RU2200165C1 - 17α-ACETOXY-3β-PHENYLPROPIONYLOXY-6-METHYL-PREGNA-4,6-DIENE-20-ONE ELICITING GESTAGENOUS ACTIVITY AND MEDICINAL AGENT BASED ON THEREOF - Google Patents

Abstract

FIELD: organic chemistry, steroids, medicine, gynecology, pharmacy. SUBSTANCE:inventionrelatestonovelcompound17α-acetoxy-3β-phenylpropionyloxy-6-methylpregna-4,6-diene-20-one of the formula (I)

Description

 The invention relates to medicine, more specifically to medicines intended to preserve pregnancy, and may find application in obstetric practice and gynecology.

 Highly active gestagen compounds are known to be used in obstetric practice, such as turinal (norsteroid) [Radar - Drug Encyclopedia. Ed. 7 recycled. Ch. Editor Yu.F. Krylov. - M .: "RLS-2000", ed. 2000, p. 942], acetomepregenol (pregnane series of compounds) [ibid., P. 116]. The latter is taken as a prototype.

 The basis of the invention is the task of expanding the range of products with the ability to maintain pregnancy.

и лекарственное средство, активным началом которого является указанное вещество, позволяют решить поставленную задачу. В дальнейшем для заявленного вещества использовано сокращенное название - фенилпропионилокс.Newly synthesized substance, which is 17α-Acetoxy-3β-phenylpropionyloxy-6-methylpregna-4,6-dien-20-one of the formula (I)

and the drug, the active principle of which is the specified substance, can solve the problem. In the future, the abbreviated name is used for the claimed substance - phenylpropionylox.

 The inventive substance belongs to the range of pregnane and contains an additional methyl group at the C-6 carbon atom, an acetoxy group at C-17 in the α configuration, a phenylpropionyloxy group at C-3 in the β configuration, and double bonds in positions 4, 5 and 6, 7.

The invention is illustrated by drawings, on which:
FIG. 1 is a photograph of a histological preparation of a cross section of the uterus to determine the effect of phenylpropionylox at a dose of 0.25 mg / kg on the endometrium in infantile rabbits, evaluated on a McPhail scale,
FIG. 2 is a graph of the dependence of the McPhail index on the administered dose in immature female rabbits with enteral administration of turinal and acetomepregenol,
FIG. 3 is a plot of the McPhail index versus the administered dose in immature female rabbits with enteral administration of phenylpropionylox.

 Compound I is a crystalline substance of white or almost white color, insoluble in water, readily soluble in chlorohydrocarbons (chloroform, methylene chloride), soluble in acetone, benzene, alcohol, sparingly soluble in hexane and ether. The structure of the compound is confirmed by elemental analysis and spectral data.

 The inventive compound of formula I was obtained from megestrol acetate II in two stages: reduction with sodium borohydride in methanol, the resulting 17α-acetoxy-3β-hydroxy-6-methylpregn-4,6-dien-20-one (III) acylated the action of phenylpropionic acid chloride in the environment of benzene and pyridine. The synthesis scheme of the claimed compound (I), see the end of the description.

To prevent adverse reactions: the hydrolysis of the acetoxy group and the reduction of the carbonyl group to form compound IV, a small excess of sodium borohydride is used, the reaction is carried out under cooling and at volume-weight ratios: 1 part by weight II, from 0.03 to 0.12 parts by weight NaBHBH ₄ and from 6 to 8 volumes of methanol at 15-30 ^o C for 30 minutes The recovery process proceeds in suspension, by-products are formed in trace amounts (TLC analysis). Stage acylation is carried out in the ratio: 1 weight. including III, 0.5-0.6 volumes of phenylpropionic acid chloride, 0.7-1 volumes of pyridine, 5-8 volumes of benzene.

 The target product is isolated by pouring the reaction solution into dilute hydrochloric acid, followed by separation of the benzene layer, washing it with water, filtering the benzene solution through an alumina layer, evaporating and crystallizing the product from methanol.

Examples illustrating the method of obtaining the claimed compounds:
Preparation of 17α-acetoxy-3β-hydroxy-6-methylpregn-4,6-dien-20-one (III)
To a suspension of 20 g of megestrol acetate in 160 ml of methanol cooled to 12-14 ^° C, 2 g of sodium borohydride are added in portions at such a rate that the reaction temperature remains in the range of 15-30 ^° C. After stirring for 30 minutes, 140- 150 ml of water containing 4-5 ml of concentrated hydrochloric acid. After stirring for 30 minutes, the precipitate is filtered off. 19.2 g of III are obtained with a melting point of 195-203 ^{° C.} , [α] $\genfrac{}{}{0ex}{}{\text{20}}{\text{D}}$ -54.3 ^o (2% solution in chloroform).

Preparation of 17α-acetoxy-3β-phenylpropionyloxy-6-methylpregna-4,6-dien-20-one (1)
A mixture of 19 g of III, 150 ml of benzene, 19 ml of pyridine, 10 ml of phenylpropionic acid chloride is stirred for 20 hours at room temperature. The reaction mass is slowly poured into dilute hydrochloric acid. The benzene layer was separated, the aqueous was extracted with benzene. The combined benzene solution is washed with water and, after concentration in vacuo, filtered through a layer of alumina. The eluate was evaporated in vacuo. Obtain 18.6 g (73% of theory.) I after crystallization from methanol, so pl. 145-146 ^o C, [α] $\genfrac{}{}{0ex}{}{\text{20}}{\text{D}}$ -73.3 ^o (C 1.25% in chloroform).

Found C, 76.27; H 8.39 C ₃₃ H ₄₂ O ₅
Calculated: C 76.41; H, 8.22.

UV spectrum: λ _max 240 nm, E 21450;
IR spectrum ν, cm ^-1 : 1705 and 1720 (С = O esters), 1250 (СО acetate), 1270 (СО phenylpropionate), 1460.

1 H-NMR spectrum (CDCl ₃ , δ ppm): 0.69 (with CH ₃ -18); 0.99 (with CH ₃ -19); 1.78 (with CH ₃ -6); 2.02 and 2.1 (with COSH ₃ ); 5.18; 5.14; 5.53 (m C ₃ -H, C ₄ -H, C ₇ -H); 7.25 (m C ₆ H ₅ ).

 Other properties and characteristics of the claimed substance 17α-acetoxy-3β-phenylpropionyloxy-6-methylpregn-4,6-dien-20-one of the formula (I) were investigated in experimental animals. The studies were carried out in comparison with the known and used in practice drugs and are given in the examples.

Example 1. Determination of gestagenic activity in the Clauberg-McFail test (Clauberg-McPhail)
Progestogenic activity was investigated by the Klauberg-Mac Feil method on immature female rabbits. Compared substances were administered in 4-6 doses. It was found that phenylpropionylox, like acetomepregenol and turinal, causes a dose-dependent increase in secretory processes in the endometrium of infantile rabbits. The degree of secretory changes in the endometrium was studied on histological preparations of transverse sections of the uterus under a binocular magnifier. According to generally accepted standards, when studying the gestagenic activity of drugs, the secretion stages are evaluated on a McPhail scale from 0 to 4 points:
- zero stage of secretion (0 points) - the endometrium and stromal glands are compact, the epithelial layer of the uterine mucosa is continuous, occasionally narrow glands are found,
- the first stage of secretion (1 point) - development of the uterine glands is noted, but they are short, deeper stroma layers are not changed
- the second stage of secretion (2 points) - the glands increase in size and quantity, the stroma is not changed, the color intensity increases from deeper layers to the lumen
- the third stage of secretion (3 points), the glands penetrate into the deeper layers of the stroma, but they are narrow, the glandular tissue loses its ability to concentrate around the lumen
- the fourth stage (4 points) is characterized by a strong development of the uterine glands and a violation of the endometrium, the glands penetrate close to the myometrium, on the sections of the stroma connective tissue sites are found in the endometrial fields.

 When examining the uterus of animals in the control group receiving estrogenic preparation, hypertrophy of the muscle layer and endometrium was noted. However, the inner surface of the uterus remains smooth, which corresponds to the zero stage on the McPhail scale.

 As seen in FIG. 1, under the influence of phenylpropionylox in the endometrium, the following changes are observed: the mucous membrane of the uterus contains numerous glands that extend through the entire thickness of the endometrium and penetrate into the deep layers of the stroma. The progestational activity of the drug that causes these changes in the endometrium of infantile rabbits is 3-3.5 points on the McPhail scale.

 The graphs presented in figure 2 and figure 3, characterize the degree of secretory transformation of the endometrium. As can be seen from the graph in figure 2, Turinal in doses of 0.004, 0.016 mg / kg had a weak effect on the endometrium, estimated at 0.125 and 0.3 points, respectively. A further increase in dose caused activation of secretory changes in the endometrium. At a dose of 0.5 mg / kg turinal showed its maximum gestagen activity, equal to 3.1 points. A further increase in the dose of the drug administered did not cause activation of secretory processes in the endometrium, which corresponded to the effect caused by the lower dose of 0.5 mg / kg. It follows from the same graph that acetomepregenol showed similar activity. At doses of 0.008-0.125 mg / kg, their activity exceeded the effect exerted by Turinal, and at doses of 0.25 mg / kg and 0.5 mg / kg it was established at a level close to the effect caused by Turinal in the same doses. As can be seen from the graph in figure 3, at a dose of 0.25 mg / kg phenylpropionylox exhibits the highest gestagenic activity, corresponding to 3.2 points on the McPhail scale. With a four-fold dose reduction, the pronounced gestagenic effect of this compound is preserved (2, 6 points). At doses less than 0.008 mg / kg, the activity of the drug was evaluated less than 1 point. The experimental data were processed by the method of regression analysis, which allowed us to calculate the regression coefficients, on the basis of which the biological activity of phenylpropionylox was determined.

Table 1 shows the biological activity of the compounds, evaluated by ED ₅₀ . The effective dose (IU ₅₀ ), at which the frequency of the pharmacological effect taken into account is 50%, the corresponding McPhail index equal to 2, for progesterone was 0.26 mg / kg, for turinal - 0.034 mg / kg, for acetomepregenol - 0.01 mg / kg, for phenylpropionylox - 0.00926 mg / kg. Given ED ₅₀ , relative progestogenic activity was calculated for each compound, taking progesterone activity as a unit.

 The calculation results showed that phenylpropionylox has a high relative gestagenic activity, 28.1 times higher than the activity of progesterone (chart). The relative gestagenic activity of turinal in the Klauberg-Mac Feil test is 7.7 times higher than the activity of progesterone. Acetomepregenol significantly exceeds the activity of turinal and at the same time it is slightly inferior to phenylpropionylox, exceeding the activity of progesterone by 26 times (Table 1).

 Example 2. Determination of gestagenic activity in the Corner Allen test.

 The study of gestagenic responses of phenylpropionylox was continued in experiments on ovariectomized rabbits to preserve pregnancy in the Corner Allen test. According to this test, mature rabbits 18 hours after mating underwent bilateral oophorectomy (under general anesthesia). Phenylpropionylox in the form of an oil solution was administered intramuscularly at a dose of 0.25 mg / kg, that is, at a minimum dose providing a pronounced progestogenic effect in the Clauberg-Mac Feil test. The drug was administered for 6 days, starting from the day of the ovariectomy, thus covering the period from the development of the egg to the formation of the blastocyst, and their number was recorded. Ovariectomized control animals received intramuscularly a solvent of 0.4 ml vegetable oil.

 The results of the study are presented in table 2. From the above data it follows that the studied new compound has progestational activity, as it causes pregnancy in some ovariectomized rabbits.

 Summarizing the data obtained in the test of Klauberg-Mac Feil and Corner Allen, we can conclude that phenylpropionylox is an active progestogen compound, superior in activity to some known steroid drugs.

 Example 3. Determination of androgenic and anabolic activity of phenylpropionylox.

 The results of studies performed on experimental castrated male rats treated with phenylpropionylox at a dose of 1.5 mg / kg daily for 7 days showed that the mass of seminal vesicles and ventral prostate was 6.42 ± 0.20 mg and 56.3 ± 3.1 mg, respectively. In control animals receiving testosterone propionate, the mass of seminal vesicles was 18.1 ± 1.1 mg, the mass of the ventral prostate was 76.2 ± 2.2 mg; in control animals treated with the solvent, 6.52 ± 0.21 mg and 58.10 ± 2.40 mg, respectively.

 The results of the studies indicate that phenylpropionylox does not cause statistically significant changes in the mass of seminal vesicles and the ventral prostate in experimental animals compared with animals of the control group treated with solvent. At the same time, the administration of testosterone propionate caused a significant increase in the mass of these organs.

 Thus, the administration of phenylpropionylox in a dose of 1.5 mg / kg to infantile castrated male rats did not cause a statistically significant increase in the mass of seminal vesicles and ventral prostate as compared to the control (vegetable oil), which indicates the absence of the androgenic effect of the claimed substance.

 To identify the possible anabolic effects of phenylpropionylox, animals were decapitated on the 8th day from the start of the experiment, m. Levator ani and muscle mass was determined.

 In experimental animals treated with phenylpropionylox, mass m. levator ani was 61.34 ± 3.36 mg. In the control group of rats treated with testosterone propionate, mass m. levator ani was 82.1 ± 3.0 mg, and in the control group receiving the solvent, 62.10 ± 3.20 mg.

 The results of the experiments show that testosterone propionate significantly increases muscle mass, while phenylpropionylox practically does not affect the mass of the target organ. Based on the data obtained, it can be concluded that phenylpropionylox in a dose of 1.5 mg / kg does not cause an increase in mass m. levator ani, which indicates the absence of anabolic effect in the claimed substance.

Example 4. Determination of the estrogenic activity of phenylpropionylox
The study of the estrogenic activity of phenylpropionylox was carried out according to the Dorfman method by recording changes in uterine mass in infantile female rats. The experimental animals were injected intramuscularly with phenylpropionylox in a dose of 1.5 mg / kg in a volume of 0.1 ml for 3 days. The control rats were injected with a solvent — vegetable oil in the same volume. Then the animals were decapitated, the uterus was isolated and weighed.

 The uterine mass of experimental rats was 44.21 ± 2.16 mg; in control animals, the uterine mass was 43.62 ± 2.81 mg.

 The results of the experiments showed that the test compound in the specified dose practically does not cause changes in the mass of the uterus of infantile female rats. Consequently, phenylpropionylox does not have an estrogenic uterotropic effect.

 Example 5 Determination of the degree of influence of phenylpropionylox on contractile activity of the uterus.

 Studies have shown that intraperitoneal administration to pregnant rats with a gestational age of 14-15 days of gestagenic drugs acetomepregenol and phenylpropionylox at a dose of 0.25 mg / kg leads to inhibition of contractile activity of the uterus, recorded by bioelectric activity. The research results are presented in table. 3.

 Already 15 minutes after the administration of acetomepregenol in the specified dose, a decrease in the amplitude of the myometrium biopotentials by 23.3% was noted. A reduced amplitude was also noted 20-30 minutes after drug administration. The frequency of contractions did not change. The restoration of the amplitude occurred 45 minutes after the administration of the drug (table. 3).

 A similar picture was observed with the introduction of the claimed substance - phenylpropionylox. As follows from the table, phenylpropionylox in a dose of 0.25 mg / kg 15 minutes after administration reduces the amplitude of biopotentials by 22%. The same level of decrease in amplitude was also noted 20-30 minutes after drug administration. The drug did not cause changes in the frequency of contractions of the myometrium (table. 3). Recovery of baseline was observed 45 minutes after administration of the drug.

 Thus, the studied gestagen drugs: acetomepregenol and phenylpropionylox in a certain dose have a similar inhibitory effect on the contractile activity of the myometrium in pregnant rats with a period of 14-15 days of pregnancy in in vivo experiments.

 Example 6. Determining the effect of phenylpropionylox on the possibility of prolonging pregnancy with threatening abortion.

 The effect of phenylpropionylox on the possibility of prolonging pregnancy with threatening abortion induced by prodigiosan was determined in experiments on mice.

Animals to prevent abortion on the 13th, 14th and 15th day of pregnancy were injected subcutaneously with gestagens at a dose of 0.25 mg / kg - acetomepregenol in one group, phenylpropionylox in another group of animals. The animals in the control group were injected with olive oil. Spontaneous abortion was recorded by the presence of stillborn fetuses. The experimental results are presented in table. 4
As can be seen from Table 4, in all animals of the 1st group after administration on the 16th day of pregnancy prodigiosan (20 μg / animal), spontaneous abortion was observed already on the 17-18th day of pregnancy, which was preceded by spotting from the genital tract. Preliminary administration of acetomepregenol to pregnant mice (group 2) prevented miscarriage in 6 out of 8 animals. With subcutaneous administration of phenylpropionylox (group 3) - in 7 out of 8 animals it was possible to maintain a pregnancy (mice 's pregnancies ended in delivery with term live fetuses). In the control group of animals treated with olive oil, pregnancy ended in childbirth at the usual time.

 Similar positive results were obtained when inducing abortion by interleukin-lβ, prostaglandin enzaprost F and other drugs.

The newly synthesized substance is a low toxic compound. Doses 10-100000 times the therapeutic dose do not cause death of animals, which confirms the low toxicity of the drug
The data obtained indicate a correlation of the pregnancy-preserving effect of substances and their gestagenic activity.

 Comparative tests of the new substance and the known substances used in obstetrics and gynecology, the detection of its gestagenic activity made it possible to determine the composition of the new drug, the active principle of which is phenylpropionylox. Traditionally, such drugs are available in tablet form. Obtained during the experiments, the characteristics of the substance made it possible to determine the proportion of the active principle (phenylpropionylox) in the drug — 0.8-3.0 wt.%.

In particular, the recommended composition, wt.%:
17α-Acetoxy-3β-phenylpropionyloxy-6-methylpregna-4,6-dien-20-one - 1.2
Lactose - 88.8
Starch - 7.3
Calcium Stearate - 0.9
Talc - 1.8
The tablet drug can be recommended for women with a clinical picture of an abortive and beginning abortion from 4 weeks of pregnancy with a continuation of the course to 20 weeks of pregnancy.

Claims (3)

1. 17α-Acetoxy-3β-phenylpropionyloxy-6-methylpregna-4,6-dien-20-one of the formula I

possessing gestagenic activity. 2. A drug having progestational activity, consisting of an active principle and pharmaceutically acceptable excipients, characterized in that the active principle is 17α-acetoxy-3β-phenylpropionyloxy-6-methylpregna-4,6-dien-20-one of the formula I

in the amount of 0.8-3.0% by weight of the drug. 3. The drug under item 2, characterized in that it includes, as pharmaceutically acceptable excipients, lactose, starch, calcium stearate and talc in the following ratio of components, wt. %:
17α-Acetoxy-3β-phenylpropionyloxy-6-methylpregna-4,6-dien-20-one - 1.2
Lactose - 88.8
Starch - 7.3
Calcium Stearate - 0.9
Talc - 1.8

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