RS20060416A - Substituted 16-ketosteroids of the estratriene and androstene series with biological activity - Google Patents

Abstract

Invention relates to new, biologically active substituted 16-ketosteroids estratriene and androstene series having the general formula IWherein R is methyl or benzyl radical, and X has the structure as the following Where the R2 hydrogen, alkyl radical from with 1 – 4 carbons, acyl radical, alkylsulphonyloxy radical or aryl radical. According to the invention, onto a 17-keto group of the appropriate 17-keto-16-oxymes is carried out additon of RLi or RMgJ where R has previously mentioned meanings, in tetrehydrofuran where obtained 16-oxymino-17-substituted estratriene and androstene derivative having the general formula as the II that can be separated or further thereto applied is acid hydro titanium thrichloride, where there comes to hydrolysis of 16-oximino group in the appropriate 16-keto group with simultaneous migration of R group with 17α to 17β position when obtained is 16-keto-17 substituted derivative in both series.

Description

SUBSTITUTED 16-KETOSTEROIDS OF THE ESTRATRIENE AND ANDROSTEN SERIES

WITH BIOLOGICAL ACTIVITY

1. TECHNICAL FIELD

The invention belongs to the field of organic chemical technology, more precisely to the field of making synthetic steroids with biological activity. According to the international classification of patents, the invention belongs to classes C 07 J 51/00; A 61 K 31/56, A 61 K 31/575, A 61 K 31/58.

This application was separated from the basic application P-604/96.

2. DEFINED PROBLEM

This invention includes new, hitherto unknown estrone and androstene derivatives with anti-estrogen and anti-androgen activity, 16-keto-17-substituted derivatives in the estrone and androstene series, as well as the process for their preparation starting from the corresponding 17-keto-16-oximes.

3. STATE OF THE ART

D-seco steroids are of great interest both because of their possible biological activity (Baran, J.S., J.Med. Chem. 1967, 10, 1039), and because of the role they may have on certain biochemical processes (Hu, Y. et al, Steroids, 1995, 60, 491). D-seco-estrone and D-seco androstene derivatives can be obtained in different ways. For the first time, when determining the structure of estrogen hormones (Marrian and Haslewood, J.Soc.Chem.Ind., 1932, 51, 279T), D-sec dicarboxylic acid with the gross formula C18H22O5 was obtained by treating estriol with alkalis. In the same way, estradiol and estrone give D-seco monocarboxylic acid (Heer et al., Helv.Chim. Acta, 1945, 28, 156).

Huffman et al. (J.Biol.Chem., 1952, 196, 367) oxidized 3-methoxyestra-1,3,5(10)-triene-17-hydroxy-16-one with lead tetraacetate in acetic acid after 24 hours and isolated 3 as the main product. -methoxy-17-oxo-16,17-sec-ester-1,3,5(10)-triene-16-carboxylic acid. D-secosteroids can also be obtained by Beckman fragmentation of oximes or their derivatives using dimethylsulfoxide and trifluoroacetic acids as reagents (Fenselau et al., J.Org.Chem. Soc. (C) 1956, 3887). D-sec cyano aldehyde in the androstene series was obtained by the action of p-toluenesulfonyl chloride at room temperature in pyridine (Miljković et al., J.Org.Chem. 1973, 38, 3585), and the corresponding derivative in the estrone series was obtained in good yield (Miljković et al., J.Org.Chem. 1977, 42, 2101), under similar conditions starting from 3-Methoxyestra-1,3,5(10)-trien-16-oximino-17(3-ol.

Various 17-alkyloxy-, 17-acyloxy-, diarylhalogenarylalkyloxy-, arylsulfonyloxy-, alkylsulfonyloxy-16,17-secoester-1,3,5(10)-triene derivatives were obtained using Beckman fragmentation under mild conditions (Pejanović et al., patent Yu 46859; Petrović et al., Steroids 1990, 27, 276).

In our earlier work, 3p,17p-dihydroxy-16-oximino-17-picolyl-5-androstene was synthesized (Miljković and Gaši, Chimica Chronica, New Series, 1980, 9, 325), which under a very mild reaction application of titanium trichloride in an acidic medium, at room temperature, gives a seco cyano derivative 30-hydroxy-16,17-seco-16-nitrile-17-keto-17-picolyl-5-androstene (Miljković and Gaši, Buli.Soc.Chim. Belgrade, 1981, 46, 263).

The compounds known so far have shown certain biological activities, which could be increased after certain modifications.

4. SOLUTION DESCRIPTION

For the above reasons, and based on the previous knowledge about the biological activity of this type of compound and the relationship between structure and biological activity, we synthesized a number of new compounds in the estrone and androstene series in the expectation that these new compounds will have improved biological activity, which was shown in the biological tests whose results we cite.

The subject of the invention are compounds 16-keto-17-substituted-estra-1,3,5(10)-triene and 16-keto-17-substituted-5-androstene derivatives of the general formula I in which R represents a methyl or benzyl residue, and X has the following structure:

where R2 is hydrogen, an alkyl radical with 1 - 4 carbons, an acyl radical, an alkylsulfonyloxy radical or an aryl radical and the procedure for their preparation.

According to the invention, the 17-keto group of the 17-keto-16-oxime of the general formula III

where X has the aforementioned meanings,

the addition of RLi or RMgJ, where R has the previously mentioned meaning, is performed in tetrahydrofuran at a temperature of -20° to -10°C, whereby 16-oximino-17-substituted steroids of the estrone and androstene series of the general formula II are obtained

in which R, X and R2 have the aforementioned meaning.

The obtained compounds can be isolated and represent new biological compounds or can be used as intermediates further in the process by treating them with acidic aqueous titanium trichloride, whereby the hydrolysis of the 16-oximino group into the corresponding 16-keto group occurs with the simultaneous migration of the R group from 17a to the 170 position when 16-keto-17-substituted derivatives are obtained in both series, general formula I, in which R and X have the previously stated meaning.

The invention will be further illustrated by the following examples.

Example 1

3p, 17a-dihydroxy-16-keto-17fi-benzyl-5-androstene

3β,17β-dihydroxy-16-oximino-17α-methyl-5-androstene (1 g) was dissolved in ethanol (100 ml). A solution of titanium trichloride (0.72 g of titanium is heated for one hour at the boiling point with 1.72 ml of hydrochloric acid) and then n-hexane (20 ml) are added to the resulting solution. The reaction mixture is left at room temperature for 12 hours, poured into water, neutralized with aqueous sodium bicarbonate solution to pH 7 and extracted with ether. The organic layer is washed with water, dried and evaporated to dryness under vacuum. The resulting crude product was recrystallized from methanol to give pure 3β,17α-dihydroxy-16-keto-17β-benzyl-5-androstene (0.78 g, 81.2%), m.p. 185°-187°C.

Example 2

3- methoxy- l 6- keto- l 7a- hydroxy- l 7p- benzyl- estra- l, 3, 5(10)- triene

5 3-Methoxy-17a-benzyl-17p-hydroxy-16-oxyrunoestra-1,3,5(10)-triene (1.0 g) was dissolved in ethanol (240 ml) and a mixture of concentrated HCl and ethanol (1:1, 73 ml) and a solution of titanium trichloride (0.7 g of titanium) was heated at the boiling point with hydrochloric acid for one hour. /1:1, 71 ml/) and then n-hexane (40 ml) was added. The reaction mixture is left at room temperature for 18 hours, poured into water, neutralized with aqueous sodium bicarbonate solution to pH 7 and extracted with ether. The organic layer is washed with water, dried and evaporated to dryness under vacuum. The obtained crude compound was purified by silica gel column chromatography (hexane : ethyl acetate /8:2/) and pure 3-methoxy-16-keto-17a-hydroxy-17P-benzyl-estra-1,3,5(10)-triene (0.57 g, 51%) was obtained.

Example 3

3p, 17a- dihydroxy- 16- keto- 17p- methyl- 5- androstene

3β,17β-dihydroxy-16-oximino-17α-methyl-5-androstene (1 g) was dissolved in ethanol (150 ml) and a mixture of concentrated HCl and ethanol (1:1, 30 ml) was added to the solution. A solution of titanium trichloride (0.6 g of titanium is heated for one hour at the boiling point with hydrochloric acid /1:1, 60 ml) and then n-hexane (30 ml) are added to the obtained solution. The reaction mixture is left at room temperature for 12 hours, poured into water, neutralized with an aqueous sodium bicarbonate solution to pH 7 and extracted with ether. The organic layer is washed with water, dried and evaporated to dryness under vacuum. The resulting crude product was recrystallized from methanol to give pure 3β,17α-dihydroxy-16-keto-17β-methyl-5-androstene (0.83 g, 87.32%), m.p. 272°-275°C.

Example 4

3- methoxy- l 6- keto- l 7a- hydroxy- l 7p- methyl- estra- l, 3, 5(10)- triene

From 3-methoxy-17a-benzyl-17P-hydroxy-16-oxyminoestra-1,3,5(10)-triene (1.0 g), in the same way as in example 2, a crude product is obtained, which after crystallization from methanol gives pure 3-methoxy-1 6-keto-17a-hydroxy-17p-methyl-estra-1,3,5(10)-triene (0.8 g, 84.2%), wt. 185°-187°C.

Example 5

The estrone and androstene derivatives obtained according to the invention were then tested in order to determine their biological activity.

As an illustration of the change in the original hormonal activity of estrone derivatives, estrogens and antiestrogens (agonistic and antagonistic effects) were tested using the uterotropic method (Bowler et al., Steroids, 1989, 54, 71).

A solution of 3-methoxy-16-keto-17α-hydroxy-17β-benzyl-estra-1,3,5(10)-triene in olive oil was injected s.c. in a dose of 400 ug/day (total dose 25 mg/kg body weight) to immature female rats (21-23 days of gestation, 35-40 g), alone or in combination with estradiol benzoate 72-75 hours after the first injection, the uterus was cleaned of fatty tissue, the intrauterine fluid was removed and the weight of the uterus was measured.

Estrone and antiestrone activity are calculated according to the following formulas:

Agonistic effect (%) = 100 x (C - A)/(B - A)

Antagonistic effect (%) = 100 x (B - D)/(B - A)

Where A, B, C and D represent the weights of the uterus (mg/100 g of body weight) of untreated animals and animals treated only with estradiol, only with the test substance, or with a combination of estradiol benzoate and the test substance. The administered dose had no residual estrogenic activity (agonistic effect - 1.5%), and showed an antagonistic activity of 17.8%.

Example 6

As an illustration of the antiandrogenic activity, which is demonstrated by various mechanisms (blockade of testosterone synthesis, competition for androgen receptors, blockade of 5a-reductase and aromatase, considering that in many tissues testosterone acts after transformation into DHT, i.e. estradiol), androstene derivatives were tested in relation to: Competition for androgen receptors in the ventral prostate of a castrated (24h) adult male rat in vitro (radioreceptor analysis; Simerd et al., Moll. Cell. Endocrinol. 1986, 44, 261).

6

7 Inhibition of aromatase activity in the homogenate of the ovaries of PMSG-pretreated female rats in the presence of testosterone as a substrate, and measurement of estradiol production by radioimmunological analysis, Inhibition of 3|3HSD, P450i7, Iiazei 17(3HSD activity in the postmitochondrial fraction of the testes or suspension of Levdig cells of adult male rats (Marić et al., J.Steroids) Biochem. Mol. Biol., in press).

Increasing concentrations of 3p,17a-dihydroxy-16-keto-17p-methyl-5-androstene were incubated in the presence of 2 nM<3>H-T (18 h, 4°C), and the postmitochondrial fraction of the prostate of a castrated male rat was used as a receptor source. Based on the competitive curve obtained for this compound, it was shown that it competes significantly for androgen receptors (IC50= 4 uM, IC50 for flutamide is 35 uM, and for hydroxy-flutamide 0.8 uM according to Simard et al., 1986). This compound was also tested for anti-aromatase activity. Ovary homogenate (0.26 mg of protein) was incubated for 15 min in the absence and presence of different concentrations of the tested substance under conditions of saturation concentration of testosterone as a substrate. Based on the obtained distribution, it was shown that this compound selectively blocks aromatase (IC50= 350 uM). It was also established that it has no significant activity on other steroidogenic enzymes.

Claims (3)

1. New 16-keto-17-substituted-estra-1,3,5(10)-trienic and 16-keto-17-substituted-5- androstene derivatives of the general formula I in which R is a methyl or benzyl radical and X has the following structure where R2 is hydrogen, an alkyl radical with 1-4 carbons, an acyl radical, an alkylsulfonyloxy radical or an aryl radical. 2. Process for obtaining new 16-keto-17-substituted steroids of the estrone and androstene series of the general formula I according to claim 1, characterized in that the 17-keto group of the 17-keto-16-oxime of the formula III in which X has the previously mentioned meaning, the addition of compounds of the type RLi or RMgJ, where R has the previously mentioned meanings, in tetrahydrofuran, at a temperature of -20° to -10°C, whereby a compound of the general formula II is obtained wherein R and X have the aforementioned meanings, which can be isolated or further treated with acidic aqueous titanium trichloride, whereby the hydrolysis of the 16-oximino group to the corresponding 16-keto group occurs with simultaneous migration of the R group from the 17a to the 17p position when the 16-keto-17-substituted derivative is obtained in both series. 3. A compound according to claim 1, for use as a drug with anti-estrogenic and anti-androgenic activity.