One-pot green synthesis and bio-assay of pyrazolylphosphonates

Res Chem Intermed

https://doi.org/10.1007/s11164-018-3319-y

One-pot green synthesis and bio-assay

of pyrazolylphosphonates

Gundluru Mohan¹Sarva Santhisudha^1,2Sudileti Murali¹

•

Nemallapudi Bakthavatchala Reddy^1,3Gundala Sravya^1,3

Grigory V Zyryanov^3,4Cirandur 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

H₂O₂radical 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 H₂O₂radical 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% NaHCO₃solution, 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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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 C₂₁H₂₃ClN₃O₆P: 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 ½ðA_cÀ A_sÞ Â 100

where A_cis the absorbance of the control and A_sis the absorbance of the tested

sample. The anti-oxidant activity was also expressed as IC₅₀. The IC₅₀values were

well deﬁned as the concentration (in lg/mL) of the compounds at which formation

of DPPH and H₂O₂radicals 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% CO₂to 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 IC₅₀values. 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)

Yield^a(%)

1.

No catalyst

24 h

30

Nr^b

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 (1)

Methanol

Ethanol

IPA

2

86

87

85

88

73

55

67

60

73

85

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

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–CH₃protons; (4) a singlet at 2.52–2.36 ppm

is due to CH₃protons; 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–CH₃protons. 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–CH₃carbons, and

at 24.6–21.4 and 12.7–11.8 ppm conﬁrmed the –CH₃carbon. The ³¹P NMR

(165.9 MHz, CDCl₃and 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 IC₅₀value is associated with a higher

radical scavenging activity. The DPPH and H₂O₂radical 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 Yield^aMelting

S. No.

(4a–o)

(Min)

(%)

Point (˚C)

1

30

96

> 300

[24]

2

3

4

5

30

96

95

> 300

94

96

> 300

6

7

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 Yield^aMelting

S. No.

(Min)

(%)

Point (˚C)

8

30

92

> 300

[24,25]

9

30

91

10

11

30

90

89

> 300

12

30

88

> 300

[24,25]

13

14

30

93

94

> 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 IC₅₀of ascorbic acid

used as standard. While nearly the same activities were revealed in the compounds

4c and 4d compared to the IC₅₀of 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 IC₅₀of 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 IC₅₀values. 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 IC₅₀values

compared to the IC₅₀of 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 H₂O₂radical 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,

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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 H₂O₂radical 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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One-pot green synthesis and bio-assay of pyrazolylphosphonates

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