6-Substituted 1H-quinolin-2-ones and 2-methoxy-quinolines: Synthesis and evaluation as inhibitors of steroid 5α reductases types 1 and 2

Article abstract of DOI:10.1016/S0223-5234(00)01167-3

A Negishi-type coupling reaction between 6-bromo-2-methoxyquinoline. (1a) and various 4-bromo-N,N'dialkyl-benzamides gave access to 6-substituted 2-methoxy-quinolines 1-3 and 1H-quinolin-2-ones 4-12. Most of these compound proved to be inhibitors of steroid 5α reductases with activity arid selectivity both being strongly dependent on the features of the heterocycle and the size of the N,N-dialkylamide substituent. The most active inhibitor for the human type 2 isozyme was 6-[4-(N,N-diisopropylcarbamoyl)phenyl]-1H-quinolin-2-one 4 (K(i) 800 ± 85 nM), showing mostly competitive inhibitory patterns. A type 1 selective inhibitor could be identified with 6-[4-(N,N-diisopropylcarbamoyl)phenyl]-N-methyl-quinolin-2-one (5, IC₅₀ 510 nM). (C) 2000 Editions scientifiques et medicales Elsevier SAS.

Full text of DOI:10.1016/S0223-5234(00)01167-3

Eur. J. Med. Chem. 35 (2000) 931–940
´
© 2000 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved
Original article
PII: S0223-5234(00)01167-3/FLA
6-Substituted 1H-quinolin-2-ones and 2-methoxy-quinolines: synthesis and
evaluation as inhibitors of steroid 5a reductases types 1 and 2
Eckhard Baston, Anja Palusczak, Rolf W. Hartmann*
Fachrichtung 8.5 Pharmazeutische und Medizinische Chemie, Universita¨t des Saarlandes, P.O. Box 15 11 50,
D-66041 Saarbru¨cken, Germany
Received 25 January 2000; revised 20 April 2000; accepted 26 April 2000
Abstract – A Negishi-type coupling reaction between 6-bromo-2-methoxyquinoline (1a) and various 4-bromo-N,N-dialkyl-benza-
mides gave access to 6-substituted 2-methoxy-quinolines 1-3 and 1H-quinolin-2-ones 4–12. Most of these compound proved to be
inhibitors of steroid 5a reductases with activity and selectivity both being strongly dependent on the features of the heterocycle and
the size of the N,N-dialkylamide substituent. The most active inhibitor for the human type 2 isozyme was 6-[4-(N,N-diisopropylcar-
bamoyl)phenyl]-1H-quinolin-2-one 4 (Ki 800985 nM), showing mostly competitive inhibitory patterns. A type 1 selective inhibitor
´
could be identified with 6-[4-(N,N-diisopropylcarbamoyl)phenyl]-N-methyl-quinolin-2-one (5, IC50 510 nM). © 2000 Editions
scientifiques et me´dicales Elsevier SAS
1H-quinolin-2-ones / methoxyquinolines / nonsteroidal inhibitors / 5a reductase isozymes 1 and 2
1. Introduction
geous for the treatment of BPH and possibly prostate
cancer and type 1 selective compounds for the sys-
temic and topical therapy of skin disorders.
Recognition of the importance of 5a dihydrotestos-
terone (DHT) in many endocrine diseases such as
benign prostatic hyperplasia (BPH) [1, 2], male pat-
tern baldness [3] or cystic acne [4] has stimulated
efforts to synthesize inhibitors of steroid 5a reductase
(EC 1.3.99.5) [5]. This enzyme catalyzes the irre-
versible reduction of testosterone (T) to the more
potent tissue-specific androgen DHT [6]. On the basis
of pathological studies of males genetically deficient
in this enzyme, selective blockade of DHT biosynthe-
sis is expected to provide a potential treatment for the
aforementioned disorders [7, 8].
The discovery of two isozymes (named type 1 and
type 2) and the determination of their tissue distribu-
tion has enhanced the prospects for the development
of more specific drugs for certain diseases [9]. Dual
acting inhibitors are now considered to be advanta-
Several categories of steroidal and nonsteroidal
compounds that inhibit 5a reductase have been re-
ported, among them Finasteride (Proscar®), Epriste-
ride or LY 191704 [4, 10, 11]. All these compounds
have structural features in common, which enable
them to interfere with the enzyme%s electrophilic
residue, that promotes enolization of T and thus
permits the transfer of an hydride into the 5a position
of the substrate. Finasteride and LY 191704, both
possessing a lactam moiety as A-ring, can compete
with the natural substrate via the enol form of this
carboxamide structure.
Our attention has been drawn to nonsteroidal in-
hibitors in view of the possible side effects of steroidal
compounds. Based on the transition state paradigm,
that besides Finasteride also led to the development
of 4-MA [5] as at least in vitro very potent inhibitor
of steroid 5a reductase, we opted for the synthesis and
biochemical evaluation of 6-aryl substituted 1H-
* Correspondence and reprints:
E-mail address: rwh@rz.uni-sb.de (R.W. Hartmann).

932
E. Baston et al. / European Journal of Medicinal Chemistry 35 (2000) 931–940
quinolin-2-ones and 2-methoxy-quinolines (figure 1).
These structures can be considered as conformation-
ally restricted analogues of pyridone derivatives of
type A, that were recently reported by our group to be
mediocre inhibitors of the human type 2 isozyme (A,
IC50 13 mM) [12].
2. Chemistry
6-Bromo-2-methoxyquinoline (1a) was chosen as
key intermediate (figure 2). This compound seemed
very attractive as a starting point, as it also allowed
the derivatization to N-nonalkylated and hydro-
Figure 1. 5a-Reductase inhibitors and general structure of title compounds.
Figure 2. Synthesis of key intermediate 1a.

E. Baston et al. / European Journal of Medicinal Chemistry 35 (2000) 931–940
933
Figure 3. Synthesis of the title compounds 1–12.
[16]. Subsequent hydrolysis with hydrochloric acid (6
M) afforded 1H-quinolin-2-ones 4, 8, 10 that were
either methylated using dimethylsulfate (5, 11) or
hydrogenated under pressure (6, 9, 12). Hydrogenation
of the methyl compound 5 resulted in compound 7.
genated analogues, that cannot easily be obtained
directly from quinoline derivatives.
E-Ethoxyacryloyl chloride, which was prepared in
excellent yield according to a method of Tietze et al.
[13] from ethylvinylether and oxalyl dichloride, was
condensed with 4-bromoaniline to give amide 1d. This
was cyclized using concentrated sulfuric acid, yielding
6-bromo-1H-quinolin-2-one (1c). Treatment of 1c with
phosphorusoxolylchloride and replacement of the
chlorine substituent in 1b by a methoxy group using
sodium methanolate, gave the desired 6-bromo-2-
methoxyquinoline (1a) in excellent overall yield (43 %,
4 steps).
From 1a a Negishi-type coupling reaction [14, 15]
with various 4-bromobenzamides was performed and
gave the 6-aryl-substituted 2-methoxyquinolines 1–3
in high yield (figure 3). The required benzamides were
synthesized according to a general procedure using
4-bromobenzoic acid and the corresponding amines
3. Pharmacology
All newly synthesized compounds were tested for
inhibitory potency versus 5a reductases using prostate
homogenates (pH 5.5, human type 2), the DU 145 cell
line (human type 1) and rat ventral prostate (pH 6.6,
type 1). The percentage inhibition values of the com-
pounds at a concentration of 10 mM are presented, in
case of highly active compounds IC50 values (table I).
Experiments using the steroidal compounds Finaste-
ride and 4-MA have been performed in parallel. The
data obtained are also presented in table I.

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E. Baston et al. / European Journal of Medicinal Chemistry 35 (2000) 931–940
4. Results and discussion
lone pair electrons from interacting with the enzyme’s
electrophilic residue.
2-Methoxyquinoline derivatives 1–3 (table I), that
can be considered as ‘frozen’ enol tautomers of e.g.
compound 5 do not display any inhibitory potency.
This result might be ascribed to a steric hindrance
caused by the methyl group, that prevents the oxygen
Quinoline derivative 4 shows a good inhibitory
potency for the human type 2 isozyme, without dis-
playing considerable inhibition of the human type 1
isoform. Surprisingly N-methylation of 4 leads to a
complete loss of type 2 activity but a strong increase
Table I. Inhibition of human and rat steroid 5a reductases by 6-substituted 2-methoxy quinolines 1–3 and 6-substituted
1H-quinolin-2-ones 4–12.
Compound
X
D
R
Human BPHa,c
type 2 % inh.
Human DU 145b
type 1 % inh.
Ratc,d % inh
(10 mM) [iC50 (nM)]e
(10 mM) [iC50 (nM)]e
(10 mM) [iC50 (nM)]e
1
2
3
–
–
–
–
–
–
isopropyl
isobutyl
cyclohexyl
n.i.f
n.i.
n.i.
n.d.g
n.d.
n.d.
n.i.
n.i.
n.i.
4
5
6
7
H
Me
H
D3,4
D3,4
isopropyl
isopropyl
isopropyl
isopropyl
83 [1700]
9
35
n.i.
27
87 [510]
28
65
55
55
28
Me
30
8
9
H
H
D3,4
isobutyl
isobutyl
70 [4850]
52
73
47
79
74
10
11
12
H
Me
H
D3,4
D3,4
cyclohexyl
cyclohexyl
cyclohexyl
63 [7660]
n.i.
30
82 [2160]
36
78 [2740]
95 [1470]
6
93 [1320]
4 MA
Finasteride
[4]
[3]
[7]
[45]
[16]
[11]
–
–
a Human prostate homogenates, pH 5.5: mainly type 2 isozyme is active.
3
b Human prostatic carcinoma of the brain (DU 145 cell line expressing type 1 isozyme), substrate H androstenedione 5 nM.
3
c Substrate 1b2b H testosterone, 210 nM.
d Rat ventral prostate, pH 6.6.
e The indicated values represent the mean of at least two experiments; relative standard deviation for IC50-values B20 %; absolute
standard deviation for percent inhibition B10 %.
f No inhibition.
g Not determined.

E. Baston et al. / European Journal of Medicinal Chemistry 35 (2000) 931–940
935
Table II. 13C-NMR chemical shifts of C-2 of 6-[4-(N,N-di-
isopropylcarbamoyl) phenyl] -substituted quinolinones 4–7a.
this observation supports the important role of the
carboxamide function for enzyme inhibitory activity.
Alkylation of the nitrogen can either disrupt or en-
hance this interaction probably due to additional
steric effects. On the basis of calculated electron pop-
ulations for the heterocycles quinolone and N-
methylquinolone [18], electronic changes caused by
N-methylation, that could disfavour amide resonance,
can be excluded. This is in accordance with the small
observed changes in 13C-NMR chemical shifts at C-2
for compounds 4–7 (table II).
In the series of compounds 8–12 we then examined
the influence of altering the size of the amide sub-
stituent. Compounds 8 and 10 with the bulkier di-
isobutylamide or dicyclohexylamide substituent are
more active for the human type 1 isoform in compari-
son to compound 4 with the smaller diisopropylamide
group. On the other hand, these structural modifica-
tions lead to a decrease of activity for the type 2
isoform. Very surprisingly N-methylation of 10 leads
not only to a reduction of activity for the type 2
isozyme, but also for the type 1 isozyme. In compari-
son to compounds 4 and 5 an increase in activity was
expected, but with 11 no potent inhibitor was
obtained.
Compound
D
Y
l C-2
(ppm)
4
5
6
7
D3,4
D3,4
H
CH3
H
164.4
162.1
170.8
170.1
CH3
a Solvent: CDCl3; concentration 25 mg/mL.
of type 1 activity (compound 5 type 1: IC50 510 nM).
However, no compound reaches the activity of the
steroidal references which are approximately two or-
ders of magnitude more potent than 4 and 5. The
corresponding hydrogenated analogues 6 and 7 are
less active for both isoforms than the parent com-
pounds. Although these findings do not correlate well
with results obtained from steroidal inhibitors [17],
The inhibitory data for the rat enzyme neither
shows a good correlation with human type 1 nor type
2 enzyme. Especially compound 5, which was a rather
Figure 4. Determination of the type of inhibition of 6-[4-(N,N-diisopropylcarbamoyl) phenyl]-1H-quinolin-2-one (4) versus
human type 2 5a-reductase. For details concerning the determination of the Ki-value and the type of inhibition, see experimental
section (Km Testosterone: 800–1000 nM, Ki, competitive (4): 800985 nM, a=5.2). The indicated data points represent the mean
of at least 3 experiments. Inhibitor concentrations: --"-- 2000 nM, -- -- 1000 nM, --ꢀ-- 500 nM, --ꢁ-- control; testosterone
concentrations: 0.21 mM, 0.51 mM, 1.01 mM, 2.01 mM.

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E. Baston et al. / European Journal of Medicinal Chemistry 35 (2000) 931–940
potent inhibitor of the human type 1 isozyme (IC50
510 nM) but exhibits only moderate activity for the
rat enzyme (IC50:10 mM). These species differences
reveal the difficulties using rat enzyme for an initial
inhibitor evaluation. The inhibitory nature of this
series of compounds for human type 2 5a reductase
was further evaluated. Quinolinone derivative 4, the
most potent inhibitor for this isozyme shows a mixed
type of inhibition (competitive/noncompetitive) with
predominant competitive inhibitory pattern (Ki =
800985 nM, Km (testosterone): 800–1000 nM, figure
4) in analogy to the 4-azasteroids [17, 19].
tetramethylsilane. Elemental analysis indicated by the
symbols of the elements were within 90.4 % of the
theoretical values and were performed in the Institut fu¨r
Anorganische Chemie, Universita¨t des Saarlandes,
Saarbru¨cken.
6.1.1. N-(4-Bromophenyl)-(E)-3-ethoxyacrylamide (1d)
(E)-3-Ethoxyacryloyl chloride (47 g, 0.35 mol) was
slowly added to a solution of 4-bromoaniline (51.6 g, 0.3
mol) in dichloromethane (400 mL) and pyridine (50 mL)
and stirring was continued for 1.5 h at 25 °C. The
precipitated yellow solid was filtered off and repeatedly
washed with water (3×100 mL). Upon evaporation of
the mother liquids further product fractions of varying
purity were obtained, that were recrystallized from
petroleum ether (40–60 °C)/acetone of increasing polar-
ity. Yield: 81 %, pale yellow needles, mp: 165–166 °C.
Physicochemical measurements with 4 reveal a pKs
value of \12, confirming that at physiological condi-
tions no ionization takes place [20].
3
1H-NMR: l 1.27 (t; 3H, J=7.08 Hz, CH3CH2); 3.95
5. Conclusions
3
3
(q; 2H, J=7.08 Hz, CH3CH2); 5.50 (d; 1H, J=12.40
Hz, OꢀCHꢁCH); 7.49 (d; 1H, 3J=12.40 Hz,
In conclusion, the present paper presents a new
class of nonsteroidal inhibitors of 5a reductase
isozymes 1 and 2. Furthermore, it was shown that the
results obtained with steroidal inhibitors (especially
with respect to modifications of the steroidal A-ring)
cannot be directly translated into this series of quino-
line derivatives. With compound 5 we identified a new
highly active type 1 selective inhibitor of steroid 5a
3
OꢀCHꢁCH); 7.46 u. 7.58 (AA%BB%; 4H, J=8.84 Hz,
aromat. H).
6.1.2. 6-Bromo-1H-quinolin-2-one (1c)
N-(4-Bromophenyl)-(E)-3-ethoxyacrylamide (1d) (54
g, 0.2 mol) was gradually added to concentrated sulfuric
acid (550 mL). After stirring at 25 °C for 3 h, the
mixture was cautiously poured onto crushed ice (1.5 kg).
The precipitated product was filtered off, washed with
water (5×100 mL) and recrystallized from ethylacetate/
methanol (95/5). Yield: 83 %, colourless crystals, mp:
reductase, which might be
development.
a
lead for drug
1
6. Experimental protocols
275 °C (Lit. [22]: mp: 269–271 °C). H-NMR: l 6.55 (d;
3
3
1H, J=9.32 Hz, H-3); 7.25 (d; 1H, J=8.84 Hz, H-8);
3
4
7.63 (dd; 1H, J=8.62 Hz, J=1.76 Hz, H-7); 7.87 (d;
6.1. Chemistry
3
4
1H, J=9.72 Hz, H-4); 7.92 (d; 1H, J=1.76 Hz, H-5);
Unless otherwise indicated, materials obtained from
commercial suppliers were used without further purifica-
tion. Solvents for reactions under anhydrous conditions
were dried according to standard procedures [21]. All
reactions, except those involving water as a reagent,
were conducted under nitrogen atmosphere. Melting
points were measured on a Reichert thermometer hot
stage microscope and are uncorrected. Silica gel TLC
and column chromatography were performed on a
Merck TLC 60F-254 (0.25 mm) and a Merck Kieselgel
60. IR spectra were determined with a Perkin Elmer
11.84 (s; 1H, NH). Anal. C9H6BrNO (C, H, N).
6.1.3. 6-Bromo-2-chloroquinoline (1b)
6-Bromo-1H-quinoline-2-one (1c) (34 g, 0.15 mol)
was suspended in phosphorusoxylchloride (120 mL) and
heated under reflux for 1 h. After cooling to 25 °C, the
solvent was evaporated and the remaining residue dis-
solved in chloroform (200 mL) and poured onto crushed
ice (500 g). The mixture was neutralized with a saturated
ammonia solution, the phases were separated and the
aqueous phase was extracted with chloroform (2×150
mL). The combined organic layers were dried over
magnesium sulfate and the solvent was evaporated to
give a solid, that was recrystallized from hexane/ethylac-
etate of increasing polarity. Yield: 83 %, colourless crys-
1
infrared spectrometer 398 (KBr). H-NMR spectra were
determined on a Bruker AM 400 at 400 MHz in d6-
Me2SO (DMSO) solution and chemical shifts are re-
ported in
l
parts per million downfield from

E. Baston et al. / European Journal of Medicinal Chemistry 35 (2000) 931–940
937
3
tals, mp: 165 °C (Lit. [23]: mp: 157 °C). 1H-NMR: l 7.66
(s; 3H, OCH3); 7.06 (d; 1H, J=8.84 Hz, H-3); 7.40 (d;
3
3
3
(d; 1H, J=8.40 Hz, H-3); 7.90 (d; 1H, J=8.84 Hz,
2H, J=7.96 Hz, 2H of AA%BB% system); 7.85 (m; 3H,
H-8 and 2H of AA%BB% system); 8.02 (d; 1H, J=8.40
Hz, H-7); 8.24 (s; 1H, H-5); 8.30 (d; 1H, J=8.84 Hz,
3
4
3
H-8); 7.95 (dd; 1H, J=9.06 Hz, J=2.20 Hz, H-7);
4
3
3
8.36 (d; 1H, J=2.20 Hz, H-5); 8.44 (d; 1H, J=8.40
Hz, H-4). Anal. C9H5BrClN (C, H, N).
H-4). Anal. C23H26N2O2 (C, H, N).
6.1.4. 6-Bromo-2-methoxyquinoline (1a)
6.1.4.2. 6-[4-(N,N-Diisobutylcarbamoyl) phenyl]-2-
methoxy-quinoline (2)
Sodium (0.5 g, 22 mmol) was dissolved in dry
methanol (20 mL) and then a suspension of 6-bromo-2-
chloroquinoline (1b) (4 g, 16.5 mmol) in methanol (20
mL) was added within 10 min. The mixture was refluxed
for 17 h and, after cooling to 25 °C, the solvent was
distilled off. The remaining residue was treated with
water (50 mL) and chloroform (120 mL), the phases
were separated and the aqueous layer was extracted with
chloroform (2×50 mL). The combined organic extracts
were dried over magnesium sulfate, the solvent was
evaporated and the remaining solid was recrystallized
from petroleum ether (40–60 °C). Yield: 77 %, colour-
less crystals, mp: 98 °C. IR: w 3140, 2880, 1695, 1680,
Prepared in a similar way as described for 1 from
6-bromo-2-methoxyquinoline (1a) and 4-bromo-N,N-di-
isobutylbenzamide. The compound was purified by a
double recrystallization from hexane. Yield: 52 %,
colourless crystals, mp: 122 °C. IR: w 2960, 2870, 1625,
1610, 1480, 1420, 1395, 1285, 1265, 1110, 1020, 830.
1H-NMR: l 0.70 and 0.95 (2s; 12H, CH(CH3)2); 1.84
and 2.07 (2s; 2H, CH(CH3)2); 3.14 and 3.32 (2s; 4H,
3
CH2CH(CH3)2); 4.01 (s; 3H, OCH3); 7.07 (d; 1H, J=
3
8.84 Hz, H-3); 7.44 (d; 2H, J=7.96 Hz, 2H of AA%BB%
system); 7.86 (m; 3H, H-5 and 2H of AA%BB% system);
3
8.03 (d; 1H, J=8.40 Hz, H-7); 8.26 (s; 1H, H-8); 8.31
1
3
1425, 1280, 1255, 1200, 930, 890, 810. H-NMR l 3.99
(d; 1H, J=8.84 Hz, H-4). Anal. C25H30N2O2 (C, H,
3
(s; 3H, OCH3); 7.08 (d; 1H, J=8.84 Hz, H-3); 7.71 (d;
N).
1H, 3J=8.84 Hz, H-8); 7.78 (dd; 1H, 3J=9.06 Hz,
4
4J=1.76 Hz, H-7); 8.17 (d; 1H, J=1.76 Hz, H-5); 8.22
6.1.4.3. 6-[4-(N,N-Dicyclohexylcarbamoyl) phenyl]-2-
methoxy-quinoline (3)
3
(d; 1H, J=8.84 Hz, H-4). Anal. C10H8BrNO (C, H,
N).
Prepared in a similar way as described for 1 from
6-bromo-2-methoxyquinoline (1a) and 4-bromo-N,N-di-
cyclohexylbenzamide. The compound was purified by
recrystallization from hexane. Yield: 58 %, colourless
solid, mp: 195 °C. IR: w 2860, 2830, 1640, 1610, 1480,
1435, 1370, 1250, 1130, 1030, 900, 840. 1H-NMR: l
1.08–1.71 (m; 20H, Cyclohexyl H); 3.30 (s; 2H,
6.1.4.1. 6-[4-(N,N-Diisopropylcarbamoyl) phenyl]-2-
methoxy-quinoline (1)
6-Bromo-2-methoxy-quinoline (1a) was dissolved in
tetrahydrofuran (40 mL) and cooled to −75 °C. Then
t-butyllithium (14 mmol, 8.75 mL of a 1.6 M solution in
pentane) was injected via a syringe within 5 min. After
10 min carefully dried zinc(II)chloride (0.94 g, 7 mmol)
was added and the mixture was warmed to 25 °C.
4-Bromo-N,N-diisopropylbenzamide (1.99 g, 7 mmol)
and tetrakis(triphenylphosphine)palladium (0) (100 mg)
were added and the mixture was heated under reflux for
2 h. After cooling, the solvent was distilled off and the
residue was treated with water (50 mL) and chloroform
(100 mL). The phases were separated and the aqueous
phase was extracted with chloroform (2×50 mL). The
combined organic layers were dried over magnesium
sulfate, the solvents were evaporated and the crude
product obtained was purified by column chromatogra-
phy using hexane/ethylacetate of increasing polarity.
Yield: 63 %, white powder, mp: 136–138 °C. IR: w 2960,
1630, 1610, 1485, 1440, 1395, 1380, 1340, 1285, 1245,
3
CH(CH2)2); 4.01 (s; 3H, OCH3); 7.07 (d; 1H, J=8.84
Hz, H-3); 7.39 (d; 2H, 3J=7.96 Hz, 2H of AA%BB%
system); 7.85 (m; 3H, H-8, 2H of AA%BB%-system); 8.04
3
(d; 1H, J=8.84 Hz, H-7); 8.27 (s; 1H, H-5); 8.31 (d;
3
1H, J=9.28 Hz, H-4). Anal. C29H34N2O2 (C, H, N).
6.1.4.4. 6-[4-(N,N-Diisopropylcarbamoyl) phenyl]-1H-
quinolin-2-one (4)
6-[4-(N,N-Diisopropylcarbamoyl)phenyl]-2-methoxy-
quinoline (1) (1.0 g, 2.8 mmol) in hydrochloric acid (6
M, 10 mL) was heated under reflux for 5 h. After being
cooled to 25 °C, water (50 mL) was added and the pH
adjusted to 9 with sodium hydroxide. The mixture was
extracted with chloroform (3×50 mL) and the com-
bined organic layers were dried over magnesium sulfate.
Evaporation of the solvent gave a residue, that was
purified by recrystallization from ethylacetate. Yield:
41 %, pale yellow crystals, mp: 246–247 °C. IR: w 3140,
1
1215, 1040, 1020, 900, 860, 840, 830, 770. H-NMR: l
1.31 (m; 12H, CH(CH3)2); 3.69 (s; 2H, CH(CH3)2); 4.01

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E. Baston et al. / European Journal of Medicinal Chemistry 35 (2000) 931–940
2960, 1660, 1620, 1430, 1375, 1335, 1260, 1210, 820, 765,
25 °C. After 1 h dimethylsulfate (150 ml, 1.5 mmol) was
injected with a syringe and stirring was continued for
1.5 h. The solvent was evaporated in vacuo, water (20
mL) was added and the mixture was extracted with
ethylacetate (3×30 mL). The combined extracts were
dried over magnesium sulfate and the solvent was evap-
orated. The compound was purified by column chro-
matography (silica gel) using ethylacetate as eluent.
Yield: 45 %, pale yellow crystals, mp: 284 °C. IR: n
2960, 1665, 1615, 1440, 1345, 1210, 1120, 855, 820, 770.
1H-NMR: l 1.23 (s; 12H, CH(CH3)2); 3.66 (m; 5H,
1
540, 520. H-NMR l 1.39 (m; 12H, CH(CH3)2); 3.68 (s;
3
2H, CH(CH3)2); 6.55 (d; 1H, J=9.76 Hz, H-3); 7.39
(m; 3H, H-8 und 2H of AA%BB% system); 7.74 (d; 2H,
3J=8.40 Hz, 2H of AA%BB% system); 7.87 (dd; 1H,
4
3
3J=8.84 Hz, J=1.76 Hz, H-7); 7.97 (d; 1H, J=9.72
Hz, H-4); 8.03 (s; 1H, H-5); 11.85 (s; 1H, NH). Anal.
C22H24N2O2 (C, H, N).
6.1.4.5. 6-[4-(N,N-Diisobutylcarbamoyl) phenyl]-
1H-quinolin-2-one (8)
3
Prepared
from
6-[4-(N,N-diisobutylcarbamoyl)-
CH(CH3)2 and N-CH3); 6.67 (d; 1H, J=9.28 Hz, H-3);
3
phenyl]-2-methoxy-quinoline (2) in a similar way, as
described for compound 4. The mixture was refluxed for
24 h and the target compound was purified by recrystal-
lization from acetone/methanol (12/1). Yield: 65 %,
colourless solid, mp: 250 °C. IR: w 3140, 2960, 1655,
1640, 1620, 1570, 1470, 1430, 1260, 1200, 1100, 935, 820.
1H-NMR: l 0.69 and 0.95 (2s; 12H, CH(CH3)2); 1.83
and 2.08 (2s; 2H, CH(CH3)2); 3.13 and 3.32 (2s; 4H,
7.39 and 7.79 (AA%BB%; 4H, J=8.40 Hz); 7.63 (d; 1H,
3J=8.84 Hz, H-8); 7.99 (2 d; 2H, H-4 and H-7); 8.12 (s;
1H, H-5). Anal. C23H26N2O2 (C, H, N).
6.1.4.8. 6-[4-(N,N-Dicyclohexylcarbamoyl) phenyl]-N-
methylquinolin-2-one (11)
Prepared from 6-[4-(N,N-dicyclohexylcarbamoyl)-
phenyl]-1H-quinolin-2-one (10) in a similar way as de-
scribed for compound 5. Yield: 38 %, colourless solid,
mp: 191 °C. IR: w 2930, 2850, 1640, 1605, 1480, 1435,
3
CH2CH(CH3)2); 6.55 (d; 1H, J=9.72 Hz, H-3); 7.41
(m; 3H, H-8 and 2H of AA%BB%-system); 7.76 (d; 2H,
3
3J=7.96 Hz, 2H of AA%BB%-system); 7.88 (d; 1H, J=
1
1370, 1270, 1250, 1190, 1130, 1030, 900, 840. H-NMR:
3
8.84 Hz, H-7); 7.98 (d; 1H, J=9.72 Hz, H-4); 8.05 (s;
l 0.77–1.72 (m; 20H, Cyclohexyl H); 3.20 (s; 2H,
1H, H-5); 11.84 (s; 1H, NH). Anal. C24H28N2O2 (C, H,
N).
3
CH(CH2)2); 4.01 (s; 3H, NꢀCH3); 7.07 (d; 1H, J=8.40
3
Hz, H-3); 7.39 (d; 2H, J=7.52 Hz, 2H of AA%BB%-sys-
tem); 7.85 (m; 3H, H-8 and 2H of AA%BB%-system); 8.04
6.1.4.6. 6-[4-(N,N-Dicyclohexylcarbamoyl) phenyl]-
1H-quinolin-2-one (10)
3
(d; 1H, J=7.52 Hz, H-7); 8.27 (s; 1H, H-5); 8.31 (d;
3
1H, J=8.40 Hz, H-4). Anal. C29H34N2O2.0.6 H2O (C,
Prepared from 6-[4-(N,N-dicyclohexylcarbamoyl)-
phenyl]-2-methoxy-quinoline (3) in a similar way, as
described for compound 4. The mixture was refluxed for
24 h in hydrochloric acid (6 M)/methanol (8/2) and the
target compound was purified by recrystallization from
ethylacetate and subsequent digestion with hexane.
Yield: 60 %, colourless solid, mp: 273–274 °C. IR w
2930, 2850, 1680, 1630, 1435, 1370, 1320, 1250, 1130,
H, N).
6.1.4.9. 6-[4-(N,N-Diisopropylcarbamoyl) phenyl]-
1H-3,4-dihydroquinolin-2-one (6)
6-[4-(N,N-Diisopropylcarbamoyl)phenyl]-1H-quino-
lin-2-one (4) (0.5 g, 1.38 mmol) was dissolved in ethanol
(150 mL) and palladium (5 %) on charcoal (50 mg) was
added. The mixture was hydrogenated for 20 h at a
pressure of 30 bars. The catalyst was filtered off and the
solvent was evaporated in vacuo. The remaining oil
crystallized after 1 day at 4 °C and was recrystallized
from ethylacetate. Yield: 72 %, colourless crystals, mp:
251–252 °C. IR: w 3230, 2980, 1690, 1610, 1520, 1495,
1
830. H-NMR: l 0.83–1.71 (2 m; 20H, Cyclohexyl H);
3.20 (m; 2H, CH(CH2)2); 6.54 (d; 1H, 3J=9.72 Hz,
H-3); 7.39 (d; 1H, 3J=8.40 Hz, H-8); 7.35 and 7.75
3
3
(AA%BB%; 4H, J=8.40 Hz); 7.88 (d; 1H, J=8.40 Hz,
3
H-7); 7.98 (d; 1H, J=9.72 Hz, H-4); 8.05 (s; 1H, H-5);
11.84 (s; 1H, NH). Anal. C28H32N2O2 (C, H, N).
1
1445, 1340, 1240, 1200, 1035, 820. H-NMR l 1.50 (s;
3
6.1.4.7. 6-[4-(N,N-Diisopropylcarbamoyl) phenyl]-N-
methylquinolin-2-one (5)
To a solution of 6-[4-(N,N-diisopropylcarbamoyl)-
phenyl]-1H-quinolin-2-one (4) (0.98 g, 2.8 mmol) in dry
N,N-dimethylformamide (5 mL) was added sodium hy-
dride (0.13 g, 3.2 mmol, 60 % solution in mineral oil) at
12H, CH(CH3)2); 2.69 (t; 2H, J=7.52 Hz, H-3); 3.04
(t; 2H, 3J=7.52 Hz, H-4); 3.70 (s; 2H, CH(CH3)2);
3
6.94 (d; 1H, J=8.40 Hz, H-8); 7.32 and 7.65 (AA%BB%;
3
3
4H, J=8.18 Hz); 7.48 (d; 1H, J=7.96 Hz); 7.53 (s;
1H, H-5); 10.18 (s; 1H, NH). Anal. C22H26N2O2 (C, H,
N).

E. Baston et al. / European Journal of Medicinal Chemistry 35 (2000) 931–940
939
6.1.4.10. 6-[4-(N,N-Diisopropylcarbamoyl) phenyl]-
N-methyl-3,4-dihydroquinolin-2-one (7)
lent to 200–300 mg human protein or 200–250 mg rat
protein, NADPH (100 mM for human and 200 mM for
rat enzyme) and testosterone (0.21 mM including 100
Prepared from 6-[4-(N,N-diisopropylcarbamoyl)-
phenyl]-N-methylquinolin-2-one (5) in a similar way as
described for compound 6. Yield: 68 %, colourless crys-
tals, mp: 266 °C. IR: w 2960, 1680, 1620, 1500, 1470,
3
nCi 1b2b H T) in a final volume of 250 ml. The test
compounds were dissolved in DMSO (2 % per incuba-
tion). After preincubation for 5 min, the enzymatic
reaction was started by addition of testosterone and
carried out at 37 °C for 30 min using sodium phosphate
buffer (40 mM, pH 6.6, rat enzyme) or sodium citrate
buffer (40 mM, pH 5.5, human enzyme). The reaction
was stopped by addition of 50 mL sodium hydroxide
solution (10 M) and the steroids were extracted with
diethylether (500 mL). Steroid separation by RP-HPLC
was performed as reported [12].
1
1435, 1365, 1340, 1130, 855, 820, 770. H-NMR l 1.40
3
(m; 12H, CH(CH3)2); 2.70 (t; 2H, J=7.28 Hz, H-3);
3
2.98 (t; 2H, J=7.52 Hz, H-4); 3.40 (s; 3H, N-CH3);
3
3.57 (s; 2H, CH(CH3)2); 7.05 (d; 1H, J=8.40 Hz, H-8);
3
7.38 and 7.57 (AA%BB%; 4H, J=8.18 Hz); 7.40 (d; 1H,
3
4
4J=1.76 Hz, H-5); 7.48 (dd; 1H, J=8.40 Hz, J=1.76
Hz, H-7). Anal. C23H28N2O2 (C, H, N).
6.1.4.11. 6-[4-(N,N-Diisobutylcarbamoyl) phenyl]-
1H-3,4-dihydroquinolin-2-one (9)
For the determination of Ki values the inhibitor was
tested at 3 concentrations near its IC50 value with vari-
able substrate concentrations (0.21, 0.51, 1.01 and 2.02
mM T). The kinetic parameters were calculated using the
following equations [19]:
Prepared from 6-[4-(N,N-diisobutylcarbamoyl)-phen-
yl]-1H-quinolin-2-one (8) in a similar way as described
for compound 6. Yield: 67 %, colourless solid, mp:
208 °C. IR: w 3200, 3050, 2960, 1680, 1635, 1500, 1465,
1
V
Km
Vmax
1
S
1
Vmax
1
1435, 1375, 1275, 1100, 825. H-NMR l 0.69 and 0.94
=
·
+
(1)
(2)
(2s; 12H, CH(CH36 )2); 1.82 and 2.06 (2s; 2H, CH(CH3)2);
3
2.50 (t; 2H, H-3); 2.96 (t; 2H, J=7.52 Hz, H-4); 3.12
1
V
Km
Vmax
I
Ki
1
S
1
Vmax
I
hKi
ꢀ ꢁ ꢀ ꢁ
3
=
1+
+
1+
and 3.27 (2s; 4H, CH2CH); 6.94 (d; 1H, J=7.96 Hz,
3
H-8); 7.36 and 7.67 (AA%BB%; 4H, J=8.18 Hz); 7.50 (d;
1H, 3J=7.96 Hz, H-7); 7.55 (s; 1H, H-5). Anal.
The first equation represents a modification of the
Lineweaver–Burk relation according to Lee and Wilson
[24] with V (reaction velocity), Vmax (maximum veloc-
ity), Km (Michaelis–Menten constant) and S (mean
substrate concentration). Equation (2) indicates the spe-
cial case of a mixed type (competitive/non competitive)
inhibitor (h=ꢀ, pure competitve inhibitor; h=1, pure
non competitive inhibitor).
C24H30N2O2 (C, H, N).
6.1.4.12. 6-[4-(N,N-Dicyclohexylcarbamoyl) phenyl]-
1H-3,4-dihydroquinolin-2-one (12)
Prepared from 6-[4-(N,N-dicyclohexylcarbamoyl)-
phenyl]-1H-quinolin-2-one (10) in a similar way as de-
scribed for compound 6. Yield: 68 %, colourless solid,
mp: 236–237 °C. IR: w 3200, 3060, 2930, 2850, 1680,
1640, 1500, 1435, 1365, 1315, 1250, 1185, 1130, 1000,
835. 1H-NMR: l 0.81–1.70 (m; 20H, Cyclohexyl H);
6.2.2. DU145 cell assay
Intact human prostatic carcinoma DU145 cells are
used as the source of type 1 5a-reductase [25, 26]. The
inhibitory potency of the compounds was determined
similar to [27] by monitoring the conversion of the
tritiated substrate androstenedione during an incubation
period of 6 h (RPMI 1640 medium containing 10 % fetal
calf serum; pH 7.2) A day before the experiment DU145
cells were seeded in a 24-multiwell-plate at a density of
200 000 cells/well and allowed to become adherent
overnight. Compounds to be tested were dissolved in
DMSO and 5 mL of each were added to the cells in a
final volume of 0.5 mL complete medium. As control of
conversion (typically about 35 % under these conditions)
served a triplicate of wells without inhibitors and as
positive control for inhibition a steroidal reference (e.g.
3
3
2.69 (t; 2H, J=7.52 Hz, H-3); 2.95 (t; 2H, J=7.48
3
Hz, H-4); 3.20 (s; 2H, CH(CH2)2); 6.94 (d; 1H, J=8.40
3
Hz, H-8); 7.30 and 7.66 (AA%BB%; 4H, J=7.96 Hz);
3
7.50 (d; 1H, J=8.40 Hz, H-7); 7.55 (s; 1H, H-5); 10.19
(s; 1H, NH). Anal. C28H34N2O2 (C, H, N).
6.2. Enzyme inhibitor e6aluation
6.2.1. Prostatic homogenate assays
Rat ventral prostate and human prostatic tissue from
BPH patients served as enzyme source and were homog-
enized as previously reported [12]. The enzyme prepara-
tion contained nuclei, mitochondria and microsomes.
Each incubation mixture contained homogenate equiva-

940
E. Baston et al. / European Journal of Medicinal Chemistry 35 (2000) 931–940
Patel G.F., Liang T., Cascieri M.A., Cheung A.H., Brooks
J.R., Nerman C., J. Med. Chem. 29 (1986) 2298–2315.
Finasteride: 80, 60, 40, 20 nM) was used. After the 6 h
incubation period in 5 % CO2 at 37 °C the medium
samples were extracted twice with 1 mL diethylether and
the steroids were separated by HPLC [12].
[11] Holt D.A., Levy M.A., Oh H.J., Erb J.M., Heaslip J.I., Brandt
M., Lan-Hargest H.Y., Metcalf B.W., J. Med. Chem. 33 (1990)
943–950.
[12] Hartmann R.W., Reichert M., Go¨hring S., Eur. J. Med. Chem.
29 (1994) 807–817.
[13] Tietze L.F., Schneider C., Pretor M., Synthesis (1993) 1079–
1080.
Acknowledgements
[14] Knochel P., Singer R.D., Chem. Rev. 93 (1993) 2117–2188.
[15] Rottla¨nder M., Palmer N., Knochel P., Synlett (1996) 573–575.
[16] Fong C.W., Grant H.G., Aust. J. Chem. 26 (1983) 50–54.
[17] Frye S.V., Curr. Pharm. Des. 2 (1996) 59–84.
Thanks are due to the Deutsche Forschungsgemein-
schaft (DFG) and to the Fonds der Chemischen Indus-
trie for financial support. We thank Wolfgang Reichert
for performing the DU 145 cell culture assay and Dr
Martina Kotthaus for fruitful discussions.
[18] Lewis F.D., Reddy G.D., Ellbert J.E., Tillberg B.E., Meltzer
J.A., Kojima M., J. Org. Chem. 56 (1991) 5311–5318.
[19] Segel I.H., Enzyme Kinetics: Behaviour and Analysis of Rapid
Equilibrium and Steady-State Enzyme Systems, Wiley, New
York, 1993.
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