DE3490728C2 - Androstane derivatives - Google Patents

Description

The present invention relates to new steroid derivatives the androstane series (hormone cytostatics), the have antitumor and hormonal activity and in medicine for cancer treatment and as contraceptives Can find use.

A steroid derivative of the androstane series is known, 2 [bis (2-chloroethyl) aminomethylene] 5 α -androstan-17 β -ol-3-one-17 β- acetate (see Burstein, H. Ringold. J. Org. Chem. , 26, p. 3085, 1961)

in which the androgenic properties of the molecule Hormone is an alkylating group bound. An anti-tumor activity is however not described.

A number of steroid derivatives of the androstane series are known, in which the anti-tumor and hormonal activity is proven. Such derivatives include for example, N-di (2-chloroethyl) aminophenylacetic acid ester (Chlorphenacyl) and the steroidal alcohol esters dehydroepiandrosterone and testosterone. These connections act on tumors that counter alkylating Reagents (Walker carcino sarcoma W-256 from rats and adenocarcinoma the mammary gland of mice Ca-755) very sensitive and some mammary gland tumors in mice and rats (HK, R-13 762), which is useful against alkylating reagents are insensitive.  

The connections point however has a toxic effect on the organism, which differ in neurotoxicity, lymphopenia and occurrence of necrosis in the liver and kidneys. (Sofiina Z.P., Lagova N.D., Valueva I.M., Kusmina Z.V., Shkodinskaya E.N., Khaletsky A.M., Trudy I Vsesoyuznoi konferentsii po khimioterapii zlokachestvennykh opukholei, Riga, 1968, p. 441-443; M. Wall, G. Abernethy, F. Carroll, D. Taylor. J. med. chem., 1969, 12, 5, p. 810-818; U. Schaeppi, J. Heyman, R. Fleischman, H. Rosenkrantz, V. Glievski, R. Phelan, D. Cooney, R. Davis. Canc. Chem. Rep., 1973, p. 3, 4, 1, p. 85-95; Lagova N.D. in: Aktuellnye voprosy eksperimentalnoi khimioterapii opukholei, Chernogolovka, 1980, vol. 1, Pp. 222-225). It should be noted that it is for the above Chorphenacyl derivatives of the androstane series (hormone cytostatics) in the literature there is no information about their specific physiological Effect caused by the structure of the hormone it contains is evoked what undoubtedly on the height and that affects the spectrum of their antitumor activity.

The invention has for its object new steroid derivatives of the Androstane range, which one Antitumor and hormonal activity, especially selective Effect on the target tissues and occurring tumors exhibit.

This object is achieved as can be seen from the preceding claim.  

The compounds according to the invention differ in the structure of the contained hormone and make metabolites of the main testosterone hormone of the androstane series.

The steroids of the androstane series according to the invention (hormone cytostatics) contain metabolites of testosterone as steroids and are esters of 5 α -androstane substituted in positions 3 and 17.

They represent amorphous or fine crystalline powdery substances of white or white-yellowish color that is water-insoluble and in organic solvents are soluble. they demonstrate long-term storage stability in the dark a temperature of plus 5 to 0 ° C and are at Daylight yellow.

The compounds according to the invention are as follows: 17 β - [N-di (2-chloroethyl) aminophenylacetate] 5 α- andro stan-17 β -ol-3-one (I); 3,3- [N-Di (2-chloroethyl) aminophenylacetate] 5 α - androstandiol-3 β , 17 β (II); 17 β - [N-Di (2-chloroethyl) amino phenylacetate] 5 α -androandiol-3 β , 17 β (III);

The compounds of the invention have one high antitumor activity and hormonal (androgenic and anabolic) activity, the nature of which Structure of the hormonal molecular part of these substances as well as the structure of the alkylating group and the The binding location of the same depends on the steroid.

Your advantage is there in the selectivity of their effect on the target tissues and tumors arising in these tissues what the peculiarities of the spectrum of their anti-tumor effects secures against the known hormone cytostatics. The proposed parenteral (subcutaneous) procedures for Introduction of the hormone cytostatics according to the invention causes lower toxicity and higher selectivity the biological effects of the compounds as that oral procedure because there is no cleavage of the molecule into a hormonal and a highly toxic alkylating Fragment caused.

The cytostatic activity of the invention Connections became more solid on a number of strains Transplantation tumors and leukoses in mice and rats namely adenocarcinoma of the mammary gland Ca-755, Lewis Lung carcinoma LL, cervical cancer ZK-5, Walker carcinosarcoma W-256, ovarian tumor OT, lymphoid leukosis L 1210 examined.

For comparison with the compounds according to the invention, dihydrotestosterone (5 α -androstan- 17 β -ol-3-one), dehydroepiandrosterone and chlorophenacyl or the preparation sarcolysin ( β - [N-di (2-chloroethyl) aminophenyl] - α - alanine hydrochloride), which belong to the molecular inventory of the compounds according to the invention, and the known hormone cytostatics, ie dehydroepiandrosterone esters with chlorophenacyl (DEA-mustard) and the preparation Östrazyt (Östra-1,3,5 (10) -trien-3,17 β -diol, 3N- [bis- (2-chloroethyl)] carbamate 17 sodium phosphate) under searches.

The compounds were subcutaneously in oil in the animals Doses of mg / kg body weight for 5 to 10 days within the one for the investigations on the tumors in question usual deadlines (24 or 48 hours after the transplant of the tumor) injected. Östrazyt was performed same way in 0.9% NaCl solution.

Spontaneous was used to study selectivity and / or carcinogenic tumors of Mice and rats that appear in the target tissues. In In these cases, treatment was started when the Tumors had reached a certain size, which precludes the possibility of their spontaneous regression.

The criteria for antitumor activity were the Extension of survival time of animals (VÜT%) and inhibiting the growth of solid tumors (HWT%)

where T is the mean survival of the treated animal,
C represents the mean survival of the control animals;

where O is the mean tumor volume (tumor mass) in the group of treated animals, K is the mean tumor volume (tumor mass) in the control group.

As criteria of the anti-tumor effect on exposure healing was performed on spontaneous and induced tumors of animals (%) and the change in tumor dimensions compared to the initial data (%).

The investigations were carried out on lineage mice and their First generation hybrids performed.

The results obtained in the investigation of the antitumor activity of the compounds according to the invention on some transplant tumors are shown in Table 1.

As can be seen from Table 1, the inventive Compounds have a strong anti-tumor effect regarding a number of transplant tumors, which results in a significant inhibition of tumor growth by 66 to 99% and / or prolongation of survival of treated animals expressed by 25 to 160%. The advantages are particularly visible  at Ca-755. The connections don't just inhibit the growth of Ca-755 but prolong it also the survival of animals. The well-known hormone cytostatic from the Androstan series DEA- Mustard and Östrazyt have no anti-tumor activity shown this tumor.

Table 1 also indicates that the invention Compounds from alkylating reagents Chlorphenacyl and sarcolysine, which belong to their molecule, in strength and spectrum of the antitumor effect and in differentiate between toxicity. For example, they are therapeutic doses of the compounds of the invention in a range of 5 to 50 mg / kg with daily injection within 5 days while the most tolerable Doses of chlorphenacyl and sarcolysine at this Treatment methods are 1.5 or 3 mg / kg.

For the compounds 17 β - [N-Di (2-chloroethyl) amino phenylacetate] 5 α -androstan-17 β -ol-3-one (I), 3 β - [N-Di (2-chloroethyl) aminophenylacetate] 5 α- androstandiol-3 β , 17 β (II) and 17 β - [N-Di (2-chloroethyl) aminophenylacetate] 5 α - androstandiol-3 β , 17 β (III) the selectivity and the differences occurred to a higher degree the effect on the tumors arising in hormone-dependent tissues transplant cervical cancer ZK-5, ovarian tumors OT and on induced and spontaneous ovarian tumors and mammary gland tumors of mice and rats. At the same time, these compounds were less active in the treatment of animals with leukosis L 1210 (Table 1), which testifies to their lower alkylating activity. As the data in Table 2 show, compound I weakly prevents the growth of the ovarian tumor, while compound III and especially compound IV cause a strong growth inhibition of this tumor. From the same table it can be seen that the cervical cancer and ovarian tumor are weak or not at all sensitive to dihydrotestosterone and chlorphenacyl, while the estrogen cytostatic agent estracyte even promotes the growth of cervical cancer ZK-5. From the information shown in Table 3 follow the advantages of the compounds I, IV and III over the known androgen testosterone propionate in their antitumor activity against induced and spontaneous ovarian and mammary gland tumors of mice and rats.

The study of the general toxic effect of Compounds according to the invention have shown that they are relative are weakly toxic. Mice tolerate five times Administration of the substances in doses of 5 to 50 mg / kg. Most substances have a broad therapeutic Dose range (Table 4). They release a high anti-tumor effect in cans, which are in their absolute Differentiate size by 2 to 6 times.

Table 2

Antitumor activity of the steroid derivatives of the androstane series on tumors originating from the target tissues

Table 3

Antitumor effect of the steroid derivatives of the androstane series on induced and spontaneous tumors originating from the target tissues

Table 4

Highly tolerated doses (HVD) and the therapeutic dose range of the steroid derivatives of the androstane series (mice, five injections)

For examination of the selectivity of action of the compounds according to the invention to the target tissue were determined their hormonal properties of biological means (androgenic and anabolic effect) and biochemical (specific binding with receptors of dihydrotestosterone and 17 β-estradiol) tests. The androgenic and anabolic activity was determined after changing the mass of the prostate gland and the m. Levator ani detected in castrated rat brood and expressed in indicators (ratio of the relative mass of the corresponding organs after exposure to the mass in the control group, which was defined as 1). The specific binding with receptors of dihydrotestosterone and 17 β- estradiol (free binding sites) was investigated in ventral prostate of normal and (2 weeks) previously castrated rats, which received a therapeutic course of preparations. The determination of the specific binding of dihydrotestosterone and 17 β-estradiol were introduced in cytosol fractions by the tissue by the proteins by means of protamine sulfate after incubation of cytosols with saturating concentrations of [³H] dihydrotestosterone and [³H] -17 β-estradiol at 0 ° C failed. Nuclear receptors were detected from the fraction of nuclei that had been previously extracted with 0.4 N KCl solution by precipitation of the hormone-receptor complex using protamine sulfate.

Table 5 contains information on the androgenic and anabolic effect of the compounds according to the invention.

Table 5 indicates that the inventive Steroid derivatives of the androstane series a weak one show anabolic activity. Your androgenic activity depends on the structure of the hormone part of the compound from. The duration of the androgenic effect is the largest for compound I. The However, androgenic activity of Compound I is significant weaker than with testosterone propionate (the known Androgen).

It is found that the steroid derivatives according to the invention the androstane series specifically with the receptors of the corresponding hormones are bound. So binds the compound I to cytoplasmic receptors of dihydrotestosterone with subsequent cooperation with the corresponding acceptor zones of chromatin both in Normal tissues (ventral prostate and uterus in rats) as well as in tumors of animals and humans, which in the androgen-specific target tissues (mammary gland) stand.

The hormonal properties of the compounds I, II and III according to the invention also appeared in the influence on the reception of 17 β-estradiol in the tissues of the ventral prostate apparent. The differences were found between the connections. For example, in intact rats, the specific binding of [3 H] -17 β- estradiol in the cytosol increased 5-fold after the introduction of compound II and 10-fold after the introduction of compound I. After the introduction of compound III, the specific binding of [3 H] -17 β- estradiol in the cytosol was not demonstrated. (She was completely inhibited). The substances according to the invention thus significantly changed the sensitivity of the animals to estrogens.

The substances I, II and III described also have a different metabolism in the androgen-specific target tissues. The differences are already apparent at the level of the main enzyme of androgen metabolism, the 5 α -reductase, and at the level of 3 β -, 3 α -, 6 α - and 7 α -hydroxylases.

The steroid derivatives of the androstane series according to the invention differ with regard to the spectrum of the Antitumor effect, the general toxic, hormonal effect as well as the metabolism in the target tissues. Each of the connections is characterized by only its own peculiarities the biological effect and is therefore of its own accord Interest.

Table 5

Androgenic and anabolic properties of hormone cytostatics, ie the steroid derivatives of the androstane series

When studying the kinetics of the hormonal effects of 17 β - [N-Di (2-chloroethyl) aminophenylacetate] 5 α -an drostan-17 β -ol-3-one (I) an interesting law was discovered. The stimulation period of target organs (prostate, testicles, seminal vesicles) was replaced by a period of strong inhibition after 1.5 months, while in animals that only received dihydrotestosterone, the mass of the organs listed returned to the norm at this time. Also noteworthy is the permanent inhibitory effect of compound I on the thyroid gland in male and female rats (analogous to the effect of dihydrotestosterone) as well as on the uterus and ovaries, although the mass of these organs does not affect the 3 months after stopping the introduction of the preparation Norm declined. The effect of the temporary "chemical castration" of compound 1 has been found in female rats with estrogen-active follicular ovarian cysts. After the treatment course with compound I, the characteristic constant heat in rats ceases. Laparotomy and ovarian hystology revealed that the ovary stopped due to follicular atresia and atrophy of the interstitial tissue of the ovary, which is estrogen-active in rats. The effect of "chemical castration" has also been demonstrated in rats during the lactation period, which after treatment with 17 β - [N-Di (2-chloroethyl) aminophenylacetate] 5 α -androstan-17 β -ol-3-one (I ) lost their reproductive ability for 5 to 7 months. This information testifies to the contraceptive properties of this substance.

Compound I is shown to also alkylate Possesses properties. It disrupts the RNA transcription and transport process, causes structural defects in the DNA, which is faster in normal cells (spleen) than in the tumor cells (sarcoma S-37). The the latter speaks for the selectivity of the antitumor effect.

The example of compound I thus shows that  the hormone cytostatics, d. H. the steroid derivatives of the androstane series, have a double mechanism of biological action. They have an alkylating and a hormone effect.

The technological design of the manufacturing process of steroid derivatives of the androstane series is simple.

The synthesis of the compounds can be represented by the following scheme put:

A: R1 = -OH, = O; R² = -OH, = O;
B: R1 = = O; D² = -OCOCH₂C₆H₄N (CH₂CH₂Cl) ₂;
R² = = O, R¹ = -OCOCH₂C₆H₄N (CH₂CH₂CL) ₂;
C: R¹ = -OH, R² = -OCOCH₂C₆H₄N (CH₂CH₂Cl) ₂;
R² = -OH, R¹ = -OCOCH₂C₆H₄-N (CH₂CH₂Cl) ₂.

The synthesis of the compounds according to the invention is carried out by reacting the N-di (2-chloroethyl) aminophenylacetic acid chloride with 5 α -androstane substituted in the 3 β or 17 β position, followed by reduction of esterification products with sodium borohydride.

Example 1 Preparation of 17 β - [N-Di (2-chloroethyl) aminophenyl acetate] 5 α -androstan-17 β -ol-3-one (I)

To a slurry of 30.0 g (0.10 mmol) of 5 α -An Drostan-17 β -ol-3-one in 500 ml of dry benzene is added the calculated quantity of triethylamine and 38.0 g (0.11 mmol) of N -Di (2-chloroethyl) aminophenylacetic acid chloride. The mixture obtained is stirred at 80 ° C. within 24 hours and triethylamine hydrochloride is separated off. The benzene solution is washed with aqueous KHCO₃ solution and H₂O and evaporated. The residue is dissolved in benzene, applied to the column filled with Al₂O₃ and eluted with benzene. The benzene eluate is evaporated, the residue is crystallized as an oil from ether. The yield of product is 27.8 g (49.2%, based on the starting hormone), the melting temperature is between 99 and 101 ° C (ether).

C₃₁H₄₃NO₃Cl₂:
Found in %:
C 67.85; H 7.89; Cl 12.96.
Calculated in%:
C 67.89; H 7.84; Cl 12.95.

Example 2 Preparation of 3 β - [N-Di (2-chloroethyl) aminophenyl acetate] 5 α- androstandiol-3 β , 17 β (II)

4.2 g of 3 β - [N-di (2-chloroethyl) aminophenylacetate] 5 α -androstane-17 β- one are mixed with 90 ml of dry methanol and 0.4 g of sodium borohydride are added at plus 5 ° C, stirring 1 Hour, acidified using acetic acid until a pH of 6 was reached, the solution was evaporated, the residue was washed with water and dried over sodium sulfate. 4.0 g of amorphous powder are obtained from the benzene solution. The yield of product is 95% (based on the starting keto compound).

C₃₁H₄₅Cl₂NO₃:
Found in %:
C 67.59; H 8.25; Cl 12.88.
Calculated in%:
C 67.90; H 8.16; Cl 12.95.

Example 3 Preparation of 17 β - [N-Di (2-chloroethyl) amino phenylacetate] 5 α- androstandiol-3 β , 17 β (III)

This connection is made analogously to the synthesis of the compound described in Example 2.

17 β - [N-Di (2-chloroethyl) aminophenylacetate] 5 α -androstan-3-one is introduced into the reduction reaction. The subsequent operations are carried out as in Example 2.

The reduction product is chromatographed on the column filled with silica gel (benzene-ethyl acetate ratio = 4: 1), fractions containing 3 α -OH isomer (Rf 0.41) and 3 β -OH isomer (Rf 0.33) to be collected. The yield of β-end isomer is 67%, based on the crude 3-keto compound.

C₃₁H₄₅Cl₂NO₃:
Found in %:
C 67.54; H 8.26; Cl 13.03.
Calculated in%:
C 67.59; H 8.25; Cl 12.88.

The steroid derivatives of the androstane series according to the invention can be found in various fields of medicine especially in gynecology and oncology as cytostatics and use contraceptives.

Claims (1)

Androstane derivatives of formulas I, II and III