrous methanol and the mixture heated to refluxing temperature, at which temperature the compound dissolves to give a clear solution. 200 mg of solid sodium carbonate are added and the reaction mixture is stirred for about 7.2 hrs. at which time TLC of the crude reaction components shows that virtually all starting 4' - deoxy - 1 - formylleurosidine has disappeared. The solvent is removed by evaporation and the residue containing 4' - deoxy - 4 - desacetyl 1 - formylleurosidine formed in the above reaction is partitioned between water and methylene dichloride. The organic layer is separated and dried and the solvent is removed by evaporation yielding 506 mg of a white solid which is substantially pure 4' - deoxy - 4 - desacetyl - 1 - formylleurosidine.

The compound had the following physical characteristics: Mass spectrum: m/e 766 (M+), 764, 735, 254, 252, 205, 138.

Infrared spectrum: v (CHO3) 3450, 1734, 1680, 1596, 1495, 1456, 1434 cm-'.

100 MHz pmr spectrum: (CDC13) includes N-formyl at a 8.80, methyl singlets at 3.89 (C,6OCH3) and 3.66 (C18-CO2CH3), broadened multiplet at 3.82 (C3-CO2CH3), and no N-CH3 around 2.75 (or OCOCH3 around 2.06).

The corresponding sulfate salt is formed as in the previous examples using acetone as a solvent and 0.26 ml of 2% sulfuric acid in ethanol. Other solvents can be used and it is preferred to use a solvent in which the base is readily soluble but the sulfate salt substantially insoluble.

4' - Deoxy - 4 - desacetylvincristine and its sulfate salt are prepared in entirely analogous fashion from 4'-deoxyvincristine.

EXAMPLE 3 Alternate Preparation of 4'-Deoxy-4-desacetyl-1 -formylleurosidine A reaction mixture was prepared containing 1.48 g of 4'-deoxy VLB "B", 1 g of sodium carbonate and 100 ml of methanol and was heated to reflux under a nitrogen atmosphere. Thin layer chromatography of an aliquot taken at two hours indicated that the hydrolysis reaction to remove the 4-acetyl group was about half completed. The reaction mixture, after standing overnight at room temperature, was heated to reflux again for eight and one-half hours. Thin-layer chromatography of an aliquot 20:1:1 ether/diethylamine/toluene/methanol using a solvent indicated that the reaction had gone to completion. The solvent was removed from the reaction mixture by evaporation and the resulting residue was dissolved in a mixture of methylene dichloride and water. The methylene dichloride phase was separated and dried. Evaporation of the methylene dichloride yielded a residue comprising by TLC a very polar substance plus the expected 4' - deoxy - 4 - desacetylleurosidine. The residue which weighed 1.33 g was dissolved in benzene. The highly polar material was substantially insoluble in benzene and was separated by filtration. The filtrate was evaporated to dryness and the residue weighing 500 mg was chromatographed on Woelm silica gel using a 20:1:1 ether/diethylamine/toluene solvent system (with increasing quantities of methanol) as the eluant. The progress of the chromatography was followed by thin-layer chromatography and fractions shown to contain 4' - deoxy - 4 desacetylleurosidine were combined and yielded 348 mg of base on evaporation of the solvent. The residue was treated with 1.28 ml of 2 percent sulfuric acid in methanol (0.36 M) and the resulting solution was filtered to yield 315 mg of 4' deoxy - 4 - desacetylleurosidine sulfate.

4' - Deoxy - 4 - desacetylleurosidine had the following physical characteristics: Mass spectrum: m/e 752 (M+), 750, 693, 691, 555, 338, 240, 138.

Infrared spectrum: v (CHO3) 3455, 1724, 1610, 1497, 1457, 1431 cm-'.

100 MHz pmr spectrum: AIM C3l3 9.43 (br s, 1, C3-OH), 7.92 (brs, 1, indole N-H), 7.47-7.63 (m, 1, C11, -H), 7.06-7.31 (m, 3, C,2,~,4,H), 6.58 (s, 1, C14-H), 6.10 (s, 1, C17-M), 5.78-5.87 (m, 2, C6,7-H), 4.10 (m, 1, C4-H), 3.83 (s, 3, C16-OCH3), 3.78 (s, 3, C3-CO2CH3), 3.70 (s, 1, C2-H), 3.58 (s, 3, C1s,-CO2CH3), 2.75 (s, 3, N-CH3), 0.76-1.06 (m, 6, C21,21,-H).

4' - Deoxy - 4 - desacetylleurosidine (834 mg) obtained from filtrates and including solid filtered material was combined. The combined material probably contained 3040 percent of the highly polar material referred to above. The combined material was dissolved in 100 ml of acetone containing 7 ml of acetic acid. The solution was stirred for 15 minutes at room temperature and then cooled to -650C in a dry-ice acetone bath under a nitrogen atmosphere. 1110 mg of chromium trioxide were dissolved in 13 ml of glacial acetic acid and 2 ml of water.

This solution was added in dropwise fashion to the solution of 4' - deoxy - 4 desacetylleurosidine. The reaction mixture was stirred in the temperature range -60 to -650C for one hour and then quenched by the addition of 35 ml of 14 M aqueous ammonium hydroxide. The reaction mixture was next poured onto ice and the resulting aqueous suspension extracted several times with chloroform.

The chloroform extracts were combined, washed with water, and dried. Removal of the chloroform in vacuo yielded 794 mg of a residue shown by thin-layer chromatography to contain essentially one-spot material besides the original very low Rf impurity. This residue was chromatographed over Woelm silica gel using an initial eluant of 20:1:1 ethyl ether/diethylamine/toluene solvent mixture containing 0.9 percent methanol. The eluant was employed in 150 ml portions.

The percent of methanol was increased for each successive 150 ml eluant portion up to 15 percent. Fractions shown to contain 4' - deoxy - 4 - desacetyl - 1 formylleurosidine by thin-layer chromatography were combined and yielded 293 mg of pure 4' - deoxy - 4 - desacetyl - I - formylleurosidine after evaporation of the solvent. The sulfate salt was prepared as before.

4' - Deoxy - 4 - desacetylvincristine can be prepared as above by hydrolysing 4'-deoxy VLB "A" to yield 4' - deoxy - 4 - desacetyl VLB and then oxidizing this compound with CrO3 in acetic acid at -600C.

The compounds of this invention, as represented by Formula I above, particularly those in which R' is acetoxy, are powerful anti-tumor agents. The compound 4' - deoxy - 4 - desacetylleurosidine is also an oncolytic agent. In demonstrating the activity of these drugs against transplanted tumors in mice, a protocol was used which involved the administration of the drug by the intraperitoneal route at a given dose level for 7-10 days after innoculation with the tumor or alternatively, on the first, fifth, and ninth days after innoculation.

Table I gives the results of several experiments in which transplanted tumors in mice were treated successfully with a compound of this invention.

In the table, column 1 gives the name of the compound; column 2, the transplanted tumor; column 3, the dose level or dose level range and the number of days the dosage was administered; column 4, the route of administration, and column 5, the percent inhibition of tumor growth or percent prolongation of survival time, e.g., B 16. (ROS is an abbreviation of Ridgeway osteogenic sarcoma; GLS for Gardner lymphosarcoma, P1534(J) and L1210 are leukemias; CA755 is an adenocarcinoma; and B16 is a melanoma).

TABLE I Percent Inhibition or Prolongation of Compound Tumor mg/kgxDays Route Survival Time 4'-Deoxy- B16 0.9x3 IP Toxic 1-formylleurosidine 0.25-0.6x3 " 98-146 free base 755 0.6x3 " 54 0.4x3 " 73 0.25x3 " 47 0.2-0.3x10 " Toxic 0.06-0.135x10 " 33-93 0.03-0.09x9 " 8-72 P1534(J) 0.18x10 " Toxic P1534(J) 0.08-0.18x10 " 9-46 GLS 0.25-0.6x3 " 73-100 4'-Deoxy GLS 0.18x3 " 100 1-formylleurosidine 0.4x3 ,, 100 sulfate 0.6x3 ,, Toxic 0.25x3 " Toxic L1210 0.4-0.6x3 " Toxic 0.25x3 ,, 65* ROS 0.4x3 " 100 0.18x3 " 93 0.25x3 " Toxic 4'-Deoxyvincristine B16 0.30-0.9x3 IP 36-143* sulfate 755 0.25-1.8x9-10 " Toxic 0.06-0.13x9-10 " 15-100 0.9x3 " Toxic 0.1-0.3x3 " 36-100 GLS 0.4-0.6x3 " Toxic 0.18-0.25x3 " 62-94 4'Deoxy-4-desacetyl- B16 0.15-0.6x3 " 31-62** 1-formyl- GLS 0.6x3 " Toxic leurosidine sulfate 0.25-0.40x3 94-100 0.18x3 95 4'-deoxy-4-desacetyl- 755 0.6x3 ,, Toxic leurosidine sulfate 0.4x3 " 37 0.25x3 ,, 32 B16 0.6x3 " Toxic** 0.3x3 " 103** 0.15x3 ,, 77** GLS 0.9x3 " Toxic 0.6x3 " 63 0.4x3 ,, Toxic *lor more indefinite survivors.

**Delayed treatment-dosed 5th, 9th and 13th days.

In utilizing the novel compounds of this invention as anti-tumor agents, either the parenteral or oral route of administration may be employed. For oral dosage, a suitable quantity of a pharmaceutically-acceptable salt of a base according to Formula II formed with a non-toxic acid, such as the sulfate salt, is mixed with starch or other excipient and the mixture placed in telescoping gelatin capsules each containing from 7.5 to 50 mg of active ingredients.

Similarly, the anti-neoplastically active salt can be mixed with starch, a binder and a lubricant and the mixture compressed into tablets each containing from the 7.5-50 mgs of salt. The tablets may be scored if lower or divided dosages are to be used. Parenteral administration is preferred however. For this purpose, isotonic solutions are employed containing 1-10 mg/ml of a salt of an indoledihydroindole of Formula II such as the sulfate salt. The compounds are administered at the rate of from 0.01 to 1 mg/kg and preferably from 0.1 to I 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 being administered.

In utilizing a compound of this invention clinically, the clinical physician would administer the compound initially by the same route and in the same vehicle and probably against the same types of tumors as are indicated for vincristine or VLB. The dose levels employed would reflect the difference in dose levels found in the treatment of experimental tumors in mice, the dose levels of the the compounds of this invention being less than those used with vincristine and VLB. In clinical tests, as with other anti-tumor agents, particular attention would be paid to the effect of the oncolytic compounds of this invention against the ten "signal" tumors set forth at page 266 of "The Design of Clinical Trials in Cancer Therapy" edited by Staquet (Futura Publishing Company, 1973).

Claims (34)

WHAT WE CLAIM IS:

1. A dimeric indole-dihydroindole of the formula

5t 7 6/ > t 'CH Formula g'3:'t- '' R4 121 Formula I z 4 ' I It H l O0 1 I e 2 R2 II 0 wherein R' is OH or

R2 is CHO or when R' is OH R2 may be C113; one of R3 and R4 is H and the other is C2H5; RS is H; and pharmaceutically acceptable salts thereof.

2. A compound of Claim 1, wherein R' is

3. A compound of Claim 1, wherein R' is OH.

4. 4'-Deoxyvincristine.

5. 4'-Deoxy-1-formylleurosidine.

6. 4'-Deoxy-4-desacetyl-l-formylleurosidine.

7. 4'-Deoxy-4-desacetyl leurosidine.

8. The sulfate salt of any compound of Claims 1 to 7.

9. An anti-tumor pharmaceutical composition comprising an inert carrier and as active ingredient a compound or its pharmaceutically acceptable salt of any of Claims 1 to 7.

10. A process for the preparation of l-formyl dimeric indole-dihydroindole of the formula

5, 7/ if - - R4 ,'/5\10 '- g , Ii' Formula II 0 H 1 S se 10N q Is ii 51X \R-- 51 AH3 CH30-- 7 X R / \3/R -0H I : CHO C-0-C II 0 wherein R' is OH or

one of R3 and R4 is H and the other C2H,; and pharmaceutically acceptable salts thereof; which comprises reacting methyl dimeric indole-dihydroindole of the formula

5B 3 II' j7)R4 S i' 3 a:1 2 13 H3 Formula III 1,1. 141 III I II H 10 I 0 e/e\ I J "I! CH30--${sIX CH2 7H3 CH3 C+CH îl o 0 wherein R', R3 or R4 are as defined above with a chromic acid oxidizing agent in a reaction mixture at low temperature, and recovering the free base or a pharmaceutically acceptable salt thereof.

11. A process of Claim 10 for preparing l-formyl dimeric indoledihydroindole of Formula II wherein the chromic acid oxidizing agent is chromium trioxide and acetic acid.

12. A process of Claim 10 or 11 for preparing a l-formyl dimeric indoledihydroindole of Formula II wherein the reaction takes place at 50 to -650C.

13. A process of Claims 10 12 for preparing a l-formyl dimeric indoledihydroindole of Formula II wherein the reaction mixture comprises acetone and acetic acid.

14. The process of any one of Claims 10 to 13 for prcparing 4'deoxyvincristine which comprises reacting 4'-deoxy VLB "A" with chromium trioxide and acetic acid.

15. The process of Claim 14 for preparing 4'-deoxyvincristine sulfate which comprises reacting 4'-deoxy VLB "A" with chromium trioxide and acetic acid and recovery as a sulfate salt.

16. The process of any one of Claims 10 to 13 for preparing 4' - deoxy - I formylleurosidine which comprises reacting 4'-deoxy VLB "B" with chromium trioxide and acetic acid.

17. The process of Claim 16 for preparing 4' - deoxy - 1 - formylleurosidine sulfate which comprises reacting 4'-deoxy VLB "B" with chromium trioxide and acetic acid and recovery as a sulfate salt.

18. The process of any one of Claims 10 td 13 for preparing 4' - deoxy - 4 desacetyl - I - formylleurosidine which comprises reacting 4' - deoxy - 4 - desacetyl - leurosidine with chromium trioxide and acetic acid.

19. The process of Claim 18 for preparing 4' - deoxy - 4 - desacetyl - 1 formylleurosidine sulfate which comprises reacting 4' - deoxy - 4 - desacetyl leurosidine with chromium trioxide and acetic acid and recovery as a salt.

20. A process for the preparation of 4-desacetyl dimeric indoledihydroindole of the formula

5J 5, 4 *-- R4 I, IV t CH3 Formula IV H 10 esy I jo" Is ii siX 9 /3 CH3 77! Lo2 \XIs\iX40H 2 CH}CH3 II 0 wherein R2 is CH3 or CHO; one of R3 and R4 is H and the other is C2Hs; and pharmaceutically acceptable salts thereof which comprises hydrolysing a 4acetoxy dimeric indole-dihydroindole of the formula

S, 71 - - - Y/ i3Iy(TIII7sf!2i a C-O-CHs Formula V H 10 S Ge I TICH2/ I 4( =CHO CH30-- 77! CCH3 R2 C+CH3 I I 2 R 6H3 II 0 wherein R2, R3 and R4 are as defined above in a reaction mixture, and recovery of the 4-desacetyl free base or a pharmaceutically acceptable salt thereof.

21. A process of Claim 20 for preparing a 4-desacetyl dimeric indoledihydroindole wherein a 4-acetoxy dimeric indole-dihydroindole is hydrolyzed by sodium carbonate in a reaction mixture.

22. A process of Claim 20 or 21 for preparing a 4-desacetyl dimeric indoledihydroindole wherein a 4-acetoxy dimeric indole-dihydroindole is hydrolyzed in anhydrous methanol at reflux temperature.

23. The process of any one of Claims 20 to 22 for preparing 4' - deoxy - 4 desacetyl - 1 - formyl leurosidine which comprises hydrolysis of 4' - deoxy - 1 formylleurosidine by sodium carbonate in anhydrous methanol.

24. The process of Claim 23 for preparing 4' - deoxy - 4 - desacetyl - 1 formylleurosidine sulfate which comprises hydrolysis of 4' - deoxy - 1 formylleurosidines by sodium carbonate in anhydrous methanol and recovery as a sulfate salt.

25. The process of any one of Claims 20 to 22 for preparing 4' - deoxy - 4 desacetyl - leurosidine which comprises hydrolysis of 4'-deoxy VLB "B" by sodium carbonate in anhydrous methanol.

26. The process of Claim 25 for preparing 4' - deoxy - 4 - desacetyl leurosidine sulfate which comprises hydrolysis of 4'-deoxy VLB "B" by sodium carbonate in anhydrous methanol and recovery as a sulfate salt.

27. A method of inhibiting a tumor by the administration to a non-human mammal host of a tumor of an anti-tumor effective dose of a dimeric indoledihydroindole of Formula I as described in Claims 1 to 7.

28. A dimeric indole-dihydroindole compound of the Formula I as defined in Claim 1 substantially as hereinbefore described with particular reference to any one of Examples 1 to 3.

29. A pharmaceutical composition as claimed in Claim 9 substantially as hereinbefore described.

30. A process for preparing a l-formyl dimeric indole-dihydroindole of the Formula II as defined in Claim 10 substantially as hereinbefore described with particular reference to Examples I and 3.

31. A l-formyl dimeric indole-dihydroindole of the Formula 11 as defined in Claim 10 when prepared substantially as hereinbefore described with particular reference to Examples 1 and 3.

32. A process for preparing a 4-desacetyl dimeric indole-dihydroindole of the Formula IV as defined in Claim 20 substantially as hereinbefore described with particular reference to Examples 2 and 3.

33. A 4-desacetyl dimeric indole-dihydroindole of the Formula IV as defined in Claim 20 when prepared substantially as hereinbefore described with particular reference to Examples 2 and 3.

34. A method of inhibiting a tumor as claimed in Claim 26 substantially as hereinbefore described.