WO1995013815A1 - Combination method for the treatment of patterned alopecia - Google Patents

Abstract

Combinations of (A) 17β-substituted-4-aza-5α-cholestan-3-ones of formula (I) and stereoisomers and pharmaceutically acceptable salts and esters thereof, administered topically or internally; and (B) a potassium channel opener, or a pharmaceutically acceptable salt thereof, administered topically, are useful for treating human patterned alopecia, including male and female pattern baldness, alopecia senilis and/or alopecia areata.

Description

TITLE OF THE INVENTION

COMBINATION METHOD FOR THE TREATMENT OF PATTERNED ALOPECIA

BACKGROUND OF THE INVENTION

The present invention is concerned with the use of 7β- substituted-4-aza-5 -cholestan-3-one compounds as 5α-reductase inhibitors in combination with a potassium channel opener such as minoxidil for the treatment of patterned alopecia, e.g., male pattern baldness.

Baldness or alopecia, in addition to male pattern alopecia, female pattern alopecia, and alopecia senilis, includes alopecia areata, and further, diseases accompanied by basic skin lesions such as cicatrix or infectious tumors, or accompanied by systemic disorders, for example, an internal secretion abnormality or nutritional disorder. Also, concerning alopecia areata, it is considered that an autoimmune phenomenon participates therein, and therefore, the administration of a substance having an immunosuppressive action can have therapeutical effect on alopecia areata.

The causes of human pattern alopecia (also called androgenic alopecia, or androgenetic alopecia, or male or female pattern baldness) and alopecia senilis are considered to be: an activation of male hormones at organs such as hair roots and the sebum gland; a lowering in the amount of blood reaching the hair follicles; a scalp abnormality caused by an excessive secretion of sebum, a formation of peroxides, or a propagation of bacteria; genetic causes; and aging. Hair revitalizing materials of the prior art generally comprise compounds having the actions of removing or alleviating the causes mentioned above formulated therein. For example, a compound having the action of inhibiting the activation of male hormones, or a compound having the action of increasing the amount of blood reaching the hair follicles, is formulated. Nevertheless, in human pattern alopecia and alopecia senilis, the epilation mechanism and the hair generation mechanism are very complicated, and by merely inhibiting an activation of male hormones or increasing the amount of blood reaching the hair follicles, as practiced in the prior art, does not sufficiently treat or prevent baldness or alopecia. Accordingly, there is a long-felt need for a hair revitalizing agent for male pattern alopecia and alopecia senilis, which provides satisfactory results.

Patterned baldness is sometimes called androgenic alopecia because male hormones are necessary for its development. It does not occur before adolescence, nor in castrates. Attempts to prevent alopecia by hormonal treatments by using anti-androgens or female hormones have not been tolerated due to side-effects or have failed. A hereditary component is also recognized since patterned alopecia runs in families. Despite intensive investigation, the mechanism whereby terminal follicles convert to vellus ones is unknown.

Minoxidil is a well-known pharmaceutical agent marketed by The Upjohn Company in the form of LONITEN® Tablets for the treatment of hypertension. The topical application of minoxidil, marketed as ROGAINE®, is a currently employed therapy for patterned alopecia. Numerous investigators have demonstrated that it can stimulate visible hair growth in some balding subjects. This compound has varying degrees of efficacy for moderating androgenic alopecia, depending on the degree of baldness, its duration, the age of the patient and, of course, on the concentration of the drug in an appropriate vehicle.

The compound minoxidil (6-amino-l ,2-dihydro-l- hydroxy-2-imino-4-piperidinopyrimidine) was approved by the FDA for the treatment of male pattern baldness in August 1988. Minoxidil was approved by the FDA for the treatment of female androgenetic alopecia on August 13, 1991. The preparation of minoxidil is described in U.S. Patent Nos. 3,382,247, and 3,644,364. U.S. Patent Nos. 4,139,619 and 4,596,812, assigned to Upjohn, disclose the structure and use of minoxidil in the topical treatment of human baldness. Similarly. an Upjohn United States Patent (U.S. Patent No. 5,026,691) discloses the use of minoxidil and an antiinflammatory agent for the treatment of human baldness. Also, WO 92/02225 discloses the use of minoxidil with finasteride for the treatment of baldness. Japanese patent Kokai 61-260010 states that topical minoxidil formulations containing other specified agents may be prepared.

It is also well known in the art that certain undesirable physiological manifestations, such as acne vulgaris, seborrhea, female hirsutism, androgenic alopecia including male pattern baldness, and benign prostatic hypertrophy, are the result of hyperandrogenic stimulation caused by an excessive accumulation of testosterone ("T") or similar androgenic hormones in the metabolic system.

Early attempts to provide a chemotherapeutic agent to counter the undesirable results of hyperandrogenicity resulted in the discovery of several steroidal antiandrogens having undesirable hormonal activities of their own. The estrogens, for example, not only counteract the effect of the androgens but have a feminizing effect as well. Non-steroidal antiandrogens have also been developed, for example, 4'-nitro-3'-trifluoromethyl-isobutyranilide. See Neri, et al., Endocrinol. 1972, 91 (2). However, these products compete with natural androgens for receptor sites, and hence have a tendency to feminize a male host or the male fetus of a female host and/or initiate feed-back effects which would cause hyperstimulation of the testes.

The principal mediator of androgenic activity in some target organs, e.g. the prostate, is 5α-dihydrotestosterone ("DHT"), formed locally in the target organ by the action of testosterone-5 - reductase (or simply 5 -reductase). Inhibitors of 5 -reductase will serve to prevent or lessen symptoms of hyperandrogenic stimulation in these organs. See especially United States Patent Nos. 4,377,584, issued March 22, 1983, and 4,760,071, issued July 26, 1988, both assigned to Merck & Co., Inc. It is now known that a second 5 -reductase isozyme exists, which interacts with skin tissues, especially in scalp tissues. See, e.g., G. Harris, et al, Proc. Natl. Acad. Sci. USA, Vol. 89, pp. 10787- 10791 (Nov. 1992). The isozyme that principally interacts in skin tissues is conventionally designated as 5 -reductase 1 (or 5 -reductase type 1), while the isozyme that principally interacts within the prostatic tissues is designated as 5 -reductase 2 (or 5 -reductase type 2).

Further described in the field are the following two publications: Proc. Natl Acad. Sci, USA, Vol. 87, pp. 3640-3645, May 1990 by S. Andersson and D.W. Russell, which describes structural and biochemical properties of cloned and expressed human and rat steroid 5-alpha reductases; and Nature, Vol 354, Nov. 1991, pp 159-161 by S. Andersson, et al, which describes the isolation of a second human enzyme, 5-alpha reductase 2, and the effect of a deletion in this gene in male pseudo-hermaphroditism.

The topical application of minoxidil has met with limited success. What is desired in the art is an improved treatment for patterned alopecia. Therefore, it is an object of this invention to provide an improved method of treating patterned alopecia, and to further provide suitable pharmaceutical compositions therefore.

SUMMARY OF THE INVENTION

The present invention involves a method for treating human patterned alopecia comprising the administration to a human host in need of such treatment of a potassium channel opener, such as minoxidil, cromakalim, pinacidil, a compound selected from the classes of s-triazine, thiane-1 -oxide, benzopyran, or pyridinopyran derivatives, or a pharmaceutically acceptable salt thereof in combination with certain 7β-substituted-4-aza-5α-cholestan-3-one compounds. The 4-aza- steroid compounds of the instant invention are inhibitors of 5α- reductase, and particularly are selective for inhibiting 5 -reductase type 1.

DETAILED DESCRIPTION OF THE INVENTION

The novel method of treatment of the present invention comprises administration of (A) a compound of structural Formula I:

I

or a stereoisomer or a pharmaceutically acceptable salt or ester thereof, wherein:

R is selected from hydrogen, methyl and ethyl; the dashed lines " z-___z " a and b independently represent a single bond or a double bond providing that when b is a double bond, the 5α hydrogen, Ha, is absent; =Z is selected from:

1 ) oxo,

2) oc-hydrogen and a β-substituent selected from: a) C1 -C4 alkyl, b) C2-C4 alkenyl, c) CH2COOH, d) -OH, e) -COOH, f) -COO(Cl -C4 alkyl), g) -OCONR ^R^ wherein R^ and R- independently are selected from: i) H, ii) C1 -C4 alkyl, iii) phenyl, and iv) benzyl, or

R and R^ together with the nitrogen atom to which they are attached represent a 5-6 membered saturated heterocycle, optionally containing one other heteratom selected from -0-, -S- and -N(R')- wherein R' is -H or methyl; h) C1-C4 alkoxy, i) C3-C6 cycloalkoxy, j) -OCOCH3, k) halo,

1) hydroxy -C1 -C2 alkyl, m) halo-Cl -C2 alkyl, n) -CF3, and

0) C3-C6 cycloalkyl;

3) =CHR3; wherein R3 is selected from -H and C1-C4 alkyl: and

4) spirocyclopropane-R^ of structure:

and

(B) a potassium channel opener, selected from the group consisting of minoxidil, cromakalim, pinacidil, a compound selected from the classes of s-triazine, thiane-1 -oxide, benzopyran, or pyridinopyran derivatives, or a pharmaceutically acceptable salt thereof.

The potassium channel opener is intended to be administered topically, while the compound of Formula I may be administered orally, systemically, parenterally or topically. The 17- substituent cholestane side chain in Formula I is in the beta configuration. Combinations of substituents and/or variables in Formula I are permissible only if such combinations result in stable compounds.

The term "C1 -C4 alkyl" as used herein, is meant to include methyl (Me), ethyl (Et), propyl (Pr), iso-propyl (i-Pr), n-butyl (n-Bu). sec-butyl (s-Bu), iso-butyl (i-Bu) and tert-butyl (t-Bu). - 7 -

The term "C2-C4 alkenyl" as used herein is meant to include vinyl, allyl, 1-propen-l -yl, 1 -propen-2-yl, 1 -buten-l-yl, 1 - buten-2-yl, and the like. Included in this invention are all E, Z diastereomers.

The term "C3-C6 cycloalkyl" as used herein is meant to include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

The term "halo" as used herein is meant to include fluoro, chloro, bromo, and iodo.

The term "OC1-C4 alkyl" or "C1 -C4 alkoxy" as used herein is meant to include methoxy, ethoxy, propoxy, iso-propoxy, n- butoxy, iso-butoxy, sec-butoxy, and tert-butoxy.

The term "OC3-C6 cycloalkyl" or "C3-C6 cycloalkoxy" as used herein is meant to include: cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, and cyclohexyloxy.

Representative examples of =Z are where the α-substituent (dashed lines) is hydrogen and the β-substituent (wedge) is e.g. methyl, ethyl, propyl, allyl, carboxymethyl, hydroxy, methoxy, ethoxy, cyclopropyloxy, cyclopentyloxy, acetoxy, fluoro, chloro, bromo, trifluoromethyl, fluoromethyl, chloromethyl, carboxy, N,N- dimethylcarbamate, hydroxymethyl, and the like.

Representative examples where =Z is an alkenyl substituent, =CH-R3, includes =CH2, =CH-CH3, =CH-CH2CH3, and the like.

Representative examples wherein =Z is the spirocyclopropyl substituent:

stereoisomers thereof and the like.

Representative examples wherein -NR' 1 R 9-" represent a heterocycle include: N-piperidinyl, N-morpholinyl. N-piperazinyl. N- (4-methyl)piperazinyl, N-thiomorpholinyl, N-pyrrolidinyl, N- imidazolidinyl and the like.

Representative compounds included in the invention wherein all of the 17- substituents are in the beta configuration are: 7β-ethyl-4-methyl-4-aza-cholest-5-en-3-one, 7β-ethyl-4-methyl-4-aza-cholestane-3-one, 7β-ethyl-4-aza-cholest-5-en-3-one, 7β-ethyl-4-aza-5α-cholestan-3-one, 7β-carboxymethyl-4-aza-cholest-5-en-3-one, 7β-carboxymethyl-4-aza-cholestan-3-one, 7β-propyl-4-methyl-4-aza-cholest-5-en-3-one, 7β-propyl-4-methyl-4-aza-5α-cholestan-3-one, 7β-propyl-4-aza-cholest-5-en-3-one, 7β-propyl-4-aza-5α-cholestan-3-one, 7β-methyl-4-aza-cholest-5-en-3-one, 7β-methyl-4-aza-cholestan-3-one, 4,7β-dimethyl-4-aza-cholest-5-en-3-one, 4,7β-dimethyl-4-aza-5 -cholestan-3-one, 4-methyl-4-aza-5oc-cholestan-3,7-dione, 7β-acetoxy-4-methyl-4-aza-5oc-cholestan-3-one, 4-methyl-4-aza-cholest-5-en-3,7-dione, 7β-hydroxy-4-methyl-4-aza-5 -cholestane-3-one, 7β-methoxy-4-methyl-4-aza-5 -cholestane-3-one, 7β-hydroxymethyl-4-aza-5α-cholestane-3-one, 7β-bromomethyl-4-aza-5 -cholestane-3-one, 7β-chloromethyl-4-aza-5α-cholestane-3-one, 7β-fluoromethyl-4-aza-5cc-choIestane-3-one, 7β-carboxy-4-aza-5oc-cholestane-3-one, 7β-trifluoromethyl-4-aza-cholest-5-en-3-one, 7.7-dimethoxy-4-methyl-4-aza-5α-cholestane-3-one, 7β-methoxy-4-methyl-4-aza-cholesta-5-en-3-one, 7β-methoxy-4-methyl-4-aza-cholesta-6-en-3-one, 7β-cyclopropyloxy-4-methyl-4-aza-5α-cholestane-3-one, 7β-cyclopropyloxy-4-methyl-4-aza-cholesta-5,7-dien-3-one, 7β-propylidene-4-methyl-4-aza-5 -cholestane-3-one, 7β-(2-ethyl)spiroethylene-4-methyl-4-aza-5α-cholestane-3-one: and 7β-methyl-4-aza-5 -cholest- 1 -en-3-one.

The 5oc-reductase inhibitor compounds of this invention can be made by procedures outlined in the following Flowsheets. Temperatures indicated are in degrees Celsius.

GENERAL FLOWSHEET

II

III GENERAL FLOWSHEET (CONT'D)

VII GENERAL FLOWSHEET (CONT'D)

20

IX

30 GENERAL FLOWSHEET (CONT'D)

X

XI

7-Beta Alkyl Series

The compounds of the instant invention comprising Z as a 7β alkyl group, e.g. methyl, ethyl, isopropyl, allyl, can be prepared by the procedure outlined in The General Flowsheet.

As seen in the Flowsheet, the starting 3-acetoxy-cholest-5- ene I (see Example 1 for synthesis) is oxidized to the corresponding 5- en-7-one II by treatment with hydrogen t-butyl peroxide and chromium hexacarbonyl in e.g. acetonitrile, at reflux. The C1 -C4 alkyl group, designated Alk, e.g. methyl, can be introduced at this point by a Grignard reaction using e.g., alkyl magnesium chloride in e.g., anhydrous THF at 0-23°C to produce the 7-alkyl-7-hydroxy adduct m. This is then oxidized with e.g. aluminum isopropoxide and cyclo- hexanone (Oppenauer oxidation conditions) in refluxing toluene solvent to produce the 7-alkyl-4,6-dien-3-one IV. This in turn is reduced via a e.g.. metal-ammonia reduction, using lithium, liquid ammonia. THF and toluene at -78°C, quenching the reaction with dibromoethane and ammonium chloride, to selectively yield the 7-beta-alkyl-5-en-3-one V. In the next step the delta-5 double bond is isomerized to the 4-ene by use of DBU (l ,8-diazabicyclo[5.4.0]undec-7-ene) in, e.g. refluxing tetrahydrofuran (THF) to produce the 7-beta-alkyl 4-en-3-one, VI. The A Ring is next cleaved by treatment with e.g. potassium permanganate, sodium periodate in t-butyl alcohol at 80°C to produce the corresponding seco-acid VII. Treatment of the seco-acid with an appropriate amine e.g., methylamine hydrochloride and sodium acetate in ethylene glycol at 180°C, yields e.g., the 4-methyl-4-aza-cholest-5-en- 3-one VIII. This in turn is selectively reduced with e.g., Ptθ2 catalyst in a hydrogen atmosphere, to remove the 5-position double bond to produce the 5α-hydrogen compound IX. The seco-acid VII can be similarly treated with ammonium acetate in acetic acid to produce the corresponding N-H compound, X, which can then be analogously treated with Ptθ2 in a catalytic hydrogenation to produce the corresponding 5oc-4N-H compound XI. Similarly, use of hydroxylamine or hydrazine for ring A closure of the seco acid will afford the corresponding delta-5 -4N-X compounds where -X can be -OH or -NH2, respectively. Reaction of the anion of saturated 4N- compound (generated from the NH precursor by NaH treatment) with methylsulfenyl chloride can provide the corresponding 4N-X compound where -X is -SCH3. Thus, R can also be -OH, -NH2 or SCH3 in the Formula.

FLOWSHEET A

FLOWSHEET A (CONT'D)

FLOWSHEET B

17 -

FLOWSHEET B (CONT'D)

→

7-Beta-Ethyl-Cholestane Analogues

The 7-ethyl substituent is introduced into the cholestane series as illustrated in Flowsheets C and D by the same analogous procedure as described in the General Flowsheets.

The starting cholesteryl acetate CA is available commercially (Aldrich). This is treated using the analogous chromium hexacarbonyl/hydrogen t-butylperoxide/acetonitrile oxidation procedure (described in JCS Perkin Trans. 1985, p. 267 by A. J. Pearson) to yield the 3-acetoxy-cholest-5-en-7-one 1. This can be reacted with an alkyl Grignard reagent, e.g. ethyl magnesium chloride to form the adduct 2. This is oxidized under Oppenauer conditions to yield the dienone 3, which then can undergo metal-ammonia reduction to yield the 7β-ethyl- 5-en-3-one, 4. This is isomerized using DBU to the 4-en-3-one, 5, which is oxidized to open Ring A to yield the seco-acid 6. This can be treated with amines, e.g. methylamine, to yield the A-ring closed 4- methyl-4-aza compound 7. This in turn can be catalytically hydrogenated to yield the 7-ethyl-5-alpha-4-methyl-4-aza-cholestan-3- one, 8_..

Similarly, by treatment of the seco-acid 6 with ammonium acetate/acetic acid, the corresponding 4-NH analog 9, is produced which can be catalytically hydrogenated to yield the 7-beta-ethyl-5α-4-aza- cholestan-3-one, 10.

Following the same procedure but using phenylmagnesium chloride as the Grignard reagent, the corresponding compounds 50 and 51 are produced.

FLOWSHEET C

13 FLOWSHEET C (CONT'D)

14

1£

7-Carboxymethyl-Cholestane Series

The 7-carboxy substituent is formed through the corresponding 7-allyl group. As seen in Flowsheet C, 7-oxo-cholesteryl acetate i is reacted with allyl Grignard reagent to form the adduct ]_]_ which is oxidized to the dienone 12 by Oppenauer conditions. Metal- ammonia reduction affords the 5-ene analog J_3, followed by DBU- catalyzed double bond isomerization to JA This in turn can be oxidized in a key step to form the 7-carboxymethyl seco-acid J_5. Treatment with amines, e.g. ammonia, forms the 4-aza derivative, 16 which is then reduced to the cholestane 17. Use of methylamine in place of ammonia can yield the corresponding 4-methyl analogs of 16 and 17.

FLOWSHEET D

14

13. 19

FLOWSHEET E

12.

7-Propyl -Cholestane Series

The 7-propyl analogs are made starting with the 7-allyl-4- en-3-one 14, which is reduced by hydrogenation using Wilkinson's catalyst to the propyl derivative J_8, oxidized to the seco-acid 19, then condensed with amines, e.g. methylamine, to form the 4-methyl analog 20 and then reduced to the cholestane 2L Corresponding treatment with ammonia is shown in Flowsheet E shows the corresponding unsubstituted 4-aza 22 and cholestane 23 analogs.

FLOWSHEET F

2≤ FLOWSHEET F (CONT'D)

23

22. FLOWSHEET G

22 30

7-Beta Methyl Cholestane Series

The 7-beta methyl cholestane series is prepared by the analogously same route as described in Flowsheets A and B for the ethyl derivatives.

The methyl Grignard reagent is used to form the adduct 24. followed by Oppenauer oxidation to form 25, metal-ammonia reduction to form 26, double bond isomerization to form 27, seco-acid oxidation to form 28, and treatment by an ammonium salt to form 29, and reduction to form 30. Corresponding treatment with methylamine produces the corresponding 4-methyI-4-aza compounds, 31 and by reduction, 32.

FLOWSHEET H

catalytic hydrogenation

+ 36

FLOWSHEET I

AC₂0/Py/DMAP/CH₂CI₂

15

25

30 7-Beta Acetoxy Cholestane Series

The 7-beta acetoxy series is prepared by the oxidation of starting 33 to the 5-en-7-one 34 by the chromium hexacarbonyl procedure described for 1 , or by pyridine-dichromate/t-butyl hydroperoxide oxidation as described in the Examples. Subsequent noble metal, e.g. platinum, ruthenium, catalyzed reduction of 34 yields two products, the reduced 7-oxo compound 35_., and 7-beta hydroxy compound 36. Acylation of 3j5 with acetic anhydride yields the 7-beta acetoxy compound 37.

FLOWSHEET J

The 7-beta ethers in the cholestane series are prepared from the 7-beta-ol (7-beta hydroxy derivative). As illustrated in Flowsheet J. the 4-Nmethyl-7-beta ol 36 can be reacted with e.g. methyl iodide and sodium hydride in e.g., dimethylformamide, to produce the corresponding methyl ether 32. The other C1 -C4 ethers can be prepared in the same manner.

The C3-C6 cycloalkyl ethers can be prepared according to the analogous procedure of Steroids, 1972, vol. 19, pp. 639-647 by R. Gardi, et al. For example, 6 can be reacted with 1 , 1 -dimethoxy- cyclohexane to produce the enol ether 3£, which can be reduced to the corresponding saturated compound by the use of palladium catalyzed hydrogenation.

FLOWSHEET K

30 FLOWSHEET L

20

25

30 FLOWSHEET M

The 7-haloalkyl series is made by the procedure illustrated in Flowsheet K.

Starting with the 7-beta-carboxy, 45, this can be treated under Hunsdiecker reaction conditions, i.e. bromination of a mercury metal salt, to yield the 7-bromo derivative 40. The chloro and iodo derivatives can be made in substantially the same fashion.

The haloethyl compounds can be made by starting with the 7-carboxymethyl analog 17_. which can be reacted with a reducing agent, e.g. borane, to produce the primary alcohol _1_. This in turn can be reacted with triphenylphosphine and carbon tetrabromide to produce the bromoethyl derivative 42.

The halomethyl compounds can be produced starting with the carboxymethyl derivative 17. This is treated with lead tetraacetate under oxidative decarboxylation/halogenation conditions, with a chloride, bromide or iodide salt to vield, e. . the 7-chloromethvl analoe 41. The carboxymethyl compound 17 can be treated with a fluorinating agent (XeF2) to yield the 7-fluoromethyl analog 44.

The 7-trifluoromethyl derivative can be made from the 7- carboxy derivative 45, by conventional Dast halogenation conditions using SF4 to yield the 7-trifluoromethyl analog 46.

FLOWSHEET N

Flowsheet N illustrates the 7-methylene series. As seen, the Wittig reaction, using e.g. Ph3PCH(CH2CH3), carried out on the 7-oxo compound 15, leads to the 7-(ethyl)methylene compound 47. Subsequent treatment of 47 with the cyclopropyl forming reagents, CH2Ϊ2 and zinc, produces the ethyl cyclopropyl spiro compound 48, which is a mixture of stereoisomers.

FLOWSHEET O

(1 ) DDQ/BSTFA/CH₃S0₃H Toluene/2372h

(2) Methylacetoacetate

2371 h, Reflux/24h

22

42

Flowsheet O illustrates the synthesis of the l -ene-7- substituted analogs. For example compound 10 is stirred with DDQ, BSTFA (bis-trimethylsilyltrifluoroacetamide) and trifluoromethyl sulfonic acid in toluene at room temperature for 24 hours, methyl acetoacetate is added and the mixture refluxed for 24 hours and purified by preparative thin layer chromatography on silica gel using 3:1 chloroform/acetone to yield 49.

Also included within the scope of this invention are pharmaceutically acceptable salts of the compounds of Formula I. where a basic or acidic group is present on the structure. When an acidic substituent is present, i.e. -COOH, there can be formed the ammonium, sodium, potassium, calcium salt, and the like, for use as the dosage form. Where a basic group is present, i.e. amino or a basic heteroaryl radical such as, e.g., 4-pyridyl, an acidic salt, i.e. hydrochloride, hydrobromide, acetate, pamoate, and the like, can be used as the dosage form.

Also, in the case of the -COOH group being present, pharmaceutically acceptable esters can be employed, e.g. acetate, maleate, pivaloyloxymethyl, and the like, and those esters known in the art for modifying solubility or hydrolysis characteristics for use as sustained release or prodrug formulations.

Representative salts include the following salts: acetate, lactobionate, benzenesulfonate, laurate, benzoate, malate, bicarbonate, maleate, bisulfate, mandelate, bitartrate, mesylate, borate, methylbromide, bromide, methylnitrate, calcium edetate, methylsulfate, camsylate, mucate, carbonate, napsylate, chloride, nitrate, clavulanate, N-methylglucamine, citrate, ammonium salt, dihydrochloride, oleate, edetate, oxalate, edisylate, pamoate (embonate), estolate, palmitate, esylate, pantothenate, fumarate, phosphate/diphosphate, gluceptate, polygalacturonate, gluconate, salicylate, glutamate, stearate, glycollylarsanilate, sulfate, hexylresorcinate, subacetate, hydrabamine, succinate, hydrobromide, tannate, hydrochloride, tartrate, hydroxynaphthoate, teoclate, iodide, tosylate, isothionate, triethiodide, lactate, and valerate.

The compounds of the present invention have asymmetric centers and may occur as racemates, racemic mixtures and as individual enantiomers or diastereomers, with all isomeric forms being included in the present invention as well as mixtures thereof. Furthermore, some of the crystalline forms for compounds of the present invention may exist as polymorphs and as such are intended to be included in the present invention. In addition, some of the compounds of the instant invention may form solvates with water or common organic solvents. Such solvates are encompassed within the scope of this invention.

The combination of a compound of Formula I and a potassium channel opener, such as minoxidil, is useful in hair revitalizing, such as in the treatment of male pattern alopecia, female pattern alopecia, alopecia senilis or alopecia areata, by providing epilation prevention, hair germination, and/or a promotion of hair generation and hair growth. The term "treating androgenic alopecia" is intended to include the arresting and/or reversing of androgenic alopecia, and the promotion of hair growth. The compounds of Formula I prepared in accordance with the method described above, are, as already described, potent and selective antiandrogens in the treatment of patterned alopecia by virtue of their ability to inhibit 5α- reductase 1.

Accordingly, the instant invention is concerned with providing a method of treating human pattern alopecia, which includes male and female pattern baldness, and alopecia senilis and alopecia areata, by administering to a patient in need of such treatment a therapeutically effective amount of a compound of Formula I and a therapeutically effective amount of a potassium channel opener, such as minoxidil, cromakalim, pinacidil, a compound selected from the classes of s-triazine, thiane-1 -oxide, benzopyran, or pyridinopyran derivatives, or a pharmaceutically acceptable salt thereof. The term "therapeutically effective amount" shall mean that amount of a drug or pharmaceutical agent that will elicit the biological or medical response of a tissue, system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician.

The compound of Formula I may be administered topically, parenterally or systemically, including orally, while the potassium channel opener, e.g., minoxidil, is administered topically, in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles. The term parenteral as used herein includes subcutaneous injections. intravenous, intramuscular, intrasternal injection or infusion techniques. In particular, the modes of administration include topical administration of minoxidil in combination with oral or topical administration of a compound of Formula I.

The present invention is thus also concerned with providing suitable topical and systemic pharmaceutical formulations for use in the novel methods of treatment of the present invention. The active agents can be administered in a single topical pharmaceutical formulation, or each active agent can be administered in a separate pharmaceutical formulation, e.g., in separate topical pharmaceutical formulations, or e.g., an oral pharmaceutical formulation of a compound of Formula I in combination with a topical pharmaceutical formulation of, e.g., minoxidil. See, e.g., U.S. Patent No.'s 4,596,812, 4,139,619 and WO 92/02225, published 20 February 1992, for dosages and formulations of potassium channel openers. For combination treatment where the active agents are in separate dosage formulations, the active agents can be administered concomitantly, or they each can be administered at separately staggered times. For example, a compound of Formula I may be administered prior to, concurrent with, or subsequent to the topical administration of minoxidil.

For topical administration, the pharmaceutical compositions of this invention can be used in the form of a pharmaceutical preparation, for example in solid, semisolid or liquid form, which contains one or more of the compounds of the present invention as an active ingredient in admixture with an organic or inorganic pharmacologically acceptable carrier or excipient suitable for external applications. In particular, the potassium channel opener and one or more compounds of Formula I may be combined in one topical pharmaceutical composition, or they may each be in separate topical pharmaceutical compositions.

Topical pharmaceutical compositions may be, e.g., in the form of a solution, cream, ointment, gel, lotion, shampoo or aerosol formulation adapted for application to the skin. Topical pharmaceutical compositions containing the compounds of Formula I ordinarily include about 0.001 % to 15% by weight of the active compound in admixture with a pharmaceutically acceptable vehicle, and more particularly about 0.01-5% by weight, of the active compound, in admixture with vehicle. For external administration, the potassium channel opener, such as minoxidil, may be formulated in a topical composition within the range of, for example, 0.1 % to 10.0% by weight, and particularly from 1 % to 5% by weight of the active compound. When either or both agents are administered topically, the hair revitilizing composition of the present invention may particularly be administered as a cream or lotion by a percutaneous administration or by spraying onto the skin.

The compounds of Formula I may be compounded, for example, with the usual non-toxic, pharmaceutically acceptable carriers for tablets, pellets, capsules, suppositories, solutions, emulsions, suspensions, and any other form suitable for use. The carriers which can be used are water, glucose, lactose, gum acacia, gelatin, mannitol, starch paste, magnesium trisilicate, talc, corn starch, keratin, colloidal silica, potato starch, urea and other carriers suitable for use in manufacturing preparations, in solid, semisolid, or liquid form, and in addition auxiliary, stabilizing, thickening and coloring agents and perfumes may be used. For example, the compounds of Formula I may be utilized with hydroxypropyl methyl-cellulose essentially as described in U.S Patent No. 4,916,138, issued April 10, 1990, or with a surfactant essentially as described in EPO Publication 0,428,169. Dosage forms for external application of compounds of Formula I and a potassium channel opener may be prepared essentially as described in EPO Publication 0,423,714 or in U.S. Patent No. 4,938,953. The active compounds are included in the pharmaceutical composition in a therapeutically effective amount, that is an amount sufficient to produce the desired effect upon the process or condition of diseases.

The compounds of Formula I can also be administered in a wide variety of therapeutic dosage forms in conventional vehicles for systemic administration, as, for example, by oral administration in the form of tablets, capsules, solutions, or suspensions, or by intravenous injection. For oral administration of compounds of Formula I, the daily dosage may be varied from 0.01 to 1,000 mg per adult human/per day, particularly from about 0.1-10 mg/day, and more particularly from about 0.2-5 mg/day per adult human. The compositions may be provided in the form of scored or unscored tablets containing 0.01 , 0.05, 0.1 , 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0 and 100 milligrams of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated. An effective amount of the drug is ordinarily supplied at a dosage level of from about 0.0002 mg to about 50 mg/kg of body weight per day. The range is more particularly from about 0.001 mg/kg to 7 mg/kg of body weight per day. Administration may be 1 to 3 times/day per person.

In the methods of the present invention, the compounds herein described in detail can form the active ingredient, and are typically administered in admixture with suitable pharmaceutical diluents, excipients or carriers (collectively referred to herein as "carrier" materials) suitably selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups and the like, and consistent with conventional pharmaceutical practices.

Capsules containing the compound of Formula I can be prepared by mixing it with lactose and magnesium stearate, calcium stearate, starch, talc, or other carriers, and placing the mixture in gelatin capsule. Tablets may be prepared by mixing the active ingredient with conventional tableting ingredients such as calcium phosphate, lactose, com starch or magnesium stearate and the like. Moreover, when desired or necessary, suitable binders, lubricants, disintegrating agents and coloring agents can also be incorporated into the mixture. Suitable binders include, without limitation, starch, gelatin, natural sugars such as glucose or beta-lactose, com sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes and the like. Lubricants used in these dosage forms include, without limitation, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like. Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum and the like.

The liquid forms in suitably flavored suspending or dispersing agents such as the synthetic and natural gums, for example, tragacanth, acacia, methyl-cellulose and the like. Other dispersing agents which may be employed include glycerin and the like. For parenteral administration, sterile suspensions and solutions are desired. Isotonic preparations which generally contain suitable preservatives are employed when intravenous administration is desired.

Topical preparations containing the active drug component can be admixed with a variety of carrier materials well known in the art, such as, e.g., alcohols, aloe vera gel, allantoin, glycerine, vitamin A and E oils, mineral oil, PPG2 myristyl propionate, and the like, to form, e.g., alcoholic solutions, topical cleansers, cleansing creams, skin gels, skin lotions, and shampoos in cream or gel formulations. See, e.g., EP 0 285 382.

The compounds of the present invention can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines.

Compounds of the present invention may also be delivered by the use of monoclonal antibodies as individual carriers to which the compound molecules are coupled. The compounds of the present invention may also be coupled with soluble polymers as targetable drug carriers. Such polymers can include polyvinyl- pyrrolidone, pyran copolymer, polyhydroxypropylmetha- crylamidephenol, polyhydroxy-ethylaspartamidephenol, or polyethyleneoxidepolylysine substituted with palmitoyl residues. Furthermore, the compounds of the present invention may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and cross-linked or amphipathic block copolymers of hydrogels.

In addition, the compositions of the present invention may be administered on an intermittent basis; i.e. at semi-daily, daily, semi- weekly, weekly, semi-monthly or monthly intervals. The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. It will be understood, however, that the specific dose level for any particular patient will depend on a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the severity of the particular disease undergoing therapy. A physician or veterinarian of ordinary skill can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the condition.

The following examples are illustrative of representative embodiments of this invention and should not be construed to be limits on the scope or spirit of the instant invention.

The Rf values cited were carried out on standard thin layer chromatographic Si gel plates. The elution solvent system used is given in the parentheses following the Rf value.

The fast atom bombardment (FAB) mass spectral values are reported as (M+l) molecular ion peaks, being the molecular weight plus one atomic mass unit. The electron impact (El) mass spectrum values are reported as molecular ion peaks and are indicated in parentheses, either being (M) or (M+2), the molecular weight, MW, or the MW plus two atomic units.

The nuclear magnetic resonance data was taken at 400 MHz in CDCI3 and is tabulated for unique proton values of each compound at the end of the Examples. The coupling constant J is given in Hertz, Hz. EXAMPLE 1

Synthesis of 7-Oxo-Cholesterol-3-acetate (1)

Cholesteryl acetate (CA) is known in the art and can be oxidized to the known 7-oxo-derivative 1 by the analogous procedure described in the JCS Perkins article by Pearson, supra.

EXAMPLE 2

Synthesis of 7-Ethyl-7-Hvdroxy-cholesterol. (2)

To a solution of 1 from Example 1 , being 5.0 g (11.32 mmol) in dry tetrahydrofuran at 0°C was added dropwise 56.6 ml ethyl magnesium bromide (1M) over 5-10 minutes. The reaction mixture was then allowed to stir at room temperature for 24 hours, then poured into saturated aqueous ammonium chloride. The THF solvent was removed under vacuum and the aqueous phase extracted with ethyl acetate. The organic layer was washed with brine, dried, concentrated to yield a yellowish-white foam. The Rf value was 0.2 (30% EtOAc/hexane). Proton NMR confirmed the assigned structure of the title compound 2 which was used in the next step without further purification.

EXAMPLE 3

Synthesis of 7-Ethyl-Cholest-4.6-Dien-3-one. (3)

The above Grignard product 2, 5.13 g (11.9 mmol) was dissolved in 50 ml toluene and cyclohexanone and about 40 ml of solvent distilled off under vacuum. To this was added 7.2 g aluminum isopropoxide and the reaction mixture refluxed overnight for 15 hours. The mixture was cooled, diluted with ethyl acetate, washed with sodium potassium tartarate, brine, and the organic layer was concentrated under vacuum and the residue steam distilled. The residue was extracted with ethyl acetate, the ethyl acetate layer, washed with brine, dried and purified by column chromatography on silica gel, eluting with 5%

EtOAc/hexane to yield the title compound 3.

Rf=0.58 (20% EtOAc/hexane).

Mass spec: 412(M=1) by FAB, Calc'd. 411.9.

EXAMPLE 4

Synthesis of 7β-ethyl-cholest-5-en-3-one. (4)

To a solution of 3.1 g of 1, from Example 3, in 46 ml ammonia, 10 ml THF, 10 milliliters toluene, was added 449 mg of metallic lithium in small pieces. After stirring the blue solution for 2 hours at -78°C, a solution of 1,2-dibromethane in 2 ml THF was added. After stirring the solution at -78°C for 10 minutes, 2.1 g of ammonium chloride was added and the mixture stirred for 10 minutes. The excess ammonia was removed by evaporation under a nitrogen stream. The reaction mixture was diluted with brine, extracted with ethyl acetate. The organic layer was washed with brine, dried and concentrated to yield crude brown viscous liquid 4 which was used as such in Example 5. Rf=0.70 (20% EtOAc/hexane). Mass Spec. 412 (El); calculated MW 412.70.

EXAMPLE 5

Synthesis of 7β-ethyl-cholest-4-en-3-one (5).

To a solution of 4, from Example 4, being 3.1 g in 30 ml THF was added 1.1 ml DBU (l ,8-diazabicyclo[5.4,0]undec-7-ene under nitrogen with stirring. The mixture was refluxed for 1.5 hours, then cooled and diluted with NH4CI. Then THF solvent was removed under vacuum and the residue extracted with ethyl acetate. The organic layer was then washed with water, brine, dried and concentrated under reduced pressure to yield a crude viscous oil. The titled product 5 was purified by chromatography on silica gel using 10% EtOAc/hexane as eluant. Mass Spec 412 (El), calc'd MW 412.70. Rf=0.6 (20% EtOAc/hexane).

EXAMPLE 6

Synthesis of 7-ethyl-17β-(6-methyl-2-heptyl)-5-oxo-A-nor-3,5- secoandrostan-3-oic acid. (6)

To a solution of 1.0 g of 5 in 18 ml t-butyl alcohol at 80°C was added 300 mg sodium carbonate in 1.8 ml water followed by a dropwise addition over 15-20 minutes of a mixture of 2.74 g sodium periodate with 20.3 mg potassium permanganate in 15 ml water. The reaction mixture was heated at 80°C for 2 hours, cooled, filtered, the residue washed with water, and then the filtrate concentrated under vacuum, acidified with aqueous HCl, extracted with ethyl acetate and the organic layer washed with aqueous NaHS03, brine, dried and concentrated to yield crude 6. The proton NMR confirmed the assigned structure. Fast atom bombardment yielded an m/z molecular ion of 434(m+2); calculated 432.69.

EXAMPLE 7

Synthesis of 7-Ethyl-4-methyl-4-aza-cholest-5-en-3-one. (7)

To a solution of 6, 500 mg in 10 ml ethylene glycol was added 1.3 g sodium acetate and 1.0 g methylamine hydrochloride.

After stirring the reaction mixture 4 hours at 180°C, the mixture was cooled, diluted with water, extracted with ethyl acetate, dried and concentrated to afford crude title compound 7. Proton NMR confiιτned the assigned structure.

Rf=0.70 (20% EtOAc/hexane).

Mass Spectral m/z ion (FAB) showed 429 (M+2). calculated, 427.72.

Analysis: calculated for C29H49NO C, 81.44; H, 1 1.55; N, 3.27

Found: C, 82.19; H, 10.92; N, 3.1 1. EXAMPLE 8

Synthesis of 7-Ethyl-4-methyl-4-Aza-Cholestan-3-one. (8)

To a solution of 7 from Example 7, being 180 mg in 5 ml acetic acid was added 54 mg platinum dioxide and the resulting mixture was evacuated and flushed with hydrogen. The reaction was shaken overnight at room temperature under hydrogen. Filtered, washed solid with EtOAc, combined EtOAc layers were washed with aqueous NaHC03, brine, dried, concentrated to yield the title compound 8.. Mass spectral analysis by FAB yielded m/z ion of 431 (m+2), calculated 429.74. Analysis: calculated for C29H51 NO

C, 81.06; H, 11.96; N, 3.26 Found: C, 81.42; H, 12.24; N, 3.16.

EXAMPLE 9

Synthesis of 7-Ethyl-4-Aza-Cholest-5-en-3-one. (9)

The seco acid 6, 0.5 g and ammonium acetate, 0.5 g, in 3.5 ml acetic acid were refluxed for 3 hours. The reaction mixture was cooled, water added and then extracted with ethyl acetate. The organic layer was dried over sodium sulfate and concentrated to yield a residue which was eluted on a silica gel column with 10% EtOAc/hexane to give pure title compound 9, mp. 147-149°C.

Mass Spec. 414 (Mtl). Calc'd; 413.69. Rf=0.45 (30% EtOAc/hexane). Analysis: calculated for C28H49NO, MW 413.69

C, 81.30; H, 11.45; N, 3.39 Found: C, 81.30; H, 1 1.87; N, 3.45.

EXAMPLE 10

Synthesis of 7β-Ethyl-4-aza-5α-cholestan-3-one, (10)

Following the general analogous procedure described in Example 8, 9 was catalytically hydrogenated to yield the titled compound, 10. Chromatography on silica gel with 50% EtOAc :hexane eluant yielded pure product, mp. 169-170°C.

Analysis: calculated for C28H49NO, MW=415.17.

Calcd: C, 80.90; H, 11.88; N, 3.37

Found: C, 81.02; H, 12.57; N, 3.47.

Mass Spec: 416 (M+l).

Rf=0.30 (30% EtOAc/hexane).

EXAMPLE 1 1

Synthesis of 7-Allyl-3.7-dihvdroxy-cholest-5-ene (11)

Following the analogous general Grignard procedure of Example 2, allyl magnesium bromide was reacted with Compound 1 in dry THF to yield the titled product FL Proton NMR confirmed the assigned structure.

Mass Spec. 441 (M+l). Calc'd. 440.71. Rf=0.25 (30% EtOAc/hexane).

EXAMPLE 12

Synthesis of 7-allyl-cholest-4.6-dien-3-one. (12)

Following the analogous general Oppenauer oxidation procedure of Example 3, compound J _. was oxidized to yield the titled compound J_2. Proton NMR confirmed the assigned structure as well the (FAB) mass spec. 423 (M+l) Calc'd. 422.35. Rf=0.78 (30% EtOAc/hexane).

EXAMPLE 13

Synthesis of 7-Allyl-cholest-5-en-3-one, (13)

Compound 12, was subjected to the analogous metal- ammonia reduction conditions of Example 4 to yield the title compound

11-

Rf=0.5 (5% EtOAc/hexane). EXAMPLE 14

Synthesis of 7-Allyl-Cholest-4-en-3-one. (14)

Following the general DBU catalyzed isomerization conditions of Example 5, compound H was analogously treated to yield the title compound 14.

Mass Spec. 425 (M+l) by FAB. Calc'd.: 424.37. Rf=0.45 (5% EtOAc/hexane).

EXAMPLE 15

Synthesis of 7-Propyl-cholest-4-en-3-one. (18)

1.0 g. of the 7-allyl-enone J_4, 5 ml. EtOAc and 50 mg triphenylphosphine rhodium chloride (Wilkinson's catalyst) were allowed to stir two hours (under H2 atmosphere). The reaction products were filtered through 25 ml silica gel, and evaporated to dryness to yield fairly pure title product, 18, as confirmed by proton NMR.

Mass Spec. 427 (M+l). Calc'd.: 426.39. Rf=0.15 (5% EtOAc/hexane).

EXAMPLE 16

Synthesis of 7-Propyl-5-oxo-A-nor-3.5-seco-cholestanoic acid, (19) Following the general procedure of Example 6 for the oxidative Ring A cleavage, compound 18 (7 -propyl analogue) was analogously treated to yield the above-titled seco-acid 19. The assigned structure was confirmed by proton NMR. Mass Spec: 447 (M+l) (FAB). Calc'd.: 446.38. Rf=0.1 (20% EtOAc/hexane). EX AMPLE 17

Synthesis of 7-Propyl-4-methyl-4-aza-cholest-5-en-3-one. (20)

Following the general procedure of Example 7, compound

19. was analogously treated with methylamine hydrochloride and sodium acetate in ethylene glycol to yield the above-titled liquid product

20. The assigned structure was confirmed by proton NMR. Mass Spec. 442 (M+l) (FAB), Calc'd.: 441.74 C,H,N. Analysis: calculated for C H N O as 0.2 H2θ, MW=441.74; Calcd: C, 80.91 ; H, 11.63; N, 3.15.

Found: C, 81.00; H, 12.06; N, 2.93. Rf=0.3 (20% EtOAc/hexane).

EXAMPLE 18

Synthesis of 7-Propyl-4-methyl-4-aza-5 -cholestan-3-one. (21 )

Following the analogous general procedure of Example 8, compound 20 was catalytically hydrogenated in HOAc to yield the title liquid compound 2L Proton NMR confirmed the assigned structure. Mass spec. 444 (M+l) (FAB), C,H,N. Analysis: calculated for for C H N; Calcd: C, 81.19; H, 12.05; N, 3.16. MW=443.41.

Found: C, 80.78; H, 12.06; N, 3.22.

Rf=0.17 (20% EtOAc/hexane).

EXAMPLE 19

Synthesis of 7-Propyl-4-aza-cholest-5-en-3-one, (22)

Following the analogous procedure of Example 9, compound J_9 was treated with ammonium acetate in acetic acid to yield the titled compound, 22. Recrystallized from EtOAc/Et2θ to yield a white crystalline solid, mp. 91-94°C, C,H,N analysis as the 0.25 H2O hydrate: Calc'd MW 427.39. Calcd: C, 80.59; H, 11.54; N, 3.24.

Found: C, 80.59; H, 11.69; N, 3.36. Mass Spec. 428 (M+l).

EXAMPLE 20

Synthesis of 7-Propyl-4-aza-5 -cholestan-3-one. (23)

Following the analogous procedure described in Example 8, compound 22 was catalytically hydrogenated to yield the title compound 23, mp. 65-68°C.

Analysis: calculated for C,H,N, calc'd as 0.25 H2θ hydrate: Calcd: C, 80.21 ; H, 11.95; N, 3.23.

Found: C, 80.20; H, 12.14; N, 3.07.

Mass Spec. = 430 (M+l) calc'd MW 429.40. Rf=0.12 (20% EtOAc/hexane).

EXAMPLE 21

Synthesis of 7-Methyl-7-Hydroxy-cholesterol. (24)

Following the analogous Grignard procedure of Example 1 , cholesteryl acetate-7-one 1 was reacted with methyl magnesium bromide under standard Grignard conditions to yield title compound 24, a solid. NMR confirmed the assigned structure and mass spectral analysis confirmed the molecular weight.

EXAMPLE 22

Synthesis of 7-Methyl-Cholest-4.6-Dien-3-one. (25)

Following the analogous procedure of Example 2, the above Grignard product 24, was subjected to Oppenauer oxidation conditions to yield the title compound, 7β-methyl-cholest-4,6-dien-3- one, 25. EXAMPLE 23

Synthesis of 7β-methyl-cholest-5-en-3-one. (26)

Following the analogous procedure of Example 4 for the metal-ammonia reduction, 25 was similarly treated with lithium in ammonia/THF/toluene to yield title compound 26.

EXAMPLE 24

Synthesis of 7β-methyl-cholest-4-en-3-one (27).

Following the general isomerization procedure of Example 5 using DBU in THF, 26 was analogously treated to yield the title compound 27.

EXAMPLE 25

Synthesis of 7β-methyl-17-(2,6-Dimethylhexyl)-5-oxo-A-nor-3,5- secoandrostan-3-oic acid. (28)

Following the general procedure of Example 6 for the oxidative Ring A cleavage, compound 27 was analogous treated to yield the above titled seco-acid 28. The proton NMR confirmed the assigned structure.

EXAMPLE 26

Synthesis of 7-Methyl-4-aza-cholest-5-en-3-one. (29)

Following the general procedure of Example 9, compound 28 was analogously treated with ammonium chloride in acetic acid to yield the above-titled product 29. Mass Spectral m/z ion (FB) showed 400.2 (M+l) (M+2) calculated, 399. EXAMPLE 27

Synthesis of 7-Methyl-4-Aza-Cholestan-3-one. (30)

Following the analogous general procedure of Example 8, compound 29 was catalytically hydrogenated in HOAc to yield the title compound 30. Mass spectral analysis by El yielded m/z ion of 401 calculated 401.

EXAMPLE 28

Synthesis of 7-Methyl-4-methyl-4-Aza-Cholest-5-en-3-one. (31 )

The seco acid 28, was treated analogously as in Example 7 to give pure title compound H. Mass Spec. 414 (m+l) by FAB, calc'd., 413.

EXAMPLE 29

Synthesis of 7β-Methyl-4-methyl-4-aza-5 -cholestan-3-one. (32)

Following the general analogous procedure described in Example 8, H was catalytically hydrogenated to yield the titled compound, 32. Chromatography on silica gel with 30% EtOAc/hexane, eluant yielded pure product. Mass Spec. (El) 415, calc'd., 415.

EXAMPLE 30

Synthesis of 4-methyl-4-aza-cholest-5-en-3,7-dione. (34)

An oxidation procedure is carried out on 4-methyl-4-aza- cholest-5-en-3-one ϋ to yield the title compound, 34- (See USP 3,264,301 by Doorenboos and J. Org. Chem. 1961, Vol. 26, p.4548.) The compound ϋ was heated at 70°C with a mixture of pyridinium dichromate/t-butyl hydroperoxide in benzene over a 3-4 hour period to produce 34- EX AMPLE 31

Synthesis of 7β-Acetoxy-4-methyl-4-aza-5 -cholestan-3-one (37)

Compound 14 is hydrogenated by the analogous procedure of Example 8 to produce the 7-H analog 35_., and the 7β-ol, 16- Acylation of 16 with acetic anhydride, in the presence of pyridine, 4- dimethylaminopyridine in methylene chloride at 23°C for 24 hrs. produces the title compound 37.

EXAMPLE 32

Synthesis of 7-Beta Methyl-4-aza-5 -cholest-l -en-3-one (49)

To a solution of 280 mg. (0.698 mmol) of 10 in 4 milliliters toluene, was added 178.8 mg. DDQ, 0.7186 mg. BSTFA and 8.163 mg. triflic acid and the reaction contents allowed to stir at room temperature for 24 hours. Methyl acetoacetate, 8.1 mg., was added and the reaction refluxed for 24 hours. The contents were cooled, diluted with ethyl acetate, washed with aqueous sodium carbonate, aqueous sodium bisulfite, brine, dried over magnesium sulfate and concentrated to yield an oil. The crude compound was purified by preparative TLC on silica gel, eluting with 3:1 CHCl3/acetone to yield pure 49, whose proton NMR confirmed the assigned structure.

The following Table lists the unique proton NMR values (400 MHz in CDCI3) for each compound. The data are reported as: s = singlet, d = doublet, m = multiplet, J = coupling constant. The absorption values are given in delta (δ) scale with a reference point signal from tetramethylsilane, and are illustrated for the C-18, C-19 and C-21 angular ring methyl protons and protons associated with unique portions of the molecule. The numbering of the 4-aza steroid is given by the following structure:

TABLE Compound No. 18-CH^ 19-CH 21-CH Others

2 s 0.660 s 1.030 d 0.940 6Hs6.120

0.662 1.060 J=7 (values given for second isomer)

3 s 0.755 s 1.061 d 0.915 4H and 6H s 5.61, - -

J=7 5.97

4 sθ.720 si.110 dθ.930 4CH2m2.83-

J=7 3.28

5 s 0.730 s 1.12 d 0.930 4Hs5.74

J=7

6 sθ.66 s 0.963 d 0.894

J=7

7 s 0.692 s 0.977 d 0.908 N-CH3 s 3.153

J=7

8 - s 0.690 _ s 0.830 _ d 0.900 N-CH3 s 2.93

J=7

9 s 0.653 s 0.991 d 0.903 6Hd4.91J=4

J=7

10 s 0.675 s 0.808 d 0.893 5H, m, 2.97 -

J=7 3.13

11 sθ.66 sθ.90 d 0.915 allylic H m(5.8-

J=7 5.94) Compound No. 18-CHλ 19-CH 21 -Oft Others

12 sθ.78 s l.07 d 0.96 allylic H m(5.73-

J=7 5.85)

13 sθ.70 s 1.08 d 0.90 6H,s(5.23)

J=7

14 sθ.73 s 1.13 d 0.93 4Hs5.72

J=7

18 sθ.71 s 1.13 dθ.93 4Hs5.71

J=7

19 sθ.65 s 0.963 dθ.91

J=7

20 s 0.691 s 0.974 d 0.902 (6H) - d , 4.92

J=7 (J=4) (N-CH3) s 3.16

21 s 0.665 s 0.795 d 0.883

J=7 (N-CH3) s 2.92 5Hm (2.96-3.00)

22 s 0.680 s 1.01 d 0.890 (6H)d4.86

J=7 J=4

23 s 0.680 s 0.808 d 0.884 5Hm (3.0-3.1)

J=7

24 s, 0.68, s, 0.94, dθ.91 6H, s, 5.19, 5.21 0.69 1.04 J=7 Compound No. 18-CH 19-CH3 21-CH Others

25 s, 0.76 s, 1.07 d 0.92 4H, 6H 5.59, 5.92

J=7

27 s, 0.70 s, 1.15 d 0.92 7-CH3, d, 1.04,

J=7 J=6.5 4H, s, 5.71

28 s, 0.69 s, 1.12 d 0.92 7-CH3, d, 0.96,

J=7 J=6.5

29 s, 0.69 s, 1.04 d θ.91 7-CH3, d, 0.97,

J=7 J=6.5 6H, d, 4.59, J=3.0

30 s, 0.67 s, 0.835 d θ.91 7-CH3, d, 1.00, J=7 J=6.5 5H, dd,

J=3.3, 12.63

31 s, 0.69 s, 1.00 d θ.95 7-CH3, d, 1.05, J=7 J=6.5 6H, d, J=3.0

32 s, 0.68 s, 0.825 d 0.91, 7-CH3, d, 1.05,

J=7H J=6.5 4-CH3, s, 3.92

33 s, 0.69 s, 1.23 d 0.91 C6 - s, 5.42

J=7 N-CH3, s, 3.14

Mass Spec (EI)=413 No. 18-CHλ 19-CH 21-CHλ Others

49 s, 0.69 s, 0.90 d 0.915 C-7CH3, 1.02,

J=7 d, J=6, C-2, IH, 5.79, dd J=2.5 J=9.1

50 s, 0.62 s, 1.01 d 0.86 C-5, IH, 3.08, J=7 dd J= 3.87 J=12.9 C-7Ph, 5H, m,

7.1-7.3

51 s, 0.63 s, 1.02 d θ.8 C-5, 1-H, 3.2, dd J=7 J= 5.88 J=10.5

C-7Ph, 5H, m,

7.08-7.3

EXAMPLE 33

A lotion comprising the composition shown below may be prepared as follows.

Ingredient (weight %)

95% Ethanol 80.0

Compound of Formula I 3.0

Minoxidil 2.0 -Tocopheral acetate 0.01

Ethylene oxide (40 mole) adducts of hardened castor oil 0.5

Purified water 14.0 perfume and dye q.s. Into 95% ethanol are added a compound of Formula I, minoxidil, -tocopherol acetate, ethylene oxide (40 mole) adducts of hardened castor oil, perfume, and a dye, and the mixture is stirred and dissolved, followed by an addition of purified water, to obtain a liquid lotion.

EXAMPLE 34

An emulsion is prepared from A phase and B phase having the following compositions as follows.

(A phase) (weight %)

Whale wax 0.5

Cetanol 2.0

Petrolatum 5.0

Squalane 10.0

Polyoxyethylene (10 mole) mono stearate 2.0

Sorbitane monooleate 1.0

Compound of Formula I 0.01

Minoxidil 0.5

(B phase) (weisht %)

Glycerine 10.0

Purified water 68.5

Perfume, dye, and preservative q.s.

The A phase and the B phase are respectively heated and melted and maintained at 80°C, both phases are mixed to be emulsified, and are cooled under stirring to normal temperature to obtain an emulsion EXAMPLE 35

A cream is prepared from A phase and B phase having the following compositions.

(A phase) (weight %)

Fluid paraffin 5.0

Cetostearyl alcohol 5.5

Petrolatum 5.5

Glycerine monostearate 3.0

Polyoxyethylene (20 mole) 2-octyldodecyl ether 3.0

Propylparaben 0.3

(B phase) (weight %)

Compound of Formula I 0.8

Minoxidil 1.0

Glycerine 7.0

Dipropylene glycol 20.0

Polyethylene glycol 4000 5.0

Sodium Hexametaphosphate 0.005

Purified water 43.895

The A phase is heated and melted, and maintained at 70°C, the B phase is added to the A phase followed by stirring, and the obtained emulsion is cooled to obtain a cream. EXAMPLE 36

A hair liquid comprising the composition shown below may be prepared.

Ingredient (weight %)

Polyoxyethlene butyl ether 20.0

Ethanol 50.0

Compound of Formula I 1.0

Minoxidil 1.0

Propylene glycol 5.0

Polyoxyethylene hardened castor oil 0.4 derivative (ethylene oxide 80 mole adducts)

Perfume q.s.

Purified water q.s.

Into ethanol is added polyoxypropylene butyl ether, propylene glycol, polyoxyethylene hardened castor oil, a compound of Formula I, minoxidil, and perfume, which are mixed under stirring, and to the mixture is added purified water, to obtain a hair liquid.

EXAMPLE 37

A hair shampoo comprising the composition shown below may be prepared.

Ingredient (weieht %)

Sodium laurylsulfate 5.0

Triethanolamine laurylsulfate 5.0

Betaine lauryldimethylaminoacetate 6.0

Ethylene glycol distearate 2.0

Propylene glycol 5.0

Compound of Formula I 1.0

Minoxidil 2.0

Ethanol 2.0

Perfume 0.3

Purified water 71.7

Into 71.1 g of purified water is added 5.0 g of sodium laurylsulfate, 5.0 g of triethanolamine laurylsulfate, 6.0 g of betaine lauryldimethylamino acetate, then a mixture obtained by adding 1.0 g of a compound of Formula I, 2.0 g of minoxidil, 5.0 g of polyethylene glycol, and 2.0 g of ethylene glycol distearate to 2.0 g of ethanol, followed by stirring, and 0.3 g of perfume, is successively added, and the mixture is heated then cooled to obtain a hair shampoo.

Preparation of minoxidil topical compositions are disclosed in U.S. Patents 4,139,619 and 4,596,812, as examples of how to prepare topical compositions for any of the potassium channel openers.

BIOLOGICAL ASSAYS

Preparation of Human prostatic and scalp 5 -reductases

Samples of human tissue were pulverized using a freezer mill and homogenized in 40 mM potassium phosphate, pH 6.5, 5 mM magnesium sulfate, 25 mM potassium chloride, 1 mM phenylmethyl- sulfonyl fluoride, 1 mM dithiothreitol (DTT) containing 0.25 M sucrose using a Potter-El vehj em homogenizer. A crude nuclear pellet was prepared by centrifugation of the homogenate at 1,500 x g for 15 min. The crude nuclear pellet was washed two times and resuspended in two volumes of buffer. Glycerol was added to the resuspended pellet to a final concentration of 20%. The enzyme suspension was frozen in aliquots at -80°C. The prostatic and scalp reductases were stable for at least 4 months when stored under these conditions.

5 -reductase assay

The reaction mixture for the type 1 5α-reductase contained 40 mM potassium phosphate, pH 6.5, 5 mM [7-^H] -testosterone, 1 mM dithiothreitol and 500 μM NADPH in a final volume of 100 μl. The reaction mixture for the type 2 5α-reductase contained 40 mM sodium citrate, pH 5.5, 0.3 mM [7-^H] -testosterone, 1 mM dithiothreitol and 500 μM NADPH in a final volume of 100 μl. Typically, the assay was initiated by the addition of 50-100 μg prostatic homogenate or 75-200 μg scalp homogenate and incubated at 37°C. After 10-50 min the reaction was quenched by extraction with 250 μl of a mixture of 70% cyclohexane: 30% ethyl acetate containing 10 μg each DHT and T. The aqueous and organic layers were separated by centrifugation at 14,000 rpm in an Eppendorf microfuge. The organic layer was subjected to normal phase HPLC (10 cm Whatman partisil 5 silica column equilibrated in 1 ml/min 70% cyclohexane: 30% ethyl acetate; retention times: DHT, 6.8-7.2 min; androstanediol, 7.6-8.0 min; T, 9.1-9.7 min). The HPLC system consisted of a Waters Model 680 Gradient System equipped with a Hitachi Model 655 autosampler, Applied Biosystems Model 757 variable UV detector, and a Radiomatic Model A 120 radioactivity analyzer. The conversion of T to DHT was monitored using the radioactivity flow detector by mixing the HPLC effluent with one volume of Flo Scint 1 (Radiomatic). Under the conditions described, the production of DHT was linear for at least 25 min. The only steroids observed with the human prostate and scalp preparations were T, DHT and androstanediol. Inhibition studies

Compounds were dissolved in 100% ethanol. IC50 values represent the concentration of inhibitor required to decrease enzyme activity to 50% of the control. IC50 values were determined using a 6 point titration where the concentration of the inhibitor was varied from 0.1 to 1000 nM. Representative compounds of this invention were tested in the above described assay for 5α-reductase type 1 and type 2 inhibition.

Human Dermal Papilla Cell Assay

The dermal papilla is a small group of cells at the base of each hair follicle, and it is presently thought that these cells are stem cells involved in hair growth. These cells have been shown to have 5 alpha reductase activity, and it is therefore possible to test inhibitors of 5 alpha reductase in these cell culture systems.

Isolated and cultured dermal papilla cells are prepared according to the methods of Messenger, A.G., "The Culture of Dermal Papilla Cells From Human Hair Follicles", Br. J. Dermatol, 770:685- 689, 1984 and Itami, S. et al, "5 -Reductase Activity In Cultured Human Dermal Papilla Cells From Beard Compared With Reticular Dermal Fibroblast", J. Invest. Dermatol, 94:150-152, 1990. Beard dermal papilla cells and occipital scalp hair of two different individuals are used throughout the study. All experiments are performed at confluency after the fourth to sixth subculture. Confluent monolayers are rinsed twice with phosphate -buffered saline, scraped from dishes by rubber policemen, and collected into a centrifuge tube. The cell suspensions are centrifuged at 1 ,500 m for 10 min at 4°C. The pellets are resuspended in 20 mM Tris-HCl buffer, pH 7.5, at 4°C, containing 250 mM sucrose, 1 mM MgCl2, and 2mM CaCl2, by vortexing and 10 passes through a 25-gauge needle. The crude homogenate is further homogenized by a teflon-glass homogenizer, and is used as the cell homogenate. For the study of subcellular localization of 5 -reductase, the cell homogenate is centrifuged at 800 x g for 10 min to yield a crude nuclear pellet. The resultant supernatant is centrifuged at 10,000 x g for 15 min to produce a crude mitochondrial pellet. The supernatant is centrifuged at 100,000 x g for 60 min to yield a microsomal pellet and cytosol. Each particulate fraction is washed twice and resuspended in the buffer.

A standard incubation mixture will consist of 50 nM [3H]- testosterone, 1 mM NADPH, 100 mM sodium citrate, pH 5.5 or 100 mM Tris-HCl, pH 7.5, and 50 ml of the cell homogenate, in a final volume of 100 ml. Each tube contains 50-100 mg of cellular protein. Incubation is carried out at 37 °C for 30 min. During this incubation, the reaction is proportional to the time. For the study of optimum pH, citrate buffer is used at pH 4.5-6.5, and the Tris HCl buffer at pH 7.0- 9.0. The protein content is determined by the method of Lowry, et al, "Protein Measurement With The Folin Phenol Reagent" J. Biol. Chem., 193:265-275, 1951.

After incubation, the reaction is stopped by adding 4 times volume of chloroform-methanol (2/l:V/V) containing 110 mg each of carrier steroids. The extracted steroids are analyzed by thin-layer chromatographyl as previously described by Gomez, et al, "In Vitro Metabloism Of Testosterone-4-14c and D-androstene-3, 17-dione-4- 14C In Human Skin" Biochem., 7:24-32, 1968, and the purity of each steroid is determined by the recrystallization method. The activity of 5 -reductase is expressed by the sum of dihydrotestosterone, androstanediol and androstanedione formed. [1,2-3H] -testosterone (55.2 Ci/mmol) is obtainable from New England Nuclear Coφoration (Boston, MA) and unlabeled steroids can be purchased from Sigma Chemical Company (St. Louis, MO). Fetal calf serum is obtainable from Hazleton (Lenaxa, Kansas). All other chemicals are of reagent grade.

While the foregoing specification teaches the principles of the present invention, with examples provided for the pmpose of illustration, it will be understood that the practice of the invention encompasses all of the casual variations, adaptations, modifications, deletions, or additions of procedures and protocols described herein, as come within the scope of the following claims and its equivalents.

Claims

WHAT IS CLAIMED IS:

1. A method of treating patterned alopecia in humans in need of such treatment comprising:

(A) administering a therapeutically effective amount of a compound of structural Formula I:

I

or a stereoisomer or a pharmaceutically acceptable salt or ester thereof, wherein:

R is selected from hydrogen, methyl and ethyl; the dashed lines " - _^ι " a and b independently represent a single bond or a double bond providing that when b is a double bond, the 5oc hydrogen, Ha, is absent; =Z is selected from:

1 ) oxo,

2) α-hydrogen and a β-substituent selected from: a) C1 -C4 alkyl, b) C2-C4 alkenyl, c) CH2COOH, d) -OH, e) -COOH, f) -COO(Cl-C4 alkyl), g) -OCONR *R2 wherein R* and R^ independently are selected from: i) H, ii) C1-C4 alkyl, iii) phenyl, and iv) benzyl, or

R! and R^ together with the nitrogen atom to which they are attached represent a 5-6 membered saturated heterocycle, optionally containing one other heteratom selected from -0-, -S- and -N(R')- wherein R' is -H or methyl; h) C1-C4 alkoxy, i) C3-C6 cycloalkoxy, j) -OCOCH3, k) halo,

1) hydroxy -C1-C2 alkyl, m) halo-Cl-C2 alkyl, n) -CF3, and 0) C3-C6 cycloalkyl;

3) =CHR3; wherein R³ is selected from -H and C1-C4 alkyl; and

4) spirocyclopropane-R3 of structure:

and

(B) topically administering a therapeutically effective amount of a potassium channel opener.

2. The method according to Claim 1 wherein the potassium channel opener is selected from the group consisting of minoxidil, cromakalim, pinacidil, a compound selected from the classes of s-triazine, thiane-1 -oxide, benzopyran, or pyridinopyran derivatives, or a pharmaceutically acceptable salt thereof.

3. The method according to Claim 1 wherein the compound of Formula I is of the structure:

4. The method according to Claim 1 wherein the compound of Formula I is of the structure:

5. The method according to Claim 1 wherein the compound of Formula I is of the structure:

6. The method according to Claim 1 wherein the compound of Formula I is of the structure:

7. The method according to Claim 1 wherein =Z is α-hydrogen and the β-substituent is Cl -C4alkyl, or C2-C4alkenyl.

8. The method according to Claim 1 wherein the compound of Formula I is selected from the group consisting of: 7β-ethyl-4-methyl-4-aza-cholest-5-en-3-one, 7β-ethyl-4-methyl-4-aza-cholestane-3-one, 7β-ethyl-4-aza-5o -cholestan-3-one, 7β-carboxymethyl-4-aza-cholest-5-en-3-one, 7β-carboxymethyl-4-aza-cholestan-3-one, 7β-propyl-4-methyl-4-aza-cholest-5-en-3-one, 7β-propyl-4-methyl-4-aza-5α-cholestan-3-one, 7β-propyl-4-aza-5oc-cholestan-3-one, 7β-methyl-4-aza-cholest-5-en-3-one,

7 β -methy l-4-aza-cholestan-3 -one ,

4,7β-dimethyl-4-aza-cholest-5-en-3-one,

4,7β-dimethyl-4-aza-5α-cholestan-3-one,

4-methyl-4-aza-5α-cholestan-3,7-dione,

7β-acetoxy-4-methyl-4-aza-5oc-cholestan-3-one,

7β-hydroxy-4-methyl-4-aza-5α-cholestane-3-one,

7β-methoxy-4-methyl-4-aza-5α-cholestane-3-one,

7β-hydroxymethyl-4-aza-5α-cholestane-3-one,

7β-bromomethyl-4-aza-5α-cholestane-3-one, 7 β-chl oromethy 1 -4-aza-5α-cholestane-3 -one ,

7β-fluoromethyl-4-aza-5α-cholestane-3-one,

7β-carboxy-4-aza-5α-cholestane-3-one,

7β-trifluoromethyl-4-aza-cholest-5-en-3-one,

7β-methoxy-4-methyl-4-aza-cholesta-5-en-3-one,

7β-cyclopropyloxy-4-methyl-4-aza-5α-cholestane-3-one,

7β-propylidene-4-methyl-4-aza-5α-cholestane-3-one,

7β-(2-ethyl)spiroethylene-4-methyl-4-aza-5α-cholestane-3-one,

7β-methyl-4-aza-5α-cholest-l-en-3-one; and the pharmaceutically acceptable salts thereof.

9. The method of Claim 8 wherein the compound of Formula I is 4,7-dimethyl-4-aza-5α-cholestan-3-one or a pharmaceutically acceptable salt thereof.

10. The method of Claim 9 wherein the potassium channel opener is minoxidil or a pharmaceutically acceptable salt thereof.

11. The method of Claim 1 wherein the potassium channel opener is minoxidil or a pharmaceutically acceptable salt thereof.

12. The method of Claim 1 wherein the compound of Formula I is administered orally.

13. The method of Claim 12 wherein the dosage of the compound of Formula I is about 0.01 to 1000 mg per adult human per day.

14. The method of Claim 13 wherein the daily dosage of the compound of Formula I is about 0.01 to 10 mg per adult human per day.

15. The method of Claim 1 wherein the compound of Formula I is administered topically.

16.^* The method of Claim 15 wherein the compound of Formula I is applied topically in a pharmaceutical composition comprising a pharmaceutically acceptable vehicle adapted for topical application and about 0.001% to 15% by weight of the compound of Formula I.

17. The method of Claim 1 wherein the potassium channel opener is applied topically to the scalp in a pharmaceutical composition comprising a pharmaceutically acceptable vehicle adapted for topical application and about 1 to 5% by weight of the potassium channel opener.

18. The method of Claim 17 wherein the potassium channel opener is minoxidil or a pharmaceutically acceptable salt thereof.

19. A pharmaceutical composition for the treatment of patterned alopecia comprising a therapeutically effective amount of each of a potassium channel opener and a compound of structural Formula I as defined in Claim 1 , and a pharmaceutically acceptable vehicle adapted for topical application.

20. The composition of Claim 19 wherein the compound of Formula I is 4,7-dimethyl-4-aza-5α-cholestan-3-one or a pharmaceutically acceptable salt thereof.

21. The composition of Claim 19 wherein the potassium channel opener is minoxidil or a pharmaceutically acceptable salt thereof.

22. The composition of Claim 21 wherein the compound of Formula I is 4,7-dimethyl-4-aza-5α-cholestan-3-one or a pharmaceutically acceptable salt thereof.