DK142815B - Analogous process for the preparation of 4-desacetoxyquincristine or acid addition salts thereof. - Google Patents

Description

s yp.a (11) PUBLICATION WRITING 1 U28 1 5 DENMARK <0,) lm Cl '0 07 0 519/04' (21) Application No 787/77 (22) Filed on 23 * f®b. 1977 (24) Race day 23 · Feb. 1977 (44) The application submitted and the petition published on 2 February. 1 981

DIRECTORATE OF

PATENT AND TRADEMARK (3 °) Priority commits * from it

Feb 24 1976, RI 586, HU

(7i) DIRECTORY OF VEGYESZETI GIAR RT., 21 Gyoeraroi u., Budapest X, HU.

(72) Inventor: Karola Jovanovies, 47 Benezur u., Budapest XII, HU: Lajos Danesi, 18 Szendro u., Budapest XII, HU: Sandor Eckhardt, 2 H & ttyu u., Budapest II, HU: Csaba Lorincz, 28 Teglaveto u. -, "Budapest X, HU:

Janos Sugar, Matroz u. B, Budapest III, HU: Zsuzsa Relle, 7-9 Rath Gy., U., Budapest XII, HU: Kalman £ zasz, 67/69 Pasareti u., Budapest II, HU: Jozsef Tamas, 2 Fuerdohely u., Budapest XVI, HU: Aron Szollosy, 13 Hogyes u., Budapest XI, HU. "" (74) Plenipotentiary in the proceedings:

Office for Industrial Excellence by Svend Schønning.

(64) Analogous process for the preparation of 4-desacetoxyvincristine or acid addition salts thereof.

The present invention relates to an analogous process for the preparation of a novel diindole alkaloid derivative, namely 4-desacetoxyquincristine having the formula set forth in the preamble of the claim, or acid addition salts thereof.

It is known that certain bialkaloids with diindole structure from the plant Vinca rosea L. have cytostatic properties (see United States Patent Nos. 3 097 137, 3 205 220, 3 887 565 and 3 890 325 and Hungarian Patent Nos. 153 200, 154 715 and 160,967). Active semi-synthetic derivatives, for example N-formylleurosine (Hungarian Patent No. 165,986) as well as various carboxamide and desacetyl derivatives (German Patent Nos. 1,795,763 and 2,415,890) as well as 4-desacetoxyvinblastine (N. Neuss, AJ Barnes and LL Huckstep, Experimentia 31/1, 18-19, 1975).

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It has now been found that 4-desacetoxyvinblastine, having the formula shown in the characterizing claim, can be converted by oxidative formylation to a novel derivative, 4-desacetoxyvinincristine; this compound exhibits excellent cytostatic properties and is less toxic than the similarly effective vineristin.

The tumor inhibitory effect of this novel compound was investigated on various transplantable tumors. The effect against the vinca-sensitive lymphoid leukemia L 1210 / VS and the vinca-refractive lymphoid leukemia L 1210 / ref in mice was tested on BDF ^ mice, the effect against Ehrlich's ascites sarcoma was tested on Swiss mice and the effect against Yoshida sarcoma and Novikoff. hepatoma was tested on rats of the Wistar strain. 4-Desacetoxyvincristine sulphate was administered in physiological saline solution once daily intraperitoneally to the test animals; the tumor inhibitory effect as well as the average life span of the animals were determined in comparison with untreated control animals. The results obtained are summarized in Table 1.

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The table shows that the average life span of the animals treated with doses of 0/1 to 1.0 mg / kg of 4-desacetylvincristine sulfate interperitoneally was 70-190% longer than the life span of the control animals infected with the same treatment with tumors but not treated with 4-desacetylvincristine sulfate. The effect of 4-desacetylvincristine sulfate depends on the size of the doses administered; the tumor inhibitory effect was first and foremost against Ehrlich's ascites sarcoma and against the Yoshida sarcoma, but significant effects could also be established against the Novikoff hepatoma. The quotient T / c in the table shows the ratio of the average survival time of the treated and the untreated animals.

When examining the toxicity of the subject compound, it was found that 4-desacetoxyvincristine is approx. 25% less toxic than the effective vineristin similar in the same doses. It is a significant advantage of the compound provided by the process that the frequent neurotoxicity associated with paralysis symptoms associated with vineristin does not even occur when lethal doses of 4-desacetoxyvincristine are administered. The approximate values for the acute toxicity (LDr_) of 4- ou desacetoxyvineristin and of vineristin are given in Table 2 below.

Table 2

Compound LD50 4-Desacetoxyquincristine (in mice, ip) 5 4-desacetoxyquincristine (in mice, iv) 6 4-desacetoxyquincristine (in rats, ip) 1.3 vineristin (in mice, ip) 4.2 vineristin (in rats, ip) 1.3 ip = interperitoneal i.v. = intravenous 5 In the human therapy, 4-desacetoxyvincristine can be administered, in particular, intravenously or by infusion and at doses of 0.01-0.03 mg / kg, advantageously 0.05-0.02 mg / kg per day, possibly. divided into smaller doses.

The process of the invention for the preparation of 4-desacetoxyvincristine or acid addition salts thereof is characterized by formylating 4-desacetoxyvinblastine oxidatively, if desired, purifying the product obtained and then, if desired, converting the obtained 4-desacetoxyvincristine into a physiologically acceptable acid addition.

In the process according to the invention, 4-desacetoxyvinblastine containing an N-methyl group is obtained by oxidative formylation 4-desacetoxyvinccristine containing an N-formyl group. By oxidative formylation here is meant simultaneous or successive execution of oxidation and formylation.

If the formylation is carried out after the oxidation, a mixture of 4-desacet-oxyvincristine and N-desmethyl-4-desacetoxyvinblastine is obtained by the oxidation as intermediate. Possibly. For example, this mixture can be subdivided into the individual components, whereupon by formulation it is possible to convert the N-desmethyl-4-desacetoxyvinblastine to the desired 4-desacetoxyvinecristine, but suitably subject immediately to the oxidation product obtained in the process whereby the 4- desacetoxyquincristine remains unchanged and the N-desmethyl-4-desacetoxyvinblastine is also converted to 4-desacetoxyquincristine.

If the reaction is carried out with the simultaneous presence of both the oxidizing agent and the formylating agent, then the desired 4-desacetoxyvincristine is immediately recovered as a single reaction product.

The oxidation may be carried out with an oxidizing active acid or with the anhydride or a salt thereof, or with oxygen or air and in the presence of a catalyst suitable for transferring oxygen.

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As oxidizing acid / eg nitric acid and especially chromic acid (CrO3) or alkali metal dichromates, eg potassium or sodium dichromate in consideration.

As catalysts suitable for the transfer of oxygen, metals of the platinum group, e.g., platinum, palladium or rhodium, can be advantageously used in a form where they are applied to a support.

In particular, formic acid is used as formylating agent.

In an appropriate embodiment of the process, 4-desacetoxyvinblastine is dissolved which, if desired, can be released immediately before the reaction from an acid addition salt - in an inert organic solvent or in the formulation agent itself, optionally in a mixture of these two agents. As the reaction medium, one can use the formylating agent itself, suitably formic acid, or a hydrocarbon of the benzene series such as toluene or xylene, particularly suitably using benzene or a mixture of formic acid and an organic solvent.

To the solution is then added a catalyst suitable for transferring oxygen, e.g., a noble metal of the platinum group to a support, advantageously palladium on activated carbon or platinum sod. In particular, the oxidation is carried out at a temperature of 0 ° -30 ° C, especially at room temperature and in the oxygen atmosphere, preferably under a pressure of 1-3 atmospheres of oxygen pressure. After completion of the reaction, the catalyst is removed by filtration and the pH of the filtrate is suitably adjusted by an inorganic base, suitably ammonia to 7-10, preferably 8.5-9. The solution thus made alkaline can be extracted with an inert organic solvent, for example benzene, toluene or o-xylene, or with a chlorinated hydrocarbon, especially dichloromethane or chloroform. The product obtained by evaporation of the extract can be purified by recrystallization, conveniently from a mixture a low boiling alcohol such as methanol, ethanol or propanol and water. The 4-desacetoxyvincristine obtained may optionally be converted into an acid addition salt, in particular sulfate salt, in the usual manner.

In another advantageous embodiment of the process of the invention, 4-desacetoxyvinblastine is dissolved in a low boiling aliphatic ketone, such as, for example, acetone, methyl ethyl ketone or methyl isobutyl ketone. As the oxidizing agent, chromic acid or an alkaline steel dichromate is suitably used; the oxidation is carried out in the presence of glacial acetic acid and acetic anhydride at temperatures below 0 ° C, preferably between -90 ° C and -30 ° C. After oxidation 142815 7

Finally, the pH of the reaction mixture is adjusted to 5-10, which is conveniently carried out in two steps, first to pH

5-7, and then to pH 8-9. In this case, as a product of oxidation, a mixture of 4-desacetoxyvinecristine and N-desmethyl-4-desacetoxyvinblastine is obtained. This mixture can be divided into individual components, for example by column chromatography, and the N-desmethyl-4-desacetoxyvinblastine is formulated separately, but suitably the mixture obtained as an oxidation product is immediately subjected to the formylation process, whereby 4-desacetoxyvincristine is obtained.

The formylation can be carried out in an inert solvent reaction, for example in a hydrocarbon of the benzene series. Suitably, however, the formylating agent itself, in particular a mixture of formic acid and acetic anhydride, is used as the reaction medium. The formylation is carried out at a temperature between 0-15 ° C advantageously at 0 ° -5 ° C and then the pH of the reaction mixture is adjusted to 7-10, advantageously 8-9.5. The 4-desacetoxyquincristine obtained can be extracted from the mixture thus made alkaline, with an inert organic solvent and suitably with a hydrocarbon of the benzene series, for example with benzene, toluene or o-xylene, or with a chlorinated hydrocarbon, advantageously dichloromethane or chloroform, and then the crude product isolated by evaporation can be purified by, for example, column chromatography or recrystallization.

For the chromatography, partially deactivated alumina (conveniently of Activity III) can be used as adsorbent and an inert organic solvent, for example benzene, toluene or o-xylene and / or a chlorinated hydrocarbon, suitably dichloromethane or chloroform as eluant. Of the eluate fractions containing the major amount of 4-desacetoxyvincristine, the product can be recovered immediately by evaporation of the combined fractions, while the eluate fractions collected before or after these main fractions can first be evaporated, after which the evaporation residues can be subjected to further chromatographic purification.

The products thus obtained may optionally, separately or combined, once again be recrystallized and / or converted into an acid addition salt, advantageous sulfate salt.

4-Desacetoxyquincristine and / or acid addition salts thereof may, in therapeutic use, generally be administered parenterally. For this purpose, injection or infusion solutions containing the active substance dissolved in a solvent suitable for parenteral administration are advantageously prepared. As a solvent, distilled water or physiological saline solution comes to mind as well as low-boiling aliphatic alcohols or glycolets. The active ingredient may also advantageously be prepared in lyophilized form, in vials for injection preparations, whereby the necessary sterile solvent is delivered by simultaneous separate ampoules, and the active substance dissolved in such a solvent immediately prior to administration. The solutions or solvents in question may contain various usual additives such as preservatives such as benzyl alcohol or p-oxybenzoic acid esters, antioxidants such as ascorbic acid or tocopherol, lactose, buffering agents and / or additional pharmacologically active substances such as local anesthetics. Solutions or solvents can be sterilized, for example, by filtration.

The process according to the invention will now be described in more detail by some exemplary embodiments. The optical turns given in the examples were measured in an Opton Polarimeter; the proton magnetic nuclear resonance spectra were recorded using the Varian EM 360 spectrometer.

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Example 4 4-Desacetoxyquinristine 12 g (0r01.59 mol) of 4-desacetoxyvinblastine are dissolved in 2640 ml of acetone and then 600 ml of chromic acid is freshly distilled off with glacial acetic acid. The mixture is cooled to -55 ° C and, with stirring, a cooled mixture of 5.94 g (0.135 mole) of chromic acid and 2000 ml of acetic anhydride is added with stirring to -55 ° C to -60 ° C. At the end of the reaction, the reaction mixture is adjusted to pH 6 by means of a concentrated ammonium hydroxide solution cooled to -50 to -40 ° C. Thereby, under further cooling, care is taken so that the temperature of the reaction mixture does not exceed 50 ° C. Then 9 liters of distilled water are added and the thus diluted solution is made alkaline to pH 8.5 with 25% ammonium hydroxide. The reaction mixture is extracted with 4 x 1500 ml of dichloromethane, the organic phases are combined and to remove the ammonium acetate formed as a by-product, wash with 3 x 100 ml of distilled water. The separated organic phase is dried over anhydrous sodium sulfate and filtered and the filtrate is evaporated to dryness under reduced pressure. The residue consisting of crude 4-desacetoxy-vin-cristine and N-desmethyl-4-desacetoxy-vinblastine is dissolved in a mixture of 60 ml of concentrated formic acid and 10 ml of acetic anhydride, the solution is poured into 300 ml of water at a temperature of 0-5 °. C and with stirring the pH is adjusted to 9 by means of a concentrated ammonium hydroxide solution having a temperature of 0-5 ° C. The alkaline solution thus obtained is extracted with 3 x 100 ml of dichloromethane, the organic phases are combined and dried and evaporated under reduced pressure to dryness. As the residue, 9.8 crude 4-desacetoxyquincristine is obtained.

This crude product is dissolved in 60 ml of benzene and placed on a 45 mm diameter chromatography column containing 500 g of alumina (the active III) and benzene. The elution is started with 1200 ml of benzene, continued with 5000 ml of 2: 1 mixture of benzene and chloroform and then with 3000 ml of 1: 1 mixture of benzene and chloroform and it is terminated with 800 ml of chloroform. 400 ml fractions are collected. The bulk of the desired product is present in fractions 15-20, which are combined and evaporated under reduced pressure to dryness. In this way, 6.5 g of 4-desacetoxyquincristine (fraction I) is obtained.

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From fractions Nos. 11 to 14 and 21 to 26, additional 4-desacetoxy-vincristine can be recovered by combining these fractions, evaporating them under reduced pressure to dryness, dissolving the residue (1.75 g) dissolved in 15 ml of benzene and applying the solution on a chromatography column containing 90 g of alumina (Activity III) of benzene. Elute with 1200 ml of 2: 1 mixture of benzene and chloroform and evaporate under reduced pressure. In this way, 1.05 g of 4-desacetoxyvinineristin (fraction II) is obtained.

The fractions I and II obtained in the manner described are combined, dissolved in 60 ml of 3: 2 mixture of methanol and water and left for 16 hours at 5 ° C for crystallization. The obtained crystals are filtered off, washed with 10 ml of a 3: 2 mixture of methanol and water at a temperature of 5 ° C and dried in vacuo. 7.0 4-desacetoxyquincristine is obtained, mp 205-209 ° C, [α] D = + 105.7 ° (c = 1 in chloroform).

Infrared spectrum; similar to that of 4-desacetoxy-vinblastine, however found at v = 1672 cm ^ the absorption band characteristic of the N-formyl group.

Mass spectrum: molecular peaks at m / e = 766. The mass number corresponds to the gross formula C44H54N3 ° 8 · PMR spectrum: in comparison with vinblastine it is determined that in the spectra of vineristin and vinblastine for the 4-acetoxy group the peaks at δ = 2, 09 ppm is missing in the spectrum of 4-desacetoxyvincristine. Characteristic peak values: δ = 3.64 ppm, methoxycarbonyl protons 3.81 ppm, aromatic -OCH + protons 3.85 ppm, methoxycarbonyl protons 8.30 ppm, N-formyl proton

Example 2 4-desacetoxyquincristine sulfate 7.0 g of 4-desacetoxyquincristine (prepared as set forth in Example 1) was dissolved in 20 ml of dichloromethane and then the solution was evaporated under reduced pressure to dryness. The dry residue was dissolved in 30 ml of anhydrous ethanol and the pH of the solution was adjusted with anhydrous ethanol containing 1% sulfuric acid on 4. The sulfate salt 11 of 4-desacetoxyvincristine immediately crystallized.

The crystallizing solution is left at room temperature and then the crystals are filtered off, washed with 30 ml of anhydrous ethanol and dried. 7.1 g of 6-desacetoxyquincristine monosulfate are obtained (63.7%). [α] D = + 58.3 ° (c = 1 in water); [α] D = + 42.0 ° (c = 1 in methanol).

Example Gel 3 4-Desacetoxyquincristine 0.50 g (0.655 mmol) of 4-desacetoxyvinblastine sulfate is dissolved in 15 ml of distilled water. The pH of the solution is adjusted with ammonia water of 8.5 to 9.0 and the released 4-desacetoxyvinblastine base is extracted with 3 x 10 ml of chloroform. The combined chloroform extracts are dried over anhydrous sodium sulfate and filtered and the filtrate is evaporated to dryness under reduced pressure. The 4-desacetoxyvinblastine base obtained as the residue (0.4 g) is dissolved in 30 ml of 98% formic acid, the solution is stirred at room temperature, the air space above the solution is flushed with oxygen and 0.5 g of platinum soda is added to the solution. During the oxidation, which lasts 18-20 hours, the oxygen atmosphere is maintained over the solution by the addition of oxygen and the progress of the reaction is followed by thin layer chromatography. After completion of the reaction, the catalyst is removed by filtration, the filtrate is diluted with five times the amount of distilled water and cooled to 5 ° C, the pH is adjusted with concentrated ammonia water of 8.5 to 9.0 and the basic solution thus obtained is extracted three times. 10 ml of chloroform. The combined chloroform extracts are dried over anhydrous sodium sulfate and filtered and the filtrate is evaporated under reduced pressure to dryness.

As the residue, 0.38 g of crude 4-desacetoxyquincristine is obtained. Optionally, the product can be recrystallized as follows: 0.38 g of crude 4-desacetoxyquincristine is dissolved in 3 ml of 3: 2 mixture of methanol and water and the solution is left for 16 hours at 5 ° C. The obtained crystals are filtered, washed and dried. In this way, 0.3 g of purified 4-desacetoxyvinocristine (75% of theoretical yield, calculated from the amount of 4-desacetoxyvinblastine base) is obtained; the physical constants of the product are the same as in Example 1 .

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Example 4 4-Desacetoxyquin cysteine

Pra 50 mg of 4-desacetoxyvinblastine sulfate is released from the base as described in Example 3 and it is isolated by extraction with chloroform and evaporation. The obtained 4-desacetoxy-vinblastine base is dissolved in 3 ml of 98% formic acid and to the solution a suspension of 200 mg of 5% palladium on activated carbon is added in 2 ml of 98-100% formic acid. The suspension container is flushed with 2 x 0.5 ml formic acid to quantitatively transfer the catalyst to the reaction vessel. From the air space above the solution, the air is replaced with oxygen and the reaction mixture is stirred for 18-20 hours at room temperature under still oxygen atmosphere. The reaction of the reaction is followed by thin layer chromatography. Then, the reaction mixture is worked up in the manner described in Example 3. 25 mg of 4-desacetoxyquin crystalline is obtained (62% based on the amount of 4-desacetoxyvinblastine base used).

EXAMPLE 5 4-Desacetoxyquincristine

As described in Example 4, the difference is that the 4-desacetoxyvinblastine base and catalyst instead of formic acid are dissolved and suspended in a 6: 1 mixture of benzene and formic acid, respectively. 20 mg of 4-desacetoxyvincristine is obtained (50% based on the amount of 4-desacetoxyvinblastine base used).

Claims (1)

13142815 Analogous Process for the Preparation of 4-Desacetoxyquincristine of Formula \ n / X ^ oh N / H / / / / / X ^ X - / χ In rVrV „2 5 CH3 ° 0 ^ | H T C00CH, ir or acid addition salts thereof, characterized in that 4-desacetoxyvinblastine of formula XX-X x ^ xX, 11, PO2CH31 'εΛ N / 2 / / / / / kJ I Jx 2H / C2H5 CH e ^; H3C C00CH3