Design and synthesis of novel steroidal imidazoles as dual inhibitors of AR/CYP17 for the treatment of prostate cancer

Article abstract of DOI:10.1016/j.steroids.2019.03.003

Both AR and CYP17 are important targets for blocking androgen signaling, and it has been accepted that multifunctional drugs have a low risk of drug resistance in the treatment of cancer. Thus, herein a series of steroidal imidazoles were designed, synthesized and evaluated as dual AR/CYP17 ligands. Several compounds displayed good biological profiles in both enzymatic and cellular assays. SAR studies showed that introducing oximino at the C-3 position of steroidal scaffold is beneficial to the enhancement of AR antagonistic activity. Among these compounds, the most potent compound 13a exhibited the best AR inhibition (IC₅₀ = 0.5 μM) that was 27-fold increase compared with the hit compound 5 as well as comparable CYP17 inhibition (IC₅₀ = 11 μM). Additionally, 13a displayed promising anti-proliferative effects on LNCap cell lines with the IC₅₀ value of 23 μM which was superior to positive control Flutamide (IC₅₀ = 28 μM). Furthermore, the docking results of 13a revealed that the oxygen atom at the position of C-3 connected to the heme of CYP17, which may be helpful for its satisfactory dual-target inhibition. In summary, this study provides an efficient strategy for multi-targeting drug discovery in the treatment of prostate cancer.

Full text of DOI:10.1016/j.steroids.2019.03.003

Accepted Manuscript
Design and synthesis of novel steroidal imidazoles as dual inhibitors of AR/
CYP17 for the treatment of prostate cancer
Qiangqiang Hou, Conghui He, Kejing Lao, Guoshun Luo, Qidong You, Hua
Xiang
PII:
S0039-128X(19)30054-6
https://doi.org/10.1016/j.steroids.2019.03.003
STE 8384
DOI:
Reference:
Steroids
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Accepted Date:
2 January 2019
26 February 2019
11 March 2019
Please cite this article as: Hou, Q., He, C., Lao, K., Luo, G., You, Q., Xiang, H., Design and synthesis of novel
steroidal imidazoles as dual inhibitors of AR/CYP17 for the treatment of prostate cancer, Steroids (2019), doi:
https://doi.org/10.1016/j.steroids.2019.03.003
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Design and synthesis of novel steroidal imidazoles as dual inhibitors of AR/CYP17
for the treatment of prostate cancer
Qiangqiang Houa, Conghui He a, Kejing Laob, Guoshun Luoa, Qidong Youa, Hua Xianga*
aJiangsu Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, 24 Tongjiaxiang,
Nanjing 210009, PR China
bShanxi Key Laboratory of Brain Disorders and Institute of Basic and Translational Medicine, Xi'an
Medical University, Xi'an, Shaanxi 710021, China
* Corresponding author. Tel.: +86 025 83271096; Fax: +86 025 83271096 (H. Xiang).
E-mail addresses: xianghua@cpu.edu.cn (H. Xiang)
Abstract
Both AR and CYP17 are important targets for blocking androgen signaling, and it has been
accepted that multifunctional drugs have a low risk of drug resistance in the treatment of cancer. Thus,
herein a series of steroidal imidazoles were designed, synthesized and evaluated as dual AR/CYP17
ligands. Several compounds displayed good biological profiles in both enzymatic and cellular assays.
SAR studies showed that introducing oximino at the C-3 position of steriodal scaffold is beneficial to
the enhancement of AR antagonistic activity. Among these compounds, the most potent compound 13a
exhibited the best AR inhibition (IC50 = 0.5 μM) that was 27-fold increase compared with the hit
compound 5 as well as comparable CYP17 inhibition (IC50 =11 μM). Additionally, 13a displayed
promising anti-proliferative effects on LNCap cell lines with the IC50 value of 23 μM which was
superior to positive control Flutamide (IC50 =28 μM). Furthermore, the docking results of 13a revealed
that the oxygen atom at the position of C-3 connected to the heme of CYP17, which may be helpful for
its satisfactory dual-target inhibition. In summary, this study provides an efficient strategy for multi-
targeting drug discovery in the treatment of prostate cancer.
Keywords: AR antagonists, CYP17 inhibitors, prostate cancer, dual target

1. Introduction
Prostate cancer (PC), one of the most common malignancies in men, is the second leading cause
of cancer-related death among males after the lung cancer in Europe and US [1]. The ligand-dependent
transcription factor androgen receptor (AR), which can be activated via binding of androgens, plays a
central role in promoting the growth and progression of PC. AR is a member of nuclear receptor
consisting of N-terminal domain (NTD), DNA binding domain (DBD), Hinge region and Ligand
binding domain (LBD) [2]. Endogenous androgen such as testosterone and dihydrotestosterone (DHT)
exhibit their biological actions by binding to the LBD of AR [3]. With no androgens binding, inactive
AR is located in cytoplasm and binds to heat shock protein 90 (HSP90). Androgens binding leads to
Hsp90 release and the following formation of homo-dimeric AR complex which subsequently
translocates to the nucleus and initiates AR-mediated responses such as the synthesis of specific
proteins like prostate-specific antigen (PSA) and triggering of cell proliferation [4]. Thus, blocking the
AR transcription or reducing androgen level could be useful therapeutic regimens in the treatment of
PC, for which androgen deprivation therapy (ADT) has been regarded as the first-line treatment.
AR antagonists are a group of ligands that can potently prevent endogenous androgens from
activating AR through binding to the same site as androgens do. A number of small molecular AR
antagonists, such as Flutamide, Bicalutamide, and Enzalutamide have been so far approved for the
treatment of PC [5-7]. Although these antiandrogens have made great achievements in the treatment of
PC, undesired resistance is still an unsolved problem which has severely limited their clinical
application [8]. Several mechanisms have been proposed for contributing to the development of
castration-resistant prostate cancer (CRPC), including AR aberrations, AR point mutations, AR gene
amplification, AR overexpression, the synthesis of intratumoral androgens and the emergence of AR
splice variants that lack of LBD [9-12]. In view of accumulating evidence showing that AR continues
to be the key molecular driver in CRPC [13], blocking androgen signaling axis remains the mainstay
treatment of CRPC. To reduce the risk of resistance and improve the therapeutic effects, combinational
drug therapy [14, 15] or development of AR-based multi-targeting antitumor agents [16, 17] offered
new opportunities.
On the other hand, CYP17 (17α-hydroxylase-17, 20-lyase) has been another promising target for
the treatment of PC [18, 19]. CYP17 is a monooxygenase and distributed in adrenal gland, tests and
prostate cancer cells [20]. The enzyme has a pivotal role for the synthesis of androgens by catalyzing
the conversion of pregnenolone and pregesterone into the corresponding androgens
dehydroepiandrosterone and androstenedione respectively [21]. Therefore, suppressing CYP17 can
attenuate the generation of androgens in PC cells, which is supposed to be an effective therapy for AR-
dependent PC. Abiraterone acetate (1) is the first approved CYP17 inhibitor of its class for the
treatment of CRPC by US Food and Drug Administration (FDA) [22]. Since then, a variety of next
generation of CYP17 inhibitors have been successively discovered, e.g. TAK-700 (2), VT-464 (3) and
so on (Fig. 1A). Most encouraging is the recent results reported in clinical trial of a multifunctional
agent TOK-001 (4), acting as both an androgen receptor antagonist and a CYP17 inhibitor, suggesting
the clinical potential of designing dual AR/CYP17 inhibitors for the treatment of CRPC [23-25].

Moreover, a single multitargeting molecule might enhance clinical benefit and result in increased
compliance, more predictable pharmacokinetic (PK) profiles and reduced risk of drug–drug
interactions compared with combined regimens [26, 27].
In our ongoing efforts to develop multitarget drug candidates in hormonal therapy area [28-31],
we reported herein the synthesis and biological evaluations of novel steroidal imidazoles as dual
AR/CYP17 inhibitors through structure modification of a previously discovered androstane-17-β-
carboxanilide-based AR-antagonist 5 (AR antagonistic activity of 17 μM) (Figure 1B). Docking
results of compound 5 within AR (PDB: 4oea) revealed no specific interactions formed by the
carboxanilide moiety, suggesting it as an amendable site for optimizing AR antagonistic activity
(Figure 1B). To achieve dual inhibition toward both targets, we therefore replaced the carboxanilide
with various imidazole-substituted alcohols based on the fact that nitrogen-containing heterocycle
moieties have been proven to be responsible for CYP17 inhibitory activity due to their ability of
coordinating with the iron at the reactive center of CYP17 [32]. Finally, we further investigated minor
varieties at C-3 position and double bond toleration in steroid ring to expand structure-activity
relationship (SAR) (Figure 1B). Preliminary biological evaluation on both CYP17 and AR proteins
identified several compounds with significantly improved AR antagonistic activity as well as excellent
CYP17 inhibition.
Figure 1. (A) Representative CYP17 inhibitors bearing nitrogen-containing heterocycles. (B) Docking results of
compound 5 within AR (PDB: 4oea) and the rational design strategy of dual AR/CYP17 inhibitors.
2. Experimental section
2.1 Chemistry
2.1.1. General.

Melting points were determined on XRC-1 a melting point apparatus and are uncorrected. IR was
recorded on Nicolet Impact-410. MS spectra were recorded on a Shimadzu GC-MS 2050 (ESI) or an
1
Agilent 1946A-MSD (ESI) Mass Spectrum. H NMR and 13C NMR spectra were recorded in CDCl3 or
DMSO with a Bruker Avance 300 MHz spectrometer at 300 K. TMS was used as an internal standard
and chemical shifts were reported in parts per million (ppm). All coupling constants (J) are in hertz
(Hz), and the signals are designated as follows: s, singlet; d, doublet; t, triplet; m, multiplet. Column
chromatography was performed with silica gel (100-200 mesh). Most chemicals and solvents were of
analytical grade and, when necessary, were purified and dried by standard methods.
2.1.2. Synthesis of intermediates 3β-hydroxy-pregnane-5-alkene-20-one-3 acetate（6a）
Compound 6b (6 g, 0.0168 mol) was dissolved in 180ml ethyl acetate and 0.6 g 10% Pd/C was
added. Then, H2 was blown in and the total reaction mixture was stirred at 30 °C for 6 h. The mixture
was filtered and the filtrate was concentrated under reduced pressure to obtain the crude product with a
yield 98%. ESI-MS m/z: 381 [M+Na]+
2.1.3. General procedure for the synthesis of intermediates 3β-hydroxy-pregnane-5-alkene-20-one
(7a) and 3β-hydroxy-pregnane-5, 16- diene-20-one (7b)
Intermediate 6a (5.92 g, 16.5 mmol) was dissolved in 118 mL methanol, and the K2CO3 (6.87 g,
49 mmol) was subsequently added. The mixture was stirred at room temperature overnight. Then, 50%
acetic acid solution was added to adjust PH 6-7. The mixed solution was concentrated under reduced
pressure to remove methanol. The mixture was filtered and washed with water to obtain the white solid
7a with a yield 93%. ESI-MS m/z: 339 [M+Na]+. Compound 7b was obtained from 6b according the
same procedure. Yield: 95%. ESI-MS m/z: 337 [M+Na]+.
2.1.4. General procedure for the synthesis of intermediates 3β-methoxymethylether-pregnane-5-
alkene-20-one (8a) and 3β- methoxymethylether-pregnane -5, 16- diene-20-one (8b)
To a solution of 7a (4.85 g, 15 mmol) in CH2Cl2 and N, N-diisopropylethylamine was added. A
solution of chloromethyl methyl ether (4.7 mL, 60 mmol) was added dropwise on an ice bath within 30
minutes under stirring. The ice bath was removed and the reaction mixture was stirred at room
temperature overnight. The mixture was poured into ice water while stirring. After about 30 min, the
mixture was shifted into separating funnel and the organic layer was separated. The aqueous layer was
extracted three times with CH2Cl2. All organic fractions were washed with saturated saline, dried over
anhydrous Na2SO4 and evaporated at reduced pressure to obtain light yellow solid 8a with a yield 98%.
ESI-MS m/z: 383 [M+Na]+. 8b was obtained from 7b in the same way. Yield: 77%. ESI-MS m/z: 359
[M+H]+.

2.1.5. General procedure for the synthesis of final compounds 3β-methoxymethylether-20-(1’-
methylimidazol-2-yl)-20-hydroxy-pregnan-5-alkene (9a) and 3β-methoxymethylether-20-(1’-
methyl imidazol-2-yl)-20-hydroxypregan-5, 16-diene (9b)
To a solution of N- methylimidazol (6.67 mmol) in dry THF was added dropwise 2.5M n-
butyllithium (3.6 mL, 8.33 mmol) at -50 °C with stirring. After 2 h, compound 8a (0.6 g, 1.67 mmol)
was added dropwise and the total reaction mixture was further stirred for 5.5 h at the same temperature.
The completion of the reaction was confirmed by TLC and saturated NH4Cl solution was added to
quench the reaction. The organic layer was separated and the aqueous layer was extracted 3 times with
ethyl acetate. All organic fractions were pooled, washed with saturated saline, dried over anhydrous
Na2SO4 and evaporated at reduced pressure to obtain compound 9a. Light yellow solid; yield: 95%.
m.p.: 223-225 ℃ ； 1H NMR(CDCl3, 300MHz), δ(ppm): 6.9(s, 1H, imidazole-4-H), 6.8(s, 1H,
imidazole-5-H), 5.4 (d, 1H, 6-H, J = 4.7 Hz), 4.7(s, 2H, 3-OCH2OCH3), 3.8(s, 3H, N-CH3), 3.7(m, 1H,
3-H), 3.4(s, 3H,3-OCH2OCH3); 13C NMR (75 MHz, CDCl3),δ(ppm): 141.1 (s), 126.4 (s), 125.57 (s),
122.7 (s), 121.27 (s), 94.6 (s), 76.91 (s), 71.6 (s), 58.3(s), 55.1 (s), 50.3 (s), 46.9 (s), 46.9 (d, J = 1.9
Hz), 39.5 (s), 37.1 (s), 36.87 (s), 34.5 (s), 34.3 (s), 31.5 (s), 30.9 (s), 30.3 (s), 29.7 (s) 28.8 (s), 28.6 (s),
20.6 (s), 19.1 (s), 16.7 (s); HRMS (ESI): m/z [M+H]+. Calcd for C27H42N2O3: 442.3195; Found:
442.3198.
9b was synthesized from 8b according to the same procedure. White solid; yield: 67%. m.p.: 205-
1
207℃； H NMR(CDCl3, 300MHz), δ(ppm): 6.9(d, 1H, imidazole-4-H, J = 1.1 Hz), 6.8(d, 1H,
imidazole-5-H, J = 1.0 Hz), 5.8(d, 1H, 16-H, J = 1.7 Hz), 5.4(d, 1H, 6-H, J = 4.4 Hz), 4.7(s, 2H, 3-
OCH2OCH3), 3.8(m, 1H, 3-H), 3.6(s, 3H, N-CH3), 3.4(s, 3H,3-OCH2OCH3); 13C NMR (75 MHz,
CDCl3),δ(ppm): 156.8 (s), 150.1 (s), 141.1 (s), 126.3 (s), 125.5 (s), 122.7 (s), 121.3 (s), 94.6 (s), 76.9
(s), 71.7 (s), 58.3 (s), 55.0 – 54.3 (m), 50.3 (s), 46.9 (s), 39.5 (s), 37.1 (s), 36.8 (s), 34.5 (s), 34.3 (s),
31.5 (s), 30.9 (s), 30.3 – 30.2 (m), 28.8 (s), 28.6 (s), 20.6 (s), 19.2 (s), 16.6 (s);HRMS (ESI): m/z
[M+H]+. Calcd for C27H40N2O3: 440.3039; Found: 440.3435.
2.1.6. General procedure for the synthesis of final compounds 20-(1’-methylimidazol-2-yl)-3β, 20-
dihydroxy-pregnan-5-alkene (10a) and 20-(1’-methylimidazol-2-yl)-3β, 20-dihydroxy-pregnan-5,
16-diene (10b)
To a solution of 9a (0.5 g, 1.13 mmol) in 10 mL methanol, 1.13 mL 6N HCl was added. The
solution was refluxed for 1 h. After cooling, the solution was poured into 100 mL ice-cold water. Then,
15% NaOH was added to adjust PH 7. The mixture was filtered and washed with water to obtain
1
compound 10a. Beige solid; yield: 69%. m.p.: 230-232℃； H NMR(DMSO-d6, 300MHz), δ(ppm):
7.6(d, 1H, imidazole-4-H, J = 1.7 Hz), 7.5(d, 1H, imidazole-5-H, J = 1.7 Hz), 6.2(s, 1H, 6-H), 5.3(s,
1H, 20-OH), 3.9(s, 3H, N-CH3); 13C NMR (75 MHz, DMSO), δ(ppm): 141.7 (s), 129.6 – 125.6 (m),
126.4 – 124.8 (m), 120.7 (s), 118.0 (s), 73.9 (s), 70.4 (s), 58.0 (s), 56.3 (s), 49.9 (s), 42.6 (s), 37.3 (s),

37.1 (s), 36.5 (s), 31.8 (s), 31.6 (s), 31.3 (s), 25.89– 25.3 (m), 23.6 (s), 20.7 (s), 19.5 (s), 13.46
(s);HRMS (ESI): m/z [M+H]+. Calcd for C25H38N2O2: 398.2933; Found: 398.2937.
10b was obtained from 9b according to the same procedure. White solid; yield 55%. m.p.: 223-
225℃； 1H NMR(DMSO-d6, 300MHz), δ(ppm): 7.7(s, 1H, imidazole-4-H), 7.6(s, 1H, imidazole-5-H),
6.0(s, 1H, 16-H), 5.3(s, 1H, 6-H), 3.8(m, 1H, 3-H), 3.6(s, 3H, N-CH3); 13C NMR (75 MHz, DMSO),
δ(ppm): 158.4 (s), 149.6 (s), 142.0 (s), 126.0 – 125.5 (m), 123.9 (s), 123.0 (s), 120.6 (s), 72.1 (s), 70.4
(s), 58.1 (s), 52.6 (s), 50.3 (s), 46.3 (s), 42.6 (s), 37.2 (s), 36.6 (s), 35.5 (s), 34.7 – 34.5 (m), 31.8 (s),
31.4 (s), 30.4 (s), 29.8 (s), 20.7 (s), 19.4 (s), 15.2 (s), 7.6 (s). HRMS (ESI): m/z [M+H]+. Calcd for
C25H36N2O2: 396.2777; Found: 396.2773.
2.1.7. General procedure for the synthesis of final compounds 20-(1’-methylimidazol-2-yl)-20-
hydroxy-pregnan-4-alkene-3-one (11a) and 20-(1’-methylimidazol-2-yl)-20-hydroxy-pregnan-4,
16-diene-3-one (11b)
To a solution of 11a (0.2 g, 0.5 mmol) in 4 mL dry toluene, cyclohexanone (2 mL) and
aluminium isopropoxide (0.3 g, 1.5 mmol) was added. The mixture was refluxed for 7 h. After cooling,
the reaction mixture was poured into 20 mL cool 15% NaOH with stirring for 20 min. The organic
layer was separated and the aqueous layer was extracted 3 times with ethyl acetate. All organic
fractions were pooled, washed with water, 10% H2SO4 and saturated saline, dried over anhydrous
Na2SO4 and evaporated at reduced pressure to obtain 11a. The crude product was purified by column
1
chromatography (CH2Cl2-CH3OH). White solid; yield: 50%. m.p.: 170-172℃； H NMR(DMSO-d6,
300MHz) δ(ppm): 6.9 (s, 1H, imidazole-4-H), 6.7(d, 1H, imidazole-5-H, J = 0.9 Hz), 5.6(s, 1H, 4-H),
5.0(s, 1H, -OH), 3.8(s, 3H, N-CH3); 13C NMR (75 MHz, DMSO), δ(ppm): 198.4 (s), 171.5 (s), 152.0
(s), 125.3 (s), 123.5 (s), 123.0 (s), 73.8 (s), 59.1 (s), 55.7 (s), 53.7 (s), 42.1 (s), 38.5 (s), 36.9 (s), 35.4
(s), 35.2 (s), 34.9 (s), 34.0 (s), 32.4 (s), 32.0 (s), 27.0 – 26.7 (m), 23.7 (s), 22.8 (s), 20.6 – 20.5 (m),
17.2 (s), 12.8 (s). HRMS (ESI): m/z [M+H]+. Calcd for C25H36N2O2: 396.2777; Found: 396.2773.
11b was obtained from 10b in the same way. White solid; yield: 80%. m.p.: 168-170℃；1H
NMR(CDCl3, 300MHz), δ(ppm): 6.9 (s 1H, imidazole-4-H), 6.8(s, 1H, imidazole-5-H), 5.8(s, 1H, 4-
H), 5.7(s, 1H, 16-H), 4.5(s, 1H, -OH), 3.6(s, 3H, N-CH3), 3.2(m, 2H, 2-H); 13C NMR (75 MHz,
DMSO),δ(ppm): 197.9 (s), 170.9 (s), 158.0 (s), 149.1 (s), 125.0 (s), 123.2 (s), 123.1 (s), 122.4 (s), 71.6
(s), 56.9 (s), 53.2 (s), 45.8 (s), 38.1 (s), 34.8 (s), 33.9 (s), 33.5 (s), 33.3 (s), 31.9 (s), 31.3 (s), 29.7 (s),
29.5 (s), 20.2 (s), 16.6 (s), 14.7 (s);HRMS (ESI): m/z [M+H]+. Calcd for C25H36N2O2: 397.2773; Found:
394.2617.
2.1.8. General procedure for the synthesis of final compounds 20-(1’-methylimidazol-2-yl)-3, 20-
dihydroxy-pregnan-4-alkene (12a) and 20-(1’-methylimidazol-2-yl)-3, 20-dihydroxy-pregnan-4,
16-diene (12b)

NaBH4 (0.0058 g, 0.75 mmol) was added to a solution of compound 17a (0.1 g, 0.25 mmol) in
methanol (4 mL). The mixture was stirred for 3 h at room temperature. 1N HCL was added to adjust
PH 7. The mixture was concentrated under reduced pressure. Ice-cool water was added with stirring for
15 min. Next, the mixture was filtered and washed with water to obtain compound 18a. White solid;
1
yield: 60%. m.p.: 160-162℃； H NMR(DMSO-d6, 300MHz) δ(ppm): 6.9 (s, 1H, imidazole-4-H),
6.7(s, 1H, imidazole-5-H), 5.2(s, 1H, 4-H), 5.0(s, 1H, 20-OH), 4.5(m, 1H, 3-H), 3.9(s, 1H, 3-OH), 3.8(s,
3H, N-CH3); 13C NMR (75 MHz, CDCl3), δ(ppm): 147.5 (s), 125.3 (s), 125.3 (s), 123.4 (s), 122.7 (s),
74.3 (s), 67.8 – 67.5 (m), 58.3 (s), 56.3 (s), 54.5 (s), 42.9 (s), 39.0 (s), 37.3 (s), 35.3 (s), 35.1 (s), 32.9
(s), 32.1 (s), 29.4 (s), 26.8 (s), 23.6 (s), 22.6 (s), 20.8 (s), 19.1 (d, J = 33.6 Hz), 13.27 (s);HRMS (ESI):
m/z [M+H]+. Calcd for C25H38N2O2: 398.2933; Found: 398.2937.
12b was obtained from 11b according to the same procedure. Beige solid; yield: 22%. m.p.: 224-
226℃； 1H NMR(DMSO-d6, 300MHz), δ(ppm): 7.0 (s, 1H, imidazole-4-H), 6.7(s, 1H, imidazole-5-H),
5.7(s, 1H, 16-H), 5.4(s, 1H, 4-H), 5.2(s, 1H, 20-OH), 4.5(m, 1H, 3-H), 3.9(s, 1H, 3-OH), 3.6(s, 3H, N-
CH3); 13C NMR (75 MHz, DMSO), δ(ppm): 158.5 (s), 149.6 (s), 144.6 (s), 125.9 (s), 125.4 (s), 123.6
(s), 122.9 (s), 72.1 (s), 66.4 (s), 57.8 (s), 54.6 (s), 46.4 (s), 37.2 (s), 35.5 (s), 34.4 (s), 34.3 (s), 32.9 (s),
32.0 (s), 30.3 (s), 29.9 (s), 29.4 (s), 20.8 (s), 18.7 (s), 15.2 (s);HRMS (ESI): m/z [M+H]+. Calcd for
C25H36N2O2: 396.2777; Found: 396.2773.
2.1.9. General procedure for the synthesis of final compounds 20-(1’-methylimidazol-2-yl)-20-
hydroxy-pregnan-4-alkene-3-oxime(13a)
To a solution of 11a (0.1 g, 0.25 mmol) in methanol (4 ml), CH3COONa (0.08 g, 1 mmol) and
hydroxylammonium chloride (0.07 g, 1 mmol) was added. The reaction mixture was refluxed with
stirring for 2 h. After cooling, the mixture was poured into ice water. The precipitate was collected by
filtration and dried to obtain compound 13a. White solid; yield: 77%. m.p.: 204-206 ℃ ； 1H
NMR(DMSO-d6, 300MHz), δ(ppm): 10.5(s, N-OH), 7.1 (s 1H, imidazole-4-H), 6.9 (s, 1H, imidazole-
5-H), 5.7 (s, 1H, 4-H), 5.3 (s, 20-OH), 3.8(s, 3H, N-CH3); 13C NMR (75 MHz, DMSO),δ(ppm): 154.0
(s), 152.5 (s), 151.3 (s), 123.2 (s), 122.4 (s), 117.8 (s), 73.4 (s), 58.2 (s), 55.4 (s), 53.2 (s), 41.9 (s), 37.3
(s), 37.0 (s), 35.3 (s), 34.6 (s), 34.2 (s), 31.7 (s), 31.6 (s), 25.8 (s), 23.4 – 23.3 (m), 22.1 (s), 20.6 (s),
18.4 (s), 17.3 – 17.2 (m), 12.6 (s);. HRMS (ESI): m/z [M+H]+. Calcd for C25H37N3O2: 411.2886; Found:
411.2886.
2.2 Biological evaluation
2.2.1. AR antagonistic activity Assay.
Hela/AR-AREs-luc cells were cultured in DMEM medium (containing 10% (v/v) FBS, (v/v) 0.2%
Hygromycin B, 0.6% (v/v) G418 sulfate, 100 U/mL Penicillin and 100 mg/mL Streptomycin) in a 5%

CO2-humidified atmosphere at 37oC. Cells were trypsinized and seeded at a density of 1 x 105/mL into
a 96-well plate (100 mL/well) and incubated at 37oC, 5% CO2 atmosphere for 12 h. Then, removing the
medium and every well was treated with 100 μL RT-1640 medium (containing 10% (v/v) FBS, 100
U/mL Penicillin and 100 mg/mL Streptomycin) containing test compounds which had been pre-
prepared to provide the concentration range of 2 x 10-5 mol/L, 1 x 10-5 mol/L, 1 x 10-6 mol/L, 1 x 10-7
mol/L and 1 x 10-8 mol/L in the presence of 0.5 nM testosterone, and they were re-incubated for a
further 36 h. Control wells were added the equivalent volume of medium containing 0.2% (v/v) DMSO.
The culture medium was removed and every well was washed twice with PBS. Then, cell lysis buffer
20 μL/well was added and oscillated for 5 minutes. Subsequently, the medium was shifted into a new
96-well plate (15 μL / well) which was non-transparent to perform fluorescence detection.
2.2.2. CYP17 preparation and assay
Tests were obtained from male rats that weighed 200-250 g (age of 8-week-old). The tests were
minced and homogenized in 0.25 M source (1:4, W/V) then centrifuged at 1000 g for 30 min at 4 oC.
The resulting supernatants were further centrifuged at 105000 g for 1 h at 4 oC. Then, the pellets were
re-suspended in 20% glycerinum – PBS buffer. The microsomal fraction was stored at -70 oC. The
microsomal protein concentrations were determined by the BCA Protein Assay Method.
The activity of CYP17 lase was determined by measuring the conversion of progesterone into
17-hydroxyl progesterone. A solution of 20 μmol / L progesterone (20 μmol / L in methanol), 80 μL
PBS and 100 μL lase (1 mg/mL) were mixed. The mixed solution was shaken for 1 min. Then, the
mixed solution, NADPH and the sample solution containing test compounds were preheated for 5 min
at 37 oC. Next, 20μL NADPH and 2 μL sample solution containing test compounds which had been
pre-prepared to provide the concentration range of 2 x 10-5 mol/L, 1 x 10-5 mol/L, 1 x 10-6 mol/L, 1 x
10-7 mol/L was added to the mixed solution. They were further incubated at 37 oC for 30min. After
centrifuging, the supernatants were detected by HPLC.
2.2.3. MTT assay for anti-proliferative activities
LNCap cells were cultured in RPMI-1640 medium (containing 10% (v/v) FBS, 100 U/mL
Penicillin and 100 mg/mL Streptomycin) at 37oC in a 5% CO2-humidified incubator. All experiments
were performed in triplicate. All cells were trypsinized and diluted to a density of 1 x 105/mL with
RPMI-1640 supplemented with 10% (v/v) FBS. The cells were seeded into 96-well plates at a volume
of 100 μL/well and incubated at 37oC, 5% CO2 atmosphere for 24 h. After this time they were treated
with 100 μL/well drug solution supplemented with serial dilutions of test compounds to provide the
concentration range of 8 x 10-5 mol/L, 4x 10-5 mol/L, 1 x 10-5 mol/L, 4x 10-6 mol/L and 1 x 10-6 mol/.
Control wells were added the equivalent volume of medium containing 1% (v/v) DMSO. Then, the

plates were re-incubated for a further 48 h. 20 μL MTT (5 mg/mL) was added to every well and cells
continued to incubate in darkness at 37oC for 4 h. Carefully removed the culture medium and 150 mL
DMSO was then added. The cells were maintained at room temperature in darkness for 20 min to
ensure thorough color diffusion before reading the absorbance. The absorbance values were read at 490
nm for determination of IC50 values.
2.2.4. Real-Time Polymerase Chain Reaction (RT-PCR).
The LNCap cells were incubated with the corresponding compounds for 24h at 37°C .RNA
samples were reverse transcribed to cDNA and the PCR reactions were performed using TaKaRa
SYBR Green Master Mix (Code. no. 638320) carried out in StepOnePlusTM Real-Time PCR instrument
(4376600, Life Technologies). The program for amplification was 1 cycle of 95°C for 2 min followed
by 40 cycles of 95°C for 10 s, 60°C for 30 s, and 95°C for 10 s. Real- time RT-PCR experiments were
performed in triplicate. The primers we used were as follows:
PSA,
forward 5′-GCAGCATTGAACCAGAGGAGTT-3′,
reverse 5′-CACGTCATTGGAAATAACATGGA-3′;
18S rRNA,
forward 5′-AGTCCCTGCCCTTTGTACACA-3′,
reverse 5′-CGATCCGAGGGCCTCACTA-3′.
2.2.5. Molecular docking
Docking studies were performed using the software Discovery Studio.3.0/CDOCK protocol
(Accelrys Software Inc.). The crystal structures of AR (PDB code: 4oea) and CYP17 (PDB ID code:
3ruk) were downloaded from Protein Data Bank. The protein and ligand were optimized and charged to
perform docking. Up to 10 conformations were retained, and binding modes presented graphically are
representative of the highest-scored conformations.
3. Results and Discussion
3.1 Chemistry.
The synthesis of 9a-13a and 9b-12b began from 6a and 6b respectively (Scheme 1). Hydrolysis
of 6a-b using potassium carbonate afforded the key intermediates 7a-b, whose hydroxy were then
protected by MOMO to yield 8a-b. According to previous report [33]， nucleophilic substitution of
8a-b with N-methylimidazole in presence of n-butyllithium afforded the final compounds 9a-b.
Literature survey revealed that nucleophilic addition resulted into the one major isomer [34, 35]. Also,
the products 9a-b are stable and can be purified by column chromatography over silica gel in good

yield. 10a-b were synthesized via deprotection of 9a-b with hydrochloric acid. Subsequently, 10a-b
underwent Oppenauer Oxidation to result in compounds 11a-b, which were then converted into 12a-b
and 13a via reduction and oximation respectively. According to previous report, the oximation
reduction offered one major isomer [36].
Scheme 1. Synthetic routes of 9a-13a and 9b-12b. Regents and conditions: (a) K2CO3, CH3OH, rt, overnight; (b)
o
MOMCl, DIEA, CH2Cl2, rt, overnight;c) n-butyllithium, N-methylimidazole, THF ,-50 C, 7.5 h; (d) 6N HCl,
CH3OH, reflux, 1 h;(e) cyclohexanone, aluminium iso-propoxide, toluene, reflux, 7 h; (f) AcONa, NH3OH.HCl,
o
CH3OH, reflux, 1h; (g) NaBH4, THF, rt, 3 h. (h) Pd/C, H2, EtOAc, 30 C, 6 h.
3.2 Biological evaluation
3.2.1. Antagonistic activity assays of AR and studies of Structure−activity relationship (SAR)
Firstly, we investigated the AR antagonistic activity of synthesized compounds by using
luciferase reporter gene assays in Hela/AR-AREs-luc cell lines [37]. Hela/AR-AREs-luc, which
contains AR gene, AREs gene and luciferase gene regulated by AREs, is a class of AR-dependent
monoclonal cell. When agonist testosterone was added to the cells, the AR was activited, triggering the
expression of luciferase gene. Addition of AR antagonists coupling with testosterone would block the
transactivation of AR, leading to a decreased luciferase activity, which is in direct proportion to the
inhibitory activity of the antiandrogens [38].
As is shown in Table 1, compounds 11a, 13a showed the best AR antagonistic activity with the
IC50 value of 3 μM and 0.5 μM, respectively. Moreover, 13a demonstrated better antagonistic effect
than positive control Fultamide. Compared with other compounds, 11a and 13a bear conjugated

structure in A ring, which may be crucial for AR inhibitory activity. Meanwhile, in contrast to 11a, 13a
contain an oximino at the position of C-3, which revealed that 3-oximino could enhance the
antagonistic efficacy. Additionally, by analyzing these data, we found that compound 10-12a exerted
antagonistic behavior stronger than 10-12b. This result confirmed that unsaturated bond at the position
of C- 16, 17 is adverse for AR inhibitory activity. Based on the result, we speculated that the
unsaturated bond might weaken the flexibility of the side chain at the position of C-17, resulting in
difficult binding of the C-20 hydroxyl with LBD of AR. In addition, from the negligible activity of
compound 9a-b，we can know that introducing long-chain alkanes at the position of C-3 is negative.
To sum up, the SAR was obtained (Table 1).
Table 1.
The AR antagonistic activity assay and SAR analysis of synthesized compounds.
AR antagonistic activity
compound
X
Double bond in ring
Inh%a at 20 μM
IC50 (μM)b
9a
-OMOM
Δ5
＜10%
NDc
10a
11a
12a
13a
9b
-OH
=O
Δ5
Δ4
50%
91%
ND
3
-OH
Δ4
68.3%
99%
ND
0.5
ND
=NOH
-OMOM
Δ4
Δ5,16
＜10%
10b
ND
ND
-OH
=O
Δ5,16
0.2%
37%
11b
Δ4,16

ND
0.7
12b
-OH
Δ4,16
20%
82%
-
-
Flutamide
aInh%=(the fluorescence value of blank control – the fluorescence value of compound)/ the fluorescence value of
blank control.
bThe IC50 value was calculated when the Inh% at 20 μM is > 90%.
cND means not determined.
3.3.2 .CYP17 inhibitory activity evaluation and cell proliferation assay
After preliminary AR antagonistic activity assay, CYP17 inhibitory activity and the potency to
inhibit the proliferation of human prostate cancer cell line LNCap were further evaluated. CYP17
inhibitory activity was determined through carrying out the CYP17 biochemical assay according to the
procedures previously reported. The CYP17 enzyme was sourced form rat tests microsomes. The
enzymatic activity of CYP17 was calculated based on the conversion of progesterone into
androstenedione. As is shown in Table 2, most compounds showed considerable CYP17 inhibitory
activity at the concentration of 20 μM compared with positive control Abiraterone. Compounds 12a,
12b, 13a manifested the best CYP17 inhibitory potency. Growth inhibitory studies showed that
compound 10a (IC50=12 μM), 11a (IC50=23 μM) and 13a (IC50=23 μM) exerted the most promising
anti-proliferative effects compared with Flutamide (IC50=28 μM) and Abiraterone (IC50=19 μM) in
the presence of 0.1 μM testosterone (T). Interestingly, compound 10a displayed surprising anti-
proliferative activity with the IC50 value of 12.01 μM though its unsatisfactory AR and CYP17
inhibition, which meant that the compound may suppress cellular proliferation via other signaling
pathways.
Table 2.
CYP17 inhibitory activity and anti-proliferative effects on LNCap cell lines
CYP17 inhibitory activitya
Cytotoxicity (IC50, μM)
LNCap c(0.1μM T)
compound
Inh% at 20 μM
IC50 (μM)
27 %
53%
52%
86%
NDb
>80
12
9a
ND
ND
3
10a
11a
12a
23
46

72%
-
11
ND
ND
ND
3
23
34
13a
9b
61%
63%
87%
100%
-
>80
>80
67
10b
11b
12b
ND
-
19
Abiraterone
Fultamide
28
aInh% means that the inhibition of each compound on the CYP17 lase.
bND means not determined.
c(0.1 μM T) means that the antiproliferative assay was performed in the presence of 0.1 μM testosterone.
To summarize, we found that compound 13a displayed the best AR antagonistic activity (IC50=0.5
μM) and excellent CYP17 inhibitory activity (IC50=11 μM), meanwhile，it also showed remarkable
growth inhibitory effects on LNCap cells (IC50=23 μM) which is better than Flutamide. Thus,
compound 13a was elected for further biological assessment.
3.3.3. Effect of 13a on the expression of PSA.
PSA gene was regulated by androgen receptor. Therefore, we adopted quantitative real-time
polymerase chain reaction (RT-PCR) to examine the ability of compound 13a to lower the expression
of PSA. The results were shown in Figure 2. In keeping with the anti-androgen activity of 13a, we can
observe the obvious decrease of PSA mRNA expression induced by 0.1μM testosterone (T), which is
superior to Fultamide under the same conditions. This result again confirmed that compound 13a was a
potent antiandrogenic agent.

8
6
4
2
0
*
**
**
***
l
T
e
a
o
r
3
d
i
t
M
1
n
μ
m
o
1
M
a
t
.
c
μ
l
0
0
u
2
F
+
T
M
μ
M
0
μ
2
1
+
.
T
0
M
μ
1
.
0
Figure 2. The increased mRNA expression of PSA induced by testosterone was reversed by 13a in LNCap cells.
The mRNA expression of PSA was examined by Real-time PCR. Values are mean ±SD (n=3). *P < 0.05, **P <
0.01, ***P < 0.001 vs. 0.1 μM T group.
3. 4. Molecular docking studies.
In order to explore the binding mode of the designed inhibitors in AR (PDB: 4oea) and CYP17
proteins (PDB: 3ruk), molecular docking studies on the compound 13a were implemented by CDOCK
in Discovery studio 3.0. As we could see from Figure 3A, the compound was perfectly located into
the ligand binding domain (LBD) of AR. Moreover, the detailed docking analysis (Figure 3B) showed
that hydroxyimino group at the position of C-3 established hydrogen bonds with Gln-711 (1.8 Å),
Met745 (2.2 Å) and Arg752 (1.7 Å) when the hydroxy group picked up hydrogen bonds with Asn705
(3.3Å) and Thr877 (2.3Å). The result just verified our initial design idea and made the potent AR
antagonistic activity rational.
A.
B.

Figure 3.(A) The binding mode of compound 13a in the cavity of AR protein.(B) The detailed docking result of
compound 13a with AR. The hydrogen bonds were indicated by red dotted lines .
The further docking study of compound 13a with CYP17 also provided evidence for its promising
CYP17 inhibitory activity. Figure 4A showed that 13a was positioned into the active site of CYP17. It
was obvious to note that the N atom of N-methylimidazole formed a hydrogen bond with Arg239
amino acid residue (1.9 Å). In addition, the O atom at the position of C-3 exactly connected to the
heme of CYP17, which was essential to inhibit the activity of CYP17. The overlap of 13a with
Abiraterone was also further performed. As depicted in Figure 4B, the binding mode of 13a was just
the opposite of Abiraterone’ and the O atom at the C-3 position of 13a was located into the same site
with N atom of Abiterone. Thus, rather than through N atom of N-methylimidazole interacting with
heme like Abiraterone, 13a coordinated with heme by the O atom at the C-3 position. Although the
unexpected binding mode ran counter to our original idea of design, the final result was not bad.
Instead, the new binding mode could indicate another way for discovering novel steroid-based dual
AR/CYP17 inhibitors.
A.
B.

Figure 4. (A) The binding mode of 13a docked into the active site of CYP17. Red imaginary line represented
hydrogen bond. (B) The overlap of 13a with Abiraterone. Yellow for 13a, pink for Abiraterone, green for heme.
4. Conclusions
In summary, a series of steroidal imidazoles targeting both AR and CYP17 were rationally
designed and synthesized as potent antiandrogenic agents that might have a low risk of drug resistance.
A series of biological evaluation in vitro and SARs were also investigated. Compound 13a which
exerted simultaneously excellent AR and CYP17 inhibitory activities was finally identified. Meanwhile,
it could also significantly reduce PSA expression induced by testosterone, which was more potent than
Flutamide. These promising pharmacological profiles of 13a indicated that it could serve as a lead
compound for further studies. In addition, the further docking study of compound 13a with CYP17
revealed a new binding mode, which might open a new avenue for discovering novel steroid-based
multifunctional AR and CYP17 inhibitors.
Supplementary data
Supplementary data (the 1H NMR, 13C NMR and HRMS spectra of target compounds associated with
this article can be found, in the online version, at.
Acknowledgements
The authors are grateful to China Pharmaceutical University. This work was supported by “Double-
First-Class” University Project CPU2018GY07.
Abbreviations
AR, androgen receptor; PSA, prostate specific antigen; CYP17, 17α-hydroxylase-17, 20-lyase; RT-
PCR, real-time polymerase chain reaction; T, testosterone; g, gram.
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



Simultaneously inhibiting AR and CYP17 benefits to the treatment of prostate cancer
A series of new steroidal imidazoles were optimized from hit AR antagonist.
13a exhibited growth inhibitory effects on prostate cancer cells by inhibiting AR and CYP17
13a could dramatically reduce PSA expression induced by testosterone, which was
comparable with Abiraterone

Docking result of 13a with CYP17 revealed a distinct binding mode compared with
Abiraterone, which might open a new avenue for discovery of novel dual AR and CYP17
inhibitors