NO173098B - ANALOGUE PROCEDURE FOR THE PREPARATION OF THERAPEUTIC ACTIVE 10-PROPYNOL ANDROSTENES - Google Patents

Description

The female sex hormones estrone and estradiol

is involved in many physiological processes. The formation of these steroids is regulated by a number of enzymes. The enzyme aromatase is the rate-limiting enzyme in the non-reversible conversion of androgens (the hormones testosterone and androstenedione) to estrogens (the hormones estradiol and estrone). Materials such as aromatase inhibitors regulate or inhibit the conversion of androgen to estrogen, and thus have therapeutic utility in the treatment of clinical conditions potentiated by the presence of estrogens. A further discussion regarding aromatase inhibitors can be found in US Patent 4,322,416.

The present invention relates to an analogue method for the production of therapeutically active compounds of general formula

wherein R 4 is C 1-4 alkyl.

The compounds produced according to the invention are optically active. The stereochemistry at the ring junction sites is the same as that observed in the natural androstane series. Thus, the configuration of the alkynyl group is 3, as the angled hydrogen at C-8 and the angled substituent at C-13. In the compounds covered by the invention, the B/C and C/D ring connection sites are trans.

The compounds produced according to the invention are inhibitors of aromatase. As such, they are useful in the treatment of hyperestrogenaemia. The compounds are useful in regulating abnormally high levels of estrogens, both when the high levels observed are relatively stable, or when there are short waves of elevated estrogen levels that occur as part of cyclic body functions. Both women and men can be treated, although of course the level of estrogen that would be considered high in men would be much lower than the amount considered high in women.

These compounds have further value in the treatment of gynecomastia, male infertility resulting from elevated estrogen levels, and hyperestrogenemia which may precede myocardial infarction. The compounds may also have value in the treatment of estrogen-dependent disease processes. The term treatment also includes application in the prevention of the course of the disease in question. The disease processes include various estrogen-induced and estrogen-stimulated tumors such as breast tumor, pancreatic tumor, endometrial tumor or ovarian cancer as well as prostatic hyperplasia and benign breast disease.

The aromatase inhibitory effect of the compounds can be determined using a radioenzymatic assay. An aromatase enzyme preparation is used from the microsomal fraction isolated from human placenta. Stereospecific elimination of 13- and 23-tritium markers from androgen substrates such as testosterone and androstenedione,

and subsequent appearance of tritiated water, are used to measure the degree of enzyme reaction during in vitro incubations.

In assessing the inhibition of aromatase activity, the compounds were tested according to

the following procedure adapted from Johnston et al.,

J. Steroid Biochem., Vol. 20, No. 6A, 1221 (1984) and Johnston, Steroids, Vol. 50, Nos. 1-3, 105 (1987). Athymic nude mice were subcutaneously injected with 1.5 x 10 human choriocarcinoma trophoblast (JAr) cells that develop tumor masses of approx. 1 g over 10 days. Tumor aromatase activity was determined in vitro by staining <3>H„0 resulting from the stereo-specific release of l-(3 3 H from l-[ 3H]-androstenedione. Cytosol (800xg) from 35 mg of tumor was the source of both aromatase and 33-steroid dehydrogenase (SDH):isomerase activity.The test compound was incubated for varying intervals (0-3 hours) with aromatase before addition of 34 pmol 1-[3H]-androstenedione to initiate a 30 minute aromatase activity assay. When 10-(2-propynyl)-19-norandrost-5-ene-33,17p-diol was tested in vitro by this procedure, the following results were observed:

Time-dependent JAr tumor aromatase activity

The biphasic response observed suggests that the reduction of the first competitive aromatase inhibition was the result of enzymatic processing of 10-(2-propynyl)-19-norandrost-5-ene-33,173-diol into a more reactive component that produced a time-dependent inhibition of aromatase.

The compound 10-(2-propynyl)-19-norandrost-5-ene-33,173-diol was also evaluated in vivo by treating nude mice with trophoblast tumors. At 6 hour post-treatment intervals, tumor aromatase activity was determined in an in vitro assay as discussed above. The vehicle for oral dosing was PEG-200.

In vivo inhibition of aromatase activity by JAr tumor xenografts in nude mice

This response indicates an increase in aromatase inhibitory activity as the dose of the compound was increased.

In the treatment of hyperestrogenemia, the compounds can be administered in different ways to the patient to be treated in order to achieve the desired effect. As used herein in connection with the treatment of hyperestrogenemia, the term "patient" is intended to refer to mammals such as primates, including humans, dogs and rodents. The compounds can be administered alone, in combination with each other or in combination with other hormone receptor antagonists. The compounds can also be administered in the form of a pharmaceutical preparation.

The compounds can be administered orally or parenterally, e.g. intravenously, intraperitoneally, intramuscularly or subcutaneously, involving injection of the active ingredient directly into tissue or tumor sites such as the mammary gland. The compounds can also be administered incorporated into extended release devices. The amount of compound administered will vary over a wide range and may be any effective amount. Depending on the patient to be treated, the condition to be treated and the method of administration, the effective amount of administered compound will vary from 1 to 1000 mg/kg body weight per day, and preferably from 40 to 200 mg/kg body weight per day.

For parenteral administration, the compounds may be administered as injectable doses of a solution or suspension of the compound in a physiologically acceptable diluent, with a pharmaceutical carrier which may be a sterile liquid such as water-in-oil, with or without the addition of a surfactant. and other pharmaceutically acceptable adjuvants. Examples of oils used in these preparations are petroleum oils and those of animal, vegetable or synthetic origin, e.g. peanut oil, soybean oil and mineral oil. In general, water, saline, aqueous dextrose and related sugar solutions and ethanols and glycols, such as propylene glycol or polyethylene glycol, are the preferred liquid carriers, particularly for injectable solutions.

The compounds according to the invention exhibit a slower onset of action and a longer half-life compared to the 10-(2-alkynyl)-steroidal aromatase inhibitors described in US patent document 4,322,416. These compounds can thus be administered in the form of a depot injection or implant. These preparations are formulated in such a way that a prolonged release of the active ingredient is enabled. The active ingredient can also be pressed into pellets or

small cylinders and are implanted subcutaneously or intramuscularly as depot injections or implants. Implants can use inert materials such as biodegradable polymers and synthetic silicones, e.g. Silastic<®>, silicone rubber manufactured by Dow-Corning Corporation. Suitable pharmaceutical carriers and formulation techniques are found in standard books such as Remington's Pharmaceutical Sciences, Mack Publishing Company, Eaton, Pennsylvania.

The analog method according to the invention is characterized by the fact that a compound of formula

reacted with sodium borohydride.

The compounds are prepared from a known compound, 3,3,17,17-bis(ethylenedioxy)-10-(2-propynyl)-19-norandrost-5-ene. This starting compound is dissolved in acetic acid, heated, treated with water, poured into NaHCO 3 solution and extracted to form a mixture of 3,3-ethylene-dioxy-10-(2-propynyl)-19-norandrost-5 -en-17-one and 10-(2-propynyl)-19-norandrost-5-en-3,17-dione. This diketone is dissolved in ethanol, treated with a borohydride reducing agent to form the 3P,17P-dihydroxy compound.

The following example illustrates the invention.

Example

A suspension of 1.3 g of 3,3,17,17-bis(ethylenedioxy)-10-(2-propynyl)-19-norandrost-5-ene in 13 ml of glacial acetic acid was placed in an oil bath at 65°C and was stirred until the steroid was dissolved. 3.3 mL of water was added and the mixture was stirred for 8 minutes, after which time the solution was poured into an ice-cold NaHCO 3 solution. The resulting product was extracted into ether. The extract was washed with bicarbonate and brine and was dried over MgSO 4 . After filtration and concentration, the residue was chromatographed on silica gel and eluted with 40% ethyl acetate in hexane to give 0.69 g of a mixture of 3,3-ethylenedioxy-10-(2-propynyl)-19-norandrost-5-en- 17-one and 10-(2-propynyl)-19-. norandrost-5-ene-3,17-dione.

Without further purification, the above diketone was dissolved in 30 ml of absolute ethanol, treated with 0.074 g of NaBH 4 and stirred for 30 minutes at room temperature.

Some precipitation occurred and 20 mL of THF was added.

After a further 2.5 hours of stirring, 0.5 ml of acetic acid was added and the solution was concentrated. The residue was taken up in a mixture of ether and ethyl acetate, washed with water,

saturated NaHCO^ and brine, and was dried over MgSO^. After filtration and concentration, the residue was twice subjected to silica gel chromatography, first by eluting with 50% ethyl acetate in hexane and then with 5% CH₂OH in Cr₂C₂ to give 0.21 g of 10-(2- propynyl)-19-norandrost-5-ene-3|3,17p<->diol. Melting point 164-166°C.

Claims (1)

Analogy method for the preparation of therapeutically active compounds of general formula wherein R 4 is C 1-4 alkyl, characterized by a compound of formula reacted with sodium borohydride.