Tetrahydronaphthalenes: Influence of heterocyclic substituents on inhibition of steroidogenic enzymes P450 arom and P450 17

Article abstract of DOI:10.1021/jm950377t

In search of new leads for selective inhibition of estrogen and androgen biosynthesis, respectively, heterocyclic substituted 2-(arylmethylene)-1- tetralones (1-4, 9-17), 2-(arylhydroxymethyl)-1-tetralones (5-8), exo- 1a,2,3,7b-tetrahydro-1H-cyclopropa[a]

Full text of DOI:10.1021/jm950377t

834
J . Med. Chem. 1996, 39, 834-841
Tetr a h yd r on a p h th a len es: In flu en ce of Heter ocyclic Su bstitu en ts on In h ibition
of Ster oid ogen ic En zym es P 450 a r om a n d P 450 17
Gerald A. Wa¨chter, Rolf W. Hartmann,* Tom Sergejew, Gertrud L. Gru¨n, and Dorothea Ledergerber
Fachrichtung 12.1 Pharmazeutische Chemie, Universita¨t des Saarlandes, P.O. Box 15 11 50, D-66041 Saarbru¨cken, Germany
Received May 22, 1995X
In search of new leads for selective inhibition of estrogen and androgen biosynthesis,
respectively, heterocyclic substituted 2-(arylmethylene)-1-tetralones (1-4, 9-17), 2-(aryl-
hydroxymethyl)-1-tetralones (5-8), exo-1a,2,3,7b-tetrahydro-1H-cyclopropa[a]naphthalenes
(18-24), and 3-alkyl substituted 4,5-dihydronaphtho[1,2-c]pyrazoles (25-27) were synthesized
and tested for inhibitory activity toward four steroidogenic enzymes, P450 arom, P450 17, P450
18, and P450 scc, as well as another P450 enzyme, thromboxane A2 (TXA2) synthase. The test
compounds inhibited human placental P450 arom, showing a wide range of inhibitory potencies.
(Z)-4-Imidazolyl compound 17 was the most potent inhibitor, with a relative potency (rp) of
110 [rp of aminoglutethimide (AG) ≡ 1, rp of fadrozole ) 359]. A competitive type of inhibition
was shown by the (E)-4-imidazolyl compound 16 (rp ) 71). On the other hand some of these
compounds inhibited rat testicular P450 17. Maximum activity was shown by the 3-pyridyl
compound 20 (rp ) 10, rp of ketoconazole ≡ 1). 20 was the only compound which exhibited a
marked inhibition of TXA2 synthase (IC50 ) 14.5 µM; IC50 of dazoxiben ) 1.1 µM). Regarding
selectivity toward the steroidogenic enzymes, compound 16 was relatively selective toward
P450 arom, whereas compound 20 was relatively selective toward P450 17. (P450 arom: Km
testosterone ) 42 nM, Ki 16 ) 33 nM, Ki 20 ) 3 µM. P450 17: Km progesterone ) 7 µM, Ki 16
) 9 µM, Ki 20 ) 80 nM). 17 and 24 were not selective since they showed strong inhibition of
P450 arom (Ki 17 ) 26 nM, Ki 24 ) 0.12 µM) and P450 17 (Ki 17 ) 0.7 µM, Ki 24 ) 0.11 µM).
Ch a r t 1
In the last few years several publications have ap-
peared dealing with the development of nonsteroidal
inhibitors of aromatase (P450 arom, CYP 19). Inhibitors
of this enzyme are potential therapeutics for the treat-
ment of estrogen dependent diseases, such as breast
cancer.1 Several structure-activity studies have re-
sulted in highly active inhibitors,2-19 some of which are
presently under clinical evaluation (fadrozole, vorozole,
letrozole, and anastrozole).20, 21 An important structural
feature of potent nonsteroidal inhibitors is an accessible
heterocyclic nitrogen atom (N). Although showing wide
variety in their structures, highly active compounds
interact with the enzyme in an identical or at least very
similar fashion. As shown by difference spectroscopic
experiments,18,22,23 they complex the heme iron of the
enzyme by means of their heterocyclic N, while the rest
of the molecule interacts with the apoprotein moiety of
the active site.
be an alternative to antiandrogens in the treatment of
androgen dependent diseases such as prostate can-
cer.27,33 The antimycotic ketoconazole, which is also an
inhibitor of P450 17,26 has been useful in the treatment
of prostate cancer in men.34 Because of its low selectiv-
ity,35 it is not commonly accepted for wide use.
The benzocycloalkenes might be appropriate tools to
elucidate the structural requirements for selective
inhibition of both P450 arom and P450 17. In the
present paper the influence of different heterocyclic
substituents on P450 arom and P450 17 inhibition of
tetrahydronaphthalenes is examined. In the following
paragraphs the syntheses of compounds 1-24 (Chart
2) and 25-27 and their inhibitory activities toward the
title enzymes (P450 arom, P450 17) and other steroido-
genic P450 enzymes (namely cholesterol side chain
cleavage enzyme (P450 scc, CYP 11A1), 18-hydroxylase
(P450 18, CYP 11B2)) as well as the P450 enzyme
thromboxane A2 (TXA2) synthase will be described.
Structure-activity studies of our group with 4-py-
ridyl-substituted benzocycloalkenes of the types shown
in Chart 1, also led to highly active inhibitors of P450
arom.9,18,19,24
Some of the compounds turned out to be selective
inhibitors of P450 arom, since they did not inhibit other
steroidogenic P450 enzymes.19 On the other hand, some
compounds showed inhibition of 17R-hydroxylase/C17,20-
lyase (P450 17, CYP 17) as well.25 In contrast to P450
arom, this enzyme has not received much attention as
a therapeutic target. Most azole-type antimycotics, i.e.
14R-demethylase inhibitors, show a more or less pro-
nounced inhibition of P450 17.26,27 Only few other
nonsteroidal compounds are known to inhibit the
enzyme.25,28-32 Inhibitors of P450 17, however, might
Ch em istr y
The substituted (E)-2-methylene-1-tetralones 1-4 and
9-16 were synthesized by condensation of 1-tetralone
with the respective aromatic aldehydes which were
either purchased or prepared according to described
methods (Scheme 1, Table 1).
* Author to whom correspondence should be addressed. Tel.: 49
681 302 3424. Fax: 49 681 302 4386.
X Abstract published in Advance ACS Abstracts, J anuary 15, 1996.
0022-2623/96/1839-0834$12.00/0 © 1996 American Chemical Society

Tetrahydronaphthalenes
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 4 835
Ch a r t 2
results in a shift of the vinyl-H singlets by approxi-
mately 0.9 ppm, from 8.04-7.62 to 7.11-6.73, indicating
formation of the corresponding Z-isomers). After ir-
radiation of (E)-2-(4-imidazolylmethylene)-1-tetralone
16 the newly formed Z-2-(4-imidazolylmethylene)-1-
tetralone 17 was isolated (method G, Table 1).
Reduction of the (E)-2-phenylmethylene-1-tetralone
1 with hydrazine hydrate and KOH is known to yield
the 1a,2,3,7b-tetrahydro-1-phenyl-1H-cyclopropa[a]naph-
thalene 18 (Scheme 2).47 We have described the ap-
plication of this method to the (E)-2-(4-pyridylmethylene)-
1-tetralone 4 recently.19 The configuration of the
resulting 1a,2,3,7b-tetrahydro-1-phenyl-1H-cyclopropa-
[a]naphthalene 21 has been determined to be exo by 1H-
and 13C-NMR spectroscopy and by X-ray analysis.19 We
extended the use of this reaction to compounds 2, 3, 12,
15, and 16, yielding the 2- and 3-pyridyl-, 3-thienyl-,
3-pyrazolyl-, and 4-imidazolyl-substituted 1a,2,3,7b-
tetrahydro-1H-cyclopropa[a]naphthalenes 19, 20, 22,
23, and 24 (method H, Scheme 2, Table 1). As observed
with 21, only one diastereomer was obtained in each
reaction which also has to be assigned the exo config-
uration (pyridyl substituent exo corresponding to the
tetrahydronaphthalene moiety) as shown by comparison
Sch em e 1
1
of the H-NMR data. However, reaction of the diazines
9-11 and the thiazoles 13 and 14 with hydrazine
hydrate and KOH gave the 3-alkyl-substituted 4,5-
dihydronaphtho[1,2-c]pyrazoles 25-27 (method H,
Scheme 2, Table 1). Obviously the 2-pyrazoline inter-
mediates formed by addition of the hydrazone nitrogen
to the double bond aromatize to yield the heterocyclic
substituted pyrazoles (Scheme 3). Under the conditions
of the reaction (high temperature and excess reductive
agent) the latter compounds decompose, leading to the
production of the corresponding alkyl-substituted pyr-
azoles. Depending on the heterocyclic substituent, the
2-pyrazoline intermediates can also isomerize to the
corresponding 1-pyrazoline compounds. The cleavage
of molecular nitrogen from the latter results in cyclo-
propane formation (Scheme 3).
The condensation of benzaldehyde, 3-thiophenecar-
boxaldehyde, and the 2-, 3-, and 4-pyridinecarbox-
aldehydes to 1-tetralone was carried out using piperi-
dine/acetic acid as a catalyst, yielding the substituted
(E)-2-methylene-1-tetralones 1-4, and 12 as described
previously (method A).9,36 4-Imidazolecarboxaldehyde,37,38
3-pyrazolecarboxaldehyde,39 and 2-amino-5-thiazole-
carboxaldehyde40 were condensed to 1-tetralone in
sulfuric acid and gave the substituted (E)-2-methylene-
1-tetralones 13, 15, and 16 (method B). 2-Pyrazine-
carboxaldehyde,41 3-pyridazinecarboxaldehyde,42,43 and
4-pyrimidinecarboxaldehyde39,44 gave the substituted
2-(hydroxymethyl)-1-tetralones 5, 6, and 8 when reacted
with the lithium enolate of 1-tetralone (method C). The
2-[hydroxy(4-pyridazinyl)methyl]-1-tetralone 7 was ob-
tained by the diethylamine-catalyzed reaction of 4-pyr-
idazinecarboxaldehyde43,45 with 1-tetralone (method D).
Only one diastereomer (racemate) was obtained in case
of 5-8. This became apparent from the analytical data
(mp, 1H-NMR) and TLC experiments. The configuration
was not determined because the compounds were either
inactive or showed only poor activity (see below). Treat-
ment of the 2-(hydroxymethyl)-1-tetralones 5-7 with
sulfuric acid yielded the (E)-2-methylene-1-tetralones
9-11 (method F). In case of 2-[hydroxy(4-pyrimidinyl)-
methyl]-1-tetralone 8, the same reaction resulted in
decomposition. (E)-2-[5-(2-Aminothiazolyl)methylene]-
1-tetralone 13 was deaminated with isopentylnitrite in
tetrahydrofuran,46 yielding the (E)-2-(5-thiazolylmeth-
ylene)-1-tetralone 14 (method E).
Biologica l P r op er ties
In h ibition of P 450 a r om . The method of Thompson
and Siiteri, i.e. human placental microsomes and [1â,2â-
3H]testosterone,49 was used for the determination of
inhibitory activities. Table 2 shows the IC50 values and
the inhibitory potencies of the compounds relative to
aminoglutethimide (AG; rp values).
In the case of the (E)-2-arylmethylene-substituted
1-tetralones, exchange of the 4-pyridyl group of com-
pound 4 (rp ) 49) by a 5-thiazolyl group (14, rp ) 17)
and a 4-imidazolyl substituent (16, rp ) 71, Ki ) 33 nM,
Km for testosterone ) 42 nM) enhanced P450 arom
inhibitory activity by factors of 4 and 18, respectively.
The change of the configuration of compound 16 led to
a further increase: the corresponding (Z)-4-imidazolyl-
methylene compound 17 showed a relative potency of
110 (Ki ) 26 nM). It reaches about 30% of the activity
shown by the well-known aromatase inhibitor fadrozole
(rp ) 35919). The other compounds with a five-
membered aryl substituent were either inactive [4-thi-
enyl (12), 5-(2-NH2)thiazolyl (13)] or showed only a
moderate activity [4-pyrazolyl (14)]. The six-membered
aryl substituents, 3-pyridyl, 2-pyrazinyl, and 4-pyrid-
The E-configuration of the compounds was deter-
mined by 1H-NMR spectroscopy according to the method
previously reported by us9,18 (irradiation with UV light

836 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 4
Wa¨chter et al.
Ta ble 1. Analytic Data for Heterocyclic Substituted 2-Methylene-1-tetralones (1-4, 9-17), 2-(Hydroxymethyl)-1-tetralones (5-8),
1a,2,3,7b-Tetrahydro-1H-cyclopropa[a]naphthalenes (18-24), and 4,5-Dihydronaphtho[1,2-c]pyrazoles (25-27)
compd
Ar/R
mp, °C
formulaa
method of prpn
recryst solvent
petroleum ether
2-propanol
2-propanol
2-propanol
acetone/petroleum ether
acetone/petroleum ether
acetone/petroleum ether
acetone/petroleum ether
acetone
acetone/petroleum ether
2-propanol/petroleum ether
2-propanol
tetrahydrofuran/petroleum ether
acetone
acetone
acetone/petroleum ether
acetone
i
petroleum ether
petroleum ether
petroleum ether
k
k, l
k
k
k
k
yield, %
1c
2d
phenyl
106-107
121-122
77-78
C17H14
O
A
A
A
A
C
C
D
C
F
F
F
A
B
E
B
B
G
H
H
H
H
H
H
H
H
H
H
44
56
45
75
54
43
54
62
62
45
68
65
68
12
46
57
29
85
72
68
60
56
57
82
44
66
7
2-pyridyl
3-pyridyl
4-pyridyl
C16H13NO
C16H13NO
C16H13NO
C15H14N2O2
C15H14N2O2
C15H14N2O2
C15H14N2O2
15H12N2O
15H12N2O
15H12N2O
15H12SO
3d
4e
113-115
107-108
158-159
124-126
132-133
144-148
112-114
135-139
97-99
5f
2-pyrazinyl
3-pyridazinyl
4-pyridazinyl
4-pyrimidinyl
2-pyrazinyl
3-pyridazinyl
4-pyridazinyl
3-thienyl
5-(2-NH2)thiazolyl
5-thiazolyl
3-pyrazolyl
4-imidazolyl
4-imidazolyl
phenyl
2-pyridyl
3-pyridyl
4-pyridyl
3-thienyl
3-pyrazolyl
4-imidazolyl
CH2CH3
(CH2)3CH3
CH(CH3)CH2CH3
6f
7f
8f
9
C
C
C
C
10
11
12
13
14
15
16
17
18g
19
20
21h
22
23
24
25
26
27
dec >200
132-133
160-162
135-137
120-124
58-60
C14H12N2OS
C
C
C
C
14H11NOS
14H12N2O
14H12N2O
14H12N2O
C17H16
80-82
C
C
16H15
16H15
C16H15
N
N
N
S
61-64
79-80
53-54
C
C
C
15H14
dec >150
14H14N2‚HBr
glass
14H14N2
b
oil
oil
oil
C12H14N2
C15H18N2
C15H18N2
b
b
a
b
C, H, and N analyses were within (0.4% of the theoretical value except footnote b. Compounds were checked for purity using HPLC.
CI mass spectra show M + 1 peaks at m/ z ) 199.1, 227.2, and 227.2 for compounds 25, 26, and 27, respectively (calculated 199.3, 227.3,
and 227.3). c See ref 48. See ref 36. e See refs 36 and 9. f One diastereomer. See ref 47. See ref 19. Purified by bulb tube distillation.
Purified by column chromatography on silica gel with mixtures of petroleum ether and ethyl acetate. HBr was bubbled through a
solution of the crude product in dry diethyl ether to yield the hydrobromide.
d
g
h
i
k
l
Sch em e 2
Sch em e 3
With the exception of the 4-pyrimidinyl compound 8,
which showed little activity, the aryl-substituted 2-(hy-
droxymethyl)-1-tetralones were inactive.
In the case of the tetrahydrocyclopropanaphthalenes,
the exchange of the 4-pyridyl group of 21 (rp value of
99; Ki ) 29 nM) by other heterocycles led to a strong
azinyl, led to compounds (3, 9, and 11) which were less
active (rp values of 2, 0.6, and 0.18, respectively) than
the parent 4-pyridyl compound 4. The phenyl, 2-pyr-
idyl, and 3-pyridazinyl compounds (1, 2, and 10) were
inactive.

Tetrahydronaphthalenes
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 4 837
Ta ble 2. Inhibition of Steroidogenic P450 enzymes (P450 arom, P450 17, and P450 18) by Heterocyclic Substituted
2-Methylene-1-tetralones (1-4, 9-17), 2-(Hydroxymethyl)-1-tetralones (5-8), and 1a,2,3,7b-Tetrahydro-1H-cyclopropa[a]naphthalenes
(18-24)
P450 aroma
IC50, µMb
P450 17f % inhibn
P450 18h % inhibn
compd
Ar
rpc
(IC50, µM)
(IC50, µM)
2-Methylene-1-tetralones
1
2
3
4
9
10
11
12
13
14
15
16
17
phenyl
2-pyridyl
3-pyridyl
4-pyridyl
2-pyrazinyl
3-pyridazinyl
4-pyridazinyl
3-thienyl
5-(2-NH2)thiazolyl
5-thiazolyl
3-pyrazolyl
4-imidazolyl
4-imidazolyl
>250
>250
<10
<10
48
<10
<10
<10
12
10
21
<10
<10
<10
<10
20
9.2
4.6
2
4d
0.6
37
38
>250
103
>250
>250
ndg
<10
<10
<10
nd
0.18
1.1
18
0.26
0.17
17
1
71
21
<10
31
43
73
110
74
2-(Hydroxymethyl)-1-tetralones
>250
5
6
7
8
2-pyrazinyl
<10
<10
<10
<10
<10
<10
<10
<10
3-pyridazinyl
4-pyridazinyl
4-pyrimidinyl
>250
>250
27
0.7
1a,2,3,7b-Tetrahydro-1H-cyclopropa[a]naphthalenes
18
19
20
21
22
23
24
phenyl
>250
>250
13
<10
14
<10
24
55
<10
19
2-pyridyl
3-pyridyl
4-pyridyl
3-thienyl
3-pyrazolyl
4-imidazolyl
<10
1.4
99e
´o
0.36
31
87 (6.3)
0.37
36
<10
nd
>250
51
0.6
90 (13)
90 (0.34)
a
b
Human placental microsomes. Concentration of inhibitor required to give 50% inhibition. Concentration of testosterone (substrate),
2.5 µM; [1â,2â-3H]testosterone, 0.225 µCi. The given values are mean values of at least three experiments. The deviations were within
(5%. c Relative potency, calculated from the IC50 values and related to AG (IC50 of AG ) 18.5 µM). See ref 9. e See ref 19. f Rat testicular
d
microsomes. Concentration of inhibitor, 125 µM; concentration of progesterone (substrate), 25 µM. Inhibition shown by ketoconazole,
62% under identical conditions (IC50 ) 65 µM). The given values are mean values of at least two experiments. The deviations were
g
h
within (5%. nd, not determined. Bovine adrenal mitochondria. Concentration of inhibitor, 1 µM; concentration of corticosterone
(substrate), 200 µM. Inhibition shown by ketoconazole, 78% under identical conditions. The given values are mean values of at least two
experiments. The deviations were within (5%.
decrease in activity: The 4-imidazolyl compound (24)
showed modest activity (rp ) 31; 0.12 µM), the 3-pyridyl
(20) and the 3-pyrazolyl compound (23) were only
slightly active [rp values of 1.4 (Ki ) 3 µM) and 0.36],
and the other compounds were inactive. The 3-alkyl-
substituted 4,5-dihydronaphtho[1,2-c]pyrazoles 25-27
were void of inhibitory activity toward P450 arom (as
well as toward the other P450 enzymes, see below; data
not given).
To get an insight into the mode of aromatase inhibi-
tion, difference spectroscopy experiments were per-
formed with 20. A type II difference spectrum was
found (minimum at 394 nm, maximum at 425 nm, data
not given), which is typical of the interaction of the
heterocyclic nitrogen with the central iron ion of the
cytochrome P450 component.22
In h ibition of P 450 17. The assay was performed
as recently described by us using rat testicular mi-
crosomes as source of the enzyme and nonlabeled
progesterone as substrate.25 The inhibition values at
the high inhibitor concentration of 125 µM are given in
Table 2. The aryl-substituted 2-methylene-1-tetralones
showed either no or slight inhibition. The only excep-
tion was the Z-isomer 17, which inhibited the enzyme
by 74% (Ki ) 0.7 µM, Km for progesterone ) 7 µM).
Interestingly, the exchange of the 4-pyridyl group of
compound 4 by a 4-imidazolyl substituent (compound
16) further decreased the moderate inhibition of P450
17 slightly [inhibition values of 37% ((4, n ) 4; Ki ) 4
µM) and 31% ((3, n ) 4; Ki ) 9 µM); the same structural
modification increased inhibition of P450 arom strongly;
see above].
In the case of the tetrahydrocyclopropanaphthalenes,
compound 21, which was the strongest inhibitor of P450
arom, exhibited only modest inhibition of P450 17 (Ki
) 3 µM). Strong inhibitors of this enzyme are the
3-pyridyl compound 20 and the 4-imidazolyl compound
24 [IC50 values of 6.3 and 13 µM corresponding to
relative potencies (rp) of 10 and 5; rp ketoconazole ≡ 1;
Ki 20 ) 80 nM; Ki 24 ) 0.11 µM], which are weak or
moderate inhibitors of P450 arom, respectively. The
other cyclopropyl compounds and the aryl substituted
2-(hydroxymethyl)-1-tetralones showed no activity.
In h ib it ion of Ot h er St er oid ogen ic P 450 E n -
zym es. An essential prerequisite for an inhibitor of sex
hormone biosynthesis to be used as a drug is selectivity
of inhibition, i.e. other steroidogenic P450 enzymes
except the target enzymes must not be inhibited.
The effects of the title compounds on inhibition of
P450 18, which catalyzes the last step in aldosterone
biosynthesis, are also shown in Table 2. Bovine adrenal
mitochondria as the source of the enzyme and cortico-
sterone as the substrate were used, and the procedure
recently described19 was applied. The 2-methylene-1-
tetralones 1-4 and 9-15 showed either no or only slight
inhibition of P450 18. Somewhat more active was the
(E)-4-imidazolyl compound 16, whereas the correspond-
ing Z-compound 17 showed a marked inhibition (inhibi-
tion values of 43% and 73%, respectively). The 2-(hy-
droxymethyl)-1-tetralones 5-8 were inactive toward

838 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 4
Wa¨chter et al.
Ta ble 3. Inhibition of Thromboxane A2 Synthase by Select
Heterocyclic Substituted 2-Methylene-1-tetralones and
1a,2,3,7b-Tetrahydro-1H-cyclopropa[a]naphthalenesa,b
Discu ssion
The 2-(arylmethylene)-1-tetralone and the 1-aryl-
tetrahydrocyclopropanaphthalene basic structures and
the variation of the heterocyclic substituents turned out
to be appropriate means for the discovery of new leads
for selective inhibitors of both P450 arom and P450 17.
In the case of the (E)-2-(arylmethylene)-1-tetralones,
the aromatase inhibitory properties of the 4-pyridyl
compound 49 could be markedly enhanced. The 4-imid-
azolyl compound 16 is a strong inhibitor of P450 arom,
which affects the other steroidogenic P450 enzymes only
in high concentrations. The corresponding Z-compound
17 is a strong inhibitor of P450 arom and also inhibits
P450 17 and P450 18 markedly. Compounds of this
kind have to be excluded as potential therapeutics, but
they might be important scientific tools for the elucida-
tion of the topography of the corresponding steroidogenic
P450 enzymes.
Unlike the results shown by the arylmethylenetetra-
lones, the tetrahydrocyclopropanaphthalenes exhibit
decreased inhibition of P450 arom but increased inhibi-
tion of P450 17 upon exchange of the 4-pyridyl group of
2119 by other heterocycles. Compound 20 turned out
to be a strong inhibitor of P450 17, which shows only
minor effects on the other steroidogenic P450 enzymes.
20 is a 3-pyridyl compound just like another very potent
inhibitor of P450 17, 17-(3-pyridyl)androsta-5,16-dien-
3â-ol.54 A compound showing interestingly strong in-
hibition of P450 arom, P450 17, and P450 18 is the
4-imidazolyl compound 24.
Taking a closer look at the structure-activity rela-
tionships, it becomes apparent that for inhibition of each
of the steroidogenic P450 enzymes, the heterocyclic
nitrogen must be sterically accessible (i.e., in the case
of a six-membered ring the N must be in the 3- or
4-position and in the case of a five-membered ring in
the 3-position). Further, an additional N next to the
heme iron complexing N is unfavorable. The decisive
criterion as to what enzyme will be inhibited and how
strongly it will be influenced is of course the three-
dimensional structure of the compounds. As the present
substances are very rigid, they are appropriate tem-
plates for the elucidation of the topography of the active
site of the corresponding steroidogenic enzymes.
The fact that structurally related compounds are able
to influence both P450 arom and P450 17 is not
surprising. P450 arom hydroxylates the steroidal 19-
methyl group, which is located in the 10â-position
between ring A and B. Consequently, the heme has to
be in a position above the steroid near the A-ring. Since
the inhibitor complexes the heme iron by means of its
N, as shown exemplarily in this as well as in other
papers,18,23 it has to be assumed that the rest of the
inhibitor molecule is located in the area of the C- and
D-rings, which means that the benzene moiety mimics
the D-ring. P450 17 hydroxylates the steroid in 17R-
position and subsequently attacks the 20-carbonyl C-
atom by means of its reactive ferric peroxy species.55,56
The heme must therefore be below the steroid near the
D-ring. After complexing the heme iron, the rest of the
inhibitor molecule is probably located in the area
occupied by the A- and B-rings of the steroid in the
course of the enzymatic reaction, the benzene nucleus
probably mimicking the A-ring. Taking this into ac-
count, it becomes obvious that the distances between
compd
% inhibnc
compd
% inhibn
IC50, µM
1
2
3
4
9
10
11
15
45
31
36
13
21
<10
<10
21
16
17
18
19
20
21
23
24
<10
26
23
40
98
42
38
17
14.5d
a
b
Human whole blood; collagen challenge (53.6 µg/mL). TxA2
synthase-catalyzed malondialdehyde formation determined by a
thiobarbituric acid assay (ref 53). c Concentration of inhibitor, 50
µM. The given values are mean values of at least two experiments.
d
The deviations were within (5%. Mean value of three experi-
ments (IC50 value).
this enzyme, as they were toward P450 arom and P450
17. The tetrahydrocyclopropanaphthalenes 18-24
showed no (19, 22), slight (18, 20, 23), or moderate (21)
inhibition. Only compound 24, which had shown strong
inhibition of P450 17, exhibited a strong inhibition of
P450 18 as well (IC50 ) 0.34 µM).
The effects of select tetrahydronaphthalenes on cor-
ticosterone (24) and aldosterone biosynthesis (16, 21,
24) were determined using the method of Ha¨usler et
al.,50 i.e. ACTH stimulated rat adrenal fragments in
potassium containing buffer. Compound 24 inhibited
corticosterone formation, showing an IC50 value of 48
µM (IC50 values: AG, 50 µM; fadrozole, 80 µM). Com-
pounds 16, 21, and 24 inhibited aldosterone formation,
showing IC50 values of 6, 3, and 0.7 µM, respectively
(fadrozole, 0.6 µM). This data corresponds very well to
the inhibition values toward P450 18 (16, 43%; 21, 55%;
and 24, 90%; Table 2).
The key enzyme of steroid hormone biosynthesis is
the cholesterol side chain cleavage enzyme (P450 scc),
which is responsible for the conversion of cholesterol into
pregnenolone. By using bovine adrenal mitochondria
and [26-14C]cholesterol (3 µM) and applying the proce-
dure of Hochberg et al.51 select compounds were tested
at a relatively high concentration of 25 µM. The
4-pyridyl-substituted compounds 4 and 21 did not
inhibit the enzyme, whereas the 4-imidazolyl com-
pounds 16 and 24 showed a weak inhibition (21% for
each of the compounds). In contrast to the tetrahydro-
naphthalenes, the reference compound AG inhibited the
enzyme strongly (53%).
In h ibition of P 450 TXA2. It has been known for
some time that 1-imidazolyl-substituted tetrahydronaph-
thalenes are strong inhibitors of thromboxane A2 (TXA2)
synthase, depending on the substituent at the benzene
nucleus.52 Since inhibition of this enzyme, which con-
tains a P450 moiety, might influence haemostatic func-
tion, select compounds were tested for inhibition of TXA2
synthase (Table 3). A high concentration of inhibitor
of 50 µM was used. With the exception of compound
20, the compounds showed inhibition values below 50%
(IC50 > 50 µM), i.e. they are only slightly active. Only
the 3-pyridyl-substituted tetrahydrocyclopropanaphtha-
lene 20 showed a strong inhibition of TXA2 synthase
(98% inhibition, IC50 ) 14.5 µM), being only 1 order of
magnitude less active than the well-known inhibitor
dazoxiben (IC50 ) 1.1 µM).

Tetrahydronaphthalenes
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 4 839
The temperature was allowed to rise to ambient temperature
over a period of 4 h. The mixture was hydrolyzed with 100
mL of a saturated solution of NH4Cl, alkalized with 2 N
NH4OH, and extracted three times with CH2Cl2. The com-
bined organic phases were washed with water, dried over
Na2SO4, and evaporated under reduced pressure. Further
purification was carried out by recrystallization from a suitable
solvent (Table 1).
the heme iron and the D-ring (P450 arom) or A-ring
(P450 17) binding sites for the corresponding substrates
might be very similar, and consequently, structurally
related compounds can inhibit both P450 arom and
P450 17. On the other hand, there are enough differ-
ences in the active sites, making a selective inhibition
possible, as also shown in this paper.
Meth od D. P r oced u r e for th e Syn th esis of 2-(Hy-
d r oxy(4-p yr id a zin yl)m eth yl-1-tetr a lon e (7). Under warm-
ing 1.08 g (10.0 mmol) of 4-pyridazinecarboxaldehyde was
dissolved in 2.0 mL of ethanol. After cooling to 4 °C, 3.00 mL
(22.5 mmol) of 1-tetralone and 0.20 mL (1.92 mmol) of
diethylamine were added. After refrigerating the mixture at
4 °C for 2 weeks, the product was precipitated with petroleum
ether. The crude product was recrystallized from acetone/
petroleum ether.
Meth od E. P r oced u r e for th e Syn th esis of (E)-2-(5-
Th ia zolyl)m eth ylen e-1-tetr a lon e (14). A 2.56 g (10.0 mmol)
sample of (E)-2-(5-(2-aminothiazolyl)methylene)-1-tetralone
(13) was suspended in 200 mL of dry tetrahydrofuran. After
addition of 10.0 mL (75.3 mmol) of isopentylnitrite, the mixture
was refluxed for 0.5 h. Evaporation of the solvent under
reduced pressure gave a residue to which 100 mL of 2 N
NH4OH was added before extraction with three portions of 100
mL of CH2Cl2. After the combined organic phases were dried
over Na2SO4, 200 g of silica gel was added. The solvent was
removed under reduced pressure, and the silica gel was filled
into a short column and extracted with 2 L of CH2Cl2. After
evaporation of the solvent under reduced pressure, the product
was purified by recrystallization from acetone (twice).
Meth od F . Gen er a l P r oced u r e for th e Syn th esis of th e
Su bstitu ted (E)-2-Meth ylen e-1-tetr a lon es 9 - 11. A solu-
tion of 20.0 mmol of the respective substituted 2-(hydroxy-
methyl)-1-tetralone was dissolved in 100 mL of 25% sulfuric
acid and refluxed for 3 h. With intense cooling from an ice
bath, the mixture was then alkalized with solid NaOH. The
mixture was extracted three times with diethyl ether. The
combined organic layers were dried over Na2SO4 and evapo-
rated under reduced pressure. Further purification was
carried out by recrystallization from a suitable solvent (Table
1).
Met h od G. P r oced u r e for t h e Syn t h esis of (Z)-2-(4-
Im id a zolyl)m eth ylen e-1-tetr a lon e (17). A solution of 224
mg (1.00 mmol) of (E)-2-(4-imidazolyl)methylene-1-tetralone
(16) in 5.0 mL of methanol was irradiated with ultraviolet light
(λ ) 200-600 nm) for 20 h. The solvent was evaporated under
reduced pressure. The residue was extracted three times with
20 mL portions of petroleum ether. Further purification of
the residue obtained in this way was carried out by recrys-
tallization from acetone in the absence of light.
Meth od H. Gen er a l P r oced u r e for th e Syn th esis of
th e Su bstitu ted exo-1a ,2,3,7b-Tetr a h yd r o-1H-cyclop r o-
p a [a ]n a p h th a len es 18-24 a n d 4,5-Dih yd r on a p h th o[1,2-
c]-p yr a zoles 25-27. A mixture of 18.0 g of KOH (322 mmol),
17.0 mL (340 mmol) of hydrazine hydrate, 300 mL of dieth-
ylene glycol, and 20.0 mmol of the respective (E)-2-methylene-
1-tetralone was heated at 150 °C for 1.5 h. Water formed
during the reaction and excess hydrazine hydrate were
removed by distillation until the temperature reached 195 °C.
The mixture was heated at this temperature for an additional
4 h. The mixture was poured on 600 g of ice and extracted
three times with CH2Cl2. The combined organic phases were
washed with water, dried over Na2SO4, and evaporated under
reduced pressure. The crude products were purified by column
chromatography or recrystallization from a suitable solvent
(Table 1).
Biologica l Meth od s. En zym e P r ep a r a tion s. The en-
zymes were prepared according to described methods: human
placental P450 arom,3 rat testicular P450 17,25 bovine adrenal
P450 18,57 and bovine adrenal P450 scc.3
En zym e Assa ys. The enzyme assays were performed as
described: P450 arom,3 P450 17,25 P450 18,19 and P450 scc.3
The Ki values and the difference spectrum were obtained
according to procedures published recently.23 The assay for
Provided that the mode of interaction of the inhibitors
with the active site is correct as suggested above, the
introduction of substituents at the benzene nucleus
should result in a further increase of activity and
selectivity. Appropriate substituents could interact with
the 17â-hydroxy or 17-keto (P450 arom) or with the
3-keto or 3â-hydroxy (P450 17) binding site of the
corresponding steroidal substrates. In the case of the
4-pyridyl compounds 4 and 21, we have shown that the
introduction of OH or OCH3 groups as a matter of fact
increased activity.9,19
In conclusion, the present study resulted in new leads
for selective inhibition of estrogen and androgen bio-
synthesis. Further optimization of the most interesting
compounds, 16 and 20, is presently under investigation.
Exp er im en ta l Section
Melting points were determined on a Kofler microscope
(Reichert; Vienna) and are not corrected. Elemental analyses
were performed at the Physical Chemistry Department of the
Universita¨t des Saarlandes and were within (0.4% of the
calculated values, except where stated otherwise (Table 1).
Mass spectra (CI) were carried out on a Finnigan MAT 90
using isobutane as reactant gas. 1H-NMR spectra were
measured on a Bruker AM 400 (400 MHz) and a Bruker AW
80 (80 MHz) and are consistent with the assigned structures.
IR spectra of KBr disks were measured on a Perkin-Elmer
Infrared Spectrometer 398. Silica gel 60 from Macherey &
Nagel was used for column chromatography. Petroleum ether
refers to petroleum ether (40-60 °C).
Meth od A. Gen er a l P r oced u r e for th e Syn th esis of th e
Su bstitu ted (E)-2-Meth ylen e-1-tetr a lon es 1-4 a n d 12. A
mixture of 2.00 g (23.5 mmol) of piperidine, 2.00 g (33.3 mmol)
of acetic acid, 14.6 g (100 mmol) of 1-tetralone, and 150 mmol
of the respective aromatic aldehyde was heated at 130 °C for
1.5 h. After removal of the lower boiling materials under
reduced pressure at temperatures not exceeding 130 °C, the
residue was dissolved in CH2Cl2. For the isolation of the
pyridine compounds 2, 3, and 4, this solution was extracted
with 2 N HCl. After neutralization of the acidic extract with
saturated NaHCO3 solution, the crude product was collected,
washed with water, and dried in vacuo. For the nonbasic
compounds 1 and 12, the CH2Cl2 solution was washed with
water, dried over Na2SO4, and evaporated under reduced
pressure. Recrystallization from a suitable solvent (Table 1)
was carried out in the absence of light.
Meth od B. Gen er a l P r oced u r e for th e Syn th esis of
th e Su bstitu ted (E)-2-Meth ylen e-1-tetr a lon es 13, 15, a n d
16. A 14.6 g (100 mmol) portion of 1-tetralone and 100 mmol
of the respective aromatic aldehyde were suspended in 75 mL
of 40% sulfuric acid. After heating at 90 °C for 20 h, the
mixture was poured on ice and neutralized with concentrated
NH4OH. The precipitate was collected, washed with water,
and dried in vacuo. The dried crude product was washed with
petroleum ether. Recrystallization from a suitable solvent
(Table 1) was carried out in the absence of light.
Meth od C. Gen er a l P r oced u r e for th e Syn th esis of
th e Su bstitu ted 2-(Hyd r oxym eth yl)-1-tetr a lon es 5, 6, a n d
8. A solution of lithium diisopropylamide prepared from 2.10
mL (15.0 mmol) of diisopropylamine in 10 mL of dry tetrahy-
drofuran and 10.0 mL (1.60 M, 16.0 mmol) of a butyllithium
solution in hexane was cooled to -70 °C. A 2.00 mL (15.0
mmol) portion of 1-tetralone was added. After stirring for 15
min
a solution of 15.0 mmol of the respective aromatic
aldehyde in 25 mL of dry tetrahydrofuran was slowly added.

840 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 4
Wa¨chter et al.
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the determination of the corticoid formation was performed
as described.23
For determination of P450 TXA2 synthase inhibition, cit-
rated human whole blood (0.5 mL) was preincubated with
inhibitor [in 10 µL of ethanol/phosphate buffer, 0.01 M
KH2PO4, 0.05 M Na2HPO4, pH 7.4 (1:1/v:v); control, vehicle;
blank, dazoxiben HCl, 100 µM] for 10 min at 37 °C. A 50 µL
portion of collagen suspension (final concentration, 53.6 µg/
mL) was added and incubation continued for another 10 min
at 37 °C. The reaction was terminated by the addition of 0.4
mL of trichloroacetic acid (20% in HCl, 0.6 M). After centrifu-
gation (10 min, 4.400g) the supernatant (0.5 mL) was pipetted
into 0.5 mL of thiobarbituric acid (TBA) solution (0.53% TBA
in phosphate buffer, 0.01 M KH2PO4, 0.05 M Na2HPO4, pH
7.4). After heating for 30 min at 70 °C and cooling for another
30 min at ambient temperature, the samples were measured
spectrofluorimetrically (λ excitation, 533 nm; λ emission, 550
nm).
Ack n ow led gm en t. Thanks are due to the Deutsche
Forschungsgemeinschaft and to the Verband der Chem-
ischen Industrie, Fonds der Chemischen Industrie, who
supported this work by grants. We thank Dr. Ulrike
Bartz for performing the difference spectroscopy experi-
ment, Dr. Christine Batzl-Hartmann and Dr. Vincent
C. O. Njar for fruitful discussions, and Martina Palzer
and Tanja Kany for technical assistance as well as
Frank Engel and Brunhilde Rischar for the preparation
of the manuscript.
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