Synthesis and biological evaluation of 4-(4-(alkyl-and phenylaminocarbonyl)benzoyl)benzoic acid derivatives as non-steroidal inhibitors of steroid 5α-reductase isozymes 1 and 2

Article abstract of DOI:10.1002/1521-4184(200203)335:2/3<83::AID-ARDP83>3.0.CO;2-3

The synthesis and biological evaluation of 4-(4-(alkyl- and phenylaminocarbonyl)benzoyl)benzoic acids (4a-4d) as non-steroidal inhibitors of steroid 5α-reductase are described. The compounds were tested in vitro for inhibitory activity toward rat and huma

Full text of DOI:10.1002/1521-4184(200203)335:2/3<83::AID-ARDP83>3.0.CO;2-3

Arch. Pharm. Pharm. Med. Chem. 2002, 2, 83–88
Non-steroidal 5α-Reductase Inhibitors 83
Ola I. A. Salem,
Tobias Schulz,
Rolf W. Hartmann
Synthesis and Biological Evaluation of 4-(4-(Alkyl-
and Phenylaminocarbonyl)benzoyl)benzoic Acid
Derivatives as Non-steroidal Inhibitors of Steroid
5␣-Reductase Isozymes 1 and 2
8.5 Pharmaceutical and
Medicinal Chemistry,
Saarland University,
P.O. Box 15 11 50,
D-66041 Saarbrücken,
Germany
The synthesis and biological evaluation of 4-(4-(alkyl- and phenylaminocarbonyl)-
benzoyl)benzoic acids (4a–4d) as non-steroidal inhibitors of steroid 5α-reductase
are described.The compounds were tested in vitro for inhibitory activity toward rat
and human 5α-reductase isozymes 1 and 2 at a concentration of 10 µM.The most
active inhibitor for the human type 2 isozyme was 4-(4-(phenylaminocarbonyl)ben-
zoyl)benzoic acid, compound 4c (IC50 = 0.82 µM).
Keywords: Non-steroidal 5α-reductase inhibitors; Substituted benzoylbenzoic
acids; Rat and human steroid 5α-reductase isozymes 1 and 2; Benign prostatic
hyperplasia; Prostate cancer
Received: August 1, 2001 [FP615]
nyl-4-carboxylic acid derivatives with alkyl- or arylamino-
carbonyl substituents show fairly strong inhibitory activi-
Introduction
Dihydrotestosterone (DHT) is the most potent androgen,
and is formed as a metabolite of the predominant circu-
lating androgen testosterone (T). Steroid 5α-reductase
catalyzes the conversion of T to DHT. Benign prostatic
hyperplasia (BPH) is a disease resulting in urinary dys-
function which is assumed to be mediated by DHT [1].
The reduction of DHT concentration by inhibition of 5α-
reductase can lead to a partial remission of BPH and
thus can improve the disease. DHT is also implicated in
the etiology of prostate cancer [2], and several disorders
of the skin including acne [3], androgenic alopecia [4],
and hirsutism [5].Steroidal inhibitors have demonstrated
clinical efficacy for the treatment of BPH, among them fi-
nasteride (Proscar®) [6] or epristeride [7] (Chart 1).How-
ever, unwanted side effects are likely to occur [8], as
most steroidal drugs show such activities. On the other
hand, non-steroidal inhibitors have not yet shown clinical
efficacy for the treatment of BPH. The discovery of two
isozymes (type 1 and type 2) and the determination of
their tissue distribution have led to the prospect of devel-
oping more specific drugs [9]. An effective dual inhibitor
of type 1 and type 2 human 5α-reductase may lower cir-
culating DHT to a greater extent than finasteride and
thus may show advantages in the treatment of BPH and
other diseases that depend on DHT [10]. A class of
benzophenone carboxylic acids with potent 5α-reduc-
tase type 2 isozyme specific inhibitory activity has been
described [11] and we recently found that some biphe-
ty against human 5α-reductase type 2, and moderate in-
hibitory activity toward human type 1 enzyme [12]. In or-
der to find more potent inhibitors, we introduced those
alkyl- or arylaminocarbonyl groups which are also
present in the 17β-position of most steroidal inhibitors in-
to the flexible benzophenone skeleton. In the following
we describe the synthesis of compounds 4a–4d and 6
(Chart 1) and the evaluation of their inhibitory activity to-
ward human and rat 5α-reductase isozymes 1 and 2.
Chart 1. Inhibitors of 5α-reductase and title compounds.
Synthesis
Correspondence: Rolf W. Hartmann, 8.5 Pharmaceutical and
Medicinal Chemistry, Saarland University, P.O. Box 15 11 50,
D-66041 Saarbrücken, Germany.Phone:+49 (0)681 302 3424,
Fax: +49 (0)681 302 4386, e-mail: rwh@mx.uni-saarland.de.
Monomethyl terephthalate was the precursor for the
synthesis of the benzoylbenzoic acids 4a–4d and 6
© WILEY-VCH Verlag GmbH, 69451 Weinheim, 2002
0365-6233/02–03/0083 $ 17.50+.50/0

84 Salem et al.
Arch. Pharm. Pharm. Med. Chem. 2002, 335, 83–88
ence of triethyl-amine as acid scavenger [14] in refluxing
acetonitrile. The same procedure delivered the amides
3b–3d, but only in poor yields.To improve the overall effi-
ciency, changing to a two-step procedure gave the corre-
sponding amides 3b–3d in appreciably higher yields:
using oxalyl chloride to transform the acid 2 to the acid
chloride and treating the crude reaction mixture with an
excess of the appropriate amine. For the synthesis of
compound 6 [11], we followed a new route. 4-(4-(Phen-
oxybenzoyl)benzoic acid methyl ester 5 was synthesized
by Friedel-Crafts acylation of diphenyl ether with the acid
chloride of monomethyl terephthalate in carbon disulfide
using AlCl3 as a catalyst in analogy to the two-step pro-
cedure of Kipper [15]. Saponification of the synthesized
esters 3a–3d and 5 using potassium carbonate in re-
fluxing methanol-water afforded the corresponding car-
boxylic acids 4a–4d and 6 in high yields.
Results and discussion
The inhibitory activity of compounds 4a–4d, 6 [11] and
finasteride as a reference was determined in vitro at a
concentration of 10 µM using human prostate homoge-
nate (BPH tissue for type 2 isozyme) and the DU 145 cell
line (for human type 1 enzyme) and rat prostate homoge-
nates (pH 6.6, type 1; pH 5.5, type 2). In case of potent
inhibition IC50 values were determined. As can be seen
in Table 1, all non-steroidal compounds were less active
than finasteride. For the human type 2 isozyme, com-
pounds with an aliphatic (tert-butyl, di-iso-propyl, and di-
cyclohexyl) aminocarbonyl substituent displayed weak
to moderate inhibitory activity (13–55%), while com-
pound 4c with a phenylaminocarbonyl substituent
showed significantly higher inhibitory activity (86% inhi-
bition; IC50 = 0.82 µM) indicating that an aromatic ring in
this position is important for potency. However, the de-
scribed compound 6 (IC50 = 0.053 µM) still is more potent
(15.5 times). All compounds which are active in human
type 2 isozyme are active in rat isozyme 2 as well. How-
ever, compounds 4c and 6 do not reach the activity
shown in the human enzyme. In the human type 1 iso-
zyme (DU 145 cell line), all compounds were inactive or
rather poor inhibitors, while in the case of rat prostatic
homogenate isozyme type 1 compounds 4c and 4d dis-
played moderate activity (59% and 66% inhibition, re-
spectively). It cannot be excluded that the weak inhibito-
ry activity in intact cells (DU 145) is due to an insufficient
permeability across the cell membrane. Further experi-
ments regarding this issue are in progress. As a conclu-
sion, we have presented in this paper a new class of sub-
stituted benzoylbenzoic acids as 5α-reductase inhibi-
tors. One of these derivatives, compound 4c, might be
further optimized to become a lead for more potent non-
steroidal 5α-reductase inhibitors. Further structural
modifications in this class of compounds are ongoing.
(a) i) SOCl2 ii) AlCl3, toluene, 25°C;
(b) CrO3, HOAc, H2SO4;
(c) 2-Chloro-1-methylpyridinium iodide, NEt3, tert-
butylamine, reflux;
(d) Oxalyl chloride, then amine, 25°C;
(e) K2CO3, methanol – water (9/1), reflux;
Scheme 1. Synthesis of 4a–4d.
a) i) (COCl)2 ii) AlCl3, diphenylether, CS2, reflux;
(b) K2CO3, methanol – water (9/1), reflux
Scheme 2. Synthesis of 5 and 6.
(Schemes 1 and 2). In analogy to the procedure of
Schäfer et al. [13], Friedel-Crafts acylation of toluene
with the acid chloride of monomethyl terephthalate using
AlCl3 as a catalyst afforded the benzoyl benzoic acid
methyl ester derivative 1 in good yield. Oxidation of the
methyl group to the corresponding carboxylic acid was
achieved using a chromic oxide/sulfuric acid/acetic acid
mixture to give the 4,4´-benzophenone dicarboxylic acid
monomethyl ester 2. The tert-butylamide derivative 3a
was synthesized in one step using the Mukaiyama rea-
gent (2-chloro-1-methylpyridinium iodide) in the pres-

Arch. Pharm. Pharm. Med. Chem. 2002, 335, 83–88
Non-steroidal 5α-Reductase Inhibitors 85
Table 1. In vitro inhibition of human and rat steroid 5α-reductase isoenzymes type 1 and type 2 by compounds 4a–4d, 6
and finasteride.
4a–4d
6
Compd.
No.
R, R´
Human
% Inhibition at 10 µM,
(IC50, µM)
RVPc
% Inhibition at 10 µM,
(IC50, µM)
Type 2a,d,g
Type 1b,g
Type 2d,e,g
Type 1d,f,g
4a
4b
4c
4d
6
H, tert-butyl
di-iso-propyl
H, phenyl
45
13
n.i.
n.i.
n.i.
10
n.i.
11
5
68
69
33
13
n.i.
59
66
53
86 (0.82)
55
dicyclohexyl
82h (0.053)
(2–3 nM)
finasteride
(0.043)
(0.011)
(0.01)
a
Human prostate homogenate; 200–300 µg protein per incubation, pH 5.5.
b
c
d
e
g
3
Human prostatic carcinoma (DU 145 cell line) expressing type 1 isozyme; Substrate H-androstenedione 5 nM.
Rat ventral prostate; 200–250 µg protein per incubation.
Substrate 1β,2β-3H-testosterone 210 nM.
f
pH 5.5.
pH 6.6.
Mean value;tests have been run in duplicate two times;standard deviation for IC50 values <20%; standard deviation
for percent inhibition <10%. n.i. no inhibition (percent inhibition <5%).
Percent inhibition at 0.4 µM.
h
IR: νmax. = 2960, 1720, 1645, 1610, 1280, 1110 cm–1. 1H-NMR
Acknowledgements
(CDCl3): δ = 2.45 (s, 3H, Ar-CH3), 3.96 (s, 3H, OCH3), 7.29 &
Thanks are due to the Fonds der Chemischen Industrie,
who supported this work by a grant.We thank Mrs. Anja
Palusczak for her help in performing the biological as-
says.
7.71 (d, 4H, J = 8.2 Hz, Ar-H), 7.81 & 8.13 (d, 4H, J = 8.4 Hz, Ar-
H).
4,4´-Benzophenone dicarboxylic acid monomethyl ester (2)
To a precooled mixture of 1 (254 mg, 1.00 mmol) and glacial
acetic acid (1.90 mL), sulfuric acid (0.15 mL) was added slowly
with stirring.The reaction mixture was cooled to 5°C and chro-
mic oxide (0.30 g, 3.00 mmol) was added in small portions at
such a rate that the temperature did not rise above 10°C. The
reaction mixture was stirred at 5–10°C for 2 hours, then at room
temperature overnight. The reaction mixture was poured onto
ice and filtered.The crude acid was purified by recrystallization
from ethyl acetate-methanol to yield 210 mg (74%) of the title
compound as white crystals. Mp 284–285°C. IR: νmax. = 3000,
1730, 1700, 1680, 1650, 1290, 1110, 940, 880 cm–1. 1H-NMR
(d6-DMSO): δ = 3.91 (s, 3H, OCH3), 7.85 & 7.87 (d, 4H, J =
8.4 Hz, Ar-H), 8.10 & 8.12 (d, 4H, J = 8.0 Hz, Ar-H), 13.26 (s,
1H, OH).
Experimental section
Solvents for reactions under anhydrous conditions were dried
according to standard procedures. All reactions, except those
involving water as a reagent, were conducted under nitrogen
atmosphere.Melting points were determined on a Kofler micro-
scope (Reichert, Austria) and are uncorrected. Mass spectra
(EI, 70 eV) were measured on a HP G1800A GCD. 1H-NMR
spectra were measured on a Bruker AM-400 (400 MHz) and
are consistent with the assigned structures.Chemical shifts are
given in parts per million relative to TMS as an internal stand-
ard. IR spectra were measured as KBr discs on a Perkin-Elmer
infrared spectrophotometer 398.
4-(4-(tert-Butylaminocarbonyl)benzoyl)benzoic acid methyl es-
4-(4-Methylbenzoyl)benzoic acid methyl ester (1)
ter (3a)
The title compound was synthesized in analogy to the two-step
procedure of Schäfer et al. [13] starting from monomethyl
terephthalate. Mp 127-28°C (Literature [13], 122–124°C).
A mixture of 2 (284 mg, 1.00 mmol), 2-chloro-1-methylpyridin-
ium iodide (260 mg, 1.00 mmol), triethylamine (200 mg,
2.00 mmol) and tert-butylamine (90.0 mg, 1.20 mmol) in an-

86 Salem et al.
Arch. Pharm. Pharm. Med. Chem. 2002, 335, 83–88
disulfide (50 mL) at ambient temperature. The mixture was re-
fluxed for 1 h, and then poured onto ice. The product was ex-
tracted with chloroform (4 × 50 mL).The combined organic lay-
ers were washed with water and brine and dried over MgSO4.
After evaporation of the solvent the residual solid was purified
by recrystallization from hexane-ethyl acetate. Yield 1.20 g
(72%) of white crystals. Mp 167–168°C. IR: νmax. = 2920, 1715,
1635, 1600, 1580, 1480, 1430, 1300, 1250, 1160, 1100, 930,
840, 750, 690 cm–1. 1H-NMR (CDCl3): δ = 3.96 (s, 3H, OCH3),
7.03 (d, 2H, J = 8.8 Hz, Ar-H), 7.08–7.42 (m, 5H, Ar-H), 7.79–
7.82 (m, 4H, Ar-H), 8.13 (d, 2H, J = 8.4 Hz, Ar-H).
hydrous acetonitrile (30 mL) was refluxed for 2 hours. After
cooling, dichloromethane (50 mL) was added and the organic
layer was washed once with hydrochloric acid (10%), then with
a saturated solution of sodium bicarbonate, water and brine.
The organic layer was dried over MgSO4, filtered and evaporat-
ed under reduced pressure to give 200 mg (59%) of 3a. Re-
crystallization from hexane-ethyl acetate yielded white crys-
tals. Mp 161–162°C. IR: νmax. = 3340, 2980, 1720, 1660, 1640,
1540, 1285, 1100, 880, 725 cm–1. 1H-NMR (CDCl3): δ = 1.50 (s,
9H, C(CH3)3), 3.97 (s, 3H, OCH3), 5.98 (s, 1H, NH), 7.81–7.83
(m, 6H, Ar-H), 8.15 (d, 2H, J = 8.4 Hz, Ar-H).MS (153°C):m/z =
339 (M+, 44%), 267 (100%), 284 (44%).
General procedure for the synthesis of carboxylic acids 4a–4d
and 6
General procedure for the synthesis of 3b–3d
4-(4-(Diisopropylaminocarbonyl)benzoyl)benzoic acid methyl
ester (3b)
A mixture of the ester 3a–3d (1.00 mmol) and K2CO3
(3.00 mmol) in methanol/water (9:1) was refluxed for 1 h. The
solution was cooled, diluted with water and acidified with hydro-
chloric acid (10%) to pH 5. The precipitated acid was filtered,
washed thoroughly with water and dried. The crude product
was purified by recrystallization from the solvent indicated in
the individual procedures.
To a suspension of 2 (568 mg, 2.00 mmol) in anhydrous dichlo-
romethane (20 mL) and a few drops of anhydrous dimethyl-
formamide, oxalyl chloride (0.19 mL, 2.20 mmol) was added
carefully.After the effervescence had ceased, the reaction mix-
ture was stirred for further 2 h at room temperature.The solvent
and unreacted oxalyl chloride were evaporated under reduced
pressure. The residual acid chloride was dissolved in anhy-
drous dichloromethane (10 mL) and added dropwise to a solu-
tion of di-iso-propylamine (510 mg, 5.00 mmol) in dichlo-
romethane (20 mL).The mixture was stirred for 1 h at ambient
temperature. Ethyl acetate (50 mL) was added and the organic
layer was washed successively with hydrochloric acid (10%),
saturated sodium bicarbonate solution, water and brine. The
organic layer was dried over MgSO4.The solvent was evaporat-
ed under reduced pressure and the solid obtained was recrys-
tallized from hexane-ethyl acetate to give 400 mg (54%) of 3b
as white crystals. Mp 153–154°C. IR: νmax. = 2945, 1710, 1640,
1620, 1430, 1270, 1100, 920, 840 cm–1. 1H-NMR (CDCl3): δ =
1.17 & 1.59 (2 br s, 12H, CH(CH3)2), 3.97 (s, 3H, OCH3), 7.43 &
7.82 (d, 4H, J = 8.0 Hz, Ar-H), 7.84 & 8.16 (d, 4H, J = 8.0 Hz, Ar-
H), MS (145°C):m/z = 367 (M+, 19%), 267 (100%), 324 (45%).
4-(4-(tert-Butylaminocarbonyl)benzoyl)benzoic acid (4a)
Recrystallized from ethyl acetate, yield: 92%. Mp 232–233°C.
IR: νmax. = 3320, 3040, 2960, 1680, 1650, 1540, 1410, 1260,
1210, 1100, 930, 870, 800, 720 cm–1. 1H-NMR (d6-DMSO): δ =
1.40 (s, 9H, C(CH3)3), 7.79 & 7.83 (d, 4H, J = 8.4 Hz, Ar-H),
7.95 & 8.11 (d, 4H, J = 8.0 Hz, Ar-H), 7.99 (s, 1H, NH). MS
(188°C): m/z = 325 (M+, 40%), 253 (100%), 270 (41%).
4-(4-(Diisopropylaminocarbonyl)benzoyl)benzoic acid (4b)
Recrystallized from hexane-ethyl acetate, yield: 89%. Mp
229–30°C. IR: νmax. = 3465, 2940, 2900, 1710, 1680, 1650,
1590, 1450, 1340, 1270, 920, 830 cm–1. 1H-NMR (d6-DMSO):
δ = 1.12 (br s, 6H, CH(CH3)2), 1.45 (br s, 6H, CH(CH3)2), 3.63
(m, 2H, CH(CH3)2), 7.47 & 7.80 (d, 4H, J = 8.4 Hz, Ar-H), 7.85 &
8.11 (d, 4H, J = 8.2 Hz, Ar-H). MS (151°C): m/z = 353 (M+,
21%), 253 (100%), 310 (49%).
4-(4-(Phenylaminocarbonyl)benzoyl)benzoic acid methyl ester
(3c)
Yield:53%.Mp 242–243°C.IR:νmax. = 3280, 1730, 1650, 1600,
1540, 1445, 1280, 910, 830 cm–1.1H-NMR (d6-DMSO):δ = 3.91
(s, 3H, OCH3), 7.11–7.80 (m, 5H, Ar-H), 7.87–7.89 (m, 4H, Ar-
H), 8.12 & 8.14 (d, 4H, J = 8.4 Hz, Ar-H), 10.43 (s, 1H, NH).MS
(207°C): m/z = 359 (M+, 61%), 267 (100%).
4-(4-(Phenylaminocarbonyl)benzoyl)benzoic acid (4c)
Recrystallized from ethyl acetate, yield: 90%. Mp 297–298°C.
IR: νmax. = 3280, 3050, 2900, 1710, 1680, 1650, 1590, 1450,
1340, 1270, 920, 830 cm–1. 1H-NMR (d6-DMSO): δ = 7.11–7.81
(m, 5H, Ar-H), 7.87 & 8.13 (d, 4H, J = 8.4 Hz, Ar-H), 7.89 & 8.12
(d, 4H, J = 8.4 Hz, Ar-H), 10.48 (s, 1H, NH). MS (270°C): m/z =
345 (M+, 73%), 253 (100%), 149 (53%), 254 (47%).
4-(4-(Dicyclohexylaminocarbonyl)benzoyl)benzoic acid methyl
ester (3d)
Yield:54%.Mp 151–152°C.IR:νmax. = 2930, 2840, 1720, 1650,
1600, 1430, 1280, 1100, 710 cm–1. 1H-NMR (CDCl3): δ = 1.02–
1.27 (br d, 6H, cyclohexyl-H), 1.60–1.83 (m, 12H, cyclohexyl-
H), 2.62 (br s, 2H, cyclohexyl-H), 3.05 (s, 1H, cyclohexyl-H),
3.29 (s, 1H, cyclohexyl-H), 3.97 (s, 3H, OCH3), 7.41 & 8.16 (d,
4H, J = 8.0 Hz, Ar-H), 7.82 & 7.85 (d, 4H, J = 8.4 Hz, Ar-H).
4-(4-(Dicyclohexylaminocarbonyl)benzoyl)benzoic acid (4d)
Recrystallized from hexane-ethyl acetate, yield: 95%. Mp
256–257°C. IR: νmax. = 2900, 2820, 1700, 1650, 1580, 1490,
1250, 920, 850 cm–1. 1H-NMR (d6-DMSO): δ = 1.07–1.70 (m,
18H, cyclohexyl-H), 3.16–3.27 (br d, 4H, cyclohexyl-H), 7.44 &
7.80 (d, 4H, J = 8.2 Hz, Ar-H), 7.84 & 8.11 (d, 4H, J = 8.4 Hz, Ar-
H).MS (177°C):m/z = 433 (M+, 14%), 253 (100%), 149 (34%).
4-(4-(Phenoxybenzoyl)benzoic acid methyl ester (5)
To a suspension of monomethyl terephthalate (910 mg,
5.00 mmol) in anhydrous dichloromethane (30 mL) and few
drops of anhydrous dimethylformamide, oxalyl chloride
(0.47 mL, 5.50 mmol) was added. The mixture was stirred for
2 h at room temperature.The solvent was evaporated under re-
duced pressure.The crude acid chloride was dissolved in anhy-
drous carbon disulfide (30 mL) and the resulting solution was
added dropwise to a mixture of diphenyl ether (850 mg,
5.00 mmol) and AlCl3 (1.67 g, 12.5 mmol) in anhydrous carbon
4-(4-Phenoxybenzoyl)benzoic acid (6)
Recrystallized from ethyl acetate, yield 270 mg (85%). Mp
232–33°C.IR:νmax. = 3020, 1680, 1630, 1580, 1480, 1250, 920,
840, 740 cm–1.1H-NMR (d6-DMSO): δ = 7.09 & 7.80 (d, 4H, J =
8.8 Hz, Ar-H), 7.16–7.50 (m, 5H, Ar-H), 7.76 & 8.06 (d, 4H, J =
8.2 Hz, Ar-H).

Arch. Pharm. Pharm. Med. Chem. 2002, 335, 83–88
Non-steroidal 5α-Reductase Inhibitors 87
nol and 25 µL was injected into the computer-controlled HPLC
system, which was checked before using labelled reference
controls. Radioactivity was measured with a Berthold LB 506C
monitor, using methanol/water (55/45, w/w) for T and DHT with
a flow of 0.4 mL/min and an additive flow of 1.0 mL for scintilla-
tor; baseline separation of T and DHT was achieved within
20 min. For the steroids androstenedione and dihydroandros-
tenedione methanol/water (50/50, w/w) was used.
Enzyme inhibition tests
Preparation of tissue
Rat prostatic enzyme was prepared according to the method of
Liang et al. [16] with slight modifications [17]. Male rats were
killed and prostates were taken within 5 min and put in ice cold
0.9% NaCl solution. All the following operations were per-
formed at 0–4°C. The prostates were dissected free from fat
and connective tissue, cut into pieces and weighed. Per 1 g of
tissue, 3 mL of 20 mM phosphate buffer, pH 6.5, containing
0.32 mM sucrose, and 1 mM DTT were added.The tissue was
homogenized by 10-s strokes at 20,500 rpm of an ultraturrax
(IKA) in 60-s intervals, filtered through cheesecloth, and centri-
fuged for 60 min at 105,000 g. The pellet obtained was resus-
pended in phosphate buffer. Centrifugation was repeated, the
final pellet resuspended in a minimum volume of phosphate
buffer and stored in 300 µL portions at –70°C. The 105,000 g
pellet contains nuclei, mitochondria, and microsomes and is re-
ferred to as the enzyme preparation. The protein content was
determined and was in the range of 15–25 mg/mL. Human pro-
static tissue from BPH patients was processed in the same way
using citrate buffer, pH 5.5.
Calculation procedure
The amount of DHT formed was calculated (% DHT).The zero
value was subtracted from the control (cv) and inhibition (iv)
values (cvcorr and ivcorr). Inhibition (I) was calculated using the
following equation: % I = (1 – ivcorr/cvcorr)100.
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The assay was performed as described [16] with modifications
[17]. All values were run in duplicate. The incubation was car-
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case of rat enzyme preparation phosphate buffer (40 mM,
pH 6.6 for type 1 and pH 5.5 for type 2) and in the case of hu-
man enzyme preparation citrate buffer (40 mM, pH 5.5) was
used. The incubation mixture contained approximately 250 µg
rat protein (200–300 µg human protein), 200 µM NADPH
(human enzyme, 100 µM NADPH), 0.21 µMT including 100 nCi
[1β,2β-3H]T and 2% DMSO with or without test compound
(10 µM).Above 60% inhibition, three concentrations were cho-
sen for the determination of IC50 values.The reaction was start-
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centrifugation was performed for 10 min at 4000 rpm.The water
layer was frozen and the ether layer was decanted in fresh
tubes and evaporated to dryness.
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of the compounds was determined by monitoring the conver-
sion of the tritiated substrate androstenedione (5 nM) to an-
drostanedione during an incubation period of 5 h. A day before
the experiment, DU 145 cells were seeded in a 24-multiwell-
plate at a density of 170,000 cells/well and allowed to become
adherent overnight. Compounds to be tested were dissolved in
DMSO and 5 µL of each was added to the cells in a final volume
of 0.5 mL complete medium.Inhibitors were screened at a con-
centration of 10 µM. As control of conversion (typically about
35% under these conditions) served a triplicate of wells without
inhibitors and as positive control for inhibition finasteride (80,
60, 40, 20 nM) was used. After the 5 h incubation period in 5%
CO2 at 37°C, the medium samples were extracted twice with
1 mL diethyl ether and the steroids were separated by HPLC.
Results are expressed as amount of formed androstanedione
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