DE2706366A1 - 4-DESACETOXY-CINCRISTINE, THE PROCESS FOR THE PREPARATION AND THE PHARMACEUTICAL PREPARATIONS CONTAINING THIS - Google Patents

Description

28 971

JUDGE GEDEON VEGYESZETI GYAR RT. IN BUDAPEST / HUNGARY

4-Desacetoxy-vincristine, process for its production, and pharmaceutical preparations containing it

The invention relates to the manufacture of a new one Dllndolalkaloldderlvat, the 4-deacetoxy-vincristine and of the acid addition salts of this new alkaloid derivative.

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It is known that the secondary alkaloids of Vinca rosea L., which have a diindole structure, have cytostatic properties (cf. US Pat. No. 3,097,137, 3,205-20, 3,887,565, 3,890,325; Hungarian PS 153,200, 154,715, 160 967). Effective semisynthetic derivatives, for example N-formylleurosine (Hungarian PS 165 986), as well as various carboxamide and deacetyl derivatives (DT-PS 1 795 763, 2 415 890) and 4-deacetoxy-vinblastine (N. Neuss, AJ Barnes and LL Huckstep, Experimentia 31/1 , 18-19, 1975).

It has now been found that 4-deacetoxy-vinblastine can be converted into a new derivative, 4-deacetoxy-vincristine, by oxidative formylation; these new compound shows excellent cytostatic properties and is less toxic than the similarly acting vincristine.

The tumor-inducing effects of this new compound was examined on various transplantable tumors. The effectiveness against vinca-sensitive lymphoid leukemia L 1210 / VS and vinca-refractive lymphoid leukemia L 1210 / ref from mice was tested on BDF. Mice against the Ehrlich's see ascites sarcoma on Swiss mice, tested against Yoshida sarcoma and Novikoff's hepatoma on Wistar rats. The 4-deacetoxy vincristine sulfate was administered intraperitoneally once a day in physiological saline Administered to experimental animals; the anti-tumor effectiveness and the average lifespan of the animals was determined in comparison with untreated control animals. The results obtained are summarized in Table 1 below:

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Table 1

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tumor Daily Duration of Average fransplan- T / c.100 After 40 Days Dose l.p. treatment Lifespan, days mg / kg (Days) after the ' control tatlon 9.0 treated 8.3 live tumor free L 1210 / VS 0.1 7th 21.3 9.0 237 f / 6 0/6 0.25 7th 16.6 8.3 200 1/6 1/6 0.4 7th 17.8 10.5 198 0/6 0/6 0.5 7th 14.2 10.5 171 0/6 0/6 L 1210 / ref 0.2 9 14.8 10.5 141 0/6 0/6 0.4 9 14.0 17.4 133 0/6 0/6 0.8 9 15.7 17.4 150 0/6 0/6 Honestly ¹ sches 0.3 9 30th 172 10/10 7/10 Ascites car 0.6 9 29 12.3 169 9/10 4/10 cinom 14.8 Yoshida- 0.125 1 23 14.8 187 0/6 0/6 Sarcoma 0.25 1 38.7 14.8 262 0/6 0/6 0.50 1 42.7 7.8 288 2/6 2/6 1.00 1 43.5 7.8 294 0/6 0/6 Novikoff 0.25 8th 19.0 243 1/6 0/6 Hepatoma 0.50 8th 25.0 320 3/6 0/6

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It can be seen from the table above that the average life of those with doses from 0.1 to 1.0 mg / kg 4-deacetyl vincristine sulfate i.p. treated animals was 70 to 190% higher than the lifespan of the Control animals infected with the tumors in the same way but not treated with 4-deacetyl vincristine sulfate. The effect of 4-deacetyl vincristine sulfate is depending on the size of the doses administered; the tumor-inhibiting effect was primarily significant against Ehrlich's ascites sarcoma and against Yoshida sarcoma, but considerable effectiveness could also be determined against Novikoff's hepatoma. The one in the table The quotient T / c given shows the ratio between the average survival times of the treated and untreated animals.

When examining the toxicity of the new compound, it was found that 4-deacetoxy-vincristine is 25% less toxic than vincristine, which is similarly effective in the same doses. It is a considerable advantage of the new compound that the neurotoxicity which often occurs with the administration of vincristine and which is accompanied by symptoms of paralysis does not occur in the case of 4-deacetoxy-vincristing even with the administration of lethal doses. The approximate values of the acute toxicity (LD ₅ values) of 4-deacetoxy-vincristine and vincristine are summarized in Table 2 below:

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Table 2

link (at Mice, i.p.) ^LD 5O 5 mg / kg 4-deacetoxy-vincristine (at Mice, IV) 6th 4-deacetoxy-vincristine (at Rats, i.p.) 1 4-deacetoxy-vincristine (at Mice, i.p.) 4th , 3 Vincristine (at Rats, i.p.) 1 , 2 Vlncristin , 3

In human therapy, 4-deacetoxy-vincristine is particularly useful intravenously or by infusion, in doses from 0.01 to 0.3 mg / kg, advantageously from 0.05 to 0.2 mg / kg, daily be administered once or in smaller divided doses.

The new compound 4-deacetoxy-vincristine is according to the invention prepared by oxidatively formylating 4-deacetoxy-vinblastine, if desired the product obtained cleans, and the 4-deacetoxy-vincristine obtained, if desired, in a physiologically acceptable acid addition salt convicted.

In the method according to the invention, the, a N-methyl group-containing 4-deacetoxy-vinblastine oxidative formylation containing an N-formyl group 4-deacetoxy-vincristine produced. Under oxidative: formylation is simultaneous or successive

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Understood oxidation and formylation.

If the formylation is only carried out after the oxidation, the oxidation gives as an intermediate product a mixture of 4-deacetoxy-vincristine and N-desmethyl-4-deacetoxy-vinblastine. If desired, this mixture can be broken down into the individual components and then also the N-desmethyl-4-deacetoxy-vinblastine convert into the desired 4-deacetoxy-vincristine by formylation; but it is expedient to subject what has been received Oxidation product directly from the formylation, the 4-deacetoxy-vincristine already present in the mixture remains unchanged and the N-desmethyl-4-deacetoxy-vinblastine is also converted into 4-deacetoxy-vincristine.

The reaction occurs in the presence of both the oxidizing agent and the formylating agent carried out, the desired 4-deacetoxy-vincristine is obtained immediately as a uniform reaction product.

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

Nitric acid, for example, is used as an oxidizing acid

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or especially chromic acid CrO ₃ or alkali dichromates, for example potassium or sodium dichromate, into consideration.

As catalysts suitable for transferring oxygen metals of the platinum group, e.g. platinum, palladium or rhodium, can advantageously be applied to a carrier Form.

Formic acid is used in particular as a formylating agent.

In an expedient embodiment of the invention Procedure is the 4-deacetoxy-vinblastine - which, if desired, can be released from an acid addition salt immediately before the reaction - in an inert organic solvent or in the formylating agent itself, or in a mixture of the two. The formylating agent itself, expediently formic acid or a hydrocarbon, can therefore also be used as the reaction medium of the benzene series, e.g. toluene or xylene, but especially benzene or a mixture of formic acid with an organic solvent can be used.

Then one becomes more suitable for transferring oxygen Catalyst, e.g. a noble metal of the platinum group applied to a support, advantageously palladium-activated carbon or platinum black, added to the solution. The oxidation is carried out at 0 ° to 30 ° C, preferably at room temperature, in oxygen atmosphere, advantageously under 1 to 3 atm oxygen pressure. After completion of the reaction will

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the catalyst is removed by filtration and the pH of the filtrate is adjusted with an inorganic base, expedient with ammonia, adjusted to 7-10, preferably 8.5-9. The solution made alkaline in this way is with an inert organic solvent, e.g. with benzene, toluene or xylene, or with a chlorinated one Hydrocarbon, such as dichloromethane, or with chloroform, extracted. The product obtained by evaporation of the extract can be expediently recrystallized from a mixture of a lower alcohol such as methanol, ethanol or propanol and water. That The 4-deacetoxy-vincristine obtained can, if desired, in the usual manner in an acid addition salt, especially into the sulfate salt.

In a further advantageous embodiment of the inventive In the process, the 4-deacetoxy-vinblastine is converted into a lower aliphatic ketone, e.g. in acetone, Methyl ethyl ketone or methyl isobutyl ketone, dissolved. Chromic acid or an alkali metal dichromate is expediently used as the oxidizing agent used; the oxidation takes place in the presence of glacial acetic acid and acetic anhydride, at temperatures below OC, preferably between -90 ° and -30 ° C. After the end of the oxidation, the pH of the reaction mixture is adjusted to 5 to 10, which is expedient in two steps, first to pH 5 to 7, then to pH 8 to 9, is carried out. As an oxidation product arises in this

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Case a mixture of 4-deacetoxy-vincristine and N-desmethyl-4-deacetoxy-vinblastine. This mixture can be broken down into its components, e.g. by column chromatography and the N-desmethyl-4-deacetoxy-vinblastin formylated separately will; however, the mixture obtained as the oxidation product immediately after the formylation is expedient subjected, whereby 4-deacetoxyvincristine is obtained as a unitary product.

The formylation can be carried out in a solvent which is inert under the reaction conditions, for example in a hydrocarbon the benzene series. However, the formylating agent itself, namely a mixture, is expedient of formic acid and acetic anhydride, used as the reaction medium. The formylation is between 0 ° and 15 ° C, preferably between 0 ° and 5 ° C, carried out and then the pH of the reaction mixture to 7 to 10, preferably 8 to 9.5, set. The 4-deacetoxy-vincristine obtained is made alkaline from that Mixture with an inert organic solvent, expediently with a hydrocarbon of the benzene series, e.g. with benzene, toluene or xylene, or with a chlorinated hydrocarbon, preferably with dichloromethane or chloroform, and the crude product isolated by evaporation of the extract, for example by column chromatography or recrystallization, purified.

Partially deactivated

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Aluminum oxide - suitably with activity III - as an adsorbent, and an inert organic solvent, e.g. benzene, toluene or xylene and / or a chlorinated hydrocarbon such as dichloromethane or chloroform, can be used as eluants. From the eluate fractions containing the majority of 4-deacetoxy-vincristine the product is obtained immediately by evaporation of the combined fractions, during the before or after these Main fractions collected eluate fractions first evaporated and then the evaporation residues are subjected to a further chromatographic purification.

The products obtained in this way can, if desired, be recrystallized again separately or in combination and / or converted into an acid addition salt, advantageously into the sulfate salt.

The 4-deacetoxy-vincristine and / or the acid addition salts thereof can generally be used parenterally in therapy administered. For this purpose, injection or infusion solutions are preferably prepared which contain the active ingredient dissolved in a solvent suitable for parenteral administration. As a solvent can distilled water or physiological saline solution, also lower aliphatic alcohols or Glycol ethers can be used. The active ingredient in lyophilized form, in dry ampoules, can also be advantageous Injection preparations containing

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the required sterile solvent is attached in separate ampoules and the active ingredient immediately before administration is dissolved in this solvent. The solutions or solvents mentioned can also be different common additives, e.g. preservatives such as benzyl alcohol, p-oxybenzoic acid esters, antioxidants such as ascorbic acid, tocopherol, etc., lactose, buffer substances and / or possibly also contain other pharmacologically active substances, e.g. local anesthetic agents. The solutions or solvents can be sterilized e.g. by filtering.

The method of the invention is carried out by the following Examples illustrated in more detail, but the invention is in no way limited to these examples is. The optical rotation values given in the examples were measured on an Opton polarimeter; the proton magnetic Nuclear magnetic resonance spectra were recorded with a Varian EM spectrometer.

example 1

12 g (0.0159 mol) of 4-deacetoxy-vinblastine are in 2640 ml Dissolved acetone, then 600 ml of chromic acid freshly distilled off glacial acetic acid are added to the solution. The mixture will cooled to -55 ° C and with stirring with a mixture of 5.94 g (0.135 mol) also cooled to -55 ° C to -60 ° C Chromic acid and 2000 ml of acetic anhydride were added. To

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At the end of the reaction, the pH of the reaction mixture is concentrated with a concentration cooled to -35 ° to -4O ° C Ammonium hydroxide solution adjusted to 6. Further cooling ensures that the temperature of the Reaction mixture does not exceed + 50 ° C. Then 9 distilled water are added and the diluted solution is made alkaline to pH 8.5 with 25% ammonium hydroxide. The reaction mixture is 1500 ml Dichloromethane extracted four times, the organic phases are combined and removed as a by-product resulting ammonium acetate with 1000 ml of distilled Washed water three times. The separated organic phase is dried over anhydrous sodium sulfate and filtered and the filtrate was evaporated to dryness under reduced pressure. The one made from raw 4-deacetoxy-vincristine and N-Desmethyl-4-deacetoxy-vinblastine existing dry The residue is dissolved in a mixture of 60 ml of concentrated formic acid and 10 ml of acetic anhydride, the solution Poured into 300 ml of water cooled to 0 ° to 5 ° C and with stirring with concentrated water cooled to 0 ° to 5 ° C Ammonium hydroxide solution adjusted to pH 9. The solution made alkaline in this way is mixed with 100 ml of dichloromethane each time extracted three times, the organic phases are combined, dried and dried under reduced pressure evaporated. 9.8 g of crude 4-deacetoxyvincristine are obtained as residue.

This crude product is dissolved in 60 ml of benzene and applied to a 500 g of aluminum oxide (activity III) and benzene containing chromatographic column of 45 mm diameter applied. Elution is started with 1200 ml of benzene,

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continued with 5 (X) O ml of a 2: 1 mixture of benzene and chloroform, then with 3000 ml of a 1: 1 mixture of benzene and chloroform and terminated with 800 ml of chloroform. Fractions of 400 ml each are collected. The bulk of the desired Product is contained in fractions 15 to 20, these are combined and evaporated to dryness under reduced pressure. This makes 6.5 g of 4-deacetoxy-vincristine (Fraction I) received.

Further 4-deacetoxy-vincristine can be obtained from fractions 11 to 14 and 21 to 26 by this Fractions combined, evaporated to dryness under reduced pressure, the residue obtained (1.75 g) in 15 ml of benzene dissolves and applies to a chromatographic column containing 90 g of alumina (activity III) and benzene. It is eluted with 1200 ml of a 2: 1 mixture of benzene and chloroform and the eluate is evaporated under reduced pressure. Be like that a further 1.05 g of 4-deacetoxy-vincristine (fraction II) were obtained.

The fractions I and II obtained in the above manner are combined, dissolved in 60 ml of a 3: 2 mixture of methanol and water and left to stand for 16 hours at 5 ° C. to crystallize. The crystals obtained are filtered off, washed with 10 ml of a 3: 2 mixture of methanol and water cooled to 5 ° C., and dried in vacuo. 7.0 g of 4-deacetoxy-vincristine are obtained, mp 2O5-2O9 ° C; 1 & J

+ 105.7 ° (c = 1 in chloroform)

IR spectrum: Similar to 4-deacetoxy-vinblastine, es but appears at "V = 1672 cm di <a characteristic absorption band.

but the one for the N-formyl group appears at S / = 1672 cm

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Mass spectrum: Molecular ion peak at m / e = 766 mass number, corresponds to the overall formula ^C 44 ^H 54 ^N 3 ° a

PMR spectrum: In comparison with vinblastine, it was found that those in the spectra of vincristine and vinblastine for the 4-acetoxy group characteristic peak at 6 = 2.09 ppm in the spectrum of 4-deacetoxy-vin cristin is missing. The characteristic peak values:

c = 3.64 ppm methoxycarbonyl protons

3.81 ppm aromatic -OCH- protons

3.85 ppm methoxycarbonyl protons

8.30 ppm N-formyl proton.

Example 2

7.0 g of 4-deacetoxy-vincristine (prepared according to Example 1) are dissolved in 20 ml of dichloromethane, then the solution is evaporated to dryness under reduced pressure. The dry residue is dissolved in 30 ml of anhydrous ethanol and the pH of the solution is adjusted to 4 with anhydrous ethanol containing 1% sulfuric acid. The sulfate salt of 4-deacetoxy-vincristine begins to crystallize out immediately. The crystallizing solution is left to stand at room temperature, then the crystals are filtered off, washed with 30 ml of anhydrous ethanol and dried. 7.1 g of 4-deacetoxy-vincristine monosulfate (63.7% of theory) are obtained; t \ 7 ^ = + 58.3 ° (c = in water); L °) J ^ = + 42.0 ° (c = 1 in methanol).

- 15 -

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Example 3

0.50 g (0.655 mmol) of 4-deacetoxy-vinblastine-sulfate become dissolved in 15 ml of distilled water. The pH of the solution is adjusted to 8.5 with concentrated aqueous ammonia adjusted to 9.0 and the released 4-deacetoxyvinblastine base extracted three times with 10 ml of chloroform each time. The combined chloroform extracts are dried over anhydrous Sodium sulfate dried, filtered and the filtrate evaporated to dryness under reduced pressure. The 4-deacetoxy-vinblastine base (0.4 g) obtained as residue 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 the solution with 0.5 g of platinum soot added. During the oxidation, which lasts about 18 to 20 hours, the oxygen atmosphere becomes maintained above the solution by the addition of oxygen and the progress of the reaction by thin layer chromatography tracked. After the reaction has ended, the catalyst is removed by filtration, the Filtrate with 5 times the amount of distilled cooled to 5 ° C Diluted water, adjusted the pH to 8.5 to 9.0 with concentrated aqueous ammonia and the solution made alkaline in this way was extracted three times with 10 ml of chloroform each time. The combined chloroform extracts are dried over anhydrous sodium sulfate, filtered and the filtrate to dryness under reduced pressure evaporated. 0.38 g of crude 4-deacetoxyvincristine are obtained as residue. If desired, the product can be converted into

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of the following catfish are recrystallized:

0.38 g of crude 4-deacetoxy-vincristine are in 3 ml of 3: 2 Dissolved mixture of methanol and water and the solution is left to stand at 5 ° C for 16 hours. The received Crystals are filtered off, washed and dried. This gives 0.3 g of purified 4-deacetoxyvincristine (75% of theory, calculated for the 4-deacetoxy-vinblastine base); the physical constants of the product are the same as in Example 1.

Example 4

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The base in released in the manner described in Example 3 and isolated by extraction with chloroform and evaporation. The 4-deacetoxy-vinblastine base obtained is dissolved in 3 ml of 98% formic acid and the solution is stirred the suspension of 200 mg of 5% palladium-activated carbon in 2 ml of 98-100% formic acid is added. That the vessel containing the suspension is rinsed twice with 0.5 ml of formic acid each time in order to remove the catalyst quantitatively to bring into the reaction mixture. The air is displaced by oxygen from the air space above the solution and the reaction mixture was stirred for 18 to 20 hours under a constant oxygen atmosphere at room temperature.

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The progress of the reaction is followed by thin layer chromatography. Then the reaction mixture is in worked up in the manner described in Example 3. 25 mg of 4-deacetoxy-vincristine (62% of theory, for the 4-deacetoxy-vinblastine base calculated).

Example 5

The procedure is as in Example 4, with the difference that both the 4-deacetoxy-vinblastine base and the catalyst is dissolved or suspended in a 6: 1 mixture of benzene and formic acid instead of in formic acid. 20 mg of 4-deacetoxy-vincristine (50% of theory, calculated for the 4-deacetoxy-vinblastine base) are obtained.

Example 6

It is an injection preparation consisting of a dry ampoule containing the active ingredient and a solvent ampoule manufactured.

(a) 5 g of benzyl alcohol and 5 g of lactose are dissolved in 500 ml of physiological saline solution, the solution is sterilized by Steril-Filtratlon and in

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Portions of 5 ml each filled into ampoules.

(b) Any 0.05 mg, 0.10 mg, 0.20 mg, 0.50 mg or 1.00 mg of 4-deacetoxy-vincristine sulfate are in the form of sterile solution filled into ampoules under sterile conditions and lyophilized.

The contents of the lyophilized dry ampoule are dissolved in the contents of the solvent ampoule prior to administration.

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Claims (13)

PATENT CLAIMS 1 I 4-deacetoxy-vincristine and its physiologically harmless acid addition salts. 2. 4-deacetoxy vincristine sulfate. 3. A process for the preparation of 4-deacetoxy-vincristine and its acid addition salts according to claim 1, characterized in that 4-deacetoxy-vinblastine is subjected to an oxidative formylation and, if desired, the product obtained is subjected to converted into an acid addition salt in a known manner. 4. The method according to claim 3, characterized in that the oxidative formylation is carried out by simultaneous oxidation and formylation in one reaction step. 5. The method according to claim 3, characterized in that the oxidation and the formylation in two separate reaction steps is carried out. 6. The method according to claim 3 or 5, characterized in that the formylation is carried out after the oxidation. 7. The method according to any one of claims 3, 5 or 6, characterized characterized in that chromic acid or alkali metal chromate are used as oxidizing agents. - 20 - 709834/1012 ORIGINAL INSPECTED 8. The method according to any one of claims 3, 5, 6 or 7, characterized in that the oxidation at temperatures below O ⁰ C, preferably between -3O ° C and -90 ⁰ C is carried out. 9. The method according to one of claims 3 to 8, characterized characterized in that formic acid is used as a formylating agent. 10. The method according to any one of claims 3, 4 or 9, characterized in that the oxidative Formylieruni will lead. Formylation at temperatures from ^{0 0} C to 30 ° C carried out 11. The method according to any one of claims 3, 4, 9 or 10, characterized in that in the oxidative formylation, the formylating agent itself or a mixture of the formylating agent with a benzene series hydrocarbon as a solvent is used. 12. The method according to any one of claims 3, 4, 9, 10 or 11, characterized in that the oxidative formylation in the presence of a metal catalyst the platinum group is carried out. 13. Pharmaceutical preparations with anti-tumor effects, containing a compound according to claim 1 in addition to customary carriers and diluents. 709834/1012