Synthesis of D-Ring Annulated Pyridosteroids from Î²-Formyl

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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 Chemical Science and Technology Division; CSIR-North East Institute of Science & Technology, Jorhat, Assam, India 785006; bBiological Science and Technology Division, CSIR-North East Institute of Science & Technology, Jorhat, Assam, India 785006. a

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 3hydroxy-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 PC3 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 3hydroxy-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’-dicarbethoxyandrost[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 Nacetyl 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 EtO N

HO

N

HO Abiraterone

Polysubstituted steroidal pyridine

N N

CO2Et CO2Et

HO Androst[17,16-b]pyridines (Our synthesized compound)

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 with CuI as the catalyst in DMF at 120 0C to afford 3-acetoxy-5-en-2’-phenyl-3’-carbethoxyandrost[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 Ph N

NHAc

Ph

CHO CO2Et

AcO

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

CuI CuI CuI CuI CuI CuI CuI CuI CuCl CuBr CuI CuI CuI CuI CuI

Base, Solvent

Ligand

3{1,1}

Solvent

Base

DMSO DCE THF 1,4-Dioxane DMF DMF Na2CO3 DMF Cs2CO3 DMF K2CO3 DMF K2CO3 DMF K2CO3 DMF K2CO3 1,10-Phen DMF K2CO3 1,10-Phen DMF Cs2CO3 DABCO DMF K2CO3 2,2’-Bipyridine DMF K2CO3 L-Proline DMF K2CO3

17 Pd(OAc)2

-

18

ZnBr2

19

ZnCl2

20

FeCl3

CO2Et

AcO

2{1}

1{1}

Entry Catalyst

Catalyst Ligand

Tb (oC)

Yieldc (%)

120 120 120 120 120 120 120 120 120 120 120 110 120 120 120 120

20 22 21 24 45 50 51 70 10 30 38 87 64 73 72 70

DMF

K2CO3

120

24

-

DMF

K2CO3

120

20

-

DMF

K2CO3

120

16

-

DMF

K2CO3

120

18

condition: 1{1} (0.1 mmol), 2{1} (0.15 mmol), base (0.15 mmol), catalysts (10 mol %) and ligand (20 mol %) in solvent (3 mL); bOil bath temperature; cisolated yields of 3{1,1}.

aReaction

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 0C. 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).



The structure of the synthesized steroidal pyridine 3{1,1} was elucidated by 1HNMR, 13CNMR, 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 C33H40NO4. The 500 MHz 1HNMR 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).

Ph N

CO2Et

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

CHO

CHO

NHAc

NHAc

CHO

CHO NHAc

AcO

1{1}

1{2}

1{3}

1{4}

Alkynes 2: H

H

CO2Me

CO2Et

2{5}

2{6}

CO2C(CH3)3 2{7}

Ph

CO2Me

CO2Et

Ph

H

CO2Et

CO2Me

CO2Et

2{1}

-Me(p) CO-C6H4

2{2}

2{3}

2{4}

CO2Me

Ph N

AcO

N

COOEt

CO2Me

AcO

3{1,1}, 87%

AcO

Ph

O

N

AcO

AcO

3{1,4}, 78%

3{1,7}, 76%

N

COOMe

Ph

3{3,2}, 78%

N

N

COOEt

4{1,1}, 80%

N

HO

COOEt

COOEt

COOMe

N

3{4,2}, 78%

Ph CO2Et

N

COOMe

3{3,3}, 76%

N

Ph

3{2,3}, 80%

COOEt

COOMe

COOEt

3{2,2}, 82%

COOMe

HO

3{1,6}, 79%

COOMe Ph

N

AcO

3{1,5}, 83%

O

Ph

CO2Et

O

N

AcO

N

CO2Me

p-Tolyl

CO2Et

3{1,3}, 80%

3{1,2}, 84%

N

CO2Et

N

CO2Me

N

CO2Me

HO 4{1,2}, 78%

COOEt

3{4,3}, 77% CO2Et CO2Et

4{1,3}, 76%

Figure 3. Synthesized androst[17,16-b]pyridines and non-steroidal pyridines.



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O N

O NH2OH.HCl K2PO4 CH3OH reflux, 2h

AcO

OH

NH CHO

POCl3/DMF CH2Cl2; 0-10 oC

AcO

AcO 1{1}

16-DPA-20-oxime

16-DPA

R1

N

R2 2{1-7}

R1 R2

CuI (10 Mol %) 1,10-Phen (20 Mol %) K2CO3, DMF, 110 oC R1

N

R2

10% K2CO3 CH3OH, rt AcO

HO 4{1,1-3}

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-1cyclohexene 1{4}, derived from cyclohexanone, reacted with alkynes 2{2-3} to yield 5,6,7,8tetrahydroquinoline 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. R3 R4

O

NH2OH.HCl, KH2PO4, CH3OH

R3

reflux, 2h

R4

Ac2O/AcOH N OH

Fe, Toluene

R3 R4

NHAc

R3 POCl3/DMF, 0-10 o C R CH2Cl2; 4

CHO NHAc

1{2-4} R1

CuI (10 Mol%), K2CO3, 1,10-Phen (20 mol%), DMF, 110oC.

R2 2{2,3} R2

R3 R4

N

R1

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).

N

R1

O

AcOH

N

OH

Ac N

R1

R2

R2

R2

OH

III N

=

N

R1

II

N N

N

Cu

I

N R2 Ac N H CHO

B

Ac N

Cu R1 I

N N

CHO

Ac R1 R2 N H

Cu

N N

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 dosedependent 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-2,3dicarbethoxy 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 DU145 did not exhibit positive result under similar conditions (Supplementary Figure S1). Overall, our invitro results revealed that compounds 3{1,3}, 4{1,3} and 3{2,3} exhibited most selective cytotoxic activity against PC-3. 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

375

218

189

501

275

645

PC-3

437

100

197

95.4

97

91


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A

B

Figure 5. Cytotoxicity study of 3{1,3}, 4{1,3}, 3{2,3} and AB at different indicated concentrations using RWPE-1 cells at 48 h treatment. (A) Representative phase contrast microscopic images showing the morphological changes of cells and (B) Graphical presentation indicating the percentage of cell viability (* p6.5 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 pyridine2,3-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.

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 hexaneEtOAc 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}) Yield: (87%) as a white solid (from ethanol); m.p. 186-189 0C. IR (KBr, cm-1): 1720 (CO); 1HNMR (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. 3-Acetoxy-5-en-2’,3’-dicarbmethoxy-androst[17,16-b]pyridine (3{1,2}) Yield: (84%) as a white solid (from methanol); m.p. 148-150 0C. IR (KBr, cm-1): 1733 (CO); 1HNMR (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, 4H), 1.77-1.58 (m, 7H), 1.11 (s, 3H), 1.02 (s, 3H). 13CNMR (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}) Yield: (80%) as a white solid (from ethanol); m.p. 108-110 0C. IR (KBr, cm-1): 1729 (CO); 1HNMR (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}) Yield: (78%) as a light yellow solid (from methanol); m.p. 190-192 0C. IR (KBr, cm-1): 1731 (CO); 1HNMR (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), 2.412.35 (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}) Yield: (83%) as a white solid (from methanol); m.p. 159-160 0C. IR (KBr, cm-1): 1727 (CO); 1HNMR (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, 11H), 1.11 (s, 3H), 1.01 (s, 3H). 13CNMR (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}) Yield: (79%) as a light yellow solid (from ethanol); m.p. 132-133 0C. IR (KBr, cm-1): 1729 (CO); 1HNMR (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,



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. 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 0C. IR (KBr, cm-1): 1724 (CO); 1HNMR (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. 5,6-Dicarbmethoxy-3-methyl-2-phenylpyridine (3{2,2}) Yield: (82%) as a light yellow solid (from methanol); m.p. 118-120 0C. IR (KBr, cm-1): 1749 (CO), 1728 (CO); 1HNMR (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, 3H), 2.37 (s, 3H); 13CNMR (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}) Yield: (80%) as a light yellow solid (from methanol); m.p. 84-86 0C. IR (KBr, cm-1): 1748 (CO), 1724 (CO); 1 HNMR (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), 2.37 (s, 3H), 1.33 (td, J = 7.2, 1.6 Hz, 6H); 13CNMR (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}) Yield: (78%) as a light yellow solid (from methanol); m.p. 118-120 0C. 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), 7.527.46 (m, 3H), 4.03 (s, 3H), 3.95 (s, 3H); 13CNMR (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}) Yield: (76%) as a light yellow solid (from methanol); m.p. 80-83 0C. IR (KBr, cm-1): 1735 (CO), 1723 (CO); 1 HNMR (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. 3-Hydroxy-5-en-2’-phenyl-3’carbethoxy-androst[17,16-b]pyridines (4{1,1}) Yield: (80%) as a white solid (from methanol); m.p. 233-235 0C. IR (KBr, cm-1): 3469 (OH), 1715 (CO), 3274 (OH); 1HNMR (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 = 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). 13CNMR (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. 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), 3275 (OH); 1HNMR (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}) Yield (76%) as a pale Yellow solid (from ethanol), m.p. 124-126 0C. IR (KBr, cm-1): 1731 (CO), 3265 (OH); 1 HNMR (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 of cold 50% trichloroacetic acid (TCA) was added to each well and the plate was kept at 4 0C for 1 hour



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

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 48 h. 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 50 μg/mL propidium iodide (Himedia) for 30 min at 4 0C 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.

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=𝛾𝛾 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 𝐸𝐸𝑔𝑔𝑔𝑔𝑔𝑔 shows that the sum of 𝐸𝐸𝑖𝑖𝑖𝑖𝑖𝑖, 𝐸𝐸𝑣𝑣𝑣𝑣𝑣𝑣 𝑎𝑎𝑎𝑎𝑎𝑎 𝐸𝐸𝑒𝑒𝑒𝑒𝑒𝑒 . The free solvation energy (𝐺𝐺𝑠𝑠𝑠𝑠𝑠𝑠 ) was broken down into two solvation energy such that, polar (𝐺𝐺𝑝𝑝𝑝𝑝𝑝𝑝 ) and nonpolar (𝐺𝐺𝑆𝑆𝑆𝑆 ) solvation energy components, and also an ACS Paragon Plus Environment


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. 𝑚𝑚𝑚𝑚𝑚𝑚 −1 and surface tension proportionality “γ” set at 0.0226778 kJ.𝑚𝑚𝑚𝑚𝑚𝑚 −1 . 𝐴𝐴̇−2.

RNA extraction and RT-qPCR

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 to the control tube, gently mixed the stain and incubated for 20 min at 4 0C 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) EUV) 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 Email: [email protected]

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"For Table of Contents Use Only" Synthesis of D-ring annulated pyridosteroids from β-formyl enamides and their biological evaluations Geetmani Singh Nongthombam, Kasmika Borah, Thingreila Muinao, Yumnam Silla, Mintu Pal, Hari Prasanna Dekaboruah and Romesh Chandra Boruah*


Synthesis of D-Ring Annulated Pyridosteroids from Î²-Formyl

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