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1. Chemical Synthesis

1.1. Materials and methods

Melting points were determined in open glass capillary using Bells India melting

point apparatus and were uncorrected. UV spectra in chloroform were recorded with

Shimadzu UV-VIS (Version 2640) spectrophotometer, Japan. The infrared (IR) spectra

were recorded with Shimadzu 8400-FTIR spectrophotometer in KBr discs. The 1H NMR

spectra in CDCl3 were recorded on Bruker-DPX 300 NMR spectrophotometer, USA

using TMS as an internal standard. Mass spectra were measured a Shimadzu QP-2010

plus spectrophotometer. Elemental analysis was performed on Elementar (VARIO EL-III

elemental analyzer, Germany) and values were within the acceptable limits of the

calculated values (within ± 0.4%). The homogeneity of the compounds was monitored by

ascending thin layer chromatography (TLC) on silica gel-G coated aluminum plates and

visualized by iodine vapor and UV light. Benzene/Diethyl ether (10 mL: 1 drop) was the

developing solvent.

1.2. General procedure for the synthesis of benzylidene cyclopentanones (1-4)

Equimolar quantities of cyclopentanone (0.1 mol) and p- substituted

benzaldehydes (0.1 mol) were dissolved in methanol (160 mL). 10% NaOH was added

and the reaction mixture was refluxed for 4-6 h. After the completion of the reaction, the

reaction mixture was cooled, poured into crushed ice and kept overnight. The precipitated

solid was filtered, washed with water and recrystallized from a mixture of methanol-

chloroform.

The physical data of the compounds are presented below. IR (KBr, νmax, cm-1); 1H-

NMR (CDCl3, 300 MHz, δ ppm) spectra and m/z of the compounds are as follows.

(E)-2-(4-Fluorobenzylidene)cyclopentanone (1)

UVmax (CHCl3): 343 nm; IR (KBr, υmax): 2952.81 (C-H str.), 1685.67 (C=O str.), 1593.09

(C=C str.), 835.12 cm-1 (C-H bend; 1H-NMR (CDCl3), δ ppm: 3.09 (s, 6H, -CH2 of

cyclopentyl), 7.10-7.16 (m, 2H, J = 9 Hz, 2-CH and 6-CH of p-fluorophenyl), 7.26-7.27

(m, 1H, vinylic C=CH), 7.56-7.61 (m, 2H, J = 8 Hz, 3-CH and 5-CH of p-fluorophenyl).;

MS: [C3H2F]+57, [C5H6O]+82, [C6H4F]+95, [C7H5F]+108, [C9H9O]+133.

(E)-2-(4-Methoxybenzylidene)cyclopentanone (2)


(C=C str.), 1249.91 (C-O-C str.), 835.21 cm-1 (C-H bend).; 1H-NMR (CDCl3), δ ppm:

3.08 (s, 6H, -CH2 of cyclopentyl), 3.86 (s, 3H, -OCH3), 6.95-6.98 (m, 2H, J = 8.7 Hz, 2-

CH and 6-CH of p-methoxyphenyl), 7.26 (s, 1H, vinylic C=CH), 7.56-7.59 (m, 2H, J =

8.7 Hz, 3-CH and 5-CH of p-methoxyphenyl).; MS: [C6H5]+77, [C6H11]+83, [C7H7]+91,

[C7H7O]+107, [C8H9O]+121, [C9H10O]+134, [C12H13O]+173, [C11H12O2]+176.

(E)-2-(4-Isopropylbenzylidene)cyclopentanone (3)

UVmax (CHCl3): 300nm; IR (KBr, υmax): 2955.04 (C-H str.), 1691.63 (C=O str.), 1599.04

(C=C str.), 1396.51 (doublet of isopropyl), 1184.53 and 1137.81 (C-H str. of isopropyl),

835.12 cm-1 (C-H bend).; 1H-NMR (CDCl3), δ ppm: 1.25-1.28 (d, 6H, J = 9 Hz, (-CH3)2

of isopropyl), 2.87-2.98 (m, 1H, -CH of isopropyl), 3.09 (s, 6H, -CH2 of cyclopentyl),

7.24-7.31 (d, 2H, J = 8.4 Hz, 2-CH and 6-CH of p-isopropyl), 7.52-7.55 (m, 2H, J = 8.4

Hz, 3-CH and 5-CH of p-isopropyl), 7.58 (s, 1H, vinylic C=CH).; MS: [C6H5]+77,

[C7H5]+89, [C7H7] +91, [C8H8]+104, [C9H10]+118, [C14H17]+185.

(E)-2-(4-Chlorobenzylidene)cyclopentanone (4)


(C=C str.), 840.37 cm-1 (C-H bend).; 1H-NMR (CDCl3), δ ppm: 1.59 (s, 1H, -CH2 of

cyclopentyl), 2.16 (s, 1H, -CH2 of cyclopentyl), 3.08 (s, 4H, -CH2 of cyclopentyl), 7.26 (s,

1H, vinylic C = CH), 7.39-7.41 (d, 2H, J = 7.8 Hz, 2-CH and 6-CH of p-chlorophenyl),

7.49-7.52 (d, 2H, J = 8.4 Hz, 3-CH and 5-CH of p-chlorophenyl).; MS: [C3H6]+42,

[C6H5]+77, [C6H10]+82, [C5H7O]+83, [C6H9O]+97, [C6H4Cl]+111, [C7H6Cl]+125

1.3. General procedure for synthesis of 3-substituted-2,3,3a,4,5,-hexahydrocyclo-

penta[c]pyrazoles (5-8)

The solution of hydrazine hydrate (0.03 mol) and appropriate p-substituted

benzylidene cyclohexanones (0.01 mol) in methanol (200 ml) was refluxed for 2-3 h. The

reaction mixture was cooled and kept at 0C for 24 h. The precipitated product was

filtered, washed with methanol and recrystallized from a mixture of methanol-hydrazine

hydrate. The physical data of the compounds are presented in Table 1. IR (KBr, νmax, cm-

1); 1H-NMR (CDCl3, 300 MHz, δ ppm) spectra and m/z of the compounds are as follows.

3-(4-Fluorophenyl)-2, 3, 3a, 4, 5, 6-hexahydrocyclopentapyrazole (5)

UVmax (CHCl3): 228 nm; IR (KBr, υmax): 3357.84 (N-H str.), 2914.24 (C-H str.), 1688.82

(C=N str.), 1593.09 (C=C str.), 1103.21 (C-N str.), 983.63 (C-C str.), 835.12 cm-1 (C-H

bend).;1H-NMR (CDCl3), δ ppm: 1.55-1.74 (m, 1H, -CH2 of cyclopentyl), 2.10-2.26 (m,

1H, -CH2 of cyclopentyl), 2.65-2.86 (br, 1H, -NH, disappeared on D2O exchange), 2.91-

3.15 (m, 2H, -CH2 of cyclopentyl), 3.18-3.35 (m, 3H, 3a-CH and -CH2 of cyclopentyl),

4.65-4.69 (d, 1H, J = 13 Hz, 3-CH), 7.02-7.08 (m, 2H, 2-CH and 6-CH of p-fluorophenyl

), 7.36-7.48 and 8.00-8.02 (m, integrating for 2H, 3-CH and 5-CH of p-fluorophenyl).;

MS: [C3H5]+41, [C4H7N]+69, [C5H7N]+81, [C6H4F]+95, [C6H9N2]+109, [C9H11N2]+147,

[C12H12F]+175, [C12H13N2]+185.

3-(4-Methoxyphenyl)-2,3,3a,4,5,6-hexahydrocyclopentapyrazole (6)


(C=N str.), 1512.24 (C=C str.), 1247.99 (C-O-C str.), 1178.55 (C-N str.), 825.56 cm -1 (C-

H bend).; 1H-NMR (CDCl3), δ ppm:1.62-1.69 (m, 1H, -CH2 of cyclopentyl), 2.15-2.19

(m, 1H, -CH2 of cyclopentyl ), 2.75-3.47 (m, 4H, -CH2 of cyclopentyl), 2.92-2.99 (m, 1H,

3a-CH), 3.74-3.89 (m, 3H, -OCH3), 4.60-4.64 (d, 1H, J = 13 Hz, 3-CH), 6.88-6.96 (m,

2H, 2-CH and 6-CH of p-methoxyphenyl), 6.79 and 7.12 (s, integrating 1H, NH,

disappeared on D2O exchange), 7.36-7.43 (m, 1H, 3-CH of p-methoxyphenyl), 7.51-7.59

and 8.27-8.33 (m, integrating 1H, 5-CH of p-methoxyphenyl).; MS: [CH3N2]+43, [C4H5O]

+69, [C6H5]+77, [C4H7N2]+83, [C7H7O]+107, [C6H9N2]+109, [C8H9NO]+135, [C9H11N2]+147.

3-(4-Isopropylyphenyl)-2,3,3a,4,5,6-hexahydrocyclopentapyrazole (7)


(C=C str.), 1550.48 (C=N str.), 1387.71 (doublet of isopropyl), 1186.59 and 1138.76 (C-

H str. of isopropyl), 1095.48 (C-N str.), 985.35 (C-C str.), 820.08 cm-1 (C-H bend).; 1H-

NMR (CDCl3), δ ppm: 1.24-1.26 (d, 6H, J = 6.9 Hz, -(CH3)2 of isopropyl), 1.61-1.69 (m,

1H, -CH2 of cyclopentyl), 2.16-2.25 (m, 1H, -CH2 of cyclopentyl), 2.86-2.97 (m, 4H, 3a-

CH and -CH2 of cyclopentyl), 3.10-3.33 (m, 1H, -CH2 of cyclopentyl), 3.36-3.39 (m, 1H,

-CH of isopropyl), 4.63-4.67 (d, 1H, J = 13.5 Hz, 3-CH), 7.15 (br, 1H, NH, disappeared

on D2O exchange), 7.21-7.29 (m, 2H, 2-CH and 6-CH of p-isopropylphenyl), 7.34-7.42

(m, 2H, 3-CH and 5-CH of p-isopropylphenyl).; MS: [C3H7]+43, [C4H5N]+67, [C6H5]+77,

[C5H7N]+81, [C6H9N2]+109, [C9H11]+119, [C10H13N]+147.

3-(4-Chlorophenyl)-2,3,3a,4,5,6-hexahydrocyclopentapyrazole (8)

UV λmax (CHCl3): 319 nm.; IR (KBr, υmax): 3387.11 (N-H str.), 2858.60 (C-H str.),

1599.04 (C=N str.), 1500.03 (C=C str.), 1182.40 (C-N str.), 1031.95 (C-C str.), 812.06

cm-1 (C-H bend). ;1H-NMR (CDCl3), δ ppm: 1.33-1.43 (m, 1H, -CH2 of cyclopentyl),

1.86-1.97 (m, 2H, -CH2 of cyclopentyl), 2.13-2.25 (m, 1H, -CH2 of cyclopentyl), 2.77-

2.92 (m, 1H, -CH2 of cyclopentyl), 3.00-3.10 (m, 1H, 3a-CH), 3.40-3.50 (m, 1H, -CH2 of

cyclopentyl), 4.27-4.31 (d, 1H, J = 11.7 Hz, 3-CH), 6.95-7.199 (m, 2H, 2-CH & 6-CH of

p-chlorophenyl), 7.38-7.51 (m, 1H, 3-CH of p-chlorophenyl), 8.09-8.11 and 8.38-8.41

(d, integrating for 1H, J = 9 Hz, 5-CH of p-chlorophenyl), 8.64 (br, 1H, -NH,

disappeared on D2O exchange).; MS: [C3H6]+42, [CH3N2]+43, [C3H2Cl]+73, [C6H5]+77,

[C5H7N]+81, [C6H9N2]+109 [C6H4Cl]+111, [C7H6ClN]+139, [C12H12Cl]+191.

General procedure for the synthesis of 2,3-disubstituted-2,3,3a,4,5,6-

hexahydrocyclopenta[c]pyrazoles (9-28)

To the solution of 3-substituted-3,3a,4,5,6,7-hexahydro-2H-indazoles (1-3) (0.004

mol) in pyridine (20 mL) was added an equimolar quantity of appropriate sulfonyl

chlorides, and the mixture was heated on a water bath for 2-4 h. The reaction mixture was

then cooled, poured into dilute HCl and the obtained precipitate was filtered, washed with

water and recrystallized from alcohol. The physical data of the compounds are presented

below. IR (KBr, νmax, cm-1); 1H-NMR (CDCl3, 300 MHz, δ ppm) spectra and m/z of the

compounds are as follows.

3-(4-Fluorophenyl)-2-[(4-nitrophenyl)sulfonyl]-2,3,3a,4,5,6-hexahydrocyclo-

penta[c]pyrazole (9)

UVmax (CHCl3): 382 nm; IR (KBr, υmax): 2941.54 (C-H str.), 1600.97 (C=N str.),

1510.31 (C=C str.), 1454.36 (NO2 str.), 1348.29 & 1157.33 (SO2 str.), 1176.62 (C-N str.),

1095.60 (C-C str.), 839.06 cm-1 (C-H bend).; 1H-NMR (CDCl3), δ ppm: 1.29-1.39 (m, 2H,

-CH2 of cyclopentyl), 2.10-2.19 (m, 1H, -CH2 of cyclopentyl), 2.84-3.10 (m, 3H, -CH2 of

cyclopentyl and 3a-CH), 3.40-3.50 (m, 1H, -CH2 of cyclopentyl), 4.28-4.32 (d, 1H, 3-CH,

J = 11.7 Hz), 7.08-7.17 (m, 2H, J = 9 Hz, 2-CH and 6-CH of p-fluorophenyl), 7.34 (s,

1H, 3-CH of p-fluorophenyl), 7.38-7.51 (m, 2H, 5-CH of p-fluorophenyl and 2-CH of p-

nitrophenyl), 7.54-7.62 (m, 1H, 6-CH of p-nitrophenyl), 8.08-8.11 (m, 1H, J = 9 Hz, 3-

CH of p-nitrophenyl), 8.38-8.41 (m, 1H, J = 9 Hz, 5-CH of p-nitrophenyl).; MS:

[C6H5]+77, [C6H4F]+95, [C6H8N2]+108, [C6H9N2]+109, [C6H4NO2]+122, [C6H4NO4S]+186,

[C7H6FN2O2S]+201, [C12H12FN2]+203.

3-(4-Fluorophenyl)-2,3,3a,4,5,6-hexahydro-2-tosylcyclopenta[c]pyrazole (10)

UVmax (CHCl3): 317 nm; IR (KBr, υmax): 2950.81 (C-H str.), 1585.18 (C=N str.), 1508.38

(C=C str.), 1332.86 & 1105.25 (SO2 str.), 1159.26 (C-N str.), 983.73 (C-C str.), 844.65

cm-1 (C-H bend).; 1H-NMR (CDCl3), δ ppm: 1.24-1.38 (m, 1H, -CH2 of cyclopentyl),

1.96-2.21 (m, 2H, -CH2 of cyclopentyl and 3a-CH), 2.43 (s, 3H, -CH3), 2.82-3.09 (m, 3H,

-CH2 of cyclopentyl), 3.33-3.47 (m, 1H, -CH2 of cyclopentyl), 4.24-4.28 (d, 1H, J = 11.7

Hz, 3-CH), 6.95-7.10 (m, 3H, 2-CH, 3-CH and 6-CH of p-fluorophenyl), 7.26 (s, 1H, 5-

CH of p-fluorophenyl), 7.31-7.37 (d, 1H, J = 7.8 Hz, 2-CH of tolyl), 7.39-7.45 (m, 2H,

3-CH and 6-CH of tolyl), 7.76-7.79 (d, 1H, J = 8.1 Hz, 5-CH of tolyl).; MS: [C6H4F]+95,

[CH2N2O2S]+106, [C7H6FN2]+137, [C7H7O2S]+155, [C6H8N2O2S]+172, [C12H12FN2]+203,

[C12H13N2O2S]+249, [C12H12FN2O2S]+267, [C14H12FN2O2S]+291.

3-(4-Fluorophenyl)-2-(methylsulfonyl)-2,3,3a,4,5,6-hexahydrocyclopenta[c]pyrazole

(11)


(C=C str.), 1336.71 and 1192.05 (SO2 str.), 1165.04 (C-N str.), 978.01 (C-C str.), 833.28

cm-1 (C-H bend).; 1H-NMR (CDCl3), δ ppm: 1.56 (s, 2H, -CH2 of cyclopentyl), 1.70-

1.81(m, 1H, -CH2 of cyclopentyl), 2.17-2.31 (m, 1H, 3a-CH), 2.95-3.20 (m, 5H, -CH3 of

sulphonyl chloride and -CH2 of cyclopentyl), 3.44-3.54 (m, 1H, -CH2 of cyclopentyl),

4.76-4.80 (d, 1H, 3-CH, J = 11.7 Hz), 7.04-7.11 (m, 2H, 2-CH and 6-CH of p-

fluorophenyl), 7.34-7.47 (m, 2H, 3-CH and 5-CH of p-fluorophenyl); MS: [CH3O2S]+79,

[C6H4F]+95, [C6H8N2]+108, [C12H13N2]+185, [C12H12FN2]+203 [C12H12N2O2S]+248.

3-(4-Fluorophenyl)-2-[(4-chlorophenyl)sulfonyl]-2,3,3a,4,5,6-hexahydrocyclo-




cm-1 (C-H bend). ; 1H-NMR (CDCl3), δ ppm: 1.24-1.43 (m, 1H, -CH2 of cyclopentyl),


2.90 (m, 1H, -CH2 of cyclopentyl), 3.00-3.08 (m, 2H, -CH2 of cyclopentyl and 3a-CH),

3.36-3.46 (m, 1H, -CH2 of cyclopentyl), 4.24-4.28 (d, 1H, J =11.7 Hz, 3-CH), 7.05-7.11

(m, 2H, 2-CH and 6-CH of p-fluorophenyl ), 7.26-7.30 (m, 1H, 3-CH of p-fluorophenyl),

7.38-7.45 (m, 2H, 5-CH of p-fluorophenyl and 2-CH of p-chlorophenyl), 7.50-7.53 (m,

1H, 6-CH of p-chlorophenyl), 7.82-7.85 (m, 2H, 3-CH and 5-CH of p-chlorophenyl).;

MS: [CH3OS]+63, [C6H5]+77, [C6H8N2]+108, [C6H5O2S]+141, [C6H4ClO2S]+175,

[C12H13N2]+185, [C12H12FN2]+203.

3-(4-Fluorophenyl)-2-(phenylsulfonyl)-2,3,3a,4,5,6-hexahydrocyclopenta[c]-pyrazole

(13)



cm-1 (C-H bend). ; 1H-NMR (CDCl3), δ ppm: 1.21-1.35 (m, 2H, -CH2 of cyclopentyl),


3.47 (m, 1H, 3a-CH), 4.24-4.28 (d, 1H, 3-CH, J=11.7 Hz), 6.80-7.16 (m, 2H, 2-CH and

6-CH of p-fluorophenyl), 7.26-7.46 (m, 4H, 3-CH and 5-CH of p-fluorophenyl and 2-CH

and 6-CH of phenyl), 7.51-7.64 (m, 2H, 3-CH and 5-CH of phenyl), 7.89-7.91 (m, 1H, 4-

CH of phenyl).; MS: [C5H7]+67, [C6H5]+77, [C6H11]+83, [N2O2S]+92, [C6H4F]+95,

[CH2N2O2S]+106, [C6H5O2S]+141, [C6H8N2O2S]+172, [C12H12F]+175.

3-(4-Methoxyphenyl)-2-[(4-nitrophenyl)sulfonyl]-2,3,3a,4,5,6-hexahydrocyclo-



(NO2 str.), 1541.16 (C=C str.), 1362.72 & 1170.83 (SO2 str.), 1253.77 (C-O-C str.),

1030.02 (C-N str.), 835.21 cm-1 (C-H bend).; 1H-NMR (CDCl3), δ ppm: 1.25-1.43 (m,

1H,-CH2 of cyclopentyl), 2.99 (s, 1H, 3a-CH), 3.02-3.09 (m, 3H, -CH2 of cyclopentyl),

3.44-3.47 (m, 2H, -CH2 of cyclopentyl), 3.83-3.88 (m, 3H, -OCH3 ), 4.21-4.24 (d, 1H, J =

11.7 Hz, 3 -CH of cyclopentyl), 6.89-6.98 (m, 2H, 2-CH and 6-CH of p-methoxyphenyl),

7.30-7.39 (m, 2H, 3-CH and 5-CH of p-methoxyphenyl), 7.56-7.59 (m, 2H, J = 8.7 Hz,

2-CH and 6-CH of p-nitrophenyl), 8.07-8.10 (d, 1H, J = 9 Hz, 3-CH of p-nitrophenyl),

8.36-8.39 (d, 1H, J = 8.7 Hz, 5-CH of p-nitrophenyl).; MS: [C6H5]+77, [C5H7N]+81,

[C7H7O]+107, [C6H8N2]+108, [C6H4NO4S]+185, [C6H4N3O4S]+213, [C14H12N2O5S]+320.

3-(4-Methoxyphenyl)-2,3,3a,4,5,6-hexahydro-2-tosylcyclopenta[c]pyrazole (15)


(C=C str.), 1348.29 & 1169.20 (SO2 str.), 1222.23 (C-O-C str.), 1084.81 (C-N str.),

811.70 cm-1 (C-H bend).; 1H-NMR (CDCl3), δ ppm: 1.24-1.35 (m, 1H, -CH2 of

cyclopentyl), 1.60 (br, 1H, -CH2 of cyclopentyl), 2.04-2.10 (m, 1H, 3a-CH), 2.42 (s,

3H, -CH3), 2.85-3.08 (m, 3H, -CH2 of cyclopentyl), 3.38-3.48 (m, 1H, -CH2 of

cyclopentyl), 3.82-3.85 (m, 3H, -OCH3), 4.16-4.20 (d, 1H, 3-CH, J = 11.7 Hz), 6.90-6.93

(m, 2H, 2-CH and 6-CH of p-methoxyphenyl), 7.25-7.58 (m, 4H, 3-CH and 5-CH of p-

methoxyphenyl; 2-CH and 6-CH of tolyl), 7.77-7.79 (m, 2H, J = 6 Hz, 3-CH and 5-CH

of tolyl).; MS: [C5H7N]+81, [C7H7]+91, [C7H7O]+107, [C6H8N2]+108, [C6H9N2]+109,

[C7H7O2S]+155, [C12H12N2]+184, [C13H15NO]+201, [C13H15N2O]+215.

3-(4-Methoxyphenyl)-2-(methylsulfonyl)-2,3,3a,4,5,6-hexahydrocyclo-penta[c]pyrazole

(16)

UVmax (CHCl3): 335 nm; IR (KBr, υmax): 2945.40 (C-H str.), 1697.11(C=N str.), 1510.31


983.85 cm-1 (C-H bend).; 1H-NMR (CDCl3), δ ppm: 1.61 (br, 1H, -CH2 of cyclopentyl),

1.69-1.76 (m, 1H, -CH2 of cyclopentyl), 2.19-2.27 (m, 1H, 3a-CH), 2.99-3.19 (m, 4H, -

CH3 and -CH2 of cyclopentyl), 3.47-3.58 (m, 1H, -CH2 of cyclopentyl), 3.82-3.87 (m, 5H,

-OCH3 and -CH2 of cyclopentyl), 4.71-4.75 (d, 1H, 3-CH, J = 12 Hz), 6.91-6.95 (m, 2H,

2-CH and 6-CH of p-methoxyphenyl ), 7.40-7.43 (m, 2H, J = 9 Hz, 3-CH and 5-CH of p-

methoxyphenyl).; MS: [C6H5]+77, [CH3O2S]+79, [C12H13N2]+185.

3-(4-Methoxyphenyl)-2-[(4-chlorophenyl)sulfonyl]-2,3,3a,4,5,6-hexahydrocyclopenta[c]

pyrazole (17)




cyclopentyl), 1.57 (br, 1H, -CH2 of cyclopentyl), 2.07-2.13 (m, 1H, 3a-CH), 2.87-2.89 (m,

1H, -CH2 of cyclopentyl), 2.99-3.08 (m, 1H, -CH2 of cyclopentyl), 3.37-3.47 (m, 1H, -

CH2 of cyclopentyl), 3.83-3.86 (m, 4H, -OCH3 and -CH2 of cyclopentyl), 4.17-4.21 (d,

1H, J = 12 Hz, 3-CH), 6.90-6.97 (m, 2H, 2-CH and 6-CH of p-methoxyphenyl), 7.25-

7.38 (m, 4H, 3-CH and 5-CH of p-methoxyphenyl; 2-CH and 6-CH of p-chlorophenyl),

7.48-7.58 (m, 1H, J = 9 Hz, 3-CH of p-chlorophenyl), 7.82-7.85 (m, 1H, J = 9 Hz, 5-CH

of p-chlorophenyl).; MS: [C3H6]+42, [C3H4N2]+68, [C6H5]+77, [C5H7N]+81, [C7H7O]+107,

[C6H9N2]+109, [C6H4Cl]+111, [C10H11N2O]+175.

3-(4-Methoxyphenyl)-2-(phenylsulfonyl)-2,3,3a,4,5,6-hexahydrocyclopenta-[c]pyrazole

(18)




cyclopentyl), 1.59 (br, 1H, -CH2 of cyclopentyl), 2.02-2.08 (m, 1H, 3a-CH), 2.79-3.01(m,

2H, -CH2 of cyclopentyl), 3.35-3.46 (m, 1H, -CH2 of cyclopentyl), 3.82-3.87 (m, 4H, -

OCH3 of methoxy benzaldehyde and -CH2 of cyclopentyl), 4.17-4.21 (d, 1H, J = 11.7

Hz, 3-CH), 6.89-6.93 (m, 3H, 2-CH, 3-CH and 6-CH of p-methoxyphenyl), 7.26-7.40 (m,

3H, 5-CH of p-methoxyphenyl; 2-CH and 4-CH of phenyl), 7.50-7.60 (m, 2H, 3-CH and

6-CH of phenyl), 7.89-7.92 (m, 1H, 5-CH of phenyl).; MS: [C6H5]+77, [C7H7O]+107,

[C6H8N2]+108, [C6H5O2S]+141, [C8H10NO3S]+200.

3-(4-Isopropylphenyl)-2-[(4-nitrophenyl)sulfonyl]-2,3,3a,4,5,6-hexahydrocyclo-



(NO2 str.), 1529.60 (C=C str.), 1397.64 (doublet of isopropyl), 1367.58 and 1172.76 (SO2

str.), 1184.33 and 1166.97 (C-H str. of isopropyl), 1089.82 (C-N str.), 983.73 (C-C str.),

827.49 cm-1 (C-H bend).; 1H-NMR (CDCl3), δ ppm: 1.20-1.35 (s, 8H, (-CH3)2 and -CH2 of

cyclopentyl), 1.50 (m, 1H, -CH2 of cyclopentyl), 1.80-2.40 (m, 1H, 3a-CH), 2.87-2.89

(m, 3H, -CH of isopropyl; -CH2 of cyclopentyl), 3.04 (s, 1H, -CH2 of cyclopentyl), 4.42-

4.46 (d, 1H, J = 11.7 Hz, 3-CH), 7.08-7.85(m, 7H, aromatic protons of p-isopropylphenyl

and p-nitrophenyl), 8.08-8.11 and 8.42-8.45 (d, J1 = J2 = 8.4 Hz, integrating for 1H, 5-

CH of p-nitrophenyl).; MS: [C3H7]+43, [C6H5]+77, [C6H8N2]+108, [C6H9N2]+109,

[C9H11]+119, [C6H4NO4S]+186.

3-(4-Isopropylphenyl)-2,3,3a,4,5,6-hexahydro-2-tosylcyclopenta[c]pyrazole (20)


(C=C str.), 1398.44 (doublet of isopropyl), 1361.79 & 1184.33 (SO2 str.), 1182.40 and

1126.47 (C-H str. of isopropyl), 1106.97 (C-N str.), 1091.75 (C-C str.), 829.42 cm -1 (C-H

bend).; 1H-NMR (CDCl3), δ ppm: 1.23-1.27 (m, 6H, (-CH3)2), 1.53 (s, 4H, -CH2 of

cyclopentyl), 2.06-2.16 (m, 1H, 3a-CH), 2.41 (s, 3H, -CH3), ), 2.87-3.00 (m, 1H, -CH of

isopropyl), 3.35-3.46 (m, 2H, -CH2 of cyclopentyl), 4.22-4.25 (d, 1H, 3-CH, J = 11.7

Hz), 7.22-7.34 (m, 5H, aromatic of p-isopropylphenyl and 2-CH of tolyl ), 7.35-7.37 (m,

2H, 3-CH and 6-CH of tolyl), 7.77-7.80 (d, 1H, J = 9 Hz, 5-CH of tolyl).; MS:

[C3H7]+43, [C6H5]+77, [C5H7N]+81, [C6H8N2]+108, [C6H9N2]+109, [C9H11]+119, [C10H12N]

+146, [C7H7O2S]+155.

3-(4-Isopropylphenyl)-2-(methylsulfonyl)-2,3,3a,4,5,6-hexahydrocyclo-penta[c]pyrazole

(21)



1160.68 (C-H str. of isopropyl), 1055.10 (C-N str.), 985.66 (C-C str.), 821.70 cm-1 (C-H

bend).;1H-NMR (CDCl3), δ ppm: 1.24-1.27 (m, 6H, (-CH3)2), 2.16-2.29 (m, 3H, 3a-CH

and -CH2 of cyclopentyl), 2.88-2.98 (m, 1H, -CH of isopropyl), 3.05 (m, 3H, -CH3), 3.14-

3.47 (m, 2H, -CH2 of cyclopentyl), 3.51-3.57 (m, 2H, -CH2 of cyclopentyl), 4.74-4.78 (d,

1H, J = 11.7 Hz, 3-CH), 7.21-7.25 (d, 2H, J = 5.1 Hz, 2-CH and 6-CH of p-

isopropylphenyl), 7.37-7.39 (m, 2H, 3-CH and 5-CH of p-isopropylphenyl).; MS:

[C3H7]+43, [C6H5]+77, [CH3O2S]+79, [C6H8N2]+108, [C6H9N2]+109 [C9H11]+119,

[C11H15NO2S]+225, [C15H19N2]+227.

3-(4-Isopropylphenyl)-2-[(4-chlorophenyl)sulfonyl]-2,3,3a,4,5,6-hexahydrocyclo-

penta[c] pyrazole (22)




bend).; 1H-NMR (CDCl3), δ ppm: 1.24-1.35 (m, 6H, (-CH3)2 of isopropyl), 1.55 (s, 2H, -

CH2 of cyclopentyl), 2.08-2.12 (m, 1H, 3a-CH), 2.87-2.96 (m, 3H, -CH of isopropyl and -

CH2 of cyclopentyl), 3.01-3.09 (m, 1H, -CH2 of cyclopentyl), 3.42-3.49 (m, 1H, -CH2 of

cyclopentyl), 4.23-4.27 (d, 1H, 3-CH, J = 11.7 Hz), 7.22-7.25 (m, 2H, 2-CH and 6-CH

of p-isopropylphenyl), 7.34-7.36 (m, 2H, 3-CH and 5-CH of p-isopropylphenyl), 7.46-

7.49 (d, 2H, J = 9 Hz, 2-CH and 6-CH of p-chloroophenyl), 7.82-7.85 (d, 2H, J = 9 Hz,

3-CH and 5-CH of p-chlorophenyl).; MS: [C3H7]+43, [C6H5]+77, [C6H9N2]+109, [C6H4Cl]

+111, [C9H11]+119, [C12H12ClN2O2S]+283, [C15H19N2O2S]+291, [C18H16ClN2O2S]+359.

3-(4-Isopropylphenyl)-2-(phenylsulfonyl)-2,3,3a,4,5,6-hexahydrocyclo-penta[c]pyrazole

(23)




bend). ; 1H-NMR (CDCl3), δ ppm: 1.23-1.27 (t, 6H, J = 6.3 Hz and 6.0 Hz, (-CH3)2), 1.54

(s, 2H, -CH2 of cyclopentyl), 2.06-2.10 (m, 1H, 3a-CH), 2.85-3.01 (m, 4H, -CH of

isopropyl and -CH2 of cyclopentyl), 3.39-3.44 (m, 1H, -CH2 of cyclopentyl), 4.23-4.27 (d,

1H, 3-CH, J = 11.7 Hz), 7.22-7.25 (m, 3H, 2-CH and 6-CH of p-isopropylphenyl and 4-

CH of phenyl), 7.34-7.38 (m, 3H, 3-CH and 5-CH of p-isopropylphenyl; 3-CH of

phenyl), 7.48-7.59 (m, 2H, 2-CH and 6-CH of phenyl), 7.89-7.92 (d, 1H, J = 9 Hz, 5-CH

of phenyl).; MS: [C6H5]+77, [C6H8N2]+108, [C9H11]+119, [C15H19N2]+227, [C18H17N2O2S]

+325, [C19H18N2O2S]+338, [C20H21N2O2S]+353.

3-(4-Chlorophenyl)-2-[(4-nitrophenyl)sulfonyl]-2,3,3a,4,5,6-hexahydrocyclo-



(NO2 str.), 1520.60 (C=C str.), 1371.58 & 1174.76 (SO2 str.), 1087.82 (C-N str.), 965.84

(C-C str.), 825.49 cm-1 (C-H bend). ; 1H-NMR (CDCl3), δ ppm: 1.25 (s, 1H, -CH2 of

cyclopentyl), 1.95-2.29 (m, 3H, 3a-CH and -CH2 of cyclopentyl), 2.95-3.10 (m, 2H, -CH2

of cyclopentyl), 3.49 (m, 1H, -CH2 of cyclopentyl), 4.65-4.69 (m, 1H, J = 12 Hz, 3-CH),

6.37-6.64 (m, 1H, 2-CH of p-chlorophenyl), 6.97-7.60 (m, 6H, 3-CH, 5-CH, and 6-CH of

p-chlorophenyl; 2-CH, 3-CH and 6-CH of p- nitrophenyl), 7.76-7.88 (m, 1H, 5-CH of p-

nitrophenyl).; MS: [CH2N2]+42, [C5H7]+67, [C6H5]+77, [C5H7N]+81, [C6H9N2]+109,

[C6H4Cl]+111, [C6H5O2S]+141.

3-(4-Chlorophenyl)-2,3,3a,4,5,6-hexahydro-2-tosylcyclopenta[c]pyrazole (25)



cm-1 (C-H bend).; 1H-NMR (CDCl3), δ ppm: 1.28-1.31 (m, 1H, -CH2 of cyclopentyl), 1.75

(s, 2H, -CH2 of cyclopentyl), 2.09-2.21 (m, 1H, 3a-CH), 2.43 (s, 3H, -CH3 of tolyl), 2.88-

3.09 (m, 2H, -CH2 of cyclopentyl ), 3.32-3.50 (m, 1H, -CH2 of cyclopentyl), 4.23-4.27 (d,

1H, J = 12 Hz, 3-CH), 7.26-7.42 (m, 6H, aromatic of chlorophenyl; 2-CH and 6-CH of

tolyl), 7.51-7.54 (d, 1H, J = 9 Hz, 3-CH of tolyl), 7.75-7.78 (d, 1H, J = 9 Hz, 5-CH of

tolyl).; MS: [C6H5]+77, [C5H7N]+81, [CH2NO2S]+92, [C6H9N2]+109, [C6H5O2S]+141,

[C7H7O2S]+155, [C13H15N2O2S]+263, [C19H19ClN2S]+342, [C19H19ClN2OS]+358.

3-(4-Chlorophenyl)-2-(methylsulfonyl)-2,3,3a,4,5,6-hexahydrocyclopenta[c]pyrazole

(26)

UV λmax (CHCl3): 315 nm; IR (KBr, υmax): 2910.68 (C-H str.), 1602.90 (C=N str.),

1508.38 (C=C str.), 1371.22 & 1174.26 (SO2 str.), 1099.35 (C-N str.), 941.69 (C-C str.),

835.70 cm-1 (C-H bend). ; 1H-NMR (CDCl3), δ ppm: 1.69-1.95 (m, 1H, -CH2 of

cyclopentyl), 2.00-2.76 (m, 5H, 3a-CH and -CH2 of cyclopentyl), 3.00 (m, 3H, -CH3),

3.30-3.51 (m, 1H, -CH2 of cyclopentyl), 4.70-5.01 (d, 1H, J = 12 Hz, 3-CH), 6.42-7.93

(m, 3H, 2-CH, 3-CH and 6-CH of p-chlorophenyl), 8.62 (s, 1H, 5-CH of p-

chlorophenyl).; MS: [C5H7N]+81, [CH3N2O2S]+107, [C6H8N2]+108, [C6H4Cl]+111,

[C12H11ClN2]+218.

3-(4-Chlorophenyl)-2-[(4-chlorophenyl)sulfonyl]-2,3,3a,4,5,6-hexahydrocyclo-





1.93-2.15 (m, 1H, 3a-CH), 2.88-3.05 (m, 1H, -CH2 of cyclopentyl), 3.44-3.49 (m, 1H, -

CH2 of cyclopentyl), 3.75-3.84 (m, 2H, -CH2 of cyclopentyl), 4.22-4.26 (d, 1H, J = 11.7

Hz, 3-CH), 6.83-6.98 (m, 2H, 2-CH and 6-CH of p-chlorophenyl), 7.26-7.38 (d, 1H, J =

8.1 Hz, 3-CH of p-chlorophenyl), 7.56-7.63 (d, 2H, J = 8.7 Hz, 5-CH of p-chlorophenyl

and 2-CH of SO2-p-chlorophenyl), 8.07-8.10 (d, 1H, J = 8.7 Hz, 6-CH of SO2-p-

chlorophenyl), 8.17-8.20 (d, 1H, J = 8.7 Hz, 3-CH of SO2-p-chlorophenyl), 8.35-8.38 (d,

1H, J = 8.4 Hz, 5-CH of SO2-p-chlorophenyl).; MS: [C6H5]+77, [C5H7N]+81,

[C6H8N2]+108, [C6H9N2]+109, [C6H4Cl]+111, [C6H4O2S]+140.

3-(4-Chlorophenyl)-2-(phenylsulfonyl)-2,3,3a,4,5,6-hexahydrocyclopenta[c]pyrazole

(28)




1.94-2.14 (m, 2H, 3a-CH and -CH2 of cyclopentyl), 2.79-3.09 (m, 3H -CH2 of

cyclopentyl), 3.33-3.46 (m, 1H, - CH2 of cyclopentyl), 4.26-4.30 (d, 1H, J = 12 Hz, 3-

CH), 7.16-7.42 (m, 5H, aromatic of p-chlorophenyl and 4-CH of phenyl), 7.51-7.56 (m,

2H, 2-CH and 6-CH of phenyl), 7.60-7.65 (m, 1H, 3-CH of phenyl), 7.88-7.91 (d, 1H, J

= 9 Hz, 5-CH of phenyl). ; MS: [C5H7]+67, [C6H5]+77, [C5H7N]+81, [CH2NO2S]+92,

[C6H4Cl]+111, [C12H12ClN]+205, [C12H12ClN2]+219, [C12H13N2O2S]+249.

2. Evaluation of anti-cancer activity

2.1. MTS assay on cancer cell lines

An MTS assay on the cancer cell lines was performed using the CellTiter 96®

AQueous One Solution (Promega, Madison, WI) Cell Proliferation Assay kit. The

cytotoxicities of the synthesized compounds were evaluated using lung (A549) and breast

(MDA-MB-231, MDA-MB-468, T-47D, MCF-7) cancer cell lines.

Stock solutions (10 mM) of test compounds were prepared in 1% DMSO and

serial dilutions were made in culture media at 100 µM, 10 µM, 1 µM, 100 nM and 1 nM

concentrations. One 96-well assay plate was used for each cell line treated with 4

compounds. Each concentration was tested in 3 replicates and each assay plate included

2 wells containing media only and untreated cells.

In order to determine the cytotoxicity, the adherent cells were plated into 9 lanes

of a 96 well plate while the tenth lane was left as a media only control. The cell number

was pre-determined. After 24 hours, cells in 8 lanes were treated with various

concentrations of different compounds whereas the cells in one lane were untreated and

served as a control. Cell were allowed to grow for the next 72 hours at which time they

were processed for cell viability assays. 20 μl of MTS solution was added to each lane

containing 100 μl of drug/media solution and incubated at 37 °C for 1-2 hours.

Absorbance was recorded at 490 nm. and the quantity of the formazan product was found

to be directly proportional to the number of cells viable in the culture.

Data was analyzed by Python to calculate the concentration of the test compound

exhibiting 50% growth inhibition (IC50). Background (no cells, media only) absorbance

was subtracted and values from replicate wells were averaged and normalized to the

absorbance of the untreated control wells.

To calculate the IC50 values a four-parameter logistic model was used, which is

ideal for data that has an initial response plateau, a transition phase, and a final response

plateau. The IC50 was fitted in base 10 logarithmic units, logIC50, then converted to IC50.

The four-parameter logistic model is given by the equation

Absorbance=I top+Ibot−I top

1+10−HILL( logIC50 − X )

where HILL is fixed at 2.0 (a measure of the steepness of the transition region),

Itop is the absorbance obtained at very low/no drug concentration and was fixed at

1,

Ibot is fixed at 0,

X is dose concentration in logarithmic units, and absorbance is the measured

absorbance at 490 nm.

A Monte Carlo simulation was applied to determine the confidence level of the

fitted parameters. Multiple synthetic data sets were randomly sampled based on a

Gaussian distribution centered on the ideal data set generated from the best-fit

parameters. Afterward, the same regression was repeated to obtain best-fit parameters for

each synthetic data set. Finally, the standard deviation of the logIC50 parameter was

determined.

2.2. Tubulin binding assay

All fluorescence measurements were performed using a HORIBA Scientific

FluoroMax-4 (Horiba Scientific, Edison, NJ, USA) supported by FluorEssence 3.5

software. A 0.3 cm path length of quartz Cuvette was used for all the fluorescence

measurements. Spectrofluorimetric titrations for determining the tubulin binding

parameters of the drug molecules were performed using a tryptophan fluorescence

quenching assay. Briefly, the compound (11 or 16) (0-70 µM) was incubated with 2 µM

tubulin (goat brain tubulin, purified as described earlier1,2 in PEM buffer (50 mM PIPES,

pH 6.8, 3 mM MgSO4, and 1 mM EGTA), pH 6.8, for 45 min at 35 °C. The samples were

excited at 295 nm and the emission peaks at 335 nm were recorded3. An inner filter

correction was performed for each sample using the formula

Fcorrected = Fobserved × antilog [(Aex + Aem) / 2]

where Aex is the absorbance at the excitation wavelength (295 nm) and Aem is the

absorbance at the emission wavelength (335 nm). The dissociation constant (KD) was

calculated by

KD = ([free ligand] + 1) / B

where B is fractional occupancy and [free ligand] is the concentration of free drug

molecule. The fractional occupancy (B) was determined by the formula

B = F/ Fmax

where F is the change in fluorescence intensity when tubulin and its ligand are in

equilibrium and Fmax is the value of maximum fluorescence change when tubulin is

completely bound with its ligand4. The experiment was performed at least three times.

2.3. Docking studies

Two different models for αβ-tubulin were built and used for blind docking in

Molecular Operating Environment (MOE) (Chemical Computing Group, Inc., Montreal,

QC, Canada). The models are based on two PDB structures, namely 1TVK and 4O2B.

The former is a crystal structure of epothilone bound to tubulin at a resolution of 2.89

Å5. The latter is a recent 2.30 Å model of the crystal structure of tubulin bound to the

drug BAL27862, which binds at the colchicine binding site producing a curved

conformation of tubulin6. Thus, the two models covered both the straight and curved

conformations, which guaranteed not to miss any possible binding site. These two

structures were chosen because of the high resolution at which they were developed and

the fact that they do not have as many missing residues, as other models do. The two

models were loaded separately to MOE and the structure preparation module was used to

add missing atoms/residues and when possible add hydrogens and assign ionization states

at physiological conditions.

To prepare the ligands for docking, 11, 16, 17 and 10 were first optimized using

B3LYP density functional7–9 and 6-31G (d,p) basis set in Gaussian 09 (Gaussian Inc.,

Wallingford, CT, United States). We first used MOE to run a blind docking of 11 and 16

on the straight and curved tubulin models. Hence, we ran four different blind docking

runs, holding the receptor rigid in all runs and retaining 100 pose from the results. The

top ten poses of each run were then analyzed and the putative binding sites were marked

accordingly. Afterwards, the four query molecules were docked to each of the putative

binding sites. However, in these last runs the side chains of the receptor within 6 Å of the

binding pockets were allowed to move under a constraint/tether retaining 50 poses for

each molecule. Tethering was to allow for any induced fit effects to take place and

optimize the binding of the ligands to the receptor. The scores of all ligands to each

pocket were compared and the most probable binding pocket was defined accordingly. In

all docking runs, the triangle matcher algorithm was used for placement, London dG

scoring function was used for first rescoring, force field MMFF94x for refinement and

GBVI/WSA dG scoring function for second rescoring.

References

1. Yenjerla, M.; Lopus, M.; Wilson, L. Methods Cell Biol. 2010, 95, 189–206.2. Lopus, M. Mol. Cell. Biochem. 2013, 382, 93–102.3. Pannu, V.; Rida, P. C. G.; Ogden, A.; Clewley, R.; Cheng, A.; Karna, P.; Lopus, M.;

Mishra, R. C.; Zhou, J.; Aneja, R. Cell Death Dis. 2012, 3, e346.4. Manchukonda, N. K.; Naik, P. K.; Santoshi, S.; Lopus, M.; Joseph, S.; Sridhar, B.;

Kantevari, S. PloS One 2013, 8, e77970.5. Nettles, J. H.; Li, H.; Cornett, B.; Krahn, J. M.; Snyder, J. P.; Downing, K. H. Science

2004, 305, 866–869.6. Prota, A. E.; Danel, F.; Bachmann, F.; Bargsten, K.; Buey, R. M.; Pohlmann, J.; Reinelt,

S.; Lane, H.; Steinmetz, M. O. J. Mol. Biol. 2014, 426, 1848–1860.7. Becke, A. D. J. Chem. Phys. 1993, 98, 5648–5652.8. Lee, C.; Yang, W.; Parr, R. Phys. Rev. B Condens. Matter 1988, 37, 785–789.9. Vosko, S. H.; Wilk, L.; Nusair, M. Can. J. Phys. 1980, 58, 1200–1211.

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