CH616937A5 - Process for producing 9-hydroxyellepticine derivatives - Google Patents

Abstract

The compounds of formula I, in which R is an alkyl group, R1 and R2 are respectively the hydrogen atom and an acyl group, an acyl group and a hydrogen atom, or an alkyl group and an acyl group, and X is a halogen atom, are prepared from 9-hydroxyellepticine by acylation in position 9 and quaternisation in position 2 using a compound RX, optionally acylation in position 6 and optionally deacylation in position 9. By exchange of ions, the derivatives in which X is an acetoxy group are obtained. The compounds of formula I and their derivatives have an antitumor activity, in particular against murine leukaemia L 1210 and murine leukaemia P 388. <IMAGE>

Description

The present invention relates to a process for obtaining new products, derivatives of hydroxy-9-ellipticine or hydroxy-9 dimethyl-5, 11 (6H) pyrido [4,3-b] carbazole, which are in particular applicable as a medicament or as a principle of pharmaceutical compositions, in particular for the treatment of cancers, and have been found to be exceptionally effective for the treatment of various forms of cancer.

The preparation and the therapeutic application of hydroxy-9-ellipticine have already been described in French patent application no. 73 38416 filed on October 29, 1973 in the name of the applicant. The preparation and therapeutic application of an ellipticine derivative, methoxy-9 ellipticine lactate, have also previously been described. This is how recent studies, reported in an article by G. Mathe et al. in Rev. Europ. Clinical Studies. Biol. 15.1970, pp. 541-545 and in an article by J Le Men et al., Op. cit. pp. 534-538, it has been shown that methoxy-9 ellipticine lactate, in addition to an on-static action on leukemia 11210 and mouse tumor BP 8, has activity against acute myelob-elastic leukemia, but is found to be ineffective for the treatment of acute lymphoblastic leukemia and Hodgkin's disease; in addition, although it has an immunosuppressive action when administered after the antigen, methoxy-9 ellipticine lactate does not exhibit this activity when administered to humans. Methoxy-9 ellipticine is an essential alkaloid in the leaves of Ochrosia.

In addition, both for 9-methoxy ellipticine lactate and for other compounds commonly used in human therapy for the treatment of certain tumors, such as for example bis-betachloroethyl nitroso urea, Soul-thopterin or Methotrexate, mercapto-6-purine, fluoro-5-uracil, or cyclophosphamide or Endoxan, the anti-tumor activity manifested by these compounds is only significant at high doses close to the lethal dose or LD 50. However, it on the contrary would be very interesting, in particular in human therapy, to be able to administer the anti-tumor active principles in doses as far as possible from the toxic doses.

It has now been found that new compounds, derived from hydroxy-ellipticine, exhibit markedly superior antitumor properties with regard to numerous tumors and in particular with respect to murine leukemia L 1210 and P 388 leukemia compared to those of the known compounds mentioned above, including hydroxy-9 allipticin itself.

These new compounds have the flat structural formula:

CH

H,

'2

R

CH

wherein R is an alkyl group, Ri, and R2 are respectively a hydrogen atom and an acyl group, an alkyl group and a hydrogen atom, or an alkyl group and an acyl group, and X is an atom halogen.

These compounds are prepared from Thyxy-9 ellipticine whose formula, characteristics and a process for its

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preparation have been described in French patent application 73 38416.

The processes according to the invention, for the preparation of these compounds, are defined in claims 1 and 5.

Among the alkyl halides which can be used to quaternize the nitrogen atom in position 2, very particularly preferred are methyl iodide and ethyl iodide.

To prepare the starting hydroxy-9 ellipticine, one can proceed as indicated in the aforementioned French patent application, that is to say by demethylation of 9-methoxy ellipticine, for example by means of pyridine hydrochloride, glacial acetic acid saturated with hydrochloric acid, hydrobromic acid gas or hydroiodic acid. However, it is preferred to demethylate 9-methoxy ellipticine in the presence of pyridine hydrochloride, advantageously pyridine hydrochloride previously distilled and recrystallized, because with this demethylating agent pure 9-hydroxy ellipticine is obtained, i.e. free from unwanted side products.

The demethylating agent should preferably be used in a large molar excess relative to the methoxy-9 ellipticine used.

The starting methoxy-9 ellipticine can be obtained by extraction from a natural source, or by synthesis (see on this subject among others the articles by JW Loder in Austr. J. Chem. 1967, 20, pp. 2715- 2727 and J. Poisson and C. Miet in Ann. Pharm. Franc. 1967, 25, p. 523).

Among the new compounds thus obtained by the process according to the invention, particular mention should be made of:

9-acetoxy ellipticinium iodomethylate,

9-acetoxy-methyl 6-ellipticinium iodomethylate, and 9-hydroxy-6 methyl-ellipticinium iodomethylate.

The compounds obtained according to the invention can contain the active principle of an anti-cancerous pharmaceutical composition by containing a therapeutically effective amount, said composition can for example be in the form of an injectable solution or of solid admini-strable preparations. by bone.

It has in fact been able to establish that the compounds obtained according to the invention, and among them more particularly 9-hydroxy-iodomethylate 7-methyl-ellipticinium and acet-9-methyl-6-ellipticinium iodomethylate, have a quite exceptional and unpredictable efficacy for cancer treatments. This efficiency is such that it allows the destruction of 99.999% of leukemic cells for an inoculum of 105 cells of murine leukemia L 1210, even though the doses required to do this are very much lower than the lethal dose, which has a considerable advantage both from a medical and an economic point of view.

In order to test the anti-tumor activity of the compounds according to the invention, the effect on murine leukemias L 1210 or P 388 was used.

The experimental tumor that is mouse leukemia L 1210 is in fact commonly used for the evaluation of all the antitumor compounds currently used in human therapy, as described, for example by C.C. Zubrod in Proc. Nat. Acad. Sci. USA 69, 1972, pp. 1042-1067. The tumor system thus constituted experimentally allows a very precise quantitative evaluation of the activity of the test compound and therefore also an objective comparison between the respective activities of different compounds, for example according to the methods described by HE Skipper, FM Schabel Jr, and WS Wilcox in Cancer Chemother. Rep., 35.1964, pp. 1-111 and 45.1965, pp. 5-28.

In practice, mice have been used which have received an inoculum of L 1210 leukemia cells intraperitoneally and have been subjected, for half of them, 24 hours after said tumor inoculation to an intraperitoneal injection of a single dose of one of the compounds to be tested; the other half of the mice received an injection of the same volume of an inactive solvent and served as a control series. The mice were randomly assigned to each experimental series. A first routine experiment made it possible to determine the mortality by toxicity and thus to fix the sub-lethal doses.

In accordance with the convention in use in this field, animals that survived more than 45 days after the inoculation of tumor cells were considered to be cured.

It was thus possible to determine the mortality by toxicity under the effect of a single injection of one of the compounds at different doses (infra-lethal doses LD 50 and LD 100) and to assess the survival rate of the group of animals to which only a sub-lethal dose of a compound according to the invention was injected.

The survival rate was calculated from a statistical analysis of the experimental results, by comparison with the results obtained with the control series, according to the well-known methods of Mann-Whitey and Wilcoxon; the percentage of leukemic cells killed by the treatment carried out was calculated by the method described by H.E. Skipper et al. in the articles cited above, taking as a basis, as the case may be, either the increase in average survival in the absence of survivors, or the percentage of survivors.

For the evaluation of the therapeutic activity of the compounds obtained according to the invention, the doses of said compound were expressed by relating them to the 30-day sub-lethal dose, which was set equal to 1. This sub-dose Lethal has indeed proved to be easier to determine for the compounds obtained according to the invention than the LD 10, of which it is moreover very close and which is usually taken as a reference for the evaluation of the therapeutic activity of compounds with antitumor effect (see HE Skipper et coli, cited works).

The therapeutic activity on mouse P 388 leukemia is comparable to that obtained on leukemia L 1210 for the same doses, the protocol being identical.

It was thus possible to determine that the compounds obtained according to the invention, and in particular the iodomethylate of 9-hydroxy-6-methyl ellipticinium and the iodomethylate of 9-acetoxy-6 ellipticinium, make it possible to kill, at doses much lower than the lethal dose, up to 99.999% of leukemia cells, for a 10s inoculum of murine leukemia 11 1210, which is quite exceptional, compared to the results obtained according to the literature on the same tumor material and with the products currently commonly used in human therapy and cited above.

However, it is naturally at doses as distant as possible from toxic doses that it is advantageous to be able to use such active compounds, in particular in human therapeutics.

The invention is described in more detail in the examples below.

EXAMPLE 7 Acetoxy-9 ellipticinium iodomethylate a) Hydroxy-9 ellepticin

Was heated at gentle reflux, for 30 to 90 minutes, a mixture of one part by weight of methoxy-9 ellipticine obtained by extraction from "Yellow wood" of Reunion Island (Ochrosia maculata) and 3 to 11 parts by weight of pyridine hydrochloride. The reaction mass was colored dark brown; it was then poured onto crushed ice, the precipitate formed was filtered off and washed several times with ice water and then recrystallized from methanol. The crystals obtained had an orange color.

The melting point of the crystalline product was significantly above 330 ° C. The raw formula of this product, which we s

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4

had been able to establish that it was crystallized with 1 mole of methanol, was C18H18N2O2, which gave the crude formula C17H14N2O for pure hydroxy-ellipticin 9; the molecular weight of the latter being 262, the elementary analysis gave:

C H N O

calculated 73.53% 5.17% 9.53% 10.88%

found 73.50% 5.35% 9.48% 10.84%

The IR spectrum of the compound obtained exhibited at 3100 cm1 the band characteristic of the -OH group.

Its mass spectrum revealed a molecular peak M = 262 and analysis by thin layer chromatography with alumina as support and as eluent a 28/2 mixture of benzene / ethanol gave an Rf of 0.44.

b) Acetoxy-9 ellipticine

1 g of 9-hydroxy ellipticine was dissolved in 20 ml of dried pyridine on potassium hydroxide and a solution of 3 g of acetic anhydride in 10 ml of pyridine was added, with magnetic stirring.

The orange-colored solution first blackened, then it became red and clear. It was diluted with water and a precipitate formed, which was drained and dissolved in CHCb. The chloroform solution was washed with water; dried over Na2SO4, filtered and the solvent was removed. Then recrystallized from benzene and 1 g of product was obtained, which was formed of fine yellow crystals melting at 282 ° C.

Analysis by thin layer chromatography with the support of Silicagel and as eluent a 24/6 mixture of benzene / ethanol gave an Rf of 0.40.

The crude formula of this compound being C10H16N2O2 +1/2 H2O and its molecular weight 304.33 + 9 = 313.33, the elementary analysis gave:

C H N

calculated -73.14% 5.47% 8.98%

found 73.07-73.21% 5.73-5.75% 8.65-8.81%

Another analytical check and differentiation between 9-hydroxy-ellipticine and 9-acetoxyl-ellipticine were also performed. The results are reported in the table below.

Test at

Detection

Detection

FeCIa

UV: 320-380 nm

Iodine fluorescence

Hydroxy-9 ellipticine positive

No

Red

fluorescence

Acetoxy-9 ellipticine negative

Fluorescence

Brown

yellow

An ellipticin solution (base), dissolved in pure anhydrous dimethylformamide, was treated with an ethereal solution of gaseous hydrochloric acid. A precipitate of the hydrochloride formed, which was drained, washed with anhydrous ether and recrystallized from absolute etanol, precipitating it again with anhydrous ether.

The fine yellow crystals formed decomposed in char-bonning, without melting, around 300 ° C. This compound had the crude formula C19H16N2O2.1 / 2 H2O, HCl.

c) Acetoxy-9 ellipticinium iodomethylate

1 g of acetoxy-9 ellipticine (base) was dissolved in 10 ml of pure and anhydrous dimethylformamide. With magnetic stirring, 3 g of methyl iodide were added. After 2 hours, the precipitate formed was filtered off and washed with anhydrous ether. Fine yellow crystals were obtained, having a melting point> 330 ° C.

The crude formula of the compound being C20H19N2O2 and its molecular weight 446,286, the elementary analysis gave:

C H N I

calculated 53.82% 4.29% 6.28% 28.52%

found 53.70-53.92% 4.15-4.18% 6.30-6.35% 28.45%

Example 2

9-acetoxy-methyl 6-ellipticinium iodomethylate a) 9-acetoxy-6-methyl ellipticine (base)

1 g of 9-acetoxy-ellipticine was dissolved in 10 ml of pure and anhydrous dimethylformamide (on molecular sieve). 0.1 g of sodium hydride (50% suspension in oil) was added, previously washed several times with anhydrous ether.

0.453 g of methyl iodide were introduced with magnetic stirring and at room temperature.

After 30 minutes of stirring, it was diluted with water and extracted twice with CHCb (25 ml each). The chloroform solution was washed with water, then dried over Na2SO4, filtered and the solvent was removed.

Was recrystallized from a mixture of 1 volume of ethanol and 4 volumes of benzene, thus obtaining fine yellow crystals.

Analysis by thin layer chromatography with the support of Silicagel and as eluent a 24/6 mixture of benzene / ethanol gave an Rf of 0.50.

The crude formula of this compound being C20H18N2O2 and its molecular weight 318 378, the elementary analysis gave:

C H N

calculated 75.46% 5.70% 8.80%

found 75.42-75.38% 5.75-5.82% 8.63-8.50%

b) Acetox-9-methyl-6 ellipticinium iodomethylate

1 g of 9-acetoxy-6 methyl-ellipticine (base) was dissolved

in 10 ml of pure and anhydrous dimethylformamide.

3 g of methyl iodide were added and left to stand for 3 hours.

The precipitate was filtered off and washed with anhydrous ether. The compound obtained consisted of fine yellow crystals, which did not melt at 330 ° C, but decomposed at around 335 ° C.

The crude formula of this compound being C21H21N2O2I and its molecular weight 460,313, the elementary analysis gave:

C H N Y I

calculated 54.83% 4.60% 6.09% 6.96% 27.59% found 54.66% 4.56% 5.88% 7.22% 27.48%

Example 3

9-hydroxy-6-methyl ellipticinium iodomethylate a) 9-hydroxy-6-methyl ellipticine (base)

1 g of 9-acetoxy-6-methyl ellipticine (base) was dissolved

in 10 ml of absolute ethanol. With magnetic stirring, a few drops of sodium hydroxide solution with a density of d: 1.33 were added, to a pH of 9.

Stirring was continued for another 30 minutes. It was precipitated with anhydrous ether; drained and washed with ether, thereby obtaining yellow crystals, having a melting point of 314-315 ° C.

The crude formula of compound obtained being C18H16N2O and its molecular weight 276 32, the elementary analysis gave:

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calculated found

78.25% 78.20-78.08%

H

5.83% 5.75-5.65%

NOT

10.13% 10.06%

O

5.79% 5.55%

Another analytical check and differentiation were made between 9-acetoxy-ellipticine, 9-acetoxy-6-methyl ellipticine and 9-hydroxy-6-methyl ellipticine. The results are reported in the table below:

TLC Benzene / ethanol 26/4

FeCl3 test

Detection

UV: 320-380 nm

Fluorescence

Detection with iodine vapor

Acetoxy-9 ellipticine negative

Yellow brown fluorescence

Acetoxy-9 methyl-6 ellipticine negative

Yellow fluorescence but migrates higher brown

Hydroxy-9 methyl-6 ellipticine positive

No fluorescence but same Rf as acetoxy-9 ellipticine dark brown

forms of leukemia; murine leukemia L 1210 and murine leukemia P 388.

The lethal dose DLo was 50 mg / kg body weight of the animals tested.

Twenty 2-3-month-old female DBA / 2 mice of the DBA / 2 strain were administered intraperitoneally with a tumor inoculum of 105 L 1210 leukemia cells. After 24 hours, half of these mice were given a single injection intraperitoneally with a variable dose of the test compound, while the same half of the mice were injected with the same volume of solvent; this last half of the group of mice therefore served as a control series. The mice were randomly assigned to each experimental series.

Animals surviving more than 45 days were considered cured. The lethal dose DLo, ie 50 mg / kg, was taken as the basis for the therapeutic activity of the compounds of the invention.

In Table I below appear the results which have been obtained by varying the dose of active compound injected.

Table I

Therapeutic effect of variable doses (single injection, intraperitoneally) of iodomethylate of hydroxy-6-methyl-6 ellipticinium for a tumor inoculum of 10 cells

• 25

b) Iodomethylate of hydroxy-6 methyl-6 ellipticinium

1 g of 9-hydroxy-6-methyl ellipticine (base) was dissolved

in 10 ml of pure and anhydrous dimethylformamide. With magnetic stirring, 3 g of methyl iodide was added and stirring was continued for 2 hours. The precipitate formed was then filtered off and washed with anhydrous ether, thus obtaining fine yellow crystals, having a melting point above 330 ° C.

The crude formula of the compound obtained being C19H19N2OI and its molecular weight 418.26, the elementary analysis gave:

Dose of 9-hydroxy-6 methyl-ellipticinium iodomethylate as a fraction of the sub-lethal dose of 50 mg / kg

Average survival in days of control mice

Average survival in days of mice treated with tumor cells killed by the treatment

0.01

9.0 ± 0.4

10.3 ± 0.4

90

0.02

9.0 ± 0.4

10.4 ± 0.4

92

0.05

9.4 ± 0.4

12.7 + 0.6

98

0.1

9.4 ± 0.4

14.0 ± 0.5

99, 96

0.2

9.0 ± 0.4

13.4 ± 0.6

99, 95

1

8.7 ± 0.5

16.1 ± 0.8

99.999

H

NOT

calculated 54.59% 4.57% 6.69%

found 54.90-55.01% 4.64-4.59% 6.31-6.39%

I

30.34% 40 30.20%

Phramacological tests 1 Iodomethylate of hydroxy-9-methyl-6-ellipticinium

In order to test it, it was put in a water-soluble form, namely in the form of 9-hydroxy-2,6-dimethyl ellipticinium acetate.

To do this, a given amount of 9-hydroxy-6 methyl ellipticinium iodomethylate, prepared as indicated in Example 3-b), was dissolved in a minimum amount of pure dimethylformamide. It was diluted to 50% by adding distilled water and it was made to absorb on a column (like Pasteur pipette) of Amberlite CG-50. A volume of distilled water equal to about 10 times the volume of the column was washed, and then eluted with 0.1 molar hydrochloric acid.

It should be noted that it is important to operate quickly, in practice over a period not exceeding 60 minutes, because the solution is metastable.

The eluent was basified with IN sodium hydroxide solution to pH 12 and allowed to stand for 30 minutes. A precipitate formed which was filtered through a special sterilization membrane and washed with 0.01 molar sodium hydroxide. This precipitate was then redissolved in 0.1 molar CH3COOH. The pH was brought to 5.5 with a 0.1 N sodium hydroxide solution and a clear solution of 9-hydroxy-2,6-dimethyl ellipticinium acetate was obtained.

We tested the biological activity of this compound on two

Reading this table, we notice that:

- the therapeutic index of 9-hydroxy-6 methyl ellipticinium iodomethylate is extremely high;

- for a dose equal to l / 100th of the sub-lethal dose, we still observed a very great activity, since 90% of the leukemic cells were then killed;

- for a dose which is still only equal to 1/10 of the sub-lethal dose, the activity was such that 99.96% of the leukemic cells were killed, which is equivalent to the therapeutic effect that the hydroxy-9 ellipticine makes it possible to obtain only at the sub-lethal dose, that is to say with quantities injected 10 times higher, in any case too close to the lethal dose to be acceptable; at the present time, it seems that no other substance has been found, having on this L 1210 leukemia experimental system such a high therapeutic index;

- under the same experimental conditions, at the sub-lethal dose, the anti-tumor activity of 9-hydroxy-6-methyl iodomethylate ellipticinium appears, with 99.999% of leukemic cells killed, much greater than that of hydroxy-9 ellipticine, which was only 99.99%.

2 Acetoxy-9-methyl-6-ellipticinium iodomethylate

It has been possible to establish, by tests similar to those reported in 1 above, that the antitumor activity of 9-acetoxy-6 methyl iodomethylate ellipticinium is comparable to that of 9-hydroxy methyl iodomethylate -6 ellipticinium. The sub-lethal dose was 50 mg / kg.

For this sub-lethal dose and under the same experimental conditions as those indicated under 1, it was possible to obtain 99.999% of leukemic cells killed.

B

Claims (6)

616,937 2. Method according to claim 1, characterized in that R is a methyl or ethyl group. 2 CLAIMS 1. Process for obtaining quaternary ammonium halides corresponding to the formula CH R CH. R in which R is an alkyl group, Ri is a hydrogen atom, R2 is an acyl group and X is a halogen atom, as well as corresponding quaternary ammonium acylates, characterized in that it has: - acylate the OH group in position 9 of the hydroxide-9-ellipticine, - quaternize the nitrogen atom in position 2, using an RX compound in which R and X are as defined, and, if necessary, - replace the anion Xe with an acylate anion. 3. Process for the preparation of quaternary ammonium halides of formula I above in which R and R 1 are each an alkyl group, R 2 is an acyl group and X is a halogen atom, as well as quaternary ammonium acylates correspondents; characterized in that it consists of - acylate the OH group in position 9 of the hydroxy-9-ellipti-cine, - alkylate the nitrogen atom in position 6, and - quaternize the nitrogen atom in position 2, using an RX compound in which R and X are as defined, and, if necessary, - replace the anion Xe with an acylate anion. 4. Method according to claim 3, characterized in that R is a methyl or ethyl group. 5. Process for the preparation of quaternary ammonium halides of formula I above in which R and R 1 are each an alkyl group, R 2 is a hydrogen atom and X is a halogen atom, as well as acylates of corresponding quaternary ammonium, characterized in that it consists of: - acylate the OH group in position 9 of the hydroxy-9-ellipti-cine, - alkylate the nitrogen atom in position 6, - release the OH group in position 9 by hydrolysis, and - quaternize the nitrogen atom in position 2, using an RX compound in which R and X are as defined, and, if necessary, - replace the anion Xe with an acylate anion. 6. Method according to claim 4, characterized in that R is a methyl or ethyl group.