Synthesis of D-Ring Annulated Pyridosteroids from β-Formyl Enamides and Their Biological Evaluations

Article abstract of DOI:10.1021/acscombsci.8b00140

Herein, we report the synthesis of a novel class of substituted androst[17,16-b]pyridines (pyridosteroids) from the reaction of β-formyl enamides with alkynes in high yields. The optimized reaction protocol was extended to acyclic and cyclic β-formyl enamides to afford nonsteroidal pyridines. Cell survival assay of all compounds were carried against prostate cancer PC-3 cells wherein 3-hydroxy-5-en-2′,3′-dicarbethoxy-androst[17,16-b]pyridine showed the highest cytotoxic activity. Phase contrast microscopy and flow cytometry studies exhibited marked morphological features characteristic of apoptosis in 3-hydroxy-5-en-2′,3′-dicarbethoxy-androst[17,16-b]pyridine and abiraterone treated PC-3 cells. The treatment of 3-hydroxy-5-en-2′,3′-dicarbethoxy-androst[17,16-b]pyridine induces G₂/M phase cell cycle arrest in prostate cancer PC-3 cells. Enhancement of apoptotic inductions of PC-3 cells by 3-hydroxy-5-en-2′,3′-dicarbethoxy-androst[17,16-b]pyridine and abiraterone through the activation of caspases-6, -7, and -8 pathways were supported by qRT-PCR. In silico study of the compound 3-hydroxy-5-en-2′,3′-dicarbethoxy-androst[17,16-b]pyridine showed stable and promising interaction with the key caspase proteins. Our studies revealed that the pyridosteroid 3-hydroxy-5-en-2′,3′-dicarbethoxy-androst[17,16-b]pyridine, bearing pyridine-2,3-dicarbethoxy pharmacophore, facilitated initiation of caspase-8 and activates downstream effectors caspase-6 and caspase-7 and thereby triggering apoptosis of PC-3 cancer cells.

Full text of DOI:10.1021/acscombsci.8b00140

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Article
Synthesis of D-ring annulated pyridosteroids from #-
formyl enamides and their biological evaluations
Geetmani Singh Nongthombam, Kasmika Borah, Thingreila Muinao, Yumnam
Silla, Mintu Pal, Hari Prasanna Dekaboruah, and Romesh Chandra Boruah
ACS Comb. Sci., Just Accepted Manuscript • DOI: 10.1021/acscombsci.8b00140 • Publication Date (Web): 21 Dec 2018
Downloaded from http://pubs.acs.org on December 23, 2018
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Synthesis of D-ring annulated pyridosteroids from β-formyl
enamides and their biological evaluations
Geetmani Singh Nongthombama, Kasmika Borahb, Thingreila Muinaob, Yumnam Sillab, Mintu Palb, Hari
Prasanna Dekaboruahb and Romesh Chandra Boruah*a
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aChemical Science and Technology Division; CSIR-North East Institute of Science & Technology, Jorhat,
Assam, India 785006; Biological Science and Technology Division, CSIR-North East Institute of Science &
Technology, Jorhat, Assam, India 785006.
b
Keywords: β-Formyl enamide; azasteriod, pyridine, alkyne, SRB assay; molecular dynamic simulation;
qRT-PCR, apoptosis.
ABSTRACT: Herein we report the synthesis of a novel class of substituted androst[17,16-b]pyridines
(pyridosteroids) from the reaction of β-formyl enamides with alkynes in high yields. The optimized
reaction protocol was extended to acyclic and cyclic β-formyl enamides to afford non-steroidal
pyridines. Cell survival assay of all compounds were carried against prostate cancer PC-3 cells wherein 3-
hydroxy-5-en-2’,3’-dicarbethoxy-androst[17,16-b]pyridine showed the highest cytotoxic activity. Phase
contrast microscopy and flow cytometry studies exhibited marked morphological features characteristic
of apoptosis in 3-hydroxy-5-en-2’,3’-dicarbethoxy-androst[17,16-b]pyridine and abiraterone treated PC-
3 cells. The treatment of 3-hydroxy-5-en-2’,3’-dicarbethoxy-androst[17,16-b]pyridine induces G2/M
phase cell cycle arrest in prostate cancer PC-3 cells. Enhancement of apoptotic inductions of PC-3 cells
by 3-hydroxy-5-en-2’,3’-dicarbethoxy-androst[17,16-b]pyridine and abiraterone through the activation
of caspases-6, -7 and -8 pathways were supported by qRT-PCR. In-silico study of the compound 3-
hydroxy-5-en-2’,3’-dicarbethoxy-androst[17,16-b]pyridine showed stable and promising interaction with
the key caspase proteins. Our studies revealed that the pyridosteroid 3-hydroxy-5-en-2’,3’-dicarbethoxy-
androst[17,16-b]pyridine, bearing pyridine-2,3-dicarbethoxy pharmacophore, facilitated initiation of
caspase-8 and activates downstream effectors caspase-6 and caspase-7 and thereby triggering apoptosis
of PC-3 cancer cells.
INTRODUCTION
The pyridine substructure is one of the most ubiquitous heterocyclic motifs in organic and bioorganic
chemistry because of its widespread distribution in medicinally important natural products,
pharmaceuticals and bioactive molecules.1–4 A great deal of attention has been directed towards
development of new synthetic strategies for these biologically important substituted pyridines.5–8
Notable examples for substituted pyridine synthesis involve cycloadditions,9 cycloisomerization,10
ketoxime ester coupling with alkenylboronic acids,11 triflic anhydride/2-chloropyridine,12 alkyne coupling
with unsaturated imines,13 multicomponent reaction,14 transition metal catalyzed reactions of
substituted ketoxime15 and Vilsmeier-Haack-based cyclocondensation reaction of conjugated
ketoxime.16
Enamides constitute the building block of many biologically active compounds and attract
considerable attention as prochiral substrates in asymmetric synthesis of amino acids.17,18 Few metal
catalyzed coupling reactions of enamides with alkynes19 have been reported for synthesis of pyridine
through amide activation reaction.20 Enamides are also utilized for the preparation of pyridosteroids by
the reaction of Vilsmeier reagent.21 Most of the reactions involve the electron deficient carbonyl of N-
acetyl group via intermolecular cycloaddition reactions.22
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On the other hand, β-Formyl enamides have emerged as interesting synthons for their potential
diversity in heterocyclic synthesis.23,24 The research effort at our laboratory afforded β-formyl enamide
which was further utilized as an important reagent for one-pot preparation of pyridines.25 Despite the
cross coupling reaction of enamide with alkyne via amide activation for synthesis of substituted
pyridines as reported,26 the scope of β-formyl enamide for such an approach is yet to be explored.
Anti-cancer drug discovery has emerged as a major clinical challenge primarily due to the existence of
multi drug resistance, poor absorption of compounds and several side effects.27 Among the established
drugs, glucocorticoids have shown considerable clinical significance in targeting various cancer cells.28
Continued efforts have been made for the development of steroid-based novel therapeutic agents for
prostate cancer.29–33 The steroidal heterocycles received particular interests because of their important
biological significance.34 The 17-(3-pyridyl)androstane derivative (abiraterone) exhibited inhibition of
17α-hydroxylase/C17-20 lyase enzyme making it a clinically employed drug for prostate cancer
treatment.35 Recently, polysubstituted steroidal pyridines were evaluated for cytotoxic potential of
prostate cancer cell lines using androgen inhibitor abiraterone as control drug (Figure 1).36
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EtO
N
N
Et
CO2
N
N
Et
CO2
HO
HO
Polysubstituted steroidal pyridine
HO
Androst[17,16-b]pyridines
(Our synthesized compound)
Abiraterone
Figure 1.Abiraterone, polysubstituted steroidal pyridine and androst[17,16-b]pyridine.
In continuation of our interests in steroidal β-formyl enamides,21,23,24 herein, we report the synthesis of a
new series of D-ring fused pyridosteroids, and evaluation of their cytotoxic activities by inducing
apoptosis against prostate cancer cells. The novel androst[17,16-b]pyridine and non-steroidal pyridine
derivatives were derived from the reaction of corresponding β-formyl enamides37–39 with alkynes.
Among all compounds studied, azasteroid 4{1,3} exhibited the highest bioactivity against PC-3 prostate
cancer cells. This finding was also supported by our computational structural biology study wherein
4{1,3} showed stable interaction and lowest binding free energy against key caspase proteins.
RESULTS AND DISCUSSION
We initiated our study by examining the coupling reaction of β-formyl enamide 1{1} with
ethylphenylacetylene 2{1} (Table 1). It was observed that coupling of 1{1} and 2{1} took place smoothly
0
with CuI as the catalyst in DMF at 120 C to afford 3-acetoxy-5-en-2’-phenyl-3’-carbethoxy-
androst[17,16-b]pyridines in 70% yield (entry 8). Other solvents and bases are less effective (entry 1-7).
It was also observed that CuI improved the efficiency of the reaction and the use of CuCl/CuBr or
absence of CuI afforded very poor yield of the product 3{1,1} (entry 9-11). To improve the yield of the
product, various ligands were used in the one-pot reaction.
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Table1. Optimization of reaction conditiona
5
6
7
8
Ph
N
NHAc
CHO
Et
CO2
Ph
Catalyst
Ligand
Base, Solvent
9
Et
CO2
AcO
AcO
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3
2
1
{1,1}
{1}
{1}
Tb
Yieldc
(%)
Entry Catalyst
Ligand
Solvent
Base
(oC)
1
2
3
4
5
CuI
CuI
CuI
CuI
CuI
CuI
CuI
CuI
-
CuCl
CuBr
CuI
CuI
CuI
CuI
CuI
-
-
-
-
-
-
-
-
-
-
-
DMSO
DCE
THF
-
-
-
-
-
120
120
120
120
120
120
120
120
120
120
120
110
120
120
120
120
120
120
120
120
20
22
21
24
45
50
51
70
10
30
38
87
64
73
72
70
24
20
16
18
1,4-Dioxane
DMF
6
DMF
DMF
DMF
DMF
DMF
DMF
DMF
DMF
DMF
DMF
DMF
DMF
DMF
DMF
DMF
Na2CO3
Cs2CO3
K2CO3
K2CO3
K2CO3
K2CO3
K2CO3
Cs2CO3
K2CO3
K2CO3
K2CO3
K2CO3
K2CO3
K2CO3
K2CO3
7
8
9
10
11
12
13
14
15
16
1,10-Phen
1,10-Phen
DABCO
2,2’-Bipyridine
L-Proline
17 Pd(OAc)2
-
-
-
-
18
19
20
ZnBr2
ZnCl2
FeCl3
aReaction condition: 1{1} (0.1 mmol), 2{1} (0.15 mmol), base (0.15 mmol),
b
catalysts (10 mol %) and ligand (20 mol %) in solvent (3 mL); Oil bath
temperature; cisolated yields of 3{1,1}.
The use of 1,10-phenanthroline with K2CO3 afforded the best result with 87% of product 3{1,1} (entry 12)
at reaction temperature 110 C. Neither the use of Cs2CO3 nor ligands such as DABCO, 2,2’-bipyridine
and L-Proline showed positive results in the yield of 3{1,1} (entry 13-16). Also, the use of transition metal
catalyst [Pd(OAc)2] or Lewis acids (ZnBr2, ZnCl2, FeCl3) led to very poor yield of the product 3{1,1} (entry
17-20).
0
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1
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The structure of the synthesized steroidal pyridine 3{1,1} was elucidated by HNMR, CNMR, HRMS,
FTIR and single crystal X-Ray crystallography. The HRMS of 3{1,1} clearly indicated a molecular ion peak
at m/z 514.2990 (M++H) which is in accordance with our calculated value for empirical formula
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C33H40NO4. The 500 MHz HNMR spectrum of 3{1,1} in CDCl3 exhibited a sharp singlet at δ 7.86 ppm
which can be accounted for the aromatic C-H of the annulated pyridine ring. The absence of the peaks at
δ 9.49 and δ 10.88 ppm indicated that the formyl (-CHO) and the amide (-NHAc) groups of formyl
enamide 1{1} had participated in the intermolecular cyclisation to form the pyridine ring system. The
aromatic protons of phenyl ring appeared as multiplets at δ 7.52-7.50 and δ 7.42-7.37 ppm integrating
for two and three aromatic protons respectively. The characteristic steroidal C-3 methine and C-5
steroidal olefinic protons were observed respectively as multiplet at δ 4.66-4.59 ppm and doublet at δ
5.44 (J = 5.1 Hz) ppm. The chemical shifts of methylene and methyl protons of carbethoxy group were
recorded as quartet at δ 4.12 (J = 7.1 Hz) ppm and triplet at δ 1.00 (J = 7.1 Hz) ppm. The 3-acetoxy
methyl group exhibited as a singlet at 2.05 ppm, while steroidal angular methyl protons at C-10 and C-13
appeared as singlets respectively at δ 1.11 and δ 1.00 ppm. The steroidal core methylene protons were
shown in the region δ 2.84 – 1.58 ppm. Furthermore, the disappearance of the characteristic formyl
carbon signal at 197 ppm in the 13CNMR spectrum of 3{1,1} supported the annulation of the alkyne 2{1}
with 1{1}. Finally, the single crystal X-ray crystallography data (CCDC 1847573) confirmed the proposed
structure of the synthesized compound of 3{1,1} (Figure 2).
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Ph
N
Et
CO2
AcO
Figure 2. Single-crystal XRD of 3{1,1} (CCDC1847573)
The scope of the optimized reaction condition was explored with a series of substituted alkynes. The
transformation displayed wide versatility and proved to be a general methodology for the preparation
of a wide range of novel substituted pyridines (Figure 3). The steroidal β-formyl enamides 1{1} acted as
an excellent substrate to react with alkynes 2{1-7} producing corresponding steroidal substituted
pyridines 3{1,1} in high yields (Scheme 1). Base catalyzed hydrolysis of 3{1,1-3} under mild conditions
afforded 3-deacetylated compounds 4{1,1-3} in 76-80%. The products were characterized by high
resolution spectral and HRMS data.
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β-Formyl enamides 1:
NHAc
5
6
7
CHO
CHO
CHO
CHO
NHAc
NHAc
8
9
NHAc
AcO
1
1
1
1
{4}
{1}
{2}
{3}
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Alkynes 2:
Ph
H
H
Me
Me
Et
Et
Ph
H
CO2
CO2
-Me(p)
Et
CO2
Et
CO2
Me
CO2
CO2
CO2
CO2C(CH3 3
)
H4
CO-C6
2
2
2
2
2
2
2
{1}
{6}
{7}
{2}
{3}
{4}
{5}
Me
Et
CO2
Ph
CO2
N
N
N
Me
CO2
Et
CO2
COOEt
AcO
AcO
AcO
3
3
3
{1,1}, 87%
{1,3}, 80%
{1,2}, 84%
Ph
O
N
N
N
Et
CO2
Me
CO2
p-Tolyl
AcO
AcO
AcO
3
3
{1,5}, 83%
{1,4}, 78%
3
{1,6}, 79%
O
N
O
COOMe
COOMe
COOEt
COOEt
Ph
N
Ph
N
AcO
3
3
{2,2}, 82%
3
{1,7}, 76%
{2,3}, 80%
COOMe
COOMe
COOEt
COOEt
COOMe
COOMe
COOEt
COOEt
3
{4,3}, 77%
Ph
N
Ph
N
N
N
3
3
3
{3,2}, 78%
{4,2}, 78%
{3,3}, 76%
Et
CO2
Ph
Me
CO2
N
N
N
Et
CO2
Et
CO2
Me
CO2
HO
HO
HO
4
{1,3}, 76%
4
4
{1,2}, 78%
{1,1}, 80%
Figure 3. Synthesized androst[17,16-b]pyridines and non-steroidal pyridines.
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3
O
OH
N
4
5
6
7
NH
O
NH
2OH.HCl
K2PO4
CHO
POCl
3/DMF
; 0-10 o
CH3OH
reflux, 2h
CH2Cl2
C
8
AcO
AcO
AcO
9
1
{1}
16-DPA-20-oxime
16-DPA
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R1
CuI (10 Mol %)
1,10-Phen (20 Mol %)
, DMF, 110
oC
K
2CO3
R2
2
{1-7}
R1
R1
N
N
R2
R2
10% K
2CO3
CH3OH, rt
AcO
HO
4
3
{1,1-3}
{1,1-7}
Scheme 1. Synthesis of substituted androst[17,16-b]pyridines.
In order to explore the generality of the reaction, alicyclic formyl enamide 1{2-3} were prepared from
propiophenone and acetophenone and reacted with alkynes under the optimized condition to afford
tetrasubstituted pyridines 3{2-3,2-3} in 76-82% yield (Scheme 2). Similarly, 1-acetamido-2-formyl-1-
cyclohexene 1{4}, derived from cyclohexanone, reacted with alkynes 2{2-3} to yield 5,6,7,8-
tetrahydroquinoline 3{4, 2-3} in 77-78% yields (Scheme 2). However, unactivated alkynes such as phenyl
acetylene and diphenyl acetylene did not react with 1{1-4} to afford the expected substituted pyridines.
NH
2OH.HCl,
R3
R4
R3
R4
R3
R4
R3
CHO
, CH
POCl
3/DMF,
KH2PO4
Ac
2O/AcOH
3OH
0-10
oC
reflux, 2h
Fe, Toluene
CH2Cl2;
NHAc
O
N
R4
NHAc
OH
1
{2-4}
R1
,
CuI (10 Mol%), K2CO3
1,10-Phen (20 mol%),
DMF, 110o
R2
C.
2
{2,3}
R3
R4
R2
R1
N
3
{2-4,2-3}
Scheme 2. Synthesis of non-steroidal pyridine derivatives.
A plausible mechanism for the annulation of the D-ring is proposed as per our optimized reaction
conditions. The abstraction of N-H proton from amide group of 1{1} is initiated by K2CO3 leading to its
nucleophilic attack on activated alkynes catalyzed by CuI(1,10-phenanthroline),40 to form intermediate I.
The attack of iodinium ion on intermediate I probably facilitates intramolecular cyclisation to afford
dihydropyridine intermediate II with simultaneous loss of the catalyst. Finally, loss of -OH group from II
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results in the formation of a pyridine quaternary salt intermediate III which further undergoes loss of an
acetic acid molecule to afford a pyridine ring (Figure 4).
6
7
8
9
O
OH
Ac
N
AcOH
R1
R2
N
R1
R2
N
R1
R2
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OH
III
II
N
N
I
Cu
N
=
N
N
N
R2
R1
N
Cu
I
R1
N
Ac
N
Ac
N
Ac
N
R2
H
N
B
Cu
N
H
CHO
CHO
O
I
I
Figure 4. Mechanism for synthesis of pyridine derivatives from β-formyl enamide.
Biological Evaluation
SRB assay
The in-vitro cytotoxicity was performed on a novel series of androst[17,16-b]pyridines (3{1,1-7} and 4{1,
1-3}) and pyridine derivatives 3{2-4,2-3} using sulforhodamine B (SRB) assay against human prostate
carcinoma cells PC-3 and anti-prostate cancer drug abiraterone (AB) as a cytotoxic control.36,41 The
cytotoxicity assay indicated that all compounds exhibited moderate activities against PC-3 cell line.
Among these, novel azasteroidal and non-steroidal compounds 3{1,3}, 4{1,3} and 3{2,3} showed strong
cytotoxic effects against PC-3 cells with IC50 values 100, 95.4 and 97 µM respectively in a dose-
dependent manner compared to standard drug AB (91 µM). The screening of these three compounds
against human non-tumorigenic prostate epithelial cell RWPE-1 (Table 2) indicated higher toxic effects
of compounds 3{1,3} (218 µM) and 3{2,3} (275 µM) in comparison to 4{1,3} and AB with low toxic values
viz. 501 µM and 645 µM respectively. Based on these results, 4{1,3} was found to be the most effective
compound having improved window of cytotoxic effect with a wider range of IC50 values in PC-3 and
RWPE-1 cells and comparable with positive control abiraterone (AB) (Table 2).
In addition, morphological changes of cells were investigated using inverted light microscopy in
exposure to these novel compounds leading to apoptosis. Phase contrast microscopic images showed
the phenotypical changes with cell shrinkage, plasma membrane blebbing, round or oval sizes and finally
collapse of the cells into small fragments (apoptotic bodies) of RWPE-1 (Figure 5A) and PC-3 (Figure 6A)
cells after treatment with compounds (3{1,3}, 4{1,3}, 3{2,3} and AB) at different concentrations (0, 33,
100, 165 and 330 µM) compared to untreated cells at 48 h. Other synthesized compounds showed
either moderate or equal cytotoxic effect to RWPE-1 and PC-3 cell under the similar treatment
conditions. In this study, it was observed that most of the PC-3 cells treated with 4{1,3} died at higher
concentration (165 µM), whereas RWPE-1 cells were not cytotoxic at that concentration (Figure 6A-B
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and 5A-B respectively). Similar pattern of cytotoxic effects were found in AB treated PC-3 and RWPE-1
cells (Figure 6A-B and 5A-B respectively). After careful observation, it is presumed that the pyridine-2,3-
dicarbethoxy pharmacophore in both 4{1,3} and 3{2,3} might have played a crucial role in displaying
potential cytotoxic activities. It was interesting to note that the pharmacophore pyridine-2,3-dimethoxy
moiety in 3{1,2} and 4{1,2} did not show encouraging activities (IC50 437 µM and 197 µM) in PC-3 cells.
However, our attempt to evaluate the cytotoxic effect of 4{1,3} with another prostate cancer cell DU-
145 did not exhibit positive result under similar conditions (Supplementary Figure S1). Overall, our in-
vitro results revealed that compounds 3{1,3}, 4{1,3} and 3{2,3} exhibited most selective cytotoxic activity
against PC-3.
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Table 2: IC50 values (µM) of novel steroidal compounds in human prostate cancer cell lines.
Cells
3{1,2}
3{1,3}
4{1,2}
4{1,3}
3{2,3}
AB
RWPE-1
PC-3
375
437
218
100
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197
501
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Figure 5. Cytotoxicity study of
48 h treatment. (
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{1,3}
,
4{1,3}
,
3{2,3} and AB at different indicated concentrations using RWPE-1 cells at
A
) Representative phase contrast microscopic images showing the morphological changes of cells
and (B
) Graphical presentation indicating the percentage of cell viability (* p<0.05).
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Figure 6. Cytotoxicity study of
treatment. (
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3{2,3} and AB at different indicated concentrations using PC-3 cells at 48 h
A
) Representative phase contrast microscopic images showing the morphological changes of cells and (B)
Graphical presentation indicating the percentage of cell viability (* p<0.05).
Molecular dynamics simulation studies
Binding mode prediction
Docking with Molecular Dynamic (MD) simulation study has become the most powerful method for
structure-based drug design and reveals more reliable protein-ligand binding complexes and accurate
binding free energies.42–44 Recent studies have revealed that few caspases most importantly initiator
caspase-8 and effectors caspase-6 and -7, are critically associated with apoptosis pathways.45 Therefore,
the molecular docking experiments for the possible interaction of compounds 3{1,3}, 4{1,3}, 3{2,3} and
AB with those aforementioned caspase proteins were performed using AutoDockVina.46 Accordingly,
complexes of 12 docked structures were generated for these four compounds. The binding affinity of all
ligands upon interaction with active site residues of the target proteins and docked poses of each ligand
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were evaluated and shown in Table 3. All compounds including AB showed similar binding affinity of
>6.5 kcal/mol against all the three targets (caspase-6, caspase-7 and caspase-8) (Supplementary Table
S2).
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A close-up view of active site region of caspase-6, caspase-7 and caspase-8 docked with these ligands
are shown in Figure 7 (A–L). The hydrogen bond network between the ligand and the active site residues
were shown in green dotted lines. It was evident that all the compounds were bound at the active site
region that includes residues Arg-220, His-219, His-121, Gln-161 of caspase-6; Gly-287, Arg-286, Ala-285,
Ser-332 and Arg-341 of caspase-7 complexes and Arg-248, Lys-253, Leu-254, Cys-360, Ser-411 and Arg-
413 of caspase-8 and formed direct hydrogen bonding network. It was also observed that the 3-hydroxy
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compounds 4{1,3} and AB showed similar binding conformation mode while 3{1,3} and 3{2,3} showed
reverse binding conformation (Figure 7).
Figure 7. A close-up view of active site region of caspase-6, caspase-7 and caspase-8 docked with ligands. The 12 docking
complexes were represented as (A) AB + caspase-6, (B) {1,3} + caspase-6, (C) 4{1,3} + caspase-6, (D) 3{2,3} + caspase-6, (E) AB
+ caspase-7, (F) {1,3} + caspase-7, (G) 4{1,3} + caspase-7, (H) 3{2,3} + caspase-7, (I) AB + caspase-8, (J) {1,3} + caspase-8, (K)
4{1,3} + caspase-8 and (L) 3{2,3} + caspase-8. Direct hydrogen bonding between the ligand and the active site residues were
3
3
3
shown in green dotted lines and ligands AB,
3
{1,3}, 4{1,3} and 3{2,3} were represented in green, red, blue and cyan respectively
in all the model.
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MD simulation and binding free energy calculation
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In order to examine the overall stability of the binding mode of each compound against the target
protein, we carried out MD simulations of the docked complexes. All complexes were simulated upto 50
ns and MD trajectory produced during the simulation were analyzed to see the flexible and reliable
binding of the ligand against the target.
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The stability of each simulation frame of the protein-ligand complex was monitored by calculating
the root mean square deviation (RMSD) from the starting structure to the end of the simulation. The
RMSD of all the starting ligand-protein configurations increased initially during the equilibration phase
and stabilized during the entire time. In Figure 8, the upper panel showed the average RMSD of all the
C-α-back bond atoms of holo forms of ligand-caspase complexes and middle panel showed ligand-RMSD
of 50 ns simulation run. As it is depicted in Figure 8, the RMSD of all docked complexes were stabilized
during the entire simulation and the binding of ligand did not affect the overall protein conformation.
The protein residue with higher flexibility was identified by calculating per residue root mean square
fluctuation (RMSF) (Figure 8, G and I). In caspase-6 and caspase-7 ligand complex models there were no
significant changes in residue fluctuation, whereas RMSF of caspase-8 ligand models fluctuated
differently. It was observed that the loop region encompassing residue 248-252, 353-377 were highly
fluctuated (almost 3-4Å) in
complexes.
4{1,3}-caspase-8 as compared to 3{1,3}-caspase-8 and AB-caspase-8
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Figure 8. Root Mean Square Deviation (RMSD) and Root Mean Fluctuation (RMSF) obtained during 50 ns of simulation time.
Upper panel showed ligand bound caspase complexes simulation. Cα-back bond atoms RMSD of all the 4 models of (A) caspase-
6, (B) caspase-7 and (C) caspase-8 complexes. Ligand RMSD plots of all the 4 ligands with respect to each target complex (D)
caspase-6, (E) caspase-7 and (F) caspase-8. Root mean square fluctuation (RMSF) of amino acid residue of each target during
the entire simulation time represented as (G) caspase-6, (H) caspase-7 and (I) caspase-8 complexes. The ligands were
represented as AB, 3{1,3}, 4{1,3} and 3{2,3} in green red, blue and cyan respectively.
The number of hydrogen bond formation between protein and ligand atoms and their occupancy during
the MD simulations were computed using g_hbond tool in GROMACS (Figure 9). In most cases, the
hydrogen bonds at the starting conformation (time 0 ns) obtained from the docking study
(Supplementary Table S2) were maintained during the MD simulations although in some time frames
the ligand was not bound to the protein. Among the caspase-ligand complexes, the hydrogen bond
occupancy was higher and stable in 4{1,3}-bound models as compared to 3{1,3} and 3{2,3} (Figure 9).
Interestingly, stable hydrogen bond occupancy was observed in AB-bound caspase complexes. This
finding was also evident from the active site mapping of direct hydrogen bond forming amino acid
residues of the
and -8 showed different patterns of binding mode against the three compounds (
{2,3}), the 3D structure extracted during different MD simulation time frame (Supplementary Table S3)
4
{1,3}-bound caspase proteins (Supplementary Figure S2, S3, S4). Although caspase-6, -7
3
{1,3}, {1,3} and
4
3
showed that the compound
4{1,3} was consistently bound against the target proteins.
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Figure 9. The number of inter-molecular hydrogen bond of AB,
upper panel (A-D) represented caspase-6 models, middle panel (E-H) showed caspase-7 models while lower panel (I-L) showed
caspase-8 complexes. The ligands are represented as AB, {1,3}, {1,3} and {2,3} in green red, blue and cyan respectively.
3{1,3}, 4{1,3} and 3{2,3} in complex with caspase proteins. The
3
4
3
The binding free energy of ligand-protein complex was calculated over 2500 frames collected
during 50 ns simulation run using g_mmpbsa tool as mentioned in methods of experimental section. In
this calculation, the unbound state of the ligand and protein was ignored and only the bound state of
ligand-protein complex was used. The total ligand-protein complex energy was influenced positively or
negatively by other individual interactions; thus we calculated Van der Waal, electrostatic, polar
solvation and non-polar solvation (SASA) energy along with the binding free binding. The total energy of
each complex model was summarized in Table-3.
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Table 3. A summary of interaction energy and binding free energy of the ligand-protein complex
obtained from g_mmpbsa analysis.
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Ligand-Protein
Complex
Van der waal
Energy (kJ/mol)
Electrostatic Energy Polar solvation Non Polar SASA Energy Binding Energy
(kJ/mol)
Energy (kJ/mol)
(kJ/mol)
(kJ/mol)
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Caspase-6+AB
-30.54 ± 1.71
-2.19 ± 0.29
20.20 ± 2.29
-3.41 ± 0.22
-33.16 ± 4.15
Caspase-6+
3
4
{1,3}
{1,3}
-78.81 ± 3.59
-133.23 ± 3.62
69.77 ± 2.86
-6.41 ± 0.32k
36.80 ± 2.36
49.11 ± 1.99
-7.66 ± 0.40
-74.44 ± 4.31
-91.63 ± 5.23
Caspase-6+
-12.68 ± 0.34
Caspase-6+
3
{2,3}
-97.03 ± 2.16
-92.32 ± 2.27
-69.96 ± 3.02
-186.36 ± 1.20
-158.56 ± 2.26
-2.71 ± 0.21
-12.48 ± 0.65
76.75 ± 3.67
-10.30 ± 0.40
-2.70 ± 0.22
40.62 ± 1.35
42.60 ± 1.68
76.93 ± 5.66
74.86 ± 0.79
44.89 ± 1.00
-9.40 ± 0.19
-9.93 ± 0.25
-7.30 ± 0.33
-17.80 ± 0.08
-14.14 ± 0.22
-53.55 ± 3.63
-86.37 ± 4.20
-133.87 ± 4.37
-160.41 ± 1.82
-149.45 ± 4.74
Caspase-7+AB
Caspase-7+
Caspase-7+
Caspase-7+
3
4
3
{1,3}
{1,3}
{2,3}
Caspase-8+AB
-129.14 ± 1.45
-48.92 ± 2.40
-79.76 ± 4.69
-119.06 ± 2.09
-12.22±0.70
43.83 ± 5.05
0.74 ± 5.26
98.48 ± 1.93
142.48 ± 7.61
199.88 ± 12.61
57.02 ± 1.81
-13.76 ± 0.15
-6.56 ± 0.34
-9.21 ± 0.55
-11.62 ± 0.21
-64.20 ± 2.18
-45.90 ± 4.66
-85.18 ± 4.73
-66.57 ± 2.61
Caspase-8+
Caspase-8+
Caspase-8+
3
4
3
{1,3}
{1,3}
{2,3}
-12.60 ± 0.50
These results offer an approximate view of the differences between the binding properties of the
compounds against the target. It was found that {1,3}-bound caspase complexes showed the lowest
binding energy of -91.63 ± 5.23, -160.41 ± 4.74 and -85.18 ± 4.73 (kJ/mol) against caspase-6, -7 and -8
respectively (Table 3). Overall our docking and MD simulation study revealed that among the three
4
compounds, 4{1,3} showed stable binding and promising interaction against all caspase proteins.
Real time polymerase chain reaction
To explain the mode of cytotoxic effect of potent compounds
3{1,3}, 4{1,3} and 3{2,3} the drug
metabolism associated with versatile mitochondrial enzymes were targeted based on in-silico data. In
particular, we asked the question whether mitochondrial enzyme induced permeability transition as well
as caspases activation (initiator caspase-8, -9, -10 and effector caspase-3, -6, -7) and thereby facilitated
apoptosis of cancer cells. As it is known that programmed cell death or apoptosis is the normal
physiological process for removal of unwanted cells from the body, it was planned to check the
expression levels of caspases linked with apoptosis. Based on previous cytotoxicity studies, compounds
3
{1,3}, 4{1,3}, 3{2,3} and anti-prostate cancer drug AB were selected to determine the mechanism of
cytotoxic effect in PC-3 and RWPE-1 cells. Therefore, the transcript expression levels of apoptosis
associated caspases were examined (Caspases-1, -3, -6, -7, and -8) using their specific primers
(sequences are in Table 4) by qRT-PCR.
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Table 4. List of Primer sequences used for qRT-PCR.
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Gene Name
Forward Primer (5՜-3՜)
Reverse Primer (5՜-3՜)
8
9
GGAACGGTGAAGGTGACAG
TTTCCGCAAGGTTCGATTTTCA
AGAGGGGATCGTTGTAGAAGTC
TGGACTTGGGAGAGGACT
GGCATCTGCGCTCTACCATC
ACAGTCCAGTTCTGTACCACG
β-actin
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Caspase-1
Caspase-3
Caspase-6
Caspase-7
Caspase-8
ATGGCGAAGGCAATCACATTT
CGGTCCTCGTTTGTACCGTC
AGAGTCTGTGCCCAAATCAAC
GTGCTGGTTTCCCCGACAT
CGCCCATACCTGTCACTTTATCA
GCTGCTTCTCTCTTTGCTGAA
Interestingly, the result demonstrated that the steroidal compound
4{1,3} strongly induced pro-
apoptotic gene caspase-8 (Figure 10) which is one of the initiator caspases that is closely coupled to pro-
apoptotic signals, activated by extrinsic pathway compared to untreated control. Once caspase-8 is
activated, it cleaves and triggers caspase cascade by activating the downstream effectors caspases-6 and
-7. The result also suggested a possible link between the compounds treated induction of apoptosis and
the activation of caspase-6 and -7 through caspase-8. However, there was no significant effect observed
in case of caspase-1 and -3 activities on induction of apoptosis.
Figure 10. Schematic diagram representing the transcript expression levels of human caspases by qRT-
PCR for the indicated five genes in PC-3 cells.
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Flow cytometer analysis
To further confirm the apoptotic induction by
Propidium iodide (PI) was performed. Data obtained from untreated and treated samples of RWPE-1
and PC-3 cells have been shown in Figure 11 and 12 respectively. During the {1,3}-induced apoptosis,
the attached flat cells become round in shape and detach from the extracellular matrix. In this study,
SSC-A vs PE-A plots were done to identify any changes in scattered population of cells. PI+ve apoptotic
cells are plotted with viable cells. In untreated (control) samples, the majority of cells were viable and
4
{1,3} in PC-3 cells, flow cytometry analysis with staining
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non-apoptotic. In contrast, PC-3 cells treated with 4{1,3} and AB at 66 µM for 48 h, showed 15.79% and
18.73% of PI+ populations indicating the induction of apoptotic pathways. Overall, this flow cytometry
data reflects the changes in the light scatter properties for the treatment of compounds compared to
untreated control consistent with previous data (shown in Figure 6) that captured the morphological
changes of the cells, suggesting that 4{1,3} is potentially cytotoxic to PC-3 prostate cancer cells.
Figure 11. Flow cytometry analysis using propidium iodide (PI) staining showing the percentage of viable
cells treated with
3{1,3},
4{1,3},
3{2,3} and AB at 66µM concentration at 48 h treatment in RWPE-1 cells.
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Figure 12. Flow cytometry analysis using propidium iodide (PI) staining showing the percentage of viable
cells treated with 3{1,3}, 4{1,3}, 3{2,3} and AB at 66 µΜ concentration at 48 h treatment in PC-3 cells.
Since the cytotoxic strategies related to cell cycle progression and the cell cycle arrest most frequently
occurs at the G1/S or G2/M boundaries, we asked the question whether {1,3} induces cell cycle arrest
and apoptosis. After treatment with {1,3} for 48 h, the PC-3 cells were fixed and stained with
propidium iodide to analyze the percentage of cells in different phases of cell cycle by flow cytometry.
We found that {1,3} inhibits completion of cell cycle by halting the cells at G2/M phase as compared to
untreated vehicle control (Figure 13A-B). The cells in each phase of the cell cycle was also quantified and
presented as shown in the bar diagram (Figure 13C). Considering 100 % of cells treated with {1,3} at
4
4
4
4
66µM for 48 h, 26.62 % of cell population were observed in the G2/M phase (Figure 13C), whereas only
10.46 % of vehicle-treated control cells in the G2/M phase (Fig. 13A-C).
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C
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0
Control
Treated
G0/G1
S
G2/M
Figure. 13: Cell cycle analysis of PC-3 cells treated with 4{1,3} (66µM) for 48 h as measured by flow
cytometry. Representative histogram showing the cell cycle distribution of (A) Vehicle control and (B)
Treated PC-3 cells. (C) Bar diagram showing percentage of cells distribution in different phases of cell-
cycle.
CONCLUSIONS
A novel class of substituted androst[17,16-b]pyridines (azasteroids) was prepared in good yields from
the reaction of steroidal β-formyl enamide 1{1} with alkynes 2{1-7} in presence of copper (I) iodide and
ligand 1,10-phenanthroline under thermal condition. The hydrolysis of 3{1,1-3} under mild basic
condition afforded 4{1,1-3} in good yields. The versatility of the reaction was extended to alicyclic and
monocyclic β-formyl enamide 1{2-4} to afford monocyclic and bicyclic pyridines 3{2-4,2-3}. A mechanism
has been proposed for the intermolecular cycloaddition reaction showing prominent catalytic role of
CuI-(1,10-Phen) system. The in-vitro anticancer activity was evaluated on the synthesized novel
azasteroids and monocyclic pyridine derivatives using Sulforhodamine B (SRB) assay of prostate cancer
cell line PC-3. The biological evaluations were performed by comparing their activity with anticancer
drug abiraterone (AB) as control. It revealed lower cytotoxic activity for compounds 3{1,3}, 4{1,3} and
3{2,3} with IC50 values (100, 95.4 and 97 µM) respectively relative to AB with IC50 (91 µM). However,
compounds 3{1,3} and 3{2,3} exhibited higher toxic effect towards human normal prostate epithelial cell
RWPE-1 with IC50 values of 218 and 275 µM respectively. On the other hand, compound 4{1,3} exhibited
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low toxic effect (501 µM) comparable to AB (645 µM) showing improved windows between cytotoxic
effect of PC-3 and RPWE-1 cell lines. Also compound 4{1,3} exhibits cytotoxic effect by inhibiting
completion of cell cycle by restricting the cells at G2/M phase. The molecular docking study of 3{1,3},
4{1,3} and 3{2,3} showed similar binding affinity of >6.5 kcal/mol against the caspase proteins as
revealed by AutoDockVina. The hydrogen bond occupancy was higher and found to be more stable in
4{1,3}-bound caspase proteins. Real-time PCR demonstrated that 4{1,3} induced pro-apoptotic gene
caspase-8 and activates the downstream effector caspases-6 and -7. Flow cytometry analysis of PC-3
cells treated with 4{1,3} and AB, showed inhibition of cell cycle proliferation indicating the induction of
apoptotic pathways. Finally, our studies revealed that the novel pyridosteroid 4{1,3}, bearing pyridine-
2,3-dicarbethoxy pharmacophore, possibly facilitated initiation of caspase-8 and activates downstream
effectors caspase-6 and caspase-7 and thereby triggering apoptosis of PC-3 cancer cells.
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EXPERIMENTAL
Melting points were determined on a Büchi B-540 melting point apparatus. The 1H and 13C NMR spectra
were recorded at ambient temperature on a 500 MHz (125 MHz for 13C) NMR spectrometer. NMR
experiments are reported in δ units, parts per million (ppm), and were referenced to CDCl3 (δ 7.26 or
77.0 ppm) as the internal standard. The coupling constants J are given in Hz. The IR spectra were
recorded in a Spectrum 100 Perkin Elmer spectrometer using KBr. Column chromatography was
performed using EM Silica gel 60 (100-200 mesh). High-resolution mass spectra (HRMS) were obtained
using a Xevo XS QT of mass spectrometer, Waters ACQUITY UHPLC. Single crystal XRD data was obtained
using AXS SMART APEX-I, Bruker.
General Procedure for substituted pyridines (3).
A 25 mL Schlenk tube equipped with a stir bar was charged with β-formyl enamide 1 (0.25 mmol), alkyne
2 (0.30 mmol, 1.2 equiv.), CuI (8.5 mg, 10 mol %), 1,10-phen (18 mg, 20 mol %), K2CO3 (28.0 mg, 0.20
mmol), and dimethylformamide (2 mL). The reaction mixture was stirred under N2 at 110 °C in an oil
bath. After 10 h, the tube was cooled to room temperature and poured into ice cold water. The mixture
was extracted with EtOAc (3 × 10 mL) and washed with water. The organic layer was collected and
concentrated in vacuum. The residue was purified by column chromatography on silica gel with hexane-
EtOAc as the eluent to give the desired product 3.
Hydrolysis of substituted pyridosteroids (4).
A 50 mL Schlenk tube equipped with a stir bar was charged with substituted pyridine 3{1,1-3} (0.09
mmol), 10% aqueous K2CO3 (5 mL) and methanol (15 mL). The reaction mixture was stirred at room
temperature for 1 h, followed by removal of methanol in rotavapor under ambient temperature. The
aqueous mixture was further diluted and the pH of the solution was adjusted to neutral using dilute HCl.
Finally, the hydrolyzed product was extracted with CH2Cl2 (3 × 10 mL) and washed with water. The
organic layer was collected and concentrated in vacuum. The residue was purified by column
chromatography on silica gel with hexane-EtOAc as the eluent to give the desired product 4.
3-Acetoxy-5-en-2’-phenyl-3’carbethoxy-androst[17,16-b]pyridines (3{1,1})
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Yield: (87%) as a white solid (from ethanol); m.p. 186-189 C. IR (KBr, cm-1): 1720 (CO); HNMR (500
MHz, CDCl3) δ 7.86 (s, 1H), 7.52-7.50 (m, 2H), 7.42-7.37 (m, 3H), 5.44 (d, J = 5.1 Hz, 1H), 4.66-4.59 (m,
1H), 4.12 (q, J = 7.1 Hz, 2H), 2.84 (dd, J = 15.1, 6.3 Hz, 1H), 2.58 (ddd, J = 14.9, 12.4, 1.0 Hz, 1H), 2.37-2.32
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(m, 3H), 2.17-2.12 (m, 1H), 2.05 (s, 3H), 1.93-1.83 (m, 3H), 1.77-1.58 (m, 8H), 1.11 (s, 3H), 1.04 (s, 3H),
1.00 (t, J = 7.1 Hz, 3H). 13CNMR (125 MHz, CDCl3): δ 175.15, 170.49, 169.02, 156.95, 140.76, 140.07,
134.40, 133.57, 128.64 (2C), 128.06, 127.91 (2C), 124.88, 121.83, 73.71, 61.13, 55.87, 50.38, 45.80,
38.01, 36.77 (2C), 33.22, 31.23, 30.71, 29.94, 27.61, 21.35, 20.39, 19.24, 17.01, 13.51. HRMS (ESI) m/z
calcd for C33H40NO4 (M+ + H) 514.2957, found 514.2990.
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3-Acetoxy-5-en-2’,3’-dicarbmethoxy-androst[17,16-b]pyridine (3{1,2})
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Yield: (84%) as a white solid (from methanol); m.p. 148-150 C. IR (KBr, cm-1): 1733 (CO); HNMR (500
MHz, CDCl3) δ 7.97 (s, 1H), 5.43 (d, J = 4.8 Hz, 1H), 4.65 - 4.58 (m, 1H), 3.97 (s, 3H), 3.90 (s, 3H), 2.84 (dd,
J = 15.3, 6.4 Hz, 1H), 2.60-2.55 (m, 1H), 2.37-2.32 (m, 3H), 2.16-2.10 (m, 1H), 2.04 (s, 3H), 1.92-1.81 (m,
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4H), 1.77-1.58 (m, 7H), 1.11 (s, 3H), 1.02 (s, 3H). CNMR (125 MHz, CDCl3) δ 176.29, 170.42, 167.57,
166.08, 149.70, 140.09, 138.16, 133.39, 123.15, 121.64, 73.62, 55.68, 52.84, 52.60, 50.22, 45.84, 37.97,
36.73 (2C), 33.00, 31.11, 30.65, 30.16, 27.56, 21.31, 20.29, 19.21, 16.89. HRMS (ESI) m/z calcd for
C28H36NO6 (M+ + H) 482.2543, found 482.2542.
3-Acetoxy-5-en-2’,3’-dicarbethoxy-androst[17,16-b]pyridine (3{1,3})
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Yield: (80%) as a white solid (from ethanol); m.p. 108-110 C. IR (KBr, cm-1): 1729 (CO); HNMR (500
MHz, CDCl3) δ 7.91 (s, 1H), 5.36 (d, J = 4.5 Hz, 1H), 4.58-4.51(m, 1H), 4.39 (qd, J = 7.1, 1.0 Hz, 2H), 4.30
(qd,J = 7.1, 1.0 Hz, 2H), 2.77 (dd, J = 15.3, 6.4 Hz, 1H), 2.53-2.47 (m, 1H), 2.30 - 2.23 (m, 3H), 2.08-2.02
(m, 1H), 1.97 (s, 3H), 1.85-1.74 (m, 4H), 1.70-1.50 (m, 7H), 1.32 (td, J = 7.1, 1.0 Hz, 3H), 1.29 (td, J = 7.1,
1.1 Hz, 3H), 1.03 (s, 3H), 0.94 (s, 3H).13CNMR (125 MHz, CDCl3) δ 176.17, 170.45, 167.30, 165.56, 150.16,
140.08, 137.89, 133.47, 123.08, 121.66, 73.64, 61.89, 61.59, 55.72, 50.22, 45.84, 37.98, 36.73 (2C),
33.01, 31.11, 30.65, 30.13, 27.56, 21.32, 20.29, 19.21, 16.98, 13.99, 13.88. HRMS (ESI) m/z calcd for
C30H40NO6 (M+ + H) 510.2856, found 510.2837.
3-Acetoxy-5-en-2’-phenyl-3’-(p-tolyl)-androst[17,16-b]pyridine (3{1,4})
0
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Yield: (78%) as a light yellow solid (from methanol); m.p. 190-192 C. IR (KBr, cm-1): 1731 (CO); HNMR
(500 MHz, CDCl3) δ 7.58-7.56 (m, 3H), 7.50 (dd, J = 8.1, 1.7 Hz, 2H), 7.22-7.16 (m, 3H), 7.09 (d, J = 7.9 Hz,
2H), 5.45 (d, J = 5.0 Hz, 1H), 4.67-4.60 (m, 1H), 2.82 (dd, J = 15.1, 6.3 Hz, 1H), 2.63-2.58 (m, 1H), 2.41-
2.35 (m, 3H), 2.32 (s, 3H), 2.05 (s, 3H), 1.96-1.85 (m, 3H), 1.81-1.69 (m, 5H), 1.66-1.59 (m, 4H), 1.13 (s,
3H), 1.09 (s, 3H). 13CNMR (125 MHz, CDCl3): δ 197.86, 174.49, 170.47, 155.23, 143.86, 140.09, 139.78,
134.38, 134.13, 132.85, 132.14, 129.98 (2C), 129.24 (2C), 128.85 (2C), 128.14, 128.05 (2C), 121.86,
73.72, 55.91, 50.49, 45.80, 38.03, 36.81(2C), 33.33, 31.27, 30.75, 30.05, 27.63, 21.55, 21.34, 20.45,
19.26, 17.15. HRMS (ESI) m/z calcd for C38H42NO3 (M+ + H) 560.3165, found 560.3194.
3-Acetoxy-5-en-3’-carbmethoxy-androst[17,16-b]pyridine (3{1,5})
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Yield: (83%) as a white solid (from methanol); m.p. 159-160 C. IR (KBr, cm-1): 1727 (CO); HNMR (500
MHz, CDCl3) δ 8.96 (s, 1H), 8.08 (s, 1H), 5.44 (d, J = 4.8 Hz, 1H), 4.65-4.59 (m, 1H), 3.93 (s, 3H), 2.82 (dd, J
= 15.0, 6.4 Hz, 1H), 2.59-2.53 (m, 1H), 2.40-2.29 (m, 3H), 2.17 - 2.12 (m, 1H), 2.05 (s, 3H), 1.93 - 1.58 (m,
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11H), 1.11 (s, 3H), 1.01 (s, 3H). CNMR (125 MHz, CDCl3) δ 177.42, 170.45, 166.31, 149.05, 140.03,
136.23, 133.21, 123.64, 121.76, 73.64, 55.66, 52.12, 50.31, 45.67, 37.99, 36.75 (2C), 33.12, 31.14, 30.64,
30.08, 27.59, 21.33, 20.42, 19.22, 16.97. HRMS (ESI) m/z calcd for C26H34NO4 (M+ + H) 424.2488, found
424.2489.
3-Acetoxy-5-en-3’-carbethoxy-androst[17,16-b]pyridine (3{1,6})
0
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Yield: (79%) as a light yellow solid (from ethanol); m.p. 132-133 C. IR (KBr, cm-1): 1729 (CO); HNMR
(500 MHz, CDCl3) δ 8.89 (s, 1H), 8.01 (s, 1H), 5.36 (d, J = 5.2 Hz, 1H), 4.58-4.51 (m, 1H), 4.33 (q, J = 7.1 Hz,
2H), 2.75 (dd, J = 15.0, 6.4 Hz, 1H), 2.51-2.45 (m, 1H), 2.32-2.22 (m, 3H), 2.10-2.06 (m, 1H), 1.97 (s, 3H),
1.86-1.74 (m, 4H), 1.70-1.50 (m, 7H), 1.32 (t, J = 7.1 Hz, 3H), 1.04 (s, 3H), 0.93 (s, 3H). 13CNMR (125 MHz,
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CDCl3) δ 177.27, 170.42, 165.78, 149.01, 140.02, 136.15, 133.16, 123.95, 121.75, 73.63, 60.99, 55.67,
50.31, 45.64, 37.98, 36.74 (2C), 33.12, 31.14, 30.63, 30.06, 27.58, 21.31, 20.41, 19.21, 16.96, 14.18.
HRMS (ESI) m/z calcd for C27H36NO4 (M+ + H) 438.2644, found 438.2667.
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3-Acetoxy-5-en-3’-tert-butylcarboxy-androst[17,16-b]pyridine (3{1,7})
Yield: (76%) as a light yellow solid (from ethanol); m.p. 128-130 C. IR (KBr, cm-1): 1724 (CO); HNMR
(500 MHz, CDCl3) δ 9.23 (s, 1H), 8.03 (s, 1H), 5.44 (d, J = 4.5 Hz, 1H), 4.65-4.59 (m, 1H), 2.82 (dd, J = 15.0,
6.5 Hz, 1H), 2.55 (t, J = 14.5 Hz, 1H ), 2.37-2.29 (m, 3H), 2.17-2.10 (m, 1H), 2.05 (s, 3H), 1.93-1.63 (m,
11H), 1.59 (s, 9H), 1.11 (s, 3H), 1.00 (s, 3H). 13CNMR (125 MHz, CDCl3) δ 176.94, 170.58, 165.04, 149.08,
140.17, 136.14, 133.25, 125.50, 121.90, 81.52, 73.79, 55.88, 50.46, 45.71, 38.12, 36.88 (2C), 33.28,
31.28, 30.77, 30.21, 29.70, 28.18 (3C), 21.44, 20.55, 19.34, 17.09. HRMS (ESI) m/z calcd for C29H40NO4
(M+ + H) 466.2957, found 466.2962.
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5,6-Dicarbmethoxy-3-methyl-2-phenylpyridine (3{2,2})
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Yield: (82%) as a light yellow solid (from methanol); m.p. 118-120 C. IR (KBr, cm-1): 1749 (CO), 1728
1
(CO); HNMR (500 MHz, CDCl3) δ 8.02 (s, 1H), 7.49-7.47 (m, 2H), 7.41-7.34 (m, 3H), 3.90 (s, 3H), 3.87 (s,
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3H), 2.37 (s, 3H); CNMR (125 MHz, CDCl3) δ 166.97, 165.70, 161.09, 148.42, 139.83, 138.60, 132.73,
128.96 (2C), 128.80, 128.25 (2C), 123.71, 52.91, 52.76, 19.97. HRMS (ESI) m/z calcd for C16H16NO4 (M+ +
H) 286.1079, found 286.1063.
5,6-Dicarbethoxy-3-methyl-2-phenylpyridine (3{2,3})
0
Yield: (80%) as a light yellow solid (from methanol); m.p. 84-86 C. IR (KBr, cm-1): 1748 (CO), 1724 (CO);
1HNMR (500 MHz, CDCl3) δ 8.05 (d, J = 7 Hz, 1H), 7.49-7.47 (m, 2H), 7.41-7.34 (m, 3H), 4.39-4.31 (m, 4H),
13
2.37 (s, 3H), 1.33 (td, J = 7.2, 1.6 Hz, 6H); CNMR (125 MHz, CDCl3) δ 166.85, 165.25, 161.12, 149.17,
140.06, 138.84, 132.52, 129.14, 129.11, 128.85, 128.33 (2C), 123.63, 62.11, 61.92, 20.06, 14.14, 14.04.
HRMS (ESI) m/z calcd for C18H20NO4 (M+ + H) 314.1392, found 314.1378.
5,6-Dicarbmethoxy-2-phenylpyridine (3{3,2})
0
Yield: (78%) as a light yellow solid (from methanol); m.p. 118-120 C. IR (KBr, cm-1): 1740 (CO), 1726
(CO); 1HNMR (500 MHz, CDCl3) δ 8.31 (d, J = 8 Hz, 1H), 8.08-8.06 (m, 2H), 7.89 (d, J = 8.5 Hz, 1H), 7.52-
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7.46 (m, 3H), 4.03 (s, 3H), 3.95 (s, 3H); CNMR (125 MHz, CDCl3) δ 166.34, 164.28, 158.95, 151.07,
137.71, 136.22, 129.34, 127.91 (2C), 126.53 (2C), 121.69, 119.84, 52.02, 51.82. HRMS (ESI) m/z calcd for
C15H14NO4 (M+ + H) 272.0923, found 278.0928.
5,6-Dicarbethoxy-2-phenylpyridine (3{3,3})
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Yield: (76%) as a light yellow solid (from methanol); m.p. 80-83 C. IR (KBr, cm-1): 1735 (CO), 1723 (CO);
1HNMR (500 MHz, CDCl3) δ 8.32 (d, J = 8 Hz, 1H), 8.09-8.06 (m, 2H), 7.88 (d, J = 8.5 Hz, 1H), 7.51-7.26 (m,
3H), 4.52 (q, J = 7 Hz,2H), 4.43 (q, J = 7.5 Hz, 2H), 1.45-1.38 (m, 6H); 13CNMR (125 MHz, CDCl3) δ 166.01,
163.81, 158.80, 151.39, 137.78, 136.34, 129.25, 127.87 (2C), 126.53 (2C), 121.78, 119.68, 61.12, 60.87,
28.68. HRMS (ESI) m/z calcd for C17H18NO4 (M+ + H) 300.1236, found 300.1223.
2,3-Dicarbmethoxy-5,6,7,8-tetrahydroquinoline (3{4,2})
Yield: (78%) as a light yellow sticky liquid. IR (KBr, cm-1): 1730 (CO); 1HNMR (500 MHz, CDCl3) δ 7.86 (s,
1H), 3.98 (s, 3H), 3.91 (s, 3H), 2.99 (t, J = 6.5 Hz, 2H), 2.84 (t, J = 6.5 Hz, 2H), 1.95-1.90 (m, 2H), 1.86-1.82
(m, 2H); 13CNMR (125 MHz, CDCl3) δ 167.21, 166.02, 161.17, 148.14, 138.04, 134.33, 123.11, 53.04,
52.74, 32.60, 28.60, 22.58, 22.17. HRMS (ESI) m/z calcd for C13H16NO4 (M+ + H) 250.1079, found
250.1078.
2,3-Dicarbethoxy-5,6,7,8-tetrahydroquinoline (3{4,3})
Yield: (77%) as a light yellow sticky liquid. IR (KBr, cm-1): 1732 (CO); 1HNMR (500 MHz, CDCl3) δ 7.88 (s,
1H), 4.46 (q, J = 7 Hz, 2H), 4.38 (q, J = 7 Hz, 2H), 3.00 (t, J = 6.5 Hz, 2H), 2.84 (t, J = 6.5 Hz, 2H), 1.94-1.89
(m, 2H), 1.86-1.81 (m, 2H), 1.40 (t, J = 7 Hz, 3H), 1.37 (t, J = 7 Hz, 3H); 13CNMR (125 MHz, CDCl3) δ 166.98,
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165.44, 161.10, 148.81, 138.15, 133.99, 122.91, 62.09, 61.73, 32.59, 28.59, 22.63, 22.22, 14.10, 14.04.
HRMS (ESI) m/z calcd for C15H20NO4 (M+ + H) 278.1392, found 278.1380.
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3-Hydroxy-5-en-2’-phenyl-3’carbethoxy-androst[17,16-b]pyridines (4{1,1})
0
Yield: (80%) as a white solid (from methanol); m.p. 233-235 C. IR (KBr, cm-1): 3469 (OH), 1715 (CO),
1
3274 (OH); HNMR (500MHz, CDCl3) δ 7.85 (s,1H), 7.53-7.50 (m, 2H), 7.43-7.37 (m, 3H), 5.42 (d, J = 5.2
Hz, 1H), 4.12 (q, J = 7.2 Hz, 2H), 3.64 (s, 1H), 3.57-3.52 (m,1H), 2.84 (dd, J = 14.9, 6.4 Hz, 1H), 2.61 (dd, J =
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12.5, 1 Hz, 1H), 2.36-2.24 (m, 3H), 2.17-2.13 (m, 1H), 1.91-1.84 (m, 3H), 1.77-1.58 (m, 8H), 1.10 (s, 3H),
1.04 (s, 3H), 1.00 (t, J = 7.2 Hz, 3H). CNMR (125MHz, CDCl3) δ 175.47, 169.56, 156.98, 141.26, 140.65,
134.51, 133.70, 128.74, 128.68, 128.27, 128.07, 128.03, 124.98, 121.0, 71.67, 61.25, 56.05, 52.20, 50.58,
45.93, 42.25, 37.13, 36.78, 33.35, 31.35, 30.84, 30.06, 20.55, 19.44, 17.12, 13.62. HRMS (ESI) m/z calcd
for C31H38NO3(M+ + H) 472.2852, found 472.2837.
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3-Hydroxy-5-en-2’,3’-dicarbmethoxy-androst[17,16-b]pyridine (4{1,2})
Yield (78%) as a pale Yellow solid (from methanol); m.p. 159-160 0C. IR (KBr, cm-1): 3450 (OH), 1725 (CO),
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3275 (OH); HNMR (500MHz, CDCl3) δ 7.97 (s,1H), 5.40 (d, J = 5.3Hz, 1H), 3.98 (s, 3H), 3.92 (s, 3H), 3.90
(s, 1H), 3.58-3.51 (m,1H), 2.84 (dd, J = 15.4, 6.4 Hz, 1H), 2.61-2.55 (m, 1H), 2.36-2.24 (m, 3H), 2.16-2.10
(m, 1H), 1.91-1.82 (m, 4H), 1.70-1.57 (m, 7H), 1.09 (s, 3H), 1.02 (s, 3H).13CNMR (125MHz, CDCl3) δ
176.49, 167.72, 166.23, 149.79, 141.30, 138.33, 133.90, 123.25, 120.83, 71.64, 55.90, 53.00, 52.75,
50.43, 45.98, 42.22, 37.10, 36.76, 33.16, 31.58, 31.24, 30.80, 30.30, 20.47, 19.42, 17.02. HRMS (ESI) m/z
calcd for C26H34NO5(M+ + H) 440.2437, found 440.2439.
3-Hydoxy-5-en-2’,3’-dicarbethoxy-androst[17,16-b]pyridine (4{1,3})
0
Yield (76%) as a pale Yellow solid (from ethanol), m.p. 124-126 C. IR (KBr, cm-1): 1731 (CO), 3265 (OH);
1HNMR (500MHz, CDCl3) δ 7.98 (s,1H), 5.40 (d, J = 4.1Hz, 1H), 4.46 (qd, J = 7.2, 3.3Hz, 2H), 4.38 (qd, J =
7.2, 3.3Hz, 2H), 3.89 (s, 1H), 3.58-3.51 (m,1H), 2.84 (dd, J = 15.3, 6.4 Hz, 1H), 2.61-2.55 (m, 1H), 2.35-2.25
(m, 3H), 1.91-1.82 (m, 3H), 1.74-1.56 (m, 9H), 1.41 (td, J = 19.4 Hz, 7.2 Hz, 3H), 1.28-1.12 (m, 3H), 1.09 (s,
3H), 1.02 (s, 3H).13CNMR (125MHz, CDCl3) δ 176.52, 167.39, 166.17, 150.31, 141.29, 138.09, 133.57,
122.85, 120.85, 71.64, 62.09, 61.72, 55.91, 52.62, 50.44, 46.00, 42.21, 37.10, 36.76, 33.15, 31.58, 31.25,
30.80, 30.28, 20.47, 19.42, 17.02, 14.06. HRMS (ESI) m/z calcd for C28H38NO5(M+ + H) 468.2750, found
468.2752.
Cell lines
Prostate cancer cells, PC-3 and DU-145 were obtained from National Centre for Cell Sciences (NCCS),
Pune, India. RWPE-1 cells were purchased from American Type Culture Collection (ATCC), USA. Cells
were grown and maintained using appropriate culture medium supplemented with 10% fetal bovine
serum and 1% penicillin/streptomycin antibiotic solution. For culturing RWPE-1 cells, we used
keratinocyte SFM that was purchased from Thermo Fischer. The cells were cultured in 5% CO2 incubator
at 37 0C in a humidified atmosphere.
Cytotoxicity Assay
Cytotoxicity study of our synthesized novel steroidal compounds and standard drug abiraterone was
determined by Sulphorhodamine (SRB) assay. For this study, we used highly aggressive prostate cancer
cells, PC-3 and compared their effects in non-tumorigenic prostate epithelial cells, RWPE-1. Cells were
seeded at a density of 5000 cells/well in 96 well microtiter plates and exposed to various concentrations
(0, 33, 100, 165 and 330 µM) of these compounds dissolved in DMSO. The percentage of cell viability of
our test compounds was calculated compared to untreated control and DMSO control. The DMSO
control did not affect the cell viability in comparison to untreated control. After 48 h treatment, 25 µL
0
of cold 50% trichloroacetic acid (TCA) was added to each well and the plate was kept at 4 C for 1 hour
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and then rinsed with water several times to remove TCA solution and serum proteins. Then, 50 µL of
0.4% SRB solution was added in each well of cells for 30 min, followed by rinsing with 1% acetic acid and
air-dried until no moisture is visible and then captured the images using a light microscope. For
quantitative measurement, the incorporated dye was then solubilised in 100 µL of 10 µM tris base and
the absorbance was spectrophotometrically measured at 565 nm. The cytotoxic effect of those
compounds in cells was evaluated by measuring the half maximal inhibitory concentration (IC50) in
comparison to the untreated control as described previously.47–49
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Morphological changes detection using Light microscopy
The phenotypical changes of the cells were observed under an inverted microscope. PC-3 and RWPE-1
cells were treated with selected four compounds 3{1,3}, 4{1,3} and 3{2,3} including AB at 0, 33 and 165,
330 µM concentration and phase contrast microscopic images with 20 x magnification were captured at
48 h treatment.
Cell cycle analysis
Cell cycle analysis was performed as previously described.50 Briefly, 1 x 105 cells were seeded into 6-cm
diameter culture dishes, then treated with 4{1,3} and harvested after 48ꢀh. The cells were washed with
cold PBS followed by fixation with 70 % ethanol for 30 min at 4 0C. After careful removal of ethanol and
washing of the cells with PBS, the fixed cells were incubated with RNase A (Invitrogen) and stained with
0
50ꢀμg/mL propidium iodide (Himedia) for 30ꢀmin at 4 C and analyzed for cell cycle using CytoFLEXflow
cytometer (Beckman Coulter).
Docking studies
Molecular docking experiments of three ligands 3{1,3}, 4{1,3} and 3{2,3} were carried against human
caspase-6, caspase-7 and caspase-8 using abiraterone (AB) as a control drug. The starting atom
coordinates of the caspase-7 were taken from the Protein Data Bank PDB id: 1GQF and cleaned the
structure. In case of caspase-6 and caspase-8, instead of 3D structure available in PDB with maximum
protein sequence coverage showing more than 10 to 15 missing residues, modeled structures
downloaded from Swiss model51 were used for the study. The model structures were subjected to 1000
steps of steepest descent and 500 steps of conjugate gradient energy minimization in order to optimize
the structure. Then all target structures were provided as receptor input and carried out docking with
the four compounds (3{1,3}, 4{1,3}, 3{2,3} and AB) using AutoDockVina.46 Compared with other docking
algorithms, this software provided significant improvements and average accuracy in the binding mode
prediction. It allowed ligand flexible docking with the active site of the target. The docking parameters of
ligand and receptor (target) preparation included the addition of hydrogen atoms, merging nonpolar
hydrogen atoms, rotatable torsion bond followed by grid box construction of each target protein.
Docking study was carried out using a grid volume of 40 x 44 x 44 Å for caspase-6, 44 x 48 x 42 Å for
caspase-7 and 44 x 48 x 50 Å for caspase-8 from each of the active sites region. UCSF Chimera (version
1.11.2)52 was used for input structure preparation, energy minimization and analysis of docking results.
Hydrogen-bonding interaction network was analyzed in order to observe the direct interaction between
ligands and the target.
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Molecular Dynamic Simulation
A total of 12 complexes listed in Table 3 were simulated in GROMACS version 4.5.553,54 with explicit
solvent and under periodic boundary conditions as described by Shrivastava et al.55 Briefly,
GROMOS53a6.ff all atom force field was used to generate the topology and coordinate file of the
protein target while ligand topology file was generated through PRODRG server (version 2.5).56 Each of
the complex system was neutralized with varied number of sodium ions as per the net charges obtained
during the initial setup of the system and wrapped up in a periodic box of TIP3P water model that was
extended to 10 Å from the solvent. To treat the long-range electrostatics, Particle Mesh Ewald method
was used57 and bond lengths within the protein were constrained using LINCS algorithm.58 In order to
optimize and relax the initial solvent and ion configuration, energy minimization of 1500 steps was
performed using steepest descent method prior to the simulation. Ligand position and protein were
restrained together while the solvent and ions around were equilibrated in two-phase as temperature
coupling step (NVT) and pressure coupling step (NPT) for 100ps steps. The conformational sampling was
started after the equilibration step at a constant pressure (1 atm) and temperature (300 K). The time
steps of 2 ps were used to collect the MD simulation trajectory.
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Trajectory analysis
GROMACS MD trajectory analysis tool-kit was used to analyse trajectory generated during the last 50 ns
of production run to determine root mean square deviation (RMSD), per residue root mean square
fluctuation (RMSF); and the number of distinct hydrogen bonds formed between protein and ligand
atoms was determined by utilizing the g_hbond. Each trajectory was visualized using VMD (version) and
3D coordinate snapshots were extracted to analyse the interaction between each ligand-protein system.
UCSF Chimera version 1.11.252 was used for structure analysis and visualization. Graphs were generating
using grace version 5.1.23, MS Excel (2013) and R statistical package.
MMPBSA calculation
The binding energy of each protein-ligand complex was determined using the g_mmpbsa tool developed
by Kumari et al.59 The snapshot of 12500 frames collected every 200 ps time frame of 50 ns production
run was used to calculate the absolute free energy of the protein-ligand complex.
The set of equations below was used to determine binding free energy (ΔGbind
)
ΔGbind=Gcomplex-(Greceptor+Gligand)….......…………..………….….......(1)
ΔGbind= Egas+ Gsol– TΔS…......……………….........................…....(2)
Egas= Eint+ Evdw+ Eele.......………..............………..………………..... (3)
Gsol= Gpol+ GSA.......……………….…......................................... (4)
GSA=ꢀ SASA+ b………………………....……......................................(5)
In this study, Free energy, Polar solvation energy and Apolar solvation energy for each protein-ligand
complex were calculated. In the above equation, the protein-ligand complex, only protein and only
ligand structures stand for Gcomplex, Greceptor, and Gligand respectively. The ΔGbind was decomposed to its
individual contributions from the equation (2) to (5). In the equation (3) Gas phase energy ꢁꢂꢃꢄ shows
that the sum of ꢁꢅꢆꢇ, ꢁꢈꢉꢊ ꢋꢌꢍ ꢁꢎꢏꢎ. The free solvation energy (ꢐꢄꢑꢏ) was broken down into two
solvation energy such that, polar (ꢐꢒꢑꢏ) and nonpolar (ꢐꢓꢔ) solvation energy components, and also an
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entropy term (TΔS). Polar solvation energies were resolved by solving the Poisson-Boltzmann linear
equation and also nonpolar solvation energy through the solvent accessible surface area with a
neutralized value for “b” is 3.84928 kJ. ꢕꢖꢗ−1 and surface tension proportionality “γ” set at 0.0226778
−1
−2
̇
kJ.ꢕꢖꢗ . ꢘ .
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RNA extraction and RT-qPCR
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Total RNA from cancer cells was extracted according to manufacturer’s instructions (Himedia). The
quality and quality of RNA were measured with NanoDrop Spectrophotometer (NanoDrop Technologies
Inc., USA). A total of 1 µg RNA was used for cDNA synthesis using Hi-cDNA synthesis kit (HiMedia).
Quantitative real-time PCR was performed as previously described.60,61 Expression of caspases-1, -3, -6, -
7 and -8 were normalized to the internal control (beta actin), a housekeeping gene. Melt curve analysis
was included to assure that only one PCR product was formed. ΔΔCt values of triplicates were
normalised using endogenous control β-actin and relative expression was calculated. List of primers
used are stated in Table 4.
Flow Cytometric Assessment of Cell Viability
Flow cytometry studies were performed using Propidium iodide (PI) staining.62,63 After exposure of the
cells to 3{1,3}, 4{1,3}, 3{2,3} and AB, these were washed once with PBS and centrifuged at 300 x g for 5
minutes followed by decanting the buffer from the cells. Resuspended cells (1 x 106 cells/mL) were then
put into FACS tubes. To adjust flow cytometer settings for PI, 5 - 10 µL of PI staining solution was added
0
to the control tube, gently mixed the stain and incubated for 20 min at 4 C in the dark and then
determined the PI fluorescence with a FACScan instrument. Following this, 5 - 10 µL of PI staining
solution was added to each sample just prior to analysis of viable cells from a dot-plot of side scatter
(SSC-A) vs PE-A. Untreated PC-3 and RWPE-1 cells were used as negative control.
Conflicts of interest
There are no conflicts to declare.
ASSOCIATED CONTENT
Supporting Information: The Supporting Information is available free of charge on the ACS Publications
website at DOI:xxxxxxxxx.
Crystallographic information for compound 3{1,1}.
General experimental procedures, 1HNMR, 13CNMR and HRMS spectra of all compounds.
Phase contrast microscopic images.
Molecular dynamics simulation images.
ACKNOWLEDGEMENTS
RCB thanks CSIR, New Delhi for award of Emeritus Scientist, CSIR (ES1516Y2144). GSN thanks CSIR, New
Delhi for award of RA and financial support under Emeritus Scientist scheme, CSIR. This work is also
financially supported by SERB-Department of Science & Technology (SERB-DST), Government of India, by
providing the Ramanujan Fellowship (SB/S2/RJN-087/2014) to MP; and CSIR/UGC JRF (21/06/2015(i) EU-
V) to TM. YS and KB acknowledge Bioinformatics Infrastructure Facility (BIF) center, DBT, Govt. of India,
for providing computational facilities to carry out In-silico study. KB also acknowledge DBT, Govt. of India
for providing the fellowship under BIF, CSIR-NEIST (project code No. GAP200). The authors are thankful
to the Director, CSIR-NEIST, Jorhat for his keen interests.
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Corresponding Author
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Email: rc_boruah@yahoo.co.in
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