Synthesis and evaluation of 17-aliphatic heterocycle-substituted steroidal inhibitors of 17α-hydroxylase/C17-20-lyase (P450 17)

Article abstract of DOI:10.1021/jm991070n

In the search for potent inhibitors of P450 17, the key enzyme in androgen biosynthesis, a series of steroidal inhibitors were synthesized and tested toward rat and human P450 17. Small aliphatic heterocycles (aziridine, oxirane, thiirane, diaziridine, diazirine, azetidine) were introduced into the 17β-position of anstrost-5-en-3β-ol. After identifying that aziridine is the most suitable functional group to coordinate with the heme iron, modifications of the steroidal skeleton were performed for further optimization. A wide range of inhibitory potencies toward P450 17 were found for the 21 test compounds. The most potent inhibitors toward the human and rat enzyme were aziridine compounds 3 (IC₅₀ rat: 0.21 μM, K_i = 3 nM; IC₅₀ human: 0.54 μM, K_i = 8 nM), 5 (IC₅₀ rat: 0.43 μM, K_i = 7 nM; IC₅₀ human: 0.29 μM, K_i = 4 nM), and 8 (21R:21S = 1:1; IC₅₀ rat: 0.53 μM, K_i = 9 nM; IC₅₀ human: 0.40 μM, K_i = 6 nM) which were more potent than the reference ketoconazole (IC₅₀ rat: 67 μM; IC₅₀ human: 0.74 μM). The inhibitory potency depends markedly on the stereochemistry at C20 of the inhibitors. This effect is more pronounced for the rat enzyme. Tested for selectivity, the highly potent inhibitors show poor inhibitory activity toward P450 arom, P450 scc, P450 TxA₂, and 5α-reductase. Tested for in vivo activity, 3 and 8 (0.019 mmol/kg) decreased the plasma testosterone concentration in rats by 81% and 84% after 2 h.

Full text of DOI:10.1021/jm991070n

J . Med. Chem. 2000, 43, 4437-4445
4437
Syn th esis a n d Eva lu a tion of 17-Alip h a tic Heter ocycle-Su bstitu ted Ster oid a l
In h ibitor s of 17r-Hyd r oxyla se/C17-20-Lya se (P 450 17)
Rolf W. Hartmann,*,† Markus Hector,† Bertil G. Wachall,† Anja Palusczak,† Martina Palzer,† Volker Huch,‡ and
Michael Veith‡
Pharmaceutical and Medicinal Chemistry and Inorganic Chemistry, University of the Saarland, P.O. Box 151150,
D-66041 Saarbru¨cken, Germany
Received May 4, 1999
In the search for potent inhibitors of P450 17, the key enzyme in androgen biosynthesis, a
series of steroidal inhibitors were synthesized and tested toward rat and human P450 17. Small
aliphatic heterocycles (aziridine, oxirane, thiirane, diaziridine, diazirine, azetidine) were
introduced into the 17â-position of anstrost-5-en-3â-ol. After identifying that aziridine is the
most suitable functional group to coordinate with the heme iron, modifications of the steroidal
skeleton were performed for further optimization. A wide range of inhibitory potencies toward
P450 17 were found for the 21 test compounds. The most potent inhibitors toward the human
and rat enzyme were aziridine compounds 3 (IC50 rat: 0.21 µM, Ki ) 3 nM; IC50 human: 0.54
µM, Ki ) 8 nM), 5 (IC50 rat: 0.43 µM, Ki ) 7 nM; IC50 human: 0.29 µM, Ki ) 4 nM), and 8
(21R:21S ) 1:1; IC50 rat: 0.53 µM, Ki ) 9 nM; IC50 human: 0.40 µM, Ki ) 6 nM) which were
more potent than the reference ketoconazole (IC50 rat: 67 µM; IC50 human: 0.74 µM). The
inhibitory potency depends markedly on the stereochemistry at C20 of the inhibitors. This
effect is more pronounced for the rat enzyme. Tested for selectivity, the highly potent inhibitors
show poor inhibitory activity toward P450 arom, P450 scc, P450 TxA2, and 5R-reductase. Tested
for in vivo activity, 3 and 8 (0.019 mmol/kg) decreased the plasma testosterone concentration
in rats by 81% and 84% after 2 h.
In tr od u ction
androgen biosynthesis in the adrenals, inhibition of
P450 17 blocks synthesis in both tissues, testes and
adrenals, decreasing plasma levels of testosterone and
DHT. For that reason, P450 17 has attracted attention
as a therapeutic target, and attempts were made to
obtain specific steroidal12-19 as well as nonsteroidal1,4,18-32
inhibitors. The first inhibitors were unsuitable for
therapeutic application because of side effects caused
by poor selectivity of the inhibitor (ketoconazole)33 or
because of poor in vivo activity (liarozole).34 Recently,
some steroidal12-17,35,36 (I (abiraterone),12 II,15 III17) and
nonsteroidal23,25,26,28,31,32 (IV,31 V28 (YM116)) (Chart 2)
inhibitors have been developed and show high in vitro
and in vivo potency. Common feature of these and most
other steroidal inhibitors is a N-containing heterocycle
at the 17-position (mostly imidazole or pyridine), which
is capable of forming a coordinate bond with the heme
iron of the prosthetic group of the enzyme.
Androgens play a critical role in the development and
maintenance of sexual characteristics in human males.
However, they are also growth factors for such severe
and widespread diseases as benign prostatic hyperplasia
(BPH) and prostatic cancer.1,2 After lung cancer, pros-
tatic cancer is the second leading cause of cancer death.
Studies of the National Cancer Institute (NCI) in the
United States show that the number of new cases of
prostatic cancer increased dramatically between 1971
and 1993 (white population 127%, black population
82%). These data show the need for a new therapeutic
concept, as an alternative to the treatment with anti-
hormones, GnRH analogues, or orchiectomy. A promis-
ing strategy could be the selective inhibition of androgen
biosynthesis.3,4 The appropriate target is 17R-hydroxy-
lase/C17-20-lyase (P450 17, Cyp 17),5-7 a key enzyme
in the androgen biosynthetic pathway. P450 17 is a
cytochrome P450 enzyme (monooxygenase), which cata-
lyzes hydroxylation of progesterone and pregnenolone
in the 17R-position as well as cleavage of the C17-C20
bond to yield androstenedione and DHEA (dehydroepi-
androsterone; Chart 1).8-11
The concept of drug design presented in this paper
combines the steroidal structure of the high-affinity
substrates pregnenolone and progesterone with het-
eroatom-containing functional groups for complexation
with the heme iron. A series of small aliphatic hetero-
cycles is introduced in the 17â-position of the steroidal
skeleton. After identification of the most suitable func-
tional group, alterations of the steroid skeleton are
performed for further optimization. We report here on
the synthesis of 21 steroidal inhibitors and their inhibi-
tory activity toward human and rat P450 17. Selectivity
(P450 arom, P450 scc, P450 TxA2, 5R-reductase) and in
vivo activity (SD-rats) of selected compounds are also
presented.
These steroids are weak androgens which subse-
quently are converted by other enzymes (17âHSD,
3âHSD, 5R-reductase) to the more potent androgens
testosterone and DHT (dihydrotestosterone). While
GnRH analogues and orchiectomy do not affect the
* To whom correspondence should be addressed. Tel: +49 681 302
3424. Fax: +49 681 302 4386. E-mail: rwh@rz.uni-sb.de.
† Pharmaceutical and Medicinal Chemistry.
‡ Inorganic Chemistry.
10.1021/jm991070n CCC: $19.00 © 2000 American Chemical Society
Published on Web 10/25/2000

4438 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 23
Ch a r t 1. Catalytic Reaction of P450 17
Hartmann et al.
Ch a r t 2. Selected Inhibitors of P450 17
aldehyde 10 was a mixture of the 17â- and 17R-isomer
1
(4:1, determined by H NMR, C17 proton), from which
the former was separated by chromatography. Ring
closure led to a 3:1 diastereomeric mixture (20R:20S)
of the 3â-ols 11/12 (yield 62%) and the corresponding
3â-methyl ethers 13/14 (yield 16%). The 3â-methoxy and
3â-hydroxy derivatives were separated by FCC. The
diastereomers were purified by HPLC. Elucidation of
1
the stereochemistry was not possible by H NMR, but
X-ray analysis of 13 revealed the R configuration at C20.
With the diastereomeric mixture 11/12 as the starting
material, a 1:3 mixture of the thiiranes 15/16 (20R:20S)
(Scheme 2) was obtained by the reaction reported by
Chan and Finkebine.41 This reaction proceeds with
inversion of the configuration at C20. The diastereomers
were purified by HPLC, and their configuration was
elucidated by X-ray analysis of 15.
Sch em e 1a
The direct synthesis of the diaziridine 18 (Scheme 3)
by reaction of pregnenolone with chloramine T or
hydroxylamine-O-sulfonic acid failed. Thus, the more
reactive imine 17 was synthesized, which upon reaction
with hydroxylamine-O-sulfonic acid gave the diaziridine
18. Interestingly, the 1H NMR signals of the C18
protons are split. This indicates that 18 is a mixture of
two conformers, which is most probably caused by the
configuration stability of the ring nitrogens. Treatment
of 18 with Br2 led to the diazirine 19, which shows, in
the 1H NMR spectrum, sharp singlets for all CH3
groups.
a
Reagents: (Method A) LiAlH4, CH3NHC(CH3)3.
Ch em istr y
Starting from the corresponding oximes (Scheme 1),
the aziridines 4-8 were obtained via the same method
as described for 2 and 3.37 For all compounds, a 1:1
diastereomeric mixture (20R:20S) was obtained, which
was separated for 4-7 by FCC (flash column chroma-
tography) and HPLC. The stereochemistry at C20 was
assigned and the presence of the 17R-OH group was
The starting point for the synthesis of the azetidine
22 (Scheme 4) was a mixture of compounds 2 and 3.
Deamination with acetic acid and NaNO2 and subse-
quent acetylation of the 3â-hydroxy group led to 20.42
Treatment with chlorosulfonyl isocyanate43,44 and reac-
tion of the resulting chlorosulfonylazetidinone interme-
confirmed by comparison of the H and 13C NMR data
1
with those of 2 and 3. In the case of the 20S compounds,
the 18-methyl singlet is shifted to higher field compared
to the corresponding 20R diastereomers.14,37 In the case
of the 1:1 diastereomeric mixture 8 separation of the
isomers was not possible.
45
diate with Na2SO3 resulted in the lactam 21, which
was determined to be a 2:1 diastereomeric mixture (C20
1
atom H NMR: singlet of 18-methyl group). Reduction
using diisobutylaluminum hydride led to a 1:1 diaster-
eomeric mixture of 22. The azetidine has poor stability,
and no efforts were made to separate the isomers and
elucidate their stereochemistry.
Selected compounds (2, 3, 8, 11, 12) were transformed
into their progesterone derivatives 23,14 24,14 and 25-
27 by a modified Oppenauer oxidation46 via N-meth-
ylpiperidone and aluminum isopropoxide as reagents
(Scheme 5). In the case of compound 25, a 1:1 mixture
The oxirane compounds 11-14 (Scheme 2) were
synthesized according to a method for steroidal 10â-
oxiranyl compounds38 by reaction of trimethylsulfonium
iodide and NaH with an aldehyde.39 Compound 10 was
obtained according to Danishefsky.40 In contrast to the
literature method, introduction of a protecting group
(THP) as well as isolation of compound 9 (E:Z ) 3:4,
1
determined by H NMR, C20 vinyl proton) was neces-
sary. The crude product obtained from the reaction with

Heterocycle Steroidal Inhibitors of P450 17
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 23 4439
Sch em e 2a
a
Reagents: (a) CH3OCH2PPh3Cl, NaH; (b) 1. HClO4, 2. PPTS; (c) NaH, S(CH3)3I; (d) Ph3PS, picric acid.
Sch em e 3a
the case of the thiiranes, R configuration at C20 results
in a higher inhibitory potency. The azetidine derivative
24 shows poor inhibition for both enzymes, which might
be due to degradation of the substance under the assay
conditions. Introduction of a OH group in the 17R-
position of aziridines 2 and 3 causes a dramatic loss of
activity (compounds 6 and 7). This result was unex-
pected, since 17R-hydroxypregnenolone is also a sub-
strate of P450 17. As expected, the S-configurated
diastereomer is more potent toward the rat enzyme than
the R isomer. A ∆14-double bond (4/5) increases inhibi-
tory activity for the human enzyme. Introduction of a
methylene spacer in the 17â-side chain (8) does not
noticeably change activity toward the rat enzyme but
increases activity toward the human enzyme. In the
case of the aziridines, exchange of the pregnenolone
skeleton by progesterone (2, 3, 8 f 23-25) results in a
moderate decrease of activity toward both enzymes. In
the case of the oxiranes, the progesterone compounds
26 and 27 as well as the 3â-OMe pregnenolones 13 and
14 showed a strong decrease of inhibitory activity
compared to the corresponding pregnenolones (11, 12).
a
Reagents: (a) C6H11NH2, F3CCOOH; (b) H2NOSO3H, MeOH,
NH3 (satd); (c) Br2, NEt3.
of diastereomers was obtained, which could not be
separarated by HPLC. The isomers 26 and 27 were
isolated by HPLC.
Biologica l Resu lts
The inhibitory activity toward the P450 17 rat and
human enzyme was tested in an experimental system
comprising testicular microsomes and progesterone in
large excess as substrate. Reverse-phase HPLC with UV
detection was used to quantify the products. In contrast
to the rat enzyme, 16R- and 17R-hydroxyprogesterone
are formed with the human enzyme (ratio 3:10), and
no products of lyase activity are found as previously
reported.47
The mode of inhibition of the aziridine derivatives was
studied using UV-vis difference spectroscopy experi-
ments. A characteristic type II difference spectrum was
induced (data not given) indicating coordination of the
steroidal aziridine nitrogen to the heme iron of P450
17.48 This effect was not reversed by high excess of
progesterone. To elucidate whether the inhibitor binds
covalently to the enzyme, enzyme activity was deter-
mined after various time intervals, following preincu-
bation of the aziridine with enzyme and removal of the
inhibitor. A recovery of the enzyme activity of 25% after
5 min and up to 75% after 90 min was observed,
indicating a reversible binding mode. A slow-binding
mechanism49 could be excluded.
The IC50 values in Table 1 show that the aziridine-
substituted compounds are very potent inhibitors of
P450 17. Compound 3 exhibits IC50 values in the
nanomolar range for both enzymes (0.21 µM rat and
0.54 µM human) and Ki values of 3 and 8 nM, respec-
tively (competitive Lineweaver-Burk plot; data not
shown). A strong sterochemical dependency was ob-
served for the rat enzyme. The potencies of the 20R
isomer 2 and the 20S isomer 3 differ by a factor of 162,
whereas in the human enzyme surprisingly this factor
is only 3. Similar differences can be seen with the other
aziridine diastereomers (4/5, 6/7, 23/24). Exchange of
the aziridine ring in the 17â-position by other three-
membered heterocycles decreases inhibitory activity
(oxirane, thiirane, diaziridine, diazirine). This effect
does not depend on the species and is more pronounced
for the rat enzyme. Besides the aziridines, only diaz-
iridine 18 is a more active inhibitor for the rat enzyme
than ketoconazole. As seen with compounds 2/3, the 20S
isomer 12 of oxiranes 11/12 is more potent than the 20R
isomer 11. In the case of the human enzyme, aziridine
3 is about 3-4 times more active than oxirane 12 (20S
isomer) and thiirane 15 (20R isomer). Interestingly, in
To evaluate the selectivity of the P450 17 inhibition,
compounds 3 and 8 were tested for inhibition of P450
scc, P450 arom, and P450 TxA2 because inhibition of
these enzymes could cause side effects. P450 scc cata-
lyzes the first step in steroid hormone biosynthesis, and
inhibition of P450 scc would affect all steroid hormones.
Inhibition of P450 arom and P450 TxA2 might increase
testosterone concentration and interfere with thromb-
ocyte aggregation, respectively. Analyses were done by
recently described procedures (P450 scc,50 P450 arom,50

4440 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 23
Hartmann et al.
Sch em e 4a
a
Reagents: (a) HNO2, HOAc; (b) 1. ClO2SNCO, 2. Na2SO3 (25%), KOH (10%); (c) DibaH.
Sch em e 5a
chemistry at C20. Interestingly this effect is more
pronounced for the rat enzyme, reflecting more stringent
spatial requirements. To elucidate why the S configu-
ration results in stronger inhibition, conformational
analyses were performed with 2-5. In the lowes-energy
conformation of 2 and 3, the aziridine ring is located in
a coplanar fashion to the steroid. This conformation is
almost identical with the X-ray structure of the 17â-
oxiranyl and 17â -thiiranyl analogues 13 and 15. In the
20S compound 3 the aziridine nitrogen is located in the
R-position, in a direction where the heme iron is
supposed to be, while in the 20R diastereomer 2 the
nitrogen is in the opposite (â) position. As expected from
this model, introduction of a double bond in position 16
(compounds 4 and 5) changes the inhibitory activity of
2 and 3 only slightly. Insertion of a methylene spacer
between the steroidal moiety and the aziridine ring does
not alter inhibitory potency strongly (2/3 f 8). For the
rat enzyme, the most potent inhibitors 3, 5, and 8 have
comparable activity to the powerful steroidal inhibitor
II (Chart 2; IC50: 0.18 µM in our test system). In case
of the human enzyme, compounds 3, 5, and 8 are
slightly less potent than compound III (IC50: 0.04 µM
in our test system) and compound I, which is described
to be more potent than ketoconazole by a factor of 9.12
Concerning the mode of inhibition of the aziridines, it
has been shown that they are reversible inhibitors,
which form a tight coordinate bond with the heme iron,
leading to a very slow dissociation from the enzyme. The
lower activities of the compounds bearing other hetero-
cycles are obviously a result of the chemical nature of
the heteroatoms because force-field calculations (con-
formational analysis, data not shown) reveal that all
C20 derivatives have very similar low-energy conforma-
tions and the coordinating heteroatom is located almost
in the same position in space as it is in compound 3.
Interestingly, the results obtained with analogous ste-
roidal inhibitors of P450 arom are different: (19R)-
aziridinylandrostenedione inhibits aromatase in a slow-
binding manner,55 whereas the (19R)-oxiranyl and
thiiranyl analogues, for which the Ki values are lower
than that of the aziridine, are reversible competitive
inhibitors of the enzyme.56 Presently no explantion for
these findings is obvious. Alterations of the steroidal
skeleton of aziridine 3 lead only to slight changes in the
biological activity (compounds 4, 5, 23, 24). In contrast,
introduction of the 17R-hydroxy group causes a loss of
activity, although it has no influence on the spatial
position of the aziridine ring (conformational analysis,
data not shown). This observation supports the model
of a bilobal hydrophobic pocket converging on the heme
group. This has been supported by molecular modeling
and site-directed mutagenesis.7,57 In this model, the
A-ring of the substrate should be capable of occupying
a
Reagents: (Method B) Al(OCH(CH3)2)3, N-methylpiperidone.
P450 TxA251). Compound 3 shows weak inhibition of
P450 arom and P450 scc and stronger inhibition of P450
TxA2, whereas compound 8 exhibits moderate inhibitory
effects toward P450 TxA2 and P450 arom as well as
marked inhibition of P450 scc (IC50 P450 TxA2: 3, 4 µM;
8, 35 µM; dazoxiben, 1.1 µM; IC50 P450 arom 3, >50
µM; 8, 7.1 µM; cyclohexylaminoglutethimide,52 0.15 µM;
IC50 P450 scc: 3, >25 µM; 8, approximately 1 µM).
Selected compounds (3, 24, 25) were tested for inhibi-
tion of 5R-reductase type I and II isozymes. The DU 145
cell line and prostate homogenate of BPH patients were
used as sources for type I and II isozyme, respectively,
and the analyses were performed by recently de-
scribed53,54 procedures. As 5R-reductase transforms
testosterone to the more potent androgen dihydrotest-
osterone, a dual inhibition of both 5R-reductase and
P450 17 could lead to a more effective blockade of
androgen biosynthesis. All tested compounds show weak
inhibition toward type II isozyme (IC50 values: 3, 10
µM; 24, 16 µM; 25, 9 µM). Only compound 25 exhibits
a relatively strong inhibition of type I isozyme (IC50: 1.6
µM).
The two most potent inhibitors of the human and rat
enzyme (compound 3 and the diastereomeric mixture
8) were evaluated for reduction of plasma testosterone
concentration in male Sprague-Dawley rats. The com-
pounds were administered intraperitoneally at equimo-
lar doses comparable to 10 mg/kg body weight of
ketoconazole. Each group consisted of 7-8 rats. Blood
samples were taken by cardiac puncture after 2 and 6
h. Testosterone concentrations were determined by RIA
using [125I]testosterone. Ketoconazole does not signifi-
cantly reduce the testosterone plasma level after 2 and
6 h. In contrast, 3 and 8 show strong reduction after 2
h of 81% and 84%, respectively. After 6 h no significant
effect was observed.
Discu ssion a n d Con clu sion
The biological data shows that the aziridine group
forms a strong coordinate bond with the heme iron,
leading to extremely potent inhibitors of the human and
rat enzyme (3, 5, 8). The inhibitory activities of the
aziridine compounds depend markedly on the stereo-

Heterocycle Steroidal Inhibitors of P450 17
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 23 4441
Ta ble 1. Inhibition of the P450 17 Rat and Human Enzyme by
Pregnenolone- and Progesterone-type Compounds
be an indication that the steroidal inhibitor is now
located in the larger lobe. Provided the model is correct
that one heme is responsible for hydroxylase and lyase
activity, the consequence of this is that a selective
inhibition of the two activities is not possible by heme-
coordinating inhibitors. Moreover selective inhibition of
lyase activity seems not to be necessary from the clinical
point of view. In trials with the P450 17 inhibitors
ketoconazole33 and liarozole,34 it has been shown that
the androgen plasma concentration was reduced without
glucocorticoid plasma levels being affected. For this
reason, it can be concluded that the aziridine compounds
3 and 8, which have shown much higher in vivo activity
than ketoconazole, might be promising drug candidates
for the treatment of androgen-dependent diseases.
Exp er im en ta l Section
Ch em ica l Meth od s. Melting points were measured on a
Kofler melting point Thermopan apparatus and are uncor-
rected. IR spectra were recorded on a Perkin-Elmer 398
infrared spectrometer as KBr pellets. 1H NMR spectra were
recorded on a Bruker AM-400 (400 MHz), 13C NMR spectra
on a Bruker AM-400 (120 MHz). Chemical shifts are given in
parts per million. TMS was used as internal standard. All J
values are given in Hz. Purity was checked by GC-MS on a
HP G1800A GCD system. Mass spectra were recorded on a
HP 1074 A (GCD) spectrometer (Hewlett-Packard). Elemental
analyses were performed at the Inorganic Chemistry Depart-
ment, University of the Saarland. Reagents and solvents were
used as obtained from commercial suppliers without further
purification. Column chromatography was performed using
silica gel 60 (50-200 µm), flash column chromatography (FCC)
using silica gel 60 (40-63 µm), HPLC using a semipreparative
RP-18 column (16 mm × 250 mm, particle size 5 µm, Nucleosil-
C18). Reaction progress was determined by TLC analysis on
ALUGRAM SIL G/UV254 (Macherey-Nagel).
Meth od A: Gen er a l P r oced u r e for Syn th esis of Azir i-
d in yl Com p ou n d s 2,14 3,14 a n d 4-8. The reaction was
performed under N2 atmosphere in a dry apparatus and
solvents. To a suspension of 338 mg LiAlH4 (8 mmol) and
catalytic amounts of N-methyl-N-butylamine in 3 mL THF was
dropped 0.81 mmol of the steroidal oxime dissolved in 9 mL
THF over 40 min at 0 °C. The reaction mixture was refluxed
for 16 h. To destroy the excess LiAlH4, small amounts of water
were added to the reaction mixture at 0 °C. The mixture was
filtered and washed twice with ethyl acetate. The organic
phase was washed twice with water and brine and dried over
MgSO4. The compounds were purified by FCC or HPLC (RP-
18, 4 mm × 250 mm, Eurospher 100-C18, particle size 5 µm,
MeOH, flow rate 1.5 mL/min).
(20R)-21-Im in op r egn a -5,14-d ien -3â-ol (4): purification
FCC (CH2Cl2:EtOH 9:1); HPLC tR ) 10.8 min; yield 30%, white
solid; mp 135-40 °C. 1H NMR (CDCl3): δ 1.06 (s, C18-Me,
3H); 1.03 (s, C19-Me, 3H); 3.53 (m, C3RH, 1H); 5.19 (s, C15
dCH-, 1H); 5.40 (s, C6, dCH-, 1H). IR (KBr) cm-1: νmax 3400,
3300, 2950, 1470/60/40, 1380, 1060/50, 800. GC-MS: m/z 313
(M+), 298, 270, 263, 237, 211, 194, 171. Anal. (C21H31NO) C,
H, N.
(20S)-21-Im in op r egn a -5,14-d ien -3â-ol (5): purification
FCC (CH2Cl2:EtOH 9:1); HPLC tR ) 11.9 min; yield 30%, white
solid; mp 145-50 °C. 1H NMR (CDCl3): δ 1.01 (s, C18-Me,
3H); 1.04 (s, C19-Me, 3H); 3.53 (m, C3RH, 1H); 5.17 (s, C15
dCH-, 1H); 5.40 (s, C6, dCH-, 1H). IR (KBr) cm-1: νmax 3400,
3300, 2950, 1470/60/40, 1380, 1060/50, 800. GC-MS: m/z 313
(M+), 298, 270, 263,,237, 211, 194, 171. Anal. (C21H31NO) C,
H, N.
a
Concentration of inhibitor required to give 50% inhibition. bRat
testicular microsomes, concentration of progesterone (substrate)
25 µM. Human testicular microsomes, concentration of progest-
erone (substrate) 25 µM. The given values are mean values of at
least two experiments. The deviations were within (5%. dSee ref
14. ni, no inhibition (at 125 µM for rat enzyme and 2.5 µM for
c
e
human enzyme, respectively); nd, not determined. 1:1 Diastere-
omeric mixture was tested. KTZ, ketoconazole.
f
two different positions. It is located in the smaller lobe
for 17R-hydroxylation. After this enzymatic step, the
A-ring moves to the larger lobe for C17-20-lyase
reaction. As a consequence of this shift, C20 is posi-
tioned closer to the heme iron enabling attack of the
ferriperoxo species.57 The decrease in activity observed
with compound 3 upon hydroxylation (compound 7) is
due to the less pronounced interaction of the aziridine
N with the heme iron because no type II difference
spectrum could be obtained (data not given). This might
(20R)-21-Im in op r egn -5-en e-3â,17r-d iol (6): purification
FCC (CH2Cl2:EtOH 9:1); yield 42%, white solid; mp 192-5 °C.
1H NMR (CDCl3): δ 1.031 (s, C18-Me, 3H); 0.85 (s, C19-Me,
3H); 3.53 (m, C3RH, 1H); 5.35 (d, C6, dCH-, 1H, J ) 5.4 Hz).
13C NMR (CDCl3): δ 141.4 (C5); 120.5 (C6); 79.4 (C17); 70.1

4442 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 23
Hartmann et al.
(C3); 23.8 (C16); 20.2 (C11); 19.3 (C19); 15.2 (C18). IR (KBr)
cm-1: νmax 3500-3300, 2950, 1470/60/40, 1385, 1055. Anal.
(C21H33NO2) C, H, N.
J ) 3.08 Hz, J ) 5.32 Hz); 2.71 (t, oxirane-CH, 1H, J ) 4.4
Hz); 2.89 (m, oxirane-CH, 1H); 3.06 (m, C3RH, 1H); 5.35 (d,
C6, dCH-, 1H, J ) 4.8 Hz). IR (KBr) cm-1: νmax 3400-3300,
3015, 2950, 1470/50/30, 1060, 860. Anal. (C21H32O2) C, H.
(20S)-21-Im in op r egn -5-en e-3â,17r-d iol (7): purification
FCC (CH2Cl2:EtOH 9:1); yield 42%, white solid; mp > 200 °C
(degradation). 1H NMR (CDCl3): δ 1.028 (s, C18-Me, 3H); 0.85
(s, C19-Me, 3H); 3.53 (m, C3RH, 1H); 5.40 (s, C6, dCH-, 1H).
13C NMR (CDCl3): δ 141.4 (C5); 120.5 (C6); 79.4 (C17); 70.1
(C3); 23.8 (C16); 20.2 (C11); 19.3 (C19); 14.6 (C18). IR (KBr)
cm-1: νmax 3500-3300, 2950, 1470/60/40, 1385, 1055. Anal.
(C21H33NO2) C, H, N.
(20S)-17â-Oxir a n ylp r egn -5-en -3â-ol (12): HPLC (aceto-
nitrile/H2O:40/60, flow rate 16 mL/min) tR ) 14.7 min; yield
1
16%, white solid; mp 165-8 °C. H NMR (CDCl3): δ 0.84 (s,
C18-Me, 3H); 1.03 (s, C19-Me, 3H); 2.42 (dd, oxirane-CH, 1H,
J ) 2.64 Hz; J ) 5.28 Hz); 2.70 (dd, oxirane-CH, 1H, J ) 4
Hz; J ) 5.28 Hz); 2.87 (m, oxirane-CH, 1H); 3.53 (m, C3RH,
1H); 5.34 (d, C6, dCH-, 1H, J ) 5 Hz). IR (KBr) cm-1: νmax
3400-3300, 3015, 2950, 1470/50/30, 1060, 860. Anal. (C21H32O2)
C, H.
(21R,21S)-22-Im in op r egn -5-en -3â-ol (8): purification FCC
(CH2Cl2:MeOH 9:1); yield 37%, white solid (1:1 21R:21S
diastereomeric mixture). 1H NMR (CDCl3): δ 0.59 (s, C18-Me,
1.5H); 0.60 (s, C18-Me, 1.5H); 1.02 (s, C19-Me, 3H); 3.53 (m,
3â-Meth oxy-(20R)-17â-oxir a n ylp r egn -5-en e (13): HPLC
(acetonitrile/H2O:25/75, flow rate 24 mL/min) tR ) 13.2 min;
yield 12%, white solid; mp 131-3 °C. 1H NMR (CDCl3): δ 0.81
(s, C18-Me, 3H); 1.02 (s, C19-Me, 3H); 2.48 (dd, oxirane-CH,
1H, J ) 2.64 Hz; J ) 5.32 Hz); 2.77 (dd, oxirane-CH, 1H, J )
5.32 Hz); 2.83 (m, oxirane-CH, 1H); 3.06 (m, C3RH, 1H); 3.35
(s, CH3-O, 1H); 5.36 (d, C4, dCH-, 1H, J ) 4.8 Hz). IR (KBr)
cm-1: νmax 3030, 2950, 1465/50/30, 1380. MS: m/z 331 (M+),
313, 288, 271. Anal. (C22H34O2) C, H.
3
C3RH, 1H); 5.35 (d, C6, dCH-, 1H, J ) 5.32 Hz). IR (KBr)
cm-1: νmax 3400-3300, 2950, 1470/60/40, 1370/60/50, 1055.
GC-MS: m/e 329 (M+), 314, 279. Anal. (C21H33NO2) C, H, N.
Meth od B: Gen er a l P r oced u r e for Syn th esis of Com -
p ou n d s 23,14 24,14 a n d 25-27. N-Methylpiperidone (3.38 mL,
29 mmol) was added to a solution of the steroid (0.31 mmol)
in 10 mL dry toluene. The mixture was heated under reflux
until 1-2 mL toluene was collected in a Dean-Stark trap.
Aluminum isopropoxide (112 mg, 0.56 mmol) was added and
the mixture was refluxed for 4 h. Aluminum isopropoxide (44.7
mg, 0.22 mmol) was added once again and refluxing was
continued for 2 h. The mixture was cooled to room temperature
and diluted with 20 mL of diethyl ether. The reaction mixture
was washed with water and brine and was dried over Na2-
SO4. The solvent was evaporated and the crude product was
purified by FCC.
3â-Meth oxy-(20S)-17â-oxir a n ylp r egn -5-en e (14): HPLC
(acetonitrile/H2O:25/75, flow rate 24 mL/min) tR ) 15.0 min;
1
yield 4%, white solid; mp 165-7 °C. H NMR (CDCl3): δ 0.84
(s, C18-Me, 3H); 1.02 (s, C19-Me, 3H); 2.41 (dd, oxirane-CH,
1H, J ) 2.64 Hz; J ) 5.28 Hz); 2.70 (dd, oxirane-CH, 1H, J )
5.28 Hz); 2.87 (m, oxirane-CH, 1H); 3.06 (m, C3RH, 1H); 3.35
(s, CH3-O, 1H); 5.35 (d, C4, dCH-, 1H, J ) 5 Hz). IR (KBr)
cm-1: νmax 3030, 2950, 1465/50/30, 1380, 1095. MS: m/z 331
(M+), 313, 288, 271. Anal. (C22H34O2) C, H.
(21R,21S)-22-Im in op r egn -4-en -3-on e (25): purification
FCC (CH2Cl2:MeOH 4:1); yield 40%, white solid (1:1 21R:21S
diastereomeric mixture). 1H NMR (CDCl3): δ 0.62 (s, C18-Me,
1.5H); 0.63 (s, C18-Me, 1.5H); 1.19 (s, C19-Me, 3H); 5.73 (s,
C4, dCH-, 1H). IR (KBr) cm-1: νmax 3600-3400, 2950, 1680,
1620. MS: m/e 328 (M+), 313, 280, 223. Anal. (C22H33NO) C,
H, N.
(20R)-17â-Oxir a n ylp r egn -4-en -3-on e (26): purification
FCC (CH2Cl2:EtOH 7:1); yield 51%, white solid; mp 144-7 °C.
1H NMR (CDCl3): δ 0.87 (s, C18-Me, 3H); 1.20 (s, C19-Me,
3H); 2.42 (dd, oxirane-CH, 1H, J ) 2.8 Hz, 5.28 Hz); 2.71 (dd,
oxirane-CH, 1H, J ) 3.96 Hz, 4.88 Hz); 2.87 (ddd, oxirane-
CH, 1H, J ) 2.87 Hz, 4.00 Hz, 6.64 Hz); 5.73 (s, C4, dCH-,
1H). IR (KBr) cm-1: νmax 2950, 1680, 1620, 1430, 1380, 1330,
1230, 1190, 910, 860. Anal. (C21H30O2) C, H.
(20S)-17â-Oxir a n ylp r egn -4-en -3-on e (27): purification
FCC (CH2Cl2:EtOH 7:1); yield 49%, white solid; mp 149-54
°C. 1H NMR (CDCl3): δ 0.83 (s, C18-Me, 3H); 1.20 (s, C19-
Me, 3H); 2.47 (m, oxirane-CH, 1H); 2.75 (dd, oxirane-CH, 1H,
J ) 4.96 Hz, 4.88 Hz); 2.81 (m, oxirane-CH, 1H); 5.73 (s, C4,
dCH-, 1H). IR (KBr) cm-1: νmax 2950, 1680, 1620, 1430, 1380,
1330, 1230, 1190, 910, 860. Anal. (C21H30O2) C, H.
Gen er a l P r oced u r e for Syn th esis of Com p ou n d s 11-
14. A suspension of 0.25 g NaH (6.16 mmol, 60%) was washed
twice with THF and dried under a N2 stream. 3.7 mL of dry
DMSO was added and the suspension was heated to 80 °C for
45 min. The resulting solution was cooled to room temperature
and 5 mL of THF was added. At -5 °C 1.02 g trimethylsul-
fonium iodide (6.4 mmol) in 20 mL of DMSO was added slowly
with a syringe. After 3 min stirring, 330 mg (1.09 mmol) of 10
in 20 mL THF was added. The reaction mixture was stirred
at -5 °C for 1 and 19 h at room temperature. The organic
phase was washed with 5% NaHCO3 and extracted with CH2-
Cl2. After washing with water and brine the organic phase was
dried over MgSO4. A 3:1 diastereomeric mixture of compounds
11/12 and 13/14 was obtained. The compounds were isolated
and purified by FCC (CH2Cl2:EtOAc 5:1) and HPLC (RP-18,
16 mm × 250 mm, Nucleosil-C18, particle size 5 µm, acetoni-
trile/H2O).
(20R)-17â-Th iir a n ylp r egn -5-en -3â-ol (15) a n d (20S)-17â-
Th iir a n ylp r egn -5-en -3â-ol (16). Under a N2 atmosphere, 890
mg triphenylphosphine sulfide (3 mmol) and 690 mg picric acid
(3 mmol) were added to a solution of 95 mg (0.3 mmol) of a
mixture of 11/12 in 50 mL toluene. The mixture was heated
at 80 °C under N2 for 5 h. The reaction mixture was cooled,
diluted with ether (40 mL) and washed four times with 10%
aqueous NaHCO3 to remove picric acid. The organic layer was
washed with water and brine, dried over Na2SO4 and evapo-
rated in vacuo. The crude product was isolated and purified
by FCC (CH2Cl2:EtOAc 5:1) and HPLC (RP-18, 16 mm × 250
mm, Nucleosil-C18, particle size 5 µm acetonitrile/H2O:40/60,
flow rate 16 mL/min) to give 15 in 21% yield and 16 in 62%
yield. Compound 15: tR ) 45.1 min); white solid; mp 158-60
°C. 1H NMR (CDCl3): δ 0.79 (s, C18-Me, 3H); 1.00 (s, C19-
Me, 3H); 2.14 (d, thiirane-CH, 1H, J ) 5.76 Hz); 2.45 (d,
thiirane-CH, 1H, J ) 6.2 Hz); 2.76 (m, thiirane-CH, 1H); 3.49
(m, C3RH, 1H); 5.32 (d, C6, dCH-, 1H, J ) 4 Hz). IR (KBr)
cm-1: νmax 3450-3400, 2950, 1380, 1060, 960, 850, 810. GC-
MS: m/z 333 (M+), 301, 299, 282. Compound 16: tR ) 39.3
1
min; white solid; mp 159-63 °C. H NMR (CDCl3): δ 0.84 (s,
C18-Me, 3H); 1.01 (s, C19-Me, 3H); 2.29 (d, thiirane-CH, 1H
J ) 5.2 Hz); 2.40 (d, thiirane-CH, 1H, J ) 5.72 Hz); 2.70 (m,
thiirane-CH, 1H); 3.50 (m, C3RH, 1H); 5.32 (d, C6, dCH-, 1H,
J ) 4.8 Hz). IR (KBr) cm-1: νmax 3450-3400, 2950, 1380, 1060,
960, 850, 810. GC-MS: m/z 333 (M+), 301, 299, 282.
20-Cycloh exylim in op r egn -5-en -3â-ol (17). 500 mg of
pregnenolone (1.58 mmol) was dissolved in 9 mL of cyclohexy-
lamine and catalytic amounts of triflouroacetic acid and
refluxed under N2 for 20 h. The excess cyclohexylamine was
evaporated in vacuo and the resulting white solid was dried
under reduced pressure (0.01 Torr): yield 98%, white solid;
1
mp 137-8 °C. H NMR (CDCl3): δ 0.58 (s, C18-Me, 3H); 1.00
(s, C19-Me, 3H); 1.79 (s, C21-Me, 3H); 3.52 (m, C3RH, 1H);
5.34 (s, C6, dCH-, 1H). IR (KBr) cm-1: νmax 3300, 2950, 1660,
1450 1065. Anal. (C27H43NO) C, H, N.
17â-Dia zir id in yl-20-m eth yla n d r ost-5-en -3â-ol (18). To
200 mg of 17 (0.5 mmol) in dry 10 mL MeOH was added 1.5
mL of NH3-saturated MeOH. Over 10 min 87 mg hydroxy-
lamine-O-sulfonic acid (0.77 mmol) was added to the solution.
The reaction mixture was stirred for 24 h and allowed to stand
for additional 48 h at room temperature. The reaction mixture
was poured onto ice and extracted trice with 50 mL ethyl
(20R)-17â-Oxir a n ylp r egn -5-en -3â-ol (11): HPLC (aceto-
nitrile/H2O:40/60, flow rate 16 mL/min) tR ) 13.4 min; yield
1
46%, white solid; mp 158-60 °C. H NMR (CDCl3): δ 0.79 (s,
C18-Me, 3H); 1.02 (s, C19-Me, 3H); 2.43 (dd, oxirane-CH, 1H,

Heterocycle Steroidal Inhibitors of P450 17
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 23 4443
acetate. The combined organic phases were washed with water
and brine, dried over Na2SO4 and evaporated in vacuo. The
crude product was purified by FCC (CH2Cl2:EtOAc 25:1): yield
azetidine, 0.5H, J ) 9.28 Hz, 3.2 Hz); 3.53 (m, C3RH, 1H);
5.34 (d, C6, dCH-, 1H, J ) 5 Hz). IR (KBr) cm-1: νmax 3300,
2950, 1740, 1440, 1380, 1050, 730. Anal. (C22H35NO) C, H, N.
1
21%, white solid; mp 137-41 °C. H NMR (CDCl3): δ 0.73 (s,
Biologica l Meth od s. 1. En zym e P r ep a r a tion s. The
enzymes were prepared according to previously described
methods: human and rat testicular P450 17,21 human pla-
cental P450 arom,50 bovine adrenal P450 scc,50 and 5R-
reductase type II.53 For the P450 TxA2 assay citrated human
whole blood was used.51
C18-Me, 0.9H); 0.82(s, C18-Me, 2.1H); 1.01 (s, C19-Me, 3H),
1.10 (d, C21-Me, 3H, J ) 9.28 Hz); 2.23-2.29 (m, 17RH, 1H);
3.53 (m, C3RH, 1H); 5.34 (s, C6, dCH-, 1H). 13C NMR (CDCl3)
δ 138,2 (C-5); 118,8 (C-6); 69,1 (C-3); 54,2; 52,5; 51,7; 47,4; 40,4;
39,7; 36,6; 34,7; 33,9; 29,2; 29,1; 29,0; 25,9; 21,2; 18,5; 18,3
(C19); 16,8 (C18); 10,6 (C21). IR (KBr) cm-1: νmax 3400, 3240,
2950, 1460/40, 1390, 1170, 1070. Anal. (C21H34N2O‚0.5H2O) C,
H, N.
2. En zym e Assa ys. The following enzyme assays were
performed as described previously: rat P450 17,21 human P450
17,31 P450 arom,50 P450 TxA2,51 P450 scc,50 DU145 cells (5R-
reductase type I),54 and 5R-reductase and type II.53
Ki and Km values were determined according to Lee and
Wilson.58 Inhibitor concentrations were between the IC40 and
IC70 value of the compound, substrate concentrations between
1.25 and 20 µM. The incubation time was 15 min. All other
parameters were identical to the regular P450 17 assay. UV
difference spectroscopy experiments were performed as previ-
ously described.14
17â-Dia zir in yl-20-m eth yla n d r ost-5-en -3â-ol (19). In 18
mL CHCl3 496 mg of 18 (1.5 mmol) was dissolved and mixed
with 0.41 mL triethylamine (3 mmol). Over a period of 10 min,
0.47 g Br2 (3 mmol) in 5 mL CHCl3, was added at 0 °C. The
reaction mixture was poured into water and evaporated in
vacuo. The crude product was diluted in 6 mL of acetone and
2.25 g NaI (1.5 mmol) was added. The reaction mixture was
stirred 16 h at room temperature before it was poured onto
ice and extracted two times with ethyl acetate (50 mL). The
combined organic phases were washed with aqueous Na2S2O3
solution, water and brine, dried over MgSO4 and evaporated
in vacuo. The crude product was purified by FCC (toluene:
ether 4:1): yield 38%, white solid; mp 125-8 °C. 1H NMR
(CDCl3): δ 0.86 (s, C18-Me, 3H); 1.00 (s, C21-Me, 3H); 1.02
(s, C19-Me, 3H); 3.51 (m, C3RH, 1H); 5.33 (d, C6, dCH-, 1H,
J ) 5 Hz). 13C NMR (CDCl3): 140.9 (C-5); 121.3 (C-6); 71.7
(C-3); 55.9 (C-17); 53.8; 50.1; 43.8; 42.3; 38.7; 37.2; 36.5 (C-
10); 316; 31.5; 31.3; 25.8; 24.0; 23.5; 21.3 (C-21); 20.7; 19.3 (C-
19); 13.1 (C-18). IR (KBr) cm-1: νmax 3500-3450, 2950, 1598,
1450/40, 1390/80/70, 1065. UV (EtOH)/nm: λmax 352, 239, 204.
Anal. (C21H32N2O) C, H, N.
3â-Acetoxy-17â-(a zetid in yl-2-on e)a n d r ost-5-en e (21). 50
µL of chlorosulfonyl isocyanate (0.57 mmol) was added to 125
mg of 20 (0.44 mmol) in 5 mL of toluene. The reaction mixture
was heated at 50 °C for 24 h and cooled to room temperature,
poured onto ice and extracted with toluene and CH2Cl2. The
combined organic phases were washed with water and brine,
dried over MgSO4 and evaporated in vacuo. The resulting
chlorosulfonylazetidinone intermediate was diluted in 1 mL
ether and dropped to 2 mL 25% aqueous Na2SO3 and 1 mL
ether. The reaction mixture was stirred for 60 min at room
temperature. During the reaction, the solution was kept at
neutral pH by addition of 10% aqueous KOH. The reaction
mixture was poured onto ice and extracted with ethyl acetate
and ether. The combined organic phases were washed with
water and brine, dried over MgSO4 and evaporated in vacuo.
The crude product was purified by FCC (CH2Cl2:EtOAc 2:1).
A 2:1 diastereomeric mixture was obtained: yield 36%, white
solid. 1H NMR (CDCl3): δ 0.67(s, C18-Me, 2H); 0.72(s, C18-
Me, 1H); 1.02 (s, C19-Me, 3H); 2.03 (s, CH3-acetyl, 3H); 2.66/
2.68 (ddd, azetidinone, 1H, J ) 15.04 Hz, 2.64 Hz, 1.32 Hz);
3.02/3.04 (ddd, azetidinone, 1H, J ) 14.6 Hz, 4.84 Hz, 2.2 Hz);
3.62/3.57 (m, azetidinone, 1H); 4.61 (m, C3RH, 1H); 5.37 (d,
C6 ) CH-, 1H, J ) 4.8 Hz); 5.84/6.03 (s, amide-H, 1H). IR
(KBr) cm-1: νmax 3200, 2950, 1760, 1750, 1730, 1465/50/40,
1380/60, 1030. MS: m/e 386 (M+), 313, 310, 285, 282.
3. Deter m in a tion of P la sm a Testoster on e Con cen tr a -
tion . Tests were performed with adult male Sprague-Dawley
rats (each group consisted of 7-8 animals). All compounds
were dissolved in a mixture of olive oil and benzyl alcohol (95:
5) and administered once intraperitoneally at equimolar doses
to 10 mg/kg ketoconazole (0.019 mmol/kg). Blood samples were
taken by cardiac puncture under diethyl ether anesthesia after
2 and 6 h. Plasma testosterone values were determined by a
commercially available RIA and are reported in ng/mL plasma.
Ack n ow led gm en t. Thanks are due to Dr. Wolfgang
Reichert for performing the DU 145 cell culture assays
and to Dr. J . Schro¨der for GC-MS experiments. The
authors thank Prof. Vincent Njar, Department of Phar-
macology and Experimental Therapeutics, University
of Maryland, Baltimore, MD, for making compound III
available and Prof. Patrick Bednarski for reading the
manuscript. We are grateful to the Hermann-Schlosser-
Stiftung for awarding a scholarship to one of the authors
(M.H.) and to the Fonds der Chemischen Industrie for
financial support.
Su p p or tin g In for m a tion Ava ila ble: X-ray structure and
crystallographic data for 13 and 15. This material is available
free of charge via the Internet at http://pubs.acs.org.
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1
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