JPH02138195A - Sterol inhibitor for teststerone-5alpha reductase - Google Patents

Abstract

NEW MATERIAL: A compounds of formula I (R is formula II, formula III).

USE: Therapy and prevention of acune vulgaris, female thin hair, benign prostatomegaly, etc.

PROCESS: By the aerobic fermentation of an assimilatory carbon source, a glyocladium stock (ATCC 20826) is cultivated and propagated in an aqueous nutritive medium at about 28°C for 96-144 hr, and the objective compound is separated by a means such as chromatography.

COPYRIGHT: (C)1990,JPO

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides novel compounds of formula (1) and testosterone-5α
- Concerning the use of such compounds as reductase inhibitors.

Certain undesirable physiological manifestations such as 1L seborrhea vulgaris, female baldness, male pattern baldness and benign prostatic hyperplasia are the result of excessive androgenic stimulation due to excessive accumulation of testosterone or similar androgenic hormones in the metabolic system. are well known in the art. Early attempts to provide chemotherapeutic agents that could counteract the undesirable consequences of excess androgens led to the discovery of several steroidal antiandrogens, which themselves have undesirable hormonal activities. For example, estrogen not only antagonizes the effects of androgens but also has feminizing effects. Nonsteroidal anti-7 androgens such as 4'-nitro3'-trifluoromethylisobutylanilide have also been developed. Neri et al., Endocrinology, No. 91
Volume, No. 2, 1972 (Neri et al., E.
ndocrinology+Vo1.91, further 2 (1
See 972)). However, these products, although nonhormonal, are peripherally active and tend to compete with natural androgens for receptor sites, resulting in feminization of male hosts or male fetuses of female hosts.

The main mediator of androgenic activity in some target organs is 5α-dihydrotestosterone, which can be formed locally in the target organ by the action of testosterone-5α-reductase and has become more recently known. Became known in the industry. As a result, inhibitors of testosterone-5α-reductase were hypothesized and demonstrated to act to prevent or alleviate the symptoms of hyperandrogenic stimulation. Neife et al., Steroids, 1st
Volume 4, page 269, 1969 (Nayfeh eta
l,,5teroids, 14.269 (1969
)) demonstrated in vitro that methyl 4-androsten-3-one-17β-carboxylate is a testosterone-5α-reductase inhibitor. Then,
Voigt and Illsia, Endocrinology, Vol. 92, p. 1216, 1973.
Canadian Patent No. 970,692, published in 1999, describes the above ester and the free acid 4-androsten-3-one-17β-
Both carboxylic acids were shown to be active inhibitors of testosterone-5α-9 ductase in vitro. They further demonstrated that topical application of either testosterone or 5α-dihydrotestosterone can increase the female hamster flank organ, an androgen-dependent epidermis-like structure. However, co-administration of 4-androsten-3-one-17β-carboxylic acid or its methyl ester inhibited the testosterone-induced response but not the 5α-dihydrotestosterone-induced response. These results were taken to indicate that the compounds are anti-androgens due to their ability to inhibit testosterone-5α-reductase.

Recently, several researchers have published on the biological activities of 5α-υ ductase inhibitors. For example, Boursokus et al.
The Prostate, Volume 9, Pages 65-75, 1986
(Brooks, et al, TheProsta
te9: 65-75 (1986); Liang et al., Endocrinology, No. 117
Vol. 2, pp. 571-579, 1985; Rass+usson et al., Journal on Medicinal Chemistry, Vol. 27, pp. 1690-1701, 1984.
l ofMedicinal Chemistry 2
7: 1690-1701 (1984)) I Lian et al., Journal on Biological Chemistry (
Journal of Biological Chem
istry), Volume 259, No. 2, No. 734-73
See p. 9, 1984. However, the compounds of the present invention have a completely different structure than those previously reported for testosterone-5α-reductase inhibitors.

Compounds of Formula I are sterol inhibitors of testosterone-5α-9 ductase. Although 4,19-oxygen-bridged cholestanes have been reported in the literature, CF, Turesev and P, Kokowski, Collection on Kzekoslowata Chemical Communications, Vol. 45, No. 2
pp. 74-293, 1980 (F.
nd P., Kocovsky, Co11ection.
ofCzechoslovak Chemical
Communications, 4 5.274
-293 (1980) ); P, Kokowski Collection on Kuzekoslowak Chemical Communications, Volume 45, No. 30083022, 198
0], these compounds are not sterols, nor have they been shown to be 5α-9 ductase inhibitors.

Compounds of formula I are derived from gliocladium (Gliocla
dium) by controlled aerobic fermentation of the genus ATCCNu 20826.

[In the above formula, R is H ]

Compounds of formula (1) are inhibitors of testosterone-5α-reductase and are useful in the treatment and prevention of 11Hf, seborrhea, female hair loss, and benign prostatic hyperplasia.

The preparation and isolation of compounds of formula 1 are described.

Additionally, the in vitro activity of the compounds of II as inhibitors of testosterone-5α-reductase is also described.

Compound 2 is produced by controlled aerobic fermentation of Gliocladium bacteria.

The fungus ATCCl1m 20826 was isolated from a soil sample in Mexico. Deposit of a biologically pure culture of this microorganism under the terms of the Budapest Treaty is with the American Type Culture Collection, Rockville, Maryland.
collection) on December 17, 1986, from which it received accession number ^TCCNl 20826.
It is available as.

Studies on culture and morphological characteristics of Gliocladium were conducted. The results are as follows: The first-eyed gamma-conidiophore is septated, tufted, normally symmetrical, and forms a single gelatinous dark head. and the conidial chains there cannot be identified.

Individual conidia are hyaline, oval to ovoid, usually 2.
4 microns by 4.8-6.0 microns, forming large globules of conidia that arise from the tip of the petite and develop in mucus. Although several chains of mucoid conidia may be seen in young cultures, the main feature is the large mucoid globules, which occur on adjacent pedicles of single conidiophores, with several The conidiophores of the conidiophores coalesce into larger clusters in close proximity to each other.

Colonies on Czapek-Dox agar, potato dextrose agar, yeast extract-malt extract agar, and Saboraud maltose agar are spread out, velvety, and with abundant aerial mycelium. However, as the colony ages, it changes from the initial sandy color to yellowish brown. The reverse is brown.

The compound of estrus IE formula I is Gliocladium ATCC Th20
It is produced by aerobic fermentation of a suitable aqueous nutrient medium under controlled conditions inoculated with 826 culture. The medium contains sources of assimilable carbon, nitrogen and inorganic salts. Generally, carbohydrates (eg, glucose, fructose, maltose, sucrose, xylose, etc.) can be used either alone or in combination as nutrient medium intermediateable carbon sources. These carbon sources may be used individually or in combination in the culture medium.

In general, a large number of protein substances can be used as nitrogen sources in fermentation processes. A suitable nitrogen source is
For example, yeast hydrolysates, secondary yeast, soybean meat, cottonseed meat,
Examples include casein hydrolyzate, corn steeping liquid, brewer's soluble ingredients, or tomato paste.

Among the nutrient mineral salts that can be incorporated into the culture medium are the conventional salts capable of releasing sodium, potassium, ammonium, calcium, phosphate, sulfate, chloride, carbonate and similar ions. Trace amounts of metals such as cobalt, manganese, iron, magnesium, etc. are also included.

It should be noted that the nutrient media described herein are merely illustrative of the various media that can be used and are not intended to be limiting.

The fermentation is carried out at a temperature of about 20-37°C and the pH of the nutrient medium for growing the Gliocladium TCCNIL 20826 culture and producing the compound of formula I is about 6.
It is within the range of 8 to 7.4.

It is to be understood that the invention is not limited to the use of Gliocladium ATCCl1m 20826 for the fermentative production of compounds of formula (1). Also included within the scope of the invention is the use of other natural or artificial variants produced or derived from the above-mentioned cultures, i.e. other variants or species of the genus Gliocladium, as long as they produce compounds of formula I. This is particularly desired and intended. ATCCIl
Artificial production of h20826-derived variants or strains can be carried out by conventional physical or chemical mutagens, such as, for example, ultraviolet irradiation or nitrosoguanidine treatment of the desired culture. Recent recombinant DNA techniques such as protoplast fusion, plasmid integration, chromosomal fragment integration, etc. have also proven useful.

In a preferred embodiment of the invention, the compound of formula I is produced by controlled aerobic fermentation of Gliocladium ATCCNct 20826. Fermentation is carried out within a temperature range of about 20-37°C, preferably about 28°C.

In general, the composition of an anabolic nutrient medium can vary over a wide range. Essential nutrients are carbon and nitrogen sources. Other essential nutrients are obtained from inorganic salts such as chlorides, nitrates, sulfates, carbonates and phosphates of sodium, potassium-11, ammonium and calcium. The nutrient medium may contain sources of inorganic trace elements such as magnesium, iron, copper, manganese, zinc, cobalt, etc.

Typical carbon sources include glucose, oils, organic acids, dextrin, starch, glycerol, and the like. Typical nitrogen sources include amino acids, plant meats, extracts (e.g.
malt, soybeans, cottonseed, figs, tomatoes, corn, etc.), animal offal, various hydrolysates (eg, casein, yeast, etc.), and industrial by-products such as lard water and brewer's soluble ingredients.

The maximum yield of compound No. 2 is approximately 24-20 under optimal conditions.
This is achieved by fermentation for 0 hours, usually about 96 to 144 hours. The inoculum for fermentation is obtained either from vegetative growth on a medium that sustains rapid growth of the microorganisms, or directly from the spores.

After fermentation, the accumulated compounds of formula I are separated from related compounds and recovered from the broth by conventional chromatographic means.

Adsorption and partition chromatography, gel filtration, reversed phase liquid chromatography, etc. can also be used to obtain the compound of formula
It can be used in combination with an eluent with appropriate polarity and solubility.

Several different nutrient media are available for Gliocladium AT
CC? Applicable during fermentation of h20B26.

Changing the medium or microorganism will change the yield of the compound of formula 1 and/or the rate of its production.

Modification of the medium or microorganisms may also increase or decrease the types and amounts of compounds present in the broth. Preferred medium compositions are shown in Table 1.

Star Table F Corn Soaking Solution Tomato Paste Oatmeal Cererose Trace Elements 2 Distilled Water pH=6.8 5ga+ 0gm 0g111 0g- 10ta1 1000m/ Corn Salt Solution A Glycerol Yeast Extract Corn Oil Autoclaving Per Flask Distilled Water Re-autoclaving 1IIl Flask 0gm 15j! 11Iβ 0,5 gm O,1mjl! 15m/additional salt addition [N MgSO4・7H20 Na tartrate Fe5Oa ・7H,0 ZnS04・7HzO Distilled water trace elemental element Pe5Oa ・7H1O MnSOa ' 48zO CuC1t・211tO CaCl z )IJOi (NHt)4 MoO□・4H20 ZnSOa・7HzO Distilled water ME Yeast extract Malt extract Dextrose agar 0.1g 0.1g 0.01g 0.01g 1000 theory 1 1000■ 1000■ 25w: 100■ 56■ 19■ 200■ 1000+/2 4.0g 10.0g 4.0g 20.0 g distilled water 1000 mff pH=7.0 The terms "seed" and "production" medium are used as technical terms. Generally, an inoculum medium supports rapid growth of microorganisms, and a portion of this medium (the inoculum) is used to inoculate the production medium for large-scale fermentation.

The following examples describe the fermentative production and isolation of compounds of Formula I and several open and linked compounds thereof. These examples are merely illustrative; they are not intended to limit the scope of the invention.

A portion of the surface growth on the agar slant of the dog 1 ■ 1 [ ATCCNl 20826 culture was transferred to KF inoculation medium 54a + 1 in a 250 ml Erlenmeyer flask! inoculated inside. This inoculum was incubated for 3 days at 25° C. and 220 rpm.

The inoculum was then added to 250 l of solid production medium F842.
used to inoculate Erlenmeyer flasks. Production flasks were incubated for 14 days at 28°C and 220 rpm. Then 40ml of 40% acetone or 50%
Methanol 40s+j! was added to each flask to disrupt the solid growth and the flask contents were collected.

Fusenmasu I Disperse the solid fermentation product from Example 1 (nominally 6 litotol in total) in acetone:water (3:2 v/v, 6 L) and -
Stirred overnight. The resulting mixture was then filtered and the filtrate was evaporated to dryness. Disperse the residue in water (2 L) and water saturate n-
Extracted three times in succession with butanol (2 L each). The pooled organic phases were evaporated and the residue was then dissolved in methanol:water (
9:1 v/ν, 2 L) and n-hexane (2 L each).
L) was degreased three times in a row. The defatted methanol fraction was evaporated and the residue was dissolved in methanol:water (9:1 v/v, 300
m/) and extracted four times in succession with n-hexane (3 QOsl each). The hexane-low methanol fraction was evaporated to dryness, dispersed in the appropriate pumped water (300II11), and extracted successively with ethyl acetate (4 times, 3001 ml each) and water-saturated n-butanol (4 times, 300 mJ each). The organic phases were pooled and evaporated. The resulting residue was triturated with acetonitrile (500if) and filtered. The filter cake was washed with acetonitrile and then the pooled filtrates and washings were diluted with rhohexane (500if). Extracted twice with 250 mj each and evaporated to dryness to obtain an active residue. The acetonitrile-insoluble filter cake was dispersed in methanol and filtered. After evaporation of the filtrate, an active residue was obtained.

Gel filtration of the acetonitrile soluble fraction was carried out using Sephadex LH-20 (Pharmacia Fine Co., Ltd.) in methanol.
Chemicals (Pharmacia Fine Chem
icals)). The sample was loaded onto a 2,5 x 28.5 cm resin column (14
Add methanol (21
0 ml), each 3.5+/! 60 fractions were collected. The active ingredient was recovered in both minutes 13-24.

Gel filtration of the methanol soluble fraction was performed on Sephadex LH-20 in methanol. The sample was added from the top of a 2.5 X 28.5 cm resin column with a minimum amount of methanol, 3.5 mjl each! 60 fractions were collected. The active ingredient was recovered in both minutes 13-22.

Thin layer chromatography comparison of the active fractions from the two gel filtration experiments showed that they were similar in composition, so the active fractions from the repeat experiments were pooled and concentrated.

Gel filtration was re-performed on Sephadex LH-20 in methanol. sample with a minimum amount of methanol
x 28.5 (add from the top of the 1 m resin column,
Elute with methanol (210ml), 3.5m each! ! 60 fractions were collected. The active ingredient was recovered in both fractions 15-22, but some was also present in both subsequent fractions.

Both fractions 15-22 were pooled, evaporated to dryness, and ethyl acetate:
Redissolved in methanol (1:2 v/v, 3 ml). Partition chromatography was then performed on this sample using a Sephadex LH-20 column (150 II single layer capacity) preswollen with methanol and equilibrated with ethyl acetate:methanol (99:1 v/v).

The sample was added to the top of the column and eluted sequentially with the following solvent mixture: Ethyl acetate:methanol (99:1 v/v) 30
0 ml;! Ethyl acetate:methanol (19:1 v/
v) 250 mp.

Ethyl acetate:methanol (9:1 v/v) 200
mA methanol 1000
Two hundred fractions of 7.5 mn each of mβ were collected and then the wash fractions were collected. The main active zone eluted in fractions 85-112 and the minor zones eluted in both fractions 17-36.

Both fractions 85-112 were pooled, evaporated and diluted with methanol (
2s+1). Partition chromatography was then performed on the sample using a Sephadex LH-20 column (300 mj! bed volume) preswollen with methanol and equilibrated with ethyl acetate:methanol (19:1 v/v). Add the sample from the top of the column,
Eluted sequentially with the following solvent mixture: ethyl acetate:methanol (19:1 v/v) 50
0ml! Ethyl acetate:methanol (9:1 v/v)
300 mff ethyl acetate:methanol (3:1ν
/v) 300 ml methanol
500++j! 200 fractions of 200 each were collected, and the active ingredient was recovered in a broad zone of 26 to 110 fractions.

Both fractions 26-110 were pooled, evaporated to dryness and redissolved in methanol (6th theory i). Prevalent reversed-phase high performance liquid chromatography (HPLC) with Lichlosorb (
Lichrosorb) RP-18 column (EM Reagents, 5 micron particle size, 1010X250, operated at ambient temperature, the following solvent gradient was applied at a flow rate of 4.7 ts R/+win: 0-2 min water 2-6 minutes Water to water; Acetonitrile (9; IV/ν
) to aqueous niacetonitrile (9:1 v/v) in 8 to 28 min. :1ν/v
) to water:acetonitrile (1:l v/v) in 28-48 min. Water:acetonitrile (1:lv/v)
Linear gradient from 48 to 60 minutes to acetonitrile Thirteen such runs of acetonitrile were performed, with 60 fractions of 1 minute each collected for each run. The active component was recovered in both minutes eluting between 25 and 33 minutes.

Active fractions were pooled, evaporated to dryness and diluted with methanol (1 mN
). isocratic
c) Preparative reverse phase 11PLc was performed on the sample using a Lycrosoap RP-18 column (10 x 250 Tsuru, 5 micron particle size) at ambient temperature, flow rate 4.7+
Elution was carried out with water niacetonitrile (3:2 v/v) at wβ/win. Perform 5 runs, 1 hour each for each run.
Sixty fractions were collected. Three active zones were observed, eluting in the 9-13 minute, 14-19 minute and 27-30 minute fractions.

Repeated prevalent IPLC of both fractions eluting between 14-15 minutes using the same column and operating conditions gave compound 2a (1 mg) as a white solid.

Lychrosorb R-18 column (4,0 x 250 m, 5
micron particle size) and a flow rate of 2 ts j! Analytical HPLC eluting with water/niacetonitrile (3:2) at /sin showed a storage time of 7.0 minutes.

Repetitive preparative HPLC of both fractions eluting between 16 and 17 minutes, using the same column and operating conditions, afforded 2 lb of compound (2■) as a white solid. Analytical HPLC using a Lycrosoap RP-18 column (4, OX 250 mm, 5 micron particle size) and eluting with water:acetonitrile (3:2 v/v) at a flow rate of 2 tm It /vain resulted in a retention time of 7.7 minutes. showed that.

Formula 1 (7) ” (The solid material obtained in Viability Example 2 was characterized as follows by high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy (i.e., protons and carbon ~13) .

Mass spectrum--The mass spectrum was analyzed by electron bombardment at 90eν using Finnigan Mat 212.
2) Recorded by the device. Trimethylsilyl derivative (T?I
S) was prepared with a 1:1 mixture of BSTFA-pyridine at room temperature. Accurate mass measurement uses perfluorokerosene (PFK) as an internal standard and a peak matching method (peak matching a+method).
) was carried out using the same apparatus during high resolution. FAB spectra were obtained on a Mat 731 instrument.

Table 1 summarizes the low resolution mass spectral data. In all cases, the molecular ion is FAB-
Confirmed by MS. High resolution data were obtained only for compound 1a and are shown in Table 2.

NMR Data NMR data were obtained on a Varian XL-400 NMR spectrometer in co3oo at ambient room temperature. The chemical shift is δ3.3013 for the 'HNMR spectrum and 49.0 for the CNR spectrum compared to tetramethylsilane of oppIIl.
Shown using ppm solvent peaks as standards.

Only 3CNMR data are shown for compound 1a.

"CltMR shift Consistent with the molecular formula C+411szo+, 34 carbon atoms are observed in the spectrum of compound 1a with the following chemical shifts: 12.6.13.2.21.1.23.5.26.2.
26.8.29.3.29.5.35, 7.41.2.
41, 4.43, 3.44, 9.45.1.45.4.
46.3.49.9.56.0.57.1.62.6.
68.1.72.4.73.22,? 3.28.75
．． 3.78.2.83, 5.88.0.101.0.1
16.9.127.3.138.8.141.8.17
The spectra of 6.3 ppm 'H NMR compounds Ta and Ib are shown in FIGS. 1 and 2.

Table 2: High resolution mass spectra of compound 1a Based on these and other data, the structure was determined.

Ib a [In the above formula, R is or H H ] The above structure includes all possible stereoisomers.

The following is a method for evaluating testosterone-5α-reductase inhibitory activity in dogs and humans in vitro. The microsomal fraction of rat liver was used as the enzyme source because this enzyme is relatively easy to prepare. Human prostate enzyme was also used to evaluate compounds of Formula I.

A. Sadamei Azusu The enzyme was prepared from rat liver microsomal fraction.

A male Wistar strain (Wist) was killed by liver decapitation.
ar) Obtained from rats (low glycogen content; - night fasted).

The livers were then soaked in water-cold phosphate buffer, washed twice to remove excess blood, and chopped into small pieces with scissors on ice in a beaker. [! 5 ml of buffer per gram was added to the minced tissue and the tissue was homogenized for 10 seconds on a Brtnkn+an Polytron on setting #4. Add the same amount of buffer and homogenate for 10.
Centrifuged at OOOxg for 10 minutes. The beret was discarded.

The supernatant was centrifuged at 100,000 x g for 30 minutes [Beckman Model L5-50 Ultracentrifuge (
Beckman Model L5-50 mouth 1 tra
centrifuge)) The supernatant was discarded and the pellet was resuspended in half the original volume of phosphate buffer and rehomogenized for 5 seconds on setting #4. The homogenate was centrifuged again at 100.000xg for 20 minutes.

Add 100 μl of protein to the pellet in water-cooled assay buffer.
(Suspended at 1/me. (Protein measurement method is as follows.
Solution ll1
1 was pipetted into each vial and stored in a liquid nitrogen freezer. The enzyme can be stored for at least 4 months.

When the assay is ready, place one enzyme vial in B5.
A final enzyme protein concentration of 2-7 μg/ml in water-cooled assay buffer containing A1/ml (prepared by dissolving B5Al0 in 10 Ill of assay buffer).
It was further diluted to 1.

B. Enzyme protein concentration determined by Groves et al.
It was evaluated by the spectrophotometric method described by 5). Each absorbance difference of 0.4 between 224 and 23 arv corresponds to 0.1 μ/ll of protein.

Add methanol 1-1 to 1 vie of mycelium-containing fermentation broth in a 13X100m test tube, shake the tube for 10 seconds,
1520x in Beckman TJ-6 Tapertsop centrifuge
Centrifuged at g for 10 minutes. 10 μl of each supernatant was used directly in the assay.

10 .mu.l of the above methanol extract of each broth to be tested was added into a 13.times.100 m test tube containing 100 .mu.l of enzyme and 100 .mu.l of 3H-testosterone. The tube contents were mixed gently and incubated at 37°C for 20 hours.

Next, 1 tan of water-saturated ethyl acetate was added directly into the tube.
Shake. After addition of ethyl acetate, the blank was processed by adding enzyme. Enzyme controls were treated with 10 μl of 50% methanol. 10μ of inhibitor instead of 50% methanol extract for positive control! Processed with.

All blanks, enzyme controls and inhibitors were run twice.

The tube contents were then centrifuged for 5 minutes at 1520xg in a Beckman TJ-6 table top centrifuge. Organic phase (
0.8 mJ of the upper layer was transferred to another tube and dried under reduced pressure in a high-speed vacuum concentrator (Savant Co.).

Add 50μl of spike solution to each drying tube and gently tap the tube to remove 25μl of this solution! Whatman (
Whatman) LK 6 DF TLC plate. The plate was developed in a 50% ethyl acetate and 50% cyclohexane solvent system until the solvent rose to 374 cm on the plate.

The plate was placed under short wavelength U ■ forward light. Two separate bands are visible. These show Rf values for androstenedione and testosterone. Place the plate between the two bands, above the top band and below the bottom band, about 3C each! Make a notch on I with a razor blade. The zone containing these two bands is scraped from the glass funnel into a separate counting vial and counted for 10 minutes in scintillation cocktail 1oII.

The uppermost band (band 1) containing labeled 3H-dihydrotestosterone as converted from 3H-testosterone by 5α-ductase is the target of measurement of enzyme activity. Enzyme activity is expressed as the conversion rate of testosterone to dihydrotestosterone.

Conversion Δ% = Control or Broth Conversion % Blank Average Conversion % Broth Extract or Inhibitor Inhibition % - Control Average Conversion Δ% C0'1lJs The compound of formula I is a compound of the testosterone-5α-9 ductase enzyme. It was found to be highly active in inhibition assays. 100% inhibition is 50μ of 50% aqueous methanol extract of whole broth! It was observed in I.C. was estimated to be 1.5 μl of broth per 11 ml of assay mix @y. The active ingredient was found to be methanol soluble. Purified components of the compound of Formula I were evaluated in both rat liver enzyme and human prostate enzyme inhibition assays. ICs of this purified component against rat liver enzymes. is 0.8ng
/mA, and the IC2° of this purified component against human prostate enzyme was estimated to be 5 μg7tel. Human prostate enzyme was prepared as described in the following example.

A.1 Thaw 1.8 grams of human prostate slice, cut into pieces,
．． Homogenized in 25 M sucrose buffer. The homogenate was centrifuged at 120 ORPM for 10 minutes and the supernatant was discarded. After washing the pellet three times with buffer, it was suspended in buffer to contain approximately 300 μg of homogenized tissue in 1.0 Pβ.

Add 0.1 ml of this suspension to 0.01+j! and 3H testosterone, unlabeled testosterone and dihydrotestosterone, glucose-6-phosphate, glucose-
It was incubated with 6-phosphate dehydrogenase and NADP-containing mixture 0.1a+1 for 30 minutes at 37°C. After incubation, the steroids were dissolved in ethyl acetate3.
0tsi! The organic phase was separated and dried under N2. This extract was spotted onto a TLC plate. After developing the TLC plate with ethyl acetate:cyclohexane 1:1, the 3H-DHT zone was scraped from the plate and counted.

B. Camellia Excellent dose response was obtained with compounds of formula I.

IC5o was calculated to be approximately 5 μg/val. The results are shown in Table 2 below: 5000 30 2039
69.11000 30 33
11 48.1) 8 30
5694 11.01.6 30
6054 5.3 Testosterone-5α
-9.The ability of compounds of formula I to inhibit ductase is
This makes these compounds useful as drugs. These compounds may be particularly useful in the treatment and prevention of disease states in which testosterone-5α-reductase is implicated, such as vulgaris vulgaris, female baldness, and benign prostatic hyperplasia.

A compound of formula I, or a pharmaceutically acceptable salt thereof, is administered to a human alone or preferably in combination with a pharmaceutically acceptable carrier or diluent in pharmaceutical practice. Compounds are administered orally or parenterally. Parenteral administration includes intravenous, intramuscular, intraperitoneal, subcutaneous and topical administration.

For oral administration of a compound of the invention, the selected compound is administered, for example, in the form of a tablet or capsule or as an aqueous solution or suspension. For oral tablets, commonly used carriers include lactose and corn starch.
Lubricants such as magnesium stearate are also commonly added. For oral administration in capsule form, useful diluents are lactose and dried cornstarch. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening and/or flavoring agents can also be added. For intramuscular, intraperitoneal, subcutaneous and intravenous use, sterile solutions of the active ingredient are usually prepared, the pH of which should be suitably adjusted and buffered. For intravenous use, the total concentration of solutes should be controlled to render the product isotonic.

When a compound of formula I or a salt thereof is used as an inhibitor of testosterone-5α-reductase in humans,
The daily dose is usually determined by the attending physician. Moreover, the dosage will vary depending on the age, weight and responsiveness of the individual patient, as well as the severity of the patient's symptoms. However, in most cases, the effective daily dose will be from about 1 to about 15 in single or divided doses.
00■, preferably within the range of 10 to 500■. On the other hand, in some cases it may be necessary to use dosages outside these limits.

[Brief explanation of the drawing]

Figure 1 shows the proton nuclear magnetic resonance ('H-N
MR) spectrum diagram. FIG. 2 is a ('H-NMR) spectrum diagram of compound 1b. Procedural amendment (method) (1) We will submit one official drawing as shown in the attached sheet. March 28, 2016

Claims (1)

[Claims] 1. Compound of the following formula I: ▲There are mathematical formulas, chemical formulas, tables, etc.▼(I) [In the above formula, R is ▲There are mathematical formulas, chemical formulas, tables, etc.▼(I a) or ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (I b) ] or a pharmaceutically acceptable salt thereof. 2. The compound according to claim 1, having the empirical formula C_3_4H_5_2O_1_0 and the NMR spectrum shown in FIG. 3. The compound according to claim 1, having the empirical formula C_3_5H_5_6O_1_1 and the NMR spectrum shown in FIG. 4. A method for producing a testosterone-5α-reductase inhibitor compound, comprising growing a Gliocladium strain in an aqueous nutrient medium by aerobic fermentation of an assimilable carbon source and recovering said compound. 5. The method according to claim 4, wherein the compound has the structure according to claim 1. 6. Gliocladium strain is ATCC No. 20826
The method according to claim 4. 7. Gliocladium ATCC No. 7 in an aqueous nutrient medium by aerobic fermentation of an assimilable carbon source. 5. The method of claim 4, comprising growing the 20826 strain and recovering the compound. 8. The method according to claim 7, wherein the compound has the structure according to claim 1. 9. A mixture of compounds according to claim 1 produced by controlled aerobic fermentation of Gliocladium bacteria in an assimilative nutrient medium. 10. Bacteria are ATCC No. 10. The mixture of claim 9, which is 20826.