SE425852B - ANALOGY PROCEDURE FOR PREPARING DERIVATIVES OF VINE CHRISTINE AND LEUROSIDINE - Google Patents

Description

781 555-0 2 where R1 represents OH or O-CO-CH3, and where one of the symbols R3 and Ru represents H while the other represents C2H5, and pharmaceutically acceptable salts thereof. This process is characterized in that a 1-methyl derivative of dimeric indole-dihydroindole of the general formula: 5 'R 6 (III) wherein R 1, R 2 and Ru have the meanings given above, is reacted with chromium trioxide and acetic acid in a reaction mixture at a temperature between - 5000 and -6500, 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 Vinca rosea, have been shown to be effective in the treatment of experimentally induced malignancies in animals. Among these alkaloids are leurosine (U.S. Pat. No. 3,370,057), vincaleukoblastine (vinblastine), hereinafter referred to as VLB (U.S. Pat. No. 5,097,137), leurosidine (vinrosidine) and leurocristine (VCE or vincristine) (both described 1). U.S. Pat. No. 3,205,220), 4'-deoxy-VLB "A" and "B", Tetrahedron Letters, 783 (1968) (also desacetylleurosine hydrazide is described in this article), 4-desacetoxivine blastin (U.S. Pat. No. 3,954,773). , 4-desacetoxy-5'-hydroxyvinylblastine (U.S. Pat. No. 3,944,554), leurocolombin (U.S. Pat. No. 3,890,525), leuroformin (N-formylleurosine; Belgian Pat. No. 811,110) and vincadioline (U.S. Pat. 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 indole-dihydroindole alkaloids obtainable from Vinca rosea can be represented by the following formula I: VLB is the compound of formula I, wherein R 1 represents acetoxy, R 2 represents methyl, R 5 represents hydroxyl, R "represents ethyl and R 5 represents ethyl and R 5 represents Vincristine is the compound of formula I, wherein R 1 represents acetoxy, R 2 represents formyl, R "represents hydroxyl, R represents ethyl and H 5 represents hydrogen. Leurosidine is the compound of formula I, wherein B1 represents acetoxy, R2 represents methyl, R3 represents ethyl, R "represents hydroxyl and R5 represents hydrogen. 4'-deoxy-VLB" A "is the compound of formula I, where R1 represents acetoxy, H2 represents methyl, R3 and R5 represent hydrogen and R "represents ethyl. 4'-deoxy4VLB "B" is the compound of formula I, wherein R1, H2 and H5 have the same meanings as 1,4'-deoxyJVLB "A", but where R) represents ethyl and R "represents hydrogen.

Leurosine is the compound of formula I, wherein R 1 represents acetoxy, R 2 represents meuyl, H 5 represents euyl and R "and H 5 together form an α-epoxide ring.

Of the above alkaloids, vincristine is the most useful but at the same time the one obtained the least amount from Vinca rosea. U.S. Pat. No. 3,899,493 discloses a method for converting the more abundant VLB to vincristine by oxidation with low temperature chromic acid (-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 directly or indirectly converted to vincristine or to a drug of comparable oncolytic value. can. 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, 785-7 (1968). Although leurosine has oncolytic activity against experimentally induced tumors in mice, clinical results have been limited. 4'-deoxy-VLB "A" and 4'-deoxy4VLB "B" are reported to lack reproducible activity against experimentally induced tumors in mice.

It is an object of the present invention to convert leurosin via 4'-deoxy-VLB "A" and "B" to oncolytically active derivatives of 4'-deoxy-VLB "A" and "B", thereby indirectly converting it relatively abundantly. the alkaloid leurosine present to a drug with greater potential clinical utility. A compound of formula I, wherein R 1 represents ethyl, R 1 represents acetoxy, H 2 represents CHO and R 3 and R 5 represent hydrogen, is called 4'-deoxyvinycristine. A compound of formula I, wherein H 1 represents hydroxy and the other symbols have Since the above meanings are called 4'-deoxy-4-desacetylvincristine, since the alkaloid corresponding to vincristine, which has the reverse configuration of hydrogen and ethyl in the 4 'position compared to vincristine, is not known, the compounds , where R represents ethyl and R "represents hydrogen, at: refers to * C111 eurosidine, which has the same configuration in the H 'position as 4'-deoxy-VLB" B ", and is called a derivative of 1-formylleurosidine, i.e. 4'-deoxy-1-formylleurosidine (or 4'-deoxypivincristine) and 4'-deoxy---desacetyl-1-formylleurosidine, where R 1 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, phenyl-substituted alkanoic acid acids, hydroxycarboxylic acids, alkanoic acid and alkanoic acid aromatic sulphones, etc. Examples of such pharmaceutically acceptable salts are sulphate, pyrosulphate, bisulphate, sulphite, bisulphite, nitrate, phosphate, hydrogen monophosphate, 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, dinitrobenzoate, 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 novel compounds of formula II, in which H1 represents acetoxy, are prepared according to the invention by oxidizing either''-deoxy-VLB "A" or 4'-deoxy-VLB "B" with chromium trioxide and acetic acid at low temperature (-5 ° C). to -65 ° C).

M'-deoxy-VLB "A" and "B" can be hydrolyzed to the corresponding--desacetyl derivative under acidic or basic conditions. These des-acetacetyl derivatives of δ-deoxy-VLE "A" and "B" can then be oxidized at low temperature (-6000) with chromium trioxide without converting the M-hydroxy group to a ketone. '' -Deoxy-H-desacetyl-vincristine and H'-deoxy-H-desacetyl-1-formylleurosidine are obtained.

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 so as not to work with such base concentrations or reaction temperatures that other hydrolyzable groups of 1'-4-deoxyvinycristine or 4'-deox1-1-formylleurosidine are affected. It is also possible to use bases which are active in only 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.

The invention is illustrated by the following examples. Example 1. Preparation of H-deoxyvinycristine and 4'-deoxy-, 1-formylleurosidine. 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" 1.58 ml of acetone and 2.9 ml of glacial acetic acid at a temperature of about -5000 ml. The reaction mixture is stirred at this temperature for about 50 minutes and then cooled to -6500, at which temperature the reaction is stopped by adding 12 ml of 14 N aqueous ammonium hydroxide solution. 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 a volume ratio of 3: 1 and then with 500 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, whereupon 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 N'-deoxivine cristin thus prepared has the following physical properties: Mass spectrum: m / e 808 (M +), 806,707 IR spectrum; 3465, 1745, 1687, 1220 cm -1 UV spectrum: 210, 222, 255, 290, 298 nm 100 MHz NMR spectrum: methyl singlets at 3.88, 3.67 and 2.07 J.

The prepared 4'-deoxivine cristine is dissolved in acetone, and the resulting acetone solution is treated with 0.96 ml of a 0.56 M solution of sulfuric acid in absolute ethanol (2% by volume). A green solution is formed, which is kept at about 0 ° C overnight. Crystallization is accomplished by scraping or inoculation, and the solid crystalline 4'-deoxyvinycristine sulfate 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 M'-deoxyvinecristine sulfate is filtered off, and the filter cake is washed with ethanol. The total yield of 4'-deoxyvinycristine sulfate is 266 mg. Similarly, 4'-deoxy-1-formylleurosidine can be prepared by oxidizing 794 mg of 4'-deoxy-VLB "B" with 900 mg of chromium trioxide to 10 ml of glacial acetic acid and 1 ml of water. The residue obtained by evaporation of the oxidation mixture is shown by thin layer chromatography on 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 of methanol, is used as eluent. The following eluents were used: System Xglvm lzl 20% 200 ml 1: 1 30% 100 ml 1: 1 45% 100 ml lzl 60% 400 ml The following eluate fractions are collected: Fraction Eluate volume l 160 ml 2 100 ml j 50 ml 4 50 ml 50 50 ml 6 120 ml 7 120 ml Fractions 4-7 are combined to give 597 mg of a yellow-brown residue, which after crystallization from ethanol gives 435 mg of white, crystalline 4'-deoxy-1-formylleurosilin, This compound has the following physical properties: less spectrum: m / e 808 (W), 806, 777, 775, 356, 138, 136. iii spectrum: Mcnciš) 3470, 1745, 1690, 1222 cm life spectrum: (cgii5o fl) 210, 222, 254, 290, 298 nm. 100 MHz NMR Spectrum: methyl singlets at 5.87, 5.65 and 2.076 - pwa = 9.0 and 4.9 (166 μm).

The sulphate salt of this compound is prepared by dissolving 435 mg of the free base in 10 ml of hot ethanol, then adding 1.5 ml of a 2% solution of sulfuric acid in ethanol. Upon cooling, crystalline 4'-deoxy-1-formylleurosidine sulfate precipitates. yvsoosss-o 8, Example 2. Preparation of 4'-deoxy-4-desacetyl-1-formyl-leurosidine.

A reaction mixture consisting of 1.48 g of 4'-deoxy-VLB "B", 1 g of sodium carbonate and 100 ml of methanol was refluxed under 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 the reaction mixture has been allowed to stand overnight at room temperature, it is allowed to cool again: at a temperature of 1 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-desacetylleurosidine. The residue, which weighed 1.33 g, was dissolved in benzene. The highly polar material was essentially insoluble in benzene and separated by filtration. The filtrate was evaporated to dryness, and the residue (500 mg) was chromatographed on woelm silica gel, eluting with a 20: 1: 1 mixture of ether, diethylamine and toluene 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% (0.36 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: Maes spectrum: m / e 752 (W), 750, 693, 691, 555, 338, 240, 138.

IR spectrum: V (CHCl 3) 3455, 1724, 1610, 1497, 1457, 1431 cm-1. ioo MHz Nx fl n-spectrafndfflfflgl 9.43 (m- e, i, cñ-on), 7.92 (br s, 1, indoi NH), 7.1 + 7-7.63 (m, 1, C11, - H), 7.06-7.31 (m, 3, cl 2, 14, -H), 6.58 (S, 1, C 14 -H), 6.10 (S, 1, C 17 -H), 5.78-5.87 (m, 2, C 6.7-H), 4.10 (m, 1, Cu-H), 3.83 (s, 3, C 16 -OCH 5), 3.78 (s , 3, C) -COQCHB), 3.70 (s, 1, C2-H), 3.58 (S, 3, C18, -002053), 2.75 (S, 5, N-CH5), 0 , 75-1.05 (N, 6, C 41). 21,21 '7800555-0 9 Combined 4'-deoxy-4-desacetylleurosidine obtained from the filters and the solid filtered material was obtained to obtain 834 mg of combined product. The combined material probably contained 30-40% of the above highly polar substance. The combined material was dissolved in 100 ml of acetone containing 7 ml of acetic acid. The resulting solution was stirred for 15 minutes at room temperature and then cooled to -65 ° C in a bath of dry ice and acetone under a nitrogen atmosphere. 110 mg of chromium trioxide was dissolved in 13 ml of glacial acetic acid and 2 ml of water. The resulting solution was added dropwise to the solution of 4'-deoxy-4-desacetylleurosidine. The reaction mixture was stirred for 1 hour at a temperature between -60 and -65 ° C, after which the reaction was quenched by the addition of 35 ml of 1M aqueous ammonium hydroxide solution. . The reaction mixture was then poured onto ice, and the resulting aqueous suspension was extracted several times with chloroform. The chloroform extracts were combined, washed with water and dried. After removal of the chloroform in vacuo. 794 mg of a residue were obtained, which on thin layer chromatography gave essentially a spot in addition to the original impurity with a very low Rf value. The residue was chromatographed on Woelm silica gel, eluting with a mixture of ethyl ether, diethylamine and toluene in a volume ratio of 20: 1: 1, which mixture originally contained 0.9% methanol. The eluent was used in 150 ml portions. The methanol content was increased for each 150 ml eluent portion, and the final methanol concentration was 15%. The fractions which on thin layer chromatography were found to contain 4'-deoxy-4-desacetyl-1-formylleurosidine were combined. After evaporation of the solvent, 293 mg of pure 4'-deoxy-4-desacetyl-1-formylleurosidine were obtained.

The compound prepared has the following physical properties: Mass spectrum: m / e 766 (M +), 764, 735, 254, 252, 205, 158.

IR spectrum: δ (cnc15) 3450, 1734, 1680, 1596, 1h95, 1456, mean 100 MHz NMR spectrum: (CDCl 3) N-formyl at δ §.80, methyl singlets at 3.89 (C16- OCH 5) and 5.66 (C 18 -COECH 3), broad multiplet at 5.82 (G 3 -COECH 3), and no N-CH; about 2.75 (or OCOCH3 about 2.06).

The corresponding sulphate salt is prepared in the same manner as in the previous example using acetone as solvent and using 0.26 ml of a 2% solution of sulfuric acid in ethanol. Other solvents may 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. H'deoxy---desacetylvincristine can be prepared in the same manner as above by first hydrolyzing Ä'-deoxy-VLB "A" to form 4'-deoxy-4-desacetyl-VLB and then oxidize the compound with CrO3 in acetic acid at -6000.

The novel compounds prepared according to the invention are potent anti-tumor agents, in particular those compounds of the formula II in which the action of the novel compounds has been demonstrated. The Rs tested represent acetoxy. in experiments with mice in which tumors have been inoculated. the compounds were administered intraperitoneally at a given dose for 7-10 days after the inoculation of the tumor or alternatively on the first, fifth and ninth day after the 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 5 the dose of the test compound and the number of days this dose was administered, and in column Ä the percentage inhibition of the tumor growth or the percentage elongation of survival time, such as in samples with the B16 tumor. The tumor designations given in the table have the following meanings. ROS is an abbreviation for Ridgeway's osteogenic sarcoma, GLS is an abbreviation for Gardner's lymphosarcoma, P1534 (J) and L121O are leukemias, CA755 is an adenocarcinoma and B16 is a melanoma. 7800555-0 mm ooH | nm w «» «fi w., Nw | fl n: m | mw w fi pw fl w oo fl lmn w fi pw fl w ooH | mH w fi pw fl w i fi dalmñ w fl wm fi w mm øo fl .mw w. nm-nn cm © wuwumc> a fl ua> m> m wø fl cmmm fl nmm H fl wsunwoona nm fifi w cwuxm> H fi wunmEsv> m wc fl cñms H fi wsn fl møomm Gmwc fl nash hwuwm cacwæø mnm MN m Q nO mN m Q xo. .

O (J OCDCJCIO CIO OOO 0 OOOOOOOOOOOOOOM www nmmmmnwmno mu> w fl & w> w n f wv WHM fl u hmaaw »am u MN mH« O et al o f f m et al ø wv fi nm fl m wvàmm fl m MMW w fl m hæë flfi m .wum mn toilet flfi nnmnwm «« VMM al SSW flfl uww | ohdwHHmEh0w | fi | HhumEww © | H | H% uwummwu |: | fi Nomø | \: ßMM fl5 w fl HUW flfi ¥ flfi> fi NOwUl \ # umm flfl wc fl n fi wonømaah fi howu fi HhwwEmwU | H | wMnmv | \ # ^ mmn wnwv nwn fl mh hm | | For the oral compounds, a suitable amount of a pharmaceutically acceptable salt formed from a base of formula II and a non-toxic acid, such as the sulfate salt, may be used with starch or some other excipient, after which the mixture is poured into gelatin capsules, each containing 7.5-50 mg of the active component.The anti-neoplastic active salt can also be mixed with starch, a binder and a lubricant, after which the resulting mixture squeezed into tablet r, 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 II, 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 of testing a therapeutic dose is based on 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 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 a 1-formyl derivative of dimeric indole-dihydroindole having the general formula: 6 '5'R3 7' N // 4, in --- n ll 8. 19 3 '10' (II) wherein R1 represents OH or O-CO-CH3, and wherein one of the symbols H3 and Ru represents H and the other represents C2H5, and pharmaceutically acceptable salts thereof, characterized in that a 1-methyl derivative of dimeric indole-dihydroindole of the general formula: (111) III - than 1; 11 / few CH; * _ .H..cH¿ / en c 12 Ilšl RI 3 nm \ N _ | 2 CH? E1-o-c 78 j 7800555-0 1H, where R1, H5 and Ru have the meanings given above, are reacted with chromium trioxide and acetic acid in a reaction mixture at a temperature between -50 ° C and -6500, after which the product formed is recovered as free base or as a pharmaceutically acceptable salt thereof. MENTIONED PUBLICATIONS: