Note: Descriptions are shown in the official language in which they were submitted.
W097/04002 ~ 2 ~ ~ ~ 2 7 3 PCT~P96/02830
EPIMERS OF (22RS)-N-(1,1,1-TRIFL~ORO-2 -~H I --~ Y~P~OP- 2-YL)-3-OXO-
4-AZA-5a-~NnRO-~T-1-ENE-17B-~RRO~MIDE
s The present invention relates to two epimers of formula (I) and
(II)
CF3
O ,NH--C{~
~"~1 CH3
~/ (I)
Od~N~
H
CH3
0~,NH--C--
~ CF3
O~N~
H
namely (22R) -N- (1,1,1-trifluoro-2-phenylprop-2-yl)-3-oxo-4-aza-
5a-androst-1-ene-17~-carboxamide [compound of formula (I)] and
(22S) -N- (1,1,1-trifluoro-2-phenylprop-2-y])-3-oxo-4-aza-5a-
androst-1-ene-17~-carboxamide [compound of formula (II)].
In the formulas of the present description the ~A~hP~ line
) indicates a substituent in a configuration, i.e.
below the plane of the ring, and the wedged line ( ~'~ )
indicates a substituent in B configuralion, i.e. above the
plane of the ring.
W097/04002 n 2 1 Q ~ 2 7 3 PCT~P96/02830 ~
Metabolites and metabolic precursors of the compounds of
formula (I) and tII) are within the scope of the present
lnvention.
The mixture of the epimers of formula (I) and (II) in ratio
1:1, namely the compound (22RS)-N-(1,1,1-trifluoro-2-
phenylprop-2-yl)-3-oxo-4-aza-5a-androst-1-ene-17~-carboxamide
(laboratory code FCE 28260), has already been described in our
previous International Patent Application WO 94/03475 (see
compound 41 at page 15 and Example 5, page 45), whereas the
o single epimer (22R) or (22S) has never been specifically
mentioned therein.
The epimer mixture proved to be a potent inhibitor of
testosterone 5a-reductase enzyme both in vitro and in vivo (see
data reported in Table (I), page 33 of WO 94/03475) and
15 therefore useful in those cases in which a reduction of
androgenic activity is desired, for example, in treating benign
prostatic hyperplasia, breast and prostate cancer and certain
skin-hair alterations, for example, in treating acne,
seborrhea, female hirsutism and male pattern baldness.
20 We have now separated the two epimers from their epimeric
mixture and evaluated their activity as inhibitors of
testosterone 5a-reductase enzyme.
The two epimers can be obt~;n~, for example, following the
method described in WO 94/03475 and subsequent separation of
25 the epimer mixture thereof. Said separation can be carried out,
for example, by means of high pressure liquid chromatography
(HPLC).
Alternatively, the single epimers can be obtained,
independently from each other, by reacting 3-oxo-4-aza-5a-
30 androst-1-ene-17~-carboxylic acid with each single enantiomer
of 1,1,1-trifluoro-2-phenylprop-2-yl amine previously
resolved.
W097/04002 n a 1 ~ ~ ~ 7 3 PCT~P96/02830
--3--
Additionally, as the corresponding epimeric amides obtained
reacting 3-oxo-androst-4-ene-17~-carboxylic acid with (+)-
- 1,1,1-trifluoro-2-phenylprop-2-yl amine are more easily
separable than the corresponding 3-oxo-4-aza-5a-androst-1-
5 ene-17~-carboxamide epimers, the single pure epimers can be
advantageously obtained by:
a) reacting 3-oxo-androst-4-ene-17~-carboxylic acid, or a
derivative thereof, with (+)-1,1,1-trifluoro-2-
phenylprop-2-yl amine;
o b) separating the two epimers of the so obtained (22RS)-N-
(1,1,1-trifluoro-2-phenylprop-2-yl)-3-oxo-androst-4-ene-
17~-carboxamide (e.g. by flash chromatography on silica
gel);
c) converting the so obt~ine~ epimers separately into the
final epimers of formulas (I) and (II), according to known
reactions.
This synthetic route is reported in the following reaction
Scheme I (the absolute configurations at C(22) are
2 0 tentative):
- . -
-
wo 97/04002 ~ 9 9 2 7 3 PcT/Erg6/02830
SCHEME I
OH
f~ r
~`~~~ d ~~J~/ e
0~ 0~
IIII) (IV)
~3 f, ~9t
~V) (V~ )
O (VIa) O (VIb)
Rf inf Rf sup
t a ~D +126 (c 1, abs. EtOH) [ ~ ]D ~187 (c 1, abs. EtOH)
d) NaOH, H2O, Dioxane / reflux 1 3he) (COCI)2, Toluene / RT / 3h
f) (~ )-PhCF3(CH3)CNH2, Py.iJine, CHCI3 / reflux / 5h
g) Cl.. ~ (G ,~ ~phic separation
h) KMnO4, NalO4, tBuOH / 35 40 C / 2h
WO 97/04002 PCT/EP96/02830
~ 0 2~ Q927 3
-5-
SCHEME I (cont'd)
N~ N~3
,, , ~ CF3 CH3 ~ CH3 ~$CF3
HO~ HOJ~
~ (VI I a ) ,~J (VI Ib)
[ a 1D +108 (C 1, abs. EtOH) [ a ]D +80 (C 1, abs. EtOH)
N~ N~
~ CF3 CH3 ~ CH3 CF3
O~N ~J (VIIIa) O~N'~J (VIIIb)
H t a ~D +48 (C 1, abs. EtOH) H [ a ~D -2 (c 1, abs. EtOH)
r : `~3~'';` 3
O N H (IXa) lNH ~l (IXb)
~ a ]D +12 (c 1, abs. EtOH) [ a ]D +62 (c 1, abs. EtOH)
m m
3 ~ , ," ,~
O N H Rfinf (FCE 29331) N H Msup (FCE 29330)
~ a ]D +46 (c 1, CHC13) [ a ]D +21 (c 1, CHC13)
i) NH3, (CH2OH)2 / -180 C / 2h
1) H2/PtO2 ~ AcOH / 40 - 50 psi / 45 - 50
m) (PhSeO)20, PhCI/ refluxl 5h
W097/04002 .~ 2 ~ 9 9 2 7 3 PCT~P96/02830 ~
--6--
As r-~orted hereinbelow, in vitro biological tests for
inhibition of human prostatic testosterone 5~-reductase
enzyme unexpectedly showed that one of the two epimers,
tested separately, is approximately two-fold more potent than
5 the other, while the epimeric mixture, as expected, has
intermediate activity.
Therefore, object of the present invention are the two epimers
of formula (I) and (II) as reported above, and their use as 5a-
reductase inhibitors.
o These epimers are useful in those cases in which a reduction of
androgenic activity is desired, for example, in the treatment
and/or chemoprevention of benign prostatic hyperplasia and
prostatic cancer. Moreover, these epimers can be used in the
treatment of breast cancer and certain skin-hair alterations,
15 for example, in the treatment of acne, seborrhea, female
hirsutism and male pattern baldness.
Furthermore, the present invention relates to pharmaceutical
compositions comprising one of the epimers of formula (I) or
(II), or a mixture thereof, wherein one of the epimers is
20 present in a prevailing amount with respect to the other
epimer, in combination with one or more pharmaceutically
acceptable carriers and/or diluents.
The pharmaceutical compositions cont~; n; ng the compounds of the
invention are usually prepared according to conventional
25 methods and are ~min; stered in a suitable pharmaceutical form,
such as, for example, one of those described in WO 94/03475.
The following examples further illustrate the invention.
ExamPle 1 _ _
(22RS)-N-(l,l,l-trifluoro-2-phenylprop-2-yl)-3-oxo-4-aza-5a-
androst-l-ene-17~-ca-~ox~mide
W097/04002 ~ ~ ~ 9 9 2 7 3 PCT~P96/02830
--7--
To a suspension of 3-oxo-4-aza-5a-androstane-17~-carboxylic
acid (7 g) in chloroform (100 mL), a solution of thionyl
- chloride (SOC12) (35 mL) in chloroform (15 mL) was added
dropwise, under nitrogen, over 30 minutes, keeping the
5 reaction mixture at about +3C in an ice-water bath. The
suspension was stirred at room temperature for 1 h, and then
the volatile compounds were evaporated under vacuum. A white
solid was obtained.
The solid was suspended in chloroform (330 mL) and the
o suspension was cooled to +3C; pyridine (1.78 mL) and then
(+)-1,1,1-trifluoro-2-phenylprop-2-yl amine (7.1 g) were
added and the reaction mixture heated to 60C for 4 h and
then stirred at room temperature overnight. The reaction
mixture was poured into an ammonium chloride saturated
aqueous solution (500 mL), the organic layer was separated
and the aqueous layer extracted with methylene chloride (2 x
100 mL). The combined organic extracts were washed with
water, dried over sodium sulfate and the solvent was removed
under vacuum. The crude was purified by flash chromatography
20 on silica gel (eluant: methylene chloride/acetone 70:30) to
yield 7.036 g of (22RS)-N-(1,1,1-trifluoro-2-phenylprop-2-
yl)-3-oxo-4-aza-5a-androst-1-ene-17~-carboxamide [(epimeric
ratio 50:50 by HPLC (column: Partisphere C8, 4.6 x 250 mm;
eluant: acetonitrile/water 50:50; flow: 1 mL/min; detector:
25 W at 210 nm?].
Example 2
Separation of the mixture into the single epimers by
preparative HPLC.
Apparatus: PrepLC/System 500 (Water Associates)
Columns: 2 PrepPAK-500/SILICA
W097/04002 ~ ~ ~ 9 9 ~ 7 3 PCT~P96/02830
Eluant: Toluene/Acetic acid/methanol 100:3:3
Flow: 150 mL/min
Detector: Refractive index
5 As the absolute configuration of the single epimers was not
established by X-ray cristallography, they were identified by
laboratory code numbers FCE 29330 for the first eluted epimer
(Rf sup.) and FCE 29331 for the second eluted compound (Rf
inf.).
FCE 29330
NMR (CDC13)~: 7.50-7.33 (m, SH, Ph), 6.78 (d, lH, H(l)), 5.87
(bs, lH, NH(21)), 5.82 (dd, lH, H(2)), 5.48 (bs, lH, NH(4)),
5 3.33 (dd, lH, H(5a)), 2.07 (s, 3H, C(CF3)CH3Ph), 0.98 (s,
3H, Me(l9)), 0.72 (s, 3H, Me(18)).
[a] D +46.7 (c 0.89, abs. EtOH)
FCE 29331
- --
NMR (CDCl3) ~: 7.50-7.33 (m,5H, Ph), 6.78 (d, lH, H(l)), 5.90
(bs, lH, NH(21)), 5.82 (dd, lH, H(2)), 5.48 (bs, lH, NH(4)),
3.33 (dd, lH, H(5a)), 2.04 (s, 3H, C(CF3)CH3Ph), 0.98 (s,
3H, Me(l9)), 0.68 (s, 3H, Me(18)).
[a]D +58.3o (c 0.061, abs. EtOH)
Example 3
Resolution of racemic (~ -trifluoro-2-phenylprop-2-yl
amine into the single enantiomers
The racemic (+)-l,l,l-trifluoro-2-phenylprop-2-yl amine was
treated with (+)-O,O'-dibenzoyl-D-tartaric acid; the
W097/04002 ~ ~ 1 9 ~ ~ 7 3 PCT~P96/02830
diastereomeric salts were crystallized from isopropanol.
After 5 crystallizations a partially resolved amine was
obtained, in which the (+)-enantiomer prevailed [enantiomeric
ratio: (+)/(-) = 88/12].
Analogously, after 5 crystallizations of the diastereomeric
salts of the racemic amine with (-)-O,O'-dibenzoyl-L-tartaric
acid from isopropanol, an amine enriched in the (-)-
enantiomer was obtained [enantiomeric ratio: (+)/(-)
o 12/88].
Example 4
Reacting 3-oxo-4-aza-5a-androstane-17~-carboxylic acid with
15 an amine enriched in the (+)-enantiomer tenantiomeric ratio
(+)/(-) = 88/12], a mixture of FCE 29331 and FCE 29330 was
obtained, in which the epimer ratio was FCE 29331/FCE 29330 =
88/12 (determined by HPLC).
Reacting 3-oxo-4-aza-5a-androstane-17~-carboxylic acid with
20 an amine enriched in the (-)-enantiomer [enantiomeric ratio
(+)/(-) = 12/88], a mixture of FCE 29331 and FCE 29330 was
obtained, in which the epimer ratio was FCE 29331/FCE 29330 =
12/88 (determined by HPLC).
ExamPle 5
(22RS)-N-(1,1,1-trifluoro-2-phenylprop-2-yl)-3-oxo-androst-4-
ene-17~-carboxamide (compound (VI))
To a suspension of 3-oxo-androst-4-ene-17~-carboxylic acid
(compound (IV)) (2.00 g) in toluene (28 mL) and pyridine
(0.640 mL), cooled to 10C, a solution of oxalyl chloride
(0.68 mL) in toluene (4 mL) was added over 10 minutes. The
W097/04002 Q 21 ~ 9 2 ~ 3 PCT~P96/02830 ~
-10-
coollng bath was removed and the mixture was stirred at room
temperature for I.5 h. The volatile compounds were removed
under reduced pressure at a temperature below 40C. The acyl
chloride so obtained (compound (V)) was dissolved in
5 chloroform (100 mL), cooled in an ice-water bath;
triethylamine (0.876 mL), and then a solution of (+)-1,1,1-
tr~~ uG~-2-phenylprop-2-yl amine (2.375 g) in chloroform (4
mL) were added. The reaction mixture was heated to reflux for
4 h. The reaction mixture was poured into a chilled saturated
0 sodium chloride aqueous solution of (200 mL) and extracted
with methylene chloride (3 x 100 mL); the organic extracts
were washed with water, dried over sodium sulfate and the
solvent was removed under reduced pressure. 2.150 g of the
title compound were obtained.
15 The two epimers were separated by flash chromatography on
silica gel (eluant: n-hexane/ethyl acetate 70:30) to yield
770 mg of the less retained (Rf sup.) epimer and 700 mg of
the most retained (Rf inf.) epimer.
20 Following an analogous procedure, using a (+)-
enantiomerically enriched amine [enantiomeric ratio (+)/(-) =
88/12) a final compound with the same epimeric ratio between
the Rf inf. and Rf sup. epimers was obtained. This fact shows
that the (+)-amine yields the Rf inf. epimer.
25 Analogously, using the (-)-enriched amine a final compound
enriched in the Rf sup. epimer was obtained.
Compound (VIb~ (Rf 9UP. )
NMR (CDC13) ~: 7.50-7.30 (m, 5H, Ph), 5.86 (bs, lH, NH(21)),
5.75 (m, lH, H(4)), 2.08 (s, 3H, C(CF3)CH3Ph), 1.20 (s, 3H,
Me(l9)), 0.76 (s, 3H, Me(18)).
ta]D +187 (c 1, abs. EtOH)
W097/04002 -11- PCT~P96/02830
Compound (VIa) (Rf inf . )
NMR (CDC13) ~: 7.50-7.30 (m, 5H, P~lJ, 5.90 (bs, lH, NH(21)),
5.75 (m, lH, H(4)), 2.04 (s, 3H, C(CF3)CH3Ph), 1.20 (s, 3H,
Me(19)), 0.72 (s, 3H, Me(18)).
5 [a]D +126 (c 1, abs. EtOH)
17~-[N-(1,1,1-trifluoro-2-phenylprop-2-yl)carbamoylJ-5-oxo-4-
nor-3,5-secoandrostan-3-oic acid (Rf in~.) (compound (VIIa)
To a solution of N-(1,1,1-trifluoro-2-phenylprop-2-yl)-3-oxo-
androst-4-ene-17~-carboxamide (Rf inf.) (compound (VIa))
(1.70 g; 3.486 mmol) in tert-butanol (40 mL) and 2M aqueous
sodium carbonate ~2.09 r,~), a 2~- po~assium permanganate
aqueous solution (1.8 mL) and a 0.75M sodium metaperiodate
15 aqueous solution (30 mL) were added dropwise
simultaneously,over about 5 minutes, at about 40C, at such a
rate that the colour of the reaction mixture remained always
pink. After stirring at 40C for 1 h and 15 minutes, the
reaction mixture was cooled to room temperature, filtered and
20 tert-butanol removed from the filtrate by evaporation under
vacuum (40 mL of solvent were collected). Then the solution
was cooled to about 0C, diluted with water, acidified with
lN hydrochloric acid and extracted with ethyl acetate (4 x 30
mL) and with methylene chloride (2 x 30 mL); the collected
25 organic extracts were washed with water (2 x 30 mL), brine
(20 mL) and anhydrified over sodium sulfate. Evaporation of
the solvent yielded a solid foam, that was purified by flash
J chromatography (eluant- n-hexane/ethy] acetate 50:50) to
yield 1.656 g of white solid compound.
NMR (CDCl3) ~: 7.30-7.50 (m, 5H, Ph), 5.90 (bs, lH, NH), 2.04
(s, 3H, C(CF3)CH3Ph), 1.12 (s, 3H, Me(19)), 0.71 (s, 3H,
Me(18)).
W097/04002 PCT~P96/02830
~ a 1 ~ 3 -12-
MS (~AB ) (m/z): 508 [M+H] , 490 [M-H2O+H]+, 336 [M-
C(CF~)CH3Ph+ 2H]+, 173 PhCH3(CF3)C
[~]D +108.2 (c 1, abs. Ethanol)
5 Following an analogous procedure starting from N-(1,1,1-
trifluoro-2-phenylprop-2-yl)-3-oxo-androst-4-ene-17~-
carboxamide (Rf SUp. ) (compound (VIb)), the epimeric compound
was obtained (compound VIIb):
NMR (CDC13) ~: 7.30-7.50 (m, 5H, Ph), 5.88 (bs, lH, NH), 2.08
o (s, 3H, C(CF3)CH3Ph), 1.14 (s, 3H, Me(19)), 0.78 (s, 3H,
Me(18)).
[~] D +80 (c 1, abs. Ethanol)
N-(1,1,1-trifluoro-2-phenylprop-2-yl)-3-oxo-4-aza-androst-5-
5 ene-17~-c~ o~Amide (Rf inf.) (compound (VIIIa))
A suspension of 17~-[N-(1,1,1-trifluoro-2-phenylprop-2-
yl)carbamoyl]-5-oxo-4-nor-3,s-secoandrostan-3-oic acid (Rf
inf.) (compound (VIIa)) (1.540 g) in anhydrous ethylene
20 glycol (35 mL) was saturated at 0C with anhydrous gaseous
ammonia: the secoacid dissolved completely. The solution so
obtained was heated slowly to 180C over 1 hour and 10
minutes and maintained at this temperature for 20 minutes.
Then the temperature was allowed to reach room temperature
25 over 0.5 hour. The yellowish solution was cooled to about 0C
under good stirring: the final compound began to
precipitate. After diluting with water (30 mL) the stirring
was continued for 0.5 hour at 0C and the precipitate was
filtered and washed with water. 1.36 g of a pale brownish
solid were obtained, which was purified by flash
chromatography on silica gel (eluant: n-hexane/ethyl acetate
W097/04002 l3 PCT~P96/02830
50:50), yielded 1.090 g of the title compound.
NMR (CDC13) ~: 7.50-7.30 (m, 5H, Ph), 5.88 (bs, lH, NH), 4.81
(m, lH, H(6)), 2.06 (s, 3H, C(CF3)~H3Ph), 1.12 (s, 3H,
5 Me(19)), 0.71 (s, 3H, Me(18)).
[a] D +48.5 (c 1, abs. Ethanol)
Following an analogous procedure starting from 17~-[N-(1,1,1-
trifluoro-2-phenylprop-2-yl)carbamoyl]-5-oxo-4-nor-3,5-
o secoandrostan-3-oic acid (Rf sup.) (compound (VIIb)) the
epimeric compound was obtained (compound (VIIIb)):
NMR (CDCl3) ~: 7.50-7.30 (m, 5H, Ph), 5.88 (bs, lH, NH), 4.78
(m, lH, H(6)), 2.09 (s, 3H, C(CF3)CH3Ph), 1.12 (s, 3H,
Me(19)), 0.74 (s, 3H, Me(18)).
5 [a] D -2 (c 1, abs. Ethanol)
N-(1,1,1-trifluoro-2-phenylprop-2-yl)-3-oxo-4-aza-5a-
androstane-17~-c~hoY~mide (Rf inf.) (co~pound (IXa))
20 A solution of N-(1,1,1-trifluoro-2-phenylprop-2-yl)-3-oxo-4-
aza-androst-5-ene-17~-carboxamide (Rf inf.) (compound
(VIIIa)) (700 mg) in glacial acetic acid (45 mL) was
hydrogenated in the presence of PtO2 (Adams' catalyst) (140
mg) under a pressure of 45 psi of hydrogen at 45C for lh.
25 The reaction mixture was cooled to room temperature, the
- catalyst was filtered off and the solvent removed under
reduced pressure. The residue was taken up with methylene
chloride, washed with lN sulfuric acid, with brine, with
aqueous sodium carbonate, with brine, with water, dried over
30 sodium sulfate and the solvent was removed under vacuum. The
crude solid so obtained was purified by flash chromatography
W097/04002 PCT~P96/02830
~i~ 9927 3 -14-
on silica gel (eluant: toluene/ethyl acetate/methanol
75:20:5) to yield 420 mg of the title compound.
NMR (CDC13) ~: 7.50-7.30 (m, 5H, Ph), 5.88 (bs, lH, NH(21)),
5 5.55 (bs, lH, NH(4)), 3.06 (dd, lH, H(5a)), 2.05 (s, 3H,
C(CF3)CH3Ph), 0.92 (s, 3H, Me(l9)), 0.68 (s, 3H, Me(18)).
[a] D +12 (c 1, abs. Ethanol)
Following an analogous procedure starting from N-(l,l,l-
o trifluoro-2-phenylprop-2-yl)-3-oxo-4-aza-5a-androst-5-ene-
17~-carboxamide (R~ sup.) (compound (VIIIb)) the epimeric
compound was obtained (compound (IXb)):
NMR (CDCl3) ~: 7.50-7.30 (m, sH, Ph), 5.88 (bs, lH, NH(21)),
5.55 (bs, lH, NH(4)), 3.06 (dd, lH, H(5a)), 2.05 (s, 3H,
15 C(CF3)CH3Ph), 0.92 (s, 3H, Me(l9)), 0.71 (s, 3H, Me(18)).
[a] D +62 (c 1, abs. Ethanol)
N-(l,l,l-trifluoro-2-phenylprop-2-yl)-3-oxo-4-aza-5a-androst-
1-ene-17~-carboxamide (R~ inf.) (FCE 29331)
To a solution of N-(l,l,l-trifluoro-2-phenylprop-2-yl)-3-
oxo-4 aza-5a-androstane-17~-carboxamide (Rf inf.) (compound
(IXa)) (106 mg) in chlorobenzene (5 mL) phenylseleninic
anhydride (108.3 mg) was added. The solution was refluxed for
25 5 h, while water was removed by a Marcusson device. The
solution was evaporated and the residue dissolved in
methylene chloride, washed with aqueous sodium carbonate,
saturated sodium chloride solution, water and dried over
sodium sulfate. After evaporating the solvent, the crude was
purified by flash chromatography (eluant: toluene/ethyl
acetate/methanol 75:20:5) to yield 70 mg of the title
W097/04002 15- PCT~P96/02830
compound.
NMR (CDC13) ~: 7.50-7.33 (m, 5H, Ph), 6.78 (d, lH, H(l)),
5.90 (bs, lH, NH(21)), 5.82 (dd, lH, H(2)), 5.48 (bs, lH,
5 NH(4)), 3.33 (dd, lH, H(5a)), 2.04 (s, 3H, C(CF3)CH3Ph),
0.98 (s, 3H, Me(l9)), 0.68 (s, 3H, Me(18)).
[a] D +46 (c 1, CHCl3)
Following an analogous procedure starting from N-(l,l,l-
trifluoro-2-phenylprop-2-yl)-3-oxo-4-aza-5a-androstane-17~-
carboxamide (Rf sup.) (compound (IXb)) the epimeric compoundFCE 29330 was obtained:
NMR (CDCl3) ~: 7.50-7.33 (m, 5H, Ph), 6.78 (d, lH, H(l)),
5.87 (bs, lH, NH(21)), 5.82 (dd, lH, H(2)), 5.48 (bs, lH,
15 NH(4)), 3.33 (dd, lH, H(5a)), 2.07 (s, 3H, C(CF3)CH3Ph),
0.98 (s, 3H, Me(l9)), 0.72 (s, 3H, Me(18)).
[a] D +21 (c 1, CHCl3)
As shown by this example, FCE 29331 was obtained from (+)-
amine, wherease FCE 29330 was obtained from (-)-amine.
IN VITRO ASSAY OF 5-R~u~ASE IN~IBITION
Inhibition of 5a-reductase was evaluated using the particulate
25 fraction from homogenates of hyperplastic human prostates as
the enzyme source. The particulate fraction was prepared
centrifuging prostate homogenates at 140,000 x g. The resulting
pellet, washed several ~imes, was resuspended in buffer and
stored at -80C. in aliquots cont~; ni ng ~ 10 mg protein/ml.
30 The assay for 5a-reductase was done in a final volume of 0.5
ml, in 40 mM TRIS-HCl buffer pH 5.5, cont~;ning 1 mM
W097/04002 ~ a 7 3 PCT~P96/02830
-16-
dithiothreitol, 5 mM NADPH, 1 ~M [14C] testosterone, an aliquot
of the enzyme preparation and various concentrations of the
inhibitors. After 30 min. incubation at 37C, the reaction was
terminated by addition of 2 ml cold diethyl ether and the
5 organic phase was separated, evaporated under N2 and
resuspended in ethyl acetate. Testosterone metabolites in this
extract were separated in TLC on silica gel F 254 plates
(Merck), using chloroform, acetone and n-hP~n~ (2:1:2) as
developing solvent system.
o Radioactivity on the plate was sc~nne~ and analyzed from
quantitative plots printed by a TLC-analyzer (Berthold). The
fractional 5a-reduction of testosterone was calculated by
relating the 14C-radioactivity in the 5a-reduced metabolites
(5a-dihydrotestosterone, 3a- and 3~-androst~n~;ols) regions to
15 the total radioactivity in the testosterone and 5a-reduced
metabolites regions. The concentration of each compound
required to reduce control 5a-reductase activity by 50~ (IC50)
was determined by plotting ~ inhibition versus log of inhibitor
concentration.
20 The results obtained on the single epimers FCE 29330 and FCE
29331, and on the racemic mixture FCE 28260 are reported in the
following Table 1.
Table 1
In vitro inhibition of human prostatic 5a-reductase
by stereoicomers of FCE 28260
Co...~o~-d IC50 (DM)
FCE 28260 12
FCE 29330 17
FCE 29331 8
W097/04002 9 ~ a 9 3 PCT~P96/02830
-17-
From the results reported in Table 1 it is evident that the
epimer FCE 29331 is approximately two-fold more potent than the
other epimer FCE 29330. The mixture FCE 28260, as expected,
shows intermediate activity.
., .
