CZ2000301A3 - Preparation for treating alopecia, hirsutism of women or seborrhea or preparation for prevention bone metastasis caused by prostate cancer - Google Patents

Abstract

The use of 3-oxo-4-aza-5a-androst-1-en-173- is described. the carboxamide compounds of formula I wherein R 1 and R 2 mean the same or different group and each one represents a hydrogen atom, a hydroxyl group protected a hydroxyl group or a lower alkoxy group, or a group thereof a pharmaceutically acceptable salt or other derivative thereof for producing a composition for treating alopecia, hirsutism in women or seborrhea or to prevent bone metastasis caused prostate cancer.

Description

A composition for treating alopecia, female hirsutism or seborrhea, or a composition for preventing bone metastasis caused by prostate cancer.

Technical field

The present invention relates to a novel composition useful for the treatment of alopecia, female hirsutism or seborrhea or for the prevention of prostate cancer bone metastasis comprising as active ingredient a 3-oxo-4-aza-5α-androst-1-ene-17β-carboxamide compound.

BACKGROUND OF THE INVENTION

Over-stimulation of androgenic hormones such as dihydrotestosterone (DHT) causes androgen-dependent alopecia (male baldness or the like), acne, vulgaris, seborrhea, female hirsutism, benign prostate hypertrophy, and prostate cancer.

Steroid antiandrogenic hormones (eg, female estrogen hormone) are compounds that have been shown to be able to treat these symptoms due to overstimulation of androgenic hormones. However, they tend to bring about undesirable activities, such as feminization, because they themselves have hormonal activity.

On the other hand, non-steroidal anti-androgenic hormones have also been developed. Despite not having a hormonal effect, they compete with the natural androgens for the receptor and thus have undesirable activities such as feminization of the male fetus in the uterus or the male or the onset of the feedback mechanism, thus overstimulating the testicles.

Since 5α-reductase acts on testosterone to produce dihydrotestosterone (DHT), if the activity of 5α-reductase can be inhibited, treatment of symptoms can be expected due to overstimulation of androgenic hormones without the mentioned side effects.

Human 5α-reductase includes two isoenzymes. It has been found that 5α-reductase type I exists in the sebaceous glands of the face and skin and that 5α-reductase type II exists in the prostate.

It is expected that a strong inhibition of type I 5α-reductase existing in hair follicles would alleviate male baldness. The effects of MK386, a selective 5α-reductase inhibitor of type I, was evaluated in a monkey, a model of animal male baldness. As a result, the level of DHT in the blood showed a 30 to 40% (w / w) decrease, but this efficacy of MK386 was not found (J. Invest. Dermatol. 1995, 104,658).

MK386

Administration of Finasteride, a selective type II 5α-reductase inhibitor, to an animal model at 1 mg / kg reduced blood DHT levels by 60-70% by weight, but was unexpectedly found to be effective in treating baldness (J. Clin. Endocrinol (Metabol. 1994, 79, 991).

The effects of Finasterod on human baldness have been demonstrated in a clinical trial. Inhibition of the factor involved in the reduction of hair follicle size leads to these effects and is believed to depend on the blood DHT level. In addition, as a result of recent clinical trials, it has been found that the intensity of decreasing DHT levels in the blood is mainly dependent on the intensity of 5a-reductase type 1f inhibitory activity and that better results can be obtained using a 5a-reductase type I inhibitor in combination with a 5a-reductase inhibitor type II (J. Clin. Endocrinol. Metabol. 1996, 81, 2942-2947).

Also in the treatment of female or seborrheic hirsutism, a compound having 5α-reductase type I and 5α-reductase type II inhibitory activity is considered to be superior to a compound having only 5α-reductase type II inhibitory activity. In order to find a more effective drug for the treatment of alopecia, female hirsutism or seborrhea, a compound is required which inhibits 5α-reductase type II more strongly than Finasteride and which at the same time strongly inhibits 5α-reductase type I.

5α-reductase type II is distributed in the prostate, such that a compound having potent type II? 5-reductase inhibitory activity is effective in treating prostate cancer. Recently, however, it has been found that during the development of prostate cancer and bone metastasis, type I 5α-reductase is in active form (Japanese Patent Application (Kokai) No. Hei 8-277220). It is also contemplated that the above compound, which is capable of inhibiting both type I and type II 5α-reductase, is also useful in the prevention and treatment of prostate disease.

The 3-oxo-4-aza-5α-androst-1-ene-17β-carboxamide compound of formula (I) of the present invention has been reported to have potent prostate enzyme inhibitory activity (Japanese Patent Application (Cocai) No. Hei 5-32693 ). Based on prostate enzyme inhibitory activity, type II inhibitory activity can reasonably be expected. However, type I 5α-reductase inhibitory activity cannot be expected because type I 5α-reductase is not distributed in the prostate.

SUMMARY OF THE INVENTION

The present inventors have for many years undertaken extensive research into the synthesis of derivatives having testosterone 5α-reductase inhibitory activity and the pharmacological activities of these derivatives. As a result, it has been found that some compounds having a specific structure strongly inhibit type II 5α-reductase and additionally strongly inhibit type I 5α-reductase and thus exhibit a strongly reduced blood DHT activity, which has not been available in the prior art. selective 5α-reductase type II inhibitors, and

they are useful in the treatment of alopecia, female hirsutism or seborrhea, or in preventing bone metastasis caused by prostate cancer.

The present invention provides a novel composition useful in the treatment of alopecia, female hirsutism or seborrhea, or the prevention of prostate cancer bone metastasis. In another aspect, the present invention provides the use of said compound for the manufacture of a composition for treating alopecia, female hirsutism or seborrhea or for preventing bone metastasis caused by prostate cancer.

In yet another aspect, the present invention provides a method of treating alopecia, female hirsutism or seborrhea, or preventing bone metastasis caused by prostate cancer, comprising administering to a warm-blooded animal in need thereof a therapeutically effective amount of said compound.

The novel composition for treating alopecia according to the present invention, female or seborrheic hirsutism, or for preventing prostate cancer bone metastasis as an active ingredient comprises a 3-oxo-4-aza-5α-androst-1-ene-17B-carboxamide compound of formula I a pharmaceutically acceptable salt or other derivative thereof for the manufacture of a composition, preferably N- [1-methyl-1- (4-methoxyphenyl) ethyl] -3-oxo-4-aza-5α-androst-1-ene-17β-carboxamide.

A new use of the present invention is the use of a 3-oxo-4-aza-5α-androst-1-ene-17β-carboxamide compound of formula I or a pharmaceutically acceptable salt or derivative thereof for the manufacture of a composition for treating alopecia, female hirsutism or seborrhea; for the prevention of prostate cancer bone metastasis, preferably using N- [1-methyl-1- (4-methoxyphenyl) ethyl] -3-oxo-4-aza-5α-androst-1-ene-17β-carboxamide for the manufacture of said means.

• ·

(wherein R ¹ and R ² are the same or different and each represents a hydrogen atom, hydroxyl group or lower alkoxy group).

The term lower alkoxy in formula (I) means straight or branched chain alkoxy of 1 to 6 carbon atoms such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert. butoxy, pentoxy, isopentoxy, 2-methylbutoxy, neopentoxy, hexyloxy, 4-methylpentoxy, 3-methylpentoxy, 2-methylpentoxy, 3,3-dimethylbutoxy, 2,2-dimethylbutoxy, 1,1-dimethylbutoxy, 1,3-dimethylbutoxy or 2,3-dimethylbutoxy, of which straight-chain or branched alkoxy of 1 to 4 carbon atoms is preferred, methoxy is more preferred.

Alopecia means baldness in men and alopecia in the head in women.

Since the compound of formula I may form a salt, the term pharmaceutically acceptable salts refers to those salts of the compound of formula I of the present invention that can be converted into salts thereof. Examples of such salts preferably include alkali metal salts such as sodium salt, potassium salt and lithium salt, alkaline earth metal salts such as calcium salt and magnesium salt, and metal salts such as aluminum salt, ferric salt and zinc salt.

When left to stand in the atmosphere, the compound of formula (I) may absorb water or form a hydrate. Such a substance is also included in the present invention.

The compound of formula (I) of the present invention can be prepared by the following procedure.

Procedure

(wherein R ¹ and R ² are as described above).

According to Procedure A, a compound of formula I is produced by condensing a carboxylic acid derivative with an amine derivative.

In step A1, a compound of formula I is prepared using a compound of formula II or a reactive derivative thereof and a compound of formula III. This step is carried out in a conventional manner used in peptide synthesis, for example by the azide process, the active ester process, the mixed anhydride process or the condensation process.

Among these processes, the azide process comprises reacting an azide compound with an amine compound of Formula III. The azide compound is produced by reacting nitrous acid with an amino acid hydrazide, which is obtained by reacting a compound of formula II or an ester thereof with hydrazine and a temperature around room temperature in an inert solvent (e.g. dimethylformamide).

Examples of the nitrous acid compound used herein include alkali metal nitrites such as sodium nitrite, and alkyl nitrites such as isoamyl nitrite.

The reaction is preferably effected in an inert solvent. Examples of the solvent used herein include amides such as dimethylformamide and dimethylacetamide, sulfoxides such as dimethyl sulfoxide, and pyrrolidones such as N-methylpyrrolidone. The two-step reaction of this procedure is usually carried out in a single vessel. The reaction temperature is in the range of -50 ° C to 0 ° C in the first stage, while in the second stage it is in the range of -10 ° C to 10 ° C. The time required for the reaction ranges from 5 minutes to 1 hour in the first stage, while in the second stage from 10 hours to 5 days.

The active ester procedure is carried out by reacting a compound of formula II with an active esterifying ester to form an active ester of a compound of formula II and then reacting the resulting compound with an amine compound of formula III.

Both reactions are preferably carried out in an inert solvent. Examples of the solvent to be used according to the invention include halogenated hydrocarbons such as methylene chloride and chloroform, ethers such as ether and tetrahydrofuran, amides such as dimethylformamide and dimethylacetamide, and nitriles such as acetonitrile.

Examples of the esterifying agent as used herein include N-hydroxy compounds such as N-hydroxysuccinimide, 1-hydroxybenzotriazole, N-hydroxy-5-norbomen-2,3-dicarboxyimide, and disulfide compounds such as dipyridyldisulfide. The esterification is preferably carried out in the presence of a condensing agent such as dicyclohexylcarbodiimide, carbonyldiimidazole or triphenylphosphine.

The reaction temperature in the esterification is in the range of -10 to 100 ° C, while in the reaction of the active ester compound with the amine of formula III it is around room temperature. The time required for the reaction is in the range of from 30 minutes to 80 minutes for each of these reactions.

In the reaction of the active ester with an amine, 4-dimethylaminopyridine or the like can be added.

The mixed anhydride process is carried out by reacting the mixed acid anhydrides of the compound of formula II with an amine.

The mixed anhydride reaction is carried out by reacting the compound of formula II with a mixed anhydride forming agent. Examples of such agents include alkyl (C 1 -C 4) halogenated carbonates, such as ethyl chlorocarbonate or isobutyl chlorocarbonate, a lower alkanoyl halide such as pivaloyl chloride, (lower alkyl) or diaryl cyanophosphoric acid such as diethyl cyanophosphoric acid or diphenylcyanophosphoric or sulfenyl cyanophosphoric such as 2,4,6-triisopropylbenzenesulfonyl chloride, p-toluenesulfonyl chloride or methanesulfonyl chloride.

The reaction is preferably carried out in the presence of an organic amine such as trimethylamine or N-methylmorpholine at -10 to 50 ° C. The time required for the reaction is from 30 minutes to 20 hours.

The reaction of the mixed anhydride and the amine of formula (III) is preferably carried out in an inert solvent (for example, a halogenated hydrocarbon, amide or ether, exemplified above) in the presence of an organic amine exemplified above. The reaction temperature ranges from 0 to 80 ° C and the reaction time required for the reaction is from 1 to 48 hours.

Process A is also carried out in a mixture of a compound of formula II, a compound of formula III and a reagent forming the corresponding mixed anhydride without isolation of the mixed anhydride.

The coupling procedure is carried out directly by reacting a compound of formula II with an amine of formula III in the presence of a coupling agent such as dicyclohexylcarbodiimide, carbonyldiimidazole, 1-methyl-1-chloro-pyridinium iodide-triethylamine. This reaction is carried out under conditions similar to those used in the above reaction to prepare the active ester.

When R ¹ or R ² contains a protected hydroxy group the protecting group may be removed in conventional manner.

The starting compound of formula II or an active ester thereof is a known compound or is prepared by a method known to the skilled artisan [e.g. J. Med. Chem. 1984, 27.1690; J. Med. Chem. 1986, 29, 2298].

The compound of formula III is a known compound or is prepared by one skilled in the art (for example, Synthesis 1976, 593; J. Org. Chem. 1971, 36, 305; Angew. Chem. 1970, 82, 138; Synthesis, 1978, 24; Synthetic Commun. 1988, 18, 777; Synthetic Commun. 1988, 18, 783; Organic Reactions 1946, 3, 337; Org. Synthesis 1971, 51, 48; Tetrahedron 1974, 30, 2151; J. Org. Chem. 1972, 37, 188). For example, H ₂ NC (Me) (Me) -Ph (R ¹ ) (R ² ), which is the starting compound of the present invention, can be prepared as outlined in the following reaction scheme:

MeMgBr £ //

Me

-f-N,

Me

R '<R ² ^' ·· · ···

Me

Me

R '.

R ^: · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

Me

JH- ^nh >

ⁱⁿ Me (wherein R ¹ and R ² are as defined above, Me is methyl and Ph is phenyl) by a Grignard reaction, a hydroxyl substitution reaction with an azide group, and then reduction of the azide group by a procedure similar to that described in Synthesis 1974, 24.

The compound of formula I is administered orally in the form of tablets, capsules, granules, powders, syrups or the like, and topically in the form of an ethanolic solution, cleansing foam, cleansing cream, skin gel, skin lotion, shampoo gel, cream shampoo or the like.

Oral pharmaceutical preparations are made by methods known to those skilled in the art using excipients (examples include organic excipients such as sugar derivatives such as lactose, sucrose, glucose, mannitol and sorbitol, starch derivatives such as corn starch, potato starch, α starch, dextrin and carboxymethylcellulose, cellulose derivatives such as crystalline cellulose, low-substituted hydroxypropylcellulose, hydroxypropylmethylcellulose, carboxymethylmethylcellulose, carboxymethylcellulose calcium and internally cross-linked sodium carboxymethylcellulose, gum arabic, dextran and silicic acid excipient, dextran and pullulanine; such as light silicic anhydride, synthetic aluminum silicate and magnesium aluminum metasilicate, phosphates such as calcium phosphate, carbonates such as calcium carbonate, and sulfates such as calcium sulfate, lubricating agents (e.g. these include stearic acid, metal stearate salts of stearic acid, such as calcium stearate and magnesium stearate, talc, colloidal silica, waxes such as beeswax and spermaceti, boric acid, adipic acid, sulfates such as sodium sulfate, glycol, · 00 00 * 0 0 0 0

0 0 0 0 000 009> 0000

Fumaric acid, sodium benzoate, DL-leucine, sodium salts of aliphatic acids, lauryl sulphates such as sodium lauryl sulphate and magnesium lauryl sulphate, silicas such as silicic anhydride and silicic acid hydrate, and the aforementioned examples of starch derivatives), binders ( examples include polyvinylpyrrolidone, Macrogol and excipients similar to the above examples of excipients), disintegrants (examples include compounds similar to the above examples of excipients and chemically modified starches and celluloses such as croscarmellose sodium, sodium carboxymethylscarb and cross-linked polyvinylpyrrolidone), stabilizers ( examples include p-oxybenzoates such as methylparaben and propylparaben, alcohols such as chlorobutanol, benzyl alcohol and phenylethyl alcohol, benzalkonium chloride, phenolic derivatives such as phenol and cresol, thimerosal, dehydroacetic acid and sorbic acid), corrector flavoring agents (examples include commonly used sweetening, acidifying and flavoring agents) and / or diluents.

Topical pharmaceutical formulations are prepared by adding said compound to a well known art skilled in the art, for example, suspending agents (examples include gum arabic, tragacanth, methylcellulose, sodium carboxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, sodium alginate and bentonite), emulsifying agents (among examples include triethanolamine, sodium lauryl sulfate, sorbitan sesquioleate, polysorbate 80, and polyoxyl (40) stearic acid), wetting agents (examples include sorbitol, ethylene glycol, propylene glycol, butylene glycol, and glycerin), preservatives (examples include methyl p-oxybenzoate, ethyl p-oxybenzoate, propyl p-oxybenzoate and butyl p-oxybenzoate) or solvents (examples include water, alcohols such as ethanol, isopropyl alcohol, propylene glycol, cetanol and isostearyl alcohol, hydrocarbons such as natural fats and oils, waxes and liquid paraffin, aliphatic acids elines such as stearic acid, isostearic acid, oleic acid, and linoleic acid, and esters such as isopropyl myristate) or a mixture thereof.

The amount of the compound of formula I to be administered orally or locally will depend on the condition, age and the like of the patient. It is desirable to administer a single dose of 0.001 mg / kg body weight (preferably 0.01 to about 10 to about 9 to 10 mg / kg). Mg / kg of weight) as a lower limit and 10 mg / kg of weight (preferably 0.5 mg / kg of weight) as a higher limit to administer these amounts in a single dose or in several divided doses per day.

The best mode of carrying out the invention will now be described.

The present invention is further described herein in more detail by means of test examples, reference examples and preparation examples.

DETAILED DESCRIPTION OF THE INVENTION

Test Example 1

Method for determining inhibitory activity on human 5α-reductases type I and II

1) Preparation of recombinant human 5α-reductase types I and II

a) Cloning of 2 cDNAs of recombinant human 5α-reductase types I and II

In order to clone the fully translated regions of human 5α-reductases type I and type II by the polymerase chain reaction (PCR) method, primers of sequence id. No. 1, 2, 3, and 4 using the Oligo 1000 DNA synthesizer (manufactured by Beckman Co., Ltd.) based on the human 5a-reductase type I and type II nucleotide sequences described in the published reports (Stefan A. et al., Proc. Natl Acad Sci USA 1990, 87, 3640-3644, Stefan A. et al., Nature 1991, 354, 159-161.)

Type I negative primer. 5'-CCAGCCCTGGCGATGGCAAC-3 '(SEQ ID NO: 1)

Type I positive primer: 5'-CAGAGCTTGATTCTGACCTGTTA-3 '(SEQ ID NO: 2)

Type II negative primer 5'-ACGGCGCGATGCAGGTTCAGTG-3 '(SEQ ID NO: 3)

Type II positive primer: 5-AGCATTGTGGGAGCTCTGCTCCT-3 '(SEQ ID NO: 4)

Human liver QUICK-Clone ™ cDNA and human prostate QUICK-Clone ™ cDNA (products of Clontech Laboratories, Inc) were used as templates for the PCR method.

PCR was performed using 1 μΙ cDNA, 5 μΙ PCR buffer, 1 μΙ 10mM dNTP mixed solution, 1 μΙ of each of the negative and positive primers at 20 μΜ, 1 μΙ (5 units / ml) of TaKaRa Taq polymerase and 40 μΙ of distilled water giving a reaction volume of 50 μΙ. For PCR, the reaction cycle at 94 ° C for 20 seconds, at 55 ° C for 1 minute and at 72 ° C for 1 minute 25 times was repeated, followed by storage at 4 ° C. When a portion of about 5 μΙ of the PCR reaction mixture was electrochromatographed on a 0.8% agarose gel, two bands (type 1: 1021 nucleotide pairs, type II: 812 nucleotide pairs) were found corresponding to those expected as described above. literature. The remainder of the PCR reaction mixture was therefore purified by electrochromatography on a 5% acrylamide gel. The purified cDNA fragment was subcloned using the TA Cloning ™ Kit (manufactured by Invitrogen Corporation). By the termination staining method, it was confirmed, as a result of full nucleotide sequence analysis of each of the subcloned type I and type II cDNAs, that they had the same sequences as those described. Type I and Type II cDNAs, whose nucleotide sequences were thus confirmed, were inserted into an expression vector to allow for their expression and obtained for use.

b) Preparation of human type I and type II expression plasmids

Escherichia coli strain DH5 (supE44, hsdR17, recA1, endA1, gyrA96, thi1, relA1) (obtained from Toyobo Co., Ltd.) was transformed using pME18sH5R1 or pME18sH5R2, i.e. human type I or type II expression plasmids, as described in document attached to the TA Cloning ™ Kit. About 10 µΙ of the primary culture solution of the recombinant strain was implanted in 50 ml of 2xLB medium (20 g Bacto-Trypton (manufactured by Difco Labs.), 10 g extract of Bacto-Yeast (manufactured by Difco Labs.), 10 g sodium chloride and 2 g glucose). 1 I) containing 50 µg / ml ampicillin (Gibco BRL product). This was followed by incubation for 40 hours at 37 ° C. The culture solution was centrifuged (5000 rpm, 10 minutes) and the cells were harvested. From the resulting

pME18sH5R1 orME18sH5R2 was prepared using a QIAGEN Plasmid Maxi Kit (manufactured by Qiagen Inc.).

c) Expression and preparation of human type I and type II proteins

10-20 µg of pME18sH5R1 or pME18sH5R2 (Gene Pluser ™, manufactured by Bio-Rad Laboratories), 960 pF, 200 Y, 300 V, was electroporated into 2 x 10 ⁶ / 0.5 ml COS-1 cells. After introduction, the mixture was incubated for 48 hours and cells were harvested. These cells were homogenized (1000 rpm, 30 seconds) with Polytron (manufactured by Kinematica GmbH) in a buffered solution (20 mM potassium phosphate buffer, pH 7.4, 10% glycerol, 0.33 M sucrose, 50 µM reduced form nicotinamide- adenine dinucleotide phosphate (NADPH) and 0.001% phenylmethylsulfonyl fluoride (PMSF)) in an ice bath. This was followed by centrifugation for 1 hour at 10,000 x g. The precipitate was resuspended in buffer and the resulting suspension was stored at 80 ° C. This suspension was used as human type I or type II 5α-reductase.

2) Measurement of protein content

Protein content was measured by the Bradford method (Bio-Rad Protein Assay of Biorad) using bovine gamma-globulin (bovine fraction il, Sigma product) as a standard product.

3) Determination of 5α-reductase activity

5α-reductase activity was determined using the conversion rate of ¹⁴ C-testosterone (Amersham product) to ¹⁴ C-5α-dihydrotestosterone as an index. The compound was dissolved in dimethylsulfoxide and diluted with dimethylsulfoxide (DMSO). A 5 µl aliquot of the resulting solution was poured into each of the two tubes, while only 5 µl of DMSO was poured into the next tube (control group). 0.5 ml of enzyme reaction buffer containing 10 to 25 µg of recombinant human 5α-reductase type I or II (type I: 40 mM calcium phosphate buffer (pH 7.5) containing

1μΜ ¹⁴ C-testosterone, 1mM dithiothreitol and 0.5mM NADPH, type II: 100mM tris-citrate buffer (pH 5.5) containing 1μΜ ¹⁴ C-testosterone, 1mM dithiothreitol and 1mM NADPH), followed by incubation for 15 minutes at 37 ° C . After incubation, 2 ml of ethyl acetate (containing testosterone, 5α-dihydrotestosterone and androstenedione, each 10 µg) were added and the resulting mixture was stirred sufficiently to terminate the enzymatic reaction and the streoid compounds were extracted into the ethyl acetate layer. The ethyl acetate phase was separated from the aqueous phase by centrifugation (3000 rpm, 5 minutes). The ethyl acetate phase was transferred to another tube, followed by evaporation to dryness under a stream of nitrogen. The tube wall was washed with 0.8 mL diethyl ether, while the steroid compounds were washed away from the bottom of the tube. After re-evaporating to dryness, the steroid compound is dissolved in 40 µl of ethyl acetate and the resulting solution is poured onto a thin layer silica gel plate (LK6DF silica gel plate, Whatman product). A thin layer silica gel plate was developed twice with a 1: 1 mixture of ethyl acetate and cyclohexane to separate each fraction containing the steroid compound. The radioactivity of each fraction was determined by a bioimage analyzer (manufactured by Fuji Film Co., Ltd.). Reductase inhibitory activity was reported as the concentration for 50% inhibition (IC 50).

The IC 50 value was calculated as follows: Assuming that the conversion ratio of the control group is taken as 100%, the inhibitory ratio of reductase activity (100 - (conversion ratio after addition of test compound: conversion group conversion ratio) x 100 (%) is calculated. Using a dilution concentration at which the inhibition ratio falls within the range of 20 to 80%, and by setting the logarithmic value of the test compound concentration as X and the inhibition ratio as Y, the least squares method is calculated. From the resulting regression curve, the concentration of the compound required for 50% inhibition is calculated and denoted as IC50 ·

The Type I IC50 is shown in Table 1, the Type II IC50 is shown in Table 2.

Table 1 (Type I)

compound IC50 compound community. vz. AND 4,9.10 ^ Finasteride 7,0.10 ' ⁷ M

Table 2 (Type II)

compound IC50 compound community. vz. AND 3,2.10®M Finasteride 1,5.10 ^

Test Example 2

Effects on reducing dihydrotestosterone levels in human blood

Orally, 1 to 10 mg / subject of a compound of Formula I or 5 mg / subject of Finasteride was administered orally. Blood levels of dihydrotestosterone (DHT) and testosterone (T) were measured. Their ratios (DHT / T) before (pre) and after passage (10 and 18 hours) were calculated from which (DHT / T) / (DHT / T) pre was calculated. The results are shown in Table 3 (n = 4-6).

Table 3

after 10 h po 18 h merge ob. vz. I (1 mg) 0.518 0.525 merge ob. vz. I (5mg) 0.411 0.376 merge ob. vz. I (10mg) 0.279 0.284 Finasteride (5 mg) 0.553 0.461

Test Example 3

Test using human papilla pili cells

Papilla pili is a cell that exists in a small number of hair follicles. Today, it is believed to be a stem cell to form the basis for hair growth. This cell was found to have 5α-reductase activity. Thus, it is possible to perform a 5α-reductase inhibition assay using a cultured cell system. Isolation and incubation of papilla pili cells is performed according to Messenger A.G. (The Culture of Dermal Papilla Cell From Human Hair Follicles, Br. J. Dermatol. 1984, 110, 685-985) or Itami S. et al. (5a-Reductase Activity in Cultured Human Dermal Papilla Cell From Beard Compared With Reticular Dermal Fibroblasts, J. Invest. Dermatol. 1990, 94, 150-152.). For this assay, avian papilla cells and hair roots were obtained from the scalp region of the head of two subjects. All assays are performed under confluent conditions after these cells have been subcultured for 4 to 6 generations. Cells that have become confluent are washed twice with phosphate buffered saline (PBS), peeled off with a rubber spatula and then collected in a centrifuge tube. Cells are centrifuged at 1500 rpm for 10 minutes. at 4 ° C. The resulting pellet was suspended in a buffered solution (20 mM Tris-HCl buffer solution, pH 7.5, containing 250 mM sucrose, 1 mM magnesium chloride and 2 mM calcium chloride) and passed through a 25G needle ten times. The suspension is then homogenized with a Teflon glass homogenizer. A suspension of broken cells is obtained. To identify the site of the 5α-reductase in the cell, the resulting broken cell solution is centrifuged at 800 x g for 10 minutes to give a crude kernel fraction. The supernatant was centrifuged for 15 minutes at 10,000 x g to give a mitochondria fraction. The supernatant is then centrifuged for a further 60 minutes at 100,000 x g to give a microsome fraction and a cytosolic fraction. The precipitated portion of each is washed twice and resuspended.

Under conventional incubation conditions, 50 μΙ of broken cell solution is added to 100 mM sodium citrate (pH 5.5) or 100 mM tris-HCl (pH 7.5) containing 50 nM [ ³ H] -testosterone and 1 mM NADPH. This gives 100 μΙ of solution. A 50-100 mg broken cell solution in terms of protein content is added to each tube. The reaction is carried out for 30 minutes at 37 ° C. During incubation, the reaction is time-dependent. In order to find the optimum pH for the reaction, the citrate buffer solution is used in the range of pH 4.5 to 6.5, while the tris-HCl buffer is used at pH 7.0 to 9.0. Protein content is measured according to the method of Lowry et al. (Protein Measurement With The Folin Phenol Reagent, J. Biol. Chem. 1951, 193, 265-275.

After the incubation is complete, the reaction is terminated by the addition of four times the chloroform-methanol mixture (2: 1, v / v) containing 110 mg of each steroid. The extracted steroid is analyzed by thin layer chromatography according to the method of Gomez et al. (In Vitro Metabolism of Testosterone-4- ¹⁴ C and D-androstene-3,17-dione-4- ¹⁴ C In Human Skin, Biochem. 1968, 7, 24 to 32). The purity of each steroid is confirmed by recrystallization. 5α-Reductase activity is given by the amount of dihydrotestosterone formed. Enzyme inhibition activity is given by percent inhibition (100 - (conversion after administration of test compound: control group conversion). 100) (in%), at 100% conversion of control group.

The compounds of formula I have been found to exhibit improved 5α-reductase inhibiting activity.

·

Test Example 4

Epilation prevention and hair growth promoting effect in short-tailed macaques (1)

The short-tailed macaque suffers from alopecia that resembles male alopecia. Macaque alopecia begins soon after puberty (about 4 years old). Alopecia attacks almost all male and female macaques and depends on androgen levels. Macaque is therefore a useful animal model of male alopecia.

Macaque male animals (3 to 16 years old) are divided into groups, each consisting of 3 to 6 animals. The macaque scalp is clearly divided into a forehead and a nape, e.g. The hair on the marked part is cut off. A solution of the test compound powder is prepared in different dosages and in various combinations and these samples are applied evenly to the area from which the hair has been removed, or the solution is administered orally. Control animals receive the same amount of solvent (eg, dimethylsulfoxide), cream (subcutaneous administration), or placebo (oral administration). The hairs of the designated area are trimmed at intervals of 4 to 6 weeks and the amount of hair cut off is weighed. Administration is continued for 6 weeks to two years. Finasteride is a 5α-reductase inhibitor and is known to prevent alopecia of the animals described above. For comparison, Finasteride (oral administration) is used in the assay.

A scalp (4 mm circle), as a sample for boipsia, is taken at the beginning and at the end of the test. The presence or absence of alopecia is assessed by analyzing the activity of 5α-reductase and examining the scalp tissue.

The compounds of formula I have been found to be effective in alopecia.

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Test Example 5

Epilation prevention and hair growth promoting effect in short-tailed macaques (2)

Male and female macaques (3 to 16 years old) are divided into groups of 3 to 6 animals. A powdered solution of the test compound is prepared in various dosages and in various combinations and these samples are uniformly applied to the frontal area of the scalp or administered orally once daily. Administration is continued for 6 weeks to 2 years. Control animals receive the same amount of solvent (eg, dimethylsulfoxide), cream (subcutaneous administration), or placebo (oral administration). Frontal hair condition is evaluated at 1 month intervals and the effect of the test compound is evaluated from the size of hair diameter, hair density and hair growth promoting area and time. At the same time, a macaque picture is taken and the effect is generally assessed from the pictures. A portion of the scalp (a 4 mm diameter wheel) in the frontal area is removed and examined microscopically at the start of the test and at 3 and 6 months after the start. The effects on the hair root growth status are studied by histological analysis.

The compounds of formula I have been found to be effective in alopecia.

Test Example 6

Test using a furry rat

The hairy rat is an androgen dependent animal model that exhibits abnormal sebaceous gland cell proliferation and secretion from sebaceous gland cells at the hair roots. Its hairy roots in the juvenile period (about 4 weeks old) are mainly in the growth stage. After sexual maturation (about 8 weeks) the roots of hair will go to the resting stage. It is therefore used as a model animal for research into the effect on hair growth.

Hairy rats (male, 2-12 weeks old) are divided into groups. Each group consists of 5 to 6 rats. A powder of the test compound is prepared in various dosages and in various combinations and is uniformly applied to the skin in the dorsal region or orally once daily. Administration is continued for 4 to 8 weeks. The control group of animals is given the same amount of solvent (eg, dimethylsulfoxide), cream (subcutaneous administration) or placebo (oral administration). They are decapitated on the day after the end of administration. Skin tissue (4 mm diameter circle) in the dorsal area is collected. Influences on the sebaceous gland and on the growth state of hair roots are studied by histological analysis.

The compounds of formula I have been found to be effective in seborrhea and alopecia.

As with the assays described above, hair growth can be evaluated according to methods described in the literature (B. de Brouwer et al., Br. J. Dermatol. 1997, 137, 699-702) using nude mice.

Reference Example 1

N- [1-Methyl-1- (4-methoxy-phenyl) -ethyl] -3-oxo-4-aza-5α-androst-1-ene-17β-carboxamide

To 30 ml of dry toluene is added sequentially 1.0 g of 3-oxo-4-aza-5α-androst-1-ene-17β-carboxylic acid, 1.6 g of triphenylphosphine and 1.4 g of 2,2'-dipyridyldisulfide. The resulting mixture was stirred overnight at room temperature. The reaction mixture is chromatographed on a column of 35 g of silica gel, eluting with a mixture of acetone and methylene chloride (1: 9 to 1: 1). 1.11 g of the 2-pyridylthioester compound are obtained. To 30 ml of dry methylene chloride was added 5.0 g of the 2-pyridylthioester compound and 5.0 g of 1- (4-methoxyphenyl) -1-methylethylamine, and the resulting mixture was stirred at room temperature for 3 days. After dilution with 100 ml of methylene chloride, the mixture is washed sequentially with 1M hydrochloric acid, water, aqueous sodium bicarbonate solution and saturated brine, dried over anhydrous magnesium sulfate, and the solvent is evaporated under reduced pressure. · • ft ftft thicken. The residue is chromatographed on a column of 15 g of silica gel, eluting with a mixture of acetone and methylene chloride (1: 9 to 1: 1). Thus 5.2 g of the title compound are obtained. NMR (CDCl _{3 R} δ, ppm): 0.68 (3H, s), 0.98 (3H, s), 0.90 to 2.20 (16 H, m), 1.70 (3 H , s), 1.72 (3H, s), 3.35 (1H, t, J = 9 Hz), 3.80 (3H, s), 5.48 (1H, broad signal), 5.76 (1H, broad signal), 5.83 (1H, d, J = 10Hz), 6.82 (1H, d, J = 10Hz), 6.88 (2H, d, J = 9 Hz), 7.32 (2H, d, J = 9 Hz). IR ( _vmax , cm ^-1 , KBr): 2969, 2938, 1672, 1599, 1514, 1455, 1248, 1181, 1035 and 825.

Preparation Example 1 Tablets (5mg tablet)

component mg / tablet the compound of Reference Example 1 5 hydroxypropylmethylcellulose 15 Dec sodium starch glycolate 9.5 crystalline cellulose 30.5 croscarmellose sodium 20 May lactose (after sieving) 38.75 yellow iron sesquioxide 0.05 magnesium stearate (after sieving) 1,2 tablets 120.0

Preparation Example 2 Alcohol solution

the compound of Reference Example 1 water ethanol 15.0 wt. 45 40 Preparation example 3 Cleaning foam the compound of Reference Example 1 10.0 wt. water 70,439 chamomilla 0.01 Aloe vera gel 0.01 alantoin 0.001 triethanolamine 0.02 Methocel ™ (Methocel ™ 4-100 (Dow)) 1.50 glycerine 3.00 sodium lauryl sulfate 15.00 vitamin A oil 0.01 vitamin E oil 0.01

Preparation Example 4 Cleansing Cream

the compound of Reference Example 1 5.0 wt. synthetic beeswax 14.0 PPG ₂ myristyl propionate 5.0 lanolin alcohol 0.5 mineral oil 36.0 propylparaben 0.15 borax 1.0 water 38.35 PPG means polyethylene glycol polypropylene glycol

Preparation Example 5 Gel for the skin the compound of Reference Example 1 2.0 wt. PPG ₂ myristyl ether propionate 45.0 PPG10 cetylether 5.0 triglyceride of 18 to 36 carbon atoms 4.0 myristyl stearate 3.0 glyceryltribehenate 2,0 cyclomethicone 34,00 polyethylene 5.00

* 4 4 «► 4 4 4

I · · 1

I 4 4 4 4

4 · 44

Preparation Example 6 Skin Lotion

the compound of Reference Example 1 1.0 wt diethanolamine-oleth-3-phosphate 1.0 emulsified wax 2,0 wax of aliphatic acids of 18 to 36 carbon atoms 1.0 PPG ₂ myristyl propionate 5.0 glycerine 3.0 triethanolamine 0.5 water 86.5

Preparation example 7 Shampoo gel the compound of Reference Example 1 2.0 wt. isopropanolamine lauryl sulfate 81.5 aliphatic acid diethanolamide of coconut oil 8.0 wax glyceryl ester of 18 to 36 carbon atoms 4,5 PPG ₅ ceteth-10-phosphate 4.0

Preparation Example 8 Cream Shampoo Compound of Reference Example 1 0.1 wt.

sodium lauryl sulphate 65.0 glyceryl tribehenate 2.0 hydrolyzed collagen 1.0 lauric acid diethanolamide 5.0 water 26.9

Industrial applicability

Finasteride is a compound that is marketed on the European market as a medicine for benign prostatic hypertrophy and is now in clinical trials as a medicine for alopecia.

The compound of formula I of the present invention and related compounds have a more potent inhibitory effect on the type II isoenzyme than finasteride. They also have a strong inhibitory activity on type I isoenzyme. They have a stronger effect on lowering DHT in the blood than Finasteride and have such low toxicity that they are useful as an active ingredient in the treatment of alopecia, female hirsutism or seborrhea or as a means of preventing bone metastasis of the prostate.

SEQ ID NO. no. 1

Description of synthesized sequence: primer designed based on cDNA sequence coding for 5α-reductase type I.

SEQ ID NO. No 2

Description of synthesized sequence: primer designed based on cDNA sequence coding for 5α-reductase type I.

SEQ ID NO. No 3

Description of the synthesized sequence: a primer designed based on the cDNA sequence encoding type II 5α-reductase.

SEQ ID NO. No. 4

Description of the synthesized sequence: a primer designed based on the cDNA sequence encoding type II 5α-reductase.

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LIST OF SEQUENCES <110> Sankyo Company, Limited <120> Device for the treatment of alopecia, female hirsutism or seborrhea or for the prevention of prostate cancer bone metastasis <130> 98062SY <150> JP HEI 9-203136 <151> 29.07.1997 <160 > 4 <170> PatentIn Ver. 2.0 <210> 1 <211> 20 <212> DNA <213> artificial sequence <223> Description of artificial sequence: Designed primer based on cDNA encoding 5alpha-reductase type 1 <400> 1 ccagccctgg cgatggcaac <210> 2 <211> 25 <212> DNA <213> artificial sequence <223> Description of artificial sequence: Proposed primer based on cDNA encoding 5alpha-reductase type 1 <400> 2 cagagctga aattctgacc tgtta ftft

ftft ftft ft ft ft ft ft ft <210> 3 <211> 22 <212> DNA <213> artificial sequence <223> Artificial sequence description: Designed primer based on cDNA encoding type 2 alpha-reductase <400 > 3 acggcgcgat gcaggttcag tg <210> 4 <211> 23 <212> DNA <213> artificial sequence <223> Artificial sequence description: Designed cDNA-based primer encoding type 2 5α-reductase <400> 4 agcattgtgg gagctctgct cct

Claims (8)

PATENT CLAIMS Use of a 3-oxo-4-aza-5α-androst-1-ene-17β-carboxamide compound of the following formula I wherein R ¹ and R ² are the same or different and each represents a hydrogen atom, a hydroxyl group, a protected hydroxyl group or a lower alkoxy group, or a pharmaceutically acceptable salt or other derivative thereof, for the manufacture of a composition for treating alopecia, female hirsutism or seborrhea, or for preventing bone metastasis caused by prostate cancer. Use according to claim 1 of the 3-oxo-4-aza-5α-androst-1-ene-17β-carboxamide compound of the general formula I, wherein R ¹ and R ² are the same or different and each represents a hydrogen atom, a hydroxyl group or a lower alkoxy group, or a pharmaceutically acceptable salt or other derivative thereof, for the manufacture of a composition for treating alopecia, female hirsutism or seborrhea, or for preventing bone metastasis caused by prostate cancer. Use according to claim 1 of N- [1-methyl-1- (4-methoxyphenyl) ethyl] -3-oxo-4-aza-5α-androst-1-ene-17β-carboxamide for the manufacture of a composition for the treatment of alopecia, female hirsutism or a seborrhea or composition for preventing bone metastasis caused by prostate cancer. Use for the manufacture of a composition for the treatment of alopecia, female hirsutism or seborrhea according to any one of claims 1 to 3. Use for the manufacture of a composition for the treatment of alopecia according to any one of claims 1 to 3. Use for the manufacture of a composition for the treatment of female hirsutism according to any one of claims 1 to 3. Use for the manufacture of a composition for treating seborrhea according to any one of claims 1 to 3. Use for the manufacture of a composition for preventing bone metastasis caused by prostate cancer according to any one of claims 1 to 3.