CH627185A5 - Process for the preparation of 4-deacetoxyvincristine and of acid addition salts of this compound - Google Patents

Description

The invention relates to a process for the preparation of 4-5 deacetoxy-vincristine and acid addition salts of this compound.

It is known that the secondary alkaloids of Vinca rosea L. which have the diindole structure have cytostatic properties (cf. US Pat. No. 3,097,137, 3,205,220,888,565, io 3,890,325; Hungarian PS 153,200,154,715, 160,967 ). Effective semi-synthetic derivatives, e.g. N.For-myl-leurosin (Hungarian PS 165 986), as well as various carboxamide and deacetyl derivatives (DT-PS 1 795 763, 2 415 890) and 4-deacetoxy-vinblastin [N. Neuss, A.J. Barnes and 15 L.L. Huckstep, Experimentia 31/1, 18-19 (1975)].

The process according to the invention for the preparation of 4-deacetoxy-vincristine and acid addition salts of this compound is characterized in that 4-deacetoxy-vinblastine is subjected to an oxidative formylation and, if appropriate, 20 compounds obtained are converted into their acid addition salts. This new compound which can be produced according to the invention shows, for example, excellent cytostatic properties and is less toxic than the similarly acting vincristine.

The tumor-inhibiting effect of this new compound which can be produced according to the invention was preferably investigated on various transplantable tumors. The effectiveness, for example, against the vinca-sensitive lymphoid leukemia L 1210 / VS and vinca-refractive lymphoid leuk-30 anemia L 1210 / ref of mice was tested on BDFi mice, against the Ehrlich's Ascites Sarcom on Swiss mice Yoshida-Sarcom and Novikoff hepatoma tested on Wistar rats. The 4-deacetoxy-vincristine sulfate was usually administered intraperito-35 neal once a day to the test animals in physiological saline; the tumor-inhibiting effectiveness and the average lifespan of the animals were determined in comparison with untreated control animals. The results obtained are summarized in Table 1 below:

Table 1

tumor

Daily can i.p.

Duration of

Average

T / c. 100

After 40 days

mg / kg

treatment

Lifespan,

(Days)

Days after the

transplantation

handles control

live tumor free

L1210 / VS

0.1

7

21.3

9.0

237

1/6

0/6

0.25

7

16.6

8.3

200

1/6

1/6

0.4

7

17.8

9.0

198

0/6

0/6

0.5

7

14.2

8.3

171

0/6

0/6

L1210 / Ref

0.2

9

14.8

10.5

141

0/6

0/6

0.4

9

14.0

10.5

133

0/6

0/6

0.8

9

15.7

10.5

150

0/6

0/6

Honest

0.3

9

30th

17.4

172

10/10

7/10

Ascites carcinoma

0.6

9

29

17.4

169

9/10

4/10

Yoshida

0.125

1

23

12.3

187

0/6

0/6

Sarcom

0.25

1

38.7

14.8

262

0/6

0/6

0.50

1

42.7

14.8

288

2/6

2/6

1.00

1

43.5

14.8

294

0/6

0/6

Novikoff

0.25

8th

19.0

7.8

243

1/6

0/6

Hepatoma

0.50

8th

25.0

7.8

320

3/6

0/6

It can be seen from the table above, for example, that the average lifespan of those with cans is 0.1 to

1.0 mg / kg 4-deacetyl-vincristine sulfate i.p. animals treated was 70 to 190% longer than the lifespan of those treated with

3rd

627185

Tumors infected in the same way but not treated with 4-deacetyl-vincristine sulfate in control animals. The effect of 4-deacetyl-vincristine sulfate is usually dependent on the size of the doses administered; the tumor-inhibiting effect was primarily important against Ehrlich's Ascites-Sarcom and against Yoshida-Sarcom, but also against Novikoff-Hepatom a considerable effectiveness was found. The quotient T / c given in the table shows the ratio between the average survival times of the treated and untreated x # animals.

When examining the toxicity of the new compound which can be prepared according to the invention, it was found, for example, that 4-deacetoxy-vincristine is 25% less toxic than vincristine, which is similarly effective in equal doses. For example, it is a significant advantage of the new compound that the neurotoxicity often associated with paralysis symptoms associated with the administration of vincristine does not even occur with the administration of lethal doses in 4-deacetoxy-vincristine. The approximate acute toxicity (LD50) values of 4-deacetoxy-vincristine and vincristine are summarized in Table 2 below:

Table 2

Compound LD50 mg / kg

4-deacetoxy-vincristin / on mice, i.p. / 5

4-deacetoxy-vincristin / on mice IV / 6

4-deacetoxy-vincristin / on rats, i.p. / 1.3

Vincristine / on mice, i.p. / 4.2 30

Vincristine / on rats, i.p. / 1.3

In human therapy, 4-deacetoxy-vincristine can be administered particularly intravenously or by infusion, in doses of 0.01 to 35 0.3 mg / kg, advantageously 0.05 to 0.2 mg / kg once a day, or in smaller divided doses will.

In the sense of the invention, the new compound 4-deacetoxy-vincristine is prepared in such a way that 4-deacetoxy-vinblastine is oxidatively formylated, if desired, the product obtained is purified and, if desired, the 4-deacetoxy-vincristine obtained is converted into a physiologically acceptable acid addition salt.

In the process according to the invention, the 4-deacetoxy-vinblastine containing an N-methyl group is prepared by ts oxidative formylation to produce the 4-deacetoxy-vincristine containing an N-formyl group. “Oxidative formylation” is usually understood to mean the simultaneous or sequential execution of oxidation and formylation. so

If the formylation is carried out only after the oxidation, a mixture of 4-deacetoxy-vincristine and N-desmethyl-4-deacetoxy-vinblastine is normally obtained as an intermediate product by the oxidation. If desired, this mixture can be broken down into the individual 55 components and then the N-desmethyl-4-deacetoxy-vinblastine can also be converted into the desired 4-deacetoxy-vincristine by formylation, but the oxidation product obtained is expediently subjected directly to the formylation, which is in the mixture Already present 4- «desacetoxy-vincristin remains unchanged and the N-desmethyl-4-desacetoxy-vinblastin is also converted into 4-deacetoxy-vincristine.

If the reaction is carried out in the simultaneous presence of both the oxidizing agent and the formylating agent, 65 the desired 4-deacetoxy-vincristine is immediately obtained 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 the transfer of oxygen.

The oxidizing acid comes e.g. Nitric acid or especially chromic acid (CrOs) or alkali dichromates, e.g. Potassium or sodium dichromate can be considered.

Platinum group metals, e.g. Platinum palladium or rhodium, advantageously in the form applied to a support, can be used.

Formic acid is particularly used as a formylating agent.

In an expedient embodiment of the process according to the invention, the 4-deacetoxy-vinblastine, which can, if desired, be released from an acid addition salt immediately before the reaction, is dissolved 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 of the benzene series, e.g. B. toluene or xylene, but especially benzene or a mixture of formic acid with an organic solvent.

Then a catalyst suitable for transferring oxygen, e.g. a platinum group noble metal applied to a carrier, advantageously palladium-activated carbon or platinum carbon black, is added to the solution. The oxidation is carried out, for example, at 0 ° to 30 ° C., especially at room temperature, in an oxygen atmosphere, advantageously under 1 to 3 atm oxygen pressure. After the reaction has ended, the catalyst is generally removed by filtration and the pH of the filtrate is adjusted to 7 to 10, advantageously 8.5 to 9 with an inorganic base, advantageously with ammonia. The solution thus made alkaline is preferably washed with an inert organic solvent, e.g. extracted with benzene, toluene or xylene, or with a chlorinated hydrocarbon, especially with dichloromethane or with chloroform. The product obtained by evaporating the extract can be recrystallized, suitably from the mixture of a lower alcohol, e.g. Methanol, ethanol or propanol and water. The 4-deacetoxy-vincristine obtained can, if desired, be converted into an acid addition salt, in particular the sulfate salt, in the customary manner.

In a further advantageous embodiment of the process according to the invention, the 4-deacetoxy-vinblastine is dissolved in a lower aliphatic ketone, e.g. dissolved in acetone, methyl ethyl ketone or methyl isobutyl ketone. Chromic acid or an alkali dichromate is expediently used as the oxidizing agent; the oxidation is carried out in the presence of glacial acetic acid and acetic anhydride, at temperatures below 0 ° C., advantageously between -90 ° and -30 ° C. After the oxidation has ended, the pH of the reaction mixture is preferably adjusted to 5 to 10, which is expediently carried out in two steps, first to pH = 5 to 7, then to pH = 8 to 9. In this case, a mixture of 4-deacetoxy-vincristine and N-desmethyl-4-deacetoxy-vinblastine is usually produced as the oxidation product. This mixture can e.g. B. broken down into its components by acid chromatography and the N-desmethyl-4-desacetoxy-vinbla-stin are separately formylated, but the mixture obtained as the oxidation product is expediently subjected directly to the formylation, whereby 4-deacetoxy-vincristine is obtained as a uniform product.

Formylation can be carried out in a solvent which is inert from the point of view of the reaction, e.g. be carried out in a hydrocarbon of the benzene series. However, the formylating agent itself is expedient, namely a mixture of

627185

4th

Formic acid and acetic anhydride used as the reaction medium. The formylation is generally carried out between 0 ° and 15 ° C., advantageously between 0 ° and 5 ° C., and then the pH of the reaction mixture is adjusted to 7 to 10, advantageously 8 to 9.5. The 4-deacetoxy-vincristine obtained is usually made from the mixture made alkaline with an inert organic solvent, suitably with a hydrocarbon of the benzene series, e.g. extracted with benzene, toluene or xylene, or with a chlorinated hydrocarbon, advantageously with dichloromethane or chloroform and the crude product isolated by evaporation of the extract z. B. purified by acid chromatography or recrystallization.

For chromatography, partially deactivated aluminum oxide, suitably of activity HI, as an adsorbent and an inert organic solvent, e.g. Benzene, toluene or xylene and / or a chlorinated hydrocarbon, suitably dichloromethane or chloroform can be used as the eluent. From the eluate fractions containing the main amount of 4-deacetoxy-vincristine, the product is normally obtained directly by evaporation of the combined fractions, while the eluate fractions collected before or after these main fractions are first evaporated and then the evaporation residues are subjected to further chromatographic purification.

If desired, the products obtained in this way can be recrystallized separately or combined once again 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 administered parenterally in therapy. For this purpose, injection or infusion solutions are advantageously produced which contain the active ingredient dissolved in a solvent suitable for parenteral administration. Distilled water or physiological saline, and also lower aliphatic alcohols or glycol ethers can be used as solvents. The active ingredient can also advantageously be produced in lyophilized form, injectable preparations containing dry ampoules, the necessary sterile solvent being added in separate ampoules and the active ingredient being dissolved in this solvent immediately before administration. The solutions or solvents mentioned can also contain various customary additives, e.g. Preservatives such as benzyl alcohol, p-oxybenzoic acid esters, antioxidants such as ascorbic acid, tocopherol etc., lactose, buffer substances and / or optionally also other pharmacologically active substances, e.g. B. contain local anesthetic agents. The solutions or solvents can e.g. can be sterilized by filtration.

The process according to the invention can be illustrated in more detail by the examples below, but the invention is in no way restricted to these examples. 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 360 spectrometer.

Example 1 4-deacetoxy-vincristine

12 g (0.0159 mol) of 4-deacetoxy-vinblastine are dissolved in 2640 ml of acetone, then 600 ml of glacial acetic acid freshly distilled off from chromic acid are added to the solution. The mixture is cooled to -55 ° C. and, with stirring, a mixture of 5.94 g (0.135 mol) of chromic acid and 2000 ml of acetic anhydride, likewise cooled to -55 to -60 ° C., is added. After the reaction had ended, the pH of the reaction mixture was adjusted to 6 with concentrated ammonium hydroxide solution cooled to -35 ° to -40 ° C. Further cooling ensures that the temperature of the reaction mixture does not exceed 50 ° C. Then 9 liters of distilled water are added and the solution thus diluted is made alkaline with 25% ammonium hydroxide to pH = 8.5. The reaction mixture is extracted four times with 1500 ml of dichloromethane each, the organic phases are combined and washed three times with 1000 ml of distilled water to remove the ammonium acetate formed as a by-product. The separated organic phase is dried over anhydrous sodium sulfate, filtered and the filtrate is evaporated to dryness under reduced pressure. The dry residue consisting of crude 4-deacetoxy-vincristine and N-desmethyl-4-deacetoxy-vinblastine is concentrated in a mixture of 60 ml. Formic acid and 10 ml acetic anhydride dissolved, the solution poured into 300 ml water cooled to 0 ° to 5 ° C and adjusted to pH = 9 with stirring, cooled to 0 0 to 5 ° C concentrated ammonium hydroxide solution. The solution made alkaline is extracted three times with 100 ml dichloromethane each time, the organic phases are combined, dried and evaporated to dryness under reduced pressure. 9.8 g of crude 4-deacetoxy-vincristine are obtained as a residue.

This crude product is dissolved in 60 ml of benzene and applied to a chromatographic column of 45 mm in diameter containing 500 g of aluminum oxide (activity III) and benzene. Elution is started with 1200 ml of benzene, with 5000 ml of 2: 1 mixture of benzene and chloroform, then with 3000 ml of 1: 1 mixture of benzene and chloroform and terminated with 800 ml of chloroform. 400 ml fractions are collected. The majority of the desired product is contained in fractions 15 to 20, these are combined and evaporated to dryness under reduced pressure. 6.5 g of 4-deacetoxy-vincristine (fraction I) are obtained in this way.

From fractions 11 to 14 and 21 to 26 further 4-deacetoxy-vincristine can be obtained by combining these fractions, evaporating to dryness under reduced pressure, dissolving the residue obtained (1.75 g) in 15 ml of benzene and placing it on a Apply 90 g of aluminum oxide (activity III) and benzene-containing chromatographic column. It is eluted with 1200 ml of a 2: 1 mixture of benzene and chloroform and the eluate is evaporated under reduced pressure. A further 1.05 g of 4-deacetoxy-vincristine (fraction II) are thus obtained.

The fractions I and II obtained in the above are combined, dissolved in 60 ml of a 3: 2 mixture of methanol and water and left to crystallize at 5 ° C. for 16 hours. 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 205-209 ° C .;

[a] 2 ° = + 105.7 ° (c = 1 in chloroform)

Infrared spectrum: similar to 4-deacetoxy-vinbla-stin, but the absorption band characteristic of the N-formyl group appears at v = 1672 cm-1.

Mass spectrum: Molecular ion peak at m / e = 766 mass number, corresponds to the overall formula C14H54N3O8.

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

5

10th

is

20th

25th

30th

35

40

45

50

SS

60

65

5

627185

Methoxycarbonyl protons aromatic-OCH3 protons Methoxycarbonyl protons N-formyl proton.

5

Example 2 4-deacetoxy-vincristine sulfate 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 10 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;

20th

[a] 2o = + 58.3 ° (c = 1 in water); [a] 2 ° = + 42.0 ° (c = 1 in methanol).

Example 3 25

4-D esacetoxy-vincristine 0.50 g (0.655 mmol) of 4-deacetoxy-vinblastine sulfate are dissolved in 15 ml of distilled water. The pH of the solution is adjusted to 8.5 to 9.0 with concentrated aqueous ammonia and the released 4-deacetoxy-vinblastine base 30 is extracted three times with 10 ml of chloroform. The combined chloroform extracts are dried over anhydrous sodium sulfate, filtered and the filtrate is evaporated to dryness under reduced pressure. The 4-deacetoxy-vinblastine base (0.4 g) obtained as residue is dissolved in 30 ml of 98% 35

Formic acid dissolved, the solution stirred at room temperature, the air space above the solution is flushed with oxygen and the solution was mixed with 0.5 g of platinum soot. During the oxidation, which lasts about 18 to 20 hours, the oxygen atmosphere over the solution is maintained by adding oxygen and the progress of the reaction is monitored by thin layer chromatography.

After the reaction has ended, the catalyst is removed by filtration, the filtrate is diluted with five times the amount of distilled water cooled to 5 ° C., the pH value is adjusted to 8.5 to 9 with concentrated aqueous ammonia, ©

adjusted and the solution made alkaline extracted three times with 10 ml of chloroform. The combined chloroform extracts are dried over anhydrous sodium sulfate, filtered and the filtrate is evaporated to dryness under reduced pressure. 0.38 g of crude 4-deacet-oxy-vincristine are obtained as a residue. If desired, the product can be recrystallized in the following manner:

0.38 g of crude 4-deacetoxy-vincristine are dissolved in 3 ml of a 3: 2 mixture of methanol and water and the solution is left to stand at 5 ° C. for 16 hours. The crystals obtained are filtered off, washed and dried.

In this way, 0.3 g of purified 4-deacetoxy-vincristine (75% of theory, calculated for the 4-deacetoxy-vinblastine base) are obtained; the physical constants of the product are the same as in example 1.

Example 4 4-deacetoxy-vincristine

The base is released from 50 mg of 4-deacetoxy-vinblastine sulfate 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 mixed with the suspension of 200 mg of 5% palladium-activated carbon in 2 ml of 98-100% formic acid. The vessel containing the suspension is rinsed twice with 0.5 ml of formic acid in order to bring the catalyst quantitatively into the reaction mixture. The air is displaced by oxygen from the air space above the solution and the reaction mixture is stirred at room temperature for 18-20 hours under a constant oxygen atmosphere. The progress of the reaction is followed by thin layer chromatography. The reaction mixture is then worked up in the manner described in Example 3. 25 mg of 4-deacetoxy-vincristine (62% of theory, calculated for the 4-deacetoxy-vinblastine base) are obtained.

Example 5 4-deacetoxy-vincristin The procedure is as in Example 4, with the difference that both the 4-deacetoxy-vinblastine base and the catalyst are dissolved or suspended in a 6: 1 mixture of benzene and formic acid instead of formic acid. 20 mg of 4-deacetoxy-vincristine (50% of theory, calculated for the 4-deacetoxy-vinblastine base) are obtained.

Ô = 3.64 ppm 3.81 ppm 3.85 ppm 8.30 ppm

B

Claims (12)

627185 1. A process for the preparation of 4-deacetoxy-vincristine and of acid addition salts of this compound, characterized in that 4-deacetoxy-vinblastine is subjected to oxy-dative formylation and the compound obtained is optionally converted into its acid addition salts. 2. The method according to claim 1, characterized in that that the product obtained is converted into an acid addition salt. 2nd PATENT CLAIMS 3. The method according to claim 1, characterized in that the oxidative formylation is carried out by simultaneous oxidation and formylation in one reaction step. 4. The method according to claim 1, characterized in that the oxidation and formylation are carried out in two separate reaction steps. 5. The method according to claim 4, characterized in that the formylation is carried out after the oxidation. 6. The method according to claim 1 or according to claim 4 or 5, characterized in that chromic acid or alkali dichromates are used as oxidizing agents. 7. The method according to claim 1 or according to claim 4, 5 or 6, characterized in that the oxidation is carried out at temperatures below 0 ° C, especially between - 30 ° C and - 90 ° C. 8. The method according to claim 1, characterized in that the oxidative formylation is carried out in the presence of an organic solvent. 9. The method according to claim 1, characterized in that formic acid is used as a formylating agent. 10. The method according to claim 1, characterized in that the oxidative formylation is carried out at temperatures from 0 ° C to 30 ° C. 11. The method according to claim 1, characterized in that in the oxidative formylation, the formylating agent itself or a mixture of the formylating agent with a hydrocarbon of the benzene series is used as a solvent. 12. The method according to claim 1, characterized in that the oxidative formylation in the presence of a metal Platinum group catalyst is carried out.