SE444684B - PROCEDURE FOR PREPARING 4-DESACETYL DERIVATIVES OF VINE CHRISTINE AND LEUROSIDINE - Google Patents

Description

Where R2 represents CH5 or CHO, and where one of the symbols RB and R represents H while the other represents C2H5, and pharmaceutically acceptable salts thereof. This process is characterized in that a 4-acetoxide derivative of dimeric indole-dihydroindole having the general formula H: (V) wherein R 2, R 3 and Ru have the meanings given above, is hydrolyzed under acidic or basic conditions in a reaction mixture, after which the product formed is recovered as a free base or as a pharmaceutically acceptable salt thereof.

Several naturally occurring alkaloids, which can be obtained from Vínca rosea, have been shown to be effective in the treatment of experimentally induced malignancies in animals. These alkaloids include leurosine (U.S. Pat. No. 3,370,057), vincaleucoblastine (vinblastine), hereinafter referred to as VLB (U.S. Pat. No. 3,097,157), leurosidine (vinrosidine) and leurocristine (VCR or vincristine) (both described in U.S. Pat. U.S. Patent 3,205,220), N'-deoxy-VLB "A" and "B", Tetrahedron Letters, 783 (1968) (also desacetylleurosine hydrazide is described in this article), 4-desacetoxivine blastine (U.S. Patent 3,954,773 ), Ä-desacetoxy-3'-hydroxyvinylblastine (U.S. Pat. No. 3, 4, 540), leurocolombin (U.S. Pat. No. 3,890,325), leuroformin (N-formylleurosine; Belgian Patent 8204238-5 811110) and vincadioline ( U.S. Patent No. 5,887,565). Two of these alkaloids, namely VLB and leurocristine, are now marketed as drugs for the treatment of malignancies in humans, especially leukemias and related diseases.

The dimeric indole-dihydroindole alkaloids obtainable from Vinca rosea can be represented by the following formula I: (I) VLB is the compound of formula I, where H1 represents acetoxy, RE represents methyl, R3 represents hydroxyl, Ru represents ethyl and R5 represents ethyl and R5 hydrogen. Vincristine is the compound of formula I, where R 1 represents acetoxy, R 2 represents formyl, R 3 represents hydroxyl, R 1 represents ethyl and R 5 represents hydrogen. Leurosidine is the compound of formula I, where R1 represents acetoxy, RE represents methyl, RB represents ethyl, Ru represents hydroxyl and R5 represents hydrogen.

H'-deoxy-VLB "A" is the compound of formula I, wherein R 1 represents acetoxy, R 2 represents methyl, R 3 and R 5 represent hydrogen and Ru represents ethyl. 4'-deoxy-VLB "B" is the compound of formula I, wherein R1, R2 and R5 have the same meanings as in 4'-deoxy-VLB "A", but where RB represents ethyl and Ru represents hydrogen. Leurosin is the compound of formula I, where Bi represents acetoxy, R 2 represents methyl, RB represents ethyl and Ru and R 5 together form an N-epoxide ring. 8204238-z 4 Of the above alkaloids, vincristine is the most useful but at the same time the one obtained in the least amount from Vinca rosea. U.S. Patent No. 5,899,495 discloses a method for converting the more abundant VLB to vincristine by oxidation with chromic acid at low temperature (-60 ° C). There are other relatively abundant alkaloids, such as leurosine, in the dimerindole dihydroindole fraction obtained from Vinca rosea, and it would be desirable if these alkaloids could be converted 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" (together with varying amounts of 4'-deoxy-VLB "A") by treatment with Raney nickel in refluxing absolute ethanol; see Neuss, Gorman, Cone, & Huckstep, Tetrahedron Letters, 783-7 (1968). Although leurosine has oncolytic activity against experimentally induced tumors in mice, clinical results have been limited. 4'-deoxy-VLB "A" and 4'-deoxy-VLB "B" are reported to have no reproducible effect against experimentally induced tumors in mice.

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

A compound of formula I, wherein H4 represents ethyl, B1 represents acetoxy, R2 represents CHO and R3 and R5 represent hydrogen, is called''-deox; - vincristine. A compound of formula I, wherein R 1 represents hydroxy and the other symbols have the meanings given above, is called 4'-deoxy-4-desacetylvincristine. Since the alkaloid corresponding to vincristine, which has the reverse configuration of hydrogen and ethyl in the i 'position compared to vincristine, is not known, the compounds in which H3 represents ethyl and n "represents hydrogen will be referred to as thiolsidine, which has the same configuration in the 4 'position as 4'-deoxy-VLB "B", and is called a derivative of 1-formylleurosidine, ie 4'-deoxy-1-formylleurosidine (or H'-deoxyepivincristine) and 4'- deoxy-4-desacetyl-1-formylleurosidine, where R1 represents acetoxy and hydroxy, respectively. In each of these compounds, of course, the 1-methyl group in leurosidine has been replaced by a formyl group, and the term "1-desmethyl" has been omitted to simplify the nomenclature.

The novel compounds prepared according to the invention may form pharmaceutically acceptable acid addition salts with non-toxic acids, e.g. inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitric acid, phosphoric acid and the like, or non-toxic organic acids such as aliphatic mono- and dicarboxylic acids, phenylsanicarboxylic acids, acids, aliphatic and aromatic sulfonic acids, etc. Specific examples of such pharmaceutically acceptable salts include sulphate, pyrosulphate, bisulphate, sulphite, bisulphite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, pyrophosphate chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, benzoate, chlorobenzoate, methyl benzoate, methyl benzoate hydroxybenzoate, methoxybenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylene sulfonate, phenyl acetate, phenylpropionate, phenylbutyrate, citrate, lactate, 2-hydroxybutyrate, glycolate, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate and naphthalene-2-sulfonate.

The compounds of formula V, wherein R represents formyl, can be prepared by oxidizing either U'-deoxy-VLB "A" or Ä-deoxy-VLB "B" with chromium trioxide and acetic acid at low temperature (-50 ° C to -65 ° c). The β-deoxy-VLB "A" and "B" and the 1-formyl derivatives thereof are hydrolyzed according to the invention to the corresponding 4-desacetyl derivatives under acidic or basic conditions.

In the preferred method of carrying out the hydrolysis reaction, sodium carbonate in methanol was used at reflux temperature.

Hydrazine hydrate can also be used. Other useful bases are potassium t-butoxide, sodium or potassium methoxide or ethoxide, pyridine, triethylamine (or any other tertiary amine), urea and the like, the hydrolysis being carried out in polar organic solvents such as lower alkanols. Diluted sodium and potassium hydroxide can also be used, e.g. in methanol, but precautions must be taken not to work with such base concentrations or reaction temperatures that other hydrolyzable groups in the molecule are affected. It is also possible to use bases which are active only in non-polar solvents, such as sodium or lithium hydride in benzene, ether, tetrahydrofuran, etc. or the sodium salt of dimethyl sulfoxide in dimethyl sulfoxide. Reaction temperatures between room temperature (25 ° C) and the boiling point of the solvent used can be used. The hydrolysis can also be carried out under acidic conditions, e.g. using absolute methanol saturated with anhydrous hydrogen chloride at 0 ° C. 8204238-3 The invention is illustrated by the following examples.

Example 1. Preparation of N'-deoxy-U-desacetyl-1-formylleurosidine and M'-deoxy-U-desacetyl-vincristine. 582 mg of chromium trioxide are dissolved in 5.8 ml of acetic acid and 0.6 ml of water.

The resulting oxidation solution is added dropwise over a period of 5 minutes 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 -5000. The reaction mixture is stirred at this temperature for about 30 minutes and then cooled to -6500, at which temperature the reaction is quenched by the addition of 12 ml of 1 N aqueous solution of ammonium hydroxide. The alkalized reaction mixture is then poured into 400 ml of a mixture of ice and water, and the aqueous layer is extracted with 150 ml of ether and then 3 times with 150 ml of chloroform each. The organic layers are combined and washed with a dilute aqueous solution of sodium bisulfite, separated and dried. After evaporation of the organic solvents, 4'-deoxyvinycristine is obtained as a residue. This product is purified by chromatography on 50 g of silica with activity I. It is eluted first with 300 ml of a mixture of ethyl acetate and methanol in the volume ratio of jl and then with 300 ml of a mixture of equal parts by volume of ethyl acetate and methanol. First, a 100 ml volume fraction is collected, and then 20 ml volume fractions are collected. Fractions 8-20 are combined, after which the solvents are evaporated. This gives 279 mg of a light yellow-brown solid, which consists of a substantially pure product (gives a single spot on thin-layer chromatography).

The free base of 4'-deoxyvincristine thus prepared has the following physical properties: Mass spectrum: m / e 808 (M +), 806.70? IR Spectrum: 3465, 1745, 1687, 1220 cm -1 1 'UV spectrum: 210, 222, 255, 290, 298 nm 10 mnz NMR Spectrum: methyl singlet at 3.88, 3.67 and 2.07 The prepared 4'-deoxyvinecristine is dissolved in acetone, and the resulting acetone solution is treated with 0.96 ml of a 0.55 M solution of sulfuric acid in absolute ethanol (2% by volume) to form a green solution, which is kept at approx. 0 ° C. overnight Crystallization is carried out by scraping or inoculation, and the solid, crystalline 4'-deoxyvinecristine sulphate is separated by filtration, the filter cake is washed with cold acetone, the sulphate salt is slightly soluble in acetone, so the filtrate is evaporated to dryness. after which the resulting residue is recrystallized from ethanol, the crystalline 4'-deoxyvinecristine sulphate is filtered off and the filter cake is washed with ethanol, the total yield of 4'-deoxivine crystalline sulphate is 266 mg.

* 8204238-3 Similarly, 4'-deoxy-1-formylleurosidine can be prepared by oxidizing 794 mg of 4'-deoxy-VLB "B" with 900 mg of chromium dioxide in 10 ml of glacial acetic acid and 1 ml of water. The residue obtained by evaporation of the oxidation mixture is found by thin layer chromatography to give a major spot and a minor spot plus traces of other components. After recrystallization of the residue in anhydrous ethanol, a crystalline material is obtained, which is isolated by filtration, after which the crystals are washed with cold ethanol. This material mainly gives a spot on thin layer chromatography.

The crystalline free base thus obtained is chromatographed on 50 g of silica. A mixture of equal volumes of methylene dichloride and ethyl acetate containing increasing amounts of methanol, namely 20, 30, 45 and 60% by volume, respectively, is used as eluent. The following elution scheme was used: System Volume 1: l 20% 200 ml 1: 1 30% 100 ml 1: 1 45% 100 ml 1: l 60% 400 ml The following eluate fractions are collected: Fraction Eluate volume 160 ml 100 ml 50 ml 50 ml 50 ml 120 ml 7 120 ml Fractions 4-7 are combined to give 597 mg of a yellow brown residue which, after crystallization from ethanol, gives 455 mg of white, crisis tallin 4 '-deoxy-1-formylleurosilin. This compound has the following physical properties: Mass spectrum: m / e 808 (M +), 806, 777, 775, 536, 158, 136.

IR_spectrum = ν (cHc15) 3470, 1743, 1690, 1222 um UV spectrum: (C2H5OH) 210, 222, 254, 290, 298 nm. 100 MHz NMR Spectrum: methyl singlets at 3.87, 3.65 and 2.076 ". PK'a = 9.0 and 4.9 (166% DMF).

The sulphate salt of this compound is prepared by dissolving 435 mg of the dfn free base in 10 ml of hot ethanol, after which 1.5 ml of a 2% solution of sulfuric acid in ethanol are added. Upon cooling, crystals precipitate 4'-deoxy-1-formylleurosidine sulfate. 8204238-38 (about 7H4 mg of 4'-deoxy-1-formylleurosidine are mixed with 10 ml of anhydrous methanol, and the resulting mixture is heated to reflux temperature, where the compound dissolves to give a clear solution. 200 mg of solid sodium carbonate is added, and the resulting reaction mixture is stirred for about 72 hours.After this time, thin layer chromatography of the crude reactants shows that virtually all of the 4'-deoxy-1-formylleurosidine has disappeared.The solvent is removed by evaporation, and the residue containing 4 '-deoxy-4-des-acetyl-1-formylleurosidine is partitioned between water and methylene dichloride.

The organic layer is separated and dried, after which the solvent is evaporated. 506 mg of a white solid are obtained, which consists of substantially pure 4'-deoxy-4-desacetyl-1-formylleurosidine.

The compound prepared has the following physical properties: Mass spectrum: m / e 766 (M +), 764, 755, 254, 252, 205, 158.

IR spectrum; v (cHc1¿) 5450, 1754, 1680, 1596, 1495, 1456, 1454 and 100 Mnz NMR spectrum: (ccci) N-formyl v1d <í8,80, methyl-singietcr at 5.89 (clo-ocnj) den 5.66 (cla-cozcnñ), broad muitipieu: at 5.82 (05-00201113), and no N-c fl j about 2.75 (or ococnj about 2.06). The corresponding sulphate salt is prepared in the same manner as above using acetone as solvent and using 0.26 ml of a 2% solution of sulfuric acid in ethanol. Other solvents can also be used, and it is preferred to use a solvent in which the base is readily soluble but the sulphate salt is substantially insoluble. ''-deoxy---desacetylvincristine and its sulphate salt are prepared in a completely analogous manner from H'-deoxyvinycristine.

Example 2. Preparation of N'-deoxy-H-desacetylleurosidine A reaction mixture consisting of 1.48 g of U'-deoxy-VLB "B", 1 g of sodium carbonate and 100 ml of methanol was refluxed in a nitrogen atmosphere. After 2 hours, a sample of the reaction mixture was taken and analyzed by thin layer chromatography. It was found that the hydrolysis reaction for removing the 4-acetyl group had proceeded to about 50%. After allowing the reaction mixture to stand overnight at room temperature, it was refluxed under reflux for 8.5 hours. A sample of the reaction mixture was then analyzed by thin layer chromatography using a mixture of ether, diethylamine, toluene and methanol in the volume ratio of 20: 1: 1: 1 as solvent system.

The analysis showed that the reaction was complete. The solvent was evaporated from the reaction mixture, and the resulting residue was dissolved in a mixture of methylene dichloride and water. The methylene dichloride phase was separated and dried. After evaporation of the methylene dichloride, a residue was obtained which on thin layer chromatography was found to consist of a highly polar substance plus the desired 4'-deoxy-4-desacetylleurosidines. The residue, which weighed 1.33 g, was dissolved in benzene. The highly polar material was essentially insoluble in benzene and was separated by filtration. The filtrate was evaporated to dryness, and the residue (500 mg) of chromium was tographed on Woelm's silica gel, eluting with a mixture of ether, diethylamine and toluene in a volume ratio of 20: 1: 1 containing increasing amounts of methanol. The course of chromatography was monitored by thin layer chromatography, joining the fractions which were found to contain 4'-deoxy-4-desacetylleurosidine. After evaporation of the solvent, 348 mg of the desired base were obtained. This residue was treated with 1.28 ml of a 2% (0.6 M) solution of sulfuric acid in methanol and the resulting solution was filtered to give 315 mg of 4'-deoxy-4-desacetylleurosidine sulfate. 4'-Deoxy-4-desacetylleurosidine had the following physical properties: Mass yield: m / e 752 (M +), 750, 693, 691, 555, 338, 240, 138.

IH spectrum: Mcuciñ) 3455, 1724, 1610, 1497, 1457, 1431 et al. 10 MHz NMR-.spexfcvumnfgmnglzà 9.43 (m-s, 1, 03-011), 7.92 (br s, 1, indole NH), 7.47-7.63 (m, 1, C11, -H ), 7.06-7.51 (m, 3, Cl 2, _lu, -H) 6.58 (S, 1, Cl 4 -H), 6.10 (s, 1, C 17 -H), 5.78 -5.87 (m, 2, 06.7-H), 4.10 (m, 1, (rr-H), 3.83 (s, 3, 0176-00115), 3.78 (s, 3 , 050020115), 3.70 (s, 1, 02-11), 3.58 (s, 3, cmfcoac fl š), 2.75 (s, 3, N-c fl j), o, 76-1, o6 (m 4'-dexy-4-desacetyl-VLB can be prepared in the same manner as above by hydrolyzing 4'-deoxy-VLB "A".

The novel compounds prepared according to the invention are potent anti-tumor agents. The effect of the new compounds has been demonstrated in experiments with mice in which tumors have been inoculated. The tested compounds were administered intraperitoneally at a given dose for 7-10 days after inoculation of the tumor or alternatively on the first, fifth and ninth days after inoculation.

The results obtained are summarized in the table below.

The table shows in column 1 the test compound, in column 2 the transplanted tumor, in column 3 the dose of the test compound and the number of days this dose was administered, and in column 4 the percentage inhibition of tumor growth or the percentage prolongation of survival , such as in samples with the tumor B16. The tumor designations given in the table have the following meanings. GLS is an abbreviation for Gardner's lymphosarcoma, CA755 is an adenocarcinoma and B16 is a melanoma. iO 8204238-3 cwwcwcnëh àwuwm cunwhv m flfifi nun wum w fi pu fl w ww w fi pw fl w «. >> ..noH ..w fi» @ fi w mn ßn w fi ph fl w mm ooH - # @ w fl uw fl w w www! HR: m-wm w fl pw fl m cwnHpwvmc> m fl p @> n> m mc fl ømcm fl pmu H fl wspcwoopm nw fifi w cmuxm> HHH »nmEsu> m mc fl c fi mc Hamsucmoonm nx âqo nx nx wx nx. .onx mH.ø mwo: .o-mm.omx @ .onx @ .o-m fi. onx mN.o | wH.omx @ .o - #. o: www x mx \ uE w fl o mm> WQO w fl m nmasæ. mn mn wn flfi wnmnmm .. ¶ uauaøwc fl v fl w | on5mHH> umumwao | n | fl Nomu | \ u pmuaswc fl o fl w nouswa fl menowu fl n fl æumemmv | fl | fi hpwomww © | zu de4 Nom 8 | either parenteral or oral administration. For oral administration, a suitable amount of a pharmaceutically acceptable salt formed from a base of formula IV and a non-toxic acid, such as the sulphate salt, is mixed with starch or other excipient, after which the mixture is poured into gelatin capsules, each containing 7.5 -50 mg of the active ingredient. The anti-neoplastic active salt can also be mixed with starch, a binder and a lubricant, after which the resulting mixture is compressed into tablets, each containing 7.5-50 mg of the active salt. The tablets can be cut if lower or divided doses are desired. However, parenteral administration is preferred. For this purpose, isotonic solutions containing 1-10 mg / ml of a salt of an indole-dihydroindole of formula IV, such as the sulfate salt, were used. The compounds are administered in an amount of 0.01-1 mg per kg of body weight, preferably 0.1-1 mg / kg, once or twice a week or every other week, depending on both the activity of the administered compound and its toxicity. An alternative method for determining a therapeutic dose is based on the body surface area, administering the active compound in an amount of 0.1-10 mg per m2 of body surface area every 7 or 14 days.

When the new compounds are used clinically, physicians can administer the compounds in the same manner and in the same vehicle hitherto used for vincristine or VLB, and the new compounds can probably be used to treat the same types of tumors that have hitherto been treated with vincristine or VLB. The doses used should reflect the differences in effective doses found in the treatment of experimental tumors in mice, and the doses of the new compounds are less than the doses of vincristine and VLB used. As with the use of other anti-tumor agents, special attention should be paid in clinical trials to the action of the new oncolytic compounds against the ten "signal" tumors listed on page 266 of the book "The Design of Clinical Trials in Cancer Therapy", published by Staquet (Futura Publishing Company, 1975).

Claims (1)

A process for the preparation of an H-desacetyl derivative of dimer indole-dihydroindole of the general formula: (IV) 1 CO-CH 3 NO wherein H 2 represents CH 3 or CHO, and wherein one of the symbols RB and Ru represents H and the other represents C2H5, as well as pharmaceutically acceptable salts thereof, characterized in that a β-acetoxy derivative of dimeric indole-dihydroindole having the general formula: 8204238-3 wherein R2, RB and Ru have the the meanings given above are hydrolyzed under acidic or basic conditions in a reaction mixture, whereupon the product formed is recovered as a free base or as a pharmaceutically acceptable salt thereof.