Synthesis and biological evaluation of new steroidal pyridines as potential anti-prostate cancer agents

Article abstract of DOI:10.1016/j.ejmech.2017.12.094

A series of new steroidal pyridines have been synthesized through the based-promoted three-component reaction and preliminarily evaluated for their antiproliferative activity against different types of cancer cell lines. SARs studies showed that the heterocyclic rings attached to the 4-position of the pyridine ring were preferred over the phenyl rings for the activity. Among these compounds, the most potent compound exhibited good growth inhibition against all the tested cancer cells, especially for PC-3 cells with an IC₅₀ value of 1.55 μM. Further mechanistic studies revealed that the most potent compound inhibited colony formation, migration and evasion of PC-3 cells in a concentration-dependent manner as well as induced apoptosis of PC-3 cells possibly through the mitochondria-related apoptotic pathways. Caspase-3/-9 and PARP were activated, finally leading to the apoptosis of PC-3 cells. For the androgen-sensitive (AR⁺) prostate cancer cell line LNCaP, the most potent compound was less potent than abiraterone with the IC₅₀ value of 8.48 and 3.29 μM, respectively. The most potent compound could be used as a starting point for the development of new steroidal heterocycles with improved anticancer potency and selectivity. The synthesized steroidal pyridines contain the functional -OEt and CN groups, which could be used for further modifications for the construction of the steroid library.

Full text of DOI:10.1016/j.ejmech.2017.12.094

Accepted Manuscript
Synthesis and biological evaluation of new steroidal pyridines as potential anti-
prostate cancer agents
Yun-Kai Shi, Bo Wang, Xiao-Li Shi, Yuan-Di Zhao, Bin Yu, Hong-Min Liu
PII:
S0223-5234(17)31125-X
10.1016/j.ejmech.2017.12.094
EJMECH 10072
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 23 October 2017
Revised Date: 29 December 2017
Accepted Date: 30 December 2017
Please cite this article as: Y.-K. Shi, B. Wang, X.-L. Shi, Y.-D. Zhao, B. Yu, H.-M. Liu, Synthesis and
biological evaluation of new steroidal pyridines as potential anti-prostate cancer agents, European
Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2017.12.094.
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ACCEPTED MANUSCRIPT

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Synthesis and biological evaluation of new steroidal pyridines as potential
anti-prostate cancer agents
Yun-Kai Shi#, Bo Wang#, Xiao-Li Shi, Yuan-Di Zhao, Bin Yu*, Hong-Min Liu*
Key Laboratory of Advanced Drug Preparation Technologies (Zhengzhou University), Ministry of
Education; Collaborative Innovation Center of New Drug Research and Safety Evaluation, Henan
Province; School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450001, PR
China; Key Laboratory of Henan Province for Drug Quality and Evaluation
Corresponding Authors: Dr. Bin Yu & Prof. Hong-Min Liu
E-mail: zzuyubin@hotmail.com (Bin Yu) & liuhm@zzu.edu.cn (Hong-Min Liu)
# These authors contributed equally to this work.
Abstract: A series of new steroidal pyridines have been synthesized through the based-promoted
three-component reaction and preliminarily evaluated for their antiproliferative activity against
different types of cancer cell lines. SARs studies showed that the heterocyclic rings attached to the
4-position of the pyridine ring were preferred over the phenyl rings for the activity. Among these
compounds, the most potent compound exhibited good growth inhibition against all the tested
cancer cells, especially for PC-3 cells with an IC50 value of 1.55 µM. Further mechanistic studies
revealed that the most potent compound inhibited colony formation, migration and evasion of PC-3
cells in a concentration-dependent manner as well as induced apoptosis of PC-3 cells possibly
through the mitochondria-related apoptotic pathways. Caspase-3/-9 and PARP were activated,
finally leading to the apoptosis of PC-3 cells. For the AR+-sensitive prostate cancer cell line LNCaP,
the most potent compound was less potent than abiraterone with the IC50 value of 8.48 and 3.29 µM,
respectively. The most potent compound could be used as a starting point for the development of
new steroidal heterocycles with improved anticancer potency and selectivity. The synthesized
steroidal pyridines contain the functional -OEt and CN groups, which could be used for further
modifications for the construction of the steroid library.
Keywords: Steroids; Pyridines; Biaryl compounds; Antiproliferative activity; Prostate cancer
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1. Introduction
Steroids, an important class of polycyclic molecules, have played key roles in regulating normal
physiological processes and targeting disease-related biological sites [1, 2]. Of particular interest are
steroidal heterocycles, which have drawn wide attention due to their interesting biological profiles
and structural features [3-6]. Some steroidal derivatives are currently used in clinic or have
advanced into clinical trials for the treatment of diseases, and two representative examples are
abiraterone [7, 8] and galeterone [9, 10]. Abiraterone is presently used in clinic for the treatment of
advanced prostate cancers, galeterone has advanced into phase 2 trial for the treatment of castration
resistant prostate cancer (ClinicalTrials.gov Identifier: NCT01709734). Abiraterone is an androgen
synthesis inhibitor by blocking the action of CYP17 enzyme, which is involved in the androgen
biosynthesis [11]. Galeterone possesses an unique dual mechanism of action by selectively
inhibiting the C17, 20-lyase activity of CYP17 and androgen receptor, which demonstrates good
tolerance and clinically meaningful reductions in levels of prostate specific antigen (PSA).
Our group have long been devoted to the synthesis of novel steroidal derivatives and their
antiproliferative evaluation [12-21], aiming to identify new steroid-based antitumor agents. Some of
these compounds were found to be able to inhibit growth of human cancer cells potently and induce
cell cycle arrest and apoptosis in a time-/concentration-dependent manner. One representative
example is the steroidal compound by241 bearing the spirooxindole moiety [22, 23], which exerts
good in vitro and in vivo anticancer efficacy through the ROS-mediated mechanisms [24]. These
findings, coupled with the anticancer profiles of abiraterone and galeterone promoted us to further
explore the antitumor potential of steroidal pyridines, which were efficiently accessed from
pregnenolone (PREG), aldehyde, malononitrile and sodium ethoxide (Fig. 1).
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Fig. 1. Abiraterone, galeterone and the target molecules synthesized in this work.
2. Results and discussion
2.1 Chemistry
The intermediates 2a-p were prepared in high yields via the Al2O3/KF-catalyzed aldol condensation
of pregnenolone (PREG) with aromatic aldehydes in ethanol following our previously reported
procedures [20, 25]. Steroidal pyridines 3a–n and 4a-l were synthesized from steroidal α, β
-unsaturated ketones 2a-p, malononitrile and sodium ethoxide in a single operation as shown in
Scheme 1. Interestingly, sodium ethoxide acted as a bifunctional species, namely the ethoxy source
and the promoter of reactions between α, β-unsaturated ketones and malononitrile. The compound
5j was also synthesized from compound 3j through acetylation reaction, and the route is shown in
Scheme 1 (See Tables 1-2 for R2 substituents in compounds 3a-n and 4a-l).
Scheme 1. Synthesis of steroidal pyridines. Reagents and conditions:(a) Al2O3/KF, EtOH, reflux; (b)
Malononitrile, NaOEt, EtOH, reflux；(c) Acetyl chloride, Et3N, DMAP, DCM, r.t.
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1
All the new compounds were characterized by H, 13C NMR and high-resolution mass spectra
1
(HRMS) as described for compound 3a (Fig. 2). In the H NMR spectrum of compound 3a, the
signals of protons attached to C-18’ and C-19’ appeared at 0.49 and 0.93 ppm, respectively as sharp
singlets. The signal of H-6’ appeared at 5.29 ppm as a doublet (J = 4.5 Hz). Two doublets at 7.77
and 7.54 ppm, respectively had the same coupling constant (J = 8.6 Hz), indicating that these four
protons existed in the 4-chloro phenyl group. The singlet at 4.60 ppm was assigned to the 3’α-H of
steroid nucleus. The triplet and the quartet at 1.33 and 4.34 ppm, respectively were assigned to the
protons attached to the ethoxy group. The proton signals attached to C-5/C-3 appeared at 7.02 and
6.86 ppm. Please refer to the 2D NMR spectra of compound 3a included in the Supporting
Information for details. The presence of a molecular ion peak at m/z = 506.2817 (M+H)+ in the
mass spectrum (calcd. 506.2826) further confirmed the structure of 3a. It should be noted that most
of the synthesized compounds were obtained as mixture of rotamers because of the twisting of
biaryl compounds, which was previously observed by us [26] and other groups [27-29].
Fig. 2. Characteristic chemical shifts of compound 3a.
Based on the interesting transformations, we also tentatively proposed the possible reaction
mechanism as shown in Scheme 2 [30, 31]. In the presence of NaOEt, the 1,4-Michael addition of
malononitrile to compound 2 produced intermediate A, in which the CN group was attacked by
NaOEt, forming the unstable intermediate B. Isomerization of intermediate B in the presence of
NaOEt afforded enamine C, which was then subjected to the intramolecular dehydration, generating
intermediate D. Intermediate D can also be oxidized under air atmosphere, forming compound 4.
Compound 3 was also generated in the reaction via the competitive decyanation reaction. The
multisubstituted pyridine ring was formed in the reaction (The newly formed bonds were
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highlighted in bold), and the –OEt and -CN group in compound 3 or 4 could be allowed for further
derivatizations for the construction of the steroidal pyridine library. It was anticipated that
application of different alkoxy species would provide access to new steroidal pyridines bearing
various alkoxy groups. Malononitrile may also be replaced with other α-substituted acetonitriles in
the reaction for the synthesis of analogs of compound 4.
Scheme 2. Possible reaction mechanisms for the formation of steroidal pyridines.
2.2. Biological evaluation
2.2.1. Antiproliferative activity
The IC50 values for the synthesized compounds against three human cancer cell lines including
human gastric cancer cell line (MGC-803), human prostate cancer cell line (PC-3) and human
esophageal cancer cell line (EC-109) and a normal human gastric epithelial cell (GES-1) were
determined using the MTT assay. The results were listed in Table 1 and the well-known anticancer
drug abiraterone was used as a positive control.
As shown in Table 1, compounds 3a-i possessing different substituted phenyl ring showed weak to
moderate antiproliferative activity against the tested cancer cells. Among these compounds,
compound 3g with the 3,5-difluro group inhibited growth of PC-3 cells with an IC50 value of 16.50
µM, around 2.8-fold less potent than abiraterone (IC50 = 5.94 µM). Interestingly, replacement of the
phenyl ring with heterocyclic rings (compounds 3j-n) significantly enhanced the antiproliferative
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activity against the tested cancer cells, highlighting the importance of the heterocyclic ring for the
activity. Compound 3j bearing the pyridine ring (IC50 = 1.55 µM) was about 20-fold more potent
than compound 3b against PC-3 cells and also showed acceptable inhibition toward EC-109,
MGC-803 cells. It is worth noting that for all the tested cancer cells, compound 3j (also named as
SYK-001) was even much more potent than abiraterone, a CYP17A1 inhibitor used in clinic for the
treatment of advanced prostate cancers [11]. The preliminary data may warrant further development
of SYK-001 as a potential anticancer lead. In contrast, compounds 3m and 3o had decreased
activity toward the tested cancer cells, underscoring the importance of the position of the nitrogen
atom for the activity. Compounds 3k and 3m with the thiophenyl and furanyl group, respectively
were found to be less potent than compounds 3j, 3l and 3n. Considering that abiraterone acetate is
used in clinic as the prodrug of abiraterone, herein we also evaluated the antiproliferative activity of
the acetate of compound 3j (compound 5j) toward the above mentioned cell lines. Compound 5j
exhibited slightly decreased antiproliferative activity against PC-3 cells compared to 3j, but showed
good selectivity to PC-3 cells over EC-109, MGC-803 cell lines and normal cell line GES-1 (IC50 >
32 µM).
Table 1. Preliminary in vitro antiproliferative activities of the steroidal pyridines 3a-n and 5j
IC50(µM)
Compound
R2
PC-3
EC-109
MGC-803
GES-1
19.21±1.28
＞32
3a
3b
3c
3d
3e
＞32
＞32
31.14±1.49
＞32
＞32
＞32
＞32
＞32
＞32
＞32
＞32
＞32
＞32
＞32
＞32
＞32
＞32
21.73±1.33
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24.59±1.39
16.50±1.21
＞32
＞32
23.66±1.37
＞32
＞32
3f
3g
＞32
＞32
31.29±1.49
＞32
＞32
＞32
＞32
3h
3i
＞32
＞32
1.55±0.19
5.32±0.72
2.75±0.44
＞32
7.76±0.89
＞32
3j
3k
3l
15.26±1.18 17.55±1.24
13.41±1.12
＞32
25.76±1.41
＞32
10.78±1.03 26.24±1.41 26.08±1.41
＞32
3m
3n
6.37±0.80 16.61±1.22
4.48±0.65
15.74±1.19
6.97±0.84
5.94±0.77
>32
>32
>32
>32
5j
Abiraterone
7.72±0.88
13.12±1.11
Next, we also examined the effect of compounds 4a-l on the antiproliferative activity against the
tested cancer cell lines. As shown in Table 2, for PC-3 cells, compounds 4a-c and 4e-g exhibited
acceptable antiproliferative activity with the IC50 values less than 14 µM and were much more
potent than the corresponding analogs 3a-c and 3e, 3g. Similarly, compounds 4j-l bearing the
heterocyclic motifs also displayed moderate antiproliferative activity. Of these compounds,
compound 4b had slightly enhanced antiproliferative activity against PC-3 cells compared to
abiraterone (IC50 = 4.39 µM vs. 5.94 µM). Compared to compound 3j, compound 4j possessing an
additional -CN group attached to the 3-position of the pyridine ring was found to have decreased
antiproliferative activity against the tested cancer cells.
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Table 2. Preliminary in vitro antiproliferative activities of steroidal pyridines 4a-l
IC50 (µM)
Compound
R2
PC-3
EC-109
MGC-803
GES-1
10.43±1.02
＞32
＞32
＞32
4a
4.39±0.64
13.31±1.12
15.76+1.19
7.80±0.89
4b
4c
13.66±1.13
＞32
＞32
＞32
4d
4e
4f
＞32
＞32
＞32
＞32
＞32
21.94±1.34
＞32
9.42±0.97
7.77±0.89
28.47±1.45
13.05±1.11
27.56±1.44
NO2
6.99±0.85
＞32
19.96±1.30
＞32
12.35±1.09
＞32
＞32
＞32
＞32
4g
4h
4i
＞32
＞32
＞32
5.84±0.76
6.98±0.84
2.84±0.45
9.84±0.99
14.70±1.16
23.61±1.37
>32
4j
4k
＞32
12.19±1.08
5.94±0.77
10.57±1.02
>32
22.30±1.34
7.72±0.88
30.09±1.47
13.12±1.11
4l
Abiraterone
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Abiraterone acts as a partial antagonist of the androgen receptor (AR) [11], while the title
compounds also possess similar structural features. Therefore, we chose prostate cancer cell line
LNCaP to further evaluate the anticancer potential of compounds 3j, 4j, and 5j using abiraterone as
the control drug. LNCaP cells are androgen-sensitive (AR+) human prostate adenocarcinoma cells
and have always been used as the most clinically relevant prostate cancer models that more closely
mimic clinical disease [32]. As depicted in Fig. 3, compounds 3j, 4j and 5j inhibited growth of
LNCaP cells with the IC50 value of 8.48, 5.18, 27.88 µM, respectively, but were less potent than
abiraterone, which suppressed growth of LNCaP cells with an IC50 value of 3.29 µM.
3j
4j
120
5j
100
Abiraterone
80
60
40
20
0
0 0.1250.25 0.5
1
2
4
8
16 32
Concentration of Theatment (µM)
Fig. 3. Cell viability of compounds 3j, 4j, 5j and abiraterone against LNCaP cells. Cells were treated with the
compounds at the indicated concentrations for 72h.
2.2.2. Cell viability test and cell cycle analysis
Inspired by the favorable potency of compound 3j toward PC-3 cells, 3j was prioritized to perform
further experiment for evaluating its antitumor potential in PC-3 cells (Fig. 4). As shown in Fig. 4A,
after the treatment with 3j, the proliferation of the PC-3 cells was significantly inhibited in a
concentration and time-dependent manner. Fig. 4B showed that compound 3j obviously inhibited
colony formation of PC-3 cells. To examine the effect of different concentrations of compound 3j
on cell cycle progression against PC-3. A flow cytometric of cell-cycle assay was performed by
treating PC-3 cells with various concentrations of compound 3j (0, 0.5, 1.0, 2 µM) for 48 h. As in
shown in Fig. 4C, compound 3j had little effect on the cell cycle arrest, indicating that the
antiproliferative activity of compound 3j was not mainly achieved by the cell cycle arrest.
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Fig. 4. Effect of compound 3j on the proliferation of PC-3. (A) The effect of 3j in inhibiting cell survival of PC-3
cells measured by the MTT assay. (B) The colony formation of PC-3 cells after treatment of compound 3k. (C)
Effect of compound 3j on the cell cycle distribution of PC-3 cells. The experiments were performed three times,
and a representative result is shown above.
2.2.3. Compound 3j inhibits migration and invasion of PC-3 cells
We next investigated whether compound 3j could inhibit the migration and invasion against PC-3
cells. Firstly, we performed the wound healing assay. As shown in Fig. 5A, after treatment of PC-3
cells with compound 3j at different concentration (0, 0.5, 1.0, 2.0 µM) for 48 hours, the wound
healings were markedly suppressed in a concentration-dependent manner. Additionally, the further
transwell assay and matrigel-coated transwell assay (Fig. 5B & 5C) showed compound 3j
significantly inhibited the ability of migration and invasion of PC-3 cells concentration-dependently.
Western blotting analysis (Fig. 5D) showed that compound 3j up-regulated the expression of
epithelial cells’ biomarkers, E-Cadherin and Occludin, while the mesenchymal cells’ biomarkers,
N-Cadherin and Vimentin, were down-regulated. These results indicated that compound 3k may
block migration and invasion of PC-3 cells through inhibiting EMT process.
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Fig. 5. Effect of compound 3j on migration and invasion of PC-3 cells. (A) The Wound healing assay. (B and C)
Migration and invasion inhibition induced by compound 3j were reflected by the transwell and matrigel-coated
transwell assays. (D) Expression of N-Cadherin, Vimentin, E-Cadherin and Occludin after treatment of compound
3j and GAPDH was used as control. *P < 0.05 and **P < 0.01 were considered statistically significant compared
with the control. Dates are mean ± SD. All experiments were carried out at least three times.
2.2.4. Effect of compound 3j on apoptosis and related mechanisms
Apoptosis is known as programmed cell death and typically characterized by distinctive cell
morphological changes. Compound 3j was chosen for evaluating its ability of inducing apoptosis.
The Hochest 33258 staining was performed to investigate morphological changes of PC-3 cells.
After 48h incubation with compound 3j at indicated concentrations, characteristic apoptotic
morphological changes were observed, including cell rounding, chromatin shrinkage and formation
of apoptotic bodies (Fig. 6A), particularly at high concentrations. To further explore the effect of
compound 3j on cell apoptosis, we performed the flow cytometry analysis by AnnexinV-FITC/PI
double staining. As shown in Fig. 6B, after treatment with compound 3j for 48h at different
concentrations (0, 0.5, 1.0, 2.0 µM), the percentage of apoptotic PC-3 cells (including early phase
and late phase apoptosis) were increased up to 13.3%, 20.8%, and 22.2%, respectively. The results
showed that compound 3j could increase the cellular apoptosis in a concentration-independent
manner. Next, the western blotting analysis was performed to examine the expression of apoptosis
related proteins. As shown in Fig. 6C, treatment of PC-3 cells with compound 3j activated caspase 9
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and caspase 3 in a concentration-dependent manner, which then finally activate PARP. The data
suggest that the apoptosis caused by compound 3j may be involved in mitochondria-related
apoptosis pathway. While the expression of p53 was almost unchanged.
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lower right quadrants are for early/priAmaCryCaEpoPpTtoEticDceMllsA, uNppUerSrCighRtIqPuTadrants are for late/secondary apoptotic
cells, while the upper left quadrants represent cells damaged during the procedure. (C) Expression of apoptosis
involved protein, caspase 9, cleaved-caspase 9, PARP, cleaved-PARP, caspase 3, cleaved-caspase 3 and p53 were
determined after treatment with compound 3j. The GAPDH was used as control. * P < 0.05 and **P < 0.01 were
considered statistically significant compared with the control. Dates are mean ± SD. All experiments were carried
out at least three times.
3. Conclusions
Following our previous work in the identification of new steroidal heterocycles for cancer therapy
and inspired by the anticancer efficacy of abiraterone and galeterone, we herein reported the
synthesis of new steroidal pyridines through the based-promoted three-component one-pot reaction,
in which a new multisubstituted pyridine ring was formed. Biological evaluation indicated that the
synthesized steroidal pyridines showed varied antiproliferative activity against the tested cancer
cells dependent on the substituents attached. SARs studies showed that the heterocyclic rings
attached to the 4-position of the pyridine ring were preferred over the phenyl rings for the activity.
Among these compounds, compound 3j possessing an additional pyridine ring at the p-position
exhibited good growth inhibition against all the tested cancer cells, especially for PC-3 cells with an
IC50 value of 1.55 µM. Further mechanistic studies revealed that compound 3j inhibited colony
formation, migration and evasion of PC-3 cells in a concentration-dependent manner. E-Cadherin
and Occludin were found to be up-regulated in PC-3 cells when treated with 3j, while the
expression of N-Cadherin and Vimentin was down-regulated accordingly. Compound 3j induced
apoptosis of PC-3 cells possibly through the mitochondria-related apoptotic pathways. Caspase-3/-9
and PARP were activated, which then finally led to apoptosis of PC-3 cells, while the expression of
p53 was almost unchanged. However, compound 3j had little effect on the cell cycle arrest of PC-3
cells. For the AR+-sensitive prostate cancer cell line LNCaP, compound 3j inhibited growth with an
IC50 value of 8.48 µM, slightly less potent than the anti-prostate cancer drug abiraterone (IC50 =
3.29 µM). The synthesized steroidal pyridines contain the –OEt and CN groups, which could be
allowed for further modifications for the construction of the steroid library. Compound 3j could be
used as a starting point for the development of new steroidal heterocycles with improved anticancer
potency and selectivity.
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4. Experimental section
4.1. General
Reagents and solvents were purchased from commercial sources and were used without further
purification. Thin-layer chromatography (TLC) was carried out on glass plates coated with silica gel
and visualized by UV light (254 nm). The products were purified by column chromatography over
silica gel. Melting points were determined on a Beijing Keyi XT4A apparatus and are uncorrected.
All the NMR spectra were recorded with a Bruker DPX 400 MHz spectrometer with TMS as
internal standard in CDCl3 or DMSO-d6. Chemical shifts are given as δ ppm values relative to TMS
(Most of the peaks due to the complexity of the steroidal skeleton are merged and therefore could
not be differentiated. Thus δ values of only those peaks that could distinguish the product are
1
reported). The H-NMR data of the major rotamer were given. High-resolution mass spectra
(HRMS) were recorded on a Waters Micromass Q-T of Micromass spectrometer by electrospray
ionization (ESI).
4.2. General procedure for the synthesis of compounds 3a-n and 4a-l
The mixture of malononitrile (1.1 mmol, 1.1 eq), appropriate steroidal α,β-unsaturated ketone
(1.0mmol, 1.0 eq) and sodium ethoxide (4.0 mmol, 4.0 eq) was refluxed for 8 h in ethanol and
monitored by TLC. After the completion of the reaction, the reaction mixture was cooled to room
temperature and filtrated with ethanol, then washed with ethanol. The filtrate was concentrated
under reduced vacuum. The residue was purified by column chromatography (PE: Acetone = 4:1) to
give the target products.
4.2.1. 2-Ethoxyl-4-(4-chlorophenyl)-6-[(3'β, 17'β)-3'-(hydroxyl) androst-5'-en-17'-yl] pyridine (3a)
1
White solid, yield 17%, m.p.89.1-90.8 . H NMR (400 MHz, DMSO-d6) δ 7.77 (d, J = 8.6 Hz,
2H), 7.54 (d, J = 8.6 Hz, 2H), 7.02 (s, 1H), 6.86 (s, 1H), 5.31 (d, J =4.6 Hz, 1H), 4.60 (s, 1H), 4.34
13
(q, J = 7.0 Hz, 2H), 1.33 (t, J = 7.0 Hz, 3H), 0.93 (s, 3H), 0.49 (s, 3H). C NMR (100 MHz,
DMSO-d6) δ 162.92, 159.55, 148.65, 141.32, 136.54, 133.81, 128.98, 128.66, 120.32, 113.77,
104.77, 69.97, 60.91, 57.15, 56.09, 49.88, 44.26, 42.21, 37.62, 36.94, 36.15, 31.72, 31.40, 29.01,
24.50, 24.29, 20.38, 19.16, 14.57, 12.86. HRMS (ESI):m/z calcd for C32H41ClNO2 (M+H)+,
506.2826 ; found, .506.2817.
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4.2.2. 2-Ethoxyl-4-phenyl-6-[(3'β, 17'β)-3'-(hydroxyl) androst-5'-en-17'-yl] pyridine (3b)
1
White solid, yield 15%, m.p.100.9-102.7 . H NMR (400 MHz, CDCl3) δ 7.58 – 7.49 (m, 2H),
7.41 – 7.29 (m, 3H), 6.90 (s, 1H), 6.74 (s, 1H), 5.30 (d, J = 5.9 Hz, 1H), 4.34 (q, J = 7.0 Hz, 2H),
3.54 – 3.38 (m, 1H), 1.40 (t, J = 7.0 Hz, 3H),0.93 (s, 3H), 0.49 (s, 3H). 13C NMR (100 MHz, CDCl3)
δ 163.38, 159.75, 150.73, 140.91, 139.18, 128.87, 128.57, 127.05, 121.63, 114.56, 105.40, 71.77,
61.46, 58.07, 56.80, 50.42, 44.84, 42.32, 38.26, 37.33, 36.65, 32.24, 32.00, 31.67, 29.72, 26.94,
25.08, 24.76, 20.97, 20.91, 19.45, 19.42, 14.83, 13.03. HRMS (ESI):m/z calcd for C32H42NO2
(M+H)+, 472.3216 ; found, 472.3213.
4.2.3. 2-Ethoxyl-4-(4-bromophenyl)-6-[(3'β, 17'β)-3'-(hydroxyl) androst-5'-en-17'-yl] pyridine (3c)
White solid, yield 18%, m.p.80.3-82.0 .1H NMR (400 MHz, CDCl3) δ 7.50 (d, J = 8.5 Hz, 2H),
7.41 – 7.36 (m, 2H), 6.77 (s, 1H), 6.71 (s, 1H), 6.60 (d, J = 9.6 Hz, 1H), 5.31 (d, J = 5.2 Hz, 1H),
4.34 (q, J = 7.0 Hz, 2H), 3.53 – 3.41 (m, 1H), 1.33 (t, J = 7.0 Hz, 3H), 0.93 (s, 3H), 0.48 (s, 3H).
13C NMR (100 MHz, CDCl3) δ 162.39, 159.04, 148.46, 139.85, 137.06, 131.00, 127.60, 121.88,
120.60, 113.15, 104.21, 70.77, 60.51, 57.02, 55.76, 49.36, 43.84, 41.28, 37.20, 36.28, 35.61, 31.19,
30.95, 30.64, 28.68, 24.05, 23.71, 21.67, 19.86, 18.42, 13.76, 13.10, 12.00. HRMS (ESI):m/z calcd
for C32H41BrNO2 (M+H)+ 550.2321 ; found, 550.2321.
4.2.4. 2-Ethoxyl-4-(4-methylphenyl)-6-[(3'β, 17'β)-3'-(hydroxyl) androst-5'-en-17'-yl]pyridine (3d)
White solid, yield 13%, m.p.85.3-87.2 .1H NMR (400 MHz, CDCl3) δ 7.50 (d, J = 7.8 Hz, 2H),
7.25 (d, J = 5.2 Hz, 2H).6.90 (s, 1H), 6.73 (s, 1H), 5.38 (s, 1H), 4.40 (q, J =7.0 Hz, 1H), 3.42-3.60
13
(m, 1H), 2.40 (s, 3H), 1.41 (t, J = 7.0 Hz, 3H),1.01 (s, 3H), 0.56 (s, 3H). C NMR (100 MHz,
CDCl3) δ 163.35, 140.88, 140.81, 129.60, 126.89, 121.65, 114.44, 71.82, 56.78, 50.40, 49.96, 44.85,
42.31, 37.31, 36.64, 36.58, 34.84, 32.62, 32.32, 32.23, 31.99, 31.93, 31.67, 29.71, 29.37, 24.75,
22.70, 21.21, 20.89, 19.44, 14.81, 14.12, 13.02. HRMS (ESI):m/z calcd for C33H44NO2 (M+H)+
486.3372 ; found, 486.3373.
4.2.5. 2-Ethoxyl-4-(3-bromophenyl)-6-[(3'β, 17'β)-3'-(hydroxyl) androst-5'-en-17'-yl]pyridine (3e)
1
White solid, yield 15%, m.p.89.2-91.3 . H NMR (400 MHz, CDCl3) δ 7.72 (t, J = 1.7 Hz, 1H),
7.54 – 7.49 (m, 2H), 7.30 (t, J = 7.9 Hz, 1H), 6.84 (s, 1H), 6.69 (s, 1H), 5.37 (d, J = 5.2 Hz, 1H),
4.40 (q, J = 7.0 Hz, 2H), 3.60 – 3.46 (m, 1H), 1.40 (t, J = 7.0 Hz, 3H),1.00 (s, 3H), 0.55 (s, 3H). 13C
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ACCEPTED MANUSCRIPT
NMR (100 MHz, CDCl3) δ 163.38, 160.10, 149.23, 141.28, 140.84, 131.51, 130.40, 130.11, 125.69,
123.01, 121.72, 121.64, 114.28, 105.48, 71.90, 61.60, 58.03, 56.77, 50.37, 44.89, 42.21, 38.23,
37.31, 36.63, 32.21, 31.98, 31.57, 29.72, 25.13, 24.75, 20.89, 19.45, 14.80, 13.05. HRMS (ESI):m/z
calcd for C32H41BrNO2 (M+H)+ 550.2321 ; found, 550.2321.
4.2.6. 2-Ethoxyl-4-(4-cyanophenyl)-6-[(3'β, 17'β)-3'-(hydroxyl) androst-5'-en-17'-yl]pyridine (3f)
White solid, yield 17%, m.p.92.2-94.4 .1H NMR (400 MHz, CDCl3) δ 7.74 (d, J = 8.4 Hz, 2H),
7.68 (d, J = 8.4 Hz, 2H), 6.86 (s, 1H), 6.71 (s, 1H), 5.38 (d, J = 5.0 Hz, 1H), 4.42 (q, J = 7.0 Hz,
13
2H), 3.60 – 3.48 (m, 1H),1.41 (t, J = 7.0 Hz, 3H),1.01 (s, 3H), 0.55 (s, 3H). C NMR (100 MHz,
CDCl3) δ 163.46, 160.51, 148.65, 143.71, 140.89, 132.69, 127.78, 121.57, 118.63, 114.18, 112.22,
105.71, 71.74, 61.68, 58.07, 56.78, 50.35, 44.92, 42.28, 38.24, 37.30, 36.62, 32.20, 31.96, 31.64,
25.08, 24.73, 20.87, 19.44, 14.76, 13.05. HRMS (ESI):m/z calcd for C33H41N2O2 (M+H)+ 497.3168 ;
found, 497.3169.
4.2.7. 2-Ethoxyl-4-(3,5-difluoropheny)-6-[(3'β,17'β)-3'-(hydroxyl) androst-5'-en-17'-yl] pyridine
(3g)
White solid, yield 16%, m.p.88.7-90.6 .1H NMR (400 MHz, CDCl3) δ 7.10 (d, J = 6.3 Hz, 2H),
6.81 (m, 2H), 6.67 (s, 1H), 5.38 (d, J = 2.9 Hz, 1H), 4.40 (q, J = 7.0 Hz, 2H), 3.63 – 3.52 (m, 1H),
13
1.39 (t, J = 7.0 Hz, 3H), 1.00 (s, 3H), 0.55 (s,3H). C NMR (100 MHz, CDCl3) δ 164.60, 164.47,
163.46, 162.13, 162.00, 160.36, 148.31, 142.50, 140.78, 121.68, 113.99, 110.12, 110.05, 109.93,
109.86, 105.48, 103.78, 72.06, 61.65, 58.04, 56.78, 50.37, 44.90, 42.10, 38.23, 37.30, 36.62, 32.21,
31.97, 31.47, 29.73, 25.09, 24.74, 20.90, 19.43, 14.75, 13.03. HRMS (ESI):m/z calcd for
C32H40F2NO2 (M+H)+ 508.3027 ; found, 508.3029.
4.2.8.2-Ethoxyl-4-(4-methylsulfonylphenyl)-6-[(3'β,17'β)-3'-(hydroxyl)androst-5'-en-17'-yl]pyridine
(3h)
White solid, yield 16%, m.p.113.1-114.9 .1H NMR (400 MHz, CDCl3) δ 8.03 (d, J = 8.4 Hz, 2H),
7.78 (d, J = 8.4 Hz, 2H), 6.84 (s, 1H), 6.73 (s, 1H), 5.36 (s, 1H), 4.41 (q, J = 7.0 Hz, 2H), 3.64 –
3.46 (m, 1H), 3.11 (s, 3H), 1.41 (t, J = 7.0 Hz, 3H), 0.99 (s, 3H), 0.98 (s, 3H). 13C NMR (100 MHz,
CDCl3) δ 163.49, 163.44, 144.52, 140.69, 140.36, 128.08, 128.00, 121.69, 114.65, 105.56, 72.05,
61.76, 56.70, 50.62, 49.94, 45.77, 44.54, 41.98, 37.16, 36.54, 34.86, 32.61, 32.28, 31.35, 29.68,
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ACCEPTED MANUSCRIPT
27.58, 26.47, 20.92, 19.39, 14.89. HRMS (ESI):m/z calcd for C33H43NO4SNa (M+Na)+ 572.2810;
found, 572.2812.
4.2.9. 2-Ethoxyl-4-(4-fluoropheny)-6-[(3'β, 17'β)-3'-(hydroxyl) androst-5'-en-17'-yl] pyridine (3i)
White solid, yield 17%, m.p.195.4-197.9 .1H NMR (400 MHz, CDCl3) δ 7.61 – 7.51 (m, 2H), 7.13
(t, J = 8.5 Hz, 2H), 6.78 (s, 1H), 6.68 (s, 1H), 5.38 (d, J = 4.8 Hz, 1H), 4.41 (q, J = 7.0 Hz, 2H),
13
3.58 – 3.50 (m, 1H), 1.40 (t, J = 7.0 Hz, 3H), 1.01 (s, 3H), 0.56 (s, 3H). C NMR (100 MHz,
CDCl3) δ 163.40, 159.91, 149.66, 140.90, 135.27, 128.76, 128.68, 121.62, 115.92, 115.71, 114.34,
105.29, 71.78, 61.49, 58.06, 56.80, 50.41, 44.84, 42.32, 38.27, 37.32, 36.64, 32.23, 31.99, 31.67,
29.71, 25.08, 24.75, 20.91, 19.44, 14.81, 13.02. HRMS (ESI):m/z calcd for C32H41FNO2 (M+H)+
490.3121 ; found, 490.3122.
4.2.10. 2-Ethoxyl-4-(pyridin-3-yl)-6-[(3'β, 17'β)-3'-(hydroxyl) androst-5'-en-17'-yl]pyridine (3j)
White solid, yield 13%, m.p.122.5-124.2 .1H NMR (400 MHz, CDCl3) δ 8.94 (d, J = 1.8 Hz, 1H),
8.74 (d, J = 5.0 Hz, 1H), 8.05 – 7.97 (m, 1H), 7.52 (d, J = 5.1 Hz, 1H), 6.86 (s, 1H), 6.72 (s, 1H),
5.37 (d, J = 5.0 Hz, 1H), 4.42 (q, J = 7.0 Hz, 2H), 3.62 – 3.47 (m, 1H), 1.40 (t, J = 7.0 Hz, 3H), 1.00
(s, 3H), 0.55 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 163.47, 160.74, 148.91, 147.62, 146.31,
140.96, 136.31, 135.95, 124.61, 121.48, 114.08, 105.66, 71.65, 61.68, 58.04, 56.77, 50.34, 44.94,
42.30, 38.20, 37.32, 36.63, 32.20, 31.95, 31.92, 31.63, 29.69, 25.09, 24.72, 20.87, 19.44, 14.73,
13.04. HRMS (ESI):m/z calcd for C31H41N2O2 (M+H)+ 473.3168 ; found, 473.3157.
4.2.11. 2-Ethoxyl-4-(thiofuran-2-yl)-6-[(3'β, 17'β)-3'-(hydroxyl) androst-5'-en-17'-yl]pyridine (3k)
White solid, yield 18%, m.p.90.2-92.1 .1H NMR (400 MHz, CDCl3) δ 7.45 – 7.40 (m, 1H), 7.34 (d,
J = 5.0 Hz, 1H), 7.11 – 7.07 (m, 1H), 6.74 (s, 1H), 6.72 (s, 1H), 5.37 (d, J = 5.3 Hz, 1H), 4.39 (q, J
13
= 6.9 Hz, 2H), 3.58 – 3.45 (m, 1H), 1.39 (t, J = 7.0 Hz, 3H), 1.00 (s, 3H), 0.54 (s, 3H). C NMR
(100 MHz, CDCl3) δ 163.42, 160.03, 143.43, 142.24, 140.89, 128.08, 126.25, 124.69, 121.63,
113.05, 103.72, 71.79, 61.50, 57.92, 56.78, 56.67, 50.57, 50.39, 49.97, 45.68, 44.86, 42.31, 38.20,
37.31, 36.63, 32.21, 31.98, 31.64, 24.99, 24.74, 20.90, 19.45, 14.81, 13.01. HRMS (ESI):m/z calcd
for C30H40NO2S (M+H)+ 478.2780 ; found, 478.2781.
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ACCEPTED MANUSCRIPT
4.2.12. 2-Ethoxyl-4-(3-bromopyridin-2-yl)-6-[(3'β,17'β)-3'-(hydroxyl) androst-5'-en-17'-yl] pyridine
(3l)
White solid, yield 13%, m.p.90.9-92.8 .1H NMR (400 MHz, CDCl3) δ 7.67 (d, J = 7.6 Hz, 1H),
7.61 (t, J = 7.7 Hz, 1H), 7.48 (d, J = 7.7 Hz, 1H), 7.26 (s, 1H), 7.08 (s, 1H), 5.43 – 5.33 (m, 1H),
4.41 (q, J = 7.1 Hz, 2H), 3.60 – 3.44 (m, 1H), 1.42 (t, J = 7.0 Hz, 3H), 1.00 (s, 3H), 0.55 (s, 3H).
13C NMR (100 MHz, CDCl3) δ 163.56, 160.37, 156.87, 147.18, 142.32, 140.89, 139.04, 127.74,
121.63, 119.59, 113.59, 105.37, 71.79, 61.62, 58.04, 56.79, 50.39, 44.92, 42.32, 38.23, 37.31, 36.63,
32.22, 31.98, 31.68, 25.13, 24.75, 20.91, 19.44, 14.77, 13.05. HRMS (ESI):m/z calcd for
C31H40BrN2O2 (M+H)+ 551.2273 ; found, 551.2274 .
4.2.13. 2-Ethoxyl-4-(furan-2-yl)-6-[(3'β, 17'β)-3'-(hydroxyl) androst-5'-en-17'-yl]pyridine (3m)
White solid, yield 15%, m.p.85.7-87.6 .1H NMR (400 MHz, CDCl3) δ 7.49 (s, 1H), 6.96 – 6.87 (m,
1H), 6.78 (m, 2H), 6.49 (s, 1H), 5.38 (d, J = 5.2 Hz, 1H), 4.40 (q, J =7.1 Hz 2H), 3.61 – 3.46 (m,
13
1H), 1.39 (t, J = 7.0 Hz, 3H), 1.00 (s, 3H), 0.54 (s, 3H). C NMR (100 MHz, CDCl3) δ 163.35,
159.93, 152.26, 143.10, 140.88, 139.65, 121.63, 111.79, 110.75, 107.81, 100.56, 71.79, 61.46,
57.96, 56.76, 50.39, 44.82, 42.29, 38.23, 37.30, 36.63, 32.20, 31.97, 31.64, 24.97, 24.73, 20.93,
19.44, 14.80, 12.99. HRMS (ESI):m/z calcd for C30H40NO3 (M+H)+ 462.3008 ; found, 462.3007 .
4.2.14. 2-Ethoxyl-4-(pyridin-4-yl)-6-[(3'β, 17'β)-3'-(hydroxyl) androst-5'-en-17'-yl]pyridine (3n)
White solid, yield 11%, m.p.95.3-97.6 . 1H NMR (400 MHz, CDCl3) δ 8.86 (d, J = 4.9Hz, 2H),
7.69 (d, J = 4.9Hz, 2H), 6.88 (s, 1H), 6.75 (s, 1H), 5.37 (d, J = 5.1 Hz, 1H), 4.43 (q, J = 7.0 Hz, 2H),
3.60 – 3.46 (m, 1H), 1.40 (t, J = 7.0 Hz, 3H) 1.00 (s, 3H), 0.55 (s, 3H). 13C NMR (100 MHz, CDCl3)
δ 163.57, 161.18, 149.35, 146.23, 140.93, 123.02, 121.47, 113.68, 105.77, 71.66, 61.85, 58.08,
56.76, 50.32, 44.99, 42.26, 38.20, 37.30, 36.61, 32.19, 31.92, 31.61, 29.69, 25.11, 24.71, 22.68,
20.85, 19.43, 14.69, 13.05. HRMS (ESI):m/z calcd for C31H41N2O2 (M+H)+ 473.3168 ; found,
473.3167.
4.2.15.
2-Ethoxyl-3-cyano-4-(4-chlorophenyl)-6-[(3'β,
17'β)-3'-(hydroxyl)
androst-5'-en-17'-yl]pyridine (4a)
White solid, yield 33%, m.p.117.8-119.6 .1H NMR (400 MHz, CDCl3) δ 7.52 (d, J = 8.5 Hz, 2H),
7.47 (d, J = 6.6 Hz, 2H), 6.77 (s, 1H), 5.37 (s, 1H), 4.54 (q, J = 7.0 Hz, 2H), 3.56-3.52 (m, 1H),
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ACCEPTED MANUSCRIPT
13
1.45 (t, J = 8.0 Hz, 3H),1.01 (s, 3H), 0.56 (s, 3H). C NMR (100 MHz, CDCl3) δ 164.75, 164.13,
153.88, 140.85, 136.05, 134.90, 129.75, 129.16, 121.47, 116.41, 115.60, 91.69, 71.85, 63.22, 58.46,
56.90, 50.25, 45.62, 42.17, 38.20, 37.28, 36.60, 32.19, 31.90, 31.54, 29.26, 25.01, 24.72, 20.86,
19.42, 14.53, 13.13. HRMS (ESI):m/z calcd for C33H39ClN2O2Na (M+Na)+ 553.2598 ; found,
553.2596.
4.2.16. 2-Ethoxyl-3-cyano-4-phenyl-6-[(3'β, 17'β)-3'-(hydroxyl) androst-5'-en-17'-yl]pyridine (4b)
White solid, yield 35%, m.p.100.9-102.4 .1H NMR (400 MHz, CDCl3) δ 7.58 (d, J = 6.6 Hz, 2H),
7.48 (d, J = 5.9 Hz, 3H), 6.81 (s, 1H), 5.36 (s, 1H), 4.53 (q, J = 7.0 Hz, 2H), 3.61-3.54 (m, 1H), 1.45
13
(t, J = 8.0 Hz, 3H), 1.00 (s, 3H), 0.56 (s, 3H). C NMR (100 MHz, CDCl3) δ 182.96, 167.30,
164.22, 155.01, 140.86, 129.70, 128.85, 128.42, 121.48, 117.06, 71.74, 63.10, 58.45, 57.20, 56.91,
50.90, 50.29, 49.96, 46.37, 45.57, 42.24, 36.59, 34.93, 32.31, 31.65, 27.42, 26.93, 26.45, 21.00,
20.88, 19.41, 14.72, 13.13. HRMS (ESI):m/z calcd for C33H41N2O2 (M+H)+ 497.3168 ; found,
497.3167.
4.2.17.
2-Ethoxyl-3-cyano-4-(4-bromophenyl)-6-[(3'β,
17'β)-3'-(hydroxyl)
androst-5'-en-17'-yl]pyridine (4c)
White solid, yield 28%, m.p.115.9-117.4 .1H NMR (400 MHz, CDCl3) δ 7.62 (d, J = 7.1 Hz, 2H),
7.45 (d, J = 8.2 Hz, 2H), 6.77 (s, 1H), 5.36 (s, 1H), 4.53 (q, J = 6.9 Hz, 2H), 3.64 – 3.53 (m, 1H),
13
1.45 (t, J = 7.0 Hz, 3H),1.00 (s, 3H), 0.56 (s, 3H). C NMR (100 MHz, CDCl3) δ 164.81, 164.14,
153.91, 140.72, 135.32, 132.12, 129.99, 124.35, 121.57, 116.36, 115.59, 91.56, 72.19, 63.25, 58.45,
56.88, 50.21, 45.62, 41.95, 38.18, 37.26, 36.58, 32.18, 31.90, 31.34, 29.71, 25.01, 24.72, 20.86,
19.43, 14.55, 13.15. HRMS (ESI):m/z calcd for C33H40BrN2O2 (M+H)+ 575.2273; found,575.2271 .
4.2.18.
2-Ethoxyl-3-cyano-4-(4-methylphenyl)-6-[(3'β,
17'β)-3'-(hydroxyl)
androst-5'-en-17'-yl]pyridine （4d）
White solid, yield 27%, m.p.110.1-111.9℃. 1H NMR (400 MHz, CDCl3) δ 7.49 (d, J = 7.8 Hz, 2H),
7.29 (d, J = 8.0 Hz, 2H), 6.80 (s, 1H), 5.36 (s, 1H), 4.53 (q, J = 7.0 Hz, 2H), 3.65 - -3.43 (m, 1H),
2.41 (s, 3H), 1.46 – 1.44 (m, 3H), 1.00 (s, 3H), 0.56 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 167.21,
164.27, 155.24, 140.74, 139.96, 133.61, 129.58, 128.31, 121.60, 116.59, 116.00, 91.34, 72.21,
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ACCEPTED MANUSCRIPT
63.05, 58.42, 56.88, 50.88, 50.25, 46.34, 45.55, 41.96, 38.19, 37.27, 36.59, 34.89, 32.62, 32.19,
31.33, 29.71, 27.43, 26.46, 24.74, 21.36, 20.88, 19.41, 14.74, 14.58, 13.13. HRMS (ESI):m/z calcd
for C34H42N2O2Na (M+Na)+ 533.3144; found, 533.3145.
4.2.19.
2-Ethoxyl-3-cyano-4-(3-bromophenyl)-6-[(3'β,
17'β)-3'-(hydroxyl)
androst-5'-en-17'-yl]pyridine (4e)
White solid, yield 29%, m.p.90.7-93.3 .1H NMR (400 MHz, CDCl3) δ 7.66 (s, 1H), 7.61 (d, J =
8.0 Hz, 1H), 7.54 (d, J = 7.8 Hz, 1H), 7.37 (t, J = 7.9 Hz, 1H), 6.77 (s, 1H), 5.37 (s, 1H), 4.54 (d, J
13
= 7.1 Hz, 2H), 3.65 – 3.49 (m, 1H), 1.45 (t, J = 8.0 Hz, 3H),1.00 (s, 3H), 0.56 (s, 3H). C NMR
(100 MHz, CDCl3) δ 167.78, 164.87, 164.07, 153.52, 140.75, 138.48, 132.70, 131.20, 130.40,
127.16, 122.86, 121.56, 116.46, 115.35, 91.79, 72.11, 63.27, 58.47, 56.89, 50.22, 45.72, 41.99,
38.19, 37.26, 36.59, 32.18, 31.41, 29.70, 29.24, 25.04, 24.72, 20.85, 19.40, 14.53, 13.15. HRMS
(ESI):m/z calcd for C33H39BrN2O2Na (M+Na)+ 597.2093; found, 597.2091.
4.2.20.
2-Ethoxyl-3-cyano-4-(3-nitrophenyl)-6-[(3'β,
17'β)-3'-(hydroxyl)
androst-5'-en-17'-yl]pyridine (4f)
White solid, yield 30%, m.p.151.9-153.7 .1H NMR (400 MHz, CDCl3) δ 8.42 – 8.32 (m, 2H), 7.97
(d, J = 7.7, 1H), 7.72 (t, J = 8.0 Hz, 1H), 6.84 (s, 1H), 5.38 (d, J = 4.9 Hz, 1H), 4.56 (q, J = 7.0 Hz,
13
1H), 3.73 – 3.64 (m, 1H), 1.47 (t, J = 7.0 Hz, 3H),1.01 (s, 3H), 0.58 (s, 3H). C NMR (100 MHz,
CDCl3) δ 165.67, 164.19, 152.61, 148.50, 140.31, 138.00, 134.45, 130.14, 124.45, 123.41, 121.93,
116.40, 115.17, 91.69, 73.33, 63.52, 58.55, 56.89, 50.15, 45.77, 41.61, 38.19, 37.18, 36.54, 32.17,
31.93, 31.87, 31.07, 29.70, 29.36, 25.08, 24.71, 22.69, 20.85, 19.39, 17.88, 14.50, 14.12, 13.19.
HRMS (ESI):m/z calcd for C33H39N3O4Na (M+Na)+ 564.2838; found, 564.2837.
4.2.21.
2-Ethoxyl-3-cyano-4-(3,5-difluoropheny)-6-[(3'β,
17'β)-3'-(hydroxyl)
androst-5'-en-17'-yl]pyridine (4g)
White solid, yield 32%, m.p.90.1-91.9 .1H NMR (400 MHz, CDCl3) δ 7.10 (d, J = 5.6 Hz, 2H),
6.98 – 6.88 (m, 1H), 6.76 (s, 1H), 5.37 (d, J = 4.4 Hz, 1H), 4.55 (q, J = 7.0 Hz, 2H), 3.59-3.53 (m,
13
1H), 1.46 (t, J = 7.0 Hz, 3H), 1.01 (s, 3H), 0.56 (s, 3H). C NMR (100 MHz, CDCl3) δ 165.18,
164.09, 161.83, 152.63, 140.76, 121.52, 116.26, 114.99, 111.83, 111.57, 105.08, 91.74, 72.45,
72.11, 63.38, 58.50, 56.90, 50.22, 45.68, 42.03, 38.19, 37.26, 36.58, 32.18, 31.92, 31.43, 29.69,
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ACCEPTED MANUSCRIPT
29.36, 25.02, 24.70, 22.68, 20.84, 19.40, 14.48, 14.10, 13.13. HRMS (ESI):m/z calcd for
C33H39F2N2O2 (M+H)+ 533.2980; found, 533.2977.
2.2.22.
2-Ethoxyl-3-cyano-4-(4-methylsulfonylphenyl)-6-[(3'β,
17'β)-3'-(hydroxyl)
androst-5'-en-17'-yl]pyridine (4h)
White solid, yield 30%, m.p.85.3-87.8 .1H NMR (400 MHz, CDCl3) δ 8.08 (d, J = 8.5 Hz, 2H),
7.77 (d, J = 8.5 Hz, 2H), 6.76 (s, 1H), 5.37 (d, J = 5.0 Hz, 1H), 4.54 (q, J = 7.1 Hz, 2H), 3.67 – 3.52
13
(m, 1H), 3.12 (s, 3H), 1.47 (t, J = 8.0 Hz, 3H),1.00 (s, 3H), 0.99 (s, 3H). C NMR (100 MHz,
CDCl3) δ 168.34, 164.24, 152.88, 141.77, 141.56, 140.68, 129.52, 128.02, 121.58, 116.87, 115.22,
91.48, 72.12, 63.45, 57.29, 54.83, 50.98, 49.94, 46.55, 44.53, 42.00, 37.16, 36.56, 34.97, 32.59,
32.27, 31.92, 31.41, 29.69, 29.66, 29.36, 27.49, 26.42, 22.69, 21.00, 20.86, 19.39, 14.66, 14.12.
HRMS (ESI):m/z calcd for C34H42N2O4SNa (M+Na)+ 597.2763; found,597.2761.
4.2.23.
2-Ethoxyl-3-cyano-4-(4-fluoropheny)-6-[(3'β,
17'β)-3'-(hydroxyl)
androst-5'-en-17'-yl]pyridine (4i)
1
White solid, yield 33%, m.p.106.1-107.7 . H NMR (400 MHz, CDCl3) δ 7.64 – 7.45 (m, 2H),
7.18 (t, J = 8.3 Hz, 1H), 6.99 (s, 1H), 6.84 – 6.71 (s, 1H), 5.34 (d, J = 9.5 Hz, 1H), 4.52 (q, J = 7.5,
13
2H), 3.57 – 3.43 (m, 1H), 1.44 (t, J = 4.0 Hz, 3H),1.00 (s, 3H), 0.56 (s, 3H). C NMR (100 MHz,
CDCl3) δ 164.61, 164.19, 164.09, 160.29, 154.77, 154.03, 141.03, 130.45, 130.37, 129.82, 129.30,
128.51, 121.28, 116.54, 116.39, 116.15, 116.08, 115.86, 115.69, 114.79, 114.48, 91.71, 91.32,
71.49, 63.63, 63.17, 63.00, 58.42, 58.38, 56.88, 50.28, 50.26, 49.96, 45.58, 45.50, 42.22, 38.19,
37.32, 36.60, 36.57, 32.19, 31.90, 31.54, 29.27, 25.00, 24.98, 24.72, 20.96, 20.86, 19.42, 14.78,
14.73, 14.57, 14.54, 13.12, 13.11. HRMS (ESI):m/z calcd for C33H40FN2O2 (M+H)+ 515.3074;
found,515.3076.
2.2.24.
2-Ethoxyl-3-cyano-4-(pyridine-3-yl)-6-[(3'β,
17'β)-3'-(hydroxyl)
androst-5'-en-17'-yl]pyridine (4j)
White solid, yield 23%, m.p.105.4-107.2 .1H NMR (400 MHz, CDCl3) δ 8.78 (s, 1H), 8.77 (s, 1H),
8.01 (s, 1H), 7.46 (s, 1H), 6.78 (s, 1H), 5.37 (d, J = 5.4 Hz, 1H), 4.55 (q, J = 7.2 Hz, 2H), 3.56 –
13
3.46 (m, 1H), 1.47 (t, J = 8.0 Hz, 3H), 1.01 (s, 3H), 0.57 (s, 3H). C NMR (100 MHz, CDCl3) δ
168.09, 165.18, 164.27, 151.54, 151.36, 150.69, 148.87, 140.92, 135.89, 132.49, 123.49, 121.43,
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116.76, 116.40, 91.73, 71.69, 63.35, 58.51, 57.27, 56.91, 50.96, 50.23, 49.95, 46.49, 45.69, 42.27,
42.23, 38.20, 37.29, 36.61, 34.98, 32.59, 32.20, 31.89, 31.61, 27.44, 26.92, 26.42, 25.01, 24.74,
20.99, 19.42, 14.50, 13.14. HRMS (ESI):m/z calcd for C32H39N3O2 Na (M+Na)+ 520.2940 ; found,
520.2941.
4.2.25.
2-Ethoxyl-3-cyano-4-(thiofuran-2-yl)-6-[(3'β,
17'β)-3'-(hydroxyl)
androst-5'-en-17'-yl]pyridine (4k)
White solid, yield 34%, m.p.91.1-93.1 .1H NMR (400 MHz, CDCl3) δ 7.88 (s, 1H), 7.50 (s, 1H),
7.18 (s, 1H), 6.90 (s, 1H), 5.37 (t, J = 5.8 Hz, 1H), 4.56 – 4.47 (m, 2H), 3.60 – 3.46 (m, 1H),1.45 (t,
J = 7.0 Hz, 3H), 0.99 (s, 3H), 0.55 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 167.48, 164.43, 146.53,
140.90, 137.90, 129.14, 128.58, 121.49, 115.44, 89.62, 71.72, 63.18, 58.34, 56.90, 50.26, 46.41,
45.61, 42.22, 38.13, 37.29, 36.57, 34.85, 32.61, 32.18, 31.91, 31.63, 27.35, 26.43, 24.91, 24.72,
20.86, 19.44, 14.55, 13.11. HRMS (ESI):m/z calcd for C31H38N2O2SNa (M+Na)+ 525.2552 ; found,
525.2553.
4.2.26.
2-Ethoxyl-3-cyano-4-(thiofuran-3-yl)-6-[(3'β,
17'β)-3'-(hydroxyl)
androst-5'-en-17'-yl]pyridine (4l)
White solid, yield 27%, m.p.86.4-88.2 .1H NMR (400 MHz, CDCl3) δ 8.18 (s, 1H), 7.54 (s, 1H),
6.87 (s, 1H), 6.81 (s, 1H), 5.37 (s, 1H), 4.51 (q, J = 7.5 Hz, 2H), 3.58 – 3.48 (m, 1H), 1.44 (t, J =
7.0 Hz, 3H),1.00 (s, 3H), 0.54 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 221.24, 167.51, 164.55,
145.52, 143.88, 142.79, 140.92, 121.98, 121.43, 120.90, 116.43, 114.97, 114.61, 109.33, 88.82,
71.68, 63.04, 58.36, 56.89, 51.77, 50.26, 45.54, 42.20, 38.17, 37.28, 36.60, 35.85, 34.85, 32.17,
31.61, 30.79, 27.42, 26.43, 24.70, 21.89, 20.85, 19.43, 14.54, 13.55, 13.09. HRMS (ESI):m/z calcd
for C31H38N2O2SNa (M+Na)+ 525.2552 ; found, 525.2551.
4.3. General procedure for the synthesis of compound 5j
A mixture of compound 3j (1.0mmol), acetic anhydride (1.2mmol), 4-dimethylaminopyridine
(DMAP, 0.02mmol) and Et3N (1.0mmol) in dichloromethane (5 mL) was stirred for about 5h at
room temperature. After completion of the reaction as evident from TLC, the organic phase
concentrated under reduced vacuum. The residue was purified by column chromatography (PE:
Acetone = 4:1) to give compound 5j, White solid, yield 90%, m.p.92.2-94.0 .1H NMR (400 MHz,
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CDCl3) δ 8.85 (d, J = 1.6 Hz, 1H), 8.64 (d, J = 4.7 Hz, 1H), 7.96 – 7.82 (m, 1H), 7.38 (d, J = 7.9,
1H), 6.82 (s, 1H), 6.71 (s, 1H), 5.41 (d, J = 4.9 Hz, 1H), 4.69 – 4.53 (m, 1H), 4.46(q, J = 7.5 Hz 2H),
13
2.03 (s, 3H), 1.41 (t, J = 4.0 Hz, 3H), 1.02 (s, 3H), 0.56 (s, 3H). C NMR (100 MHz, CDCl3) δ
170.52, 163.43, 160.35, 149.64, 148.18, 147.39, 139.77, 134.75, 134.38, 123.65, 122.52, 114.21,
105.53, 73.93, 61.58, 58.03, 56.69, 50.26, 44.88, 38.18, 38.12, 37.04, 36.71, 32.16, 31.94, 27.77,
25.05, 24.72, 21.42, 20.82, 19.34, 14.76, 13.03. HRMS (ESI):m/z calcd for C33H43N2O3 (M+H)+
515.3274; found, 515.3272.
4.4. Cell proliferation inhibition by MTT assay
About 4×103 exponentially growing cells per well were seeded into 96-well plate. After 24 h
incubation, the medium was removed and replaced with 200 µL fresh medium containing different
concentration of candidate compounds for another 72 h incubation. Then, 20 µL MTT solution
(5mg/mL) was added to each well and the cells were then incubated for additional 4 h. Removal of
the medium containing MTT, followed by addition of 150 µL DMSO to each well and agitation
until the dark blue crystal was dissolved completely. The absorbance was measured using an ELx
800 Universal Microplate Reader (Bio-Tek, Inc.) at a wavelength of 570nm. The date of IC50 was
analyzed by the software of SPSS 20.
4.5. Colony formation assay
Only about 2000 exponentially growing cells per well were seeded into 6-welll plate. After 24 h
incubation, the medium was removed and replaced with 3 mL fresh medium containing different
concentration of candidate compounds for 7 days incubation. Then, the cell colonies were washed
twice by PBS, fixed with 4% paraformaldehyde for 30min, and stained by crystal violet for another
30min. After that, wash away the staining with PBS until the colonies were clear enough.
4.6. Flow cytometric analysis of cell cycle distribution and apoptotic assay
For cell cycle distribution assay, cells were seeded into 6-well plate at 3×105 cells per well. After
24h incubation, the cultured medium was replaced with fresh medium containing different
concentrations of candidate compound for 48h incubation. Then, the cells were harvested, washed
and fixed with 70% ice-cold ethanol at -20 overnight. Before analyzed by Instrument
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LSRForteassa (BD Bioscience, USA), the cells were washed by PBS and stained with RNase and PI
( KeyGEN BioTECH, USA) for 30min in dark condition according to the protocol.
For apoptotic assay, after incubation with condidate compound, the cells were harvested and the
AnnexinV-FITC/PI apoptosis kit (KeyGEN BioTECH, USA) was used according to the
manufacturer’s instructions to detect apoptosis cells. About ten thousand events were collected for
each sample and analyzed by Instrument LSRForteassa (BD Bioscience, USA).
4.7. Migration and invasion assay
Wound healing assay, transwell assay and matrigel-coated transwell assay were conducted to
evaluate the cell migration and invasion ability. For the wound healing assay, exponentially
growing cells were seed in 6-well plate at 3×105 cells per well. Until the cells density reached
80%~90%, the cell surface was scratched using a 200µL pipet tip. Then, the cells were washed and
incubated with medium containing different concentration of candidate compound for 48 h, and the
cells were photographed on an inverted microscope.
For the transwell assay, exponentially growing cells suspending in 1% FBS and condidate
compound contained medium were seed in the upper chamber of Transwell 24-well plate, while
fresh 20% FBS and compound contained medium in the lower chamber. After 24 h incubation,
remove all the medium, wash the chambers with PBS and the migrating cells were fixed with
methanol for 15 min and stained with Hoechst-33258. Each chamber was photographed using
Thermo Fisher Cellomics High Content System.
For the matrigel-coated transwell assay, cells were seeded into the Transwell 24-well plate with
Matrigel. The medium, attractant, staining and cell counting method were the same as those of the
Thranswell assay.
4.8. Hoechst 33258 staining
Exponentially growing determined cells were seeded in a 6-well plate at 3×105 per well. After
48 h incubation, the medium was replaced with fresh medium containing different concentrations of
candidate compound for another 48 h. Then, the cells were washed and fixed with methanol for
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about 20 min. Cells were stained with 5ug/mL Hoechst-33258 in PBS containing 0.5% Triton
X-100 in the dark condition for at least 30min. Apoptosis cells were examined and identified
according to the condensation and fragmentation of nuclei by fluorescence microscopy under UV
excitation.
4.9. Western blotting analysis
After treated with different concentration of candidate compound for 48 h, the cells were harvested
and lysed with cell lysis buffer [1% NP-40, 0.1% sodium dodecyl sulfate (SDS), 150 mM NaCl, 25
mM Tris-HCl, 1% deoxycholic acid sodium salt, 1% PMSF]. Equal amount of cell lysates were
denatured, sepatated by sodium dodecyl sulfate polycrylamide gel electrophoresis (SDS-PAGE),
and transferred to 0.22µm nitrocellulose membrane. The membrane was blocked with PBS
containing 5% nonfat milk for 2 h, and then incubated with the primary antibody overnight at 4 ,
followed by incubation with a horseradish peroxidase-conjugated secondary antibody. The
immunoblots were visualized by enhanced chemiluminescence kit.
Acknowledgement
This work was supported by National Natural Science Foundation of China (Project No. 81430085,
No. 21372206 and No. 81703326); National Key Research Programs of Proteins (No.
2016YFA0501800); Key Research Program of Henan Province (No. 1611003110100); the Starting
Grant of Zhengzhou University (No. 32210533).
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