NO135828B - - Google Patents

Description

The present invention relates to an analogous process for the production of dialkoxides of 17g-estradiol with topical action on skin, hair and mucous membranes, as well as with anti-seborrheic action, but without hormonal effects in therapeutically effective doses.

The compounds produced are thus dialkoxides of 17 3-estradiol with the general formula:

where R 1 denotes a lower alkyl group selected from ethyl and methyl, and R 2 denotes an alkyl group with 3-5 carbon atoms.

This class thus includes six compounds, namely:

(I) 3-propyloxy-17-methyloxy-1,3,5-(10)estratriene,

(II) 3-propyloxy-17S-ethyloxy-1,3,5-(10)estratriene,

(III) 3-butyloxy-17 6-methyloxy-1,3,5-(10)estratriene,

(IV) 3-butyloxy-17B-ethyloxy-1,3,5-(10)estratriene,

(V) 3-pentyloxy-17-methyloxy-1,3,5-(10)estratriene,

(VI) 3-pentyloxy-17B-ethyloxy-1,3,5-(10)estratriene.

These compounds are prepared according to otherwise known methods from estradiol, first preparing the alkoxide in the 3-position by the action of alkyl bromide with C^-C^ on estradiol and then introducing the methyl or ethyl group in the 17-position by means of methyl or ethyl sulfate. The following example illustrates the preparation of the first compound in the above list, namely 3-propyloxy-17g-ethyloxy-1,3,>5-(10) estratriene.

Example

First step: Preparation of the 3-propyl oxide of estradiol.

An alcoholic sodium ethylate solution is prepared by reacting 3 g of sodium with 300 ml of absolute ethanol. 30 g of estradiol is dissolved in the first-mentioned solution. 30 ml of n-propyl bromide are then added while stirring. The reaction is continued for 3 hours with stirring at 60°C, and evaporated in vacuum at 30°C to approx. 50 ml. The residue is taken up in 500 ml of benzene, and washed twice with 250 ml of 0.25N sodium carbonate, then with distilled water to neutrality. It is dried over sodium sulphate and evaporated in vacuo at 30°C.

You get 32 g of crude product (yield = 93%), and with recrystallization from 100 ml of methanol you get 31 g of pure substance (yield = 89%), m.p. = 100-101°C.

Second step: Preparation of 3-propyloxy-170-methyloxy-1, 3, 5- (10)

Austraria.

40 g of the above 3-propyl oxide is dissolved in 400 ml of anhydrous dimethyl sulphoxide. A few crystals of triphenylmethane are added as a color indicator and then freshly prepared dimethylsulfinylcarbanion (Na) until a permanent red color appears. A surplus of approx. 50% of dimethylsulfinylcarb anion (Na): The mixture is left for approx. 15 min. at room temperature and then cools it in an ice bath.. 400 ml of freshly distilled methyl sulphate is slowly added and stirred for 15 min. at room temperature. The surplus of methyl sulphate is destroyed with approx. 2 1 2N sodium carbonate, and stirring for 2 hours. The pH should be alkaline at the end of the operation. The solution is extracted with benzene (3 1, then 1.5 1), and the benzene fraction is washed with distilled water to neutrality. It is dried over sodium sulfate and evaporated in vacuo at 35°C. You get 42 g of raw product. Repeated recrystallization from ethanol gives a pure white substance with a yield of 78% calculated on the basis of the 3-propyl oxide, which melts at 66.67°C (Reichert). Purity is checked by thin-layer chromatography and gas chromatography.

It is obvious that one can prepare the other members of the class by replacing the two reactants, namely the propyl bromide and the methyl sulfate with respective amounts of the equivalent butyl or pentyl bromides, or ethyl sulfate, otherwise using the same procedure.

One thus arrives at the aforementioned substances with the following characteristic physical properties:

These compounds are new in themselves and have in common the characteristic that when they act on the skin or mucous membranes they exhibit very clear topical local effects and anti-seborrhoeic effects, while the compounds are practically free of estrogenic activity.

According to the Allen and Doisy test, total estrus is obtained in castrated rats by injection of an oil solution (0.2 ml) containing 120 µg + 10 of 3-propyloxy-17£-methyloxy-estratriene, 210 µg + 10 of 3- butyloxy-173-methyloxyestratriene or 310 µg + 10 of 3-pentyloxy-17g-methyloxyestratriene.

In general, the longer the side chain in the 3-position is, the weaker the estrogenic activity. Thus, 2-hexyloxy-173-methyloxyestratriene is inactive in the same experiment in a dose of 500 µg.

The four compounds indicated above, subjected to the same test by cutaneous application, give resp. as active doses 2-4 yg, 2-3 yg, 20-30 yg and 100 yg, and by oil injection the first compound gives activity at a dose of 70-100 yg, while the latter is reactive in an amount of 300 yg.

Replacement in the 17-position of the methyl radical with an ethyl radical does not significantly change the biological effects of the corresponding ether oxides.

The sensitivity of the same castrated Whistar rats when it comes to 17g-estradiol is under the same conditions equal to 0.6 yg + 0.05, and one can conclude from this that in the aforementioned test 3-propyloxy-17B-methyloxyestradiol is 200 times weaker in action and the 3-butoxy derivative is 300 times weaker than 170-estradiol.

The estrogenic activity, measured according to the uterotopic test, in immature guinea pigs aged 21-23 days, is the following:

Estrogen is injected cutaneously in a 5% aqueous-ethanolic solution for 3 consecutive days, and the animals are slaughtered 72 hours after the last administration.

The uterotopic effect for the three dialkoxides in the table is approx. 200 times weaker than for 17g-estradiol.

Contrary to what has been established with castrated rats, the uterotopic activity is not a function of the chain length of the radical in the 3-position.

In case of overdose, the three alkoxides behave like weak estrogens, as the uterotopic effect of estradiol flattens out when the uterine weight reaches 2-2.5 times the control animals' uterine weight, while for strong estrogens such as estradiol, 6 times the uterine weight can be achieved in relation to the control animals.

As a conclusion, double etherification in the 3- and 17-position of 17g-estradiol gives products with very low estrogenic activity towards animals, and no effect towards humans in doses much greater than the effective therapeutic doses.

The second experimental fact characterizing the described dialkoxides of estradiol is their relative potency according to the Allen and Doisy test when applied to the skin as an alcoholic 95% solution, tested on Whistar rats.

With this method of application, estrus is obtained for castrated rats at doses of 1.5 yg< + 0.5 for 3-propyloxy-17g-methyloxyestratriene and for 3-butyloxy-17(3-methyloxyestratriene, while by applying estradiol to the skin under precise the same conditions achieve an effect of 0.6 µg. If one sets up the ratio "effect threshold for cutaneous application/effect threshold for subcutaneous injection in oil solution", one obtains for estradiol a unit ratio equal to 1 and for 3-propyloxy-17f3-methyloxyestratriene a ratio equal to 80, and for 3-butyloxy-17g-methyloxyestratriene a ratio of 150.

If you extend the chain in position 3, this ratio drops to approx. 10 for 3-hexyloxy-17g-methyloxyestratriene.

This unexpected behavior for the dioxides of estradiol can be explained by the fact that the skin of the rats contains an enzyme that can hydrolyze the estradiol alkoxides, which is lacking in the other animal tissues. This hypothesis explains the very weak effect of the dialkoxides according to the Allen and Doisy test when administered by injection, and the relatively strong effect when the products are applied to the skin.

Another unexpected phenomenon that characterizes the estradiol dialkoxides also calls attention: when increasing the amount of 3-propyloxy-17g-methyloxyestratriene or 3-butyloxy-17g-methyloxyestratriene from 1-5 µg, no significant increase in the duration of estrus is found, which lasts less than 24 hours. This is different with 17g-estradiol, which in a dose of 5 yg, applied as an ethanolic solution to the skin, produces an estrus lasting an average of 5 days.

One can explain these results by the fact that, due to saturation of the enzyme, the amount of 17g-estradiol released on the skin by hydrolysis of the alkoxides is relatively constant when the applied doses vary between 1 and 5 µg, although the hormone thus released has a relatively high concentration in the skin, the concentration will be insufficient to produce a general powerful effect.

By etherifying the hydroxyl group in the 17-position with a methyl or ethyl radical and the hydroxyl group in the 3-position with a propyl or butyl radical, a relatively strong effect is thus achieved in situ (intradermally), but an effect that is negligible on the whole organism. However, this overall effect is sufficient to induce vaginal estrus in the castrated rat at a fraction of a microgram of 17-g estradiol.

The double etherification thus makes it quite unexpectedly possible to "direct" the effect of 176-estradiol towards the epithelial tissue.

With reference to more specific pharmacological investigations with 2-propyloxy-173-methyloxyestradiol (compound no. 1), the following can be summarized:

I. Gonadoinhibitory action

Compound No. 1 is nearly 80 times less gonadoinhibitory than ethinylestradiol by injection, and nearly 20 times less gonadoinhibitory than the same standard compound by oral administration.

The method consists in finding the degree of inhibition of testicular weight in young rats (and androgen receptors) as a function of the administration of a possible gonado-inhibiting compound. The results are expressed as DE 50, which denotes the dose which in the treated animals leads to a weight reduction of the testicles of 50% in relation to the testicle weight of the control animals.

The treatments are daily and last for 14 days; the application takes place subcutaneously or orally and all substances are given in identical volumes in the same medium.

The value DE 50 for gonadoinhibition of the test compounds is compared with the DE 50 for comparison compounds: for example ethinylestradiol.

Subcutaneous administration

The DE 50 of compound J-015 is 19 yg/animal/day, the DE 50 of ethinylestradiol is 0.25 yg/animal/day.

By oral route

The DE 50 compound J-015 is 20 yg/animal/day. The DE 50 for ethinylestradiol is 1.2 yg/animal/day.

II. Anti-seborrheic effect

It is known that androgens cause certain changes in various skin parameters (development of secretory activity in the sebaceous glands for example or activation of epithelial epidermis) while castration and estrogens affect the same parameters in the opposite direction.

These principles serve as the basis of a method which is considered the best and most harmonized of all tests with regard to the anti-seborrhoeic action of a given compound, (F.J. Ebling - J. Embryol. Exp. Morph. 1957, 5, 1 : 74-82, and several other following publications).

The technique uses castrated male rats that are resting

for 3-4 weeks and then on day 0 insert a pellet containing 12 pg of testosterone.

You are divided into random groups. On day 8, the animals are bathed in a solution of 10% sodium lauryl sulfate and dried in a warm air stream. They are bathed again on day 9 and after drying, a sample piece of the fur is removed, weighed and placed in ethyl ether. On day 17, a new piece of skin is removed.

At time 0 on day 17, all animals receive an intraperitoneal injection of colchicine. 5 hours later on the same day, the animals are slaughtered, and the skin is immediately removed. Likewise, the seminal vesicles and prostate gland are removed and weighed.

From day 0 to day 17, all the animals that are not control animals received a daily treatment. The following table summarizes the results, having grouped the animals into 3 control groups and 6 treatment or experimental groups. 1. Sebum decreases with castration and is rebuilt with the implantation of testosterone, even if this does not restore the androgen moth cells,

decreases with E2 (27%) for a quantity of 10 y and 41% for 30 y, the latter dose neutralizing the reaction of androgen receptors to the pellets,

decreases with connection no. I S.C. in a similar way and very much

progressive with the doses, little at P.C.

2. Number of mitoses in the sebaceous gland cells

Decreases with castration and is recovered by testosterone, does not decrease with E2 in amounts up to 30 y per day (S.C), but

decreases with 270 µg of compound I, S.C.

3. Number of mitoses in the epithelial cells

Decreases with castration,

recovered with testosterone,

decreases ++ with E2 in amounts of 30 yg.

4. Number of cores in the foam cells

Decreases with castration and is rebuilt with testosterone,

decreases with E2, proportional to the doses.

5. The thickness of the epithelium

Decreases with castration and is recovered with testosterone, decreases with E2 proportionally to the dose,

decreases with compound I, P.C. in quantities of at least 30 y and S.C.

in larger quantities.

6. Relationship between sebum and the number of mitoses in the gland cells Decreases with E2 by exclusively reducing sebum, proportional to the doses,

decreases with compound I, S.C. and likewise with connection

I, P.C.,

the number of mitoses decreases, but not the amount of sebum, in the latter two cases the ratio increases.

Conclusion

Compound No. I (3-propyloxy-17g-methyloxy-estradiol) behaves qualitatively analogously to E2, but is quantitatively superior (stronger action in the same dose).

Connection I, S.C. oestragen has an effect of 5% of E2, and this shows the additional advantage of compound No. I in relation to E2.

These features are reinforced when applied to humans

in that the two dialkoxides above have no feminizing effect when applied to the skin as oil-in-water or water-in-

oil, or as an alcoholic solution, or as an oil-

ning, in daily doses exceeding 20 mg, even after treatment for six months with application every day. The result is the same whether the products are sprayed on the nasal mucous membranes or the oral mucosa.

either in solution in perhydrosqualene, or applied to hairy skin as an ethanolic solution or a water-ethanolic solution,

or introduced into the vagina as a cream or as gynecological capsules.

In contrast, they have two alkoxides, as their derivatives

with an ethyloxy radical in the 17B position, a powerful topical effect on the skin, on hairy skin and on the mucous membrane and a significant anti-seborrhoeic effect. One can thus summarize the most frequent therapeutic uses of the dialkoxides:

1. Effects on the skin

Acne, seborrhea, skin laxity, dryness and other signs of aging on the skin.

2. Gynecological effects

The dialkoxides have a strong topical effect on the vaginal mucous membranes without affecting the secretion of cervical mucus. This effect can be utilized in the treatment of various forms of vulvitis and vaginitis, possibly secondarily infected, by stimulating healing after surgical or obstetric interventions, in vaginal discomfort and dysparynia by oral contraceptives and in the menopause etc. The steroids produced according to the invention can optionally be used together with anti-fungal drugs and/or bactericides and/or antiparasitic agents.

3. Ear-nose-throat effects

Infections (in connection with an antibiotic), antrophy of the nasal mucous membranes, pseudo-allergic conditions of the upper respiratory mucous membranes, bad breath, stomatitis, sore throat.

4. Seborrhea on hairy skin, with or without hair loss

The above alkoxides also affect the softness and quality of the skin and not only against seborrhea-hyper-secretion.

Below are the results of the clinical trials with compound no. I, with the code number J-015.

A. Dermatology

a) A first experimenter applied a cream containing 1-2% J-015, daily or every other day. It concerns a trial of 61 patients of both sexes and all ages who either had dermo-epidermal abnormalities or seborrhoeic tendencies.

The results are determined as a function of the recorded reactions to 86 pathological symptoms. The results are summarized in the following table.

b) Another experimenter applied the same type of cream to 83 patients of both sexes and in the age group 13-47 years, who

had all signs of seborrhea with or without acne.

The treatments are daily, the doctor has calculated the active doses received each day by these patients: the amount varied between 3.3 and 26 mg/day, with the majority of patients (103 out of 128) receiving 7-13 mg/day.

The treatment lasted:

less than 6 weeks for 13 patients

between 2 and 3% month for 25 patients between 4 and 6k month for 28 patients between 7 and 12 months for 17 patients.

in

It should be emphasized that, despite the quantities used and the long treatment times, in most cases none of the 53 women and girls who were treated experienced difficulties with menstruation and none of the 30 men complained of disturbances in the sexual drive or other signs of estrogen (gynaecomastia). or anti-androgen signs.

The table below summarizes the overall results of the above experiments.

The overall results are:

- favorable in 46 cases (61%), of which 34 indisputably outstanding (44.7%), - unfavorable in 30 cases (39.4%), of which 17 absolutely no effect (22%).

It should be noted that in all cases the sick judgment had developed over several months and most often over the course of several years.

Among the patients during these experiments, many had previously received other treatments (contraceptive agents, antibiotics, gonadotrophin in small serial doses, etc.): - in 15 previously unsuccessful cases of such treatments, the cream J-015 was used 19 times: 5 times with success of which 3

excellent results and 15 times without success,

- in 5 other cases of failed treatment, the cream J-015 gave 2 clear improvements, - in 11 incomplete results from other treatments, the cream gave

J-015 healing 8 times.

B. Gynecology

A broad-based trial of 50 patients to confirm the topical effects of compound J-015 on the vulvo-vaginal epithelium and to determine the possible estrogenic action of this steroid when applied to a susceptible recipient.

The medication was given both as the same cream as during dermatology or as gynecological gelatin capsules filled with an emulsion containing 5 mg/capsule (formula B) or 10 mg/capsule (separate A).

The table below summarizes the results of the first gynecological examination as a function of the indications:

As for the estrogenic effects, these were practically non-existent judging by:

- absence of secretion of cervical mucus,

- negligible change in the cytohormonal compositions of the vaginal epithelium.

One observation particularly illustrates the difference that exists for the steroids in question between their outstanding topical effects and the hormonal effects that are not very pronounced and do not progress either with the doses or with extended treatment time.

It concerns a topical vaginitis where the contraction only made it possible to insert a finger and where there was a secondary infection.

After 4 months of application of skin cream, once per day for the first 2 months and then once every other day, two fingers could be inserted into the vagina, and the patient could have sexual relations. The infection resolved spontaneously without further treatment. After 4 months and despite this development, it was found that

only 5 of 100 surface cells had pyknotic nuclei (instead of 0 before treatment).

In connection with the effect on the genital area in women, no disruption of the menstrual cycle has ever been found after gynecological treatment with J-015.

Claims (1)

Analogous process for the production of dialkoxides of 173-estradiol with topical action on skin, hair and mucous membranes, as well as with anti-seborrheic action, but without hormonal effects in therapeutically effective doses, and with the general formula: where R-^ denotes methyl and ethyl, and R 2 denotes an alkyl group with 3-5 carbon atoms, characterized in that the alkoxide in the 3-position is first prepared from estradiol by the action of an alkyl bromide corresponding to R£ on estradiol, where -after the alkyl group is introduced in the 17-position by means of an alkyl sulphate corresponding to R^.