Design, synthesis and biological evaluation of novel 3-oxo-4-oxa-5α-androst-17β-amide derivatives as dual 5α-reductase inhibitors and androgen receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2017.05.078

Prostate cancer (PCa) is the second leading cause of death in men. Recently, some researches have showed that 5α-reductase inhibitors were beneficial in PCa treatment as well. In this study, a series of novel 3-oxo-4-oxa-5α-androst-17β-amide derivatives have been designed and synthesized in a more simple and convenient method. Most of the synthesized compounds displayed good 5α-reductase inhibitory activities and androgen receptor binding affinities. Their anti-proliferation activities in PC-3 and LNCaP cell lines were also evaluated and the results indicated that most of the synthesized compounds exhibited potent anti-proliferative activities. It is obvious that the androgen-dependent cell line LNCaP was much more sensitive than the androgen-independent cell line PC-3. Among all the synthesized compounds, 11d and 11k displayed the best inhibition activity with 4-fold more sensitive toward LNCaP than PC-3, which was consistent with their high affinities observed in AR binding assay. Molecular modeling studies suggested that 11k could bind to AR in a manner similar to the binding of dihydrotestosterone to AR. Compared to the finasteride, 11k showed a longer plasma half-life (4 h) and a better bioavailability. Overall, based on biological activities data, compound 11d and 11k can be identified as potential dual 5α-reductase inhibitors and AR antagonists which might be of therapeutic importance for prostate cancer treatment.

Full text of DOI:10.1016/j.bmcl.2017.05.078

Accepted Manuscript
Design, Synthesis and Biological Evaluation of Novel 3-oxo-4-oxa-5α-an-
drost-17β-amide Derivatives as Dual 5α-reductase Inhibitors and Androgen
Receptor Antagonists
Kejing Lao, Jie Sun, Chong Wang, Ying Wang, Qidong You, Hong Xiao, Hua
Xiang
PII:
S0960-894X(17)30576-0
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.05.078
BMCL 25023
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
11 February 2017
25 April 2017
26 May 2017
Please cite this article as: Lao, K., Sun, J., Wang, C., Wang, Y., You, Q., Xiao, H., Xiang, H., Design, Synthesis
and Biological Evaluation of Novel 3-oxo-4-oxa-5α-androst-17β-amide Derivatives as Dual 5α-reductase Inhibitors
and Androgen Receptor Antagonists, Bioorganic & Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/
10.1016/j.bmcl.2017.05.078
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Design, Synthesis and Biological Evaluation of Novel
3-oxo-4-oxa-5α-androst-17β-amide Derivatives as Dual
5α-reductase Inhibitors and Androgen Receptor Antagonists
Kejing Laoa,b, Jie Suna,b, Chong Wanga,b, Ying Wangc, Qidong Youa,b,
Hong Xiaoc, Hua Xianga,b*
aJiangsu Key Laboratory of Drug Design and Optimization, China Pharmaceutical
University, 24 Tongjiaxiang, Nanjing 210009, PR China
bDepartment of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical
University, 24 Tongjiaxiang, Nanjing 210009, PR China
cNanjing Brain Hospital Affiliated to Nanjing Medical University, 264 Guangzhou
Road, Nanjing 210029, PR China
* Corresponding author. Tel.: +86 025 83271096; Fax: +86 025 83271096(H. Xiang).
E-mail addresses: xianghua@cpu.edu.cn (H. Xiang)
Abstract
Prostate cancer (PCa) is the second leading cause of death in men. Recently, some researches
have showed that 5α-reductase inhibitors were beneficial in PCa treatment as well. In this study, a
series of novel 3-oxo-4-oxa-5α-androst-17β-amide derivatives have been designed and
synthesized in a more simple and convenient method. Most of the synthesized compounds
displayed good 5α-reductase inhibitory activities and androgen receptor binding affinities. Their
anti-proliferation activities in PC-3 and LNCaP cell lines were also evaluated and the results
indicated that most of the synthesized compounds exhibited potent anti-proliferative activities. It
is obvious that the androgen-dependent cell line LNCaP was much more sensitive than the
androgen-independent cell line PC-3. Among all the synthesized compounds, 11d and 11k
displayed the best inhibition activity with 4-fold more sensitive toward LNCaP than PC-3, which
was consistent with their high affinities observed in AR binding assay. Molecular modeling studies
suggested that 11k could bind to AR in a manner similar to the binding of dihydrotestosterone to
AR. Compared to the finasteride , 11k showed a longer plasma half-life (4 h) and a better
bioavailability. Overall, based on biological activities data, compound 11d and 11k can be
identified as potential dual 5α-reductase inhibitors and AR antagonists which might be of
therapeutic importance for prostate cancer treatment.
Key words
Prostate cancer; AR antagonists; 5α-Reductase inhibitors; Synthesis; Steroids
Prostate cancer (PCa) is the second leading cause of death in men only less than lung cancer.
It was estimated that around 220,800 cases were diagnosed in the United States in 2015 alone 1. In
2
China, that number was 60,300 and has increased rapidly over the last 10 years . Androgens,
including testosterone (T) and dihydrotestosterone (DHT), and androgen receptor (AR) signaling

are necessary for prostate development and homeostasis 3. T is synthesized in testicles and adrenal
glands, and can be further converted to more potent DHT by 5α-reductase in prostate 4. Activated
via the binding of androgens, AR are crucial in the development, maintenance, and regulation of
the male phenotype and reproductive biology. In the absence of hormone ligands, AR is located in
the cytoplasm and sequestered into heat shock protein (HSP) complexes. Upon binding of
hormones, the HSPs dissociate from the complex and the AR dimerizes and translocates to the
nucleus. AR dimers bind to AR-response elements (AREs) in the DNA, recruit coactivator
complexes that modify chromatin structure, recruit RNA polymerase II and induce transcription 5,6
.
Huggins et al. introduced androgen deprivation as a therapy for advanced and metastatic PCA in
7
1941 . Thereafter, androgen ablation therapy has been shown to produce the most beneficial
responses in multiple settings in PCA patients 8.
5α-reductases, which are NADPH-dependent enzymes, are responsible for the reduction of
4-ene-3-oxosteroids to the corresponding 5α-3-oxosteroids. The 5α-reductase family is composed
of three isozymes, with the types 1 and 2 being the most known. Type 1 5α-reductase (5αR-1) is
mainly expressed in the skin and liver at an optimal pH range of 6.0~8.5, while the type 2
5α-reductase (5αR-2) at an optimal pH 5.5, is mainly found in prostate and other genital tissues 9.
More recently, type 3 isozyme was identified in castration-resistant prostate cancer cells as well as
in other tissues such as the pancreas, brain, skin and adipose tissues 10, 11. 5α-Reductase inhibitors,
like finasteride (1) and dutasteride (2), were used in the clinic for the treatment of BPH and were
also proposed for chemoprevention and treatment of prostate cancer. It is found that progressive
castration resistant prostate cancer is characterized by increased 5αR-1 and decreased 5αR-2 levels.
Dutasteride blocks both type 1and type 2 isoenzymes and has an inhibitory effect in prostate
cancer 12. It is currently being studied as a chemopreventive agent and in combination with other
second-line androgen-reducing agents in PCA treatment 13. Unfortunately, different clinical trials
have demonstrated that these 5α-reductase inhibitors increased the risk of high-grade prostate
cancer, which prevent FDA approval of finasteride and dutasteride usage in prostate cancer
treatment 14. This warrants the investigation of other potent and specific molecules with fewerside
effects.
Azasteroids are widely used as 5α-reductase inhibitors. In recent decades, a number of
4-azasteroids have been synthesized and studied. Interestingly, 4-azasteroids have also been found
to be androgen receptor antagonists. Compound 3 (Figure 1) was reported with an IC50 of 23μM
for human androgen receptor (hAR), and values of 410nM and 15nM for type 1 and type 2
5α-reductase respectively 15. Considering the fact that -NH- and -O- are bioisosteres, we set out to
design and synthesize a series of 3-oxo-4-oxa-5α-androst-17β-amide derivatives as dual
5α-reductase and AR antagonists. The biochemical activity of the synthesized compounds was
evaluated for their 5α-reductase inhibitory activities and AR binding affinities. Their
anti-proliferative effects of were also investigated in a human prostate cancer cell line LNCaP
cells and PC-3 cells.

Figure 1. The structure of finasteride, dutasteride and lead compound
Taking commercial available compound 16-dehydropregnenolone acetate (4) as starting
material, the compounds 11a~11k have been easily prepared through 7 steps (Scheme 1). Being
treated with Raney Ni, compound 4 could be selective reduced to compound 5. After hydrolysis in
methanol containing 20% K2CO3, compound 7 was provided by Oppenauer oxidation. The
17-amide derivatives 9a~9k were obtained by combining different amide with key intermediate
compound 8, which was prepared by haloform reaction. The 17-amide derivatives 9a~9k was than
oxidized under the condition of NaIO4/KMnO4 to obtain ring-opening product 10a~10k
The synthetic method of 4-oxasteroids was first reported in 1998 16. Being cyclized by Ac2O, the
4-oxasteroid products could be synthesized by Ru/C catalyzed hydrogenation. Considering the
high expense of Ru/C, we developed a new route to obtain 4-oxasteroids. By treated with NaBH4,
the ring-opening products 10a~10k can be directly converted to 4-oxasteroid products 11a~11k.
1
The 5α-H isomers have been confirmed by H NMR spectrum. 5α-H can form the aa and ae
coupling with 6-H and shows the classical dd splitting with the chemical shift (CDCl3, 300MHz)
at 3.95~3.98. Compared with the old one, this route is not only much more economical and
convenient, but also can get 5α-H isomers simply through column chromatography.

Scheme 1. The synthesis of the compound 11a~11k
All of the eleven synthesized compounds (11a-11k) were evaluated for their 5α-reductase
inhibitory activities and the result was shown in Table 1. Generally speaking, all of the tested
compounds exhibited good inhibition towards 5α-reductase at 50μM. In general, aniline
derivatives seem to be a bit more active than the rest of the series. Among them, compound 11k
presented the most potent inhibitory activity with the IC50 of 0.27μM, better than that of positive
control finasteride. Compared with compound 11h, with an unsubstituted phenyl ring,
4-methylaniline compound 11k displayed much higher inhibition rate, which indicated that
presence of electron-donation substitution on benzene is beneficial for the activity. Moreover,
among the aliphatic amine derivatives, piperidine derivative (compound 11c) is the most potent
with the inhibition rate of 89.28%, much better than the small sterically hindered derivatives
(compounds 11a and 11b). However, the further increase of the steric hindrance of amine
derivatives, such as compounds 11d, 11e and 11f, led to the decrease of inhibitory activity. This
might suggested that cyclic substitutions might be availed to the inhibition activity, but too much
steric hindrance reduced the activity.

Table 1 The inhibition of 5α-ruductase and the affinity of AR by compound 11a~11k
5α-reductase
hAR
Compound
11a
R
I% at 50μM IC50(μM) I% at 50μM IC50(μM)
48.31
39.55
89.28
69.79
36.48
-
3.73
-
11b
-
33.73
17.35
57.06
11.78
-
11c
6.01
-
16.84
-
11d
-
-
11e
11f
33.44
-
11.30
-
11g
11h
11i
46.87
67.29
15.46
84.19
98.54
-
-
18.85
7.09
-
-
-
3.29
-
-
11j
11k
0.89
0.27
14.35
55.50
47.61
Finasteride
-
-
94.29
-
0.59
-
-
-
-
Mesterolone
18.28
The AR binding affinities of synthesized compounds were then assessed by following
fluorescence polarization procedure 17 by taking mesterolone as the positive control. As shown in
Table 1, the majority compounds displayed moderate affinity rate at the concentrations of 50μM.
Compound 11d and 11k presented the best potency toward AR, in which compound 11d exhibited
the IC50 of 16.84μM, comparable with positive control. Compared with the aliphatic amine
derivatives, compounds with aromatic amine substitutions showed better binding affinities.
In vitro anti-proliferative activities of all the synthesized compounds (11a~11k) was
evaluated against 2 human prostate cancer cell lines (LNCaP and PC-3) by taking finasteride and

flutamide as positive control. As shown in Table 2, all the synthesized compounds exhibited
potent anti-proliferative activities. It is obvious that the androgen-dependent cell line LNCaP was
much more sensitive than the androgen-independent cell line PC-3. It was found compounds 11d,
11e and 11k exhibited the most potent inhibitory activity toward LNCaP with IC50 better than the
positive control flutamide. Among them, compounds 11d and 11k presented about 4-fold more
sensitive toward LNCaP than PC-3, which was consisted with their high affinity observed in AR
binding assay,
Table 2 The anti-proliferation activities by compound 11a~11k
LNCaP
Inhibition%
PC-3
Inhibition%
Compound
IC50(μM)
IC50(μM)
(80μM)
37.32
42.72
12.35
81.79
88.94
13.80
39.71
24.19
31.41
30.03
82.31
NA
(80μM)
28.10
27.87
26.23
12.88
89.89
32.02
46.33
31.68
35.42
35.07
18.06
0.40
11a
11b
70.08
68.15
85.37
127.35
40.13
74.34
50.03
86.17
75.17
62.19
113.64
>80
11c
11d
29.78
32.41
11e
11f
11g
11h
11i
11j
11k
26.47
NA
Finasteride
Flutamide
62.98
47.55
21.24
67.34
To further rationalize the prospective activities of 3-oxo-4-oxa-5α-androst-17β-amide
A
C
B
derivatives against AR, molecular docking studies were performed using the Discovery Studio
2.5/CDOCKER protocol. The docking orientation and interactions of 11k, DHT and finasteride
within the ligand binding domain (LBD) of AR (4k7a, PDB) are shown in Figure 2. In the docking
study, steroidal-core of 11k was favorably positioned similar to DHT. The 4-O of 11k played the
role of 3-carbonyl of DHT and forms hydrogen bonds with Gln711. 11k formed a second
hydrogen bond with Met780, which was very close to the second hydrogen bond formed between
DHT and Asn705. The binding position of finasteride with AR is in an opposite direction to that of
DHT with AR. Thus, finasteride is unable to form any hydrogen bonds with AR. This Docking
simulation suggested possible basis for the observed activities.

Figure 2. Molecular docking mood of compounds DHT (A) , 11k(B)and finasteride (C) with AR.
2D diagram (D) of DHT (left) and 11k (right) showing hydrogen bond interactions with key amino
acids; Asn705, Arg752, Gln711, and Thr780.
The in vivo evaluation of compound 11k, the determination of pharmacokinetic properties
was performed in male SD rats using finasteride as reference compound. The plasma
concentration-time curves after oral administration (0.2 mg/kg) to male SD mice are shown in
Figure 3. After administration, the observed plasma concentration in mice reached peak levels 4h

post dose and was not detected 6 h after administration. The calculated pharmacokinetic
parameters based on the plasma concentration profile following oral administration are shown in
Table 3. Despite the concern that lactone might be more susceptible to hydrolysis than lactam,
compound 11k exhibited a plasma half-life of 4.74h, slightly better than finasteride. The
superiority of compound 11k observed when comparing the AUCs of the two compounds, leading
to the conclusion that the bioavailability of compound 11k is better than finasteride.
Figure 3. Concentration curve of finasteride and 11k in plasma versus time. Error bars show the
value of SD (n=6).
Table 3. Pharmacokinetic Parameters for 11 k and Finasteride in Rats after po Administrationa
T1/2β(h) b
4.74±0.83
3.91±0.55
Tmax(h) b
3.83±0.41
2.67±0.52
Cmax(ug/L) b AUC(0-tn)(ug/L*h) b
AUC(0-∞)(ug/L*h) b
54.11±9.45
11k
6.89±1.22
4.19±0.91
52.49±9.20
23.48±8.11
finasteride
23.79±8.29
a Compound 11k and finasteride was administrated at a dose of 0.2mg/kg body weight. Six intact
adult male rats were employed for each treatment group; each sample was tested for three times.
b T1/2β, terminal half-life; Tmax, time of maximal concentration; Cmax, maximal concentration; AUC,
area under the curve;
In this study, a series of novel 3-oxo-4-oxa-5α-androst-17β-amide derivatives have been
designed and synthesized in a simple and convenient route avoiding the usage of expensive
catalyst. Biological evaluations were performed on their 5α-reductase inhibitory activities and AR
binding affinities. Compared with the aliphatic amine derivatives, compounds with aromatic
amine substitutions showed higher inhibition rate to 5α-reductase as well as better binding
affinities to AR. Results of anti-proliferation effects toward LNCaP and PC-3 cell lines reveled
that most of the synthesized compounds exhibited potent anti-proliferative activities. It is obvious

that the androgen-dependent cell line LNCaP was observed to be much more sensitive than the
androgen-independent cell line PC-3. Among them, compounds 11d and 11k presented about
4-fold more sensitive toward LNCaP than PC-3, which was consistent with their high affinity
observed in AR binding assay. Molecular modelling studies suggested that 11k could binding
with AR in a similar mood with DHT. Moreover, compound 11k showed a long plasma half-life
and a high bioavailability. Overall, based on biological activities data, compound 11d and 11k can
be identified as a potential dual 5α-reductase inhibitors and AR antagonists lead molecule which
might be of therapeutic importance for prostate cancer treatment.
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