One-pot green synthesis and bio-assay of pyrazolylphosphonates

Article abstract of DOI:10.1007/s11164-018-3319-y

An efficient one-pot synthesis of bioactive pyrazolylphosphonates is accomplished by the reaction of 3-methyl-1-phenyl-2-pyrazoline-5-one with various aryl aldehydes and diethyl phosphite in the presence of β-cyclodextrin (β-CD) as a catalyst. The compounds were tested for their anti-oxidant activity by DPPH and H₂O₂ radical scavenging assay and for anticancer activity against breast cancer (MCF-7), prostate cancer (DU-145) and lung cancer (A-549) cell lines with sulfarodamine-B (SRB) assay. Most of the synthesized compounds showed promising anti-oxidant activity and significant anticancer activity when compared with the standard drugs.

Full text of DOI:10.1007/s11164-018-3319-y

Res Chem Intermed
https://doi.org/10.1007/s11164-018-3319-y
One-pot green synthesis and bio-assay
of pyrazolylphosphonates
Gundluru Mohan1 Sarva Santhisudha1,2 Sudileti Murali1
•
•
•
Nemallapudi Bakthavatchala Reddy1,3 Gundala Sravya1,3
Grigory V Zyryanov3,4 Cirandur Suresh Reddy1
•
•
•
Received: 4 November 2017 / Accepted: 2 February 2018
Ó Springer Science+Business Media B.V., part of Springer Nature 2018
Abstract An efficient one-pot synthesis of bioactive pyrazolylphosphonates is
accomplished by the reaction of 3-methyl-1-phenyl-2-pyrazoline-5-one with various
aryl aldehydes and diethyl phosphite in the presence of b-cyclodextrin (b-CD) as a
catalyst. The compounds were tested for their anti-oxidant activity by DPPH and
H2O2 radical scavenging assay and for anticancer activity against breast cancer
(MCF-7), prostate cancer (DU-145) and lung cancer (A-549) cell lines with sul-
farodamine-B (SRB) assay. Most of the synthesized compounds showed promising
anti-oxidant activity and significant anticancer activity when compared with the
standard drugs.
Keyword Pyrazolylphosphonates Á Green synthesis Á b-cyclodextrin Á Anti-oxidant
activity Á Anticancer activity
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s11164-
018-3319-y) contains supplementary material, which is available to authorized users.
&
Cirandur Suresh Reddy
csrsvu@gmail.com
1
Department of Chemistry, Sri Venkateswara University, Tirupati, Andhra Pradesh 517 502,
India
2
3
Department of Chemistry, Dravidian University, Kuppam, Andhra Pradesh 517 426, India
Department of Organic and Biomolecular Chemistry, Chemical Engineering Institute, Ural
Federal University, 19 Mira Street, Yekaterinburg, Russian Federation 620002
4
I.Ya. Postovskiy Institute of Organic Synthesis, Ural Division of the Russian Academy of
Sciences, 22 S. Kovalevskoy Street, Yekaterinburg, Russian Federation 620219
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G. Mohan et al.
Introduction
In recent years, significant effort has been devoted to the synthetic studies of
organophosphorus compounds since they have several biological properties include
antitumor [1], anti-oxidant [2], antimicrobial [3], anticoagulant [4], anti-inflamma-
tory [5], antiviral [6], enzyme inhibition [7], plant growth regulation, herbicidal and
pesticidal activities [8–10]. Owing to their potential bioactivity, low toxicity and the
ease of substitution with conventional heterocyclic groups, phosphoryl compounds
are perfect substrates for use in drug design. The molecular modifications involving
the introduction of organophosphorus functionalities in simple synthons is a very
promising route for the preparation of the new generation of biologically active
phosphoryl compounds [11].
Pyrazole and its derivatives are a class of important aza-heterocyclic compounds
[12] with a variety of bioactivities such as antimicrobial [13], anticancer [14],
anticonvulsant [15], analgesic, antipyretic, anti-inflammatory [16], antibacterial
[17], antifungal [18], CNS regulating [19], enzyme inhibition [20], hypoglycemic,
anti-oxidant [21] and antihypertensive [22] activities.
Due to the tremendous synthetic and bio-potent value of phosphoryl and pyrazole
motifs, a variety of pyrazolylphosphonates (4a–o) are synthesized by the
condensation of 3-methyl-1-phenyl-2-pyrazoline-5-one (1) with various aryl
aldehydes (2a–o) and diethyl phosphite (3), expecting them to have good
bioactivities. However, very few synthetic protocols are available for the syntheses
of pyrazolylphosphonates by a three-component reaction of pyrazolones, with
various aryl aldehydes and dialkyl phosphite [23–25]. They have drawbacks such as
long reaction times, requirement of stoichiometric quantities of toxic catalysts, low
product yields and generation of wastage. Thus, there remains a need for novel and
improved synthetic routes that are elegant, inexpensive and environmentally benign.
The art of performing green and efficient chemical transformations involves non-
toxic reaction conditions. Cyclic oligosaccharides possessing hydrophobic cavities
are well known as supramolecular catalysts, which, by reversible formation of host–
guest complexes, activate the organic molecules and thus efficiently catalyze the
reactions under green conditions [26]. b-CD is the example of these biomimetic
catalysts used extensively in many novel transformations with high yield and good
selectivity, includes the synthesis of important classes of biologically active
heterocycles like pyrroles [27], thiazoles [28], quinolones [29], quinazolines [30],
and oxindoles [31]. In the present work, a green procedure for the synthesis of
pyrazolylphosphonates (4a–o) catalyzed by b-CD in solvent-free conditions has
been reported (Scheme 1). The title compounds were screened for their anti-oxidant
activity by DPPH and H2O2 radical scavenging assay and for anticancer activity
against breast cancer (MCF-7), prostate cancer (DU-145) and lung cancer (A-549)
cell lines with sulfarodamine-B (SRB) assay.
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One-pot green synthesis and bio-assay of…
Scheme 1 Synthesis of diethyl((5-hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)(aryl)methyl)
phosphonates(4a–o)
Experimental
Analysis and instruments
All the required chemicals were purchased from Sigma Aldrich and the solvents
from Merck and were used without further purification. The completion and purity
of the reactions were monitored by TLC, performed on silica gel aluminum 60
F-254 thin layer plates procured from Merck, and visualization on TLC was
achieved by UV light and iodine indicator. Melting points of the compounds were
determined on Guna digital melting point apparatus using open capillary tubes and
are uncorrected. Infrared spectra were recorded on FT-IR Bruker ALPHA
Interferometer and wave numbers are given in cm-1. NMR spectra were recorded
on a Bruker instrument operating at 400 MHz for 1H, 100 MHz for 13C and
161.9 MHz for 31P in CDCl3. TMS was used as an internal standard. Assignments
of the signals are based on the chemical shifts and intensity patterns. Chemical shift
(d) and coupling constant (J) are expressed in ppm and Hz, respectively. Mass
spectra were recorded on a LC–MS/MS-TOF API QSTAR PULSAR spectrometer;
samples were introduced by the infusion method using the electrospray ionization
technique (ESI).
General procedure for synthesis of pyrazolylphosphonate derivatives (4a–o)
To a mixture of 3-methyl-1-phenyl-2-pyrazoline-5-one (1.0 mmol), aryl aldehydes
(1.2 mmol), and diethylphosphite (2.0 mmol) were added the b-CD (10 mol%) in a
round-bottomed flask. The reaction mixture was stirred at room temperature under
neat conditions. The progress of the reaction was indicated with observation of new
spots in the TLC after 10 min. The reaction was run with stirring of the reaction
mixture for 30 min. After completion of the reaction, the reaction mixture was
poured into cold water and extracted three times with ethyl acetate (3 5 mL). The
organic layer was washed with 10% NaHCO3 solution, dried over anhydrous
Na2SO4, filtered and concentrated in a rota-evaporator to leave crude product which
was purified by recrystallization from ethanol. The aqueous layer was frozen to
0–10 °C to precipitate b-CD as a white solid. This was collected by filtration,
washed with water and dried for reuse.
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Diethyl((4-ethoxyphenyl)(5-hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)methyl)phos-
phonate(4a) Brown solid; Yield: 96%. M.p. [300 °C. FT-IR (neat, cm-1) m 3382 (–
OH), 2968 (Ar–H), 1240 (–P=O), 1027 (–P–O–C–), 746 (–P–C–). 1H-NMR (CDCl3): d
7.80 (2H, d, J = 8.0 Hz), 7.40 (2H, t, J = 12.0 Hz), 7.32 (1H, t, J = 12.0 Hz), 7.11
(2H, d, J = 8.0 Hz), 6.84 (2H, d, J = 8.0 Hz), 5.91 (1H, s), 4.90 (1H, d, J = 8.0 Hz),
4.14-3.97 (4H, m), 2.28 (3H, s), 1.40 (3H, t, J = 12.0 Hz), 1.33 (3H, t, J = 12.0 Hz),
1.21 (3H, t, J = 12.0 Hz). 13C-NMR (CDCl3): d 164.13, 158.40 (d, Jc-p = 12.0 Hz),
147.81 (d, Jc-p = 52.0 Hz), 138.96, 130.04, 129.98, 128.71(d, Jc-p = 52.0 Hz), 121.77,
114.75, 94.25, (d, Jc-p = 16.0 Hz), 64.40 (d, Jc-p = 36.0 Hz), 50.49 (d, Jc-
p = 140.0 Hz), 16.44, 14.82, 12.65. 31P-NMR (CDCl3): d 18.58. MS (ESI): m/z
445.18 Anal. Calcd. for C23H29N2O5P: C, 62.15; H, 6.58; N, 6.30; O, 18.00; P, 6.97.
Found: C, 62.06; H, 6.43; N, 6.22; O, 17.91; P, 6.88.
Diethyl((5-hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)(p-tolyl)methyl)phospho-
nate(4b) [24] Yellow solid; Yield: 96%. M.p. [ 300 °C. FT-IR (neat, cm-1) m 3404
(–OH), 2980 (Ar–H), 1247 (–P=O), 1024 (–P–O–C–), 756 (–P–C–). 1H-NMR
(CDCl3): d 7.74 (2H, d, J = 8.0 Hz), 7.38 (2H, t, J = 12.0 Hz), 7.24 (1H, t,
J = 12.0 Hz), 7.08 (2H, d, J = 8.0 Hz), 6.82 (2H, d, J = 8.0 Hz), 5.76 (1H, s),
4.98 (1H, d, J = 10.0 Hz), 4.17-3.88 (4H, m), 2.24 (3H, s), 1.39 (3H, t,
J = 12.0 Hz), 1.24 (3H, t, J = 12.0 Hz), 1.16 (3H, t, J = 12.0 Hz). 13C-NMR
(CDCl3): d 166.18, 159.42 (d, Jc-p = 12.0 Hz), 148.76 (d, Jc-p = 48.0 Hz),
139.96, 130.08, 127.58, 126.72(d, Jc-p = 54.0 Hz), 122.71, 112.78, 96.22, (d, Jc-
p = 24.0 Hz), 64.42 (d, Jc-p = 48.0 Hz), 52.42 (d, Jc-p = 128.0 Hz), 16.42,
14.81, 12.68. 31P-NMR (CDCl3): d 20.45. MS (ESI): m/z 415.17 Anal.Calcd. for
C22H27N2O4P: C, 63.76; H, 6.57; N, 6.76; O, 15.44; P, 7.47. Found: C, 63.69; H,
6.50; N, 6.68; O, 15.34; P, 7.30.
Diethyl((3,4-dimethoxyphenyl)(5-hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)methyl)phos-
phonate(4c) Brown solid; Yield: 96%. M.p. [300 °C. FT-IR (neat, cm-1) m 3397 (–OH),
2980 (Ar–H), 1264 (–P=O), 1014 (–P–O–C–), 752 (–P–C–). 1H-NMR (CDCl3): d 7.77 (2H,
d, J = 8.0 Hz), 7.73 (2H, t, J = 8.0 Hz), 7.37 (2H, t, J = 12.0 Hz), 6.69 (1H, d,
J = 8.0 Hz), 6.62 (1H, d, J = 8.0 Hz), 6.55 (1H, d, J = 8.0 Hz), 5.98 (1H, s), 4.94 (1H, d,
J = 10.0 Hz), 3.90-3.72 (4H, m), 2.90 (3H, s), 2.83 (3H, s), 2.34 (3H, s), 1.27 (3H, t,
J = 12.0 Hz), 1.22 (3H, t, J = 12.0 Hz). 13C-NMR (CDCl3): d 162.33, 153.60, 151.07,
148.75, 14.31, 146.24, 138.48, 137.66, 130.44, 128.79, 126.83, 124.82, 121.10, 119.37,
115.58, 110.72 (d, Jc-p = 172.0 Hz), 64.23 (d, Jc-p = 156.0 Hz), 61.38, 56.31 (d, Jc-
p = 184.0 Hz), 16.54, 13.36. 31P-NMR (CDCl3): d 19.57. MS (ESI): m/z 461.23 Anal.
Calcd. for C23H29N2O6P: C, 59.99; H, 6.35; N, 6.08; O, 20.85; P, 6.73 Found: C, 59.87; H,
6.26; N, 5.97; O, 20.76; P, 6.64.
Diethyl((5-hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)(3,4,5-trimethoxyphenyl)methyl)
phosphonate(4d) Yellow solid; Yield: 96%. M.p. [300 °C. FT-IR (neat, cm-1) m 3396 (–
1
OH), 2982 (Ar–H), 1256 (–P=O), 1033 (–P–O–C–), 757 (–P–C–). H-NMR (CDCl3): d
7.76 (2H, d, J = 8.0 Hz), 7.38 (2H, t, J = 12.0 Hz), 7.20 (1H, t, J = 12.0 Hz), 6.94 (1H,
s), 6.78 (1H, s), 5.90 (1H, s), 4.90 (1H, d, J = 12.0 Hz), 4.18–3.99 (4H, m), 3.84–3.73
(9H, m), 2.89 (3H, s), 1.22 (3H, t, J = 12.0 Hz), 1.15 (3H, t, J = 12.0 Hz). 13C-NMR
(CDCl3): d 164.13, 158.40 (d, Jc-p = 12.0 Hz), 147.81 (d, Jc-p = 52.0 Hz), 138.96,
131.23 (d, Jc-p = 24.0 Hz), 128.78, 128.57, 121.77, 114.76 (d, Jc-p = 12.0 Hz), 94.25
(d, Jc-p = 16.0 Hz), 64.44, 63.47 (d, Jc-p = 24.0 Hz), 61.90 (d, Jc-p = 24.0 Hz), 45.33
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One-pot green synthesis and bio-assay of…
(d, Jc-p = 244.0 Hz), 16.44, 14.82, 12.65. 31P-NMR (CDCl3): d 21.29. MS (ESI): m/z
491.90 Anal. Calcd. for C24H31N2O7P: C, 58.77; H, 6.37; N, 5.71; O, 22.83; P, 6.31
Found: C, 58.70; H, 6.31; N, 5.64; O, 22.74; P, 6.24.
Diethyl((4-(diethylamino)phenyl)(5-hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-
yl)methyl) phosphonate(4e) Brown solid; Yield: 96%. M.p. [ 300 °C. FT-IR (neat,
cm-1) m 3397 (–OH), 2924 (Ar–H), 1245 (–P=O), 1018 (–P–O–C–), 742 (–P–C–).
1H-NMR (CDCl3): 7.56 (2H, d, J = 8.0 Hz), 7.52 (2H, t, J = 12.0 Hz), 7.46 (1H,
m), 7.02 (1H, d, J = 8.0 Hz), 6.96 (2H, d, J = 8.0 Hz), d 5.82 (1H, s), 4.76 (1H, d,
J = 8.0 Hz), 4.14–3.36 (4H, m), 3.60–3.56 (2H, m), 3.26–3.22 (2H, m), 2.64–2.60
(2H, m), 2.48 (3H, s), 1.97 (3H, s), 1.32 (6H, m), 1.20 (3H, t, J = 12.0 Hz), 1.06
(3H, t, J = 12.0 Hz). 13C-NMR (CDCl3) d 151.42, 147.46, 145.61 (d, Jc-
p = 54.0 Hz), (d, Jc-p = 56.0 Hz), 138.24, 130.59 (d, Jc-p = 68.0 Hz), 129.81,
126.75, 122.40, 111.56 (d, Jc-p = 76.0 Hz), 97.11 (d, Jc-p = 7.0 Hz), 64.08 (d, Jc-
p = 84.0 Hz), 46.32 (d, Jc-p = 186.0 Hz), 44.48, 16.42 (d, Jc-p = 20.0 Hz), 13.81
(d, Jc-p = 84.0 Hz), 13.26. 31P-NMR (CDCl3): d 21.48. MS (ESI): m/z 472.68
Anal. Calcd. for C25H34N3O4P: C, 63.68; H, 7.27; N, 8.91; O, 13.57; P, 6.57. Found:
C, 63.62; H, 7.16; N, 8.86; O, 13.45; P, 6.49.
Diethyl((3-fluorophenyl)(5-hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)methyl)phos-
phonate(4f) Brown solid; Yield: 91%. M.p. [300 °C. FT-IR (neat, cm-1) m 3386 (–
OH), 2980 (Ar–H), 1225(–P=O), 1031 (–P–O–C–), 751 (–P–C–). 1H-NMR (CDCl3): d
7.60 (2H, d, J = 8.0 Hz), 7.34 (2H, t, J = 12.0 Hz), 7.10 (1H, d, J = 8.0 Hz), 6.96
(1H, d, J = 8.0 Hz), 6.0 (1H, d, J = 8.0 Hz), 6.75 (1H, m), 5.88 (1H, s), 4.90 (1H, d,
J = 16.0 Hz), 3.95–3.72 (4H, m), 2.76 (3H, s), 1.16 (3H, t, J = 12.0 Hz), 1.12 (3H, t,
J = 12,0 Hz). 13C-NMR (CDCl3): d 164.13, 156.96, 147.73 (d, Jc-p = 48.0 Hz),
146.37, 138.67, 137.83, 128.76 (d, Jc-p = 20.0 Hz), 125.66, 123.15, 121.39, 118.6,
114.36. (d, Jc-p = 88.0 Hz) 112.87 (d, Jc-p = 84.0 Hz), 104.25, 93.46, 63.56 (d, Jc-
p = 78.0 Hz), 44.44 (d, Jc-p = 144.0 Hz), 16.31 12.59, 11.92. 31P-NMR (CDCl3): d
18.99. MS (ESI): m/z 419.05 Anal.Calcd. for C21H24FN2O4P: C, 60.28; H, 5.78; F,
4.54; N, 6.70; O, 15.30; P, 7.40. Found: C, 60.19; H, 5.71; F, 4.46; N, 6.64; O, 15.22; P,
7.31.
Diethyl((4-chlorophenyl)(5-hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)methyl)phos-
phonate(4g) [23] White solid; Yield: 96% M.p. [300 °C. FT-IR (neat, cm-1) m 3416 (–
OH), 2992 (Ar–H), 1253 (–P=O), 1032 (–P–O–C–), 748 (–P–C–). 1H-NMR (CDCl3): d
7.86 (2H, d, J = 8.0 Hz), 7.54 (2H, t, J = 12.0 Hz), 7.28 (2H, t, J = 12.0 Hz), 7.16
(1H, m), 7.08 (2H, m), 5.87 (1H, s), 4.84 (1H, d, J = 12.0 Hz), 4.08–3.76 (4H, m), 2.24
(3H, s), 1.38 (3H, t, J = 12.0 Hz), 1.26 (3H, t, J = 12.0 Hz). 13C-NMR (100 MHz,
CDCl3): d 156.78, 146.90, 143.25, 138.95, 1136.23, 131.55 (d, Jc-p = 78.0 Hz), 130.68
(d, Jc-p = 74.0 Hz), 129.69, 128.64 (d, Jc-p = 12.0 Hz), 121.78 (d, Jc-p = 54.0 Hz),
119.21, 103.30, 63.24 (d, Jc-p = 76.0 Hz), 61.62 (d, Jc-p = 48.0 Hz), 44.93 (d, Jc-
p = 184.0 Hz), 16.68 14.33, 12.58. 31P-NMR (CDCl3): d 20.28. MS (ESI): m/z 439.12.
Anal.Calcd. for C21H24ClN2O4P: C, 58.00; H, 5.56; Cl, 8.15; N, 6.44; O, 14.72; P, 7.12.
Found: C, 57.95; H, 5.48; Cl, 8.04; N, 6.32; O, 14.68; P, 7.06.
Diethyl((4-bromophenyl)(5-hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-
yl)methyl)phosphonate(4h) Brown solid. Yield: 92%. M.p. [ 300 °C. FT-IR (neat,
cm-1) m 3397 (-OH), 2980 (Ar–H), 1214 (–P=O), 1072 (–P–O–C–), 752 (–P–C–).
1H-NMR (400 MHz, CDCl3) d 7.74 (2H, d, J = 8.0 Hz), 7.45 (2H, t, J = 12.0 Hz),
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G. Mohan et al.
7.32 (2H, t, J = 12.0 Hz), 7.15(1H, m), 7.13 (2H, m), 5.93 (1H, s), 4.93 (1H, d,
J = 12.0 Hz), 4.15-3.99 (4H, m), 2.28 (3H, s), 1.36 (3H, t, J = 12.0 Hz), 1.24 (3H,
t, J = 12.0 Hz). 13C-NMR (100 MHz, CDCl3): d 155.80, 146.90, 143.25, 138.95,
1136.23, 131.55 (d, Jc-p = 78.0 Hz), 130.63 (d, Jc-p = 74.0 Hz), 129.69, 128.76
(d, Jc-p = 12.0 Hz), 121.76 (d, Jc-p = 54.0 Hz), 119.27, 103.37, 63.28 (d, Jc-
p = 76.0 Hz), 61.86 (d, Jc-p = 48.0 Hz), 44.59 (d, Jc-p = 228.0 Hz), 16.42 14.37,
12.53. 31P-NMR (CDCl3): d 21.78. MS (ESI): m/z 480.05 Anal. Calcd. for
C21H24BrN2O4P: C, 52.62; H, 5.05; Br, 16.67; N, 5.84; O, 13.35; P, 6.46. Found: C,
52.54; H, 4.94; Br, 16.56; N, 5.78; O, 13.22; P, 6.38.
Diethyl((5-hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)(4-nitrophenyl)methyl)phos-
phonate(4i) [24, 25] Light yellow solid; Yield: 91%. M.p. [300 °C. FT-IR (neat,
cm-1) m 3412 (–OH), 2996 (Ar–H), 1238 (–P=O), 1017 (–P–O–C–), 757 (–P–C–). 1H-
NMR (CDCl3): d 7.86 (2H, d, J = 8.0 Hz), 7.42 (2H, t, J = 12.0 Hz), 7.38 (1H, t,
J = 12.0 Hz), 7.06 (2H, d, J = 8.0 Hz), 6.90 (2H, d, J = 8.0 Hz), 5.88 (1H, s), 4.84
(1H, d, J = 12.0 Hz), 4.04–3.86 (4H, m), 2.56 (3H, s), 1.34 (3H, t, J = 12.0 Hz), 1.16
(3H, t, J = 12.0 Hz). 13C-NMR (CDCl3): d 164.18, 158.57 (d, Jc-p = 12.0 Hz),
146.23 (d, Jc-p = 52.0 Hz), 137.96, 134.82, 123.36, 128.76 (d, Jc-p = 52.0 Hz),
121.88, 114.64, 94.34, (d, Jc-p = 24.0 Hz), 64.82 (d, Jc-p = 36.0 Hz), 48.46 (d, Jc-
p = 136.0 Hz), 16.64, 14.24, 12.16. 31P-NMR(CDCl3): d 23.24. MS (ESI): m/z 446.14
Anal. Calcd. for C21H24N3O6P: C, 56.63; H, 5.43; N, 9.43; O, 21.55; P, 6.95 Found: C,
56.54; H, 5.33; N, 9.36; O, 21.48; P, 6.90.
Diethyl((5-chloro-2-fluorophenyl)(5-hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)methyl)
phosphonate(4j) Yellow solid; Yield: 90%. M.p.: [300 °C. FT-IR (neat, cm-1) m 3428 (–
1
OH), 2987 (Ar–H), 1230 (–P=O), 1046 (–P–O–C–), 738 (–P–C–). H-NMR (CDCl3) d
7.68 (2H, d, J = 8.0 Hz), 7.46 (2H, t, J = 12.0 Hz), 7.34 (1H, t, J = 12.0 Hz), 7.28 (1H, t,
J = 12.0 Hz), 7.13 (1H, m), 6.84 (1H, m), 5.86 (1H, s), 4.88 (1H, d, J = 12.0 Hz), 4.07-
3.76 (4H, m), 2.28 (3H, s), 1.36 (3H, t, J = 12.0 Hz), 1.16 (3H, t, J = 12.0 Hz). 13C-NMR
(CDCl3): d 158.32, 157.96, 144.82, (d, Jc-p = 14.0 Hz), 136.24, 132.56, 131.67 (d, Jc-
p = 20.0 Hz), 128.68, 127.86 (d, Jc-p = 69.0 Hz), 125.94, 116.68 (d, Jc-p = 24.0 Hz),
63.68 (d, Jc-p = 52.0 Hz), 43.84 (d, Jc-p = 152.0 Hz), 16.48, 13.42, 11.56. 31P-NMR
(CDCl3) d 21.04. MS (ESI): m/z 453.11 Anal. Calcd. for C21H23ClFN2O4P: C, 55.70; H,
5.12; Cl, 7.83; F, 4.20; N, 6.19; O, 14.13; P, 6.84. Found: C, 55.62; H, 5.02; Cl, 7.76; F,
4.12; N, 6.10; O, 14.03; P, 6.76.
Diethyl((5-bromo-2-fluorophenyl)(5-hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)methyl)
phosphonate(4k) Brown solid; Yield: 90%. M.p. [300 °C. FT-IR (neat, cm-1) m 3396 (–
1
OH), 2978 (Ar–H), 1186 (–P=O), 1036 (–P–O–C–), 760 (–P–C–). H-NMR (CDCl3): d
7.87 (1H, d, J = 8.0 Hz), 7.77 (1H, d, J = 8.0 Hz), 7.64 (2H, d, J = 8.0 Hz), 7.28 (2H, t,
J = 8.0 Hz), 7.13 (1H, t, J = 8.0 Hz), 6.84 (1H, t, J = 12.0 Hz), 5.78 (1H, s), 5.27 (1H, d,
J = 12.0 Hz), 4.07–3.82 (2H, m), 3.81–3.76 (1H, m), 3.74–3.56 (1H, m), 2.27 (3H, s), 1.32
(3H, t, J = 12 Hz), 1.20 (3H, t, J = 12 Hz). 13C-NMR (CDCl3): d 160.35, 157.91, 145.81,
(d, Jc-p = 24.0 Hz), 137.13, 132.04, 131.64 (d, Jc-p = 60.0 Hz), 128.74, 127.84 (d, Jc-
p = 56.0 Hz), 125.96, 116.68 (d, Jc-p = 78.0 Hz), 63.64, 43.65 (d, Jc-p = 184.0 Hz),
16.24, 13.48, 11.50. 31P-NMR (CDCl3): d 18.19. MS (ESI): m/z 497.06 Anal. Calcd. for C,
50.72; H, 4.66; Br, 16.07; F, 3.82; N, 5.63; O, 12.87; P, 6.23. Found: C, 50.64; H, 4.58; Br,
16.00; F, 3.74; N, 5.52; O, 12.78; P, 6.15.
123

One-pot green synthesis and bio-assay of…
Diethyl((4-chloro-3-nitrophenyl)(5-hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)methyl)
phosphonate(4l) White solid; Yield: 88%. M.p. [300 °C, FT-IR (neat, cm-1) m 3425 (–
1
OH), 2979 (Ar–H), 1246 (–P=O), 1036 (–P–O–C–), 755 (–P–C–). H-NMR (CDCl3): d
7.93 (2H, d, J = 8.0 Hz), 7.66 (2H, t, J = 12.0 Hz), 7.62 (1H, d, J = 8.0 Hz), 7.44 (1H,
t, J = 12.0 Hz), 7.33(1H, m), 7.26 (1H, m), 5.89 (1H, s), 4.97 (1H, d, J = 12.0 Hz),
4.08–3.74 (4H, m), 2.33 (3H, s), 1.19 (3H, t, J = 12.0 Hz), 1.15 (3H, t, J = 12.0 Hz).
13C-NMR (CDCl3): d 147.67, 145.93, 142.91, 138.49, 137.51, 132.63, 131.46 (d, Jc-
p = 64.0 Hz), 128.84 (d, Jc-p = 20.0 Hz), 126.21, 124.57 (d, Jc-p = 20.0 Hz), 121.63
(d, Jc-p = 192.0 Hz), 103.65, 63.44 (d, Jc-p = 20.0 Hz), 61.90 (d, Jc-p = 12.0 Hz),
44.66 (d, Jc-p = 240.0 Hz), 16.39, 12.64, 11.86. 31P-NMR (CDCl3): d 21.78. MS (ESI):
m/z 480.10 Anal. Calcd. for C21H23 ClN3O6P: C, 52.56; H, 4.83; Cl, 7.39; N, 8.76; O,
20.01; P, 6.45. Found: C, 52.45; H, 4.74; Cl, 7.31; N, 8.66; O, 19.94; P, 6.36.
Diethyl((5-hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)(naphthalen-2-yl)methyl)phos-
phonate(4m) [24, 25] Light yellow solid; Yield: 93%. M.p. [300 °C. FT-IR (neat,
1
cm-1) m 3436 (–OH), 2935 (Ar–H), 1235 (–P=O), 1041 (–P–O–C–), 738 (–P–C–). H-
NMR (CDCl3): d 8.25 (1H, d, J = 8.0 Hz), 7.90 (3H, d, J = 7.0 Hz), 8.0 (2H, m), 7.64
(1H, t, J = 7.0 Hz), 7.57–7.43 (4H, m), 7.29–7.24 (1H, m), 5.25 (1H, d, J = 27.9 Hz),
5.80 (1H, s), 4.16–3.78 (4H, m), 2.42 (3H, s), 1.62 (3H, t, J = 12.0 Hz), 1.14 (3H, t,
J = 12.0 Hz). 13C-NMR (CDCl3): d 151.76 (d, Jc-p = 54.70 Hz), 148.1 (d, Jc-
p = 124.28 Hz), 139.34, 134.42, 132.36 (d, Jc-p = 48.0 Hz), 131.51 (d, Jc-
p = 28.0 Hz), 129.16 (d, Jc-p = 28.0 Hz), 128.40 (d, Jc-p = 24.0 Hz), 126.66,
126.08 (d, Jc-p = 24.0 Hz), 125.9 (d, Jc-p = 32.0 Hz), 123.50, 122.96, 94.12 (d, Jc-
p = 48.0 Hz), 64.5 (d, Jc-p = 54.0 Hz), 63.6 (d, Jc-p = 76.0 Hz), 42.60 (d, Jc-
p = 137.0 Hz), 16.54, 13.79, 12.16. 31P-NMR (CDCl3): d 21.16. MS (ESI): m/z 451.17
Anal. Calcd. for C25H27N2O4P: C, 66.66; H, 6.04; N, 6.22; O, 14.21; P, 6.88. Found: C,
66.58; H, 5.96; N, 6.13; O, 14.12; P, 6.79.
Diethyl(anthracen-9-yl(5-hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)methyl)phos-
phonate(4n) Brown solid; Yield: 94%. M.p. [300 °C. FT-IR (neat, cm-1) m 3391 (-
OH), 2986 (Ar–H), 1247 (–P=O), 1047 (–P–O–C–), 726 (–P–C–). 1H-NMR (CDCl3):
8.29 (1H, d, J = 8.0 Hz), 7.95 (2H, d, J = 8.0 Hz), 7.91 (2H, t, J = 12.0 Hz), 7.58
(2H, d, J = 8.0 Hz), 7.51 (2H, t, J = 12.0 Hz), 7.44-7.38 (5H, m), d 5.82 (1H, s), 5.12
(1H, d, J = 8.0 Hz), 4.22–3.82 (4H, m), 2.64 (3H, s), 1.35 (3H, t, J = 12.0 Hz), 1.12
(3H, t, J = 12.0 Hz). 13C-NMR (CDCl3): d 145.86 (d, Jc-p = 58.0 Hz), 145.25 (d, Jc-
p = 12.0 Hz), 138.16, 133.42, 130.48 (d, Jc-p = 24.0 Hz), 129.58 (d, Jc-
p = 48.0 Hz), 129.16 (d, Jc-p = 48.0 Hz), 127.45 (d, Jc-p = 24.0 Hz), 126.90,
126.46 (d, Jc-p = 24.0 Hz), 124.86 (d, Jc-p = 36.0 Hz), 124.36 (d, Jc-p = 84.0 Hz),
124.19 (d, Jc-p = 24.0 Hz), 122.08, 98.42 (d, Jc-p = 76.0 Hz), 64.49 (d, Jc-
p = 24.0 Hz), 63.46 (d, Jc-p = 24.0 Hz), 45.62 (d, Jc-p = 158.0 Hz), 16.51, 13.80,
12.14. 31P-NMR (CDCl3): d 19.26. MS (ESI): m/z 501.30 Anal.Calcd. for
C29H29N2O4P: C, 69.59; H, 5.84; N, 5.60; O, 12.79; P, 6.19. Found: C, 69.48; H,
5.76; N, 5.51; O, 12.70; P, 6.11.
Diethyl((5-hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)(pyren-1-yl)methyl)phos-
phonate(4o) Brown solid; Yield: 94%. M.p. [ 300 °C. FT-IR (neat, cm-1) m 3386
(OH), 2998 (Ar–H), 1224 (–P=O), 1043 (–P–O–C–), 746 (–P–C–). 1H-NMR
(CDCl3): 8.29 (1H, d, J = 8.0 Hz), 7.93 (2H, t, J = 12.0 Hz), 7.78 (2H, d,
J = 8.0 Hz), 7.73 (1H, d, J = 8.0 Hz), 7.71-7.68 (4H, m), 7.62 (1H, t,
123

G. Mohan et al.
J = 12.0 Hz), 7.49 (2H, d, J = 8.0 Hz), 7.43 (1H, t, J = 12.0 Hz), 7.37–7.34 (1H,
m), d 5.88 (1H, s), 4.51 (1H, d, J = 8.0 Hz), 4.18–4.10 (2H, m), 4.06-3.80 (4H, m),
2.56 (3H, s), 1.24 (3H, t, J = 12.0 Hz), 1.12 (3H, t, J = 12.0 Hz). 13C-NMR
(CDCl3): d 147.36 (d, Jc-p = 48.0 Hz), 145.52 (d, Jc-p = 57.0 Hz), 138.10, 135.21
(d, Jc-p = 120.0 Hz), 131.43, 130.52 (d, Jc-p = 54.0 Hz), 129.76 (d, Jc-
p = 58.0 Hz), 129.44 (d, Jc-p = 28.0 Hz), 129.34 (d, Jc-p = 20.0 Hz), 128.26
(d, Jc-p = 58.0 Hz), 127.86 (d, Jc-p = 54.0 Hz), 127.64 (d, Jc-p = 28.0 Hz),
127.48, (d, Jc-p = 28.0 Hz), 126.45 (d, Jc-p = 48.0 Hz), 125.06 (d, Jc-
p = 36.0 Hz), 122.62 (d, Jc-p = 54.0 Hz), 98.18 (d, Jc-p = 84.0 Hz), 64.6 (d,
Jc-p = 24.0 Hz), 63.4 (d, Jc-p = 12.0 Hz), 48.6 (d, Jc-p = 154.0 Hz), 16.54,
13.86, 11.61. 31P-NMR (CDCl3): d 22.16. MS (ESI): m/z 525.19 Anal. Calcd. for
C31H29N2O4P: C, 70.98; H, 5.57; N, 5.34; O, 12.20; P, 5.90. Found: C, 70.91; H,
5.48; N, 5.22; O, 12.15; P, 5.82.
Anti-oxidant assay
The anti-oxidant activity of the title compounds (4a–4o) was evaluated by DPPH
[33] and H2O2 [34] methods,. Ascorbic acid was used as standard control. The
radical scavenging activity (RSA) was calculated by using the equation:
%RSA ¼ ½ðAc À Asފ  100
where Ac is the absorbance of the control and As is the absorbance of the tested
sample. The anti-oxidant activity was also expressed as IC50. The IC50 values were
well defined as the concentration (in lg/mL) of the compounds at which formation
of DPPH and H2O2 radicals was inhibited by 50%.
In vitro anticancer assay
The in vitro anticancer activity of the title compounds (4a–o) was tested against the
proliferation of MCF-7 (breast) DU-145 (prostate) and A-549 (lung cancer) cell
lines by performing a sulforhodamine-B (SRB) assay [35]. The cell line of interest
was seeded in disinfected flat-bottomed 96-well plate (5000 cells/100 lL) in a
medium containing 10% fetal bovine serum and antibiotics (penicillin and
streptomycin). After incubation for 18–20 h in an incubator continuously supplied
with 5% CO2 to ensure appropriate adherence of the cells to the surface bottom of
the wells, the cells were treated with the compounds or the reference standard
doxorubicin. To treat cells, working dilutions of concentrations of the compounds
were prepared, of which a 2-lL aliquot was added to every well, thereby creating
the final concentration of the compounds of 0–100 lM. Each compound was tested
in triplicate and the cytotoxicity was determined as the average of that triplicate.
DMSO and doxorubicin (as standard control anti-cancer drug) were taken as vehicle
and positive controls respectively. Further, the cells were allowed to grow for
another 48 h in an incubator maintained at 37 °C with a constant supply of 5% CO2.
The plates were then air-dried and 100 lL of 10 mM Tris Base was added to each
well to solubilize the SRB before reading the absorbance using Perkin-Elmer
123

One-pot green synthesis and bio-assay of…
Multimode Reader at 510 nm. The amount of absorbance is directly relative to cell
growth and is thus used to calculate the IC50 values. In this study, for initial
screening, three types of cancer cell lines, i.e. human breast cancer (MCF-7), lung
cancer (A-549), and prostate cancer (DU-145) cell lines were tested for the
cytotoxic effect of the series of compounds.
Results and discussion
Chemistry
A strategic approach has been made for the C-P bond formation in the one-pot
synthesis of pyrazolylphosphonates (4a–o) by the reaction of 3-methyl-1-phenyl-5-
pyrazolone (1) with various aryl aldehydes (2a–o) and diethyl phosphite (3) under
green conditions using b-CD as a catalyst, which offered good to excellent yields
after a simple work-up procedure.
In order to determine the best experimental conditions, a model reaction has been
carried out by taking 3-methyl-1-phenyl-5-pyrazolone (1; 1 mmol), 4-ethoxyben-
zaldehyde (2a; 1.2 mmol) and diethyl phosphite (3; 2 mmol) for the synthesis of
diethyl((5-hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)(4-
ethoxyphenyl)methyl)phosphonate (4a). Initially, we have run the reaction under
room temperature (r.t.) and solvent-free conditions without a catalyst which resulted
in no reaction, even after 24 h (Table 1, entry 1). Then, we used different catalysts
such as FeCl3, InCl2, ZnCl2, ZnBr2, CeCl3.7H2O, BF3.SiO2, Amberlyst-15, p-TSA,
PS-PTSA, TMG, DABCO, DBU and b-CD (Table 1, entries 2–15). Among all of
them, b-CD in 10 mol% showed better catalytic activity; the reaction proceeded
very smoothly and gave the product 4a in 96% yield (Table 1, entry 15).
The reaction proceeded smoothly in solvent-free conditions. However, to study
the effect of solvent on the reaction, various polar solvents like methanol, ethanol,
isopropanol, tetrahydrofuran, and acetonitrile were tried. The reaction in methanol,
ethanol and isopropanol (Table 2, entries 1–3) decreased the product yield, but the
rate of reaction was increased due to the solubility factor. In THF and acetonitrile
(Table 2, entries 4, 5) to obtain 88 and 73% yields, the reaction has to be run for 4
and 3 h, respectively, owing to the aprotic polar nature of the solvent. When the
reaction was run in non-polar solvents such as toluene, CH2Cl2, and CHCl3
(Table 2, entries 6–8), both the rate of the reaction and product yields were
decreased.
To determine the optimum catalyst b-CD loading, model reactions were carried
out using 1, 3, 5, 10, and 15 mol% (Table 2, entries 9–13) of b-CD at room
temperature under solvent-free conditions and obtained the product yields of 60, 73,
85, 96 and 96%, respectively. Increasing the amount of catalyst beyond 10 mol%
had no additional effect either on the reaction rate or the product yield.
After establishing the optimum reaction conditions, we investigated the scope of
the reaction by condensing 3-methyl-1-phenyl-2-pyrazoline-5-one (1) with various
commercially available substituted aryl aldehydes having different electronically
activating or deactivating substituents (2a–o) and diethyl phosphite (3) to form
123

G. Mohan et al.
Table 1 Effect of the various catalysts on the synthesis of diethyl((5-hydroxy-3-methyl-1-phenyl-1H-
pyrazol-4-yl)(4-ethoxyphenyl)methyl)phosphonate (4a)
Entry
Catalyst (mol%)
Time (min)
Yielda (%)
1.
No catalyst
24 h
30
30
30
30
30
30
30
30
30
30
30
30
30
30
Nrb
32
38
35
43
54
55
62
69
75
82
85
87
90
96
2.
FeCl3 (10)
3.
InCl3 (10)
4.
ZnCl2 (10)
5.
ZnBr2 (10)
6.
BF3-OEt2 (10)
CeCl3Á7H2O (10)
BF3ÁSiO2 (10)
Amberlyst 15 (10)
p-TSA (10)
PS-PTSA (10)
TMG (10)
7.
8.
9.
10.
11.
12.
13.
14.
15.
DABCO (10)
DBU (10)
b-CD (10)
Reaction of 3-methyl-1-phenyl-5-pyrazolone (1 mmol), 4-ethoxybenzaldehyde (1.2 mmol) and diethyl
phosphite (2 mmol) using various catalysts along with b-CD in different concentrations under r.t. and
neat conditions
aIsolated yield
bNo reaction
corresponding pyrazolylphosphonates (4a–o). The details of the physical data, such
as yield and melting points, are illustrated in Table 3.
The plausible reaction sequences taking place in the synthesis of pyrazolylphos-
phonats is shown in Scheme 2. b-CD favors the Knoevenagel condensation through
activation of the carbonyl group of aldehyde (a) via hydrogen bonding
(supramolecular interaction) [32] and form intermediate (b), which can react with
3-methyl-1-phenyl-1H-pyrazol-5-ol (d) (enol form of 3-methyl-1-phenyl-2-pyrazo-
line-5-one (c)) to form intermediate (e) which on dehydration forms (f).
Phosphorylation reaction of (f) with diethyl phosphite leads to the formation of
corresponding pyrazolylphosphonate (g).
123

One-pot green synthesis and bio-assay of…
Table 2 Optimization of
Entry Catalyst (mol %) Solvent
Time (h) Yield (%)a
reaction, conditions on solvent
and catalyst loading for the
synthesis of 4a
1.
b-CD (10)
b-CD (10)
b-CD (10)
b-CD (10)
b-CD (10)
b-CD (10)
b-CD (10)
b-CD (10)
b-CD (1)
Methanol
Ethanol
IPA
2
86
87
85
88
73
55
67
60
60
73
85
96
96
2.
5
3.
6
4.
THF
4
5.
CH3CN
Toluene
CH2Cl2
CHCl3
3
6.
18
24
15
7.
Reaction conditions: 3-methyl-
1-phenyl-5-pyrazolone
(1.0 mmol),
8.
9.
Solvent-free 0.5
Solvent-free 0.5
Solvent-free 0.5
Solvent-free 0.5
Solvent-free 0.5
4-ethoyybenzaldehyde
(1.2 mmol), and
diethylphosphite (2.0 mmol) in
10.
11.
12.
13.
b-CD (3)
b-CD (5)
the presence of b-CD
aYield of the isolated product.
Solvent-free and r.t. conditions
b-CD (10)
b-CD (15)
The title compounds (4a–o) were fully characterized by physical and spectral
(FT-IR, NMR, and mass) data. In the FT-IR spectra of the title compounds, the
following bands were detected: (1) –OH stretching vibrations at 3442–3364 cm-l;
(2) –P=O stretching vibrations at 1254–1180 cm-1; (3) –P–O–C stretching
vibrations at 1048–1006 cm-l; and (4) P–C stretching vibrations at
780–720 cm-l. In the 400 MHz 1H–NMR spectra of 4a–o (in CDCl3), the
following signals were detected: (1) the chemical shifts in the region of
8.24–6.48 ppm are due to aromatic protons; (2) a singlet at d 5.96–5.62 confirmed
the –OH proton and the doublet at 5.64–4.46 ppm corresponds to HC–P proton; (3)
the multiplet in the region of 3.98–3.82 ppm and multiplet in the region of
3.56–3.32 ppm are due to the O–CH2–CH3 protons; (4) a singlet at 2.52–2.36 ppm
is due to CH3 protons; and (5) a triplet at 1.38–1.18 ppm and a triplet at
1.24–1.12 ppm are due to the O–CH2–CH3 protons. 13C–NMR (100 MHz, CDCl3,
ppm) reveals that the chemical shifts in the region of 184.5–101.3 ppm are assigned
to carbons of aromatic ring, the signals in the region of 64.8–58.6, 59.2–48.8 and
16.3–16.2 ppm confirmed the –O–CH2–CH3, HC–P and –O–CH2–CH3 carbons, and
at 24.6–21.4 and 12.7–11.8 ppm confirmed the –CH3 carbon. The 31P NMR
(165.9 MHz, CDCl3 and ppm) chemical shifts of the title compounds appeared in
the range from 18.7 to 24.6 ppm.
Pharmacology
In vitro anti-oxidant activity
The synthesized pyrazolylphosphonates (4a–o) were screened for their free radical
scavenging activity by the DPPH [33] and H2O2 [34] methods. Ascorbic acid was
used as standard control (Table 4). A lower IC50 value is associated with a higher
radical scavenging activity. The DPPH and H2O2 radical scavenging activities of the
123

G. Mohan et al.
Table 3 Synthesis of pyrazolylphosphonates (4a–o) on b-CD catalyst by r.t. and neat conditions via
Scheme 1
Structures of aryl
aldehydes (2a–o)
Structures of products
Time Yielda Melting
S. No.
(4a–o)
(Min)
(%)
Point (˚C)
1
30
96
> 300
> 300
[24]
2
3
4
5
30
30
30
30
96
95
> 300
> 300
94
96
> 300
> 300
6
7
30
30
91
92
> 300
[23]
123

One-pot green synthesis and bio-assay of…
Table 3 continued
Structures of aryl
aldehydes (2a–o)
Structures of products
(4a–o)
Time Yielda Melting
S. No.
(Min)
(%)
Point (˚C)
8
30
92
> 300
> 300
[24,25]
9
30
91
10
11
30
30
90
89
> 300
> 300
12
30
88
> 300
> 300
[24,25]
13
14
30
30
93
94
> 300
> 300
15
30
94
Reaction conditions: 3-methyl-1-phenyl-2-pyrazoline-5-one (1.0 mmol), various substituted aryl alde-
hydes (1.2 mmol), and diethyl phosphite (2.0 mmol) in the presence of 10 mol% of b-CD as catalyst at
solvent-free and r.t. conditions in 30 min
aIsolated yields
123

G. Mohan et al.
Scheme 2 Plausible mechanism for the catalytic activity of b-CD in the formation of
pyrazolylphosphonates (4a–o)
title compounds (4a–o) are shown in Table 4. Compounds 4g, 4i, 4h and 4f
exhibited the lowest anti-oxidant activities, compared to the IC50 of ascorbic acid
used as standard. While nearly the same activities were revealed in the compounds
4c and 4d compared to the IC50 of ascorbic acid. The compounds 4m, 4n, 4o, 4j, 4k
and 4l showed moderate to good anti-oxidant activity and the compounds 4e, 4b and
4a showed potent anti-oxidant activity compared to the IC50 of ascorbic acid.
In vitro anticancer activity
The synthesized pyrazolylphosphonates (4a–o) were subjected to the well-known
SRB cytotoxic assay [35] against human breast (MCF-7), prostate (DU-145), and
lung (A-459) cancer cell lines to investigate the effectiveness of the in vitro cell
cytotoxic properties (Table 5). All the data were expressed as IC50 values. The
obtained data revealed that the compounds 4o, 4n and 4m have excellent cell
growth inhibitory effects on MCF-7, DU-145 and A-549 cell lines with IC50 values
compared to the IC50 of the doxorubicin standard used. While compounds 4l, 4f, 4h,
4g and 4i showed moderate cytotoxic activity, the remaining compounds, 4k, 4j, 4b,
4c, 4b, 4a and 4e, could not show effective cytotoxic activity on the three cell lines.
Structure activity relationship (SAR) studies
Generally, the presence of electron-donating substituents such as N-alkyl, alkoxy
and alkyl groups enhances the anti-oxidant property while electron-withdrawing
aryl and halogen groups suppress the DPPH and H2O2 radical scavenging ability.
The structure activity relationship (SAR) studies regarding anti-oxidant activity of
the synthesized compounds with electron donor substituents like 4e, 4d, 4c, 4b and
4a bearing 4-(diethylamino)phenyl, 3,4,5-trimethoxyphenyl, 3,4-dimethoxyphenyl,
123

One-pot green synthesis and bio-assay of…
Table 4 Free radical scavenging activity of pyrazolylphosphonates (4a–o)
S. no.
Tested
samples
DPPH radical scavenging
activity IC50 (lg/mL)
Hydrogen peroxide scavenging
activity IC50 (lg/mL)
1.
4a
12.35
11.39
13.57
13.88
10.24
41.26
28.81
38.67
32.93
19.85
20.32
21.45
15.77
18.63
19.24
13.67
23.55
22.76
24.47
24.88
19.57
53.18
40.81
48.67
42.93
30.64
33.12
29.25
27.47
26.83
27.24
24.52
2.
4b
3.
4c
4.
4d
5.
4e
6.
4f
7.
4g
8.
4h
9.
4i
10.
11.
12.
13.
14.
15.
16.
4j
4k
4l
4m
4n
4o
Ascorbic acid
Table 5 In vitro anticancer
activity of
pyrazolylphosphonates (4a–o)
S. no.
Tested samples
MCF-7
DU-145
A-549
IC50 (lg/mL)
1.
4a
46.885
12.634
32.672
29.387
52.632
13.855
17.678
15.284
20.987
11.673
10.453
26.423
9.867
36.673
10.187
26.773
18.166
38.401
10.869
13.144
11.824
15.543
9.316
42.959
10.892
34.515
26.134
48.964
11.863
16.514
13.039
19.120
10.230
9.646
2.
4b
3.
4c
4.
4d
5.
4e
6.
4f
7.
4g
8.
4h
9.
4i
9.
4j
10.
11.
12.
13.
14.
15.
4k
8.861
4l
16.826
9.839
20.763
8.113
4m
4n
9.187
7.672
6.483
4o
7.854
6.753
5.967
Doxorubicin
9.652
7.114
8.340
123

G. Mohan et al.
4-ethylphenyl and 4-methyl phenyl groups were found to be the most active
derivatives. Compounds with electron withdrawing groups 4g, 4i, 4h, 4f, 4j and 4k
showed moderate radical scavenging activity, whereas compounds 4m, 4n and 4o
with poly aromatics like naphthalene, anthracene and pyrene ring systems revealed
less activity.
SAR studies showed that the anticancer activity increased with the increase of the
electron-withdrawing nature of groups on the phenyl ring. The compounds having
poly aromatics like naphthalene, anthracene and pyrene ring systems (4m, 4n and
4o) showed pronounced anticancer activity, followed by the compounds having halo
and nitro substituted phenyl groups (4g, 4i, 4h, 4f, 4j, 4k and 4l). the compounds
with electron donor substituents like 4-(diethylamino) phenyl, 3,4,5-trimethox-
yphenyl, 3,4-dimethoxy phenyl, 4-ethylphenyl and 4-methylphenyl groups (4e, 4d,
4c, 4b and 4a) were found to be the least active anticancer agents.
Conclusion
In summary, we have demonstrated an efficient and eco-friendly protocol for the
synthesis of pyrazolylphosphonates through the b-CD catalyzed reaction of
pyrazolone with aryl aldehydes and diethyl phosphite. This new method is endowed
with green reaction conditions such as low cost, use of non-toxic catalyst, solvent-
free medium, easy work-up process and good yields. The title compounds were
screened for anti-oxidant activity by DPPH and H2O2 radical scavenging assay and
anticancer activity against breast cancer (MCF-7), prostate cancer (DU-145) and
lung cancer (A-549) cell lines with sulfarodamine-B assay. The compounds 4e, 4d,
4c, 4b and 4a showed promising anti-oxidant activity and the compounds 4m, 4n
and 4o showed significant anticancer activity.
Acknowledgements The authors are grateful to Prof. C. Devendranath Reddy, Department of Chemistry,
S.V. University, Tirupati, for his helpful discussions and acknowledge the funding support from DST-
SERB, New Delhi, India, through Project No. SB/S1/OC-96/2013, Dt. 05-11-2014. One of the authors Dr.
S. Santhisudha expresses sincere thanks to the Science and Engineering Research Board (SERB), New
Delhi, India, for providing financial assistance (DST No: PDF/2016/000102, Dt: 11-07-2016). The
corresponding author, Prof. C. Suresh Reddy, being an associate member of Institute of Food Security and
Sustainable Agriculture, University Malaysia Kelantan, Jeli Campus, Malaysia, expresses his sincere
thanks to University Malaysia Kelantan for their timely encouragement in our research work.
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