Cyclohex-1-ene carboxylic acids: Synthesis and biological evaluation of novel inhibitors of human 5α reductase

Article abstract of DOI:10.1002/ardp.200390001

In search of novel nonsteroidal mimics of steroidal inhibitors of 5α reductase, 4-(2-phenylethyl)cyclohex-1-ene carboxylic acids 1-5 were synthesized with different substituents in para position of the phenyl ring (1: N,N-diisopropylcarbamoyl, 2: phenyl,

Full text of DOI:10.1002/ardp.200390001

Arch. Pharm. Pharm. Med. Chem. 2003, 1, 31–38
Cyclohex-1-ene Carboxylic Acids 31
Eckhard Baston,
Ola I. A. Salem,
Rolf W. Hartmann
Cyclohex-1-ene Carboxylic Acids: Synthesis and
Biological Evaluation of Novel Inhibitors of Human
5␣ Reductase
Pharmaceutical and Medicinal
Chemistry, Saarland University,
Saarbrücken, Germany
In search of novel nonsteroidal mimics of steroidal inhibitors of 5α reductase, 4-(2-
phenylethyl)cyclohex-1-ene carboxylic acids 1–5 were synthesized with different
substituents in para position of the phenyl ring (1:N,N-diisopropylcarbamoyl, 2:phe-
nyl, 3: phenoxy, 4: benzoyl, and 5: benzyl).The principal synthetic approach for the
desired compounds consisted of a Wittig olefination between 1,4-dioxaspiro[4.5]-
decane-8-carbaldehyde (4 g) and the appropriate phosphonium salts. The com-
pounds were tested for inhibition of human 5α reductase isozymes 1 and 2 using DU
145 cells and preparations from prostatic tissue, respectively.They turned out to be
good inhibitors of the prostatic isozyme 2 with compound 1 being the most potent
one (IC50 = 760 nM). Isozyme 1 was only slightly inhibited. It is concluded that the
novel structures are appropriate for being further optimized, aiming at the develop-
ment of a novel drug for the treatment of benign prostatic hyperplasia.
Keywords: Steroid 5α reductase; Isozymes 1 and 2; DU 145 cells; Nonsteroidal in-
hibitors; Steroidomimetics; Benign prostatic hyperplasia; Cyclohex-1-ene carboxyl-
ic acids
Received: March 13, 2002 [FP684]
cause of the side effects of steroidal drugs which in part
are due to their steroidal structure, we and other groups
have been trying to develop nonsteroidal compounds
Introduction
The enzyme steroid 5α reductase (EC 1.3.99.5) irre-
versibly catalyzes the reduction of testosterone (T) to the
tissue-specific
[9]. In different classes we found highly active com-
pounds in vitro.One class of compounds turned out to be
very potent in vivo as well [10].In this paper, we describe
a novel approach to the synthesis of conformationally
flexible mimics of Epristeride.This investigation was initi-
ated by our finding that the more rigid nonsteroidal mim-
ics MK73 [11] as well as EB2 and 16 [12] (Chart 1) had
shown strong inhibition of human 5α reductase type 2
(IC50 values of 410 nM, 460 nM, and 200 nM, respective-
ly). In the following paragraphs we describe the synthe-
sis of novel 4(2-phenylethyl)cyclohex-1-ene carboxylic
acids 1–5 (Chart 1) and the evaluation of their biological
activity toward human 5α reductase isozymes 1
and 2.
androgen
5α-dihydrotestosterone
(DHT). T, the androgen secreted by the testes, is the
main circulating androgen in man, responsible for the in-
crease in muscle mass, spermatogenesis, and libido. In
some tissues, such as the skin or the prostate, T acts as
a prehormone, which is converted to the more potent an-
drogen DHT through local 5α reductase [1]. DHT is be-
lieved to be the causative agent in a variety of conditions,
including benign prostatic hyperplasia (BPH), acne, hir-
sutism, and androgenic alopecia [2–5].Two different 5α
reductase isozymes, named type 1 and type 2, were
identified, which are characterized by distinct molecular
genetics, structural, and biochemical properties and by
different tissue localization [6].Isozyme 2 is mainly locat-
ed in the prostate, whereas isozyme1 is found in the pe-
riphery [6]. Finasteride and Epristeride (Chart 1) are
highly potent steroidal inhibitors, the former having been
used for the treatment of BPH for some years [7, 8]. Be-
Chemistry
For the preparation of the cyclohex-1-ene carboxylic ac-
id derivatives 1–5 a convergent synthetic approach was
chosen, including a Wittig reaction as key step. The
phosphonium salt 1 e (Scheme 1) was synthesized from
4-methylbenzoic acid via aminolysis of the acid chloride
with diisopropylamine (1 g), bromination using N-bromo-
Correspondence: Rolf W. Hartmann, Pharmaceutical and
Medicinal Chemistry, Saarland University, P.O. Box 151150,
D-66041 Saarbrücken, Germany. Phone: +49 681 302 3424,
fax: +49 681 302 4386. e-mail: rwh@mx.uni-saarland.de.
© 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
0365-6233/01/0031 $ 17.50+.50/0

32 Baston et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 31–38
Scheme 1a

Arch. Pharm. Pharm. Med. Chem. 2003, 336, 31–38
Cyclohex-1-ene Carboxylic Acids 33
succinimide (1 f), and finally reaction with triphenylphos-
phine. In a similar way the phosphonium salt 4 e was ob-
tained from 2-(4-bromomethylphenyl)-2-phenyl-[1,3]-
dioxolane [13] and subsequent treatment with triphenyl-
phosphine. The 4-phenoxy and 4-phenyl substituted
benzyltriphenylphosphonium bromides were prepared
in very good yields according to published procedures
[14, 15].
Deprotection of 1 c–5 c with diluted hydrochloric acid
gave the cyclohexanone derivatives 1 b–5 b in high
yields. Conversion into the enol triflates 1 a–5 a was
achieved using 2,6-di-tert-butyl-4-methylpyridine as a
sterically hindered base and trifluoromethanesulfonic
acid [20].
Subsequent Heck-type carboxylation with palladium(II)
acetate, triphenylphosphine, and potassium acetate un-
der a carbon monoxide atmosphere according to a re-
cent procedure developed by Cacchi [21] gave direct ac-
cess to the desired carboxylic acids 1–5 in good yields.
This direct procedure is superior to the strategy via the
corresponding carboxylic acid ester intermediates [22,
23].
The preparation of the aldehyde 4 g started from com-
mercially available 1,4-dioxaspiro[4.5]decan-8-one,
which was converted into 8-methylene-1,4-dioxa-
spiro[4.5]decane using methyltriphenylphosphonium
bromide in dimethyl sulfoxide with sodium hydride as a
base [16].The obtained exocyclic olefin was subjected to
a hydroboration, directly followed by an oxidation using
pyridinium chlorochromate, resulting in 4 g in an accept-
able yield [17, 18].
Biological results and discussion
With compound 4 g and the corresponding phosphoni-
um salts aWittig reaction was performed to yield the ole-
fins 1 d–4 d (Scheme 2).E-Z isomeric mixtures were ob-
tained, which were not separated but directly hydrogen-
ated using palladium on charcoal to give quantitatively
the saturated analogs 1 c–4 c. Upon hydrogenation of
compound 4 d for 24 h in ethyl acetate additional deketal-
ization and subsequent reduction of the carbonyl group
of the benzophenone moiety was oberved (5 c) [19].
Inhibition of human 5␣ reductase isozymes
The synthesized compounds were tested for inhibitory
potency using prostate homogenates for the human type
2 isozyme and the DU 145 cell line – a prostate cancer
metastasis of the brain – for the human type 1 isozyme
[24, 25].As can be seen fromTable 1, compound 1 bear-
ing the diisopropylcarboxamide moiety turned out to be
the most potent compound toward the prostatic isozyme.
Scheme 2b

34 Baston et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 31–38
Table 1. Inhibition of human steroid 5α reductase iso-
zymes by 4-substituted cyclohex-1-enecarboxylic acids
1–5.
Conclusion
From our results it can be concluded that the phen-
ylethylcyclohex-1-ene carboxylic acid moiety is a good
mimic of the basic structure of the potent steroidal inhibi-
tor Epristeride.The phenyl ring introduced into this mole-
cule results in an active inhibitor (compound 2). As this
group can easily be further substituted, the synthesis of
more potent inhibitors is feasible.We are currently work-
ing on the preparation of such compounds.
Compd
R
Human
Human
Acknowledgements
Type 2a,c
Type 1b,d
Thanks are due to the Fonds der Chemischen Industrie,
who supported this work by a grant, and to Mrs. Anja
Palusczak for her help in performing the biological as-
says.We are grateful to the Egyptian Government and to
Assiut University for the scholarship to Mrs. Ola Salem.
% inh. 10 µM% inh. 10 µM
[IC50, µM]
[IC50, µM]
1
2
3
4
CON(i-Propyl)2
Phenyl
[0.76]
[1.00]
[1.50]
[2.10]
[4.10]
[0.045]
[1.10]
16
57
n.d.
48
Phenoxy
Benzoyl
Benzyl
[0.005]
[0.003]
5
n.d.
Experimental
Finasteride
Epristeride
General
Materials obtained from commercial suppliers were used with-
out further purification. Solvents for reactions under anhydrous
conditions were dried according to standard procedures.All re-
actions, except those involving water as a reagent, were con-
ducted under nitrogen atmosphere. Melting points were meas-
ured on a ReichertThermometer hot stage microscope and are
uncorrected. Silica gel TLC and column chromatography were
performed on Merck TLC 60F-254 (0.25 mm) and Merck
Kieselgel 60. IR spectra were determined with a Perkin Elmer
Infrared Spectrometer 398 (KBr). 1H NMR spectra were meas-
ured on a Bruker AM 400 at 400 MHz in CDCl3 or d6-DMSO so-
lution and chemical shifts are reported in δ parts per million
downfield from tetramethylsilane. Elemental analyses indicat-
ed by the symbols of the elements were within 0.4 % of the
theoretical values and were performed by the Institute of Inor-
ganic Chemistry, Saarland University, Saarbrücken, Germany.
a
Human prostate homogenates; 200–300 µg protein,
pH 5.5.
b
c
d
DU 145 cell culture.
3
Substrate 1β2β H testosterone 210 nM.
3
Substrate H androstene-dione 5 nM. n.d: not deter-
mined.
This is not a surprise as it is in closest structural analogy
to Epristeride. Although showing an IC50 value in the na-
nomolar range, compound 1 is approximately two orders
of magnitude less potent than the parent steroidal inhibi-
tor. The fact that the aromatic substituted compounds
2–5 are only a little less potent than 1 is encouraging,
since they might be lead compounds for the develop-
ment of more potent derivatives. It is striking that the
phenyl compound 2 is more active than the phenoxy,
benzoyl, and benzyl derivatives (in decreasing order of
activity, compounds 3–5). Obviously the increase in the
length of the molecule and/or its conformational flexibility
has a negative effect on inhibitory potency. Towards the
human isozyme 1, the tested compounds were less ac-
tive. Compound 1 only showed a marginal inhibitory po-
tency.Compounds 2 and 4 were more active, but showed
less inhibition than toward type 2 isozyme. It cannot be
excluded that this phenomenon is due to pharmacoki-
netic effects, as in the assay whole cells are used and the
carboxylic acids might not easily permeate the cell wall
at the pH value of the assay.
(E/Z) 4-[2-(1,4-Dioxaspiro[4.5]dec-8-yl)-vinyl]-N,N-diisopropyl-
benzamide (1 d)
[4-(N,N-Diisopropylcarbamoyl)benzyl]triphenylphosphonium
bromide (1 e, 10.76 g, 19.2 mmol) was suspended in tetrahy-
drofuran (100 L) and butyllithium (12.5 mL, 1.6 M in hexane)
was injected via a syringe at 0 °C.The red mixture was warmed
to 25 °C and stirred for 15 min. 1,4-Dioxaspiro[4.5]decane-8-
carbaldehyde (4 g) (3.40 g, 20 mmol)) in tetrahydrofuran
(15 mL) was added and stirring was continued for 12 h. After
evaporation of the solvent, dichloromethane (200 mL) was add-
ed and the organic layer was washed with brine (2 × 50 mL),
dried (magnesium sulfate), and evaporated in vacuo.The resi-
due was chromatographed on silica gel using petroleum ether
(40–60 °C)-diethyl ether (1:1) as an eluent.Yield: 59 %, color-
less solid, mp 97–98 °C. IR (KBr) 2930, 2880, 1630, 1445,
1340, 1090, 1035, 830 cm–1. 1H NMR (CDCl3) δ 1.33 (m; 12 H);
1.43–1.82 (m; 8 H); 2.19 (m; 1 H, Ar-CH=CH-CH, E-Isomer);
2.60 (m; 1 H Ar-CH=CH-CH, Z-Isomer); 3.69 (m; 2 H); 3.95 (s;
4 H); 5.54 (dd; 1 H, J = 11.54 Hz, Ar-CH=CH-, Z-Isomer); 6.35
(d; 1 H, J = 11.82 Hz, Ar-CH=CH-, Z-Isomer); 6.20 (dd; 1 H, J =

Arch. Pharm. Pharm. Med. Chem. 2003, 336, 31–38
Cyclohex-1-ene Carboxylic Acids 35
8-[2-(4-Benzylphenyl)ethyl]-1,4-dioxaspiro[4.5]decane (5 c)
15.94 Hz, Ar-CH=CH-, E-Isomer); 6.38 (d; 1 H, J = 16.04 Hz,
Ar-CH=CH-, E-Isomer); 7.23 and 7.33 (dd; 4 H, J = 8.18 Hz).
E:Z ratio 73:27.
The reaction mixture was hydrogenated for 24 h using ethanol
as a solvent.Yield: 80 %, colorless oil. 1H NMR (CDCl3) δ 1.01–
2.11 (m; 11 H); 2.41–2.67 (m; 2 H); 3.89 (s; 4 H); 3.94 (s; 2 H);
7.04–7.18 (m; 9 H).
Compounds 2 d–4 d were prepared accordingly:
(E/Z) 8-(2-Biphenyl-4-yl-vinyl)-1,4-dioxaspiro[4.5]decane (2 d)
N,N-Diisopropyl-4-[2-(4-oxocyclohexyl)ethyl]benzamide (1 b)
Yield: 34 %, colorless solid, mp 65–75 °C. 1H NMR (CDCl3) δ
1.54–1.84 (2 m; 8 H); 2.21 (m; 1 H, Ar-CH=CH-CH, E-Isomer);
2.67 (m; 1 H, Ar-CH=CH-CH, Z-Isomer); 3.96 (s; 4 H); 5.54 (dd;
1 H, J = 11.50 Hz , Ar-CH=CH-, Z-Isomer); 6.39 (d; 1 H, J =
11.92 Hz, Ar-CH=CH-, Z-Isomer); 6.22 (dd; 1 H, J = 15.92 Hz,
Ar-CH=CH-, E-Isomer);6.42 (d;1 H, J = 16.36 Hz, Ar-CH=CH-,
E-Isomer); 7.30–7.33 (m; 9 H). E:Z ratio 53:47.
Compound 1 c (3.92 g, 10.5 mmol) was dissolved in ethanol
(20 mL) and diluted hydrochloric acid (120 mL, 3 %) was add-
ed.The mixture was stirred at 90–95 °C for 1 h and was then ex-
tracted with ethyl acetate (3 × 150 mL) after being cooled to
25 °C.The combined organic layers were washed with water (2
× 50 mL), dried (magnesium sulfate) and the solvent was evap-
orated. An oil was obtained, that crystallized upon standing.
Yield: 87 %, colorless solid, mp 75–80 °C. IR (KBr) 2920, 1710,
1620, 1435, 1365, 1335, 1160, 840 cm–1. 1H NMR (CDCl3) δ
1.21–1.46 (d; 12 H); 1.49–2.11 (m; 7 H); 2.18–2.51 (m; 4 H);
2.55–2.87 (m; 2 H); 3.54–3.95 (m; 2 H); 7.20 (s; 4 H).
(E/Z) 8-[2-(4-Phenoxy-phenyl)vinyl]-1,4-dioxaspiro[4.5]decane
(3 d)
Yield: 73 %, colorless solid, mp 97–98 °C. 1H NMR (CDCl3) δ
1.61 and 1.79 (2 m; 8 H); 2.18 (m; 1 H, Ar-CH=CH-CH, E-Iso-
mer); 2.62 (m; 1 H, Ar-CH=CH-CH, Z-Isomer); 3.96 (s; 4 H);
5.48 (dd; 1 H, J = 11.52 Hz , Ar-CH=CH-, Z-Isomer); 6.32 (d;
1 H, J = 11.52 Hz, Ar-CH=CH-, Z-Isomer); 6.10 (dd; 1 H, J =
15.92 Hz, Ar-CH=CH-, E-Isomer); 6.36 (d; 1 H, J = 15.92 Hz,
Ar-CH=CH-, E-Isomer); 6.92–7.10 (m; 9 H). E:Z ratio 80:20.
Compounds 2 b–5 b were prepared accordingly:
4-(2-Biphenyl-4-ylethyl)cyclohexanone (2 b)
Yield: 77 %, colorless solid, mp 72–74 °C. 1H NMR (CDCl3) δ
1.42–2.18 (m; 7 H); 2.34 (m; 4 H); 2.72 (t; 2 H, J = 7.94 Hz);
7.25–7.60 (m; 9 H).
(E/Z) 8-{2-[4-(2-Phenyl-[1.3]dioxolan-2-yl)-phenyl]-vinyl}-1,4-
dioxaspiro[4.5]decane (4 d)
4-[2-(4-Phenoxyphenyl)ethyl]cyclohexanone (3 b)
Yield: 85 %, colorless solid, mp 63–67 °C. 1H NMR (CDCl3) δ
1.51–1.81 (2 m; 8 H); 2.10 (m; 1 H, Ar-CH=CH-CH, E-Isomer);
2.60 (m; 1 H, Ar-CH=CH-CH, Z-Isomer); 3.95 (s; 4 H); 4.05 (s;
4 H); 5.49 (dd; 1 H, J = 11.48 Hz, Ar-CH=CH-, Z-Isomer); 6.32
(d; 1 H, J = 11.52 Hz, Ar-CH=CH-, Z-Isomer); 6.16 (dd; 1 H, J =
15.92 Hz, Ar-CH=CH-, E-Isomer); 6.36 (d; 1 H, J = 15.92 Hz,
Ar-CH=CH-, E-Isomer); 7.19–7.56 (m; 9 H). E:Z ratio 75:25.
Yield: 58 %, colorless solid, mp 59–60 °C. 1H NMR (CDCl3) δ
1.38–2.17 (m; 7 H); 2.38 (m; 4 H); 2.66 (t; 2 H, J = 7.94 Hz);
6.93–7.34 (m; 9 H).
4-[2-(4-Benzoylphenyl)ethyl]cyclohexanone (4 b)
Yield: 85 %, colorless solid, mp 103–107 °C. 1H NMR (CDCl3) δ
1.43–2.14 (3 m; 7 H); 2.29–2.47 (m; 4 H); 2.77 (t; 2 H, J = 7.74
Hz); 7.29–7.80 (m; 9 H).
4-[2-(1,4-Dioxaspiro[4.5]dec-8-yl)-ethyl]-N,N-diisopropylbenz-
amide (1 c)
4-[2-(4-Benzylphenyl)ethyl]cyclohexanone (5 b)
Yield: 52 %, colorless solid, mp 38–40 °C. 1H NMR (CDCl3) δ
1.43–2.26 (m; 7 H); 2.28–2.40 (m; 4 H); 2.72 (t; 2 H, J = 7.96
Hz); 3.95 (s; 2 H); 7.08–7.19 (m; 9 H).
Compound 1 d (4.0 g, 10.8 mmol) was hydrogenated using pal-
ladium on charcoal (10 %) in ethanol (100 mL) for 1 h.The cata-
lyst was filtered off and the solvent was evaporated and the
pure product was obtained.Yield: 98 %, colorless oil. IR (Film)
2920, 2880, 1630, 1440, 1340, 1005 cm–1. 1H NMR (CDCl3) δ
1.05–1.41 (d; 12 H); 1.47–1.89 (m; 11 H); 3.55–3.83 (m; 4 H);
3.92 (s; 4 H); 7.18 (s; 4 H).
Trifluoromethanesulfonic acid 4-{2-[4-(diisopropyl-carbamoyl)-
phenyl]-ethyl}-cyclohex-1-enyl ester (1 a)
Trifluoromethanesulfonic anhydride (0.6 g, 0.35 mL,
2.13 mmol) was injected into a solution of compound 1 b (0.5 g,
1.5 mmol) and 2,6-di-tert-butyl-4-methylpyridine (0.33 g, 1.6
mmol) in dichloromethane (20 mL) at 0 °C. The mixture was
warmed to 25 °C and stirred for 12 h. Ethyl acetate (100 mL)
was added and the organic layer was washed with diluted hy-
drochloric acid (5 %, 25 mL) and brine (25 mL), dried (magnesi-
um sulfate), filtered over celite and evaporated. Yield: 73 %,
colorless oil. IR 2910, 1625, 1440, 1365, 1330, 1210, 1130,
1100, 1030, 925 cm–1. 1H NMR (CDCl3) δ 1.3 (d; 12 H); 1.51–
1.88 (m; 9 H); 3.92 (m; 4 H); 6.28 (m; 1 H); 7.24 (s; 4 H).
Compounds 2 c–5 c were prepared accordingly:
8-(2-Biphenyl-4-ylethyl)-1,4-dioxaspiro[4.5]decane (2 c)
The reaction mixture was hydrogenated for 30 min using ethyl
acetate as a solvent.Yield: 85 %, colorless oil. 1H NMR (CDCl3)
δ 1.01–1.80 (m; 11 H); 2.28–2.45 (m; 2 H); 3.89 (s; 4 H); 6.93–
7.70 (m; 9 H).
8-[2-(4-Phenoxyphenyl)ethyl]-1,4-dioxaspiro[4.5]decane (3 c)
The reaction mixture was hydrogenated for 2 h using ethyl ace-
tate as a solvent. Yield: 85 %, colorless oil. 1H NMR (CDCl3) δ
0.83–1.79 (m; 11 H); 2.34–2.74 (m; 2 H); 3.91 (s; 4 H); 6.62–
7.46 (m; 9 H).
Compounds 2 a–5 a were prepared in a similar way:
Trifluoromethanesulfonic acid 4-(2-biphenyl-4-ylethyl)cyclohex-
1-enyl ester (2 a)
Yield: 79 %, pale yellow oil. 1H NMR (CDCl3) δ 1.25–2.35 (m;
9 H); 2,69 (t; 2 H, J = 7.52 Hz); 5.74 (s; 1 H); 7.23–7.59 (m; 9 H).
8-{2-[4-(2-Phenyl-[1,3]dioxolan-2-yl)phenyl]ethyl}-1,4-dioxa-
spiro[4.5]decane (4 c)
Trifluoromethanesulfonic acid 4-[2-(4-phenoxyphenyl)ethyl]-
cyclohex-1-enyl ester (3 a)
The reaction mixture was hydrogenated for 30min using ethyl
acetate as a solvent.Yield: 98 %, colorless solid, mp 93–94 °C.
1H NMR (CDCl3) δ 1.06–1.88 (m; 11 H); 2.59 (t; 2 H, J =
6.80 Hz); 3.88 (s; 4 H); 4.01 (s; 4H); 6.93–7.59 (m; 9 H).
The reaction mixture was stirred for 3 h at 25 °C and the com-
pound was isolated by column chromatography (silica gel)

36 Baston et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 31–38
2.73 (s; 2 H); 6.83 (s; 1 H); 7.38 (d; 2 H, J = 7.52 Hz); 7.54–7.71
(m; 7 H); 11.91 (s; 1 H). Anal. (C22H22O3) C, H, N.
using hexane-ethyl acetate (4:1) as an eluent.Yield: 63 %, pale
yellow solid, mp 68–70 °C. 1H NMR (CDCl3) δ 1.49–2.34 (m;
9 H); 2.63 (t; 2 H, 3J = 7.74 Hz); 5.73 (s; 1 H); 6.94 and 7.13 (dd;
4 H, J = 8.84 Hz); 6.98–7.10 and 7.30–7.34 (2 m, 5 H).
4-[2-(4-Benzylphenyl)ethyl]cyclohex-1-ene carboxylic acid (5)
Yield: 32 %, colorless crystals, mp (petroleum ether
(40–60 °C)/ethyl acetate): 177–178 °C. IR (KBr) 3000, 2940,
1680, 1640, 1515, 1490, 1450, 1430, 1420, 1280 cm–1.1H NMR
(DMSO-d6) δ 1.15–1.73 (m; 9 H); 2.57 (t; 2 H, J = 7.74 Hz); 3.88
(s; 2 H); 6.83 (s; 1 H); 7.10–7.33 (m; 9 H); 12.10 (s; 1 H).
Trifluoromethanesulfonic acid 4-[2-(4-benzoylphenyl)ethyl]-
cyclohex-1-enyl ester (4 a)
The product was isolated by column chromatography (silica
gel) using hexane-ethyl acetate (7:3) as an eluent.Yield: 53 %,
pale yellow oil. 1H NMR (CDCl3) δ 1.51–2.37 (m; 9 H); 2.74 (t;
2 H, J = 7.74 Hz); 5.74 (s; 1 H); 7.28 and 7.75 (dd; 4 H, J = 8,40
Hz); 7.46–7.81 (m; 5 H).
Inhibition of 5α reductase in vitro
Enzyme inhibition test
Trifluoromethanesulfonic acid 4-[2-(4-benzylphenyl)ethyl]cy-
clohex-1-enyl ester (5 a)
Preparation of tissue
Prostatic enzyme was prepared according to the method of
Liang et al. [24] with slight modifications [26]. All the following
operations were performed at 0–4 °C. The human prostates
were dissected free from fat and connective tissue, cut into
pieces and weighed. Per 1 g of tissue, 3 mL of 20 mM citrate
buffer, pH 5.5, containing 0.32 mM sucrose and 1 mM dithio-
threitol (DTT) were added. The tissue was homogenized by
10-s strokes at 20,500 rpm of an ultraturax (IKA) in 60-s inter-
vals, filtered through cheesecloth and centrifuged for 60 min at
105,000 g.The pellet obtained was resuspended in citrate buff-
er.The centrifugation was repeated, the final pellet resuspend-
ed in a minimum volume of citrate buffer and stored in 300 µL
portions at –70 °C. The 105,000 g pellet contains nuclei, mito-
chondria, and microsomes and is referred to as the enzyme
preparation. The protein content was determined and was in
the range of 15–25 mg/mL.
The reaction mixture was stirred for 4 h at 25 °C and the product
was isolated by column chromatography (silica gel) using
hexane-ethyl acetate (4:1) as an eluent.Yield: 36 %, colorless
oil. 1H NMR (CDCl3) δ 1.44–2.36 (m; 9 H); 2.61 (t; 2 H, J =
7.74 Hz); 3.95 (s; 2 H); 5.72 (s; 1 H); 7.07–7.30 (m; 9 H).
4-{2-[4-(N,N-Diisopropylcarbamoyl)phenyl]ethyl}cyclohex-1-ene
carboxylic acid (1)
Compound 1 a (0.50 g, 1 mmol), potassium acetate (0.39 g,
4 mmol), palladium(II) acetate (9 mg) and triphenylphosphine
(21 mg) were stirred in N,N-dimethylformamide (10 mL) under
CO atmosphere at 25 °C for 14 h.Water (30 mL) was added and
the solution was acidified with diluted hydrochloric acid (20 mL,
0.5 N). The mixture was extracted with dichloromethane (3 ×
25 mL) and the combined organic layers were washed with
brine (25 mL), dried (magnesium sulfate) and the solvent was
evaporated.The remaining brownish oil was dissolved in dilut-
ed sodium hydroxide solution (0.5 N, 50 mL) and extracted with
diethyl ether (2 × 25 mL). The aqueous layer was acidified
(hydrochloric acid, 2 N, 30 mL) and extracted with diethyl ether
(3 × 25 mL), dried (magnesium sulfate) and evaporated.The re-
maining residue was recrystallized from petroleum ether
(40–60 °C)/ethyl acetate of increasing polarity. Yield: 42 %,
colorless crystals, mp 175–177 °C. IR (KBr) 3000, 2930, 1685,
1640, 1450, 1345, 1275, 1040 cm–1.1H NMR (DMSO-d6) δ 1.23
(s; 12 H); 1.83–2.37 (3 m; 9 H); 2.64 (t; 2 H, J = 7.74 Hz); 3.62
(m; 2 H); 6.84 (s; 1 H); 7.17 and 7.24 (dd; 4 H, J = 7.96 Hz);
12.07 (s; 1 H). Anal. (C22H31NO3) C, H, N.
Incubation procedure
The assay was performed as described [24] with modifications
[26]. All values were determined in duplicate. The incubation
was carried out for 30 min at 37 °C in a total volume of 250 µL
using citrate buffer (40 mM, pH 5.5). The incubation mixture
contained approximately 200–300 µg human protein, 100 µM
NADPH, 0.21 µM T including 100 nCi [1β,2β-3H]T and 2 %
DMSO with or without test compound (10 µM). In case of ex-
ceeding 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 ad-
dition 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.
Compounds 2–5 were prepared accordingly:
4-(2-Biphenyl-4-ylethyl)cyclohex-1-ene carboxylic acid (2)
Yield: 40 %, colorless crystals, mp (petroleum ether (40–
60 °C)/acetone):225–226 °C.IR (KBr) 3000, 2920, 1670, 1645,
1485, 1430, 1280 cm–1. 1H NMR (DMSO-d6) δ 1.24–2.34 (m;
9 H); 2.67 (t; 2 H, J = 7.74 Hz); 6.85 (s; 1 H); 7.30 and 7.57 (dd;
4 H, J = 8.18 Hz); 7.33–7.46 and 7.63 (2 m; 5 H); 12.06 (s; 1 H).
Anal. (C21H22O2) C, H, N.
Human type 1 inhibition: DU 145-assay [27]
Intact human prostatic carcinoma DU145 cells were used as
the source of type1 5α reductase [28].The inhibitory potencies
of the compounds were determined by monitoring the conver-
sion of the tritiated substrate androstenedione (5 nM) to an-
drostanedione during an incubation period of 6 h. A day before
the experiment, DU145 cells were seeded in a 24-multiwell-
plate at a density of 180,000 cells/well and allowed to become
adherent overnight. Compounds to be tested were dissolved in
DMSO and 5 µL of each were added to the cells in a final vol-
ume of 0.5 mL complete medium.Inhibitors were first screened
at concentrations of 10 µM in an initial test and in cases exceed-
ing 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 as a positive control for inhibition Fin-
asteride (80, 60, 40, 20 nM). After the 6h incubation period in
5 % CO2 at 37 °C the medium samples were extracted twice
4-[2-(4-Phenoxyphenyl)ethyl]cyclohex-1-ene carboxylic acid (3)
Yield: 24 %, colorless crystals, mp (petroleum ether (40–
60 °C)/acetone):206–207 °C.IR (KBr) 3000, 1680, 1640, 1590,
1505, 1490, 1430, 1280, 1235, 1165, 870, 850 cm–1. 1H NMR
(DMSO-d6) δ 1.22–2.31 (m; 9 H); 2.61 (t; 2 H, J = 7.74 Hz); 6.84
(s; 1 H); 6.92 and 7.22 (dd; 4 H, J = 8.62 Hz); 6.95–7.12 and
7.35–7.39 (2 m; 5 H); 12.05 (s; 1 H). Anal. (C21H22O3) C, H, N.
4-[2-(4-Benzoylphenyl)ethyl]cyclohex-1-ene carboxylic acid (4)
Yield: 18 %, white powder, mp (hexane/ethyl acetate): 197–
198 °C. IR (KBr) 3000, 2930, 1680, 1660, 1610, 1320, 1280,
940, 930, 700 cm–1. 1H NMR (DMSO-d6) δ 1.25–2.32 (m; 9 H);

Arch. Pharm. Pharm. Med. Chem. 2003, 336, 31–38
Cyclohex-1-ene Carboxylic Acids 37
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with 1 mL of diethyl ether and the steroids were separated by
HPLC. Results are expressed as amount of formed andros-
tanedione as percentage of control values.
HPLC procedure
Steroid separation was performed [26] similarly to the method
of Cook et al. [29].The steroids were dissolved in 50 µL metha-
nol and 25 µL was injected into the computer-controlled HPLC
system, which was checked before using labelled reference
controls. Radioactivity was measured using 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 dihydro-
androstenedione methanol/water (50/50, w/w) was used.
Calculation procedure
The amount of DHT formed was calculated (% DHT).The zero
value was subtracted from the control (cv) and inhibition (iv)
values (cvcorr and ivcorr). Inhibition (I) was calculated using the
following equation: % I = (1 – ivcorr/cvcorr)100.
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International Union of Pure and
Applied Chemistry (IUPAC)
P. Heinrich Stahl / Camille G.Wermuth (Eds.)
Handbook of Pharmaceutical Salts
An essential step
in the preclinical phase
of drug development
Properties, Selection, and Use
2002. Approx 400 pages. Hardcover. Approx E 149.00* / £ 75.00 / sFr 250.00.
ISBN 3-906390-26-8. Publication date: March 2002
*The e-Price is only valid for Germany.
O–
Cl
Na+
O
NH
Cl
Contents:
The majority of medicinal chemists in pharmaceutical industry whose primary focus is the
design and synthesis of novel compounds as future drug entities are organic chemists for whom
salt formation is often a marginal activity restricted to the short-term objective of obtaining
crystalline material, and, because a comprehensive resource that addresses the preparation,
selection, and use of pharmaceutically active salts has not been available, may forego the
opportunities for increased efficacy and improved drug delivery provided by selection of an
optimal salt.To fill this gap in the pharmaceutical bibliography, we have gathered an international
team of seventeen authors from academia and pharmaceutical industry who, in the contributions
to this volume, present the necessary theoretical foundations as well as a wealth of detailed
practical experience in the choice of pharmaceutically active salts.
The Physicochemical Background: Fundamentals of Ionic
Equilibria • Solubility and Dissolution of Weak Acids, Bases,
and Salts • Evaluation of Solid-State Properties of Salts •
Pharmaceutical Aspects of the Drug Salt Form • Biological
Effects of the Drug Salt Form • Salt-Selection Strategies • A
Procedure For Salt Selection and Optimization • Large-Scale
Aspects of Salt Formation: Processing of Intermediates and
Final Products • Patent Aspects of Drug-Salt Formation •
Regulatory Requirements for Drug Salts in the European
Union, Japan, and the United States • Selected Procedures
for the Preparation of Pharmaceutically Acceptable Salts of
Acids and Bases • Monographs on Acids and Bases
For customers in all other countries: John Wiley & Sons, Ltd., Baffins Lane
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