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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)
UVmax (CHCl3): 362 nm; IR (KBr, υmax): 2900.47 (C-H str.), 1629.90 (C=O str.), 1529.60
(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)
UVmax (CHCl3): 346 nm; IR (KBr, υmax): 2914.54 (C-H str.), 1683.74 (C=O str.), 1560.48
(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)
UVmax (CHCl3): 275 nm; IR (KBr, υmax): 3300.00 (N-H str.), 2929.67 (C-H str.), 1606.76
(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)
UVmax (CHCl3): 306 nm; IR (KBr, υmax): 3357.84 (N-H str.), 2950.24 (C-H str.), 1600.81
(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)
UVmax (CHCl3): 305 nm; IR (KBr, υmax): 2945.81 (C-H str.), 1593.23 (C=N str.), 1506.46
(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-
penta[c]pyrazole (12)
UVmax (CHCl3): 320 nm; IR (KBr, υmax): 2947.33 (C-H str.), 1697.41 (C=N str.), 1587.47
(C=C str.), 1359.86 & 1170.83 (SO2 str.), 1084.03 (C-N str.), 981.80 (C-C str.), 827.49
cm-1 (C-H bend). ; 1H-NMR (CDCl3), δ ppm: 1.24-1.43 (m, 1H, -CH2 of cyclopentyl),
1.53-1.55 (m, 1H, -CH2 of cyclopentyl), 2.08-2.17 (m, 1H, -CH2 of cyclopentyl), 2.82-
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)
UVmax (CHCl3): 315 nm; IR (KBr, υmax): 2960.83 (C-H str.), 1599.04 (C=N str.), 1500.67
(C=C str.), 1356.00 & 1155.40 (SO2 str.), 1089.82 (C-N str.), 980.00 (C-C str.), 825.56
cm-1 (C-H bend). ; 1H-NMR (CDCl3), δ ppm: 1.21-1.35 (m, 2H, -CH2 of cyclopentyl),
2.05-2.13 (m, 2H, -CH2 of cyclopentyl), 2.85-3.18 (m, 2H, -CH2 of cyclopentyl), 3.34-
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-
penta[c]pyrazole (14)
UVmax (CHCl3): 384 nm; IR (KBr, υmax): 2964.69 (C-H str.), 1683.91 (C=N str.), 1558.16
(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)
UVmax (CHCl3): 327 nm; IR (KBr, υmax): 2935.76 (C-H str.), 1597.11 (C=N str.), 1508.38
(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
(C=C str.), 1354.07 & 1172.04 (SO2 str.), 1246.23 (C-O-C str.), 1083.32 (C-N str.),
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)
UVmax (CHCl3): 339 nm; IR (KBr, υmax): 2926.11 (C-H str.), 1624.12 (C=N str.), 1552.75
(C=C str.), 1384.94 & 1145.75 (SO2 str.), 1273.06 (C-O-C str.), 1045.45 (C-N str.),
804.34 cm-1 (C-H bend).; 1H-NMR (CDCl3), δ ppm: 1.26-1.36 (m, 1H, -CH2 of
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)
UVmax (CHCl3): 330 nm; IR (KBr, υmax): 2960.83 (C-H str.), 1595.81 (C=N str.), 1512.24
(C=C str.), 1356.50 & 1100.69 (SO2 str.), 1224.84 (C-O-C str.), 1026.16 (C-N str.),
831.35 cm-1 (C-H bend).; 1H-NMR (CDCl3), δ ppm: 1.22-1.29 (m, 1H, -CH2 of
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-
penta[c]pyrazole (19)
UVmax (CHCl3): 322 nm; IR (KBr, υmax): 2931.90 (C-H str.), 1600.97 (C=N str.), 1548.89
(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)
UVmax (CHCl3): 324 nm; IR (KBr, υmax): 2980.83 (C-H str.), 1683.91 (C=N str.), 1558.54
(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)
UVmax (CHCl3): 312 nm; IR (KBr, υmax): 2933.83 (C-H str.), 1685.84 (C=N str.), 1580.46
(C=C str.), 1387.62 (doublet of isopropyl), 1350.22 & 1186.26 (SO2 str.), 1183.47 and
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)
UVmax (CHCl3): 325 nm; IR (KBr, υmax): 2929.84 (C-H str.), 1654.07 (C=N str.), 1579.59
(C=C str.), 1383.45 (doublet of isopropyl), 1361.79 & 1176.62 (SO2 str.), 1191.36 and
1158.96 (C-H str. of isopropyl), 1095.08 (C-N str.), 988.00 (C-C str.), 770.67 cm-1 (C-H
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)
UVmax (CHCl3): 322 nm; IR (KBr, υmax): 2960.83 (C-H str.), 1689.70 (C=N str.), 1543.10
(C=C str.), 1386.67 (doublet of isopropyl), 1300.65 & 1168.90 (SO2 str.), 1182.40 and
1168.90 (C-H str. of isopropyl), 1106.97 (C-N str.), 900.98 (C-C str.), 829.42 cm-1 (C-H
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-
penta[c]pyrazole (24)
UVmax (CHCl3): 323 nm; IR (KBr, υmax): 2950.83 (C-H str.), 1606.97 (C=N str.), 1542.23
(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)
UVmax (CHCl3): 333 nm; IR (KBr, υmax): 2974.33 (C-H str.), 1597.11 (C=N str.), 1508.38
(C=C str.), 1354.07 & 1170.83 (SO2 str.), 1081.75 (C-N str.), 981.80 (C-C str.), 829.42
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-
penta[c]pyrazole (27)
UVmax (CHCl3): 334 nm; IR (KBr, υmax): 2956.97 (C-H str.), 1676.13 (C=N str.), 1543.36
(C=C str.), 1361.78 & 1162.40 (SO2 str.), 1095.82 (C-N str.), 1053.17 (C-C str.), 831.35
cm-1 (C-H bend).; 1H-NMR (CDCl3), δ ppm: 1.25-1.68 (m, 2H, -CH2 of cyclopentyl),
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)
UVmax (CHCl3): 345 nm; IR (KBr, υmax): 2983.98 (C-H str.), 1697.41 (C=N str.), 1527.67
(C=C str.), 1356.00 & 1172.76 (SO2 str.), 1091.75 (C-N str.), 956.78 (C-C str.), 875.26
cm-1 (C-H bend).; 1H-NMR (CDCl3), δ ppm: 1.22-1.36 (m, 1H, -CH2 of cyclopentyl),
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
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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.;
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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.