CS214793B2 - New method of making the dimeric disulphides of indol-dihydroindole derivatives genus vinca connected by the bridge - Google Patents

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KPATENT (22) Registered 14 05 79 (21) (PV 6476-80) (32) (31) (33) Priority from 15 05 78 (905Θ67) United States (40) Posted 09 09 81 (45) Released 15 09 84

OFFICE FOR INVENTION DISCOVERIES 214793 (U | (B2 |

(EI) IJ. Part C 07 D 519/04 // A 61 K 31/475 (72)

The inventor of CONRAD ROBERT ALLEN and GERZON KOERT, INDIANAPOL-IS, INDIANA (Sp. St.) 173)

Proprietor of ELI LILLY AND COMPANY, INDIANAPOLIS, INDIANA (Sp. A.) (54) New method of producing dimeric diesulphides of indole-dihydroindole derivatives of the genus Vinca, linked by bridge 1

It has been found that certain natural alkaloids obtained from Vinca rosea (Catharanthus roseus don. Alkaloids of Catharanthus or Vinca) are effective in the treatment of experimental tumor diseases in animals. These compounds include leurosine (see U.S. Patent No. 3,370,057), vincaleukoblastine (vinblastine) - hereinafter referred to as VLB (U.S. Pat. No. 3,097,137), leucoroform (see Belgian Patent No. 811110), leurosidine (vinrosidine) and leurocristine, hereinafter referred to as vincristine (both see U.S. Patent No. 3,205,220), deoxy-VLB "A" and "B" [see Tetra-hedron Letters, 783 (1958)], 4-deacetoxyvin- blastine (U.S. Pat. No. 3,954,773), 4-deacetoxy-3'-hydroxyvinblastine (U.S. Pat. No. 3,944,554), leurocolombine (U.S. Pat. No. 3,890,325) and vincadioline (U.S. Pat. 3,887,565). Two of these alkaloids, VLB and vincristine, are currently on the market as agents for the treatment of tumoral diseases, in particular leukemia and related human diseases. These two commercial alkaloids are usually administered intravenously.

Chemical modification of alkaloids from genus 2 nostmi. First of all, it should be borne in mind that the structure of the molecule of these substances is uncommonly complex and that it is therefore very difficult to develop chemical reactions by which a particular functional group in the molecule would belamodified without affecting other groups. Secondly, dimeric alkaloids lacking the desired chemotherapeutic properties have been isolated or produced from the fractions obtained from Vinca rosea plants or those from those derived from these plants. concludes that these compounds are closely related to the active alkaloids from which they often differ only in one carbon. The anti-neoplastic efficacy of the set seemed to be limited to very specific basic structures, and hence the hope of obtaining more effective drugs by modifying these structures seemed very small. Successful modifications of physiologically active alkaloids include the preparation of 6,7-dihydro-VLB (see U.S. Pat. No. 3,352,868) and the replacement of the acetyl group on Ci (carbon at position 4 of the cyclic system VLB - see below by numbering) with a higher alkanoyl group or non-acyl group Groups (see U.S. Pat. No. 3,392,173). Some of these

One of these C-derivatives, in which the C4-acetyl group of VLB is replaced by a chloroacetyl group, is also a useful intermediate for the preparation of structurally modified VLB derivatives in which the C4-acetyl group VLB replaces N, N-dialkylglycyl- group (see U.S. Patent No. 3,387,001).

Cs-carboxamide and carboxhydrazide derivatives of VLB, vincristine, vincadioline and the like have also been prepared, and have been found to be effective against cancer (see Belgian Patent No. 813,168J. These compounds are of particular interest since, for example, The 3-carboxamides VLB are more potent against the Ridgeway osteogenic carcinoma and Gardner's lymphosarcoma than the VLB alone, that is, the basic alkaloid from which they were derived, and some of these amino derivatives approach efficacy of vihcristine against the same tumors. C3-carboxamide 4-deacetyl-VLB (vindesine) is currently in human clinical trials and appears to be less neurotoxic than vincristine and is effective against leukemias, including vincristine-resistant leukaemias In Belgian Patent Specification No. 858,451, bis-derivatives of diene compounds of the genus Vinca are described and protected. which may be described by the general formula R — NH — CH 2 - (CH 2) n —S—

- S — CH 2 - (CH 2) n —NH — R wherein R is a Vinca dimeric alcaloid residue containing a carbonyl group at C 3 and n is 1 to 5. The compounds of this structure have been found to be the main products of the reaction of the Cs-carboxazide of the compound of the genus Vinca (R — N3Ja of the compound of formula NH2 — CH2 — CH2 — SH, and are believed to be formed by the oxidation of the carboxamidoethylmercaptan group to C3 by air, such as the cysteine - cystine system. Of course, oxidation is limited to the formation of a disulfide bond from mer-captoalkylamide and is not generally applicable to the synthesis of other bis-derivatives of dimer compounds of the genus Catharanthus, linked by bridge.

The present invention provides an improved process for the production of certain tetramers of the Vinca compounds described and protected in Belgian Pat. No. 858,451. The latter improved method is a process for the preparation of compounds of general formula II, R6_NH — CH2— (CH2) n — Q— - (CH 2) n -CH 2 —NH — R 6 (Π), wherein each of R 6 is a radical of the in-dol-dihydroindole alkaloid of general formula III,

wherein R 2 represents a methyl group or a hydrogen atom with a hydroxyl group or a hydrogen atom and the other represents an ethyl group, and R 5 represents a hydrogen atom, or R 4 and R 5 together with a ϊ 7 214793 6

S is a β-epoxide ring and R 3 is an ethyl group, and Q is an S-S group, and pharmaceutically acceptable acid addition salts thereof, characterized in that the general formula R 6 -N 3 is reacted with a diamine of the general formula NH 2 —CH 2 - (CH 2) n —Q— (CH 2) n —CH 2 —NH 2 wherein Q is S — S, at a ratio of 2 moles of azide R 6 —N 3 to each mole of the diamine, where appropriate recovered the product is converted into its pharmaceutically acceptable acid addition salt.

Non-toxic acids useful in the preparation of pharmaceutically acceptable acidic salts of the compounds of Formula II include inorganic acids such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, nitrous and phosphoric acids, as well as non-toxic organic acids, including aliphatic acids. non-and dicarboxylic acids, phenyl substituted alkanoic acids, hydroxyalkanoic and alkanedioic acids, aromatic acids and aliphatic and aromatic sulfonic acids. Thus, such pharmaceutically acceptable salts include, for example, sulfates, pyrosulfates, bisulfates, sulfites, bi-sulfites, nitrates, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates. , caprylates, acylates, formates, isobutyrates, capronates, heptanoates, propiolates, oxalates, malonates, succinates, succates, sebacates, fumarates, maleates, benzoates, chlorobenzoates, methylbenzoates, di-triobenzoates, hydroxybenzoates, methoxybenoates , phthalates, terephthalates, benzenesulfonates, toluenesulfonates, chlorobenzenesulfonates, xylene sulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, 2-hydroxybutyrates, glycolates, salts with malic acid, tartrates, methanesulfonates, propanesulfonates, 1-naphthalenesulfonates and 2- naphthalenesulfonates.

The first reaction step for the preparation of the general formula (II) is carried out by mixing the azide of the general formula R6-N3 with a diamine of the formula NH2 --CH2 - (CH2) n - Q-- - in a suitable inert solvent, usually methylene chloride. n - CH 2 -NH 2 wherein Q is as defined above, in an amount of 2 moles of azide per mole of diamine. The diamine is also dissolved in an inert solvent, usually methylene dichloride, prior to addition. As mentioned below, the starting azide is usually not purified prior to this reaction, but is used as it was isolated from the reaction mixture, and therefore the same solvent as used to isolate the diamine is expediently used in the reaction with the diamine. to azide. The resulting Vinca bis-amides are isolated by standard procedures and purified by chromatography. For example, using VLB as the starting material in this synthesis is as follows: The VLB is reacted with anhydrous hydrazide in an inert solvent dissolving both of these reactants, such as in a lower alkanol, in a closed reaction vessel. Typically, the reaction mixture is heated to a temperature in the range of from 40 to 100 ° C for a period of from 12 to 48 hours, during which time the reaction is virtually complete. The product of the reaction is 4-deacetyl-VLB-C3-carboxhydrazide, since the reaction with hydrazine leads not only to the conversion of the ester group to C3 to the hydrazide group, but also to basic hydrolysis of the acetyl group to C4 to hydroxyl. Other ester groups in the molecule are not attacked under the above reaction conditions, only by the fact that a certain amount of 18'-demethoxycarbonyl derivative is usually formed. However, the main product of the reaction is 4-deacetyl-VLB-C3-carboxhydrazide. In an analogous manner, 4-deacetyl-C3-carboxhydrazes of deoxy-VLB 'A' and 'B', leurosidine aleurosine are prepared. The carboxhydrazides thus formed are converted into the corresponding azides by the action of sodium nitrite in the acid. Typically, the product reaction is used as it is isolated from the reaction mixture, since it has been found that it is not necessary to purify the azide to obtain the desired bisamide.

By reacting 2 moles of 4-deacetyl-C3-carboxamides VLB, leurosidine, deoxy-VLB " A " and " B ", leurosine and other alkaloids of formula R6-OCH3, wherein R6 is a radical of formula II With the exception that R2 represents only a methyl or hydrogen atom, with a diamine of the general formula NH2-CH2- (CH2) n-Q- (CH2-CH2-CH2-NH2 where Q is as defined above, standard reaction conditions of reaction and amine) described in Belgian Patent Specification No. 858 451 for the preparation of monoamides of the same Vinca alkaloid, to obtain the desired products of the formula R6-NH-CH2- (CH2-N-Q- (CH2) n-CH2-NH-R6 Preparation of starting materials 4-deacetyl-VLB-C3-carboxhydrazide VLB is heated in anhydrous ethanol with an anhydrous hydrazine of about 18 hours in a sealed reaction vessel to a temperature of about 60 DEG C. The starting materials used in the above syntheses are prepared as follows. The reaction vessel was cooled, opened, its contents removed, and the volatiles were evaporated in vacuo, the 4-deacetyl-VLB-C3-carboxhydrazide residue was taken up in methylene dichloride, the methylene dichloride solution was washed with water, separated and dried the methylene dichloride is evaporated in vacuo. The residue was dissolved in a mixture of equal portions of chloroform and benzene and chromatographed on silica gel. The eluent is a mixture of benzene, chloroform and triethylamine. Initial chromatographic fractions contain unreacted starting materials along with 4-deacetyl-VLB by-product reaction. Other fractions contain 4-deacetyl-18‘-demethoxycarbonyl-VLB-C3-carboxhydrazide, previously described by Neuss et al. in Tetrahedron Letters, 1968, 783. The following fractions, which contain 4-deacetyl-VLB-C3-carboxhydrazide by thin layer chromatography, are combined in vacuo to remove the solvent. The solid residue melts at about 2119 ° C to 220 ° C with decomposition.

Alternatively, 4-deacetyl-VLB can be reacted with hydrazine to give 4-deacetyl-VLB-C3-carboxhydrazide and leurocristine or 4-deacetyl-leurocristine [which can be prepared according to the method of U.S. Pat. patent specification. No. 3,392,173] can be reacted with anhydrous hydrazine in anhydrous ethanol to yield 4-deacetyl-1-deformylleurocristine-C3-carboxhydrazide isolated in the form of a morphine powder. Analogously, also 4-deacetyl-C3-carboxhydrazides of deoxy-VLB, leurosidine and leurosine. 4-deacetyl-VLB-C3-carboxazide are prepared in 15 ml of anhydrous methanol to dissolve 678 mg of 4-deacetyl-VLB-C3-carboxhydrazide to the solution. 50 ml of 1 N aqueous hydrochloric acid are added, the resulting solution is cooled to about 0 DEG C., 140 mg of sodium nitrite are added thereto, and the reaction mixture is stirred for 10 minutes while maintaining the temperature at about 0 DEG. After adding sodium nitrite, the solution turns dark reddish brown. The resulting mixture was basified by the addition of an excess of cold 5% aqueous sodium bicarbonate and extracted three times with methylene dichloride. The 4-deacetyl-VLB-C3-carboxazide, resulting from the above-described reaction, is converted into methylene dichloride and the organic layer separated.

B li. The thus obtained methylene dichloride solution of 4-deacetylvinblast-C3-icarboxazide is usually used without further purification.

In an analogous manner, 4-de-acetyl-C3-carboxazid derivatives of 1-deformyl-vincristine, leurosidine, 4'-deoxy-VLB 'A' and 'B' and leurosine are prepared.

The invention is illustrated by the following non-limiting Examples. Example 1 Preparation of bis [β- (4-deacetyl-VLB-C3-carboxamido] ethyl] disulfide 8 g of 4-deacetyl-VLB-C3-carboxhydrazide in aqueous hydrochloric acid was not reacted with sodium nitrite to give 4-deacetyl-VLB The azide formed in this reaction is isolated in the form of a solution of 400 ml of methylene dichloride.

22.5 g of cystamine dihydrochloride are added to 500 ml of methanol in which 10.8 g of sodium methoxide is dissolved, the reaction mixture is stirred for about 4 hours and the solvent is evaporated. The residue was stirred with ether for about 1 hour and then filtered. Evaporation of the ether filtrate in vacuo gave about 10 g of oily residual cystamine as the free base.

To a solution of 4-deacetyl-VLB-C3-carboxazide in methylene dichloride is added 760 mg of cystamine free base in methylene dichloride. Addition of 75 ml of tetrahydrofuran causes the reaction mixture to stir at room temperature for about 16 hours, then is filtered and the filtrate is evaporated to dryness under vacuum. The residue containing bis [3- (4-deacetyl-VLB-C3-carboxamido) ethyl] disulfide is dissolved in methylene dichloride, the solution is washed with water and dried. The methylene dichloride evaporation residue contains two main products according to thin layer chromatography in methylene dichloride-methanol-ethyl acetate (1: 1: 1) solvent system. The residue is subjected to silica gel chromatography using the above solvent system as eluent. Thin-layer fractions containing bis- [β- (4-deacetyl-VLB-C3-carboxamido) ethyl] disulfide were combined and evaporated from the solvent. 2.6 g of the desired product are obtained. The next major reaction product is also isolated and purified. This product yields 1 mol of 4-deacetyl-VLB-C3-carboxazide and cystamine, i.e. 4-deacetyl-VLB-C3-N-2- (2-aminoethyldithio) ethylcarboxamide. Higher yields of this product are obtained when 1 molezide is reacted with 1 mole of cystamine.

The bis [3- (4-deacetyl-VLB-C3-carboxamido) ethyl] disulfide thus prepared is identical to the product obtained by the process described in Belgian Patent No. 813,168.

The bis [beta- (4-acetyl-VLB-C3-carboxamido) ethyl] disulfide disulfate is prepared in ethanol 214793 10 by the addition of a stoichiometric amount of 2% (v / v) ethanolic sulfuric acid. The sulfate is also prepared by titrating the base solution with 2% ethanolic sulfuric acid to a point where an aliquot of the solution, diluted with 5 volumes of water, has a pH between 3.0 and 3.5. Any of these procedures give an off-white amorphous salt upon evaporation of the ethanol.

Part A

By the above procedure, the reaction of 4-deacetyl-VLB-C3-carboxide with bis (γ-amino) -propyl disulfide yields bis [γ- (4-deacetyl-VLB-C3-carboxamido) propyl] disulfide with the following physical characteristics :

Elemental analysis: calculated: 3.72% S, found: 3.87% S.

Molecular Spectrum: m / e 841, 855.

Titration (66% dimethylformamide): pKa ,20 5.20, 721. IR (v, CHCl 3): 1730 and 1670 cm -1. 13 CNMR: the spectrum is the same as for vindesine because it contains a signal for secondary amide at 173.7 ppm and other signals at 42.0, 36.00 and 39.2 ppm. The yield is 10-20%.

Part B

By the above-described procedure, the reaction of 4-deacetylleurosine-C3-carboxazide with cystamine in a 2: 1 molar ratio gives bis [3- (4-deacetylleurine-C3-carboxamido) ethyl] disulfide with the following physical characteristics:

Mass Spectrum: m / e 825. IR (v, CHCl 3): 1730 and 1670 cm -1.

Titration (66% dimethylformamide): pKa ,3 5.3 and 7.0.

Molecular weight (osmometry): 1479 (calculated 1620). The 13 C NMR spectrum is analogous to that of vindesine with the signal for secondary amide at 173.7 ppm and other signals at 39.2 and 37.5 ppm. The yield is 10-20%. Example 2 Preparation of salts

The sulphates of the above amides are prepared by dissolving the corresponding amide in absolute ethanol and adjusting the pH of the resulting solution to about 3.0 to 3.5 with 2% ethanolic sulfuric acid. with inorganic ions such as chlorides, bromides, phosphates, nitrates and the like, as well as salts with organic ions such as benzoates, methanesulfonates, maleates, tartrates and the like.

The compounds of formula (II) are active against transplanted tumors in mouse invivo. To demonstrate the efficacy of the compounds of the present invention, a method is employed in which the test substance is applied, usually intraperitoneally, at different predetermined doses every day for 9 to 10 days or every fourth day after tumor inoculation. Table I shows the results of experiments in which mice with transplanted Gardner lymphosarcoma (GLS), C3H mammary tumor, melanoma B16, P 1534 (J) leukemia, P 388 leukemia, or 755 adenocarcinoma were treated with compounds of formula II. In the first column of this table, the name of the test compound, the second column of the tumor, the third column of the dosing, and the fourth column the percentage of tumor growth inhibition or prolongation. The use of the novel compounds of general formula (II) as anti-mammalian tumor agents is expediently carried out by parenteral administration, with intravenous administration being preferred, although intraperitoneal administration may also be used in smaller mammals. Isotonic solutions containing 1 to 10 mg of the alkaloid base salt of the invention in 1 ml are used for intravenous administration. The active compounds are administered at a dose of 0.01 to 1 mg / kg body weight of the mammal, preferably 0.1 to 1 mg / kg, once or twice weekly or every two weeks, depending on both the potency and the toxicity of the active ingredient. The alternative way to arrive at a therapeutic dose is based on the body surface area, with a dose of 0.1 to 10 mg / m 2 of body surface area of the mammal being administered every 7 or 17 days.

Claims (1)

21 / 4'7f9 ^ 11 12 TABLE I Compound Name 'Tumor Dose' Percentage of lilhibtoe in mg / kg x after- or prolonged survival -number of days bis [β- (4-deacety lleur osine-C3- P 388 30 - x 3 -35-carboxamide ethyl] disulfide-15 x 3 46 disulfate bis [β- (4-deacetyl-VLB-C3- GLS 0.4 x 9 43 to 100-carboxamido) ethyl] disulfide-1,2 x 3 62 to 93-disulfate 0.9 x 3 34 to 46 C3H 1.2 x 3 toxic dose bis [; (3- (4-deacetyl-VLB-C3- P 388 0.9 X 3 1.2 x 3 : 31; to 36 53 to 61 carboxamido) ethyl] disulfide-9 × 3 39 to 47 disulfate 0.6 x 33 to 44 755 Q.} 6 x 3 60 P 1 S 34 (1 J) 1.2 x 3 0.9 x 3 0.6 x 3 67 to 68 34 to 54 29 to 34 big [| Si (4-deacetyl-VLB-C3- B16 1.6 x 3 1.2 x 3 0.9 x 3 0.6 x 3 0.6 x 3 90 to 99 62 or toxic doses 50-to 53 20 to 39 118 (7 µ-carboxamide J ethyl) disulfide-0.9 x 86 86 (7) disulfate 1 , 2 x 3 24 (3.) bisj.y- (4-deacetyl 1-VLB-C3- B16 0.6 x 3 99 (8) ikarboxainic) propyl] (1 - 0.9 x 3 61 :( 5. ) su Ifidd i sulphate 1,2 x 3 68 (8:] Legend: * p The number of indeterminate surviving specimens is indicated in parentheses. In the clinical use of the compound of Formula II ', the treating physician will initially administer the active ingredient of the invention by the same route, in the same vehicle, and possibly against the same types of tumors as vincristine or VLB. 'The variation in dosage should be taken into account in the difference in weight. the dosage levels found in experimental tumors will be ruled out, with the dose levels of the compounds of formula (II) generally being comparable to those used when administering vibiblistin and VLB against single-susceptible tumors or these compounds. In clinical trials, as with other anti-tumor agents, particular attention should be paid to the effect of the oncolytic compounds of formula (II) against "ten" "signaling tumors" on page 266 in "The Design N ° CMaloTrials in Cancer Therapy" (New Statuet, Publishing Publishing Company, 1S73J. New Method for Producing Di-C11 disulphide-indole-dihydroindole derivatives of the genus VENILA Vinca, bridged with the formula II, -R6-NH-CH 2 - (CH 2 N-Q -) (CH 3 -CH 3 -NH-R 6, (Mj, wherein the indole-dihydroindole alkaloid of the general R 6 is a residue of formula IIIa, 214793 13 14) wherein R 2 is methyl or hydrogen, one of R 3 and R 4 is hydroxy or hydrogen and the other is ethyl, and R 5 is hydrogen, or R 4 and R 5 together form an α-epoxide ring and R 3 is an ethyl group, and Q is an S-S group, and pharmaceutically acceptable acid addition salts thereof, characterized in that the azide of formula R6-N3 is reacted with a diamine of the formula NH2-CH2- (CH2) n-Q- (CH2) n Wherein Q is S-S, in a ratio of 2 moles of the compound of formula R6-N3 to each mole of the diamine of the above formula, and optionally converting the product into a pharmaceutically acceptable acid addition salt thereof. Severografia, n. P., Plant 7, Most