Synthesis and evaluation of novel steroidal oxime inhibitors of P450 17 (17α-hydroxylase/c17-20-lyase) and 5α-reductase types 1 and 2

Article abstract of DOI:10.1021/jm001008m

17α-Hydroxylase/C17-20-lyase (P450 17, CYP 17) and 5α-reductase are the key enzymes in androgen biosynthesis and targets for the treatment of prostate cancer and benign prostatic hyperplasia. In the search of inhibitors for both enzymes, 23 pregnenolone- or progesterone-based steroids were synthesized bearing an oxime group connected directly or via a spacer to the steroidal D-ring. Tested for inhibition of human and rat P450 17, some pregnenolone (9, 11, 14) and a series of progesterone compounds (17-20) turned out to be highly active inhibitors of the human enzyme. The most active compound was Z-21-hydroxyiminopregna-5,17(20)-dien-3β-ol (9) showing K(i) values of 44 and 3.4 nM for the human and rat enzymes, respectively, and a type II UV-difference spectrum indicating a coordinate bond between the oxime group and the heme iron. In contrast to the pregnenolones which showed no inhibition of 5α-reductase isozymes 1 and 2, the progesterones 16, 17, 20, 21, and 23 showed marked inhibition, especially toward the type 2 enzyme. Compounds 17 and 20 were identified as potent dual inhibitors of both P450 17 and 5α-reductase. Tested for selectivity, the most potent P450 17 inhibitors 9, 10, and 14 showed no or only marginal inhibition of P450 arom, P450 scc, and P450 TxA₂. Selected compounds were tested for inhibition of the target enzymes using whole-cell assays. Compounds 9-11 strongly inhibited P450 17 being coexpressed with NADPH-P450 reductase in E. coli cells, and 16, 20, and 23 markedly inhibited 5α-reductase expressed in HEK 293 cells. Tested for in vivo activity, 9 (0.019 mmol/kg) decreased the plasma testosterone concentration in rats after 2 and 6 h by 57% and 44%.

Full text of DOI:10.1021/jm001008m

4266
J . Med. Chem. 2000, 43, 4266-4277
Syn th esis a n d Eva lu a tion of Novel Ster oid a l Oxim e In h ibitor s of P 450 17
(17r-Hyd r oxyla se/C17-20-Lya se) a n d 5r-Red u cta se Typ es 1 a n d 2
Rolf W. Hartmann,* Markus Hector, Samer Haidar, Peter B. Ehmer, Wolfgang Reichert, and J oachim J ose
Pharmaceutical and Medicinal Chemistry, University of the Saarland, P.O. Box 151150, D-66041 Saarbru¨cken, Germany
Received J une 19, 2000
17R-Hydroxylase/C17-20-lyase (P450 17, CYP 17) and 5R-reductase are the key enzymes in
androgen biosynthesis and targets for the treatment of prostate cancer and benign prostatic
hyperplasia. In the search of inhibitors for both enzymes, 23 pregnenolone- or progesterone-
based steroids were synthesized bearing an oxime group connected directly or via a spacer to
the steroidal D-ring. Tested for inhibition of human and rat P450 17, some pregnenolone (9,
11, 14) and a series of progesterone compounds (17-20) turned out to be highly active inhibitors
of the human enzyme. The most active compound was Z-21-hydroxyiminopregna-5,17(20)-dien-
3â-ol (9) showing Ki values of 44 and 3.4 nM for the human and rat enzymes, respectively, and
a type II UV-difference spectrum indicating a coordinate bond between the oxime group and
the heme iron. In contrast to the pregnenolones which showed no inhibition of 5R-reductase
isozymes 1 and 2, the progesterones 16, 17, 20, 21, and 23 showed marked inhibition, especially
toward the type 2 enzyme. Compounds 17 and 20 were identified as potent dual inhibitors of
both P450 17 and 5R-reductase. Tested for selectivity, the most potent P450 17 inhibitors 9,
10, and 14 showed no or only marginal inhibition of P450 arom, P450 scc, and P450 TxA2.
Selected compounds were tested for inhibition of the target enzymes using whole-cell assays.
Compounds 9-11 strongly inhibited P450 17 being coexpressed with NADPH-P450 reductase
in E. coli cells, and 16, 20, and 23 markedly inhibited 5R-reductase expressed in HEK 293
cells. Tested for in vivo activity, 9 (0.019 mmol/kg) decreased the plasma testosterone
concentration in rats after 2 and 6 h by 57% and 44%.
Ch a r t 1. Target Enzymes
In tr od u ction
Two enzymes involved in androgen biosynthesis are
targets for the treatment of prostatic diseases: 17R-
hydroxylase/C17-20-lyase (P450 17, CYP 17) and 5R-
reductase (Chart 1).1,2 P450 17 catalyzes the 17R-
hydroxylation of pregnenolone and progesterone (P) and
the subsequent cleavage of the C20,21-acetyl group to
yield the corresponding androgen.3 The antimycotic
ketoconazole, which is not only an inhibitor of fungal
14R-demethylase but also of human P450 17,4 has
already been successfully used in the treatment of
prostate cancer (PC) in men.5 Because of its side effects,
some of which are related to its low selectivity, it is not
commonly accepted for wide use.6 A number of
steroidal1,2,7-12 and nonsteroidal1,2,13-25 inhibitors of
P450 17 have been described as potential drugs for the
treatment of PC. All of them contain a functional group,
mostly a nitrogen-bearing heterocycle, capable to form
a coordinate bond with the heme iron of the enzyme.
Regarding steroidal compounds, a 3-pyridyl group and
a 4-imidazolyl group in position 17 lead to potent
inhibitors I and II (Chart 2),7,10 the former of which is
in clinical trial.2
Ch a r t 2. Selected Inhibitors of P450 17
5R-Reductase catalyzes the conversion of testosterone
(T) to the more potent androgen dihydrotestosterone
(DHT).26 High concentrations of the latter are associated
with benign prostatic hyperplasia (BPH),27 prostatic
cancer,28 and diseases such as male pattern baldness.29
There are two isozymes (types 1 and 2) with different
tissue distributions.30 The most currently used 5R-
reductase inhibitor in BPH treatment is finasteride, a
steroidal compound inhibiting mainly isozyme 2. The
long-term pharmacotherapy with finasteride generally
has been well-tolerated.31 Nevertheless, its limited
activity and its side effects32 have caused us and others
to look for new classes of steroidal33-35 and non-
steroidal36-45 inhibitors.
* Corresponding author. Tel: +49 681 302 3424. Fax: +49 681 302
4386. E-mail: rwh@rz.uni-sb.de.
10.1021/jm001008m CCC: $19.00 © 2000 American Chemical Society
Published on Web 09/26/2000

Novel Steroidal Oxime Inhibitors of P450 17
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22 4267
Sch em e 1a
Starting from 3â-acetoxyandrost-5-en-17-one, the tri-
flate 8c was synthesized.7 This triflate was transferred
with ethyl vinyl ether and catalytic amounts of tetra-
(triphenylphosphine)palladium.49 Two products were
obtained resulting from the R- or â-attack of the pal-
ladium intermediate on the double bond. Subsequent
â-elimination and hydrolysis with HClO4 yielded 8b and
10b in a ratio of 2:9 which were separated by liquid
1
chromatography (LC). H NMR data show that 8b is a
1:1 mixture of the E- and Z-isomers. Treatment of 10b
with NH2OH and deprotection yielded the oxime 10. 8b
was converted in the same way into a mixture of the
acetylated oximes 8a and 9a . At this stage the E- and
Z-isomers were separated by LC. Using 1H NMR-
NOESY technique, 8a was classified as the E-isomer
and 9a as the Z-isomer. In the spectra of 8a a coupling
of the C20 proton and the C18 methyl protons was
observed, while the C21 proton showed a signal with
the C16 methylene group. Deprotection led to the
oximes 8 and 9, respectively. The ∆14,∆16-ketone 11b
was obtained according to the method of Solo and
Singh.50 After protection of the ∆5-double bond by
addition of Br2, the allylic 14-position was brominated
(AIBN, NBS). Treatment of the tribromide according to
Finkelstein gave the ∆14,∆16-ketone 11b. Treatment
of 11b with NH2OH and deprotection of 11a gave the
oxime 11. For the preparation of compound 12 the
method of Templeton and Yan51 was applied, treating
11b with tri-n-butyltin hydride under irradiation (sun-
beam lamps). TLC monitoring revealed the formation
of a side product, which was determined by 1H NMR as
the 17R-isomer of the desired ∆14-ketone. The ratio
between the â- and R-isomers was 11:2. After LC
separation, each isomer was transferred into the cor-
responding oxime 12 and 13, respectively. The protected
oxime 13a was not isolated but directly deprotected to
13.
A methylene spacer was introduced into the 17â-side
chain by the following synthetic route (Scheme 3). 3â-
THP-androst-5-en-17-one was transferred to the nitrile
14c using Wittig-Horner reaction. Selective reduction
of the exo-configurated double bond with Mg/MeOH52
yielded the 17â-derivative 14b, which was reduced to
the aldehyde 14a using DibaH. The latter was converted
to the oxime 14 by NH2OH treatment and deprotection
or was transferred by Grignard reaction (Mg, MeI) and
subsequent oxidation of the resulting alcohol to the
ketone 15a . The oxidation was performed with PCC
adsorbed on Al2O3, which simplifies the purification of
15a . NH2OH treatment of 15a gave the corresponding
oxime 15.
a
Reagents and conditions: Method A: NH2OH, NaOAc; (a)
n-amyl nitrile, K; (b) NH2NH2‚H2O, CH3COOH; (c) KOH.
As it cannot be expected that P450 17 inhibition leads
to a complete blockade of androgen formation, it might
be advantageous to additionally inhibit 5R-reductase.
A dual inhibitor of both enzymes, however, must not
inhibit aromatase, P450 arom, another P450 enzyme
necessary for the metabolism of testosterone (Chart 1).
Several compounds which inhibit both P450 17 and 5R-
reductase have already been reported.1,2,11
There are few nonsteroidal22 and steroidal1,2,11 oximes
described in the literature, some of which are dual
inhibitors of P450 17 and 5R-reductase.2,11 In this study
we report on the synthesis of 23 steroidal pregnenolone-
and progesterone-based oximes and the evaluation of
their biological activity. Inhibition of the target enzymes
(P450 17 rat and human; human 5R-reductase isozymes
1 and 2) using in vitro as well as cellular assays was
determined as well as selectivity toward other P450
enzymes (P450 scc, P450 arom, P450 TxA2). In vivo
P450 17 inhibition of compound 9 was measured in rats.
Ch em istr y
Compounds 1, 4, and 5 (Scheme 1) were synthesized
as previously described.46,47 The oximes 2, 3, 6,8
7
Selected compounds (6, 9-11, 14, 15) were trans-
ferred into their progesterone derivatives (16-21) by a
modified Oppenauer oxidation using N-methylpiperi-
done and aluminum isopropoxide as reagents (Scheme
4). The 5-en-3â-ol AB-ring of compounds 6 and 15 was
converted to a 4-ene-3,6-dione system (22, 23; Scheme
4) by treatment with PDC in DMF as previously
described.53
(Scheme 1), 10,11,48 11, 12, 13 (Scheme 2), and 15
(Scheme 3) were obtained by reaction of the correspond-
ing steroidal ketones; the oximes 8, 9 (Scheme 2), and
14 (Scheme 3) were obtained by reaction of the corre-
sponding steroidal aldehydes with NH2OH. The syn-
thesis of oximes 1-7, 14, and 15 (Schemes 1-3) started
from the pregnenolone derivatives; the synthesis of
compounds 8-13 started from the corresponding 3â-
protected steroids, which gave after cleavage of the
protecting group the desired pregnenolone derivatives.
Introduction of a ∆14-double bond, ∆16-double bond,
or ∆14,∆16-double bond system into the D-ring was
performed following the described route (Scheme 2).
Biologica l P r op er ties
The inhibitory activity toward P450 17 rat and human
enzymes was tested using testicular microsomes. Proges-
terone was used as substrate, and RP-HPLC with UV

4268 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22
Hartmann et al.
Sch em e 2a
a
Reagents and conditions: (a) 2,6-di-tert-butyl-4-methylpyridine, (F3CSO2)2O; (b) P(Ph3)4Pd, CH3CH2OCHdCH2; (c) 1. Br2, CH3COOH,
2. NBS, AIBN, 3. NaI, acetone; (d) HSn(n-butyl)3, h; Method A: NH2OH, NaOAc; Method B: deacetylation, KOH (10%), MeOH.
Sch em e 3a
a
Reagents and conditions: (a) (EtO)2POCH2CN, NaH; (b) Mg, MeOH; (c) DibaH, -76 °C; (d) 1. CH3I, Mg, 2. PCC-Al2O3; Method A:
NH2OH, NaOAc; (e) deprotection, PPTS.
Sch em e 4a
of the P450 17 rat and human enzymes (data not given).
In contrast to these compounds the C20-oxime 6 shows
a rather good inhibition of the human enzyme, exhibit-
ing an IC50 of 1.65 µM (Table 1). Introduction of a 17R-
hydroxy group into compound 6 causes a loss of activity
(compound 7), whereas introduction of a ∆16-double
bond increases activity toward the human enzyme
dramatically (Table 2, compounds 10 and 11). The
increase of activity is probably due to the conjugation
of the oxime group with the D-ring double bond, because
compound 12, bearing an isolated ∆14-double bond,
shows only poor activity. Exchanging the side chain of
compound 12 from the â-position into the R-position
(compound 13, Scheme 2) does not increase activity (IC50
values toward rat and human enzyme: >125 and >2.5
µM, respectively). It is striking that all pregnenolone
C20-oximes exhibit almost no inhibition of the rat
enzyme.
a
Reagents and conditions: Method C: Al(OCH(CH3)2)3, N-
methylpiperidone; Method D: PDC, DMF.
detection was employed for product determination as
recently described by us.14,24 Compounds 1-5 (Scheme
1) bearing one or two oxime groups connected directly
to the steroidal D-ring show only poor or no inhibition
The C21-oximes 14, 15, 8, and 9 show moderate (8)
to excellent (9) inhibitory activity toward the human
enzyme (Tables 1 and 3) and reasonable activity toward

Novel Steroidal Oxime Inhibitors of P450 17
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22 4269
Ta ble 1. Inhibition of P450 17 Rat and Human Enzymes by 17-Substituted Steroidal Oximes
a
b
Concentration of inhibitor required to give 50% inhibition. Rat testicular microsomes, concentration of progesterone (substrate) 25
µM. c Human testicular microsomes, concentration of progesterone (substrate) 25 µM. The given values are mean values of at least two
experiments, deviations within (5%. See ref 8. ni ) no inhibition (at 125 µM for rat and 2.5 µM for human enzymes, respectively).
d
Ta ble 2. Inhibition of P450 17 Rat and Human Enzymes by Pregnenolone and Progesterone Oximes: Influence of Saturation on the
D-Ring
IC50 (µM)a
humanc
IC50 (µM)a
humanc
>2.5
0.10
D-ring
compd
ratb
ni
>125
125
compd
ratb
saturated
∆16
∆14,16
∆14
6d
10e
11
1.65
0.17
0.20
16
>125
>125
>125
18e
19
0.20
12
>125
>2.5
ketoconazole
67
0.74
ketoconazole
67
0.74
a
Concentration of inhibitor required to give 50% inhibition. ni ) no inhibition. b Rat testicular microsomes, concentration of progesterone
(substrate) 25 µM. c Human testicular microsomes, concentration of progesterone (substrate) 25 µM. The given values are mean values
of at least two experiments, deviations within (5%. See ref 8. e See ref 11.
d
Ta ble 3. Inhibition of P450 17 Rat and Human Enzymes by Steroidal Oximes and Ketoconazole (descending order of activitya)
rat enzyme
human enzyme
compd
type
D-ring
R
IC50 (µM)b
compd
type
D-ring
R
IC50 (µM)b
17
20
9
14
21
15
prog
prog
preg
preg
prog
preg
satd
satd
satd
satd
satd
satd
CHCHNOH
CH2CHNOH
CHCHNOH
CH2CHNOH
CH2CCH3NOH
CH2CCH3NOH
0.14
0.30
0.52
2.76
3.6
9.8
67
125
9
preg
prog
preg
prog
preg
prog
preg
prog
sat
CHCHNOH
CCH3NOH
CCH3NOH
CHCHNOH
CCH3NOH
CCH3NOH
CH2CHNOH
CH2CHNOH
0.077
0.10
0.17
0.18
0.20
0.20
0.27
0.30
0.74
1.18
1.65
18
10
17
11
19
14
20
∆16
∆16
satd
∆14,16
∆14,16
satd
ketoconazole
11
preg
∆14,16
CCH3NOH
satd
ketoconazole
15
6
preg
preg
satd
satd
CH2CCH3NOH
CCH3NOH
a
b
IC50 values of compounds not listed were >125 and >2.5 µM for the rat and human enzymes, respectively. See Tables 1 and 2.
the rat enzyme (compound 14 and 9). Compound 9 is
the most active inhibitor of the human enzyme in this
study, being 64 times more potent than its E-isomer
(compound 8). The progesterone compounds with a
saturated D-ring show reduced inhibition of the human
enzyme compared to the corresponding pregnenolone

4270 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22
Hartmann et al.
F igu r e 1. Inhibition of human P450 17 by compound 9. (A) Lee-Wilson plot of enzyme activities at various substrate and inhibitor
concentrations (see Experimental Section). The Km for progesterone was 4.3 µM. (B) Slope of each reciprocal plot against inhibitor
concentration. The given values are mean values of at least two experiments. The deviations were within (5%.
analogues, whereas inhibitory activity toward the rat
enzyme is enhanced (Tables 1 and 3). Thus, compounds
20 and 17 are potent inhibitors of the rat enzyme,
exhibiting IC50 values of 0.30 and 0.14 µM, respectively.
In case of the unsaturated D-ring compounds, however,
the change of the pregnenolone to the progesterone
skeleton does not influence inhibitory activity toward
both enzymes strongly (Table 2). The introduction of a
keto group into the 6-position of compounds 16 and 21
is not suitable for increasing inhibition (compounds 22
and 23, Table 1). It is worth mentioning that several
compounds show stronger inhibition of the two P450 17
enzymes than the reference ketoconazole (Table 3).
For compound 9 Ki values of 44 nM (human enzyme:
Km progesterone ) 4.3 µM, Figure 1) and 3.4 nM (rat
enzyme: Km progesterone ) 1.4 µM) were determined.
The chemical nature of the complex formed was studied
using UV-vis difference spectroscopy following the
procedure previously described.8 Figure 2 shows the
characteristic type II spectrum (trough: 392 nm, peak:
419 nm) indicating the formation of a coordinate bond
between the oxime nitrogen and the heme iron of the
P450 17 enzyme. This effect is concentration-dependent
(data not shown). Interestingly the type II spectrum was
not reversed by the addition of high substrate concen-
trations, suggesting that 9 forms a rather tight complex
with the heme iron.
Selected compounds were tested for inhibitory potency
versus human 5R-reductase isozymes 1 and 2. Prostate
homogenate of BPH patients was used as the source for
the type 2 enzyme (pH 5.5) and the DU 145 cell line as
the source for isozyme 1. Inhibition assays were per-
formed with radiolabeled substrate, and HPLC was
applied for product separation. While the pregnenolone
compounds do not show inhibitory activity toward both
isozymes (data not given), some of the progesterone
compounds exhibit marked inhibition (Table 4). The
C20-oxime 16 is a strong inhibitor of both isozymes (IC50
values toward types 1 and 2: 1.63 and 0.58 µM,
respectively). The introduction of one or two double

Novel Steroidal Oxime Inhibitors of P450 17
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22 4271
F igu r e 2. Rat enzyme: type 2 difference spectrum of compound 9 (A: 31 µM). Addition of excess substrate (progesterone) does
not reverse the effect (B: 300 µM, 5 min; C: 600 µM, 10 min).
Ta ble 4. Inhibition of Human 5R-Reductase Isozymes 1 and 2
by Progesterone-Derived Oximes
of the reference finasteride (IC50 values in our tests: 45
and 3 nM for types 1 and 2, respectively). Interestingly
the structure modifications performed in this study
resulted in selective inhibitors of 5R-reductase (com-
pounds 16, 21, and 23) as well as dual inhibitors of 5R-
reductase and P450 17 (compounds 20 and 17).
The selectivity of the most potent P450 17 inhibitors
was tested toward P450 arom, P450 TxA2, and P450 scc
taking into account that inhibition of these enzymes
could cause side effects. P450 scc catalyzes 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 con-
centration and interfere with thrombocyte aggregation,
respectively. High concentrations of compounds 9, 10,
and 14 were tested using the procedures described
recently (P450 scc,54 P450 arom,54 and P450 TxA255).
None of the compounds inhibit P450 arom (inhibitor
concentration: 25 µM, reference CHAG56 IC50: 0.15
µM). Only compound 9 shows weak inhibition of P 450
TxA2 (35% inhibition, 50 µM, reference dazoxiben IC50:
1.1 µM). Tested for inhibition of P450 scc, compound 14
inhibits the enzyme by 74% at an inhibitor concentra-
tion of 25 µM, while the other compounds were not
active.
A precondition for in vivo activity is the ability of the
compounds to permeate cell membranes. Therefore
selected inhibitors were tested for inhibition of the
target enzymes using the whole-cell assays recently
developed by our group57,58 Employing E. coli cells
coexpressing P450 17 and NADPH-P450 reductase,57
compounds 9-11, which were the most potent inhibitors
of the microsomal P450 17 enzyme, turned out to be very
potent in this assay as well. Exhibiting IC50 values from
0.23 to 0.53 µM, the compounds are again more potent
than the reference ketoconazole (IC50: 2.8 µM, Table
5). Tested on HEK293 cells expressing isozymes 1 and
258 of 5R-reductase, compounds 16, 20, and 23 are
similarly active, showing IC50 values between 0.86 and
2.9 µM (Table 6).
a
Human DU-145 cell assay, concentration of androstenedione
b
5 nM. Enzyme preparation from human BPH tissue, concentra-
tion of testosterone 210 nM, pH ) 5.5. The given values are mean
values of at least two experiments, deviations within (5%. ni )
no inhibition, nd ) not determined.
bonds (∆16 and ∆14,∆16) into the D-ring decreases
activity dramatically (compounds 18 and 19). A keto
group in position 6 leads to an almost complete loss of
activity toward type 1 isozyme and reduces type 2
inhibition (compound 22). Transferring the oxime group
from position 20 to 21 enhances type 2 inhibitory
potency. Thus, compound 20 is the most potent inhibitor
toward type 2 isozyme. The C22-steroids 21 and 23 show
an increased inhibition of type 1 isozyme and are strong
inhibitors of type 2 isozyme. The Z-configurated ∆17-
compound 17 is also a strong inhibitor of isozyme 2.
However, none of the compounds reaches the activity

4272 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22
Hartmann et al.
Ta ble 5. Inhibition of Human P450 17 by Selected Compounds
Using E. coli Cells Coexpressing P450 17 and NADPH-P450
Reductase
ones are similarly active as the corresponding preg-
nenolones. In rat enzyme, 9 and its progesterone
analogue 17 are very potent inhibitors. Compound 17
exceeds the activity of ketoconazole, being 478 times
more potent, and in addition is superior to the steroidal
inhibitor II10 (IC50: 0.18 µM in our assay). In human
enzyme, 9 and the D-ring unsaturated C20-oximes 10,
11, 18, and 19 exhibit strong inhibitory potency. Com-
pound 9, being 10 times more potent than ketoconazole,
shows an activity close to that of II (IC50: 0.04 µM in
our assay).
a
compd
IC50 (µM)
9
10
11
0.23
0.52
0.42
2.8
ketoconazole
a
Concentration of inhibitor required to give 50% inhibition.
Recombinant E. coli cells were used; concentration of progesterone
(substrate) 25 µM. The given values are mean values of at least
two experiments, deviations within (10%.
Inhibition of related P450 enzymes (P450 arom, P450
TxA2, P450 scc) was determined as an indication of
possible side effects. Especially inhibiton of P450 scc,
the key enzyme of steroid biosynthesis, could result in
dramatic effects by blockade of aldo- and glucocorticoid
formation. The compounds tested turned out to be
rather selective: i.e., they show either no effect or only
inhibition in high concentrations. In vivo compound 9
is active showing a strong reduction of the testosterone
plasma concentration in rats. There is no cause for
concern that inhibition of P450 17 could reduce adrenal
17R-hydroxylation and thus glucocorticoid synthesis. In
clinical trials with P450 17 inhibitors ketoconazole59 and
liarozole,60 it has been shown that the androgen plasma
concentration was reduced without glucocorticoid plasma
levels being affected.
Some of the title compounds show potent inhibition
of the second target enzyme 5R-reductase, compounds
16, 17, 20, 21, and 23. They have a saturated D-ring in
common and are progesterone-based. Compounds 16,
20, and 23, tested for activity using whole cells, are
highly active as well.
Potent dual inhibitors of both target enzymes are
compounds 17 and 20. They might be leads for the
development of drugs for the treatment of androgen-
dependent diseases. Being active in vivo, compound 9
might be a lead for further development of a prostate
cancer drug.
Ta ble 6. Inhibition of Human 5R-Reductase Isozymes 1 and 2
by Selected Compounds Using HEK293 Cells Expressing
Isozymes 1 and 2
a
IC50 (µM)
compd
16
20
23
HEK293-5R1
HEK293-5R2
1.77
2.44
2.90
0.54
1.17
0.89
0.86
0.06
finasteride
a
Concentration of inhibitor required to give 50% inhibition;
300 000 cells/well transfected with type 1 5R-reductase expression
plasmid pRcCMV-I were used and 300 000 cells/well transfected
with type 2 5R-reductase expression plasmid pRcCMV-II were
used; concentration of [3H]androstenedione 5 nM; incubation time
30 and 13 min for types 1 and 2, respectively. The given values
are mean values of at least three experiments, deviations within
(10%.
A prerequisite for performing in vivo experiments in
the rat is the effectiveness of the compounds toward the
rat enzyme. It is apparent from Tables 1 and 2 that
several highly potent inhibitors of human P450 17 do
not show satisfactory inhibition of the rat enzyme to be
tested in vivo. Fortunately the most potent inhibitor of
human P450 17, compound 9, inhibits the rat enzyme
sufficiently. It was administered intraperitoneally to
adult SD rats equimolar to 10 mg/kg ketoconazole (0.019
mmol/kg). In contrast to the reference compound, which
is not active at this dose, 9 decreases the plasma
testosterone concentration after 2 and 6 h by 57% and
44%, respectively (plasma testosterone concentration ng/
mL: control (2 h) 1.51, (6 h) 0.71; ketoconazole (2 h)
1.60, (6 h) 0.64; 9 (2 h) 0.66, (6 h) 0.39; n ) 7-8).
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 disks. 1H NMR spectra were
recorded on a Bruker AM-400 (400 MHz) or DRX 500 (500
MHz) instrument. 13C NMR spectra were recorded on a Bruker
AM-400 (120 MHz) instrument. Chemical shifts are given in
parts per million, and TMS was used as internal standard for
spectra obtained in DMSO-d6 and CDCl3. 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 in the Inorganic Chemistry Department,
University of the Saarland. Reagents and solvents were used
as obtained from commercial suppliers without further puri-
fication. Column chromatography (LCC) was performed using
silica gel 60 (50-200 µm), flash-column-chromatography (FCC)
using silica gel 60 (40-63 µm), and HPLC using a semi-
preparative RP-18-column (16 mm × 250 mm, particle size:
5 µm, Nucleosil-C18), and reaction progress was determined
by TLC analysis on ALUGRAM SIL G/UV254 (Macherey-
Nagel).
Discu ssion a n d Con clu sion
The present study shows that some of the synthesized
steroidal oximes are selective inhibitors of either human
P450 17 (16, 9, 10,11 11, 14, 15, 18,11 19) or 5R-reductase
(16, 21, 23). Interestingly, a few compounds are dual
inhibitors of both enzymes (17, 20).
It is obvious that the oxime group is appropriate to
coordinate with the heme iron of P450 17. It might also
be a suitable functional group for the design of inhibitors
of other P450 enzymes. A high species difference is
apparent from the differing inhibition values for the P
450 17 isozymes shown by many compounds. Although
17R-OH-pregnenolone is a high-affinity substrate of P
450 17, introduction of a OH group into position 17R of
compound 6 leads to complete loss of inhibitory activity
toward the human enzyme 7. The present data shows
that the inhibitory potency strongly depends on the
position of the oxime group: the C21-oximes are more
active than the C20-oximes. Inhibition, however, is not
related to the basic steroidal structure: the progester-
Meth od A. Gen er a l P r oced u r e for th e Syn th esis of
Oxim e Com p ou n d s 2, 3, 6, 7, 8a -12a , 14, a n d 15. 983 mg
NaOAc (12 mmol) and 421 mg NH2OH‚HCl (6 mmol) were
dissolved in 100 mL MeOH and the solution was refluxed for
10 min. The hot solution was dropped to the steroidal ketone

Novel Steroidal Oxime Inhibitors of P450 17
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22 4273
(5.95 mmol) and stirred for 2 h at 4 °C. The reaction mixture
was diluted with 65 mL water and extracted four times with
65 mL EtOAc. The organic phase was washed twice with
water, brine and dried over MgSO4. The compounds were
purified by FCC and recrystallization from EtOH/H2O (1:1).
21-Hyd r oxyim in o-21-m eth ylp r egn -5-en -3â-ol (15): pu-
rification recrystallization (H2O:EtOH 1:1); yield 97%, white
1
solid, mp 146-50 °C; H NMR (DMSO-d6) δ 0.59 (s, C18-Me,
3H); 0.94 (s, C19-Me, 3H); 1.70 (s, C22-Me, 3H); 3.25 (m, C3RH,
3
1H); 5.26 (d, C6, dCH-, 1H, J ) 5.26 Hz); 10.16 (s, dNOH,
1H); IR (KBr) cm-1 νmax 3300, 2950, 1730, 1440, 1370, 1250,
1050; GC-MS m/e 345 (M+), 330, 328, 313, 260, 234. Anal.
(C22H35NO2) C, H, N.
16,17-Dih yd r oxyim in oa n d r ost-5-en -3â-ol (2): purifica-
tion FCC (dichloromethane:ethyl acetate 1:1); yield 80%, white
1
crystals, mp 248-50 °C; H NMR (DMSO-d6) δ 0.86 (s, C18-
Meth od B. Gen er a l P r oced u r e for th e Syn th esis of
Com p ou n d s 8-13. 1 mmol 3â-acetoxy compound was dis-
solved in 3% KOH (MeOH) and heated to 70 °C for 10 min.
The reaction mixture was cooled to room temperature, poured
onto ice and extracted two times with CH2Cl2. The combined
organic phases were washed with water and brine, dried over
MgSO4 and evaporated in vacuo. The crude product was
purified by FCC and recrystallization from EtOH/H2O (1:1).
21-Hyd r oxyim in op r egn a -5,17(20)-d ien -3â-ol (8, E-iso-
m er ): purification recrystallization (H2O:EtOH 1:1); yield
Me, 3H); 0.95 (s, C19-Me, 3H); 5.28 (m, dCH-, 1H); 10.73 (s,
dNOH, 1H); 11.11(s, dNOH, 1H); IR (KBr) cm-1 νmax 3525,
2910, 1430, 1460, 1060, 960. Anal. (C19H27N2O3) C, H, N.
17-Hyd r oxyim in oa n d r ost-5-en -3â-ol (3): purification re-
crystallization (EtOH:H2O 1:1); yield 80%, white crystals, mp
1
200-2 °C; H NMR (DMSO-d6) δ 0.83 (s, C18-Me, 3H); 0.96
(s, C19-Me, 3H); 5.28 (m, dCH-, 1H); 10,06 (s, dNOH, 1H);
IR (KBr) cm-1 νmax 3400, 2900, 1660, 1450, 1060, 950. Anal.
(C19H29NO2) C, H, N.
20-Hyd r oxyim in op r egn -5-en e-3â,17R-d iol (7): purifica-
tion recrystallization (H2O:EtOH 1:1); yield 94%, white solid,
mp 258-9 °C; 1H NMR (DMSO-d6) δ 0.65 (s, C18-Me, 3H);
1.01 (s, C19-Me, 3H); 1.99 (s, C21-Me, 3H); 3.67 (m, C3RH,
1H); 5.35 (d, C6, dCH-, 1H, 3J ) 4.8 Hz); 10.38 (s, dNOH,
1H); IR (KBr) cm-1 νmax 3500-3200, 2950, 1460, 1370, 1050.
Anal. (C21H33NO3) C, H, N.
1
98%, white solid, mp 229-33 °C; H NMR (DMSO-d6) δ 0.78
(s, C18-Me, 3H); 1.01 (s, C19-Me, 3H); 3.25 (m, C3RH, 1H);
5.27 (d, C6, dCH-, 1H, 3J ) 5.0 Hz); 5.69 (d, C20, dCH-,
1H, 3J ) 10.1 Hz); 5.69 (d, C21 dCH-, 1H, 3J ) 10.0 Hz);
10.71 (s, dNOH, 1H); IR (KBr) cm-1 νmax 3500-3200, 2950,
1650, 1440, 1320, 1050. Anal. (C21H31NO2) C, H, N.
21-Hyd r oxyim in op r egn a -5,17(20)-d ien -3â-ol (9, Z-iso-
m er ): purification recrystallization (H2O:EtOH 1:1); yield
3â-Acetoxy-21-h yd r oxyim in op r egn a -5,17(20)-dien e (8a ,
E-isom er ): purification FCC (toluene:ether 8:1); yield 38%,
1
98%, white solid, mp 207-11 °C; H NMR (DMSO-d6) δ 0.78
1
white solid, mp 197-9 °C; H NMR (500 MHz, DMSO-d6) δ
(s, C18-Me, 3H); 1.08 (s, C19-Me, 3H); 3.25 (m, C3RH, 1H);
0.78 (s, C18-Me, 3H); 1.01 (s, C19-Me, 3H); 1.98 (s, CH3COO-,
3
5.27 (s, C6, dCH-, 1H); 6.23 (d, C20, dCH-, 1H, J ) 10.0
3H); 4.43 (m, C3RH, 1H); 5.36 (d, C6, dCH-, 1H, 3J ) 4.0
3
Hz); 7.13 (d, C21 dCH-, 1H, J ) 9.6 Hz); 10.83 (s, dNOH,
3
Hz); 5.70 (d, C20, dCH-, 1H, J ) 10.0 Hz); 7.76 (d, C21 d
1H); IR (KBr) cm-1 νmax 3500-3200, 2950, 1650, 1440, 1320,
1050. Anal. (C21H31NO2) C, H, N.
3
CH-, 1H, J ) 10.5 Hz); 10.67 (s, dNOH, 1H); IR (KBr) cm-1
νmax 3520, 2950, 1750, 1650, 1370/60, 1230, 1040, 940. Anal.
20-Hyd r oxyim in op r egn a -5,16-d ien -3â-ol (10): purifica-
tion recrystallization (H2O:EtOH 1:1); yield 99%, white solid,
mp 244-7 °C (lit. mp 217-8 °C48); 1H NMR (DMSO-d6) δ 0.90
(s, C18-Me, 3H); 0.98 (s, C19-Me, 3H), 1.86 (s, C21-Me, 3H);
3.30 (m, C3RH, 1H); 5.28 (d, C6, dCH-, 1H, 3J ) 4.8 Hz);
6.03 (m, C16 dCH-, 1H); 10.70 (s, dNOH, 1H); IR (KBr) cm-1
νmax 3500-3200, 2950, 1440, 1370, 1050. Anal. (C21H31NO2‚
0.6H2O) C, H, N.
(C23H33NO3) C, H, N.
3â-Acetoxy-21-h yd r oxyim in op r egn a -5,17(20)-d ien e (9a ,
Z-isom er ): purification FCC (toluene:ether 8:1); yield 38%,
1
white solid, mp 152-6 °C; H NMR (500 MHz, DMSO-d6) δ
0.79 (s, C18-Me, 3H); 1.01 (s, C19-Me, 3H); 1.98 (s, CH3COO-,
3H); 4.43 (m, C3RH, 1H); 5.40 (d, C6, dCH-, 1H, 3J ) 4.2
Hz); 6.23 (d, C20, dCH-, 1H, 3J ) 9.6 Hz); 7.14 (d, C21 d
3
CH-, 1H, J ) 9.6 Hz); 10.84 (s, dNOH, 1H); IR (KBr) cm-1
20-Hyd r oxyim in op r egn a -5,14,16-tr ien -3â-ol (11): puri-
fication recrystallization (H2O:EtOH 1:1); yield 99%, white
νmax 3520, 2950, 1750, 1650, 1370/60, 1230, 1040, 940. Anal.
(C23H33NO3) C, H, N.
1
solid, mp 175-80 °C; H NMR (DMSO-d6) δ 1.13 (s, C18-Me,
3â-Acetoxy-20-h yd r oxyim in op r egn a -5,16-d ien e (10a ):
purification LCC (petrol ether:EtOAc 1:1); yield 55%, white
solid, mp 180-5 °C; 1H NMR (CDCl3) δ 0.95 (s, C18-Me, 3H);
1.05 (s, C19-Me, 3H); 2.00 (s, C21-Me, 3H); 2.03 (s, CH3COO-,
3H); 4.62 (m, C3RH, 1H); 5.38 (d, C6, dCH-, 1H, 3J ) 5.1
3H); 1.20 (s, C19-Me, 3H); 2.09 (s, C21-Me, 3H); 3.53 (m, C3RH,
1H); 5.46 (d, C6, dCH-, 1H, 3J ) 5.0 Hz); 6.96 (s, C16 dCH-,
1H); 7.15 (s, C15 dCH-, 1H); 10.70 (s, dNOH, 1H); IR (KBr)
cm-1
νmax 3500-3300, 2950, 1530, 1440, 1040, 1060, 980. Anal.
(C21H29NO2‚0.75H2O) C, H, N.
Hz); 6.06 (m, C16 dCH-, 1H); IR (KBr) cm-1
νmax 3480, 2950,
20-Hyd r oxyim in op r egn a -5,14-d ien -3â-ol (12): purifica-
tion recrystallization (H2O:EtOH 1:1); yield 99%, white solid,
mp 180-5 °C; 1H NMR (DMSO-d6) δ 0.77 (s, C18-Me, 3H);
0.96 (s, C19-Me, 3H); 1.77 (s, C21-Me, 3H); 2.57 (dd, C17RH,
1H, 3J ) 7.52; 10.16 Hz); 3.26 (m, C3RH, 1H); 5.20 (s; C15
dCH-, 1H); 5.32 (s, C6, dCH-, 1H); 10.42 (s, dNOH, 1H);
IR (KBr) cm-1 νmax 3400-3200, 2950, 1450/40, 1380/70, 1050.
Anal. (C21H31NO2‚0.5H2O) C, H, N.
20-Hyd r oxyim in o-17R-p r egn a -5,14-d ien -3â-ol (13): pu-
rification recrystallization (H2O:EtOH 1:1); yield 99%, white
solid, mp 103-5 °C; 1H NMR (CDCl3) δ 1.13 (s, C18-Me, 3H);
0.97 (s, C19-Me, 3H); 1.70 (s, C21-Me, 3H); 2.75 (dd, C17RH,
1H, 3J ) 4.8 Hz, 8.4 Hz); 3.24 (m, C3RH, 1H); 5.17 (s; C15
dCH-, 1H); 5.32 (s, C6, dCH-, 1H); 10.30 (s, dNOH, 1H);
IR (KBr) cm-1 νmax 3400-3200, 2950, 1460/40, 1370, 1050;
GC-MS m/e 329 (M+), 312, 297, 284, 271. Anal. (C21H31NO2)
C, H, N.
1725, 850. Anal. (C23H33NO3) C, H, N.
3â-Ace t oxy-20-h yd r oxyim in op r e gn a -5,14,16-t r ie n e
(11a ): purification FCC (petrol ether:EtOAc 1:1); yield 26%,
white solid, mp 165-8 °C; 1H NMR (DMSO-d6) δ 1.09(s, C18-
Me, 3H); 1.15 (s, C19-Me, 3H); 1.95 (s, C21-Me, 3H); 1.99 (s,
CH3COO-, 3H); 4.47 (m, C3RH, 1H); 5.46 (m, C6, dCH-, 1H);
5.96 (m, C16 dCH-, 1H); 7.62 (d, C15 dCH-, 1H, 3J ) 2.2
Hz); 10.72 (s, dNOH, 1H); IR (KBr) cm-1
νmax 3470, 2950, 1720,
850. Anal. (C23H31NO3) C, H, N.
3â-Acetoxy-20-h yd r oxyim in op r egn a -5,14-d ien e (12a ):
purification recrystallization (H2O:EtOH 1:1); yield 99%, white
1
solid, mp 183-6 °C; H NMR (DMSO-d6) δ 0.78 (s, C18-Me,
3H); 1.00 (s, C19-Me, 3H); 1.77 (s, C21-Me, 3H); 1.98 (s, CH3-
COO-, 3H); 4.44 (m, C3RH, 1H); 5.21 (m, C15 dCH-, 1H);
5.41 (s, C6, dCH-, 1H); 10.42 (s, dNOH, 1H); IR (KBr) cm-1
νmax 3300, 2950, 1730, 1440, 1370, 1250, 1050.
21-Hyd r oxyim in op r egn -5-en -3â-ol (14): purification FCC
(CH2Cl2:EtOH 10:1); yield 62%, white solid, mp 185-8 °C; 1H
NMR (DMSO-d6) δ 0.59 (s, C18-Me, 3H); 0.95 (s, C19-Me, 3H);
3.24 (m, C3RH, 1H); 5.26 (d, C6, dCH-, 1H, 3J ) 4.8 Hz); 6.63
(t, C21 CHdNOH, 0.5H, 3J ) 5.2 Hz); 7.26 (t, C21 CHdNOH,
21-Meth ylp r egn -5-en -3â-ol-21-on e (15a ). 1 mmol 3â-THP
compound61 (330.5 mg) was dissolved in 100 mL EtOH and
300 mg (1.2 mmol) pyridinium p-toluenesulfonic acid and
stirred for 3 h at 55 °C. The reaction mixture was cooled to
room temperature and 100 mL EtOAc was added. The organic
phase was washed with water and brine, dried over MgSO4
and evaporated in vacuo. The crude product was purified by
FCC and recrystallization from EtOH/H2O (1:1): purification
FCC (CH2Cl2:EtOAc 3:1); yield 99%, white solid, mp 168-70
3
0.5H, J ) 6.4 Hz); 10.30 (s, dNOH, 0.5H); 10.71 (s, dNOH,
0.5H); IR (KBr) cm-1
νmax 3500-3200, 2950, 1440/50/70, 1050;
GC-MS m/e 331 (M+), 314, 298, 296, 246, 220. Anal. (C21H33
NO2) C, H, N.
-

4274 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22
Hartmann et al.
1
°C (lit. mp 177-8 °C61); H NMR (CDCl3) δ 0.60 (s, C18-Me,
20-Hyd r oxyim in op r egn -4-en e-3,6-d ion e (22): purifica-
3H); 1.01 (s, C19-Me, 3H), 2.14 (s, C22-Me, 3H), 2.46-2.48 (dd,
tion FCC (CH2Cl2:EtOAc 5:1); yield 50%, white solid, mp
3
1
17RH, 1H, J ) 15.44 Hz, 4.02 Hz); 3.53 (m, C3RH, 1H); 5.35
124-9 °C; H NMR (CDCl3) δ 0.59 (s, C18-Me, 3H); 1.11 (s,
(t, C6, dCH-, 1H, 3J ) 2.08 Hz); 13C NMR (CDCl3) δ 209 (C21);
140.9 (C5); 121.6 (C6); 71.8 (C17); 55.6; 50.5; 50.3; 46.0; 44.8;
42.4; 42.0; 37.4; 36.7; 36.6; 32.1; 31.9; 30.2; 28.4; 24.7; 20.8;
19.4 (C19); 12.6 (C18); IR (KBr) cm-1 νmax 3520, 2950, 1700,
1440, 1380/70/60, 1250, 1060; GC-MS m/e 330 (M+), 312, 297,
245, 239. Anal. (C22H34O2) C, H, N.
C19-Me, 3H); 1.74 (s, C21-Me, 3H); 5.91 (s, C4, dCH-, 1H);
10.40 (s, dNOH, 1H); IR (KBr) cm-1 νmax 3500-3300, 2950,
1740, 1690, 1670, 1450, 1370, 1250/40, 1220. Anal. (C21H29
NO3) C, H, N.
-
21-Hydr oxyim in o-21-m eth ylpr egn -4-en e-3,6-d ion e (23):
purification FCC (CH2Cl2:EtOAc 5:1); yield 26%, white solid,
mp 70-5 °C; 1H NMR (CDCl3) δ 0.67 (s, C18-Me, 3H); 1.11 (s,
C19-Me, 3H); 1.71 (s, C22-Me, 3H); 5.90 (s, C4, dCH-, 1H);
10.19 (s, dNOH, 1H); IR (KBr) cm-1 νmax 3500-3300, 2950,
1740, 1690/80, 1265/20. Anal. (C22H31NO3‚EtOH) C, H, N.
20-Ca r bon itr ilop r egn a -5,17(20)-d ien e 3â-Tetr a h yd r o-
p yr a n yl Eth er (14c). Under a N2 atmosphere 3.6 g diethyl
cyanomethylphosphonate (20 mmol) in 20 mL DME was
dropped to 834 mg NaH (60% suspension in oil) in 25 mL
DME. The clear solution was stirred and refluxed for 10 min.
To this solution 1.93 g (5.19 mmol) THP-androstenolone was
added and refluxing was continued for 5 h. The reaction
mixture was cooled to room temperature and diluted with 50
mL ether and 25 mL water. Using additional 25 mL ether the
mixture was extracted. The organic phase was washed with
water and brine, dried over MgSO4 and evaporated in vacuo.
The crude product was purified by FCC toluene/ether (8:1).
yield 99%. Compound 14c was converted to 14b without
further analysis.
20-Car bon itr ilopr egn -5-en e 3â-Tetr ah ydr opyr an yl Eth er
(14b). 15 g 14c (38 mmol) was dissolved in 750 mL MeOH. 73
g Mg turnings were added (3 mol). After 30 min the reaction
started. The reaction mixture was stirred for 2 h, and the
reaction temperature kept below 25 °C. Once again 18.7 g Mg
turnings (0.77 mol) and 450 mL MeOH were added and the
reaction mixture was stirred at room temperature for 24 h.
At 0 °C, ether and 6 N HCl was added and the organic phase
was washed with aqueous NaHCO3, water and brine. After
drying over MgSO4, the solvent was evaporated in vacuo. The
crude product was purified by FCC toluene/EtOAc (12:1). yield
77%. Compound 14b was converted to 14a without further
analysis.
Meth od C. Gen er a l P r oced u r e for th e Syn th esis of
Oxim e Com p ou n d s 16-21. 3.38 mL methylpiperidone (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 via a Dean-Stark trap. Alu-
minum 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 ether. The reaction mixture was
washed with water and brine, dried over Na2SO4 and was
evaporated. The crude product was purified by FCC.
20-Hydr oxyim in opr egn -4-en -3-on e (16): purification FCC
(CH2Cl2:EtOAc 7:1); yield 72%, white solid, mp 208-11 °C; 1H
NMR (DMSO-d6) δ 0.59 (s, C18-Me, 3H); 1.14 (s, C19-Me, 3H);
1.72 (s, C21-Me, 3H); 5.63 (s, C4, dCH-, 1H); 10.36 (s, dNOH,
1H); IR (KBr) cm-1 νmax 3500, 2950, 1670, 1620, 1640, 1370,
1230, 970, 920. Anal. (C21H31NO2‚0.3toluene) C, H, N.
21-Hyd r oxyim in op r egn a -4,17(20)-d ien -3-on e (17, Z-iso-
m er ): purification FCC (CH3Cl:MeOH 40:1); yield 25%, white
1
solid, mp 172-9 °C; H NMR (DMSO-d6) δ 0.82 (s, C18-Me,
3H); 1.17 (s, C19-Me, 3H); 5.64 (s, C4, dCH-, 1H); 6.23 (d,
3
3
C20, dCH-, 1H, J ) 9.6 Hz); 7.14 (d, C21 dCH-, 1H, J )
9.6 Hz); 10.85 (s, dNOH, 1H); IR (KBr) cm-1
νmax 3500-3200,
2950, 1660, 1050/30/10; GC-MS m/e 328 (M+), 313, 310, 285,
282. Anal. (C21H29NO2) C, H, N.
20-Hyd r oxyim in op r egn a -4,16-d ien -3-on e (18): purifica-
tion FCC (CH2Cl2:EtOH 10:1); yield 83%, white solid, mp
253-8 °C (lit. mp 254-9 °C11); 1H NMR (DMSO-d6) δ 0.92 (s,
C18-Me, 3H); 1.17 (s, C19-Me, 3H); 1.89 (s, C21-Me, 3H); 5.84
(s, C4, dCH-, 1H); 6.02 (s, C16 dCH-, 1H); 10.74 (s, dNOH,
1H); IR (KBr) cm-1
νmax 3340, 2950, 1660/50, 1640, 1610, 1440,
P r egn -5-en -21-a l 3â-Tet r a h yd r op yr a n yl E t h er (14a ).
23.5 g 14b (59 mmol) was diluted in 250 mL dry toluene and
cooled to -76 °C. 170 mL DibaH (20% in toluene) was added
and the reaction mixture was stirred at -76 °C for 1 h. 94 mL
MeOH and 47 mL water were added and the reaction mixture
was stirred for 3 h at room temperature. After the extraction
with ether, the organic phase was washed with aqueous 5%
citric acid, aqueous NaHCO3, water and brine. After drying
over MgSO4, the solvent was evaporated in vacuo. The crude
product was purified by FCC: purification FCC (toluene:ether
9:1); yield 42%, white solid, mp 142-6 °C; 1H NMR (CDCl3) δ
0.62 (s, C18-Me, 3H); 1.02 (s, C19-Me, 3H), 3.47-3.54 (m, THP,
1370/60, 1285, 1245, 1190, 1000. Anal. (C21H29NO2) C, H, N.
20-Hyd r oxyim in op r egn a -4,14,16-tr ien -3-on e (19): pu-
rification FCC (CH2Cl2:EtOAc 15:2); yield 70%, white solid,
mp 282-4 °C; 1H NMR (DMSO-d6) δ 1.18 (s, C18-Me, 3H);
1.26 (s, C19-Me, 3H); 1.95 (s, C21-Me, 3H); 5.67 (s, C4, dCH-,
1H); 5.94 (t, C15, dCH-, 1H, 3J ) 2 Hz); 6.61 (d, C16, dCH-,
1H, 3J ) 2 Hz); 10.76 (s, dNOH, 1H); IR (KBr) cm-1
νmax 3320,
2950, 1660/50, 1615, 1450, 1360, 1290/40, 1190, 990/80/30, 900,
850, 725. Anal. (C21H27NO2) C, H, N.
21-Hydr oxyim in opr egn -4-en -3-on e (20): purification FCC
(CH3Cl:MeOH 40:1); yield 54%, white solid, mp 149-51 °C;
1H NMR (DMSO-d6) δ 0.63 (s, C18-Me, 3H); 1.15 (s, C19-Me,
3H); 5.62 (s, C4, dCH-, 1H); 6.63 (t, C21 CHdNOH, 0.7H, 3J
3
2H); 3.91 (m, THP, 1H); 4.71 (d, THP, 1H, J ) 4.4 Hz); 5.35
3
3
(t, C6, dCH-, 1H, J ) 5.72 Hz); 9.77 (t, C21, CHO, 1H, J )
3
2.24 Hz); IR (KBr) cm-1
νmax 2950, 1730, 1200, 1120, 1060/40.
) 5.2 Hz); 7.25 (t, C21 CHdNOH, 0.3H, J ) 5.2 Hz); 10.31
(s, dNOH, 0.7H); 10.71 (s, dNOH, 0.3H); IR (KBr) cm-1 νmax
3300, 2950, 1680/60, 1620; GC-MS m/e 330 (M+), 313, 288,
217. Anal. (C21H31NO2) C, H, N.
3â-Acetoxyp r egn a -5,14-d ien -20-on e (12b)51 a n d 3â-Ace-
toxy-17R-p r egn a -5,14-d ien -20-on e (13b). 200 mg 11b (0.56
mmol) was diluted in 40 mL xylene. 2 mL (0.75 mmol) tri-n-
butyl-SnH was added under a N2 stream to this solution. The
reaction mixture was stirred under irradiation (2 × 150 W
Osram-Sunbeam lamps) for 1 h at room temperature and 12
h under reflux. After that 20 mL MeOH was added and the
mixture was refluxed for an additional hour to terminate the
reaction. The reaction mixture was cooled to room tempera-
ture, diluted with water and extracted with CH2Cl2. The
organic phase was washed with aqueous NaHCO3, water and
brine, dried over Na2SO4 and evaporated in vacuo. The crude
product was purified by FCC (toluene:ether 10:1). A 11:2
mixture of compounds 12b and 13b was obtained. Compound
12b: yield 39%, white solid, mp 156-8 °C (lit. mp 158-61
21-Hyd r oxyim in o-21-m eth ylp r egn -4-en -3-on e (21): pu-
rification FCC (CH2Cl2:EtOAc 8:1); yield 53%, white solid, mp
159-60 °C; H NMR (DMSO-d6) δ 0.82 (s, C18-Me, 3H); 1.15
1
(s, C19-Me, 3H); 1.70 (s, C22-Me, 3H); 5.76 (s, C4, dCH-, 1H);
10.13 (s, dNOH, 1H); IR (KBr) cm-1 νmax 3320, 2950, 1650,
1615; MS m/e 344 (M+), 327. Anal. (C22H33NO2) C, H, N.
Meth od D. Gen er a l P r oced u r e for th e Syn th esis of
Oxim e Com p ou n d s 22 a n d 23. 5.53 mmol pyridinium
dichromate (PDC) in 20 mL dry MeOH was added to 250 mg
(0.79 mmol) pregnenolone compound in 20 mL dry DMF. The
reaction mixture was stirred for 6 h at room temperature. The
reaction mixture was poured into 350 mL water and was
extracted twice with EtOAc. The organic phase was washed
with water and brine, dried over Na2SO4 and evaporated in
vacuo. The crude product was purified by FCC and recrystal-
lization from EtOH/H2O (1:1).
1
°C51); H NMR (CDCl3) δ 0.88 (s, C18-Me, 3H); 1.02 (s, C19-
Me, 3H); 2.03 (s, C21-Me, 3H); 2.17 (s, CH3COO-, 3H); 2.93
(dd, 17RH, 1H, 3J ) 9.76 Hz, 8.4 Hz); 4.61 (m, C3RH, 1H);
3
5.18 (d, C15 dCH-, 1H, J ) 2 Hz); 5.43 (t, C6, dCH-, 1H,

Novel Steroidal Oxime Inhibitors of P450 17
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22 4275
3J ) 2 Hz). Compound 13b: yield 7%, white solid, mp 148-
difference spectroscopy experiments were performed as previ-
ously described.8
1
50 °C; H NMR (CDCl3) δ 1.33 (s, C18-Me, 3H); 1.04 (s, C19-
Me, 3H); 2.04 (s, C21-Me, 3H); 2.17 (s, CH3COO-, 3H); 3.08
E. coli (P 450 17/NADP H-P 450 Red u cta se) Assa y.57 To
test the inhibitory activity of compounds on human P450 17
coexpressed with rat NADPH-P450 reductase, 0.1 M sodium
phosphate, pH 7.4, was preincubated with 25 µM 1,2-[3H]-
progesterone and an appropriate concentration of inhibitor at
37 °C for 10 min. The reaction was started by the addition of
a suspension of recombinant E. coli XL1 pJ L17/OR.57 A578 was
3.0. After 45 min of vigorous shaking of the horizontally
positioned cups at 37 °C, the reaction was stopped by heating
at 95 °C for 5 min. Steroids were extracted for 5 min with ethyl
acetate. The samples were evaporated, dissolved in methanol
and analyzed by HPLC as described.57
(dd, 17RH, 1H, 3J ) 8.84 Hz, 5.32 Hz); 4.61 (m, C3RH, 1H);
3
5.17 (d, C15 dCH-, 1H, J ) 2.68 Hz, 4.44 Hz); 5.43 (t, C6,
3
dCH-, 1H, J ) 2 Hz).
3â-Acetoxyp r egn a -5,17(20)-d ien -21-a l (8b) a n d 3â-Ace-
toxyp r egn a -5,16-d ien -20-on e (10b).50 11.5 g 8c (25 mmol)
in 125 mL dry DMSO was treated with 6.21 g (8.63 mL, 61
mmol) triethylamine and 520 mg (PPh3)4Pd. 8.95 g (12.05 mL,
124 mmol) ethyl vinyl ether was dropped to the reaction
mixture. After heating to 60 °C, the reaction mixture was
stirred for 4 h. 0.5 M HCl was added and the aqueous solution
was extracted twice with EtOAc. The combined organic phases
were washed with aqueous NaHCO3, water and brine, dried
over MgSO4 and evaporated in vacuo. The crude product was
purified by FCC petrol ether/EtOAc (7:1). A 2:9 mixture of
compounds 8b and 10b was obtained. Compound 8b: yield
11%, white solid, 1:1 E/ Z-mixture; 1H NMR (CDCl3) δ 0.88
(s, C18-Me, 3H); 1.04 (s, C19-Me, 3H); 2.03 (s, Acetyl-Me, 3H);
4.60 (m, C3RH, 1H); 5.38 (d, C6, dCH-, 1H, 3J ) 4.96 Hz);
5.75 (t, C20, dCH-, 0.5H, 3J ) 2.64 Hz); 5.77 (t, C20, dCH-,
HEK293-5R1 a n d -5R2 Assa ys.58 The 5R-reductase expres-
sion plasmids pRcCMV-I and pRcCMV-II were constructed by
insertion of the full-length human cDNA encoding the 5R-
reductase type 1 or 2, respectively. Human embryonic kidney
cells HEK293 were transfected with either pRcCMV-I or
pRcCMV-II using the lipid transfection reagent Roti-Fect
(Roth, Karlsruhe, Germany). By selection of stable transfected
cells (using G418-sulfate), clones with high 5R-reductase
activity were identified and named HEK293-5R1 and HEK293-
5R2, respectively. For the inhibition assay, 300 000 cells were
seeded in each well of a 24-well tissue culture plate and
incubated overnight in a humidified 95% O2 and 5% CO2
atmosphere at 37 °C to allow attachment of the cells. The
medium (DMEM with 10% FCS) was removed and replaced
by 0.5 mL of fresh medium containing substrate (5 nM [3H]-
androstenedione) and inhibitor. Inhibitors were dissolved in
dimethyl sulfoxide (DMSO). DMSO concentration in control
and inhibitor incubations was 1%. After incubation, the
supernatant was removed and extracted with ether. The
organic phase was evaporated and the residue dissolved in
methanol and subjected to HPLC analysis as described.36
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 equimolar 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
double-antibody testosterone [125I]RIA (ICN Eschwege; detec-
tion limits: 0.1 ng/mL under assay conditions) and are given
in ng/mL plasma.
3
0.5H, J ) 2.64 Hz); 9.86 (s, C21-CHO, 0.5H); 9.88 (s, C21-
CHO, 0.5H); IR (KBr) cm-1
νmax 2950, 1740, 1680, 1620, 1380/
60, 1260, 1140, 1040. Anal. (C23H32O3) C, H, N. Compound 10b:
1
yield 62%, white solid, mp 170-3 °C (lit. mp 166-8 °C11); H
NMR (CDCl3) δ 0.92 (s, C18-Me, 3H); 1.05 (s, C19-Me, 3H);
2.03 (s, COOCH3, 3H); 2.26 (s, C21-Me, 3H); 4.59 (m, C3RH,
1H); 5.38 (d, C6, dCH-, 1H, 3J ) 5.1 Hz); 6.69 (dd, C16, d
CH-, 1H, 3J ) 3.52 Hz, 1.76 Hz); IR (KBr) cm-1 νmax 2950,
1730, 1665, 850.
16-Hyd r oxyim in oa n d r ost-5-en -3â-ol-17-on e (1). 0.5 g
potassium was dissolved in 20 mL tert-butyl alcohol and 2 g
androstenolone (6.93 mmol) was added under a N2 atmosphere.
It was kept overnight, then diluted with water, acidified and
extracted with chloroform: purification recrystallization (EtOH:
H2O 10:1); yield 80%, white crystals, mp 247-9 °C (lit. mp
248-9 °C46); 1H NMR (DMSO-d6) δ 0.86 (s, C18-Me, 3H); 0.98
(s, C19-Me, 3H); 5.29 (m, C6, dCH-, 1H); 12.3 (s, dNOH, 1H);
IR (KBr) cm-1
NO3) C, H, N.
νmax 3400, 3200, 1745, 1690, 950. Anal. (C19H27-
16-Hyd r oxyim in oa n d r ost-5-en -3â-ol-17-on e Hyd r a zon e
(4). 0.5 g 1 (1.57 mmol) in 250 mL ethanol and 5 mL hydrazine
hydrate were refluxed for 1 h in the presence of a few drops of
glacial acetic acid: purification recrystallization (EtOH:H2O
10:1); yield 75%, white crystals, mp 279-82 °C (lit. mp 280-3
°C47); 1H NMR (DMSO-d6) δ 0.83 (s, C18-Me, 3H); 0.97 (s, C19-
Me, 3H); 5.29 (m, C6, dCH-, 1H); 7.6 (s, dNNH2, 2H); 11.19
(s, dNOH, 1H); IR (KBr) cm-1 νmax 3385, 3200, 1600, 1450,
1360, 1130, 1055, 950. Anal. (C19H29N3O2) C, H, N.
Ack n ow led gm en t. Thanks are due to Dr. J . Schro¨-
der for GC-MS experiments and Mrs. Anja Palusczak
and Martina Palzer for technical assistance in the
biological tests. We also thank Prof. Vincent Njar,
Department of Pharmacology and Experimental Thera-
peutics, University of Maryland, Baltimore, MD, for
making compound II available to us. We are grateful
to the Hermann-Schlosser-Stiftung for awarding a
scholarship to M.H. and to the Fonds der Chemischen
Industrie for financial support.
16-Hyd r oxyim in oa n d r ost-5-en -3â-ol (5). 1 g 4 (2.88
mmol), 0.8 g KOH and 40 mL ethylene glycol were refluxed
for 2 h, poured into water, and extracted with dichlo-
romethane: purification FCC (dichloromethane: ethyl acetate
1:1); yield 50%, white crystals, mp 200-2 °C (lit. mp 202-5
°C47); 1H NMR (DMSO-d6) δ 0.75 (s, C18-Me,3H); 0.96 (s, C19-
Me, 3H); 5.28 (m, dCH-, 1H); 10.19 (s, dNOH, 1H); IR (KBr)
cm-1 νmax 3375, 2930, 1660, 1450, 1070, 950. Anal. (C19H29
NO2) C, H, N.
-
Refer en ces
Biologica l Meth od s. 1. En zym e P r ep a r a tion s. The
enzymes were prepared according to described methods:
human and rat testicular P450 17,14 human placental P450
arom,54 bovine adrenal P450 scc,54 and 5R-reductase type 2.36
For the P450 TxA2 assay citrated human whole blood was
used.55
2. En zym e Assa ys. The following enzyme assays were
performed as described: rat P450 17,14 human P450 17,24 P450
arom,54 P450 TxA2,55 P450 scc,54 DU145 cells (5R-reductase
type 1),62 and 5R-reductase type 2.36
Ki and Km values were determined according to Lee and
Wilson.63 Inhibitor concentrations were between the IC40 and
IC70 values 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-
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