Novel 5α-reductase inhibitors: Synthesis, structure-activity studies, and pharmacokinetic profile of phenoxybenzoylphenyl acetic acids

Article abstract of DOI:10.1021/jm050728w

Novel substituted benzoyl benzoic acids and phenylacetic acids 1-14 have been synthesized and evaluated for inhibition of rat and human steroid 5α-reductase isozymes 1 and 2, The compounds turned out to be potent and selective human type 2 enzyme inhibitors, exhibiting IC₅₀ values in the nanomolar range. The phenylacetic acid derivatives were more potent than the analogous benzoic acids. Bromination in the 4-position of the phenoxy moiety led to the strongest inhibitor in this class (12; IC₅₀ = 5 nM), which was equipotent to finasteride. Since oral absorption is essential for a potential drug, 12 was further examined. In the parallel artificial membrane permeation assay (PAMPA) it turned out to be a good permeator, whereas it was a medium permeator in Caco2 cells. After oral administration (40 mg/kg) to rats a high bioavailability and a biological half-life of 5.5 h were observed, making it a promising candidate for clinical evaluation.

Full text of DOI:10.1021/jm050728w

748
J. Med. Chem. 2006, 49, 748-759
Novel 5r-Reductase Inhibitors: Synthesis, Structure-Activity Studies, and Pharmacokinetic
Profile of Phenoxybenzoylphenyl Acetic Acids
Ola I. A. Salem,† Martin Frotscher,† Christiane Scherer,† Alexander Neugebauer,† Klaus Biemel,‡ Martina Streiber,†
Ruth Maas,‡ and Rolf W. Hartmann*,†
8.2 Pharmaceutical and Medicinal Chemistry, Saarland UniVersity, P.O. Box 15 11 50, D-66041 Saarbru¨cken, Germany, and
Pharmacelsus CRO, Science Park 2, D-66123 Saarbru¨cken, Germany
ReceiVed July 28, 2005
Novel substituted benzoyl benzoic acids and phenylacetic acids 1-14 have been synthesized and evaluated
for inhibition of rat and human steroid 5R-reductase isozymes 1 and 2. The compounds turned out to be
potent and selective human type 2 enzyme inhibitors, exhibiting IC50 values in the nanomolar range. The
phenylacetic acid derivatives were more potent than the analogous benzoic acids. Bromination in the 4-position
of the phenoxy moiety led to the strongest inhibitor in this class (12; IC50 ) 5 nM), which was equipotent
to finasteride. Since oral absorption is essential for a potential drug, 12 was further examined. In the parallel
artificial membrane permeation assay (PAMPA) it turned out to be a good permeator, whereas it was a
medium permeator in Caco2 cells. After oral administration (40 mg/kg) to rats a high bioavailability and a
biological half-life of 5.5 h were observed, making it a promising candidate for clinical evaluation.
Introduction
we and other groups have focused on the synthesis and
evaluation of nonsteroidal inhibitors.16-29 Recently, we have
discovered a novel type of dual inhibition of 5R-reductase 1
and 2, a concept which is called hybrid inhibition.30 Esters of
substituted benzylidene-4-carboxylic acids are inhibitors of type
1 enzyme.30 They act in the periphery and are cleaved in the
prostate to the corresponding acids, which are potent type 2
inhibitors.30 In a previous work,28 we used compound A (Figure
1), described by Holt et al.,29 as a potent 5R-reductase inhibitor
as starting point for structural modifications aimed at the
optimization of the compound. The most potent inhibitor
obtained was 4-(4-(phenylaminocarbonyl)benzoyl)benzoic acid,
showing an IC50 value of 820 nM for human type 2 enzyme.
As this might not be sufficient for clinical efficacy, we describe
in this paper the continuation of the former study, i.e., the
synthesis, biological evaluation, and molecular modeling of
novel phenoxybenzoyl benzoic and phenylacetic acids 1-14
(Figure 1). For the determination of biological activity in vitro,
studies were performed using human and rat isozymes 1 and 2.
Since besides potency the pharmacokinetic properties of a NCE
are crucial, the most potent compound of this series was further
investigated. To predict the oral availability of the most potent
compound, the parallel artificial membrane permeation assay
(PAMPA) and the Caco2 assay were used. Finally, the in vivo
pharmacokinetic profile was determined in rats.3
5R-Reductase is a membrane-bound, NADPH-dependent
enzyme (EC 1.3.99.5) that irreversibly catalyses the reduction
of 4-ene-3-oxosteroids to the corresponding 5R-3-oxosteroids,
particularly, testosterone (T) to the most potent androgen 5R-
dihydrotestosterone (DHT).1 Two isozymes, 5R-reductase 1 and
2, have been characterized2 and confirmed by molecular
cloning.3 They show high contents of hydrophobic amino acids
and a similar hydrophobicity pattern. However, the identity of
their amino acid sequence is only about 50%, and different tissue
distribution and biochemical properties were found for both
isozymes. The type 1 isoform is the predominant enzyme in
sebaceous glands and liver, whereas the type 2 enzyme is
dominant in the prostate. An acidic pH of 5.5 is optimal for
type 2 activity, whereas a pH between 6.0 and 8.5 is favorable
for isozyme 1. The affinity for the substrate T is higher for
isozyme 2 (Km = 0.4 µM) than it is for isozyme 1 (Km =10
µM).4 5R-Reductase was shown to be involved in human
disorders, e.g. benign prostatic hyperplasia (BPH),5,6 alopecia,7
acne,8 and hirsutism,9 and is proposed to play a role in prostate
cancer.10 BPH is the most common proliferative disease that
affects males and causes obstructive uropathy. The use of
surgery is effective in the treatment, but its high costs and
postoperative complications leave the need for other treatments.
Finasteride (Proscar, Propecia) and more recently dutasteride
(Avodart, Avolve) are two steroidal 5R-reductase inhibitors
already used for the treatment of BPH and male pattern baldness.
Finasteride is a relatively selective 5R-reductase type 2 inhibitor
capable of reducing the circulating DHT levels by approximately
70%,11,12 whereas dutasteride, a potent dual inhibitor of both
isozymes, reduces serum DHT by more than 90%; thus, it is a
more efficient steroid 5R-reductase inhibitor than finasteride.13
It is well-tolerated and side effects, e.g., sexual disorders and
gynecomastia, are transient.14,15 Fewer side effects might be
exhibited by appropriate nonsteroidal inhibitors. Consequently,
Supporting the experimental findings, we applied a pharma-
cophore modeling approach that describes nicely the biological
activities of the inhibitors investigated by us.
Chemistry
Terephthalic acid monomethyl ester was the precursor for
the acids 1-4, which were synthesized via two different
routes: First, for the preparation of compounds 1-3, the
precursor was converted to the acid chloride, which then was
reacted with anisole in a Friedel-Crafts acylation in the presence
of AlCl3 to yield the ketone 1c as described.32 Ether cleavage
with AlCl3 in benzene afforded the phenol 1b, which was
subjected to alkylation with different alkyl bromides in the
presence of a base (NaH or K2CO3, respectively) to give the
esters 1a-3a. Alternatively, following the procedure of Goossen
* To whom correspondence should be addressed. Tel: +49 681 302
3424. Fax: +49 681 302 4386. E-mail: rwh@mx.uni-saarland.de.
† Saarland University.
‡ Pharmacelsus CRO.
10.1021/jm050728w CCC: $33.50 © 2006 American Chemical Society
Published on Web 12/27/2005

NoVel 5R-Reductase Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2 749
Figure 1. Lead compound (A) and structural optimizations (1-14).
Scheme 1. Synthesis of Compounds 1-4a
a Conditions: (a) 80%, (i) (COCl)2, CH2Cl2/DMF, room temperature for 1 h; (ii) anisole, AlCl3, CH2Cl2, room temperature for 24 h; (b) 81%, AlCl3,
benzene, reflux, 15-45 min; (c) 39-62%, R-Br, NaH, DMF, rt, 3-24 h; (d) 73%, diphenylmethyl bromide, K2CO3, DMF, 90 °C for 24 h; (e) 31-87%,
K2CO3, MeOH/H2O (9:1), reflux; (f) 37%, 3-methoxyphenylboronic acid, DSC, Pd(F6-acac)2, Pcy3, Na2CO3, THF, 60 °C overnight.
et al.,33 the palladium-catalyzed cross-coupling reaction of
3-methoxybenzeneboronic acid and terephthalic acid mono-
methyl ester using DSC (di-N-succinimidyl carbonate) as a
coupling reagent proceeded smoothly and afforded the ester 4c.
Demethylation of 4c and alkylation of the obtained phenol 4b
in analogy to the above-mentioned procedure provided the ester
4a. Saponification of the esters 1a-4a led to the target acids
1-4 (Scheme 1).
For the synthesis of compounds 5 and 6, 4-(4-nitrobenzoyl)-
benzoic acid 5d was subjected to acid-catalyzed methylation
to produce the ester 5c. A mild reduction of the nitro group
using stannous chloride following the procedure of Bellamy et

750 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2
Scheme 2. Synthesis of Compounds 5 and 6a
Salem et al.
a Conditions: (a) 75%, glacial acetic acid, sulfuric acid, CrO3, room temperature for 3 h; (b) 78%, MeOH, sulfuric acid, reflux for 18 h; (c) 93%,
SnCl2·2H2O, EtOH, 70 °C for 1 h; (d) 30%, benzyl bromide, DMF, room temperature, 20 h; (e) 48-54%, K2CO3, MeOH/H2O (9:1), reflux for 1-2 h; (f)
49%, benzoyl chloride, triethylamine, CH2Cl2, room temperature for 2 h.
Scheme 3. Synthesis of Compounds 7, 8 and 11, 12a
a Conditions: (a) 15-64%; (i) (COCl)2, AlCl3, CH2Cl2, 5-10 °C for 15 min; (ii) 4-(un)substituted-diphenyl ether, AlCl3, CH2Cl2, 24-40 °C for 1 h; (b)
31-83%, K2CO3, MeOH/H2O (9:1), reflux for 1-2 h.
Scheme 4. Alternative Pathway for the Synthesis of Compound
al.34 afforded the amino derivative 5b in excellent yield.
7a
Reaction of the amino group of 5b with benzyl chloride or
benzoyl chloride, followed by saponification of the formed esters
5a and 6a, gave access to the acids 5 and 6, respectively
(Scheme 2).
The phenylalkanoic acid methyl esters 7b, 8b, and 11b were
subjected to acylation35 using oxalyl chloride/AlCl3. The
obtained acid chlorides were reacted with the corresponding
a Conditions: (a) 44%, NBS, benzoyl peroxide, CCl4, reflux; (b) 47%,
NaCN, 1,4-dioxane/H2O, reflux; (c) 63%, aq NaOH (40%), EtOH, reflux
then 1 N HCl.
diphenyl ethers in the presence of AlCl3 to afford the alkanoic
acid esters 7a, 8a, 11a, and 12a. The overall yield in this two-
step reaction was in some cases below 20%, most probably due
to the formation of positional isomers. Saponification of these
esters resulted in the acids 7, 8, 11, and 12 (Scheme 3).
Alternatively, bromination of the methyl group of (4-
methylphenyl)(4-phenoxyphenyl)methanone 7e using NBS in
the presence of benzoyl peroxide, followed by sodium cyanide
treatment of the obtained bromo derivative 7d, provided the
acetonitrile derivative 7c, which after hydrolysis under basic
conditions and subsequent acidification afforded the acid 7 in
good yield (Scheme 4).
Scheme 5. Synthesis of Compounds 9 and 10a
Heck coupling36 of the iodide 9b with methyl acrylate
provided the cinnamic acid derivative 9a in good yield.
Hydrogenation of 9a with H2/Pd for 18 h resulted in both
reduction of the keto group and saturation of the olefinic double
bond, thus furnishing the propanoic acid methyl ester 10a.
Saponification of the esters 9a and 10a afforded the corre-
sponding acids 9 and 10 (Scheme 5).
The synthesis of compounds 13 and 14 started from the
benzoic acids 13c and 14c. Compound 13c was afforded
previously by oxidation of 4-bromo-2-fluorobenzaldehyde using
a modified Lindgren reaction.37,38 Friedel-Crafts acylation of
a Conditions: (a) 84%, methyl acrylate, Pd(OAc)2, TBAC, DMF, 50 °C
for 3 h; (b) 68%, Pd/C, H2, MeOH, rt, 18 h; (c) 89%, K2CO3, MeOH/H2O
(9:1), reflux.
diphenyl ether with the acid chlorides of 13c and 14c respec-
tively yielded the ketones 13b and 14b. The latter were subjected
to a cross-coupling reaction with ethynyltrimethylsilane in the
presence of PdCl2‚2PPh3 and cuprous iodide39 to provide 13a
and 14a in excellent yield. The hydroboration of the produced

NoVel 5R-Reductase Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2 751
Scheme 6. Synthesis of Compounds 13 and 14a
Table 2. Inhibition of Human and Rat Steroid 5R-Reductase Types 1
and 2 in Vitro by Compounds 7-11
% inhibn (10 µM) or [IC50, µM]
human
RVP
compd
X, Y
type 2a,d type 1b,e pH 5.5c,d pH 6.6c,d
7
8
9
10
11
-CH2-, CdO
[0.023]
[2.80]
ni
ni
ni
ni
nd
77
44
63
nd
nd
42
49
61
nd
nd
-(CH2)2-, CdO
a Conditions: (a) 60-70%; (i) (COCl)2, CH2Cl2/DMF, room temperature
for 1 h; (ii) diphenyl ether, AlCl3, CH2Cl2, 24-40 °C for 1 h; (b) 84-
88%, triethylamine/THF (5:1), PdCl2·2PPh3, CuI, trimethylsilylacetylene,
80 °C for 2-3 h; (c) 29-39%, BH3, 1M in THF, 3 h at room temperature,
MeOH, 3 N NaOH, 35% H2O2 at 40 °C.
-CHdCH-, CdO [0.47]
-(CH2)2-, CH2
-(CH2)3-, CdO
12
10
a-e See Table 1.
Table 1. Inhibition of Human and Rat Steroid 5R-Reductase Types 1
and 2 in Vitro by Compounds 1-6
Table 3. Inhibition of Human and Rat Steroid 5R-Reductase Types 1
and 2 in Vitro by Compounds 12-14
% inhibn (10 µM) or [IC50, µM]
% inhibn (10 µM) or [IC50, µM]
human
RVP
human
RVP
type 2a,d
type 1b,e
pH 5.5c,d
pH 6.6c,d
compd
R
type 2a,d type 1b,e pH 5.5c,d pH 6.6c,d
compd
Z, R
1
2
3
4
5
6
A
4-(Ph)CH2O
4-(2,5-(CF3)2Ph)CH2O [0.113] 12
[0.119] ni
45
29
71
55
nd
nd
33
64
69
73
35
nd
nd
53
7
12
13
14
H; H
H, Br
F, H
[0.023]
[0.005]
[0.045]
[0.027]
ni
ni
ni
ni
77
55
73
78
42
59
57
[1.45]
4-(Ph)2CHO
3-(Ph)CH2O
4-(Ph)CH2NH
4-(Ph)CONH
4-(Ph)O
[0.79]
9
[0.131] ni
CH3, H
60
51
nd
nd
a-e See Table 1.
[0.053] ni
finasteride
[0.005] [0.051] [0.011]
[0.01]
finding confirms our previous observation27 that the hydrophobic
pocket, which also accommodates the substituents in the 17-
position of the steroidal inhibitors, is limited in size. Introduction
of two trifluoromethyl substituents into the benzyl group of 1,
mimicking the steroidal dual type 1 and 2 inhibitor dutasteride,
does not have a significant impact on inhibitory activity (IC50
values: 1, 119 nM; 2, 113 nM). The displacement of the
benzyloxy group from the para to the meta position decreases
activity only marginally (IC50 values: 1, 119 nM; 4, 131 nM).
On the other hand, exchange of the ether oxygen of compound
1 by an isosteric NH group reduces activity drastically. The
resulting benzylamine compound 5 is similarly active as the
benzoylamine 6 showing a reduction of activity by a factor of
100 compared to compound 1 (IC50 values: 1, 119 nM; 6, ca.
10 µM). From these results it has to be concluded that the
phenoxy group in the para position of the benzoyl benzoic acid
moiety should not be changed. In this class of compounds, this
group is obviously appropriate for interaction with the lipophilic
pocket of the human type 2 isozyme.
In the second step in the optimization process, variations in
the carboxylic acid side chain by insertion of different spacers
between the benzene ring and the carboxylic group were
performed. These modifications were inspired by recent results
of our group27 and others45 indicating that the protein shows
conformational flexibility in the corresponding part of the active
site. It becomes apparent from Table 2 that the insertion of a
methylene spacer resulted in the phenylacetic acid compound
7 and an increase in inhibitory activity by a factor of 2 (IC50
values: compound A, 53 nM; compound 7, 23 nM). Further
elongation of the side chain with an ethylene (compound 8) or
propylene (compound 11) spacer reduced inhibitory activity
a Human prostate homogenates, 125 µg protein, pH 5.5. b DU 145 cell
line. c Rat ventral prostate, 200-250 µg protein. d Substrate was [1â,2â-
3H]testosterone (210 nM). e Substrate was [3H]androstenedione (5 nM). ni,
no inhibition; nd, not determined.
alkynes 13a and 14a, followed by oxidation with aqueous NaOH
and hydrogen peroxide, led to the corresponding carboxylic acids
13 and 14 (Scheme 6).40
Biological Results
Inhibition of Human and Rat 5r-Reductase Isozymes 1
and 2 in Vitro. The inhibitory activities of compounds 1-14
were determined using rat prostate homogenates (pH 6.6 for
type 1 and pH 5.5 for type 2), human prostate homogenate (BPH
tissue for type 2) according to the method of Liang et al.,41 and
the DU145 cell line (for human type 1 enzyme) as described.42-44
The percent inhibition values at a concentration of 10 µM or,
for more potent compounds, the IC50 values are presented in
Tables 1-3.
In a previous work,28 we exchanged the phenoxy group of
the potent and selective benzoyl benzoic acid A29 (Table 1) by
alkyl- and aryl carbamoyl groups to afford novel active and
selective 5R-reductase inhibitors. However, the activities ob-
tained did not reach the potency of the lead compound. In this
study, we first performed further modifications of the phenoxy
group, all of which are based on the insertion of a second atom
between the two benzene nuclei, thus leading to an elongation
of the molecule (compounds 1-6, Table 1). Replacement of
the phenyl group of compound A by a benzyl or diphenylmethyl
group reduces inhibitory activity toward human type 2 enzyme
by a factor of 2 and 15, respectively (compounds 1 and 3). This

752 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2
Salem et al.
dramatically (IC50 value: 8, 2.8 µM) and led to an inactive
compound (compound 11, 10% inhibition at a concentration of
10 µM). Interestingly, enhancing the rigidity of the ethylene
spacer in compound 8 by dehydrogenation, resulting in the
cinnamic acid derivative 9, increased activity by a factor of 6
(IC50 values: compound 8, 2.8 µM; compound 9, 0.47 µM).
Reduction of the carbonyl group in the benzoyl moiety of
compound 8 resulted in a loss of activity (compound 10, 12%
inhibition at a concentration of 10 µM). This indicates that a
planar structure provided by the sp2 carbon of the carbonyl linker
between the two benzene nuclei in compound 8 is favorable as
compared to the tetrahedral structure in compound 10. Similar
results have been obtained in the class of benzylidenepiperidine
5R-reductase inhibitors.23
Finally, the influence of aromatic substitution on rings A and
C of compound 7 was investigated. Fluorine and methyl were
selected, since both increase lipophilicity but differ in their
electronic effects. They were introduced into the 3-position as
substitution ortho to the carboxylic group is known to diminish
inhibitory activity. As shown in Table 3, the fluoro substituent
reduced inhibition by a factor of 2 (IC50 values: compound 13,
45 nM; compound 7, 23 nM). The methyl group did not increase
the inhibitory activity either (compound 14, 27 nM). Introduction
of bromine into the 4-position of the C ring, however, markedly
enhanced type 2 enzyme inhibitory activity. The resulting
compound 12 exceeded compound 7 by a factor of 5 (IC50
values: compound 12, 5 nM; compound 7, 23 nM) and was
equipotent to the steroidal inhibitor finasteride (IC50 value: 5
nM).
Figure 2. The mean profile ((SEM; n ) 5) of absolute plasma levels
(µg/mL) in rats versus time after a single oral dose (40 mg/kg) of
compound 12.
Table 4. Pharmacokinetic Parameters of Compound 12 in Male Rats
after Oral Application (40 mg/kg)
parametersa
value
parameters
1/2z (h)
value
dose po (mg/kg)
40
t
5.5
0.12
314.5
336.1
C
max obs (µg/mL)
Cz (µg/mL)
max obs (h)
tz (h)
22.7
2.7
6.0
24
CL (L/kg/h)
AUC0-t (µg h/mL)
z
t
AUC0-∞ (µg h/mL)
a Cmax obs, maximal measured concentration; Cz, last analytical quantifiable
concentration; tmax obs, time to reach the maximum measured concentration;
tz, time of the last sample which has an analytical quantifiable concentration;
t
1/2z, half-life of the terminal slope of a concentration-time curve; AUC0-t ,
z
area under the concentration-time curve up to the time tz of the last sample;
AUC0-∞, area under the concentration-time curve extrapolated to infinity;
Cl, body clearance.
Regarding inhibition of the human type 1 enzyme, it is
striking that the title compounds did not show inhibitory activity
in this cellular assay (Tables 1-3). This cannot be explained
by an insufficient permeation across the cell membrane of the
DU145 cells due to their carboxylate group. As shown below,
compound 12 shows a good permeation of artificial membranes
(PAMPA assay), and other carboxylic acids have been shown
to be able to inhibit the enzyme in the very same assay. Thus,
it has to be concluded that the title compounds are selective
type 2 inhibitors.
similarity in the extent of gastrointestinal absorption between
human and rat.48
After a single po administration of compound 12 to male rats
(40 mg/kg), plasma samples were collected over 24 h and
plasma concentrations were determined by HPLC-MS/MS.
Individual profiles of five rats showed a continuous increase of
plasma levels over time. In these rats, maximal plasma
concentrations (Cmax) were in the range between 21 and 48 µg/
mL. The time of maximal plasma concentration (tmax) was
measured 6-8 h postdosing in four out of five rats. In one rat,
the Cmax value was measured 4 h after application. Subsequently,
plasma concentrations decreased again. Plasma levels were low
but still measurable 24 h after application in all rats. The mean
profile of absolute plasma levels of 12 is illustrated in Figure
2.
The pharmacokinetic parameters of 12 evaluated on absolute
group mean values are presented in Table 4. After oral
administration, 12 was slowly absorbed, as shown by the tmax
of 6 h. In addition, the elimination was medium, as indicated
by a half-life (t1/2) of 5.5 h and by a clearance rate of 0.12 L/kg/
h, which was considered as low, relative to an estimated hepatic
plasma flow of 2.16 L/kg/h in the rat.49
With respect to the inhibition of the rat isozymes, most of
the compounds turned out to be dual inhibitors for both isoforms.
However, the inhibitory activity was only weak. The most potent
inhibitors toward rat type 2 enzyme were compounds 3, 7, 13,
14 (71-78% inhibition at a concentration of 10 µM), whereas
the most active inhibitor toward rat type 1 enzyme was
compound 3 (73% inhibition). Due to this lack of activity for
the rat enzymes, rats are not appropriate for the determination
of the biological activity of the title compounds. However, they
can be used for the determination of phamacokinetic properties.
Pharmacokinetic Evaluation in Vitro and in Vivo. Com-
pound 12 showed a high permeability in PAMPA (flux rate )
91 ( 3% and recovery 81%). Thus, it is most likely that
compound 12 is able to cross biological membranes, indicating
a good oral absorption. Evaluation of compound 12 on Caco2
cells revealed a Papp value of 6.6 ( 0.7 cm/s, which is indicative
of a medium permeator.46 In this assay, ketoprofen was used
as a “high permeator” (Papp ) 28.9 ( 3.0 cm/s) and ranitidine
as a “low permeator” reference (Papp ) 0.07 ( 0.02 cm/s)
according to the FDA guidance.47
Discussion and Conclusion
Aiming at the discovery of a potent, selective, and in vivo
active 5R-reductase type 2 inhibitor, compound A was structur-
ally modified. An important structural feature for the enhance-
ment of inhibitory activity was the extension of the carboxylic
acid side chain with a methylene spacer (compounds 7 and 12-
13). However, further elongation of the side chain reduced the
activity markedly (compounds 8-11).
To get an idea about the in vivo properties of compound 12,
the pharmacokinetics was determined after oral administration
in the rat. These results should be well transferable to the human
situation, since it has been demonstrated that there is a great
For structural elucidation, we employed the 5R-reductase type
2 pharmacophore model, which was recently described by Chen

NoVel 5R-Reductase Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2 753
et al.50 The model consists of five pharmacophoric features: two
hydrogen-bond acceptors (HBA1-2) and three hydrophobic
groups (HP1-3). For any two of the five features a distance
contraint is given (see Supporting Information, Table S1). In
this context, a compound fits well to the pharmacophore model
if it has all features present and if the distance constraints are
within the range claimed by the model. In this work, we applied
our inhibitors 7-9 and 12 to the pharmacophore model of Chen
et al. Therefore, we used the lowest energy conformations of
compounds 7-9 and 12 to calculate the distances between any
two of the five features of the pharmacophore model. These
conformations were calculated by a simulated annealing mo-
lecular dynamics approach followed by an ab initio electronic
structure geometry optimization. This approach allows us to get
highly reliable minimum structures of our inhibitors. For more
details, see the molecular modeling procedure in the Experi-
mental Section below. The results are summarized in Table S1
located in the Supporting Information. This table shows that
the calculated values of the highly active inhibitors 7 and 12
(IC50 values: 23 and 5 nM) fit very well in the model, whereas
in case of the less active inhibitors 8 and 9 (IC50 values: 2800
and 470 nM) marked deviations were observed. This means that
the structure-activity relationship of our compounds are very
well described by the pharmacophore model of Chen et al.
12 in vitro and in vivo demonstrate that it is a very promising
new 5R-reductase inhibitor. Further preclinical and clinical
studies will elucidate whether this compound is an alternative
to existing steroidal and nonsteroidal inhibitors. Presently,
structure modifications are being performed to examine whether
our hybrid inhibition concept30 can also be introduced into this
class of compounds.
Experimental Section
Chemicals and solvents obtained from commercial suppliers
(Aldrich, Acros, Fluka, Lancaster) were used without further
purification. Solvents for reactions under anhydrous conditions were
dried according to standard procedures. All reactions, except those
involving water as a reagent, were performed under nitrogen
atmosphere. All reactions were monitored by thin-layer chroma-
tography (TLC) using Alugram silica G/UV254 (purchased from
Macherey-Nagel). Column chromatography (CC) was performed
on Merck Kieselgel 60 [40-63 µm (flash column chromatography,
FCC) or 50-200 µm, respectively]. Melting points were determined
on a Reichert Thermometer hot stage microscope and are uncor-
1
rected. H NMR spectra were measured on a Bruker AM 400 at
400 MHz or DRX 500 (500 MHz) instrument using the indicated
solvent. Chemical shifts are reported in δ (ppm) downfield from
TMS (δ: 0 ppm). IR spectra were performed with KBr disks or
films, as indicated, on a Perkin-Elmer 398 infrared spectrometer.
Wavenumbers are given in cm-1. The purity of the compounds
was verified using HPLC (Agilent 1100, Agilent, Waldbronn,
Germany) under the following conditions: UV-DAD; Nucleodur
100-3 C18ec colum, 3 µm, 125 × 3 mm; solvent A, H2O with
0.1% TFA; solvent B, acetonitrile; gradient, 0.0-3.0 min, 90% A
and 10% B; 3.0-10.0 min from 90% A to 0% A; 10-13 min from
0% A to 100% A; flow rate, 0.7 mL/min, temperature, 40 °C;
pressure, 18.0 MPa ( 0.1. Elemental analyses (C, H, N) were within
(0.4% of the theoretical values and were performed by the Institute
of Inorganic Chemistry, Saarland University, Saarbru¨cken, Ger-
many. 1H NMR and IR spectra as well as elemental analysis (Table
S2) are available in the Supporting Information.
In the context of the finding that the compounds of this study
did not show a significant inhibition of the type 1 enzyme, it is
worth mentioning that in a recent QSAR study we have obtained
results suggesting a different hydrogen-bonding pattern in the
binding pockets of both isoforms as well as a lower content of
aromatic amino acids in the binding site of the type 2 isozyme.51
Another important finding was the observation that a bromo
substituent in the 4-position of the phenoxy group of compound
7 leads to a further increase in activity (compound 12). This
was a little surprising since in case of compound 1 disubstitution
in the 2,5-positions with CF3, which like Br is also lipophilic
and electronegative, did not enhance activity (compound 2).
Being as active as finasteride, compound 12 is one of the most
potent human type 2 inhibitors known so far.
4-(4-Methoxybenzoyl)benzoic Acid Methyl Ester (1c). Com-
pound 1c was synthesized as previously described.32 as colorless
crystals, mp 161-162 °C (lit.32 mp 158 °C).
4-(3-Methoxybenzoyl)benzoic Acid Methyl Ester (4c). A
mixture of terephthalic acid monomethyl ester (0.9 g, 5.0 mmol),
palladium(II)-1,1,1,5,5,5-hexafluoroacetylacetonate (78 mg, 0.15
mmol), tricyclohexylphosphine (0.126 g, 0.45 mmol), Na2CO3 (1.04
g, 10 mmol), and di-N-succinimidyl carbonate (1.67 g, 6.5 mmol)
in anhydrous THF (15 mL) was purged with N2, degassed, and
stirred at 60 °C for a few minutes until the formation of CO2 ceased.
The solution was cooled to room temperature and a solution of
3-methoxybenzeneboronic acid (0.91 g, 6.0 mmol) in anhydrous
THF (15 mL) was added. The reaction mixture was stirred at 60
°C overnight, poured into water (300 mL), and extracted with ethyl
acetate (100 mL × 3). The combined organic layers were dried
over MgSO4, and the solvent was removed under reduced pressure.
The crude product was purified by FCC using hexane/ethyl acetate
(17:3) as eluent to give light brown crystals in 37% yield, mp 94-
95 °C.
4-[4-(Hydroxybenzoyl)]benzoic Acid Methyl Ester (1b). A
mixture of 1c (1.0 g, 3.70 mmol) and AlCl3 (1.97 g, 14.8 mmol) in
benzene (25 mL) was stirred at room temperature for 15 min and
then heated under reflux for further 15 min. The mixture was cooled
to room temperature, poured on ice, and extracted with ethyl acetate.
The organic layer was washed with aqueous NaHCO3 (5%, 20 mL),
water, and brine. After drying over MgSO4, the solvent was
evaporated under reduced pressure and the residue was purified
by FCC eluting with hexane/ethyl acetate (4:6) to give colorless
crystals in 81% yield, mp 184-185 °C.
To evaluate its potential for gastrointestinal absorption,
compound 12 was passed through three assays.31 The assays
were developed to identify those compounds out of a series that
are able to permeate artificial and biological membranes and to
enter blood circulation in animals. Compound 12 was predicted
to be a high permeator in the PAMPA and a medium permeator
in Caco2 cells. These results were verified by the pharmaco-
kinetic evaluation in the rat. After peroral administration of
compound 12 at the dose of 40 mg/kg, plasma levels of the
compound were measurable for at least 24 h. This showed that
compound 12, given orally, was able to cross the gastrointestinal
tract and reached the general circulation, as predicted in the
preceding in vitro tests. The pharmacokinetics of compound 12
was characterized by both a slow absorption and a slow
elimination. The slow elimination rate of 12, resulting in a long
plasma half-life, might be explained by a strong binding to
plasma proteins and/or by a high metabolic stability, which are
presently being further investigated. It is worth mentioning that
no apparent signs of discomfort could be observed in the rats
after injection of 12 during the duration of the experiment.
As the compound is less potent in vitro on the rat enzymes,
it is not feasible to investigate its in vivo potency in our in
vivo rat model.27 However, since a great similarity between the
gastrointestinal absorption between human and rat has been
demonstrated, it can be expected that 12 shows favorable
pharmacokinetic properties in humans. The results of compound
Compound 4b was prepared following the same procedure.
4-[3-(Hydroxybenzoyl)]benzoic Acid Methyl Ester (4b). Com-
pound 4b was synthesized starting from the ester 4c. The mixture
was heated under reflux for 45 min. The residue was purified using

754 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2
Salem et al.
FCC eluting with hexane/ethyl acetate (7:3) to give light yellow
crystals in 71% yield, mp 157-158 °C.
5b as a yellow powder in 93% yield, mp 135 °C. It could be used
in the next synthetic step without further purification.
4-[4-(Benzylamino)benzoyl]benzoic Acid Methyl Ester (5a).
A solution of 5b (0.1 g, 0.39 mmol) and benzyl bromide (0.14
mL, 1.17 mmol) in DMF (10 mL) was stirred at room temperature
for 20 h. The reaction was quenched by the addition of water and
the product was extracted with ethyl acetate (50 mL × 3). The
combined organic layers were washed with brine and dried over
MgSO4. After evaporation of the solvent, the residue was purified
by FCC using hexane/ethyl acetate (3:1) as eluent to give a yellow
powder in 30% yield, mp 113-114 °C.
4-[4-(Benzoylamino)benzoyl]benzoic Acid Methyl Ester (6a).
To an ice-cooled solution of 5b (0.50 g, 1.96 mmol) and
triethylamine (0.26 mL, 1.96 mmol) in anhydrous CH2Cl2 was
added benzoyl chloride (0.23 mL, 1.96 mmol). The mixture was
stirred for 2 h at room temperature, poured on ice, and extracted
with ethyl acetate (75 mL × 3). The combined organic layers were
washed with brine and dried over MgSO4. After evaporation of
the solvent under reduced pressure, the compound was purified by
recrystallization from hexane/ethyl acetate to give colorless crystals
in 49% yield, mp 199 °C.
4-[4-(Benzyloxy)benzoyl]benzoic Acid Methyl Ester (1a). A
solution of 1b (0.512 g, 2 mmol) and NaH (60% in mineral oil, 88
mg, 2.2 mmol) in anhydrous DMF (15 mL) was stirred at room
temperature for 15 min. Benzyl bromide (0.376 g, 2.2 mmol) was
added and stirring was continued for 24 h at room temperature.
The mixture was poured on ice and extracted with ethyl acetate (3
× 50 mL). The combined organic layers were washed with water
and brine. After drying over MgSO4, the solvent was evaporated
under reduced pressure. The residue was purified using FCC, eluting
with hexane/ethyl acetate (17:3) to give colorless crystals in 62%
yield, mp 169-170 °C.
Compounds 2a and 4a were prepared following the same
procedure as for 1a.
4-(4-{[2,5-Bis(trifluoromethyl)benzyl]oxy}benzoyl)benzoic Acid
Methyl Ester (2a). Compound 2a was synthesized starting from
1b and 2-(bromomethyl)-1,4-bis(trifluoromethyl)benzene. The mix-
ture was stirred at room temperature for 3 h. Purification was
achieved by FCC using hexane/ethyl acetate (19:1) as eluent to
give colorless crystals in 79% yield, mp 120-121 °C.
4-[3-(Benzyloxy)benzoyl]benzoic Acid Methyl Ester (4a).
Compound 4a was synthesized starting from 4b and benzyl
bromide. The mixture was stirred at room temperature for 3 h. The
residue was purified by FCC using hexane/ethyl acetate (9:1) as
eluent. Further purification was achieved by recrystallization from
hexane to give colorless crystals in 39% yield, mp 109-110 °C.
4-[4-(Benzhydryloxy)benzoyl]benzoic Acid Methyl Ester (3a).
A solution of diphenylmethyl bromide (0.31 g, 1.25 mmol) in
anhydrous DMF (5 mL) was added to a mixture of 1b (0.32 g,
1.18 mmol) and K2CO3 (0.189 g, 1.31 mmol) in DMF (5 mL).
The mixture was heated at 90 °C for 24 h, cooled to room
temperature, poured on ice, and extracted with ethyl acetate (50
mL × 4). The combined organic layers were washed with water
and brine and were dried over MgSO4. After evaporation of the
solvent under reduced pressure, the residue was purified by FCC
using hexane/ethyl acetate (8:2) as eluent to give a white powder
in 73% yield, mp 150-151 °C.
4-(4-Nitrobenzoyl)benzoic Acid (5d). To an ice-cooled mixture
of (4-methylphenyl)(4-nitrophenyl)methanone49 (5e, 2.2 g, 9.12
mmol) in glacial acetic acid (17 mL) was added sulfuric acid (1.4
mL) slowly to keep the temperature below 5 °C. Chromium(VI)
oxide (2.74 g, 27.4 mmol) was added in small portions at such a
rate that the temperature did not exceed 10 °C. The reaction slurry
was stirred at room temperature for 3 h, poured on ice, and extracted
with ethyl acetate (3 × 100 mL). The combined organic layers
were washed with water to neutral pH and dried over MgSO4. After
evaporation of the solvent under reduced pressure, a crude product
was obtained which was purified by recrystallization from ethyl
acetate to give a yellowish white powder in 75% yield, mp 254 °C
(lit.50 mp 257-258 °C).
4-(4-Nitrobenzoyl)benzoic Acid Methyl Ester (5c). A solution
of 5d (6.0 g, 22.1 mmol) in anhydrous methanol (50 mL) and
sulfuric acid (5 mL) was heated under reflux for 4 h. After cooling
to room temperature, the mixture was concentrated under reduced
pressure. It was poured into a cold aqueous solution of sodium
bicarbonate (5%) and extracted with ethyl acetate (200 mL × 3).
The combined organic layers were washed with small amounts of
water and brine and were dried over MgSO4. After evaporation of
the solvent under reduced pressure, 5c was obtained as light yellow
crystals in 78% yield, mp 174-175 °C. It was pure enough to be
used in the next synthetic step without further purification.
4-(4-Aminobenzoyl)benzoic Acid Methyl Ester (5b). A mixture
of 5c (5.0 g, 17.5 mmol) and SnCl2‚2H2O (19.7 g, 87.5 mmol) in
ethanol (60 mL) was heated at 70 °C for 1 h. After cooling to room
temperature, the mixture was poured on ice and the pH was adjusted
to slightly alkaline (pH 7-8) using NaHCO3 solution (5%). The
aqueous phase was extracted with ethyl acetate (50 mL × 3). The
combined organic layers were washed with brine and dried over
Na2SO4. Evaporation of the solvent under reduced pressure yielded
(4-Methylphenyl)(4-phenoxyphenyl)methanone (7e). Com-
pound 7e was prepared as previously described52 as colorless
crystals, mp 80-81 °C (lit.52 mp 79.8-80 °C).
[4-(Bromomethyl)phenyl](4-phenoxyphenyl)methanone (7d).
A solution of 7e (2.0 g, 7.0 mmol), NBS (1.25 g, 7.0 mmol), and
a catalytic amount of benzoyl peroxide in anhydrous CCl4 (30 mL)
was stirred at room temperature for 10 min and then heated under
reflux for 4 h. Succinimide was filtered off and washed with CCl4.
The combined organic phases were washed with a small amount
of water and dried over MgSO4. The solvent was evaporated under
reduced pressure and the compound was purified by FCC eluting
with hexane/ethyl acetate (19:1) to give colorless crystals in 44%
yield, mp 85-86 °C.
[4-(4-Phenoxybenzoyl)phenyl]acetonitrile (7c). A solution of
NaCN (0.049 g, 1.0 mmol) in H2O (5 mL) was added to 7d (0.180
g, 0.5 mmol), dissolved in 1,4-dioxane (5 mL). The resulting
mixture was heated under reflux for 1 h. After cooling to room
temperature the solution was acidified cautiously with diluted
hydrochloric acid and extracted with ethyl acetate (50 mL × 4).
The combined organic layers were washed with aqueous NaHCO3
solution (5%) and brine. After drying over MgSO4 and removing
the solvent under reduced pressure, the compound was purified by
FCC eluting with hexane/ethyl acetate (7:3) to give colorless crystals
in 47% yield, mp 119-120 °C.
[4-(4-Phenoxybenzoyl)phenyl]acetic Acid (7). A mixture of 7c
(0.27 g, 0.862 mmol), aqueous sodium hydroxide solution (40%, 6
mL), and ethanol (5 mL) was heated under reflux for 1 h. After
cooling to room temperature, the mixture was acidified with 1 N
hydrochloric acid and extracted with ethyl acetate (75 mL × 3).
The combined organic layers were washed with water and brine
and dried over MgSO4. After evaporation of the solvent, the
compound was purified by recrystallization from hexane/ethyl
acetate to give a white powder in 63% yield, mp 137-138 °C, tR
) 11.3 min.
4-[4-(4-Phenoxybenzoyl)phenyl]butanoic Acid Methyl Ester
(11a). To an ice-cooled solution of 4-phenylbutanoic acid methyl
ester (11b, 0.18 g, 1.0 mmol) and oxalyl chloride (0.17 mL, 2.0
mmol) was added AlCl3 (0.4 g, 3.0 mmol) in portions to keep the
temperature below 5 °C. The mixture was stirred for 15 min at
5-10 °C and then poured on ice. The aqueous phase was extracted
with dichloromethane (3 × 30 mL). The combined organic phases
were washed with brine and dried over MgSO4. After concentrating
the organic layer under reduced pressure, the residue was dissolved
in dichloromethane (30 mL) and added dropwise to a mixture of
diphenyl ether (0.17 g, 1.0 mmol) and AlCl3 (0.6 g, 4.5 mmol) in
dichloromethane (30 mL). The mixture was heated under reflux
for 1 h. After cooling, the reaction slurry was poured on ice and
extracted with ethyl acetate (3 × 50 mL). The organic layers were
combined, washed with water and brine, and dried over MgSO4.
After evaporation of the organic solvent, a crude product was

NoVel 5R-Reductase Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2 755
obtained which was purified by FCC using hexane/ethyl acetate
(8:2) as eluent to give white waxy crystals in 64% yield, mp 32-
33 °C.
Compounds 7a, 8a, and 12a were synthesized following the
procedure for compound 11a.
[4-(4-Phenoxybenzoyl)phenyl]acetic Acid Methyl Ester (7a).
Compound 7a was synthesized starting from phenylacetic acid
methyl ester 7b. The reaction mixture was stirred at room
temperature for 1 h. The product was purified by FCC using hexane/
ethyl acetate (8:2) to give a colorless oil in 18% yield.
3-[4-(4-Phenoxybenzoyl)phenyl]propanoic Acid Methyl Ester
(8a). Compound 8a was synthesized starting from 3-phenylpro-
panoic acid methyl ester 8b. The mixture was stirred at room
temperature for 1 h. Purification of the product was achieved by
FCC using hexane/ethyl acetate (17:3) to give colorless crystals in
18% yield, mp 96 °C.
{4-[4-(4-Bromophenoxy)benzoyl]phenyl}acetic Acid Methyl
Ester (12a). Compound 12a was synthesized starting from 7b and
1-bromo-4-phenoxybenzene. The crude product was purified by
FCC using hexane/ethyl acetate (17:3) as eluent to give colorless
crystals in 15% yield, mp 67-68 °C.
(4-Iodophenyl)(4-phenoxyphenyl)methanone (9b). A solution
of 4-iodobenzoyl chloride (2.66 g, 10 mmol) in anhydrous dichlo-
romethane (50 mL) was added dropwise over 30 min to a stirred
mixture of diphenyl ether (1.70 g, 10 mmol) and AlCl3 (2.67 g, 20
mmol) in anhydrous dichloromethane (50 mL). The mixture was
stirred under reflux for a further 2 h. After cooling, the reaction
slurry was poured on ice and extracted with ethyl acetate (3 × 100
mL). The combined organic layers were washed with aqueous
NaHCO3 solution (5%), water, and brine and were dried over
MgSO4. The solvent was evaporated under reduced pressure and
the residue was purified by recrystallization from ethyl acetate to
give colorless crystals in 68% yield, mp 161-162 °C.
(2E)-3-[4-(4-Phenoxybenzoyl)phenyl]acrylic Acid Methyl Es-
ter (9a). To a degassed solution of 9b (0.527 g, 1.0 mmol) in DMF
(4 mL) were successively added palladium(II) acetate (11.0 mg,
0.05 mmol), methyl acrylate (0.17 g, 2.0 mmol), tetrabutylammo-
nium chloride (0.28 g, 1.0 mmol), and K2CO3 (0.346 g, 2.5 mmol).
The reaction mixture was stirred under nitrogen at 50 °C for 3 h.
After cooling, the reaction mixture was concentrated under reduced
pressure and diluted with diethyl ether (20 mL). The mixture was
washed with water (10 mL), aqueous NaHCO3 solution (5%), and
brine. After drying the organic layer over MgSO4, the solvent was
removed under reduced pressure. Purification was achieved by FCC
using hexane/ethyl acetate (8:2) as eluent to give colorless crystals
in 84% yield, mp 184-185 °C.
3-[4-(4-Phenoxybenzyl)phenyl]propanoic Acid Methyl Ester
(10a). Compound 9a (0.519 g, 1.5 mmol), dissolved in methanol
(30 mL), was subjected to hydrogenation under atmospheric
pressure for 18 h using Pd/C 10% as a catalyst. After filtration, the
solvent was evaporated under reduced pressure. The crude product
was purified by FCC using hexane/chloroform (9:1) as eluent to
give a colorless oil in 68% yield.
for 1 h at room temperature. The solvent and the unreacted oxalyl
chloride were removed under reduced pressure. The residual acid
chloride was dissolved in anhydrous dichloromethane (20 mL) and
added dropwise to a mixture of diphenyl ether (1.39 g, 8.19 mmol)
and AlCl3 (2.73 g, 20.4 mmol) in anhydrous dichloromethane (50
mL) at room temperature. The mixture was refluxed for 1 h, cooled
to room temperature, poured on ice, and extracted with diethyl ether
(3 × 200 mL). The combined organic layers were washed with
aqueous NaHCO3 (5%, 30 mL), water, and brine. After drying over
MgSO4, the solvent was evaporated under reduced pressure.
Purification by FCC using hexane/dichloromethane (7:3) as eluent
gave colorless crystals in 60% yield, mp 74-75 °C.
Compound 14b was synthesized following the procedure of
compound 13b.
(4-Bromo-2-methylphenyl)(4-phenoxyphenyl)methanone (14b).
The starting material was 4-bromo-2-methylbenzoic acid 14c. The
reaction mixture was stirred at room temperature for 1 h. The crude
product was purified by FCC using hexane/chloroform (9:1) to give
a colorless oil in 70% yield.
[2-Fluoro-4-(trimethylsilanylethynyl)phenyl](4-phenoxy-
phenyl)methanone (13a). Under nitrogen, ethynyltrimethylsilane
(0.1 g, 1.0 mmol) was added to a mixture of 13b (0.371 g, 1.0
mmol), bis-(triphenylphosphine)palladium(II) chloride (14.0 mg,
0.02 mmol) and copper(I) iodide (8.0 mg, 0.04 mmol) in anhydrous
triethylamine/THF (4:1). The reaction mixture was heated to 80
°C for 2 h. After cooling to room temperature, the reaction mixture
was filtered and the filtrate was evaporated under reduced pressure.
The residue was dissolved in chloroform (60 mL). The solution
was washed with water (2 × 3 mL) and dried over Na2SO4. The
solvent was removed under reduced pressure. Flash column
chromatography using hexane/ethyl acetate (9:1) and subsequent
crystallization from hexane yielded colorless crystals in 84% yield,
mp 98-99 °C.
Compound 14a was synthesized following the procedure for
compound 13a.
[2-Methyl-4-(trimethylsilanylethynyl)phenyl](4-phenoxy-
phenyl)methanone (14a). 14b was used as starting material. The
reaction mixture was heated at 80 °C for 3 h. The crude slurry was
purified by FCC using hexane/dichloromethane (8:2) as eluent to
give a colorless oil in 88% yield.
[3-Fluoro-4-(4-phenoxybenzoyl)phenyl]acetic Acid (13). To a
solution of 13a (0.32 g, 0.81 mmol) in anhydrous THF was added
BH3 (1 M in THF, 0.89 mL, 0.89 mmol) at 0 °C. After stirring for
3 h at room temperature, methanol (0.41 mL) was added carefully
and the solution was oxidized at 40 °C with H2O2 (35%, 0.41 mL)
in aqueous NaOH solution (3 N, 0.41 mL). The mixture was stirred
at room temperature for 1 h, acidified with diluted hydrochloric
acid, and extracted with ethyl acetate (75 mL × 3). The combined
organic layers were washed with water and brine. After drying over
MgSO4, the solvent was evaporated under reduced pressure. Column
chromatography of the residue using hexane/ethyl acetate (9:1)
afforded the analytically pure product as pale yellow crystals in
39% yield; mp 109-110 °C, tR ) 11.3 min.
Compound 14 was synthesized following the procedure of
compound 13.
[3-Methyl-4-(4-phenoxybenzoyl)phenyl]acetic Acid (14). 14a
was used as starting material. The product was purified by CC using
hexane/ethyl acetate (8:2) as eluent to give cream colored crystals
in 29% yield; mp 113-114 °C, tR ) 11.7 min.
4-[4-(Benzhydryloxy)benzoyl]benzoic Acid (3). A mixture of
3a (0.297 g, 0.70 mmol) and K2CO3 (0.290 g, 2.10 mmol) in
methanol/H2O (9:1, 15 mL) was heated under reflux for 4 h. After
cooling to room temperature, the mixture was acidified with diluted
hydrochloric acid and extracted with ethyl acetate (50 mL × 3).
The combined organic layers were washed with water and brine.
After drying over MgSO4, the solvent was evaporated under reduced
pressure. The residue was recrystallized from ethyl acetate to afford
the title compound as a white powder in 87% yield; mp 204-205
°C, tR ) 12.3 min.
4-Bromo-2-fluorobenzoic Acid (13c). To an ice-cooled mixture
of 4-bromo-2-fluorobenzaldehyde (2.0 g, 0.01 mol) in acetonitrile
(30 mL) were slowly added, one by one, aqueous NaH2PO4 (0.24
g in 10 mL of water, 0.002 mol), H2O2 (1.36 mL, 0.014 mol), and
a solution of NaClO2 (1.3 g, 0.014 mol) in H2O (20 mL). The
mixture was stirred overnight at room temperature. At the end of
the reaction, a voluminous precipitate was formed. A small amount
of Na2SO3 was added to destroy unreacted HOCl and H2O2. After
acidification of the mixture with aqueous HCl (10%), the product
was extracted with ethyl acetate (3 × 100 mL). The organic layer
was washed with water and dried over MgSO4. After evaporation
of the solvent, the product, a white powder (82% yield, mp 211
°C), was used without further purification.
(4-Bromo-2-fluorophenyl)(4-phenoxyphenyl)methanone (13b).
To a suspension of 13c (1.80 g, 8.19 mmol) in anhydrous
dichloromethane (25 mL) containing a few drops of DMF was
added oxalyl chloride (0.79 mL, 9.0 mmol) carefully via syringe.
After the effervescence has ceased, the reaction mixture was stirred
Compounds 1, 2, 4-8, 11, and 12 were synthesized by following
the procedure for compound 3.

756 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2
Salem et al.
4-[4-Benzyloxy)benzoyl]benzoic Acid (1). The starting material
was 1a. The procedure gave colorless crystals in 57 % yield; mp
251-252 °C, tR ) 11.6 min.
4-(4-{[2,5-Bis(trifluoromethyl)benzyl]oxy}benzoyl)benzoic Acid
(2). The starting material was 2a. The mixture was heated under
reflux for 3 h. Purification was achieved by recrystallization from
ethyl acetate to give colorless crystals in 31% yield; mp 183-184
°C, tR ) 12.7 min.
4-[3-(Benzyloxy)benzoyl]benzoic Acid (4). The starting material
was 4a. The mixture was refluxed for 2 h. The residue was purified
by recrystallization from methanol to give a white powder in 57%
yield; mp 285-286 °C, tR ) 11.6 min.
4-[4-(Benzylamino)benzoyl]benzoic Acid (5). The starting
material was 5a. The mixture was heated under reflux for 2 h. The
residue was purified by recrystallization from hexane/ethyl acetate
to give yellow crystals in 54% yield; mp 173-174 °C, tR ) 10.7
min.
250 µL. In the case of rat enzyme preparation, phosphate buffer
(40 mM, pH 6.6 for type 1) or citrate buffer (40 mM, pH 5.5 for
type 2) was used. In the case of human enzyme preparation, citrate
buffer (40 mM, pH 5.5) was employed. The incubation mixture
contained approximately 250 µg of rat protein (125 µg human
protein), 200 µM NADPH (human enzyme, 100 µM NADPH), 0.21
µM T including 100 nCi [1â,2â-3H]T, and 2% DMSO with or
without test compound (10 µM). In cases exceeding 60% inhibition,
three concentrations were chosen for the determination of IC50
values. The reaction was started by adding the prostatic enzyme
preparation and terminated by addition of 50 µL aqueous solution
of NaOH (10 M). The steroids were extracted with diethyl ether
(500 µL) by shaking for 10 min. Subsequent 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.
Human Type 1 Inhibition: DU 145-Assay.43,44 Intact human
prostatic carcinoma DU145 cells were used as the source of type
1 5R-reductase.42 The inhibitory potencies of the compounds were
determined by monitoring the conversion of the tritiated substrate
androstenedione (5 nM) to androstanedione during an incubation
period of 6 h. A day before the experiment, DU145 cells were
seeded in a 24-well plate at a density of 180 000 cells/well and
allowed to become adherent overnight in 1 mL of RPMI-1640
medium (with 10% FCS). Appropriate concentrations (10 µM final
concentration at initial tests) of inhibitors dissolved in dimethyl
sulfoxide (DMSO) were applied in duplicates. Growth medium was
replaced by 500 µL of fresh medium containing 5 µL of the inhibitor
and 5 nM [3H]androstenedione as substrate. Inhibitors were first
screened at concentrations of 10 µM in an initial test, and in cases
exceeding 80% inhibition, three concentrations were chosen for
measurement of IC50 values. As control of conversion (typically
about 35% under these conditions) a triplicate of wells without
inhibitors was used and finasteride (80, 60, 40, 20 nM) was a
positive control for inhibition. After a 6-h incubation period in 5%
CO2 at 37 °C, the medium samples were extracted twice with 1
mL of diethyl ether and the steroids were separated by HPLC.
Results are expressed as the amount of formed androstanedione as
a percentage of control values.
HPLC Procedure. Steroid separation was performed20 similarly
to the method of Cook et al.56 The steroids were dissolved in 50
µL of methanol, and 25 µL was injected into the computer-
controlled HPLC system, which was checked before using labeled
reference controls. Radioactivity was measured by employing 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 scintillator. Baseline separation of T and DHT was achieved
within 20 min. For the steroids androstenedione and androstanedi-
one, methanol/water (50/50, w/w) was used and the retention times
were 11.2 and 17.5 min, respectively.
4-[4-(Benzoylamino)benzoyl]benzoic Acid (6). The starting
material was 6a. The mixture was heated under reflux for 1 h. The
residue was purified by recrystallization from ethyl acetate to give
a white powder in 48% yield, mp 278-279 °C.
4-(4-Phenoxybenzoyl)phenylacetic Acid (7). The starting mate-
rial was 7a. The mixture was heated under reflux for 2 h to give a
white powder in 76% yield; mp 137-138 °C, tR ) 11.3 min.
3-[4-(4-Phenoxybenzoyl)phenyl]propanoic Acid (8). The start-
ing material was 8a. The mixture was heated under reflux for 2 h.
The product was purified by recrystallization from hexane/ethyl
acetate to give colorless crystals in 83% yield; mp 161 °C, tR
11.7 min.
)
4-(4-(4-Phenoxybenzoyl))phenylbutyric Acid (11). The starting
material was 11a. The mixture was heated under reflux for 2 h.
Purification of the product was achieved by recrystallization from
hexane/ethyl acetate to give colorless crystals in 76% yield; mp
114-115 °C, tR ) 12.0 min.
{4-[4-(4-Bromophenoxy)benzoyl]phenyl}acetic Acid (12). The
starting material was 12a. The mixture was heated under reflux
for 1 h to give colorless crystals in 31% yield; mp 168 °C, tR
12.1 min.
)
3-[4-(4-Phenoxybenzyl)phenyl]propanoic Acid (10). A mixture
of 10a (0.21 g, 0.6 mmol) in methanol/aqueous KOH (2 M) (1:1,
10 mL) was heated under reflux for 1 h. The crude product was
purified by recrystallization from hexane to give colorless crystals
in 89% yield; mp 105-106 °C, tR ) 12.3 min.
Compound 9 was synthesized following the procedure of
compound 10.
(2E)-3-[4-(4-Phenoxybenzoyl)phenyl]acrylic Acid (9). The
starting material was 9a. The mixture was refluxed for 2 h. The
crude product was purified by recrystallization from ethyl acetate
to give colorless crystals in 89% yield; mp 235 °C, tR ) 11.8 min.
Enzyme Inhibition Test. Preparation of Tissue. Rat prostatic
enzyme was prepared according to the method of Liang et al.41
with slight modifications.20 Male rats were sacrificed and prostates
were taken within 5 min and put in ice-cold 0.9% aqueous NaCl
solution. All the following steps were performed 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
dithiothreitol (DTT) was added. The tissue was homogenized by
10-s strokes at 20 500 rpm of an ultraturax (IKA) in 60-s intervals,
filtered through cheesecloth, and centrifuged for 60 min at 105 000g.
The pellet obtained was resuspended in phosphate buffer. The
centrifugation was repeated, and the final pellet was resuspended
in a minimum volume of phosphate buffer and stored in 300-µL
portions at -70 °C. The 105 000g pellet contains nuclei, mito-
chondria, and microsomes and is referred to as the enzyme
preparation. The protein content was determined55 and was in the
range of 15-25 mg/mL. Human prostatic tissue from BPH patients
was processed in the same way using citrate buffer, pH 5.5.
Incubation Procedure. The assay was performed as described41
with modifications.20 All values were run in duplicate. The
incubation was carried out for 30 min at 37 °C in a total volume of
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.
Parallel Artificial Membrane Permeation Assay (PAMPA).
The procedure according to Zhu et al. 57 was used. The membrane
was built up by pipetting a solution of lipids in an organic solvent
on a supporting filter material in 96-well plates. For all compounds,
stock solutions at concentrations of 5 mM were prepared (test
compounds in DMSO, reference compounds in ethanol). The stock
solutions were subsequently diluted in Tris-buffer (0.05 M, pH 7.4)
to a final concentration of 250 µM. Permeation rates of all test
compounds were measured in triplicates. Diffusion time across the
artificial membrane was 16 h. Reference values without lipid layer
were individually determined for all compounds. The concentrations
in the acceptor compartments were measured by UV difference
spectroscopy using a microtiter plate reader (Spectramax Plus384
,
Molecular Devices). For the compounds the λmax values were
determined in a previous run. The permeation rates are expressed

NoVel 5R-Reductase Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2 757
as flux rates, which were calculated according to
flux (%) ) OD(test well)/OD(control well) × 100
Quantum (ThermoFinnigan) triple quadrupole mass spectrometer
equipped with an electrospray interface (ESI).
The HPLC for the Caco2 samples was run isocratically on a
EC50/3 Nucleodur 110-3 C18 ec-column (Macherey Nagel) with
a flow rate of 300 µL/min. The HPLC for the serum samples was
run on a CC 30/2 Nucleosil 100-3 C18 HD column (Macherey
Nagel) in gradient mode with a flow rate of 300 µL/min. The mobile
phase for the Caco2 samples consisted of a mixture of methanol/
water (75/25) containing 0.1% formic acid and 10 mM ammonium
acetate. For the pharmacokinetic study, the mobile phase consisted
of 10 mM ammonium acetate in water and methanol containing
10 mM ammonium acetate. The proportion of methanol was
programmed to linearly increase from 20 to 80% over 7 min and
subsequently to decrease within 0.5 min to 20% again. For
re-equilibration of the column, the proportion was subsequently held
at this concentration for 1.5 min. MS detection was run in positive
electrospray mode. The tandem mass spectrometer was used in
multiple reaction mode (MRM) to detect parent and product ions
of compound 12 and its internal standard.
Sample Preparation. Primary stock solutions in methanol
containing up to 20% of DMSO were diluted in buffer or rat plasma
to prepare calibration standards in the range from 0.15 to 75 µM
for Caco2 samples or from 0.05 to 500 µM for serum samples. All
samples were extracted for LC-MS/MS analysis to avoid ion
suppression effects by matrix components during MS detection.
Consequently, samples were acidified using hydrochloric acid and
extracted with diethyl ether.
For measuring recovery, the OD of the starting solution was
determined and the recovery calculated according to
recovery (%) )
2 × OD(control well) × OD (starting solution) × 100
Three internal standards were used, one with low and two with
high flux rates (chlorothiazide, metoprolol, and coumarin, respec-
tively57).
Caco2 Assay. Cell Culture. Caco2 cells were obtained from
DSMZ, Braunschweig, Germany. Cells were maintained in 25 cm2
flasks in Dulbecco’s modified Eagle medium (ccpro, Neustadt,
Germany) containing high glucose (4.5 g/L), 10% FCS, 0.1 mM
nonessential amino acids, 2 mM l-glutamin, 0.1 mg/mL strepto-
mycin, and 100 U/mL penicillin at 37°C, 5% CO2, and 95% relative
humidity. Cells utilized for the transport studies were always
between passage 4 and 15 after receipt from the culture collection.
For transport studies, Caco2 cells were grown on Costar Transwell
Plates (polycarbonate membrane, 0.4 µm pore size) for 21 days.
Growth and differentiation were monitored by the measurement of
the transepithelial electrical resistance (TEER) during culture. The
permeation assay was performed after the TEER of a monolayer
had reached a value around 400 Ω/cm2.
Validation. For validation of the liquid/liquid extraction method,
recoveries in triplicates at three different concentrations (high,
medium, low) in the range of the calibration curve were determined.
The precision of the measurements was high, which was expressed
by correlation coefficients higher than 0.96. The lowest calibration
standards were chosen as the limit of quantification.
Molecular Modeling Procedure. Geometry Optimization. The
geometry optimization (minimization) of compounds has been
carried out using the Molecular Mechanics ff99 force field
implemented in the AMBER 7 suite of programs.59 The simulation
systems were minimized by 5000 steps of steepest descent followed
by 10 000 steps of conjugate gradient.
Transport Assay. The transport assay was performed as
described.46 The appropriate drug solutions (50 µM) were added
to the apical side at time zero, and samples from the apical side
were taken at 0 and 60 min and from the basolateral side at 15, 30,
and 60 min. The removed sample volume was replaced each time
by fresh basolateral buffer. The concentrations of the compounds
in the samples were determined using LC-MS/MS. The integrity
of the monolayer was checked at the end of the experiment by
assessing the transport of the paracellular marker lucifer yellow at
a concentration of 100 µM. Each experiment was performed in
triplicate.
The apparent permeability coefficients (Papp) for each well were
calculated using the following equation
Simulated Annealing Molecular Dynamic Simulation. After
the minimization described above, the following simulated anneal-
ing steps were repeated 10 times: First, the simulation system was
heated from 200 to 1500 K at run time of 10 ps (step size 0.5 fs,
20 000 steps). Afterward, 30 ps of molecular dynamic simulation
(step size 0.5 fs, 60 000 steps) at constant temperature (1500 K)
was performed. Finally, the system was cooled to 200 K (step size
0.5 fs, 60 000 steps). Temperature was regulated by coupling to an
external bath (Berendsen’s method60) using a bath coupling constant
of 1.2 ps. The generalized Born model was applied to account for
solvation effects.61 This procedure led to 10 low-energy structures
which were superimposed and grouped into families by their rms
values. An average structure of the family containing the most
members was calculated and used for further investigations.
Ab Initio Electronic Structure Calculations. The average
structure of the family obtained in the last step was further enhanced
by performing an ab initio geometry optimization using the B3LYP
density functional method.62 The 6-31G** basis set of Pople et al.63
with d-polarization functions for non-hydrogens and p-polarization
functions for hydrogens was used. All ab initio electronic structure
calculations were performed by the Gaussian 98 (Rev. A.7)64 suite
of programs.
P
app (cm/s) ) (∆cVR/∆tA)co
where ∆c/∆t is the cumulative amount transported as a function of
time (seconds), VR is the volume of the receiver chamber (mL), A
is the surface area of the cell monolayer (cm2), and co is the
concentration of compound at t ) 0.
In Vivo Pharmacokinetics. Male Wistar rats weighing 300-
330 g (Harlan Winkelmann) were housed in a temperature-
controlled room (20-22 °C) and maintained in a 12h light/12h dark
cycle. Food and water were available ad libitum. They were
anaesthetized with a ketamine (135 mg/kg)/xylazine (10 mg/kg)
mixture and cannulated with silicone tubing via the right jugular
vein and attached to the skull with dental cement.58 Prior to the
first blood sampling, animals were connected to a counterbalanced
system and tubing to perform blood sampling in the freely moving
rat.
The oral administration of 12 at the dose of 40 mg/kg body
weight was performed in awake rats (n ) 5) by using a feeding
needle. The compound was dissolved in 0.5% CMC and given at
a volume of 5 mL/kg. Blood samples (0.3 mL) were taken at 0,
0.5, 1, 2, 4, 6, 8, and 24 h postdose and collected in heparinized
tubes. They were centrifuged at 3000g for 10 min, and plasma was
harvested and kept at -20 °C until analyzed.
The mean of absolute plasma concentrations ((SEM) was
calculated for the five rats and the regression was performed on
group mean values. The pharmacokinetic analysis was performed
using a noncompartment model (PK Solutions 2.0, Summit
Research Services).
The overall procedure leads to a most likely lowest energy
conformation of the inhibitors studied in this publication.
Acknowledgment. O.S. is grateful to the Egyptian Govern-
ment for a scholarship. Thanks are due to the Fonds der
Chemischen Industrie, which supported this work by a grant.
We thank Mrs. Anja Palusczak for her help in performing the
biological assays. Thanks are due to Dr. J. Zapp for his help in
collecting the 2D-NMR spectra and to Dr. T. Hutschenreuter
for performing the HPLC purity tests for the compounds.
HPLC-MS/MS analysis and quantification of the samples was
carried out on a Surveyor-HPLC-system coupled with a TSQ

758 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2
Salem et al.
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Supporting Information Available: Calculation of distances
between five features of compounds 7-9 and 12, using simulated
annealing techniques and ab initio calculations (Table S1), results
from elemental analysis (Table S2), and routine H NMR and IR
spectroscopic data. This material is available free of charge via
the Internet at http://pubs.acs.org.
1
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