IE46308B1 - Vincristine derivatives - Google Patents

Abstract

Dimeric indolodihydroindoles have the following formula In this formula: R<1> denotes the group OH or , 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

This invention relates to vincristine derivatives which are dimeric indole-dihydroindoles.

The present, invention provides a dimeric indoledihydroindole of the formula ’· «Άί , .// W •f Li·’,·..;.

Formula I ?/Κ Fl 1 A- ...... Ύ15 CHsO—JC*. . ti—R1 si.....CHs \3/ x®—OH C-O-CHa II wherein R1 is OH or O-C-CH,; Π «3 0 12 R is CHO or when R is OH R may be CH3; 4 one of R and R is H and the other is C2H5; R5 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 pharma15 ceutically acceptable salt. -24 6 3 6 8 The invention also provides a process for the preparation of 1-formyl dimeric indole-dihydroindole of the formula s' .ZvVV , If γ^-0-CHa Formula II CHaO—·* ΡΠ ’! W cu / μ FX 57 \χ·\/ tua.

I CHO •i*0H • C-O-CHa II wherein R is OH or O-C-CH,; II 3 4 one of R and R is H and the other C2H5; and pharmaceutically acceptable salts thereof; which comprises reacting l-methyl dimeric indole-dihydroindole of the formula II ο S' wherein R3-, 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 -446368 . 2 3 4 wherein R is CH3 or CHO; one of R and R is H and the other is C2H5; and pharmaceutically acceptable salts thereof which comprises hydrolysing a 4-acetoxy dimeric indoledihydroindole of the formula -546308 12ia#i . . . // /// . γ? ”,·.· Formula V 9/8' CHsO—' Z\ W\ / /' ft* sp··— \8 o in ^ixaO-l CHs C-CHs II C-O-CHs 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 indo'lfe-dihydroindole 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 /5 -646368 therein); 4-desacetoxy vinblastine (U.S. Patent No. 3,954,773); 4-deaacetoxy-3'-hydroxyvinblastine (U.S. Patent No. 3,944,554); leurocolombine (U.S. Patent No. 3,890,325), leuroformine (N-formylleurosine, 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 ccmpounds analogous 3 bo those of formula I where R is acetoxy, R is methyl, R is . hydroxyl, R is ethyl and R is H, VLB is represented; where R^ is acetoxy, R2 is formyl, R3 is hydroxyl, R^ is ethyl and 12 R is H, vincristine is represented; where R is acetoxy, R 4 5 /S is methyl, R is ethyl, R is hydroxyl, and R is H, leuro1 2 3 sidine is represented; where R is acetoxy, R is methyl, R 4 and R are H and R is ethyl, 4'-deoxy VLB A is repre12 5 sented; where R , R and R are the same as in 4'-deoxy VLB 3 4 A but R is ethyl and R is hydrogen, 4'-deoxy VLB B is 1 2 3 represented; and where R is acetoxy, R is methyl, R is 4 5 ethyl and R and R taken together form an α-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 acid oxidation at low (-60°C.) temperatures. There are other relatively abundant alkaloids such as leurosine in the dimeric indole-dihydro-746308 indole 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 ύ varying amounts of 4'-deoxy VLB A) by treatment with-Raney nickel in refluxing absolute ethanol—see Neuss, Gorman, Cone and Huokstep, 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 onoolytioally active derivatives of 4'-deoxy VLB A and B”, thereby /3 converting indirectly the relatively abundant alkaloid leurosine into a drug of greater potential clinical utility. 1 A compound of formula I in which R is ethyl, R 2 3 5 is acetoxy, R is CHO, and R and R are hydrogen is named 4'-deoxyvincristine; a compound where R^ is hydroxy but the other groups are the same is named 4'-deoxy-4-desacetylvinoristine. 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 3 4 in which R is ethyl, and R is hydrogen will be referred .SS back to leurosidine which has the same configuration at 4' as 4'-deoxy VLB B and will be referred to as derivatives of 1-formylleurosidine; i.e., 4'-deoxy-1-formylleurosidine (or 4'-deoxyepivincristinej and 4'-deoxy-4-desaoetyl-lformylleurosidine where R^ is acetoxy or hydroxy, respectively. -8In each of the above names, it will be understood that the 1-methyl group of leurosidine has been replaced by a formyl group and that the 1-desmethyl term has been omitted to simplify the nomenclature.

The pharmaceutically-acceptable salts of the carpounds of this 5 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-aad phosphorus acid, as well as non-toxic organic acids including aliphatic mono and di10 carboxylic 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, mono15 hydrogenphosphate, 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, naphthalene-2-sulfonate. -9^46308 The compounds of this invention according to Formula I above wherein R* is formyl may be prepared by low temperature chromic acid oxidation of either 4'-deoxy VLB A or 4'-deoxy VLB 9 -under acidic condition,, e.g. chromium trioxide 5 and acetic acid. The reaction temperature is preferably in the range -50 to -65°C. The reaction· mixture preferably comprises acetone and acetic acid. 4'-Deoxy VLB A and B or their 1-formyl derivatives can be hydrolyzed to the corresponding 4-desacetyl derivatives under acidic or basic conditions. These 410 desacetyl derivatives of 4'-deoxy VLB A and B can then be oxidised at low temperature (-60°C) with chromium trioxide without converting the 4-hydroxy group to a ketone to yield 4'-deoxy-4-desacetylvincristine and 4'-deoxy-4desacetyl-l-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-l-formylluerosidine are affected. 3ases 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 -1046308 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 0°C.

B The invention will be further understood from the fallowing examples.

Example 1 Preparation of 4'-Deoxyvincristine 582 mg. of chromium trioxide are dissolved in .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 /S' 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 ZS of the residue over 50 gm. of activity 1 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. -11Fractions 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~l Ultraviolet’spectrum: 210, 222, 255, 290, 298 nm 100 MHz: NMR spectrum: methyl singlets at 3.88, 3.67 and 2.07 6., *-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 I'S 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 41-deoxyvincristine .25- sulfate is 266 mg. -1246308 /5 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'-deoxyl-£ormylleurosidine. 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 1:1 20% 200 1:1 30% 100 1:1 45% 100 1:1 60% 400 yields the following fractions: Fraction Volume of Eluate 1 160 ml. 2 100 ml. 3 50 ml. 4 50 ml. 5 50 ml. 6 120 ml. 7 120 ml. -1346308 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: υ (CHCl^) 3470, 1743, 1690, 1222 cm-1.

Ultraviolet, spectrum; (CjHgOH) 210, 222, 254, 290, 298. 100 MHzNMR spectrum: methyl singlets 3.87, 3.65 and 2.07 6. pK' = 9.0 and 4.9 (in 66% DMF). a 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-l-formylleurosidine sulfate deposits on cooling.

EXAMPLE 2 Preparation of 4'-Deoxy-4-desacetyl-1-formylleurosidine About 744 mg. of 4’-deoxy-l-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 than virtually all starting 4'deoxy-Ifformylleurosidine has disappeared. The solvent is removed by evaporation and the residue containing 4'deoxy-4-desacetyl-l-formylleurosidine formed in the above reaction is partitioned between water and methylene di-14chloride. 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-desacetyl1-formylleurosidine.

S The compound had the following physical characteristics : Mass spectrum: m/e 766(M+), 764, 735, 254, 252, 205, 138 Infrared spectrum: v (CHClj) 3450, 1734, 1680, 1596, 1495, 1456, 1434 cm-1. 100 MHz pmr spectrum: (CDClj) includes N-formyl at 68.80, methyl singlets at 3.89 (C^g-OCHj) and 3.66 (Ci8-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-l-formyl-85 leurosidine A reaction mixture was prepared containing 1.48 g. of 4'-deoxy VLB Β, 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 -1546308 indicated that the hydrolysis reaction to remove the 4acetyl 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 /S’ 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 3S methanol (0.36M) 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 (CHClj) 3455, 1724, 1610, 1497, 1457, 1431 cm1 S' 100 MHz pmr spectrum: δ ^^13 9.43 (br s, 1, C3-OH), 7.92 (brs, 1, indole N-H), 7.47-7.S3 (m, 1, C.^, -H), 7.06-7.31 (m, 3, C12,_14,-H), 6.58 (s, 1, C14»H), 6.10 (s, 1, C1?-H), 5.78-5.87 (m, 2, Cg ?-H), 4.10 (m, 1, C4»H), 3.83 (s, 3, C16-OCH3), 3.78 (s, 3, Cg-COjCHj), 3.70 (s, 1, C2-H), 3.58 (s, 3, C18,-CO2CH3), 2.75 (s, 3, N-CH3), 0.76-1.06 (m, 6, C2l,21,-H)· 4'-Deoxy-4-desacetylleurosidine (834 mg.) obtained from filtrates and including solid filtered material was combined. The combined material probably contained 30-40 /5 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 -65°c. 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 -65°C. 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 -1746303 794 mg. of a residue shown by thin-layer chromatography to contain essentially one-spot material besides the original very low R^ impurity. This residue was chromatographed over Woelm silica gel using an initial eluant of 20:1:1 ethyl I 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 41-deoxy-4-desacetyl-l-formylleuro10 sidine by thin-layer chromatography were combined and yielded 293 mg. of pure 4'-deoxy-4-desacetyl-l-formylleurosidine after evaporation of the solvent. The sulfate salt was prepared as before. 4'-Deoxy-4-desacetylvincristine can be prepared as IS above by hydrolysing 4'-deoxy VLB A to yield 4'-deoxy4-desacetyl VLB and then oxidizing this compound with CrO3 in acetic acid at -60°C.

The compounds of this invention, as represented by Formula I above, particularly those in which R3 is acetoxy, are powerful anti-tumor agents. The compound 4'-deoxy4-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 2a 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. -18In 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 admini5 stration, and column 5, the percent inhibition of tumor growth or percent prolongation of survival time, e.g., B16. (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,). -1946308 β ΜΗ ο Ο •rH +> a Λ·Α ·Α •Μ Α Εη fl (β Β Επη Η β 18 ο > fl Η ·Α β ο > (DMA Ο ft 3 Α W Ί) Α ft 0 VO · © 0 «r ϋ rt cm 0 vo © ο υ υ υ •rt rt »rt c\ t** *rt rt •rt »rt •rt *rt X i X 1 1 X 1 1 O O X x X* Ο χ 0 oo m r* 0 rt oo 0 © rt O O 0 0 0 in o rt o σν in i·*- fr* rt &+ C* rtrtfrfr E-ι vo rt C\ tf ft = = Η ο Η σι ο X η χ ιη σι J? X cn ε Xncioffl VO rt rt © rt rt VO rt rt rt rt rt rt · rt rt rt • · · · rt rt vo rt o X • OOXOO X X • X X X X 1 X X o II II X X © X ΙΛ in 1 vo rt co co in co m 1 in co in © rt VO *4* rt rt © © rt © rt rt ’ί V0 rt *4* rt ST rt rt ©οοοοοοοοοο ο ο α ο Η) © «3· φ , η η vo in in in co cn rt CQ rt in rt rt tf fl rt O tf r* Oi tf © 0 tf tf (U β •Α Ό Α ω Α 1 ιΑ (D Saia ω X >ι <8 ο ε Λ V Μ Q 0 (D ι μη ω - I Α Α· |Α Μη (D β •Α Ό •Α (0 Α '3 hSi 4630S TABLE I (continued) β X 0 0 X AG® π o ε 43 -rl -rl H A 43 10 C 5Ή H fi 10 A A r-i ·Η GOA ® k k 0 ft 3 k ω A k ft 0 * * * rn O O * O * Tf ϋ o o o ϋ TP cm 0 o ϋ 0* •k X •rf Η Ή H •rf © vo •rf p-f •H rf ·# * 1 ft 1 ft i ft 1 | ft 1 ft ft m r*· vo 0 in o vo 0 iM X 0 tp in 0 > ni OOh rn EH X Eh m Eh vo rn Eh © © Eh m m Η A om o Ik ifi Eh fts s W © © X X I I © cn © ft t*> ft m ft m X N m o © co X CM VO τρ m rn m • « a rn m vo · » m » rn m m rn Ο X o • • o o ο M X 1 1 1 o O 1 1 X 1 X X © m vo I 1 co in in co in m m e\j © © X Tp X X vo CM X VO Tp CM vo m X ooooo oooooo oo© ω m rn ft ft ft © VO Tp O O O vo in cn VO W in VO cn x m A X h! in X Hi CQ Γ* o CQ 0 CQ o Q X X 0 >» 0 >1 0 β X x X X •rf 0 ro 0 ro 4J υ μη o X 0 ro x ro x •rf ω ro 0 ro μ 0 0 0 0 ϋ *0 rtf β i 0 1 0 •rf tp c tp ro i> 1 1 X 1 X >hX fchXf ft 0 ft £.X ftX 0 -P 0 e w 0 0 α ro 0 μ o 0 O O X oo μ ό μ ( X t x ro ι ro - ro - I 0 - 0 Tp 0 Tp X χ Tp X &1 (ϋ XJ s M en k » >43 •r) A > in k rH 3 tJ tn ® co ® 0 A d •rl | fi A rl fi <14 ® J! 3 Ό A fi fl •rl ® k ffl A k o -a g J) A k fl O A ffl a a * « * -2146308 In utilizing1 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 S 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 /5 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. 1 mg,/kg. of mammalian body weight once or twice a week or every two weeks de20 pending 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. 3S 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 -22reflect the difference in dose levels found in the treatment of experimental tumors in mice, the dose levels of the compounds of this invention being less than those used with vincristine and VLB. In clinical tests, as with other S 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)

1. A dimeric indole-dihydroindole of the formula wherein R 1 is OH or O-C-CH,; H 3 0 2. 12 R is CHO or when R is OH R may be CH 3 ; one of R 3 and R 4 is H and the other is C 2 H 5 ; R 5 is H; and pharmaceutically acceptable salts thereof.

2. A compound of claim 1 wherein R 1 is O-C-CH 3

3. A compound of claim 1 wherein R^ is OH.

4. 4'-Deoxyvincristine.

5. 4'-Deoxy-l-formylleurosidine.

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

7. 4'-Deoxy-4-desacetyl leurosidine. -2446308

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 com5 pound or its pharmaceutically acceptable salt of any of claims 1 to 7.

10. A process for the preparation of 1-formyl dimeric indole-dihydroindole of the formula /<0 wherein R^ is OH or O-C-CH,; fl ' 3 4 one of R and R is H and the other C 2 H 5 ; and pharmaceutically acceptable salts thereof; which comprises reacting 1-methyl dimeric indole-dihydroindole of the formula -2546308 s' - m\/ s V \Z^_ » A * Formula III 'f-C-O-CHa 1 II 0 7ft CHaO—< ,Z\, \A,Z ., K C FJ CHa δ-0-CHa II 13 4 wherein R , R or R 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 5 salt thereof.

11. A process of claim 10 for preparing 1formyl dimeric indole-dihydroindole of Formula ll wherein the chromic acid oxidizing agent is chromium trioxide and acetic acid. /O

12. A process of claim 10 or 11 for preparing a 1formyl dimeric indole-dihydroindole of Formula IX wherein the reaction takes place at -50° to -65°C.

13. A process of claims 10-12 for preparing a 1formyl dimeric indole-dihydroindole of Formula II wherein the /5 reaction mixture comprises acetone and acetic acid. -2646368

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

15. The process of claim 14 for preparing 4'5 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-l-formylleurosidine which comprises reacting 4'10 deoxy VLB B with chromium trioxide and acetic acid.

17. The process of claim 16 for preparing 4'deoxy-l-formylleurosidine sulfate which comprises reacting 4'-deoxy VLB B with chromium trioxide and acetic acid and recovery as a sulfate salt. /5

18. The process of any one «£ claims 10 to 13 for preparing 4'-deoxy-4-desacetyl-l-formylleurosidine which comprises reacting 4'-deoxy-4-desacetyl-leurosidine with chromium trioxide and acetic acid.

19. The process of claim 18 for preparing 4'20 deoxy-4-desacetyl-l-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-desaoetyl dimeric indoledihydroindole of the formula -2746308 ,, H/fy\ 10 'k' \ / Ύ \—\ e ' \a* >· Π 17 'Ί > 4' Y f-O-O-CHa Formula IV CHaO—< Fl/ zk SV „ /* i: FT £1 ' ι : C-O-CHa II 5 3 4 wherein R rs CHj or CHO; one of R and R is H and the Other is CjHg; and pharmaceutically acceptable salts thereof S which comprises·hydrolysing a 4-acetoxy dimeric indoledihydroindole of the formula 284-6 308 s' H 0 Formula V 2 3 4 wherein R , R and R are as defined above in a reaction mixture, and recovery of the 4-desacetyl free base or a pharmaceu5 tically acceptable salt thereof.

21. A process of claim 20 for preparing a 4desaoetyl dimeric indole-dihydroindole wherein a 4-acetoxy dimeric indole-dihydroindole is hydrolyzed by sodium carbonate in a reaction mixture. 10

22. A process of claim 20 or 21 for preparing a 4desacetyl dimeric Indole- dihydroindole 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 /5 preparing 4 1 -deoxy-4-desacetyl-l-formyl leurosidine vhich comprises hydrolysis of 4 1 -deoxy-l-formylleurosidine by sodium carbonate in anhydrous methanol. -2946308

24. The process 6f claim 23 for preparing 4'-deoxy4-desacetyl-l-formylleurosidine sulfate which comprises hydrolysis of 4'-deoxy-l-formylleurosidines by sodium carbonate in anhydrous methanol and recovery as a sulfate £ salt.

25. The process of any one of claims 20 to»2z 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 4'10 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 manmal-host of a tumor of an anti-tumor /5 effective dose of a dimeric indole-dihydroindole of Formula I as described in claims 1 to 7. -3028. 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.

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

29. 30. A process for preparing a 1-formyl dimeric indole-dihydroindole of the Formula II as defined in claim 10 substantially as hereinbefore described with particular 10 reference to examples 1 and 3.

30. 31. A 1-formyl dimeric indole-dihydroindole of the Formula II as defined in claim 10 when prepared substantially as hereinbefore described with particular reference to examples 1 and 3. /S'

31. 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.

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

33.

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