RU2292209C2 - Antitumor remedy taken per os and method for producing the remedy - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: preparation belongs to gestagen class as 17^α-acetoxy-3β-butanoyloxy-6-methyl-pregna-4,6-dien-20-on allowing peroral application. The compound shows no toxic activity and has no androgenic side effect. It is found to be better than Depo-Provera gestagen used in clinical practice and possesses chemosensitizing activity. The preparation is obtained by megestrol acetate reduction with sodium borohydride with following product etherification with butyric acid anhydride. The reaction mixture is treated with ammonia solution after etherification with butyric acid excess being removed to increase output and target product separation.

EFFECT: enhanced effectiveness of treatment.

3 cl, 3dwg, 1 tbl

Description

Antitumor agent for oral administration and method for its preparation.

The invention relates to medicine, in particular to the chemistry of steroids, and relates specifically to progestogens with antitumor activity.

The use of progestogens in oncology for the treatment of hormone-dependent tumors such as uterine cancer, breast cancer, and endometrial cancer is known. As drugs based on progestogens, such as megees, depot provera, depostate are used [Mashkovsky M.D. “Medicines”, Moscow, “Medicine”, part 2, 1993, pp. 547-548]. The most widely used drug was depot prover, in which medroxyprogesterone acetate is used as a progestogen for the treatment of breast cancer, endometrial cancer ["Federal guidelines for the use of medicines for doctors." Issue I, 2000, p. 517].

The progestogens indicated in these preparations contain Δ ⁴ -3-keto grouping, characteristic not only for progesterone, but also for testosterone. The latter may be the cause of the presence of side androgenic properties in these gestagens that cause verification in women.

In this application, as an antitumor agent, a gestagen of the formula 1-3β-butanoyloxy-6-methyl-17α-acetoxy-pregna-4,6-dien-20-one (compound 1) and a method for its preparation are provided.

The claimed compound has a high antitumor activity, does not detect side androgenic properties (Hershberger test), and its structure does not contain Δ ⁴ -3-keto groups.

Figure 1 - the effect of compound 1 in combination with vincristine on the growth of transplantable acute vincristine-resistant mouse lymphocytic leukemia P388;

figure 2 - cytostatic activity of gestagens against MCF-7 cells;

figure 3 - cytostatic activity of gestagens against HeLa cells.

The antitumor activity of the claimed drug is shown in experiments on mice of the CBA line with transplantable cervical cancer RShM-5. The depot prover (MPA, medroxyprogesterone acetate) was used as a comparison drug.

Example 1

The study of the antitumor activity of the test compound was carried out on transplantable cervical cancer of the cervix cervical cancer-5, sensitive to progestins during orthotopic inoculation. The tumor was initially induced by methylcholanthrene in the subcutaneous autograft of the cervix of the mouse - CBA in 1970. The tumor was transplanted during laparotomy with a 1 mm ³ fragment into one of the uterine horns using a trocar to CBA mice under hexenal anesthesia. For transplantation, female mice of the CBA line, weighing 18-20 grams, were used. Treatment was started on day 3 after tumor inoculation. The course of treatment was 14 days with daily administration of the studied gestagens. Mice were killed 17 days after the start of the experiment. The antitumor effect was evaluated by inhibition of tumor growth (change in tumor mass), calculating the TPO coefficient (inhibition of tumor growth). In the experimental groups, 8 mice were used, in the control - 13 mice.

Scheme of introduction:

Compound 1 was administered in doses of 0.5; 1 and 2 mg / mouse orally through a tube daily for 14 days. Solvent - 1% starch paste. Depo-proofers were administered at doses of 1 and 2 mg / mouse orally through a tube daily for 14 days. The solvent is distilled water.

The results of a study of the effect of compound 1 on cervical cancer growth 5 are presented in table 1.

Table 1.
The antitumor activity of compound 1 when administered orally for 14 days to mice with cervical cancer of cervical cancer-5. Group Dose (mg / mouse) Tumor mass, M ± m (mg.) SRW (inhibition of tumor growth),% The control - 417 ± 93.5 - 0.5 265.4 ± 126.85 36 Compound 1 one 113.6 ± 27.76 73 *, ** 2 285.3 ± 128.99 32 MPA one 205 ± 71.76 55 * 2 187.1 ± 73.7 55 * Note: * - significant differences from control for p≤0.01; ** - significant difference from SRW for MPA (p≤0.05).

The experimental results indicate that compound 1 at a dose of 1 mg / mouse inhibits the growth of cervical cancer-5 by 73%, while MPA in the same dose - by 55%. Thus, the claimed agent when administered orally has antitumor activity against cervical cancer-5, and the therapeutic effect is superior to the comparison drug MPA.

Example 2

The inventive compound has a pronounced chemosensitizing effect, increasing the antitumor effect of vincristine in relation to transplantable acute vincristine resistant P388 mouse lymphocytic leukemia.

To evaluate the chemosensitizing effect of compound 1, the antitumor activity of vincristine as an independent compound was compared with the antitumor activity of vincristine in combination with compound 1.

The mice of the DBA2 line were vaccinated with vincristine-resistant lymphocytic leukemia P388, 2 million cells / mouse, the next day the Vincristine group (7 mice, the middle column in FIG. 1) was injected with vincristine 1 μg / g intraperitoneally (20 μg / mouse), the group "Vincristine + compound 1" (7 mice, the right column from the center in figure 1) vincristine 1 μg / g intraperitoneally (20 μg / mouse) and after 2 hours compound 1 1 mg / mouse (50 mg / kg) orally oil.

The control group (7 mice, the first column in figure 1) received the oil orally. On day 7 after inoculation, tumor growth was calculated by the number of ascites fluid cells. Figure 1 presents the results of an experiment evaluating the effect of compound 1 in combination with vincristine on the growth of transplantable acute vincristine resistant P388 mouse lymphocytic leukemia.

On the ordinate axis - tumor growth (percentage increase,%), * - a significant difference in the number of cells in the Vincristine + Compound 1 group from the Vincristine Group.

In the control group, the tumor grew in 7 days by 185%, in the Vincristine group - by 80%, in the Vincristine + compound 1 group - by 35%. Thus, compound 1 at a dose of 1 mg / mouse (50 mg / kg) exhibits a chemosensitizing effect, increasing the antitumor effect of vincristine by 16% and inhibiting tumor growth in combination with vincristine by 53%. Vincristine as an independent compound inhibits tumor growth by 37%.

Compound 1 significantly (P <0.001) enhances the antitumor effect of vincristine in relation to hormone-independent resistant acute lymphocytic leukemia of the mouse P388, therefore, is a chemosensitizer in relation to this tumor.

Example 3

The inventive tool has cytostatic activity against human breast cancer cells (MCF-7 line) and human cervical cancer (HeLa line).

Cytostatic activity was defined as suppression of cell viability (%) in the presence of the studied compounds. The effect of progestogens on tumor cell viability was assessed by the MTT method (3- (4,5-dimethylthiazolyl-2) -2,5-diphenyltetrazolium bromide) [Mealey K.L., Barhoumi R., Burghardt R.C. et al. Doxycycline induces expression of P glycoprotein in MCF-7 breast carcinoma cells // Antimicrob. Agents Chemother. - 2002 - V.46. N 3. - P.755-761]. MPA and progesterone were used as comparison compounds.

Data on the cytostatic activity of gestagens against MCF-7 cells are presented in figure 2. The cytostatic activity of gestagens against HeLa cells is shown in FIG. 3.

In the figures along the ordinate axis is the percentage of living cells,%; in each concentration given in figures 1 to 4, respectively: progesterone, compound 1, MPA, control; * - significant difference from control at P <0.001, ◆ - significant difference from control at P <0.05.

For MCF-7 human breast cancer cells, compound 1 has a more pronounced cytostatic effect than progesterone and a similar cytostatic effect with MPA (P <0.05). Compound 1 has a more pronounced cytostatic effect than MPA and progesterone in HeLa cell culture (P <0.05).

Example 4

The claimed compound is not toxic. This is shown in studies of acute toxicity in experiments on rats with the introduction of doses of the compound that are 50-100 times higher than the therapeutic (taking into account the wide range of recommended doses).

Example 5

The subject of the invention is also a process for the preparation of compound 1 by borohydride reduction of megestrol acetate followed by esterification of the reduction product with butyric acid anhydride.

The esterification of steroids containing a hydroxyl group is carried out, as a rule, in pyridine using an excess of anhydride [T.W. Greene. Protective groups in organic synthesis. New York, Wiley-Interscience, 1981, p. 110]. This General method is used in the described method for producing the claimed compound (Pat. Ross. 2099347). However, the butyric acid formed upon decomposition of the excess anhydride makes it difficult to isolate the target product in its pure form. When reproducing the production method described in the analogue, additional purification by recrystallization or chromatography on alumina is required to isolate the product, which reduces the yield of the product.

In the present invention, to free from excess butyric acid, the reaction mixture is treated with an ammonia solution after esterification.

The proposed method allows to obtain the target product without additional purification with a yield of up to 90% (calculated on megestrol acetate).

Get 3β-butanoyloxy-6-methyl-17α-acetoxy-pregna-4,6-dien-20-one as follows.

To a cooled suspension of 100 g of megestrol acetate in 600 ml of methanol, 9.8 g of sodium borohydride are added portionwise. The mixture is stirred for 30 minutes, after which, 250 ml of water are added during cooling and the solution is neutralized by the addition of diluted HCl to pH 7.0. The precipitated C ₃ -carbinol precipitate was filtered, washed with water and dried. 97.5 g of carbinol are obtained, which are dissolved in 195 ml of pyridine, 85 ml of butyric acid anhydride are added, stirred at 30 ° C for 1 hour and then stirring is continued at room temperature for 5 hours. To decompose the excess butyric acid, the solution is stirred with 40 ml of water for 1 h, after which it is slowly added to an aqueous solution of ammonia (44 ml of 25% ammonia in 1.1 l of water). The mixture was stirred for 1 h, the precipitate was filtered off and washed with water, then with methanol. Get 102.8 g of product (86.6% based on megestrol acetate), so pl. 118-120 ° С, [α] _D - 78 ° (with 1.0; CHCl ₃ ), identical with the known sample [mp. 117-118.5 ° C, [α] _D - 77 ° (Pat. Ross. 2099347)].

The claimed invention is not obvious to specialists working in the field of chemotherapy of tumors.

Despite the abundance of currently antitumor drugs with the most diverse structure and action of drugs, the search for new drugs in the fight against malignant neoplasms is still an urgent task both in terms of increasing the effectiveness of antitumor drugs and reducing their toxicity, as well as the possibility use in a wide variety of combinations.

In connection with the great socio-economic significance of the problem of treating malignant growth, the creation of new compounds is an urgent problem in both experimental and clinical medicine.

The creation of the compound required lengthy scientific research, a large team of chemists, pharmacologists, biologists, and other specialists who sometimes performed a significant amount of not only creative, but also quite laborious and original technical work.

It is completely unobvious to date is the possibility of creating means for oral administration containing gestagens - the claimed tool is the first domestic gestagen suitable for oral administration.

Thus, the claimed compound, the progestogen 3β-butanoyloxy-6-methyl-17α-acetoxypregn-4,6-diene-20-one, is competitive with the known progestogens that are part of the widely used anticancer drugs, and surpasses them in some indicators, which, of course, is very valuable in clinical, social, and economic terms.

Claims (3)

1. An antitumor agent for oral administration, characterized in that it is 3β-butanoyloxy-6-methyl-17α-acetoxy-pregna-4,6-dien-20-one of the formula 1

2. The method of obtaining the compound of formula 1 according to claim 1 by reducing megestrol acetate with sodium borohydride followed by esterification of the product of reduction with butyric acid anhydride, characterized in that the reaction mixture after etherification is treated with 1% aqueous ammonia solution to bind the excess of butyric acid anhydride formed during decomposition with subsequent separation of the precipitate formed. 3. The method according to claim 2, characterized in that the ammonia solution is used in a ratio of 3: 1 to the volume of the reaction mass.

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