Synthesis and 5α-reductase inhibitory activity of C21 steroids having 1,4-diene or 4,6-diene 20-ones and 4-Azasteroid 20-oximes

Article abstract of DOI:10.3390/molecules17010355

The synthesis and evaluation of 5α-reductase inhibitory activity of some 4-Azasteroid-20-ones and 20-oximes and 3β-hydroxy-, 3β-Acetoxy-, or epoxy-substituted C₂₁ steroidal 20-ones and 20-oximes having double bonds in the A and/or B ring are described. Inhibitory activity of synthesized compounds was assessed using 5α-reductase enzyme and [1,2,6,7-³H]testosterone as substrate. All synthesized compounds were less active than finasteride (IC₅₀: 1.2 nM). Three 4-Azasteroid-2-oximes (compounds 4, 6 and 8) showed good inhibitory activity (IC₅₀: 26, 10 and 11 nM) and were more active than corresponding 4-Azasteroid 20-ones (compounds 3, 5 and 7). 3β-Hydroxy-, 3β-Acetoxyand 1α,2α-, 5α,6α- or 6α,7α-epoxysteroid-20-one and -20-oxime derivatives having double bonds in the A and/or B ring showed no inhibition of 5α-reductase enzyme.

Full text of DOI:10.3390/molecules17010355

Molecules 2012, 17, 355-368; doi:10.3390/molecules17010355
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and 5α-Reductase Inhibitory Activity of C21 Steroids
Having 1,4-diene or 4,6-diene 20-ones and 4-Azasteroid
20-Oximes
Sujeong Kim 1, Yong-ung Kim 2,* and Eunsook Ma 1,*
1
College of Pharmacy, Catholic University of Daegu, Hayang, 712-702, Korea
2
College of Herbal Bio-industry, Daegu Hany University, Gyeongsangbuk-do 712-715, Korea
* Authors to whom correspondence should be addressed; E-Mails: ykim@dhu.ac.kr (Y.-U.K.);
masook@cu.ac.kr (E.M.); Tel.: Tel. +82-53-819-1404 (Y.-U.K.); +82-53-850-3621 (E.M.);
Fax: Fax: +82-53-819-1272 (Y.-U.K.); +82-53-850-3602(E.M.).
Received: 1 November 2011; in revised form: 14 December 2011 / Accepted: 16 December 2011 /
Published: 30 December 2011
Abstract: The synthesis and evaluation of 5α-reductase inhibitory activity of some
4-azasteroid-20-ones and 20-oximes and 3β-hydroxy-, 3β-acetoxy-, or epoxy-substituted
C21 steroidal 20-ones and 20-oximes having double bonds in the A and/or B ring are
described. Inhibitory activity of synthesized compounds was assessed using 5α-reductase
enzyme and [1,2,6,7-3H]testosterone as substrate. All synthesized compounds were less
active than finasteride (IC50: 1.2 nM). Three 4-azasteroid-2-oximes (compounds 4, 6 and 8)
showed good inhibitory activity (IC50: 26, 10 and 11 nM) and were more active than
corresponding 4-azasteroid 20-ones (compounds 3, 5 and 7). 3β-Hydroxy-, 3β-acetoxy-
and 1α,2α-, 5α,6α- or 6α,7α-epoxysteroid-20-one and -20-oxime derivatives having double
bonds in the A and/or B ring showed no inhibition of 5α-reductase enzyme.
Keywords: 5α-reductase inhibitor; BPH; 4-azasteroid-20-oxime
1. Introduction
It has long been established that prostatic growth is stimulated by androgens and more recent
investigations suggest that this tropic support is provided by 5α-dihydrotestosterone (DHT), rather than
by the classic testicular hormone testosterone (T) [1]. Metabolically DHT is made from T by the action

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of the enzyme steroid 5α-reductase (5AR). This enzyme also catalyzes the NADPH-dependent
reduction of the ∆4 double bond of several other steroid substrates [2,3]. A deficiency of 5AR in males
results in an incomplete differentiation of external genitalia at birth [4]. On the other hand, abnormally
high 5AR activity in humans results in excessively high DHT levels in peripheral tissues, which is
implicated in the pathogenesis of prostate cancer, benign prostatic hyperplasia (BPH), acne and male
pattern baldness [5,6]. It is presently recognized that there are two genes encoding two distinct
isozymes of 5AR that are differentially expressed in human tissues and referred to as type 1 5AR
(5AR1) and type 2 5AR (5AR2) [7-9]. Although 5AR1 is predominantly expressed in the skin and
liver, 5AR2 is mainly expressed in prostate, seminal vesicles, liver and epididymis [10].
Various steroidal [10-12] and non-steroidal [13-15] inhibitors have been synthesized and tested
against 5AR. Of these, finasteride (PROSCAR®, Figure 1), a type II-selective 5α-reductase inhibitor,
was the first 5AR inhibitor approved in the USA for the treatment of BPH and prostate cancer. It has
also been demonstrated that dutasteride (Figure 1) acts as a 5AR1 and 5AR2 inhibitor [16] and
turosteride (Figure 1) is selective for the type II isoform of 5α-reductase. However, since finasteride is
slow acting and produces side effects affecting sexual function [17], this has caused us and others to
seek new classes of steroidal and non-steroidal inhibitors.
Figure 1. Representative 5α-reductase inhibitors.
The steroidal pharmacophore provides as an anchor between the key A-ring lactam amide and C-17
substitutent while the former acts as an enzyme transition state mimic of intermediate enolate, the
latter significantly enhances potency via binding a pocket largely lipophilic in nature [18]. On the other
side, the introduction of polar groups in C-17 is proved to modulate and modify the activity of these
testosterone-derived inhibitors [19]. Taking into consideration that β-substitution at C-17 could
dramatically affect the potency, a large number of modifications were carried out to find inhibitors [18].
The stereoselective synthesis [20] of mono- and dialkylcarboxamides as 17-β side chains were
reported to enhance inhibiting activity [21].
Of reported C21 steroid derivatives, some steroidal 20-hydroxyimino derivatives were reported as
dual inhibitors of CYP 17 (17α-hydroxylase/C17,20-lyase) and 5α-reductase [10,22]. Recently, 3- and
19-oximes of 16α,17α-cyclohexanoprogesterone derivatives were synthesized and evaluated to probe
the surfaces of progesterone receptor’s and two other protein ligand binding pockets neighboring to 3-
and 19-positions of steroid core [23]. Furthermore, 3-acetoxy-5α,6α-epoxyprogn-16-en-20-one, which
doesn’t have a double bond in the A or B ring [18] and 6α,7α-epoxy progesterone [11] were reported
to exhibit higher 5AR inhibitory activity than finasteride. Furthermore, steroidal epoxy compounds

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have been used as important intermediates to synthesize potent inhibitors of 5AR, since their facile
ring opening allows the introduction of various functionalities in a stereospecific manner. Also, it was
reported that the 5AR inhibitory activity was tightly dependent on the presence, position, and number
of unsaturations on the A-C rings of steroid skeleton [24].
In order to evaluate the combined effect of the hydroxyimino group, epoxy ring, and additional
double bond on the 5AR inhibitory activity of the steroidal compound, on the basis of the facts
described in these references, we report here the synthesis and 5AR inhibitory activity of some
4-azasteroids bearing a hydroxyimino group at C-20 and 3-hydroxy- or 3-acetoxy-, or epoxy-
substituted C21 steroidal 20-one and 20-oxime derivatives having double bond in the A or B ring.
2. Results and Discussion
2.1. Chemistry
The syntheses of 4-azapregnene or 4-azapregnane 20-oxime and pregnane or 5-pregnene 20-oxime
derivatives are shown in Schemes 1 and 2. Progesterone (1) was treated with sodium periodate and
potassium permanganate to form 5,20-dioxo-A-nor-3,5-secopregnane-3-oic acid (2) [25]. This
compound was treated with ammonium formate to afford 4-aza-5-pregnene-3,20-dione (3) in 45%
yield, which was converted into its 20-oxime analog 4 in 71% yield using hydroxylamine. The
20-C=O carbon peak (209.4 ppm) of compound 3 was not observed in 13C-NMR spectrum of compound 4,
but the 20-C=NOH carbon peak was identified at 158.9 ppm. In the IR spectrum of 4, the characteristic
oxime bands were observed at 3,575 cm−1 (OH), 1,668 cm−1 (C=N) and 953 cm−1 (N-O), and N-H and
3-C=O bands of A ring of 4-azasteroid skeleton were observed at 3,200 and 1,660 cm−1, respectively.
To assign the configuration of the 20-oxime, an analysis of its COSY spectrum were used. The
COSY spectrum of 20-oxime 4 did not exhibit correlation peaks of the H-16 and the proton 20-oxime
group, consequently, 20-oxime 4 was identified as the E-isomer.
The catalytic hydrogenation reaction of 3 with 10% Pd-C in ethanol afforded 4-aza-pregnane-3,20-
dione (5) in 81% yield, which was refluxed with DDQ in the presence of BSTFA to give the
1
1,2-position dehydrogenated compound, 4-aza-1-pregnene-3,20-dione (7) in 66% yield. The H-NMR
spectrum of 7 showed peaks at δ 6.80 (H-1) and 5.82 (H-2) ppm for two double bond hydrogens. Two
carbonyl carbons and double bond carbons were assigned at 209.2 (C-20), 166.5 (C-3), 150.9 (C-1)
and 123.0 (C-2) ppm in the 13C-NMR spectrum. The compounds 5 and 7 were treated with
hydroxylamine to the synthesize 20-oxime analogs 6 and 8 and the structures of 6 and 8 were
identified by same method described for compound 4.
The syntheses of 1,2-epoxy- or 6,7-epoxy-pregnadiene or pregnatriene derivatives were shown
in Scheme 2. Pregnenolone was refluxed with four equivalents of DDQ in dioxane to form
1,4,6-pregnatriene-3,20-dione (9). This trienone was treated with 30% hydrogen peroxide under basic
conditions to synthesize 1α,2α-epoxy-4,6-pregnadiene-3,20-dione (10) and the reaction of 9 and
m-chloroperoxybenzoic acid (mCPBA) at room temperature afforded 6α,7α-epoxy-1,4-pregnadiene-
3,20-dione (13), stereoselectively and regioselectively. According to the same manner reported [26],
the stereochemistry of α-epoxidation of compound 10 was confirmed by irradiation of H-1 (3.47 ppm)
which showed a NOE to C-19 methyl proton (1.19 ppm) in 1D-NOESY spectrum and α-epoxidation of

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compound 13 was also confirmed by irradiation of H-6 (3.37 ppm) which showed a NOE to C-19
methyl proton (1.21 ppm) in the 1D-NOESY spectrum.
Scheme 1. Synthesis of 4-azasteroid 20-oxime derivatives.
O
O
NaIO4, KMnO4
CH3COONH4
CH3COOH, 4 h
Reflux, 45%
Na2CO3, t-BuOH
Reflux, 3 h, 47%
O
O
O
O
N
HO
H
3
Progesterone, 1
2
NH2OH HC
Et3N, EtOH
Reflux, 12
81%
10%Pd-C, H2
EtOH, 45 C
5 h, 81%
OH
OH
OH
N
N
N
DDQ, Dioxane
BSTFA, Reflux
5 h, 66%
O
O
N
H
N
O
N
H
5
H
7
4
NH2OH HCl
Et3N, EtOH
NH2OH HCl
Et3N, EtOH
Reflux, 12 h,
78%
Reflux, 12 h,
74%
OH
OH
N
N
O
O
N
N
H
H
Scheme 2. Synthesis of epoxypregnadiene-3,20-dione-20-oximes.

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The reaction of 10 and hydroxylamine under reflux conditions for 6 h gave 20-oxime 11 and
3,20-dioxime 12. The two oxime hydrogen peaks (H-3 and H-20) of compound 12 were identified at
1
11.34 and 10.39 ppm in its H-NMR spectrum and the two carbon peaks (C-3 and C-20) were
13
identified at 147.3 and 149.5 ppm in the C-NMR spectrum. The structure of 11 was identified by
observing one carbonyl carbon peak (C-3) at 183.2 ppm in the 13C-NMR spectrum and one oxime
1
hydrogen peak at 10.28 ppm in the H-NMR spectrum. The configuration of 20-oxime 12 was
identified as E- by observing no correlation peak of the H-16 and the proton 20-oxime group in the
COSY spectrum. The 3-oxime 12 showed no correlation peak of the H-4 or H-2 and the proton
3-oxime group in the COSY spectrum. To support the configuration of the 3-oxime, it should be noted
that the suggestions about anisotropic influence of N-O bond in E- an Z-isomeric 3-steroidal oxime on
chemical shifts differences of 4-H signal were experimentally confirmed in this study [27,28]. The
chemical shift of H-4 peak (6.15 ppm) of 12 was shifted further downfield than H-4 peak (5.87 ppm)
of 11, thus confirming the E-configuration of the 3-oxime group. Compound 13 was reacted with
hydroxylamine under reflux conditions for 6 h to yield 6α,7α-epoxypregna-1,4-diene-3,20-dione-20-
oxime (14). One carbonyl carbon peak (C-3) of 14 at 185.4 ppm was observed in the corresponding
13C-NMR spectrum. The structures of compounds 15–22 are shown in Figure 2.
Figure 2. Previous synthesized compounds [29].
2.2. Testosterone 5-Reductase Assay
The series of C21 steroids 1–22 and finasteride as a reference were evaluated for their inhibitory
activity against testosterone 5-reductase prepared from rat prostates. It has been known that both the
type 1 and the type 2 isozymes are present in the ventral prostate of the rat [8]. Since the rat prostate
extracts for measuring testosterone 5-reductase inhibition were used in a neutral pH buffer, both
isozymes were assayed in this study due to differences in its biochemical properties. The inhibition
(IC50 values or % inhibition at constant concentration for the less active compounds) determined
against rat prostatic testosterone 5-reductase are summarized in Table 1.
To compare the inhibitory activity, progesterone (1) was evaluated and showed the potent inhibitory
activity with IC50 value of 6.5 nM in our assay. Compound 2 exhibited only a weak inhibitory activity
toward the enzyme, which indicated the presence of steroid ring A is important for the inhibitory
activity. The introduction of lactam ring in steroid ring A showed a broad range of inhibitory activities

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from moderate inhibition for compound 3 to potent inhibition for compounds 5 and 7. The 4-azasteroid
5, the lactam analogue of progesterone, was as active as progesterone, indicating that the replacement
of sp2 carbon atoms at C-4 and C-5 position into sp3 nitrogen and sp3 carbon atom at C-4 and C-5
position, respectively, has little effect on the activity. The presence of a double bond between C-5 and
C-6, as shown in compound 3, significantly reduced the activity, while the introduction of a double
bond between C-1 and C-2, as shown in compound 7, led to a moderate decrease in the activity. The
20-oxime analogues (compounds 4, 6 and 8) showed potent inhibition, indicating that the replacement
of oxygen atom on C-20 with an oxime functional group has a positive effect on the activity. The
20-oxime analogue of compounds 3 and 5, as exemplified by compounds 4 and 6, showed a substantial
increase in activity, while the 20-oxime analogue of compound 7, as shown in compound 8, almost did
not affect the activity. The 17-hydroxyimino analog 8 (IC50: 11 nM), which has same steroidal skeleton
as finasteride, except for the substituent at the C-17 position, showed 9.2 times less inhibitory activity
than finasteride (IC50: 1.2 nM). This result again confirms that the presence of a lipophilic group at
C-17 is very important.
1,4,6-Pregnatriene-3,20-dione (9) showed moderate inhibition, indicating that the introduction of
additional double bonds at 1,2- and 6,7-position of progesterone resulted in the diminished activity
compared to the progesterone. While it was already reported that a conjugated system of C-3, C-4, and
C-5 position showed good activity [30,31], our result showed bad activity. The epoxy compounds 10
and 13 showed no inhibitory activity, indicating that the presence of hydrophilic epoxy moiety in 1,2-
or 6,7-position results in a loss of activity.
Table 1. Screening results of testosterone 5α-reductase inhibitory activity.
Compound No.
IC50 (M)
6.5 × 10−9
>1 × 10−5
2.3 × 10−6
2.6 × 10−8
6.1 × 10−6
1.0 × 10−8
1.4 × 10−8
1.1 × 10−8
7.6 × 10−7
>1 × 10−5
Compound No.
IC50 (M)
>1 × 10−5
>1 × 10−5
>1 × 10−5
>1 × 10−5
>1 × 10−5
>1 × 10−5
1.4 × 10−8
2.1 × 10−6
1.4 × 10−7
1.2 × 10−9
1
2
3
4
5
6
7
8
9
13
15
16
17
18
19
20
21
22
10
Finasteride
The experiments were done in triplicate.
3β-Acetoxypregnenolone (15) and 20-oxime derivative (16), 3β-hydroxy-5α,6α-epoxypregnane and
20-oxime (17 and 18), 3β-hydroxy pregn-4-ene-20-oxime (19) exhibited no inhibition for 5α-reductase
enzyme. This result could indicate that the carbonyl group directly bonded at C-3 position is critical for
the inhibitory activity. Compound 20, the 20-oxime analogue of progesterone (1), was less active than
corresponding carbonyl compound. 1,4-Pregnadiene-20-oxime (22) exhibited inhibitory activity
similar to that of compound 9 and was more active than 6α,7α-epoxy-1,4-pregnadiene-20-one (13)
having an epoxy ring at the 6,7 position. 1,2- or 6,7-Epoxy rings and a double bond at C-1-C-2 in
addition to C-4 seems to be detrimental to the activity.

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3. Experimental
3.1. General Methods
All non-aqueous reactions were performed under a dry atmosphere of nitrogen. The commercial
reagents were purchased from Aldrich, Fluka, or Sigma. Solvents were purified and dried prior to use.
Melting points were measured on Thomas-Hoover melting point apparatus and not corrected. 1H-, 13C-
NMR, HSQC, HMQC and NOESY spectra were taken on a Varian 400 MHz spectrometer in CDCl3
and DMSO-d6. Chemical shifts (δ) are in parts per million (ppm) relative to tetramethylsilane, and
coupling constants (J) are in Hertz. IR spectra were determined on FT-IR JASCO 4100 spectrometer.
GC/MS spectra were obtained on a Shimadzu QP 5050 and JEOL GC Mate 2 mass spectrometers.
Analytical TLC was performed on pre-coated silica gel 60 F254 plates (Merck). Solvent systems
for TLC were ethyl acetate/n-hexane mixtures and 10% MeOH in dichloromethane. Column
chromatography was carried out on Merck silica gel 9385 (230–400 mesh) eluting with ethyl
acetate/n-hexane or methanol/dichloromethane mixtures.
3.2. Synthesis
The synthesis of steroids 2–14 (Schemes 1 and 2) was briefly described below and the preparation
of the compounds 15–22 (Figure 2) was previously reported [29].
3.2.1. 5,20-Dioxo-A-nor-3,5-secopregnane-3-oic acid (2)
To a solution of 4-pregnene-3,20-dione (1, 2 g, 6.36 mmol) in tert-butanol (20 mL), anhydrous
sodium carbonate (1.1 g, 10.13 mmol) and H2O (2 mL) were added and the reaction mixture was
refluxed. A solution of sodium periodate (11.4 g, 53.25 mmol) and potassium permanganate (140 mg,
0.89 mmol) in H2O (30 mL) was dropwise added to refluxed reaction mixture that was refluxed for an
additional 3 h. The precipitate was filtered, washed with water and the filtrate was acidified with 5M
HCl and extracted with dichloromethane. The organic phase was dried with anhydrous MgSO4, filtered,
eliminated in vacuum. The crude product was purified by silica gel column chromatography (ethyl
acetate:n-hexane = 1:3) to give 2 as a pale yellow solid. Yield: 1 g (47%), m.p.: 176–178 °C; 1H-NMR
13
(CDCl3) δ: 2.13 (3H, s, 21-CH3), 1.13 (3H, s, 19-CH3), 0.69 (3H, s, 18-CH3); C-NMR (CDCl3) δ:
214.8 (C-20), 209.5 (C-5), 179.0 (COOH), 63.6, 56.1, 50.6, 48.1, 44.2, 38.6, 38.2, 35.0, 31.7, 31.4,
29.4, 29.2, 24.6, 23.0, 21.7, 20.5, 13.6; GC-MS (EI) m/z: 334 [M]+, 316 [M-H2O]+, 262
[M-CH2CH2COO]+; IR, cm−1: 3148 (OH), 1734 (20-C=O), 1698 (3-C=O and COO).
3.2.2. 4-Aza-5-pregnene-3,20-dione (3)
To a mixture of 5,20-dioxo-A-nor-3,5-secopregnane-3-oic acid (2, 200 mg, 0.60 mmol) in acetic
acid (2 mL) was added ammonium acetate (138 mg, 1.80 mmol) and refluxed for 4 h. The reaction
mixture was evaporated to remove acetic acid and washed with water, neutralized with saturated
NaHCO3 and extracted with dichloromethane. The organic phase was dried with anhydrous MgSO4,
filtered, and eliminated under vacuum. The crude product was purified by silica gel column
chromatography (methanol-dichloromethane = 1:49) to give 3 as a pale yellow solid. Yield : 85 mg

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1
(45%), m.p.: 158–160 °C; H-NMR (CDCl3) δ: 7.34 (1H, s, NH), 4.81 (1H, t, J = 2.8 Hz, H-6), 2.55
(1H, t, J = 9.0 Hz, H-17), 2.14 (3H, s, 21-CH3), 1.10 (3H, s, 19-CH3), 0.66 (3H, s, 18-CH3); 13C-NMR
(CDCl3) δ: 209.4 (C-20), 169.3 (C-3), 140.0, 103.0, 63.5, 56.6, 47.8, 44.0, 38.5, 34.3, 31.6, 31.5(2),
29.6, 28.5, 24.4, 22.8, 21.0, 18.7, 13.3; GC-MS (EI) m/z: 315 [M]+, 300 [M-NH]+; IR, cm−1: 3198
(N-H), 1702 (20-C=O), 1658 (3-C=O).
3.2.3. 4-Aza-5-pregnene-3,20-dione-20-oxime (4)
4-Aza-5-pregnene-3,20-dione (3, 200 mg, 0.63 mmol) was dissolved in ethanol (10 mL) and
NH2OH·HCl (66 mg, 0.95 mmol), triethylamine (1 mL) were added at room temperature and the
reaction mixture was refluxed for 12 h. The reaction mixture was cooled down to room temperature,
concentrated and added with H2O and extracted with dichloromethane. The combined organic layers
were dried with anhydrous MgSO4, filtered, and concentrated to crude oily product which was purified
by column chromatography (ethyl acetate-n-hexane = 1:3) to give 4 as a white solid. Yield: 147 mg
1
(71%), m.p.: 208–210 °C; H-NMR (CDCl3) δ: 7.36 (1H, s, NH), 4.82 (1H, t, J = 3.4 Hz, H-6), 1.87
(3H, s, 21-CH3), 1.10 (3H, s, 19-CH3), 0.67 (3H, s, 18-CH3); 13C-NMR (CDCl3) δ: 169.6 (C-3), 158.9
(C=NOH), 140.2 (C-5), 103.3 (C-6), 56.9, 56.1, 48.2, 44.1, 38.6, 34.6, 32.0, 31.8, 29.8, 28.7, 24.3,
23.3, 21.1, 19.0, 15.4, 13.5; GC-MS (EI) m/z: 330 [M]+, 315 [M-NH]+; IR, cm−1: 3575 (OH), 3200
(N-H), 1668 (C=N), 1660 (3-C=O), 953 (N-O).
3.2.4. 4-Aza-pregnane-3,20-dione (5)
10% Pd-C (180 mg) was added to a solution of 4-aza-5-pregnene-3,20-dione (3, 300 mg 0.95 mmol)
in 95% ethanol (100 mL) and the mixture shaken in a Parr hydrogenation apparatus (50 psi) at 45 °C
for 5 h. The reaction mixture was filtered on a Celite pad and the filtrate was evaporated under reduced
pressure to form a white precipitate. The crude product was purified by silica gel column
chromatography (methanol-dichloromethane = 1:49) to give 5 as a white solid. Yield: 244 mg (81%),
m.p.: 275–277 °C; 1H-NMR (CDCl3) δ: 5.69 (1H, s, NH), 3.06 (1H, dd, J1 = 4.0 Hz, J2 = 12.0 Hz, H-5),
2.53 (1H, t, J = 8.8 Hz, H-17), 2.12 (3H, s, 21-CH3), 0.90 (3H, s, 19-CH3), 0.63 (3H, s, 18-CH3);
13C-NMR (CDCl3) δ: 209.4 (C-20), 172.1 (C-3), 63.6, 60.7, 56.1, 51.1, 44.3, 38.7, 35.8, 35.0, 33.4,
31.5, 29.5, 28.6, 27.4, 24.3, 22.8, 21.1, 13.5, 11.3; GC-MS (EI) m/z: 317 [M]+, 302 [M-NH]+; IR, cm−1:
3578 (OH), 3195 (N-H), 1705 (20-C=O), 1668 (3-C=O), 1675 (C=N), 954 (N-O).
3.2.5. 4-Aza-pregnane-3,20-dione-20-oxime (6)
4-Aza-pregnane-3,20-dione (5, 100 mg, 0.32 mmol) was dissolved in ethanol (10 mL) and
NH2OH·HCl (48 mg, 0.47 mmol), triethylamine (0.5 mL) were added at room temperature and the
reaction mixture was refluxed for 12 h. The reaction mixture was cooled down to room temperature,
concentrated and added with H2O and extracted with dichloromethane. The combined organic layers
were dried with anhydrous MgSO4, filtered, and concentrated to crude oily product which was purified
by column chromatography (ethyl acetate-n-hexane = 1:3) to give 6 as a white solid. Yield: 83 mg
1
(78%), m.p.: 194–196 °C; H-NMR (CDCl3) δ: 7.68 (1H, s, NH), 5.54 (1H, br s, NOH), 1.87 (3H, s,
21-CH3), 0.91 (3H, s, 19-CH3), 0.64 (3H, s, 18-CH3); 13C-NMR (CDCl3) δ: 172.1 (C-3), 158.8

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(C=NOH), 60.8, 56.7, 55.4, 51.3, 44.1, 38.7, 35.9, 35.3, 33.4, 29.5, 28.6, 27.5, 24.1, 23.0, 21.2, 15.1,
13.4, 11.4; GC-Mass (EI) m/z: 332 [M]+, 317 [M-NH]+; IR, cm−1: 3565 (OH), 3221 (N-H), 1665
(3-C=O).
3.2.6. 4-Aza-1-pregnene-3,20-dione (7)
To a solution of 4-aza-pregnane-3,20-dione (5, 100 mg, 0.31 mmol) in 1,4-dioxane (10 mL) was
added 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ, 113 mg, 0.50 mmol) and refluxed. Bis
(trimethylsilyl)trifluoroacetamide (BSTFA, 0.3 mL) was added dropwise to the refluxing reaction
mixture and reacted for an additional 5 h at same temperature. The reaction mixture was filtered,
washed with 1% Na2SO3 (aq.), 5% Na2SO3 (aq.) and H2O and extracted with dichloromethane. The
organic phase was dried with anhydrous MgSO4, filtered, and concentrated in vacuum to afford the
crude residue product which was purified by silica gel column chromatography (ethyl acetate-n-hexane
1
= 1:3) to give 7 as a white solid. Yield: 64 mg (66%), m.p.: 174–176 °C; H-NMR (CDCl3) δ: 6.80
(1H, d, J = 10.0 Hz, H-1), 5.82 (1H, dd, J1 = 2.0 Hz, J2 = 10.0 Hz, H-2), 5.48 (1H, s, NH), 3.34 (1H, dd,
J1 = 7.2 Hz, J2 = 9.2 Hz, H-5), 2.54 (1H, t, J = 9.0 Hz, H-17), 2.13 (3H, s, 21-CH3), 0.98 (3H, s,
13
19-CH3), 0.64 (3H, s, 18-CH3); C-NMR (CDCl3) δ: 209.2 (C-20), 166.5 (C-3), 150.9 (C-1), 123.0
(C-2), 63.5, 59.7, 56.1. 47.5, 44.3 39.5, 38.7, 35.3, 31.5, 29.4, 26.0, 24.3, 22.9, 21.3, 13.6, 12.0;
GC-MS (EI) m/z: 315 [M]+, 300 [M-NH]+; IR, cm−1: 3190 (N-H), 1665 (3-C=O).
3.2.7. 4-Aza-1-pregnene-3,20-dione-20-oxime (8)
4-Aza-1-pregnene-3,20-dione (7, 100 mg, 0.32 mmol) was dissolved in ethanol (10 mL) and
NH2OH·HCl (48 mg, 0.48 mmol), triethylamine (0.5 mL) were added at room temperature and the
reaction mixture was refluxed for 12 h. The reaction mixture was cooled down to room temperature,
concentrated and added with H2O and extracted with dichloromethane. The combined organic layers
were dried with anhydrous MgSO4, filtered, and concentrated to crude oily product which was purified
by column chromatography (ethyl acetate-n-hexane = 1:3) to give 8 as a white solid. Yield: 78 mg
(74%), m.p.: 235–237 °C; 1H-NMR (CDCl3) δ: 6.78 (1H, d, J = 11.0 Hz, H-1), 5.67 (1H, dd, J1 = 2.2 Hz,
J2 = 11.0 Hz, H-2), 5.48 (1H, s, NH), 3.34 (1H, dd, J1 = 7.2 Hz, J2 = 9.2 Hz, H-5), 2.54 (1H, t,
J = 9.0 Hz, H-17), 2.13 (3H, s, 21-CH3), 0.98 (3H, s, 19-CH3), 0.64 (3H, s, 18-CH3); 13C-NMR (MHz,
CDCl3) δ: 168.8 (C-3), 156.3 (C=NOH), 151.8 (C-1), 124.5 (C-2), 62.5, 59.7, 55.1. 46.5, 44.3 39.5,
36.7, 34.3, 30.2, 28.4, 24.0, 22.3, 20.9, 21.3, 18.3, 14.0; GC-MS (EI) m/z: 330 [M]+; IR, cm−1: 3576
(OH), 3123 (N-H), 1673 (C=N), 1660 (3-C=O), 954 (N-O).
3.2.8. 1,4,6-Pregnatriene-3,20-dione (9)
To a solution of 5-pregnen-3β-ol-20-one (pregnenolone, 3.0 g, 9.46 mmol) in 1,4-dioxane (40 mL),
2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ, 8.6 g, 37.90 mmol) was added and refluxed for 4 h.
Yield : 1.3 g (44%), m.p.: 144–146 °C (145–147 °C) [29].

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3.2.9. 1α,2α-Epoxypregna-4,6-diene-3,20-dione (10)
1,4,6-Pregnatriene-3,20-dione (9, 500 mg, 1.61 mmol) was dissolved in methanol (25 mL) and 30%
hydrogen peroxide (4.5 mL) and 5% NaOH-MeOH (1.2 mL) were added and stirred at room
temperature for 4 h. Yield: 360 mg (68%), m.p.: 183–185 °C (184–187 °C) [29].
3.2.10. 1α,2α-Epoxypregna-4,6-diene-3,20-dione-20-oxime (11) and 1α,2α-epoxypregna-4,6-diene-
3,20-dioxime (12)
1α,2α-Epoxypregna-4,6-diene-3,20-dione (10, 100 mg, 0.31 mmol) was dissolved in ethanol
(10 mL) and NH2OH·HCl (32.3 mg, 0.47 mmol), triethylamine (0.5 mL) were added at room
temperature and the reaction mixture was refluxed for 6 h. The reaction mixture was cooled down to
room temperature, concentrated and added with H2O and extracted with dichloromethane. The
combined organic layers were dried with anhydrous MgSO4, filtered, and concentrated to crude oily
product which was purified by column chromatography (ethyl acetate-n-hexane = 1:1) to give two
compounds 11 and 12 as a white solid.
1
Compound 11: Yield: 41 mg (39%), m.p.: 228–230 °C; H-NMR (DMSO-d6) δ: 10.28 (1H, s,
20-NOH), 6.10 (1H, d, J = 9.2 Hz, H-7), 6.01 (1H, d, J = 9.2 Hz, H-6), 5.87 (1H, s, H-4), 3.60 (1H, s,
13
H-2), 3.41 (1H, s, H-1), 1.71 (3H, s, 21-CH3), 1.05 (3H, s, 19-CH3), 0.68 (3H, s, 18-CH3); C-NMR
(CDCl3) δ: 189.4 (C-3), 156.2 (C-5), 148.5 (C-20), 134.7 (C-7), 128.1 (C-6), 117.3 (C-4), 58.0 (C-1),
55.9 (C-2), 53.1, 52.9, 45.3, 43.3, 37.5, 37.4, 36.4, 22.8, 22.0, 21.5, 18.2, 15.8, 13.6; GC-MS (EI) m/z:
341 [M]+; IR, cm−1: 3569 (OH), 1673 (C=N), 1661 (3-C=O), 950 (N-O).
1
Compound 12: Yield : 23 mg (21%), m.p.: >230 °C (dec.); H-NMR (DMSO-d6) δ : 11.34 (1H, s,
3-NOH), 10.39 (1H, s, 20-NOH), 6.15 (1H, s, H-4), 6.11 (1H, d, J = 9.6 Hz, H-6), 5.87 (1H, d,
J = 9.6 Hz, H-7), 3.63 (1H, s, H-2), 3.50 (1H, s, H-1), 1.78 (3H, s, 21-CH3), 1.01 (3H, s, 19-CH3), 0.64
(3H, s, 18-CH3), 13C-NMR (CDCl3) δ: 155.6 (C-5), 149.5 (C-20), 147.3 (C-3), 135.9 (C-7), 129.0
(C-6), 109.4 (C-4), 58.1 (C-1), 56.4 (C-2), 53.2, 52.5, 46.0, 44.3, 38.5, 38.4, 37.4, 23.9, 23.2, 21.3,
18.2, 15.8, 13.6; GC-MS (EI) m/z: 356 [M]+; IR, cm−1: 3558 (OH), 1670 (C=N), 953 (N-O).
3.2.11. 6α,7α-Epoxypregna-1,4-diene-3,20-dione (13)
To a solution of 1,4,6-pregnatriene-3,20-dione (9, 200 mg, 0.64 mmol) in chloroform (20 mL),
m-chloroperoxybenzoic acid (276.1 mg, 1.60 mmol) was added and stirred at room temperature for 24
h. Water was added to the reaction mixture which was washed with FeSO4 (aq.), H2O and extracted
with dichloromethane. The organic layer was washed with H2O and dried with anhydrous MgSO4,
filtered, concentrated to crude precipitate which was purified by column chromatography (ethyl
acetate-n-hexane = 1:3) to give 10 as a pure white solid. Yield: 98 mg (48%), m.p.: 206–208 °C; 1H-NMR
(CDCl3) δ: 7.01 (1H, d, J = 10.4 Hz, H-1), 6.49 (1H, s, H-4), 6.24 (1H, dd, J1 = 1.8 Hz, J2 = 10.2 Hz,
H-2), 3.65 (1H, d, J = 3.2 Hz, H-7), 3.37 (1H, d, J = 3.2 Hz, H-6), 2.59 (1H, t, J = 9.4 Hz, H-17), 2.14
(3H, s, 21-CH3), 1.21 (3H, s, 19-CH3), 0.73 (3H, s, 18-CH3); 13C-NMR (CDCl3) δ: 208.9 (C-20), 185.2
(C-3), 159.6 (C-5), 153.1 (C-1), 131.2 (C-2), 127.9 (C-4), 62.9 (C-7), 53.8 (C-6), 51.7, 51.3, 44.3, 41.0,

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38.2, 35.3, 31.5, 23.7, 23.0, 21.9, 20.6, 13.3; GC-MS (EI) m/z: 326 (M+), 308 (M+-H2O); IR, cm−1:
1671 (20-C=O), 1666 (3-C=O).
3.2.12. 6α,7α-Epoxypregna-1,4-diene-3,20-dione-20-oxime (14)
6α,7α-Epoxypregna-1,4-diene-3,20-dione (10, 100 mg, 0.31 mmol) was dissolved in ethanol
(10 mL) and NH2OH·HCl (32.3 mg, 0.47 mmol), and triethylamine (0.5 mL) were added at room
temperature and the reaction mixture was refluxed for 6 h. After cooling down to room temperature,
the reaction mixture was concentrated, H2O added and the mixture extracted with dichloromethane.
The combined organic layers were dried with anhydrous MgSO4, filtered, and concentrated to crude
oily product which was purified by column chromatography (ethyl acetate-n-hexane = 1:3) to give 14
1
as a white solid. Yield: 67 mg (63%), m.p.: 245–247 °C; H-NMR (DMSO-d6) δ: 10.29 (1H, s,
20-NOH), 7.15 (1H, d, J = 10 Hz, H-1), 6.23 (1H, s, H-4), 6.07 (1H, d, J = 10.0 Hz, H-2), 3.70 (1H,
J = 3.2 Hz, H-7), 3.34 (1H, J = 3.2 Hz, H-6), 1.65 (3H, s, 21-CH3), 1.29 (3H, s, 19-CH3), 0.56 (3H, s,
13
18-CH3); C-NMR (CDCl3) δ: 185.4 (C-3), 160.4 (C-5), 157.7 (C=NOH), 155.4(C-1), 130.7 (C-2),
126.3 (C-4), 72.6 (C-7), 63.3 (C-6), 56.5, 49.1, 43.4, 43.3, 41.9, 38.1, 34.3, 23.6, 23.0, 22.7, 21.9, 15.6,
13.4; GC-MS (EI) m/z: 341 [M]+; IR, cm−1: 3567 (OH), 1673 (C=N), 945 (N-O).
3.3. Testosterone 5-Reductase Assay
The enzyme suspension of testosterone 5-reductase was prepared from the homogenate of the
ventral prostate of male Sprague-Dawley rats using the methods previously reported [32]. Testosterone
5-reductase inhibition was measured according to the methods previously reported [32]. A brief
description follows: Sprague-Dawley male rats 7-8 weeks old were sacrificed by diethyl ether, and the
ventral prostates were excised and minced with a pair of scissors. The prostates were homogenized
with a glass-glass homogenizer in medium A (0.32 M sucrose, 0.1 mM dithiothreitol and 20 mM
sodium phosphate, pH 6.5) to about 20% (w/v). The homogenate was filtered over 8 layers of surgical
gauze, and centrifuged at 3,000  g for 15 min. The resulting pellets were resuspended in medium A to
about 25% (w/v) by triturating the suspension sequentially through an 18-gauge and then a 20-gauge
needle. The assay mixture containing the test sample in ethanol (10 L), reaction solution [525 L,
1 mM dithiothreitol, 40 mM sodium phosphate (pH 6.5), 50 µM NADPH and 2.2 mM [1,2,6,7-
3H]testosterone, 3.15-3.89 TBq/mmol, PerkinElmer (Waltham, MA, USA)] and the enzyme
suspension prepared above (40 L), was incubated at 37C for 1 h. After extraction with ethyl acetate
(1 mL), a sample of the ethyl acetate phase (50 L) was chromatographed on a silica plastic sheet
(Kieselgel 60 F254, Merck KGaA (Darmstadt, Germany)) using the developing solvent system ethyl
acetate-cyclohexane (1:1). Each of the testosterone and 5-dihydrotestosterone areas was cut from the
3
sheet. The H radioactivity of each fragment was counted in 5 mL of Aquasol-2 (PerkinElmer) with a
liquid scintillation counter [LS 6500, Beckman Coulter (Brea, CA, USA)]. Testosterone 5-reductase
inhibitory activity was calculated as follows:
Reaction rate (r, %) = DHT/(T + DHT)  100
Inhibiton (%) = (1 − (rsample − rblank)/(rcontrol − rblank))  100

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3
where DHT and T are the H radioactivity recovered in the areas of 5-dihydrotestosterone and
testosterone, respectively. rsample and rcontrol are reaction rates of the test sample and the enzyme control
in which reaction rates are measured in the presence and absence of test sample, respectively, and rblank
is reaction rate of the substrate blank in which reaction rate is measured in the absence of test sample
and enzyme suspension. IC50 was calculated from the inhibitory activity values at several
concentrations using linear regression analysis. Each enzyme reaction was carried out in duplicate.
4. Conclusions
In this paper we have described the synthesis of novel 20-hydroxyimino substituted C21 steroids
having additional double bonds in the A and/or B ring and 20-hydroxyimino-4-azasteroid derivatives
and their evaluation as inhibitors against 5α-reductase. All synthesized compound showed less activity
than finasteride. While the hydroxyimino group at the 20-position of a C21 steroid skeleton enhanced
the inhibitory activity in some 4-azasteroid derivatives (compounds 4, 6 and 8), the presence of an
additional double bond and an epoxy group at the 1,2-position in the molecule might significantly
reduce the inhibitory activity regardless of steroid 20-one or 20-oxime analogs.
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Sample Availability: Samples of the compounds are available from the authors.
© 2012 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).