Synthesis of 1,2,3-triazole and isoxazole-linked pyrazole hybrids and their cytotoxic activity

Article abstract of DOI:10.1007/s00044-017-1884-z

Abstract: A series of novel 1,2,3-triazole and isoxazole-linked pyrazole derivatives have been synthesized from pyrazole and triazole/isoxazole scaffolds. The synthesized congeners were evaluated for their anti-proliferative efficacy in four cancer cell lines (MCF7, HEPG2, IMR32, and HELA). Majority of the compounds demonstrated potent cytotoxic activities, among them 4k and 4l showed significant cytotoxicity in all the tested cell lines. Graphical Abstract: [InlineMediaObject not available: see fulltext.].

Full text of DOI:10.1007/s00044-017-1884-z

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-1884-z
ORIGINAL RESEARCH
Synthesis of 1,2,3-triazole and isoxazole-linked pyrazole hybrids
and their cytotoxic activity
B. Madhavilatha1 Narjis Fatima1 Gowravaram Sabitha1 B. V. Subba Reddy1
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J. S. Yadav2 Debanjan Bhattacharjee3 Nishant Jain3
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Received: 28 October 2016 / Accepted: 22 March 2017
© Springer Science+Business Media New York 2017
Graphical Abstract
Abstract A series of novel 1,2,3-triazole and isoxazole-
linked pyrazole derivatives have been synthesized from
pyrazole and triazole/isoxazole scaffolds. The synthesized
congeners were evaluated for their anti-proliferative effi-
cacy in four cancer cell lines (MCF7, HEPG2, IMR32, and
HELA). Majority of the compounds demonstrated potent
cytotoxic activities, among them 4k and 4l showed sig-
nificant cytotoxicity in all the tested cell lines.
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Keywords Azides Alkynes 1,2,3-triazoles Isoxazoles
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Click chemistry Cytotoxicity
Electronic supplementary material The online version of this article
(doi:10.1007/s00044-017-1884-z) contains supplementary material,
which is available to authorized users.
Introduction
* Gowravaram Sabitha
gowravaramsr@yahoo.com
sabitha@iict.res.in
Pyrazole derivatives are an important class of heterocyclic
compounds that are known to possess interesting biological
and pharmacological activities (Liu et al. 2013; Madsen
et al. 2000; Zhang et al. 2000; Selwood et al. 2001) such as
antitumor (Farag et al. 2008; Insuasty et al. 2010; El-Deeb
and Lee 2010;Raffa et al. 2004; Rostom 2010), antiviral
(Rashad et al. 2008; Genin et al. 2000), and anti-
inflammatory (Ragavan et al. 2010). Also pyrazole deriva-
tives have a long history of application in the agrochemical
* B. V. Subba Reddy
basireddy@iict.res.in
1
Natural Products Chemistry Division, CSIR-Indian Institute of
Chemical Technology, Tarnaka, Hyderabad 500007, India
2
Center for Semiochemicals, CSIR-Indian Institute of Chemical
Technology, Tarnaka, Hyderabad 500007, India
3
Centre for Chemical biology, CSIR-Indian Institute of Chemical
Technology, Tarnaka, Hyderabad 500007, India

Med Chem Res
Fig. 1 Pyrazofurin and 1-(4-
Chlorophenyl)-4-hydroxy-3-
substituted-1H-pyrazoles
industry as herbicides and insecticides (Miller and
Molyneaux 1999; Gaede and Mcdermott 1993). 4-
Hydroxypyrazole derivatives pyrazomycin (pyrazofurin) is
a highly potent broad-spectrum antiviral drug and also has
been used clinically as an anticancer agent. 1-(4-Chlor-
ophenyl)-4-hydroxy-3-substituted-1H-pyrazoles (Fig. 1)
were reported by the U.S. National Cancer Institute (NCI) to
have pronounced anticancer activity.
Furthermore, 1,2,3-triazole and isoxazole motifs have
gained considerable interest in recent years due to their
occurrence in diverse natural products and notable biolo-
gical activity. It is reported that, 1,2,3-triazoles can act as
anticancer agents (Rostom et al. 2003; Al-Saadi et al. 2005;
Wan et al. 2006), antibacterials (Ferreira et al. 2010), HIV-1
protease inhibitors (Whiting et al. 2006), and anti-
histaminics (Buckle et al. 1986). While, isoxazoles are core
components of many natural products such as ibotenic acid
(Sperry and Wright 2005), muscimol (Shin et al. 2005) and
drugs like valdecoxib (Rozman et al. 2002), leflunomide
(Lawrence et al. 2005). The significant biological activities
observed for pyrazoles and 1,2,3-triazoles/isoxazoles sug-
gested potential bioactivity and pharmaceutical activity if
these two scaffolds could be combined in one molecule.
This has led us to report a series of novel 1,2,3-triazole and
isoxazole-linked pyrazole hybrids and study their anticancer
activities.
~250 °C). Organic solutions were concentrated on a rotary
evaporator at 40–45 °C. Infrared (IR) spectra were recorded
on a Perkin–Elmer 683 spectrometer. 1H NMR (nuclear
magnetic resonance) and 13C NMR spectra were recorded in
CDCl3 on Varian Gemini 200, Bruker AV-300, and Varian
Innova 500 NMR spectrometers. Chemical shifts are repor-
ted in parts per million (ppm) with respect to internal TMS.
Coupling constants (J) are quoted in Hertz (Hz). The mul-
tiplicities: s, br s, d, dd, ddd, dt, t, q, and m refer to singlet,
broad singlet, doublet, doublet of doublets, doublet of
doublet of doublets, doublet of triplets, triplet, quartet, and
multiplet, respectively. Mass spectra (ESI and EI) were
recorded on a Micromass VG-7070H mass spectrometer.
High-resolution mass spectra (HRMS) were obtained using
either a MAT1020B TOF or a double focusing spectrometer.
Typical procedure for the synthesis of 1-aryl/1,3-diaryl-1H-
pyrazol-4-ols (2a–d)
Step 1 To a solution of 1,3-diphenylpyrazole-4-carbox-
aldehyde (1b, 10 g, 0.04 mol) in chloroform (100 mL) was
added mCPBA (13.91 g, 0.08 mol) and the reaction mixture
was heated to 60–70 °C for 2 h. After completion of the
reaction as followed by TLC, the reaction mixture was cooled
to room temperature (RT) and washed with 10% Na2CO3
solution (2 × 50 mL) (to remove excess mCPBA). The
organic layer was separated, dried over anhydrous sodium
sulphate and concentrated in vacuo to give 9.5 g of residue.
Experimental
General
Step 2 To the above residue was added water (50 mL),
MeOH (50 mL) and conc HCl (90 mL). The mixture was
heated on a water bath for 1 h. After cooling to RT, cold
water was added, filtered the solid and recrystalized from
methanol to give a solid 2b (7.5 g).2a: orange solid, 2b:
pale yellow solid, 2c: white crystals, 2d: white solid.
Melting points were recorded on electro thermal melting
point apparatus and are uncorrected. All the solvents and
reagents were purified by standard techniques. Reactions
were performed in oven-dried round-bottomed flasks. Crude
products were purified by column chromatography on Acme
Chemical Co (India) silica gel (60–120 mesh). Thin-layer
chromatography was performed using Merck 60 F-254
plates; the samples were made visual by exposure to ultra-
violet light and/or by exposure to a methanolic acidic solu-
General procedure for the synthesis of 1-aryl/1,3-diaryl-4-
(prop-2-ynyloxy)-1H-pyrazoles (3a–d)
To a suspension of NaH (3.46 mmol, 2 eq) in anhydrous
Tetrahydrofuran (THF) (5 mL) was added a solution of 1-
aryl/1,3-diaryl-1H-pyrazol-4-ols (2a–d)(1.73 mmol, 1 eq) in
tion of p-anisaldehyde (developed on
a
hot-plate,

Med Chem Res
anhydrous Dimethyl formamide (DMF) (5 mL) in dropwise
manner at 0 °C. After 30 min, propargyl bromide (2.76
mmol, 1.6 eq) was added at 0 °C and allowed to stir for 30
min at room temperature. After completion, followed by
TLC, the mixture was quenched with ice H2O (10 mL) and
extracted with EtOAc (2 × 50 mL). The combined organic
layer was dried over anhydrous Na2SO4, concentrated, and
purified by silica gel column chromatography (hexane:ethyl
acetate, 9:1) to afford the pure O-propargylated compounds
3a–d.
HRMS (ESI) for C19H16FN2O [M+H]+ found 307.1249,
calcd 307.1246.
3-(4-Chlorophenyl)-1-(4-fluorophenyl)-4-(prop-2-yn-1-
yloxy)-1H-pyrazole (3d)
Brown solid; Yield 92%; mp: 99 °C; IR (KBr): 2920, 1511,
1
1213, 830 cm−1; HNMR (400 MHz, CDCl3):δ 8.02–7.98
(m, 2H, Ar–H), 7.74 (s, 1H, pyrazol-H), 7.70–7.65 (m, 2H,
Ar–H), 7.40–7.37 (m, 2H, Ar–H), 7.18–7.12 (m, 2H,
Ar–H), 4.71 (d, J = 2.2 Hz, 2H, Ar–H), 2.59 (t, J = 2.2 Hz,
1H, ≡CH), 13C NMR (75 MHz, CDCl3): δ 143.1 (C4),
130.4 (C3), 113.1 (C5), 77.9 (C7), 76.4 (C8), 59.6 (C6),
161.5 (d, JC-F = 245.2 Hz), 139.9, 136.5 (d, JC-F = 2.2 Hz),
133.4, 128.5, 128.5, 127.6, 127.6, 120.0 (d, JC-F = 8.1 Hz,
2C), 119.9, 116.3 (d, JC-F = 23.5 Hz, 2C),(Aromatic);
HRMS (ESI) for C18H13ClFN2O [M+H]+ found 339.0700,
calcd 339.0698.
4-(Prop-2-yn-1-yloxy)-1-(p-tolyl)-1H-pyrazole (3a)
Brown solid; Yield 98%; mp 58 °C; IR (KBr): 2921, 1578,
1519, 1407, 1038, 817, 653, 501 cm−1 1H NMR (500
;
MHz, CDCl3): δ 7.67 (d, J = 0.7 Hz, 1H, pyrazol-H),
7.53–7.49 (m, 3H, pyrazol-H and Ar–H), 7.24–7.21 (m, 2H,
Ar–H), 4.61 (d, J = 2.4 Hz, 2H, O-CH2), 2.57 (t, J = 2.2
Hz, 1H, ≡CH), 2.37 (s, 3H, Ar–CH3); 13C NMR (100 MHz,
CDCl3): δ 145.6 (C4), 129.9 (C3), 112.9 (C5), 78.3 (C7),
75.9 (C8), 59.4 (C6), 20.8 (C15), 138.0, 135.8, 129.8,
129.8, 118.4, 118.4 (aromatic); HRMS (ESI) for,
C13H13N2O [M+H]+ found 213.1030, calcd 213.1027.
General procedure for the synthesis of 1-benzyl-4-((1-aryl/
1,3-diaryl-1H-pyrazol-4-yloxy)methyl)-1H-1,2,3-triazoles
(4a–m)
To a mixture of (Azidomethyl)-benzenes (0.2 mmol), pro-
pargyl compounds (3a–d) (0.18 mmol) in tBuOH:H2O (1:1,
8 mL), CuSO4.5H2O (0.036 mmol) and sodium ascorbate
(0.17 mmol) were added at room temperature. The mixture
was stirred for 2 h at the same temperature. The mixture was
poured into ice-water (5 mL) with stirring. The solution was
extracted with EtOAc (3 × 20 mL), and washed with brine,
dried over MgSO4 and concentrated in vacuo. The crude
product was purified by column chromatography (pet.ether:
ethyl acetate, 5:5) to afford the pyrazolo-triazole hybrid
compounds (4a–m) (Table 1).
1,3-Diphenyl-4-(prop-2-yn-1-yloxy)-1H-pyrazole (3b)
Brown solid; Yield 96%; mp: 59 °C; IR (KBr): 2924, 2854,
1594, 1501, 1456, 1239, 753, 690 cm−1 1H NMR(500
;
MHz, CDCl3): δ 8.09–8.05 (m, 2H, Ar–H), 7.77 (s, 1H,
pyrazol-H), 7.73–7.69 (m, 2H, Ar–H), 7.46–7.38 (m, 5H,
Ar–H), 7.34–7.28 (m, 1H, Ar–H), 4.67 (d, J = 2.4 Hz, 2H,
Ar–CH2), 2.56 (t, J = 2.3 Hz, 1H, ≡CH); 13C NMR (75
MHz, CDCl3): δ 143.7 (C4), 127.6 (C3), 113.0 (C5), 78.2
(C7), 76.1 (C8), 59.5 (C6), 140.9, 140.2, 132.0, 129.3,
129.3, 128.4, 128.4, 126.5, 126.5, 125.8, 118.1, 118.1
(Aromatic); HRMS (ESI) for C18H15N2O [M+H]+ found
275.1189, calcd 275.1184.
Spectral data
1-(4-Fluorophenyl)-4-(prop-2-yn-1-yloxy)-3-(p-tolyl)-1H-
1-(2-Chlorobenzyl)-4-(((1-(p-tolyl)-1H-pyrazol-4-yl)oxy)
pyrazole (3c)
methyl)-1H-1,2,3-triazole (4a)
Brown solid; Yield 95%; mp: 95 °C; IR (KBr): 2910, 1511,
Brown solid; Yield 82%; mp: 100 °C; IR (KBr): 2921,
1
1402, 1213, 1063, 830, 581 cm−1
;
1H NMR (300 MHz,
1509, 1240, 1024, 750, 683 cm−1; H NMR (500 MHz,
CDCl3): δ 7.94–7.91 (m, 2H, Ar–H), 7.61 (s, 1H, Pyrazol-
H), 7.66–7.61 (m, 2H, Ar–H), 7.22 (d, J = 7.7 Hz, 2H,
Ar–H), 7.13–7.08 (m, 2H, Ar–H), 4.65 (d, J = 2.4 Hz, 2H,
Ar–CH2), 2.55 (t, J = 2.2 Hz, 1H, ≡CH), 2.37 (s, 3H,
Ar–CH3); 13C NMR (100 MHz, CDCl3): δ 143.5 (C4),
128.9 (C3), 113.0 (C5), 78.2 (C7), 76.1 (C8), 59.5 (C6),
21.2 (C21), 160.6 (d, JC-F = 245.2 Hz), 141.1, 137.5, 136.6
(d, JC-F = 1.8 Hz), 129.1, 129.1, 126.3, 126.3, 119.8 (d, JC-
F = 8.2 Hz, 2C), 116.0 (d, JC-F = 22.7 Hz, 2C), (Aromatic);
CDCl3): δ 7.66–7.62 (m, 2H, C5’ and C3’-H), 7.51–7.41
(m, 4H, C5-H and Ar–H), 7.35–7.19 (m, 5H, Ar–H), 5.68
(s, 2H, N–CH2),5.14 (s, 2H, O–CH2), 2.37 (s, 3H,
Ph–CH3); 13C NMR (CDCl3, 75 MHz): δ 146.2 (C4), 144.0
(C4’), 129.9 (C3’), 127.6 (C5’), 112.4 (C5), 65.4 (C13),
51.5 (C6), 20.8 (C14), 138.0, 135.8, 133.5, 132.1, 130.4,
130.3, 129.8 (2C), 129.7, 123.0, 118.3 (2C), (Aromatic);
HRMS (ESI) for C20H19ClN5O [M+H]+ found 380.1278,
calcd 379.1280.

Med Chem Res
Table 1 Pyrazolo-triazole hybrid compounds (4a–m)
1-(2-Bromobenzyl)-4-(((1,3-diphenyl-1H-pyrazol-4-yl)oxy)
methyl)-1H-1,2,3-triazole (4d)
Compounds
R
R1
R2
Mp (°C) Yield (%)
4a
4b
4c
4d
4e
4f
H
p-CH3 o-Cl
p-CH3 o-Br
p-CH3 p-OMe
100
118
82
85
75
90
89
76
89
90
85
80
85
95
92
Brown solid; Yield 90%; mp: 131 °C; IR (KBr): 3110,
1
2900, 1584, 1480, 1235, 1045, 750, 680 cm−1; H NMR
H
H
102
(500 MHz, CDCl3): δ 8.06–8.03 (m, 2H, Ar–H), 7.79 (s,
1H, C5’-H), 7.70 (dd, J = 8.6, 1.0 Hz, 2H, Ar–H), 7.66(s,
1H, C5-H), 7.61 (dd, J = 7.7, 1.2 Hz, 1H, Ar–H), 7.46–7.37
(m, 4H, Ar–H), 7.33–7.20 (m, 4H, Ar–H), 7.16. (dd J = 7.4,
1.6 Hz, 1H, Ar–H), 5.66 (s, 2H, N–CH2), 5.23 (s, 2H,
O–CH2); 13C NMR (CDCl3, 75 MHz) : δ 144.2 (C4), 144.1
(C4’), 128.2 (C3’), 125.8 (C5’), 112.8 (C5), 65.7 (C13), 53.9
(C6), 140.7, 140.2, 133.8, 133.2, 132.1, 130.5, 130.4, 129.3
(2C), 128.3 (2C), 127.5, 126.4 (2C), 123.5, 123.2, 118.1
(2C), (Aromatic); HRMS (ESI) for C25H21BrN5O [M+H]+
found 426.1731, calcd 426.1733.
Ph
Ph
Ph
H
H
H
o-Br
131
o-F
119
p-NO2
o-Cl
110
4g
4h
4i
p-CH3C6H4- p-F
p-CH3C6H4- p-F
p-CH3C6H4- p-F
p-CH3C6H4- p-F
p-CH3C6H4- p-F
p-CH3C6H4- p-F
p-CH3C6H4- p-F
173
o-Br
189
p-OMe
p-NO2
H
161
4j
162
4k
4l
Liquid
172
m-Cl
p-NO2
4m
210
*Yields are given after purification by silica gel column chromatogr-
phy and their purity is over 95% based on 1H NMR and 13C NMR
spectra
4-(((1,3-Diphenyl-1H-pyrazol-4-yl)oxy)methyl)-1-(2-
fluorobenzyl)-1H-1,2,3-triazole (4e)
1-(2-Bromobenzyl)-4-(((1-(p-tolyl)-1H-pyrazol-4-yl)oxy)
methyl)-1H-1,2,3-triazole (4b)
Brown solid; Yield 89%; mp: 119 °C; IR (KBr): 2926,
1
1593, 1499, 1238, 1055, 756, 690 cm−1; H NMR (500
MHz, CDCl3): δ 8.06–8.03 (m, 2H, Ar–H), 7.79 (s, 1H,
C5’-H), 7.71–7.67 (m, 2H, Ar–H), 7.63 (s, 1H, C5-H),
7.45–7.22 (m, 8H, Ar–H), 7.15–7.07(m, 2H, Ar–H), 5.57
(s, 2H, N–CH2), 5.23 (s, 2H, O–CH2); 13C NMR (CDCl3,
75 MHz); δ 144.2 (C4), 144.1 (C4’), 127.5 (C3’), 125.7
(C5’), 112.8 (C5), 65.6 (C13), 47.8 (d, JC-F = 4.4 Hz, C6),
160.5 (d, JC-F = 247.8 Hz), 140.6, 140.2, 132.1, 130.9 (d,
JC-F = 8.1 Hz), 130.5 (d, JC-F = 2.9 Hz), 129.2 (2C), 128.3
(2C), 126.4 (2C), 124.8 (d, JC-F = 2.9 Hz), 123.1, 121.6 (d,
JC-F = 14.7 Hz), 118.1 (2C), 115.8 (d, JC-F = 21.3 Hz)
(Aromatic); HRMS (ESI) for [M+H]+ C25H21FN5O found
448.1549, calcd 448.1550.
Pale brown solid; Yield 85%; mp: 108 °C; IR (KBr): 2918,
1
1595, 1503, 1242, 1049, 751, 687 cm−1; H NMR (500
MHz, CDCl3): δ 7.68–7.59 (m, 3H, C5’ and C3’-H and C5-
H), 7.51–7.45 (m, 3H, Ar–H), 7.33–7.15 (m, 5H, Ar–H),
5.67 (s, 2H, N–CH2), 5.14 (s, 2H, O–CH2), 2.36 (s, 3 H,
Ph–CH3); 13C NMR (CDCl3, 75 MHz): δ 146.2 (C4), 143.9
(C4’), 129.6 (C3’), 128.2 (C5’), 112.4 (C5), 65.4 (C13), 53.8
(C6), 20.8 (C14), 138.0, 135.8, 133.8, 133.2, 130.5, 130.4,
129.8 (2C), 123.4, 123.1, 118.3 (2C), (Aromatic); HRMS
(ESI) for C20H19BrN5O [M+H]+ found 424.0772, calcd
424.0770.
1-(4-Methoxybenzyl)-4-(((1-(p-tolyl)-1H-pyrazol-4-yl)oxy)
4-(((1,3-Diphenyl-1H-pyrazol-4-yl)oxy)methyl)-1-(2-
methyl)-1H-1,2,3-triazole (4c)
nitrobenzyl)-1H-1,2,3-triazole (4f)
Pale yellow solid; Yield 75%; mp: 102 °C; IR (KBr): 2915,
Pale yellow solid; Yield: 76%; mp: 110 °C; IR (KBr): 2927,
1
1
1590, 1500, 1239, 1040, 758, 680 cm−1; H NMR (500
1592, 1518, 1349, 1125, 1052, 752, 692 cm−1; H NMR
MHz, CDCl3): δ 7.63–7.62 (m, 1H, C5’-CH), 7.50–7.44 (m,
4H, C3’ and C5-H and Ar–H), 7.25–7.19 (m, 4H, Ar–H),
6.91–6.86 (m, 2H, Ar–H), 5.46 (s, 2H, N–CH2), 5.11 (s,
2H, O–CH2), 3.80 (s, 3H, Ph–O–CH3), 2.36 (s, 3H,
Ph–CH3); 13C NMR (CDCl3, 75 MHz): δ 146.2 (C4), 144.0
(C4’), 129.6 (C3’), 126.2 (C5’), 112.3 (C5), 65.4 (C13), 55.2
(C15), 53.7 (C6), 20.8 (C14), 159.9, 138.0, 135.8, 129.8
(2C), 129.6 (2C), 122.5, 118.3 (2C), 114.4 (2C), (Aro-
matic); HRMS (ESI) for C21H22N5O2 [M+H]+ found
376.1773, calcd 376.1770.
(500 MHz, CDCl3): δ 8.21–8.15 (m, 2H, Ar–H), 8.02 (d, J
= 7.7 Hz, 2H, Ar–H), 7.78 (s, 1H, C5’-H), 7.68 (d, J = 7.7
Hz, 2H, Ar–H), 7.59 (s, 1H, C5-H), 7.47–7.23 (m, 8H,
Ar–H), 5.61 (s, 2H, N–CH2), 5.25 (s, 2H, O–CH2); 13C
NMR (CDCl3, 75 MHz); δ 144.7 (C4), 144.0 (C4’), 127.6
(C3’), 125.9 (C5’), 112.8 (C5), 65.6 (C13), 53.1 (C6), 148.0,
141.2 140.6, 140.0, 132.0, 129.3 (2C), 128.5 (2C), 128.3
(2C), 126.3 (2C), 124.2 (2C), 123.1, 118.0 (2C), (Aro-
matic); HRMS (ESI) for [M+Na] C25H20N6O3Na found
475.1496, calcd 475.1494.

Med Chem Res
1-(2-Chlorobenzyl)-4-(((1-(4-fluorophenyl)-3-(p-tolyl)-1H-
pyrazol-4-yl)oxy)methyl)-1H-1,2,3-triazole (4g)
HRMS (ESI) for C27H25FN5O2 [M+H]+ found 470.1990,
calcd 470.1992.
Yellow solid; Yield: 89%; mp: 173 °C; IR (KBr): 2919,
4-(((1-(4-Fluorophenyl)-3-(p-tolyl)-1H-pyrazol-4-yl)oxy)
methyl)-1-(4-nitrobenzyl)-1H-1,2,3-triazole (4j)
1
1514, 1400, 1349, 1225, 1052, 752 cm−1; H NMR (500
MHz, CDCl3): δ 7.90 (d, J = 8.2 Hz, 2H, Ar–H), 7.72 (m,
1H, C5’-H), 7.66–7.62 (m, 3H, C5-H and Ar–H), 7.43 (dd,
J = 7.9, 1.1 Hz, 1H, Ar–H), 7.33–7.30 (m, 1H, Ar–H), 7.24
(dd, J = 7.3, 1.2 Hz, 1H, Ar–H), 7.22–7.18 (m, 3H, Ar–H),
7.14–7.10 (m, 1H, Ar–H), 5.66 (s, 2H, N–CH2), 5.21 (s,
2H, O–CH2), 2.37 (s, 3H, Ph–CH3); 13C NMR (CDCl3, 75
MHz); 144.1 (C4), 144.0 (C4’), 127.6 (C3’), 123.3 (C5’),
113.0 (C5), 65.7 (C13), 51.5 (C6), 21.3 (C14), 160.7 (d, JC-
F = 245.2 Hz), 141.0, 137.4, 136.6 (d, JC-F = 1.8 Hz),
133.5, 132.2, 130.4, 130.3, 129.9, 129.2, 129.0 (2C), 126.3
(2C), 119.7 (d, JC-F = 8.2 Hz, 2C), 115.9 (d, JC-F = 22.7
Hz, 2C), (Aromatic); HRMS (ESI) for C26H22ClFN5O [M
+H]+ found 474.1496, calcd 474.1499.
Yellow solid; Yield: 80%; mp: 162 °C; IR (KBr):2919,
1
1516, 1346, 1218, 1122, 826 cm−1; H NMR (500 MHz,
CDCl3): δ 8.19 (d, J = 8.7 Hz, 2H, Ar–H), 7.89 (d, J = 8.2
Hz, 2H, Ar–H), 7.71 (s, 1H, C5’-H); 7.65–7.61 (m, 2H,
Ar–H), 7.59 (s, 1H, C5-H), 7.36 (d, J = 8.7 Hz, 2H, Ar–H),
7.19 (d, J = 8.6 Hz, 2H, Ar–H), 7.14–7.10 (m, 2H, Ar–H),
5.61 (s, 2H, N–CH2), 5.22 (s, 2H, O–CH2), 2.37 (s, 3H,
Ph–CH3); 13C NMR (CDCl3, 75 MHz) : δ 144.5 (C4), 143.8
(C4’), 128.8 (C3’), 122.9 (C5’), 112.7 (C5), 65.4 (C13), 52.9
(C6), 21.0 (C15), 160.5 (d, JC-F = 245.2 Hz), 147.8, 141.0,
140.6, 137.3, 136.3 (d, JC-F = 1.8 Hz), 128.8 (2C), 128.3
(2C), 126.0 (2C), 124.0 (2C), 119.5 (d, JC-F = 8.2 Hz, 2C),
115.8 (d, JC-F = 23.6 Hz, 2C), (Aromatic); HRMS (ESI) for
C26H22FN6O3 [M+H]+ 485.1740 found, calcd 485.1737.
1-(2-Bromobenzyl)-4-(((1-(4-fluorophenyl)-3-(p-tolyl)-1H-
pyrazol-4-yl)oxy)methyl)-1H-1,2,3-triazole (4h)
1-Benzyl-4-(((3-(4-chlorophenyl)-1-(4-fluorophenyl)-1H-
pyrazol-4-yl)oxy)methyl)-1H-1,2,3-triazole (4k)
Pale orange solid; Yield: 90%; mp: 189 °C; IR (KBr):2933,
1
1518, 1421, 1349, 1222, 1002, 752 cm−1; H NMR (500
Pale brown liquid; Yield: 85%; IR (KBr): 2916, 1616, 1558,
1225, 1122, 1022, 744 cm−1; 1H NMR (500 MHz, CDCl3):
δ 7.97 (d, J = 8.4 Hz, 1H, Ar–H), 7.74 (s, 2H, C5’-H,
Ar–H), 7.64–7.61 (m, 4H, C5-H, Ar–H), 7.44 (d, J = 7.9,
1.2 Hz, 1H, Ar–H), 7.36–7.30 (m, 2H, Ar–H), 7.27–7.21
(m, 3H, Ar–H), 7.13 (t, J = 8.1 Hz, 2H, Ar–H), 5.65 (s, 2H,
N–CH2), 5.19 (s, 2H, O–CH2); 13C NMR (CDCl3, 75
MHz): δ 144.5 (C4), 144.2 (C4’), 128.7 (C3’), 123.1 (C5’),
112.9 (C5), 65.1 (C13), 53.1 (C6), 160.5 (d, JC-F = 245.0
Hz), 141.2, 137.0, 136.7 (d, JC-F = 1.8 Hz), 135.8, 130.4
(2C), 129.8 (2C), 129.2, 129.0 (2C), 126.4 (2C), 119.9 (d,
JC-F = 8.2 Hz, 2C), 116.2 (d, JC-F = 22.9 Hz, 2C), (aro-
matic); HRMS (ESI) for C25H20ClFN5O [M+H]+ 460.1340
found, calcd 460.1342.
MHz, CDCl3): δ 7.91 (d, J = 8.3 Hz, 2H, Ar–H), 7.73 (s,
1H, C5’-H), 7.67–7.60 (m, 4H, C5-H and Ar–H), 7.32–7.10
(m, 7H, Ar–H), 5.66 (s, 2H, N–CH2), 5.22 (s, 2H, O–CH2),
2.38 (s, 3H, Ph–CH3); 13C NMR (CDCl3, 75 MHz): δ 144.1
(2C, C4 and C4’), 128.2 (C3’), 123.5 (C5’), 113.1 (C5), 65.7
(C13), 53.9 (C6), 21.3 (C14), 160.6 (d, JC-F = 245.0 Hz),
141.0, 137.4, 136.6 (d, JC-F = 1.8 Hz), 133.8, 133.2, 130.5,
130.4, 129.2. 129.1 (2C), 126.3 (2C), 123.3, 119.8 (d, JC-F
= 8.1 Hz, 2C), 116.0 (d, JC-F = 22.7 Hz, 2C), (Aromatic);
HRMS (ESI) for C26H22BrFN5O [M+H]+ found 518.0989,
calcd 518.0991.
4-(((1-(4-Fluorophenyl)-3-(p-tolyl)-1H-pyrazol-4-yl)oxy)
methyl)-1-(4-methoxybenzyl)-1H-1,2,3-triazole (4i)
1-(3-Chlorobenzyl)-4-(((3-(4-chlorophenyl)-1-(4-
fluorophenyl)-1H-pyrazol-4-yl)oxy)methyl)-1H-1,2,3-
triazole (4l)
Pale yellow solid; Yield: 85%; mp: 163 °C; IR (KBr): 2950,
1513, 1464, 1252, 829 cm−1; 1H NMR (500 MHz, CDCl3):
δ 7.90 (d, J = 8.2 Hz, 2H, Ar–H), 7.71 (s, 1H, C5’-H),
7.65–7.61 (m, 2H, Ar–H), 7.49 (s, 1H, C5-H), 7.24–7.18
(m, 4H, Ar–H), 7.12 (t, J, = 9.0 Hz, 2H, Ar–H), 6.88 (d, J
= 8.6 Hz, 2H, Ar–H), 5.45 (s, 2H, N–CH2), 5.18 (s, 2H,
O–CH2), 3.80 (s, 3H, Ph–CH3), 2.37 (s, 3H, Ph–O–CH3),
13C NMR (CDCl3, 75 MHz): δ 144.2 (C4), 144.1 (C4’),
126.2 (C3’), 122.7 (C5’), 112.9 (C5), 65.7 (C13), 55.3
(C14), 53.8 (C6), 21.3 (C15), 160.7 (d, JC-F = 245.2 Hz),
160.0, 140.9, 137.4, 136.6 (d, JC-F = 1.8 Hz), 129.7 (2C),
129.2, 129.0 (2C), 126.3 (2C), 119.7 (d, JC-F = 8.2 Hz, 2C),
116.1(d, JC-F = 23.6 Hz, 2 C), 114.5 (2C), (aromatic);
Orange solid; Yield: 85%; mp: 172 °C; IR (KBr): 2923,
1
1516, 1458, 1258, 1122, 858, 744 cm−1; H NMR (500
MHz, CDCl3): δ 7.96 (d, J = 8.5 Hz, 2H, Ar–H), 7.76 (s,
1H, C5’-H), 7.66–7.62 (m, 3H, C5-H, Ar–H), 7.44 (d, J =
7.9,1.1 Hz, 1H, Ar–H), 7.36–7.30 (m, 2H, Ar–H),
7.28–7.20 (m, 3H, Ar–H), 7.13 (t, J = 8.2 Hz, 2H, Ar–H),
5.67 (s, 2H, N–CH2), 5.21 (s, 2H, O–CH2); 13CNMR
(CDCl3, 75 MHz): δ 144.3 (C4), 144.1 (C4’), 127.8 (C3’),
123.6 (C5’), 112.8 (C5), 67.0 (C13), 52.1 (C6), 160.5 (d, JC-
F = 245.2 Hz), 141.0, 137.4, 136.5 (d, JC-F = 1.7 Hz),

Med Chem Res
Table 2 Pyrazolo-isoxazole hybrid products (5a–g)
135.5, 132.2, 131.1, 130.2, 129.8, 129.2, 128.9 (2C), 126.1
(2C), 119.3 (d, JC-F = 8.1 Hz, 2C), 115.2 (d, JC-F = 22.8
Hz, 2C), (Aromatic); HRMS (ESI) for C25H19Cl2FN5O [M
+H]+ found 494.0950, calcd 494.0949.
Compounds
R
R1
R2
Mp (°C) Yield
(%)
5a
5b
5c
5d
5e
5f
H
p-CH3 p-OMe
149
109
79
86
80
92
80
94
82
Ph
Ph
H
H
p-OMe
4-(((3-(4-Chlorophenyl)-1-(4-fluorophenyl)-1H-pyrazol-4-
yl)oxy)methyl)-1-(4-nitrobenzyl)-1H-1,2,3-triazole (4m)
3,4,5-triOMe Liquid
p-OMe 150
3,4,5-triOMe 134
p-CH3C6H4- p-F
p-CH3C6H4- p-F
p-CH3C6H4- p-F
p-CH3C6H4- p-F
Yellow solid; Yield: 90%; mp: 210 °C; IR (KBr):2923,
p-F
210
189
1
1516, 1358, 1228, 858 cm−1; H NMR (500 MHz, DMSO
5g
p-OMe
CDCl3): δ 8.21 (d, J = 8.7 Hz, 1H, Ar–H), 7.98 (d, J = 8.6
Hz, 1H, Ar–H), 7.80 (d, J = 6.7 Hz, 2H, C5’-H and Ar–H),
7.72–7.66 (m, 2H, C5-H and Ar–H), 7.49–7.43 (m, 4H,
Ar–H), 7.35 (d, J = 8.6 Hz, 2H, Ar–H), 7.16 (t, J = 8.3 Hz,
*Yields are given after purification by silica gel column chromato-
graphy and their purity is over 95% based on 1H NMR and 13C NMR
spectra
2H, Ar–H), 5.71 (s, 2H, N–CH2), 5.25 (s, 2H, O–CH2); 13
C
NMR (CDCl3, 75 MHz): δ 144.6 (C4), 144.5 (C4’), 128.8
(C3’), 123.1 (C5’), 112.7 (C5), 66.1 (C13), 53.1 (C6), 160.7
(d, JC-F = 245.3 Hz), 151.4, 147.7, 143.6, 141.1, 140.7,
137.3, 136.5 (d, JC-F = 1.7 Hz), 128.6 (2C), 128.1 (2C),
126.2 (2C), 124.7 (2C), 119.7 (d, JC-F = 8.3 Hz), 115.9 (d,
(C14), 159.2, 137.7, 130.0, 129.0 (2C), 128.2 (2C), 126.7
(2C), 114.6 (2C), 113.7 (Aromatic); HRMS (ESI) for
C21H20N3O3 [M+H]+ found 362.1504, calcd 362.1498.
JC-F = 23.7 Hz),
(Aromatic);
HRMS
(ESI)
for
5-(((1,3-Diphenyl-1H-pyrazol-4-yl)oxy)methyl)-3-(4-
methoxybenzyl)isoxazole (5b)
C25H19ClFN6O3 [M+H]+ 505.1189 found, calcd 505.1191.
General procedure for the synthesis of 3-benzyl-5-((1-aryl/
1,3-diaryl-1H-pyrazol-4-yloxy)methyl)isoxazoles (5a–g)
Yellow solid; Yield: 86%; mp: 109 °C; IR (KBr): 2922,
1
1575, 1498, 1228, 1129, 828 cm−1, H NMR (500 MHz,
CDCl3): δ 8.01 (d, J = 8.4 Hz, 2H, Ar–H), 7.68–7.60 (m,
4H, C5’-H and Ar–H), 7.41 (d, J = 8.6 Hz, 3H, Ar–H),
7.17–7.11 (m, 4H, Ar–H), 7.01 (s, 2H, Ar–H), 6.70 (s, 1H,
Propargylated compounds (3a–d) (0.18 mmol), triethyla-
mine (0.18 mmol) were taken in Dichloromethane (5 ml)
and resulting mixture was cooled to 0 °C. Addition of 2 mL
aqueous sodium hypochlorite (11%, 2 mL) over 30 min at 0
°C followed by aryl aldehyde oximes (0.36 mmol) and
maintained the reaction mixture at room temperature. After
completion of reaction monitored by TLC, the reaction
mixture was concentrated under vacuum and extracted with
ethyl acetate. The organic layers was separated and dried
over anhydrous sodium sulfate. The resulting crude product
was washed with n-hexane to obtain pyrazolo-isoxazole
hybrid products (5a–g) (Table 2).
C4-H), 5.21 (s, 2H, O–CH2); 3.89 (s, 3H, Ar–O–CH3); 13
C
NMR (CDCl3, 75 MHz): δ 166.5 (C3), 162.1 (C5), 143.6
(C4’), 141.8 (C3’), 129.1 (C5’), 101.5 (C4), 67.1 (C12), 53.6
(C13), 158.9, 138.9, 132.7, 132.0 (2C), 130.0, 129.0 (2C),
128.2 (2C), 127.8, 126.7 (2C), 120.1 (2C), 114.6 (2C),
113.7 (Aromatic); HRMS (ESI) for C26H22N3O3 [M+H]+
found 424.1661, calcd 424.1668.
5-(((1,3-Diphenyl-1H-pyrazol-4-yl)oxy)methyl)-3-(3,4,5-
trimethoxybenzyl)isoxazole (5c)
Spectral data
Pale brown liquid; Yield: 80%; IR (KBr): 2935, 1585, 1518,
1228, 1122, 852 cm−1; 1H NMR (500 MHz, CDCl3): δ 8.01
(d, J = 8.5 Hz, 2H, Ar-H), 7.68–7.63 (m, 3H, C5’-H and
Ar–H), 7.40 (d, J = 8.6 Hz, 3H, Ar–H), 7.17–7.13 (m, 3H,
Ar–H), 7.01 (s, 2H, Ar–H), 6.66 (s, 1H, C4-H), 5.21 (s, 2H,
O–CH2), 3.92 (s, 6H, Ar–O–CH3), 3.89 (s, 3H,
Ar–O–CH3); 13C NMR (CDCl3, 75 MHz): δ 167.9 (C3),
162.4 (C5), 143.7 (C4’), 141.1 (C3’), 123.7 (C5’), 101.6
(C4), 65.3 (C12), 60.9 (C14), 56.2 (2C, C13 and C15),
156.7, 153.5 (2C), 136.7, 135.6, 132.2, 129.2 (2C), 126.4
(2C), 129.9 (2C), 125.3 (2C), 113.6, 113.5, 105.2 (2C),
(Aromatic); HRMS (ESI) for C28H26N3O5 [M+H]+ found
484.1872, calcd 484.1866.
3-(4-Methoxybenzyl)-5-(((1-(p-tolyl)-1H-pyrazol-4-yl)oxy)
methyl)isoxazole (5a)
Brown solid; Yield: 79%; mp: 149 °C; IR (KBr): 2953,
1
1600, 1458, 1258, 1122, 858, 744 cm−1; H NMR (500
MHz, CDCl3): δ 7.77–7.73 (m, 2H, C5’ and C3’-H),
7.65–7.63 (m, 1H, Ar–H), 7.53–7.48 (m, 3H, Ar–H),
7.25–7.21 (m, 2H, Ar–H), 7.00–6.96 (m, 2H, Ar–H), 6.63
(s, 1H, C4-H), 5.13 (s, 2H, O–CH2), 3.86 (s, 3H,
Ar–O–CH3), 2.37 (s, 3H, Ar–CH3); 13C NMR (CDCl3, 75
MHz): δ 166.9 (C3), 162.9 (C5), 143.9 (C4’), 131.2 (C3’),
128.9 (C5’), 101.1 (C4), 65.3 (C12), 53.1 (C13), 20.8

Med Chem Res
5-(((1-(4-Fluorophenyl)-3-(p-tolyl)-1H-pyrazol-4-yl)oxy)
HRMS (ESI) for C25H17ClF2N3O2 [M+H]+ found
methyl)-3-(4-methoxybenzyl)isoxazole (5d)
464.0977, calcd 464.0980.
Yellow solid; Yield: 92%; mp: 150 °C; IR (KBr):2919,
5-(((3-(4-Chlorophenyl)-1-(4-fluorophenyl)-1H-pyrazol-4-
yl)oxy)methyl)-3-(4-methoxybenzyl)isoxazole (5g)
1516, 1430, 1258, 1022 cm−1
;
1H NMR (500 MHz,
CDCl3): δ 8.68 (d, J = 9 Hz, 1H, Ar–H), 8.50 (d, J = 9 Hz,
1H, Ar–H), 7.95 (d, J = 8 Hz, 2H, Ar–H), 7.74 (d, J = 8.8
Hz, 2H, Ar–H), 7.68–7.63 (m, 2H, C5’-H and Ar–H),
7.16–7.06 (m, 3H, Ar–H), 6.98 (d, J = 8.8 Hz, 2H, Ar–H),
6.62 (s, 1H, C4-H), 5.18 (s, 2H, O–CH2), 3.87 (s, 3H,
Ar–O–CH3), 2.40 (s, 3H, Ar–CH3); 13C NMR (CDCl3, 100
MHz): δ 167.6 (C3), 162.8 (C5), 143.9 (C4’), 141.2 (C3’),
128.6 (C5’), 101.0 (C4), 65.1 (C12), 55.3 (C13), 21.3
(C14), 162.7 (d, JC-F = 244.3 Hz), 162.2, 137.7, 136.5,
White solid; Yield: 82%; mp: 189 °C; IR (KBr): 2935,
1
1584, 1518, 1405, 1126, 858 cm−1; H NMR (300 MHz,
CDCl3): δ 8.01 (d, J = 8.7 Hz, 2H, Ar–H), 7.74 (d, J = 8.9
Hz, 2H, Ar–H), 7.69–7.61 (m, 3H, C5’-H), 7.40 (d, J = 8.7
Hz, 2H, Ar–H), 7.19–7.10 (m, 2H, Ar–H), 6.98 (d, J = 8.9
Hz, 2H, Ar–H), 6.62 (s, 1H, C4-H), 5.19 (s, 2H, O–CH2),
3.86 (s, 3H, O–CH3); 13C NMR (CDCl3, 75 MHz): δ 167.3
(C3), 162.1 (C5), 143.8 (C4’), 141.1 (C3’), 128.7 (C5’),
101.4 (C4), 65.3 (C12), 53.7 (C13), 21.0 (C14), 162.5 (d,
JC-F = 245.2 Hz), 161.8, 137.8, 136.7 (d, JC-F = 1.8 Hz),
129.2 (2C), 128.2 (2C), 126.3 (2C), 120.0 (d, JC-F = 8.3 Hz,
2C),116.0 (d, JC-F = 22.6 Hz, 2C), 114.4 (2C), 113.3
(Aromatic); HRMS (ESI) for C26H20ClFN3O3 [M+H]+
found 476.1177, calcd 476.1182.
130.0, 129.2 (2C), 128.2 (2C), 126.3 (2C), 119.9 (d, JC-F
=
8.1 Hz, 2C), 116.1 (d, JC-F = 22.7 Hz, 2C), 114.3 (2C),
113.1(Aromatic); HRMS (ESI) for C27H23FN3O3 [M+H]+
found 456.1723, calcd 456.1729.
5-(((1-(4-Fluorophenyl)-3-(p-tolyl)-1H-pyrazol-4-yl)oxy)
methyl)-3-(3,4,5-trimethoxybenzyl)isoxazole (5e)
Orange solid; Yield: 80%; mp: 134 °C; IR (KBr): 2935,
Materials and methods
1
1584, 1513, 1435, 1256, 1128 cm−1; H NMR (400 MHz,
CDCl3): δ 7.96 (d, J = 8.2 Hz, 2H, Ar–H), 7.67–7.63 (m,
3H, C5’-H and Ar–H), 7.24 (m, 1H, Ar–H), 7.15–7.11 (m,
3H, Ar–H), 7.01 (s, 2H, Ar–H), 6.63 (s, 1H, C4-H), 5.21 (s,
2H, O–CH2), 3.92 (s, 6H, Ar–O–CH3), 3.89 (s, 3H,
Ar–O–CH3), 2.39 (s, 3H, Ar–CH3); 13C NMR (CDCl3, 75
MHz): δ 168.1 (C3), 162.5 (C5), 143.9 (C4’), 141.2 (C3’),
123.9 (C5’), 101.7 (C4), 65.2 (C12), 60.9 (C14), 56.3 (2C,
C13 and C15), 21.3 (C16), 161.3 (d, JC-F = 245.3 Hz),
156.2, 153.6 (2C), 137.7, 136.6, 129.2 (2C), 126.4 (2C),
119.9 (d, JC-F = 8.1 Hz, 2C), 116.1(d, JC-F = 22.8 Hz, 2C),
113.2, 113.1, 104.1 (2C), (Aromatic); HRMS (ESI) for
C29H27FN3O5 [M+H]+ found 516.1934 calcd 516.1937.
Cell cultures, maintenance, and antiproliferative
evaluation
The cell lines, MCF7, HEPG2, IMR32 and HELA (breast,
liver, neuroblastoma, and cervical), which were used in this
study were procured from American Type Culture Collec-
tion (ATCC), United States. The synthesized test com-
pounds were evaluated for their in vitro antiproliferative
activity in these four different human cancer cell lines. A
protocol of 48 h continuous drug exposure was used, and a
Sulforhodamine B (SRB) cell proliferation assay was used
to estimate cell viability or growth. All the cell lines were
grown in Dulbecco’s modified Eagle’s medium (containing
10% fetal bovine serum in a humidified atmosphere of 5%
CO2 at 37 °C). Cells were trypsinized when sub-confluent
from T25 flasks/60 mm dishes and seeded in 96-well plates
in 100 μL aliquots at plating densities depending on the
doubling time of individual cell lines. The microtiter plates
were incubated at 37 °C, 5% CO2, 95% air, and 100%
relative humidity for 24 h prior to addition of experimental
drugs and were incubated for 48 h with different doses
(0.01, 0.1, 1, 10, 100 µM) of prepared derivatives. After 48
h incubation at 37 °C, cell monolayers were fixed by the
addition of 10% (wt/vol) cold trichloroacetic acid and
incubated at 4 °C for 1 h and were then stained with 0.057%
SRB dissolved in 1% acetic acid for 30 min at room tem-
perature. Unbound SRB was washed with 1% acetic acid.
The protein-bound dye was dissolved in 10 mM Tris base
solution for OD determination at 510 nm using a microplate
5-(((3-(4-Chlorophenyl)-1-(4-fluorophenyl)-1H-pyrazol-4-
yl)oxy)methyl)-3-(4-fluorobenzyl)isoxazole (5f)
Violet solid; Yield: 94%; mp: 210 °C; IR (KBr): 2929,
1
1516, 1438, 1255, 1122, 828 cm−1; H NMR (500 MHz,
CDCl3): δ 8.00 (d, J = 8.5 Hz, 2H, Ar–H), 7.69–7.62 (m,
3H, C5’-H and Ar–H), 7.60–7.50 (m, 2H, Ar–H), 7.47–7.43
(m, 1H, Ar–H), 7.41 (d, J = 8.4 Hz, 2H, Ar–H), 7.19–7.11
(m, 3H, Ar–H), 6.67 (s, 1H, C4-H), 5.22 (s, 2H, O–CH2);
13C NMR (CDCl3, 75 MHz); δ 167.1 (C3), 162.6 (C5),
143.5 (C4’), 141.3 (C3’), 128.9 (C5’), 101.1 (C4), 65.1
(C12), 53.4 (C13), 162.5 (d, JC-F = 245.2 Hz), 162.2 (d, JC-
F = 244.3 Hz), 137.8, 136.7(d, JC-F = 1.9 Hz), 136.3 (d, JC-
F = 1.7 Hz), 129.2 (2C), 128.2 (2C), 120.0 (d, JC-F = 8.2
Hz, 2C), 119.8 (d, JC-F = 8.1 Hz, 2C), 116.0 (d, JC-F = 22.6
Hz, 2C), 115.8 (d, JC-F = 22.7 Hz, 2C), 113.3 (Aromatic);

Med Chem Res
reader (Enspire, Perkin Elmer, USA). Using the seven
absorbance measurements [time zero, (Tz), control growth,
(C), and test growth in the presence of drug at the five
concentration levels (Ti)], the percentage growth was cal-
culated at each of the drug concentrations levels. Percentage
growth inhibition was calculated as:
pyrazole-4-ols 2b–d were prepared. Next, these 4-
hydroxypyrazoles 2a–d were O-alkylated with propargyl
bromide in THF/DMF (1:1) using NaH as a base at 0 °C to
RT to give new O-propargylated pyrazole derivatives 3a–d
in 85–96% yields, respectively (Scheme 1).
These alkyne substituted pyrazoles (3a–d) underwent
Copper(I)-catalyzed Azide-Alkyne Cycloaddition (CuAAC)
with diverse substituted benzyl azides in THF CuSO4·5H2O
and sodium ascorbate, in t-butyl alcohol and water (1 : 1) at
room temperature affording the corresponding regioselec-
tive 1,4-disubstituted-1,2,3-triazole-linked pyrazole hybrids
4a–m in excellent yields (Scheme 2). Similarly, compounds
3a–d on reaction with aryl aldoximes (Duncan et al. 2011;
Hansen et al. (2005)) yielded isoxazole functionalized
pyrazole derivatives 5a–g (Scheme 2).
[(Ti−Tz)/(C−Tz)] × 100 for concentrations for which
Ti>/=Tz
[(Ti−Tz)/Tz] × 100 for concentrations for which Ti<Tz.
The dose response parameter, GI50 was calculated for
each experimental agent. Growth inhibition of 50% (GI50)
was calculated from [(Ti−Tz)/(C−Tz)] × 100 = 50, which
is the drug concentration resulting in a 50% reduction in the
net protein increase (as measured by SRB staining) in
control cells during the drug incubation. Values were cal-
culated for this parameter if the level of activity is reached;
however, if the effect is not reached or is exceeded, the
value for that parameter was expressed as greater or less
than the maximum or minimum concentration tested.
Spectral data for the important compounds were pro-
vided in Supporting Information.
Biology
Anti-proliferative activity
All the synthesized compounds 1-benzyl-4-((1-aryl/1,
3-diaryl-1H-pyrazol-4-yloxy)methyl)-1H-1,2,3-triazoles
(4a–m) and 3-benzyl-5-((1-aryl/1,3-diaryl-1H-pyrazol-4-
yloxy)methyl)isoxazoles (5a–g) were evaluated for their
anti-cancer activity against four human cancer cell lines,
HELA (cervix), HEPG2 (liver), MCF-7 (breast), and IMR-
32 (neuroblastoma). Nocodazole and Paclitaxel were
employed as positive controls. Anti-mitotic drugs are rou-
tinely used as frontline therapies; hence, we chose to
compare the activities of the congeners with anti-mitotic
agents. The compounds were treated at five dose con-
centrations for 48 h according to the NCI-60 protocol. The
anti-proliferative efficacy of the compounds was evaluated
using SRB assays and expressed as GI50. Based on the GI50
values, we deduced the structure–activity relationship
(SAR) of the compounds. Based on the SAR Studies we
assumed that compounds containing halogen-substituted
Results and discussion
Chemistry
The known 1-aryl-1H-pyrazole-4-carbaldehyde (Finar and
Godfrey 1954, Maria et al. 2009), and 1,3-diaryl-1H-pyr-
azole-4-carbaldehydes (Prakash et al. 2006) were used as
starting materials. Initially, p-methylphenyl-1H-pyrazole-4-
carbaldehyde 1a was converted to p-methylphenyl-1H-
pyrazole-4-ol 2a by a new route. Thus, p-methylphenyl-1H-
pyrazole-4-carboxaldehyde was treated with mCPBA in
CHCl3 between 60–70 °C for 2 h, and quenching with 10%
Na2CO3 solution yielded p-methylphenyl-1H-pyrazole-4-ol
2a in 91% yield (Scheme 1). Similarly, other 1,4-diaryl-1H-
Scheme 1 Synthesis of 1-aryl/
1,3-diaryl-1H-pyrazol-4-ols
(2a–d) and 1-aryl/1,3-diaryl-4-
(prop-2-ynyloxy)-1H-pyrazoles
(3a–d)

Med Chem Res
Scheme 2 Synthesis of 1-
benzyl-4-((1-aryl/1, 3-diaryl-1H-
pyrazol-4-yloxy)methyl)-1H-
1,2,3-triazoles (4a–m) and 3-
benzyl-5-((1-aryl/1,3-diaryl-1H-
pyrazol-4-yloxy)methyl)
isoxazoles (5a–g). All the
intermediates and target
molecules were characterized
using spectroscopic techniques
(1H and 13C NMR, Mass and
HRMS) to confirm their
chemical structure
Table 3 Growth Inhibition 50
(GI50)/µM values of compounds
(4a–m, 5a–g) in selected cancer
cell lines
Compounds
HELA
HEPG2
2.5 0.02
MCF-7
IMR-32
4.8 0.5
4a
3.6 0.4
3.3 0.3
5.5 0.5
4b
5.2 0.5
4.2 0.1
4.6 0.15
6.9 0.3
2.7 0.02
6.3 0.25
12.3 1.1
5.1 0.4
7.3 0.5
5.9 0.5
7.3 0.4
8.2 0.7
2.3 0.02
4.9 0.06
15 1.2
4c
3.5 0.08
8.7 0.5
3
0.2
4d
9.8 0.7
10 0.4
17.4 0.8
4.8 0.05
6.6 0.09
4.8 0.2
3.7 0.2
5.1 0.2
3.8 0.5
6.1 0.8
20.7 1.7
10.5 1.1
5.1 0.3
5.6 0.9
6.1 0.5
7.3 0.8
7.2 0.6
9.5 0.5
0.05 0.003
0.02 0.002
4e
10.7 1.2
3.1 0.35
3.3 0.05
2.4 0.02
4.4 0.1
14.4 1.4
5.2 0.2
4f
4g
8.8 0.3
4h
6.3 0.07
5.2 0.05
3.6 0.6
4i
4j
9.7 1.2
4k
1.8 0.04
2.7 0.07
14.7 1.3
8.3 0.6
2.1 0.01
2.9 0.2
4l
4m
11.8 0.9
7.9 0.8
5a
8.4 0.6
6.3 0.7
8.3 0.6
11 1.4
5b
4.9 0.08
10.7 1.1
10.8 1.2
11.1 1.3
13.9 1.6
10.2 1.2
0.02 0.002
0.01 0.002
3.7 0.4
5c
5.2 0.08
9.1 0.9
5d
5e
9.08 1.1
6.05 0.8
9.6 1.1
9.3 1.1
10 1.1
5f
5g
8.7 0.9
0.04 0.002
<0.01 0.001
Paclitaxel
Nocodazole
<0.01 0.002
0.01 0.001

Med Chem Res
with combined H,-antihistamine and mast cell stabilizing prop-
erties. J Med Chem 29:2262
aromatic rings on pyrazole moiety such as 4k, 4l, 4h
showing potent activity especially in HELA cells.
Nitro group present on the benzyl moiety attached to
triazole ring increased the potentiality of the compound 4g
towards anti-cancer activity, as well as halogens and
methoxy groups (Cl, F, Br, OMe) decreased the potentiality
of the compound towards anti-cancer activity this was
observed in the case of 4k, 4l, 4d. Groups present at the 3rd
position of the pyrazole ring also effected the potentiality of
the compounds towards anti-cancer activity, such as halo-
gen substituted aromatic group at 3rd position showed
potent activity (ex 4k, 4l) and p-Me containing aromatic
groups as well as no substitution at the 3rd position of the
pyrazole ring slightly decreased the potentiality of the
compounds (ex 4i, 4g, 4a, 4b, 4c) and Ph-substitution on
pyrazole ring decreased the potentiality and showed mod-
erate activities (ex 4d, 4e).
If both the halogens present on the aromatic ring attached
to pyrazole moiety and NO2 group on the aromatic ring
attached to the triazole moiety greatly effected the potenti-
ality of the compounds (ex 4m, 4j). By considering above
all the information 4k and 4l are the most potent of the
congeners.
Based on the GI50 values, 5b was found to be the most
potent in the isoxazole series. However, as compared to the
triazole series, isoxazoles showed moderate activities
towards anti-cancer activity against four human cancer cell
lines. These SAR assumptions were made based on the
preliminary GI50 results (Table 3).
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Conclusion
In this study, we have described a synthesis of new pyrazole
derivatives containing 1,2,3-triazole and isoxazole motifs
(4a–m and 5a–g). These compounds were evaluated for
their cytotoxic activity against four human tumor cell lines
(MCF7, HEPG2, IMR32, and HELA). The results showed
that compounds 4k and 4l having fluoro, chloro groups
strongly inhibited the growth of cell lines.
Acknowledgements N.F. thanks DST (SR/WOS-A/CS-124/2012),
India for the award of fellowship. G.S. and B.V.S.R thank CSIR, India
for financial support as part of XII Five Year plan programmed under
title ORIGIN (CSC-0108).
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