Aqueous phase synthesis, crystal structure and biological study of isoxazole extensions of pyrazole-4-carbaldehyde derivatives

Article abstract of DOI:10.1016/j.molstruc.2017.08.094

A series of novel isoxazol derivatives was synthesized by green route in aqueous phase at room temperature by the reaction of 3-methyl-4H-isoxazol-5-one with 3-(substituted phenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde by one-pot Knoevenagel condensation method using sodium benzoate as a catalyst. Compounds were characterized on the basis of IR, ¹H NMR, mass spectroscopy and melting point determination. Crystal structures of five compounds were determined by X-ray diffraction. The compounds formed were screened for antibacterial and antifungal activity. Some compounds showed activity close to ampicillin against E. coli, S. aureus, and S. pyogenus. Two compounds showed antifungal activity against C. albicans close to standard greseofulvin.

Full text of DOI:10.1016/j.molstruc.2017.08.094

Accepted Manuscript
Aqueous phase synthesis, crystal structure and biological study of isoxazole
extensions of pyrazole-4-carbaldehyde derivatives
Sachin S. Wazalwar, Anita R. Banpurkar, Franc Perdih
PII:
S0022-2860(17)31174-2
10.1016/j.molstruc.2017.08.094
MOLSTR 24232
DOI:
Reference:
To appear in:
Journal of Molecular Structure
Received Date:
Revised Date:
Accepted Date:
11 June 2017
09 August 2017
29 August 2017
Please cite this article as: Sachin S. Wazalwar, Anita R. Banpurkar, Franc Perdih, Aqueous phase
synthesis, crystal structure and biological study of isoxazole extensions of pyrazole-4-carbaldehyde
derivatives, Journal of Molecular Structure (2017), doi: 10.1016/j.molstruc.2017.08.094
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ACCEPTED MANUSCRIPT
1] Series of novel isoxazole derivatives is synthesized by green route in aqueous phase.
2] Effect of the catalyst was monitored.
3] 5 new crystal structures are reported with hydrogen bonding and π-π interactions.
4] Hydrogen bonding diversity was observed in 3-hydroxy and 4-hydroxy derivatives.
5] Antibacterial and antifungal studies are carried out for all compounds.

ACCEPTED MANUSCRIPT
Aqueous phase synthesis, crystal structure and biological study of isoxazole extensions of
pyrazole-4-carbaldehyde derivatives
Sachin S. Wazalwara, Anita R. Banpurkara, Franc Perdihb
a- Rajiv Gandhi College of Engineering, Research & Technology, Chandrapur, INDIA
b- Faculty of Chemistry and Chemical Technology, University of Ljubljana, Slovenia
Abstract: A series of novel isoxazol derivatives was synthesized by green route in aqueous phase at room
temperature by the reaction of 3-methyl-4H-isoxazol-5-one with 3-(substituted phenyl)-1-phenyl-1H-pyrazole-4-
carbaldehyde by one-pot Knoevenagel condensation method using sodium benzoate as a catalyst. Compounds were
characterized on the basis of IR, 1H NMR, mass spectroscopy and melting point determination. Crystal structures of
five compounds were determined by X-ray diffraction. The compounds formed were screened for antibacterial and
antifungal activity. Some compounds showed activity close to ampicillin against E. coli, S. aureus, and S. pyogenus.
Two compounds showed antifungal activity against C. albicans close to standard greseofulvin.
Keywords: Aqueous phase synthesis, One-pot synthesis, Isoxazol derivatives, 3-Methyl-4H-isoxazol-5-one, 3-
(substituted phenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde
Introduction:
Aqueous phase synthesis of organic compounds is environmentally friendly, safe, non-toxic, clean, green and
inexpensive [1-3]. Many important transformations can be carried out in water [1-3]. Multicomponent reactions in
water is the best method for the synthesis of a variety of organic compounds under eco friendly conditions [4,5]. Use
of water decreases the risk associated with organic solvents and also maintains a healthy laboratory atmosphere for
the workers. Thus, water as a reaction media is attractive from both the economical and environmental point of view
[6]. Isoxazole derivatives have served as a versatile building block in organic synthesis [7]. Isoxazol-5-one
derivatives have attracted increasing interest because of their significant pharmaceutical and therapeutic properties,
such as hypoglycaemic, immunosuppressive, anti-inflammatory, and anti-bacterial activities [8-11]. In addition,
isoxazole core is a structural element in many commercial pharmaceuticals [12-15].
The present work was carried out using conventional method to synthesize 4-[3-(substituted phenyl)-1-phenyl-1H-
pyrazole-4-ylmethylene]-3-methyl-4H-isoxaol-5-ones. At first step, ethyl acetoacetate reacts with hydroxylamine
hydrochloride to afford oxime, and further ring closure occurs to form product 3-methyl-4H-isoxazol-5-one. And
then, the Knoevenagel reactions between 3-methyl-4H-isoxazol-5-one and 3-(substituted phenyl)-1-phenyl-1H-
pyrazole 4-carbaldehydes produces final product.
In recent years, some new methods such as solid state grinding [16], solid state heating [16] and multicomponent
reactions [17,18] have been reported, with variable yields of condensation compounds in solution or under solvent-
free conditions. Recently, synthesis of these compounds was reported to be carried out using ultrasound irradiation
[19]. A study on effect of solvent and mole percent of sodium benzoate as catalyst was also reported earlier [20].
In present study, the conventional method of aqueous phase stirring was adopted for the synthesis of 3-methyl-4H-
isoxazol-5-one. The second step i.e. Knoevenagel reaction was carried out in situ by adding appropriate pyrazole-4-

ACCEPTED MANUSCRIPT
carbaldehyde derivative along with a catalyst. Knoevenagel reaction was carried out without catalyst and also by
using sodium benzoate as catalyst, separately. Second step was also carried out as room temperature stirring only.
Present paper reports the antibacterial and antifungal study of all synthesized products along with crystal structure of
five compounds. Some compounds were found to have activity comparable to ampicillin against E. coli and one
compound was found to have activity comparable to S. pyogenus. This is the first known report of crystal structure
of this class of molecules.
Experimental:
Materials and method: All the starting material and the solvents were purchased from commercial sources and
were used without further purification. Melting points were determined in open capillaries using electro thermal
melting point apparatus and are uncorrected. Progress of reactions was monitored by TLC. Infrared (IR) spectra
(4000–600 cm–1) of the samples were recorded using a Perkin–Elmer Spectrum 100, equipped with a Specac Golden
Gate Diamond ATR as a solid sample support. 1H NMR spectra were recorded with a Bruker Avance III 500 nuclear
magnetic resonance spectrometer with TMS as internal reference. MS spectra were recorded with an Agilent 6624
Accurate Mass TOF LC/MS instrument (ESI ionization).
General procedure for synthesis of 4-[3-(substituted phenyl)-1-phenyl-1H-pyrazole-4-ylmethylene]-3-methyl-
4H-isoxaol-5-one: The starting materials, 3-(substituted phenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde 4a–h were
prepared by using Vilsmeier–Haack reaction of acetophenone phenylhydrazone 3a–h, which was obtained from
substituted acetophenone 1a–h and phenyl hydrazine 2 in the presence of ethanol containing a catalytic amount of
HCl (Scheme I) [21].
0.015 mol hydroxylamine hydrochloride (1.04 g) was dissolved in 20 ml of water and cooled to about 10°C, and
then 0.015 mol ethyl acetoacetate (1.95 g) was added. The mixture was shaken thoroughly till the appearance of pale
yellow color. To this solution 0.015 mol 3-(substituted phenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde (4a-h) was
added and stirred thoroughly without any catalyst till completion of the reaction for about 2.5hours. Completion of
the reaction was monitored by TLC. Dark yellow colored product was obtained which was filtered, dried and
washed with hot ethanol (80%, mixture of 80 ml absolute ethanol and 20 ml water) to remove unreacted pyrazol
carbaldehyde if any, and recrystallized using absolute ethanol (99.9% minimum assay). The same procedure was
repeated using sodium benzoate as a catalyst (0.0015 mol) which showed increase in yield compared to that without
using catalyst (Scheme-II) [19]. Crystals of five compounds were obtained by slow evaporation using mixture of
ethanol and chloroform (1:1 v/v) as solvent. Crystals suitable for crystallographic study could not be obtained for 8c,
8e and 8h.
Spectral Data for compounds 4a–h:
1]1,3-Diphenyl-1-H Pyrazole-4-carbaldehde (4a): Off white needle like crystals, yield 89%, Melting point 1350C,
1
IR:(υmax/cm-1); 3121 (Ar-CH), 2828 (C–H in CHO), 1677(C=O), 1519 (C=N), 1443(C=C); H NMR (500 MHz,

ACCEPTED MANUSCRIPT
CDCl3, δppm): 9.96 (s, 1H, –CHO); 8.46 (s, 1H, Pyrazole-CH); 7.72-7.68 (m, 4H, Ar-H); 7.56-7.55 (m, 2H, Ar-H);
7.46-7.43(m, 2H, Ar-H); 7.35-7.32(m, 1H, Ar-H).
2]3-(4-Chloro-phenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde (4b)- Off white needle like crystals, yield 87%,
Melting point 1220C, IR: (υmax/cm-1); 3124 (Ar-CH), 2835 (C–H in CHO), 1667(C=O), 1513 (C=N), 1449(C=C),
678 (C-Cl); 1H NMR (500 MHz, CDCl3, δppm): 9.97 (s, 1H, –CHO); 8.46 (s, 1H, Pyrazole-CH); 7.76(d, 2H, Ar-H);
7.72(d, 2H, Ar-H); 7.47-7.44(t, 2H, Ar-H); 7.41(d, 2H, Ar-H); 7.35-7.32 (t, 1H, Ar-H).
3]3-(4-Bromo-phenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde (4c)- Off white needle like crystals, yield 88%,
Melting point 1300C, IR:(υmax/cm-1); 3124 (Ar-CH), 2828 (C–H in CHO), 1677(C=O), 1519 (C=N), 1443(C=C); 1H
NMR (500 MHz, CDCl3, δppm): 9.99 (s, 1H, –CHO); 8.48 (s, 1H, Pyrazole-CH); 7.76-7.72(m, 4H, Ar-H); 7.46-
7.43 (t, 4H, Ar-H); 7.34-7.31 (t, 1H, Ar-H).
4]3-(4-Methoxy-phenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde (4d)-Off white, yield 88%, Melting point 1100C,
1
IR: (υmax/cm-1); 3123 (Ar-CH), 2834 (C–H in CHO), 1667(C=O), 1515 (C=N), 1455(C=C); H NMR (500 MHz,
CDCl3, δppm): 9.97 (s, 1H, –CHO); 8.45 (s, 1H, Pyrazole-CH); 7.72(d, 4H, Ar-H); 7.45-7.42(t, 2H, Ar-H); 7.33-
7.30(t, 1H, Ar-H); 6.96 (d,2H,Ar-H); 3.80 (S, 3H, OCH3).
5]1-Phenyl-3-p-tolyl-1H-pyrazole-4-carbaldehyde (4e)- Off white, yield 89%, Melting point 1000C, IR: (υmax/cm-
1); 3125 (Ar-CH), 2838 (C–H in CHO), 1673(C=O), 1518 (C=N), 1450(C=C); 1H NMR (500 MHz, CDCl3, δppm):
9.98 (s, 1H, –CHO); 8.46 (s, 1H, Pyrazole-CH); 7,72(d, 2H, Ar-H); 7.63(d, 2H, Ar-H); 7.45-7.42(t, 2H, Ar-H); 7.30-
7.33(t, 1H, Ar-H); 7.24 (d, 2H, Ar-H); 2.36(s, 3H, Ar-CH3).
6]3-(4-Hydroxy-Phenyl)-1-phenyl-1-H-pyrazole-4-carbaldehde (4f)- Off white, yield 76%, Melting point 1500C,
1
IR:(υmax/cm-1); 3337Ar-OH), 3110 (Ar-CH), 2815 (C–H in CHO), 1680(C=O), 1513 (C=N), 1445(C=C); H NMR
(500 MHz, CDCl3, δppm): 9.95 (s, 1H, –CHO); 9.79(s, 1H, Ar-OH); 9.28(s, 1H, Pyrazole-CH); 7.98(d, 2H, Ar-H);
7.78 (d, 2H, Ar-H); 7.58-7.55(t, 2H, Ar-H); 7.43-7,40(t, 1H, Ar-H); 6.88(d, 2H, Ar-H).
7]3-(3-Hydroxy-Phenyl)-1-phenyl-1-H-pyrazole-4-carbaldehde (4g)- Off white, yield 72%, Melting point 900C,
1
IR:(υmax/cm-1): 3443(Ar-OH), 3124(Ar-CH), 2896(C–H in CHO), 1660(C=O), 1519(C=N), 1443(C=C); H NMR
(500 MHz, CDCl3, δppm): 9.98(s, 1H, –CHO); 9.66 (s,1H,Ar-OH); 9.29(s, 1H, Pyrazole-CH); 7.99-7.98(m, 2H, Ar-
H); 7.59-7.56(m, 2H, Ar-H); 7.45-7.42(m,1H,Ar-H); 7.35-7.29(m, 2H, Ar-H); 6.89-6.88 (m,1H, Ar-H).
8]3-(3-Nitro-phenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde (4h)-Greenish yellow, yield 87%, Melting point
1
1400C, IR: (υmax/cm-1): 3131(Ar-CH), 2835(C–H in CHO), 1680(C=O), 1596(C=N), 1525(C-NO2) 1425(C=C); H
NMR (500 MHz, CDCl3, δppm): 10.02 (s, 1H, –CHO); 8.51(s, 1H, Pyrazole-CH); 8.27-8.24(m, 2H, Ar-H); 7.75-
7.74 (m,2H, Ar-H); 7.63-7.59(m, 1H, Ar-H); 7.50-7.46(m, 2H, Ar-H); 7.38-7.35(m, 1H, Ar-H).
Spectral data for compounds 8a–h:

ACCEPTED MANUSCRIPT
1] 4-(1,3-Diphenyl-1H-pyrazol-4-ylmethylene)-3-methyl-4H-isoxazole-5-one (8a): Yellow crystalline solid, yield
65%, melting point 1800C, MS (ESI+) m/z 330.1233 (MH+). HRMS calcd. for C20H16N3O2: 330.1237. Found:
330.1233. IR: (υmax/cm-1): 3151(Ar-CH), 1745(C=O), 1615(HC=N), 1501(C=C-C) 1247(C-O). NMR (500 MHz,
CDCl3, δ/ppm): 9.94 (s, 1H, pyrazolic H); 7.82- 7.81 (m, 2H, Ar-H); 7.57-7.56(m, 2H, Ar-H); 7.49-7.44 (m, 5H, Ar-
H); 7.42(s, 1H, CAr=CH); 7.36-7.33 (m, 1H, Ar-H); 2.14 (s, 3H, -CH3 isoxazol).
2] 4-[3-(4-chloro-phenyl)-1-phenyl-1H-pyrazole-4-ylmethylene]-3-methyl-4H-isoxaol-5-one (8b): Yellow
crystalline solid, yield 50%, melting point 2100C, MS (ESI+) m/z 364.085(MH+). HRMS calcd. for
C20H15ClN3O2: 364.0847. Found: 364.0850. IR: (υmax/cm-1): 3151(Ar-CH), 1737(C=O), 1602(HC=N),
1498(C=C-C), 1218 (C-O); NMR (500 MHz, CDCl3, δ/ppm): 9.92 (s, 1H, pyrazolic H); 7.81 (d, 2H, Ar-H); 7.52-
7.42 (m, 7H, Ar-H); 7.34 (s, 1H, C=CH); 2.15 (s, 3H, -CH3isoxazol).
3] 4-[3-(4-Bromo-phenyl)-1-phenyl-1H-pyrazole-4-ylmethylene]-3-methyl-4H-isoxaol-5-one (8c): Yellow
crystalline solid, yield 51%, melting point 2150c, MS (ESI+) m/z 408.0337 (MH+). HRMS calcd. for
C20H15BrN3O2: 408.0342. Found: 408.0337. IR: (υmax/cm-1): 3145(Ar-CH), 1731(C=O), 1612(HC=N), 1495(C=C-
C), 1227(C-O); NMR (500 MHz, CDCl3, δ ppm) 9.92 (s, 1H, pyrazolic H); 7.81 (d, 2H, Ar-H); 7.63 (d, 2H, Ar-H);
7.48-7.35 (m, 5H, Ar-H); 7.33 (s, 1H, C=CH); 2.15 (s, 3H, -CH3).
4] 4-[3-(4-Methoxy-phenyl)-1-phenyl-1H-pyrazole-4-ylmethylene]-3-methyl-4H-isoxaol-5-one (8d): Yellow
crystalline solid, yield 57%, melting point 1900c, MS (ESI+) m/z 360.1343 (MH+). HRMS calcd. for
C21H18N3O3: 360.1343. Found: 360.1343. IR: (υmax/cm-1): 3151(Ar-CH), 1728(C=O), 1605(HC=N), 1498(C=C-
C), 1227(C-O). NMR (500 MHz, CDCl3, δ/ppm) 9.92 (s, 1H, pyrazolic H) 7.81 (d, 2H, Ar-H); 7.51 (d, 2H, Ar-H);
7.48-7.44 (m, 2H, Ar-H); 7.42 (s, 1H, C=CH); 7.36-7.32 (m, 1H, Ar-H); 7.01 (m, 2H, Ar-H); 3.83 (s, 3H, O-CH3);
2.15 (s, 3H, -CH3isoxazol).
5] 3-Methyl-4-(1-phenyl-3-p-tolyl-1H-pyrazol-4-ylmethylene)-4H-isoxazol-5-one (8e): Yellow crystalline solid,
yield 58%, melting point 1850c, MS (ESI+) m/z 344.1396 (MH+). HRMS calcd. for C20H18N3O2: 344.1396.
Found: 344.1394. IR: (υmax/cm-1): 3145(Ar-CH), 1737(C=O), 1608(HC=N), 1504(C=C-C) 1224(C-O). NMR (500
MHz, CDCl3, δ/ppm): 9.92 (s, 1H, pyrazolic H); 7.82 (d, 2H, Ar-H); 7.47-7.43 (m, 5H, Ar-H); 7.35-7.29 (m, 3H,
Ar-H); 2.39 (s, 3H, Ar-CH3); 2.15 (s, 3H, -CH3 isoxazol).
6] 4-[3-(4-Hydroxy-phenyl)-1-phenyl-1H-pyrazole-4-ylmethylene]-3-methyl-4H-isoxaol-5-one (8f): Yellow
crystalline solid, yield 47%, melting point 2250C, MS (ESI+) m/z 346.1192 (MH+). HRMS calcd. for
C20H16N3O3: 346.1186. Found: 346.1192. IR: (υmax/cm-1): 3145(Ar-CH), 1745(C=O), 1594(HC=N), 1501(C=C-
C) 1216(C-O); NMR (500MHz, DMSO, δ ppm): 9.97(s, 1H, Ar-OH); 9.85 (s, 1H, pyrazol H); 7.92 (d, 2H, Ar-H);
7.64-7.58 (m, 5H, Ar-H); 7.49-7.46 (m, 1H, Ar-H); 6.96 (d, 2H, Ar-H); 2.24 (s, 3H, -CH3 isoxazol).
7] 4-[3-(3-Hydroxy-phenyl)-1-phenyl-1H-pyrazole-4-ylmethylene]-3-methyl-4H-isoxaol-5-one (8g): Yellow
crystalline solid, yield 46%, melting point 2300C, MS (ESI+) m/z 346.1188 (MH+). HRMS calcd. for

ACCEPTED MANUSCRIPT
C20H16N3O3: 346.1186. Found: 346.1188. IR: (υmax/cm-1): 3150(Ar-CH), 1708(C=O), 1600(HC=N), 1496(C=C-
C), 1221(C-O). NMR (500MHz, DMSO, δ/ppm) 9.85 (s, 1H, Ar-OH); 9.82 (s, 1H, pyrazole H); 7.93-7.92 (m, 2H,
Ar-H); 7.67(s, 1H, Ar-H); 7.64-7.61(m, 2H, Ar-H); 7.51-7.48(m, 1H, Ar-H); 7.41-7.38(m,1H,Ar-H); 7.17-7.13(m,
2H, Ar-H); 6.96-6.94 (m, 1H, Ar-H); 2.24(s, 3H, CH3 isoxazol).
8] 3-Methyl-4[-3-(3Nitrophenyl)-1-phenyl-1H pyrazole-4-ylmethylene]-4H-isoxazol-5-one (8h): Yellow
crystalline solid, yield 65%, melting point 1900C, MS (ESI+) m/z 375.1085 (MH+). HRMS calcd. for
C20H15N4O4: 375.1088. Found: 375.1085. IR: (υmax/cm-1): 3148(Ar-CH), 1737(C=O), 1600(HC=N), 1522(C=C-
C), 1343(N-O Stretch), 1216 (C-O). NMR (500MHz, CDCl3, δ/ppm) 9.96 (s, 1H, pyrazole H); 8.47 (s, 1H, Ar-H);
8.33-8.31 (m, 1H, Ar-H); 7.96-7.95 (m, 1H, Ar-H); 7.83-7.81 (m, 2H, Ar-H); 7.72-7.68 (m, 1H, Ar-H); 7.50-7.47
(m, 2H, Ar-H); 7.39-7.35 (m, 2H, Ar-H ); 2.17 (s, 3H, -CH3 isoxazol).
X-ray crystallographic study: Single crystal X-ray diffraction data were collected on an Agilent Technologies
SuperNova Dual diffractometer with an Atlas detector using monochromated Cu-Kα radiation (λ = 1.54184 Å) (8a–
b) or Mo-Kα radiation (λ = 0.71073 Å) (8d, 8f–g) at room temperature. The data were processed using CrysAlis Pro
[25]. The structures were solved by direct methods using the program SIR97 [26] or Superflip [27] and refined on F2
using full-matrix least-squares procedures using SHELX2014 [28]. All non-hydrogen atoms were refined
anisotropically. Hydrogen atoms were readily located in a difference Fourier maps and were subsequently treated as
riding atoms in geometrically idealized positions, with C–H = 0.93 (aromatic and alkenyl) or 0.96 Å (CH3), O–H =
0.82 Å and with Uiso(H) = kUeq(C or O), where k = 1.5 for OH and methyl groups, which were permitted to rotate
but not to tilt, and 1.2 for all other H atoms. Crystallographic data is listed in Table 1.
Table 1: Crystallographic data for some 4-[3-(substituted phenyl)-1-phenyl-1H-pyrazole-4-ylmethylene]-3-methyl-
4H-isoxazol-5-one derivatives
Compound code
8a
8b
8d
8f
8g
CCDC number
Molecular formula
Molecular weight
1553040
C20H15N3O2
329.35
1553042
C20H14ClN3O2
363.79
1553043
C21H17N3O3
359.38
1553044
C20H15N3O3
345.35
1553041
C20H15N3O3
345.35
Crystal System
Space group
orthorhombic
Pbca
monoclinic
P21/n
orthorhombic
Pbca
monoclinic
I2/a
monoclinic
P21
Unit Cell dimensions
(Å and °)
a
b
c
α
7.5316(5)
20.6808(14)
21.2837(16)
90
12.4540(5)
7.4050(3)
19.2385(6)
90
13.0953(8)
7.3785(5)
36.670(2)
90
23.6058(10)
3.9439(2)
34.9489(13)
90
9.6545(8)
7.4121(8)
12.0115(14)
90
β
γ
90
90
103.942(3)
90
90
90
93.858(4)
90
106.248(10)
90
Z
8
4
8
8
2
V (Å3)
3315.1(4)
1.320
0.708
1721.94(11)
1.403
2.131
3543.2(4)
1.347
0.092
1504
3246.3(2)
1.413
0.098
825.21(15)
1.390
0.096
Dcalc (g cm-3)
μ (mm-1)
F(000)
1376
752
1440
360

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Reflections collected
Independent
reflections
8106
3144
10506
3265
14656
4069
18485
3705
4409
3130
Rint
0.0405
227
0.0251
237
0.0460
247
0.0294
237
0.0403
238
0.0534, 0.1185
Parameters
R1, wR2 [I > 2σ(I)]a
0.0738, 0.2093 0.0428, 0.1188
0.0537, 0.1275 0.0433, 0.1126
R1, wR2 (all data)a
0.1068, 0.2425 0.0494, 0.1259
0.0921, 0.1493 0.0573, 0.1219
0.0943, 0.1401
Goodness-of-fitb
1.066
–0.236, 0.276
1.042
–0.266, 0.216
1.029
–0.139, 0.165
1.010
–0.185, 0.221
1.023
–0.184, 0.289
Δρmin, Δρmax (eÅ-3)
a R = ∑||Fo| – |Fc||/∑|Fo|; wR2 = {∑[w(Fo2 – Fc2)2]/∑[w(Fo ) ]}1/2. b S = {∑[(Fo2 – Fc2)2]/(n/p)}1/2 where n is the number of
2 2
reflections and p is the total number of parameters refined.
Determination of minimal inhibition concentrations by micro broth dilution for antibacterial and antifungal
activity: All the synthesized drugs were used for antibacterial test procedures. All necessary controls like drug
control, vehicle control, agar control, organism control, known antibacterial drugs control, all MTCC cultures were
tested against above mentioned known and unknown drugs. Mueller Hinton Broth was used as nutrient medium to
grow and dilute the drug suspension for the test bacteria. Inoculum size for test strain was adjusted to 108 Cfu
[Colony Forming Unit] per milliliter by comparing the turbidity. Common standard strains were used for screening
of antibacterial and antifungal activities. The strains were procured from Institute of Microbial Technology,
Chandigarh: E. coli (MTCC443), P. aeruginosa (MTCC1688), S. aureus (MTCC96), S. pyogenus (MTCC442), C.
albicans (MTCC227), A. niger (MTCC282), A. clavatus (MTCC1323). DMSO was used as diluents /vehicle to get
desired concentration of drugs to test upon standard bacterial strains.
Minimal Inhibition Concentration: The main advantage of the ‘Broth Dilution Method’ for MIC determination lies
in the fact that it can readily be converted to determine the MIC as well [29-33].


Serial dilutions were prepared in primary and secondary screening.
The control tube containing no antibiotic is immediately sub cultured (before inoculation) by spreading a
loopful evenly over a quarter of plate of medium suitable for the growth of the test organism and put for
incubation at 37 0C overnight. The tubes are then incubated overnight.



The MIC of the control organism is read to check the accuracy of the drug concentrations.
The lowest concentration inhibiting growth of the organism is recorded as the MIC.
The amount of growth from the control tube before incubation (which represents the original inoculums) is
compared.
Methods Used For Primary And Secondary Screening: Each synthesized drug was diluted obtaining 2000 μg/mL
concentration, as a stock solution.
Primary screening: In primary screening 1000 μg/mL, 500 μg/mL, and 250 μg/mL concentrations of the
synthesized drugs were taken. The active synthesized drugs found in this primary screening were further tested in a
second set of dilution against all microorganisms.

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Secondary screening: The drugs found active in primary screening were similarly diluted to obtain 200 μg/mL 100
μg/mL, 50 μg/mL, 25 μg/mL, 12.5 μg/mL, 6.250 μg/mL, and other concentrations.
The highest dilution showing at least 99 % inhibition zone is taken as MIC.
Results and discussion:
Carrying out organic synthesis in aqueous phase is highly challenging both from synthetic view point and also from
impact of environmental pollution [22]. In order to investigate the rate of reaction in aqueous phase using different
catalyst, the reaction was carried out in water as a solvent without any catalyst, and using catalyst sodium benzoate.
Pronounced electron withdrawing effect of N atom in 7 (3-methyl-4H-isoxazol-5-one) along with electron donating
effect of O atom and presence of electron releasing CH3 and keto groups makes it easily reactive. The reaction
between 4a and 7 was selected as a model without using catalyst and by using sodium benzoate as a catalyast. Better
yield and purity of product was obtained using sodium benzoate (Table 2). Enhancing the rate of the reaction and
purity of the product in water could be attributed to the increment of basicity of sodium benzoate and hydrophobic
interactions [22]. Our observations contradicts to the earlier reports by Liu et al. [20], that aromatic aldehyde with an
electron withdrawing group failed to give desired condensation reaction. We didn't observe any effect due to the
presence of electron withdrawing group on yield of the compound. No steric hindrance was observed in case of
pyrazole-4 carbaldehydes substituted with electron-withdrawing group. Of all the synthesized compounds 8e, 8f and
8g are totally new and were not yet reported.
The mechanism for the formation of desired product 8a–h involves two steps. In first step ethyl acetoacetate reacts
with hydroxyl amine hydrochloride to give oxime which further looses ethanol molecule to give cyclic compound 7,
which in aqueous phase remains in enolic form 7a. In second step deprotonation of alcoholic OH of 7a takes place
to form alkoxide ion which form a carbanion which further develops C=C bond by Knoevenagel condensation with
pyrazole carbaldehyde to form targeted product 8 (Scheme-II). Spectral data confirms the formation of compounds
8a–h. The overall mechanism for formation of isoxazole and subsequent Knoevenagal reaction is given in Scheme-
III [23-24].
Table 2: Effect of the catalyst on the yield of the products 8a–h.
Compound
R
Melting
point in
°C
%yield
without
catalyst
%yield in
presence of
Sodium
benzoate
78
68
69
8a
8b
8c
H
4-Cl
4-Br
180
210
215
32
28
29
8d
8e
8f
8g
8h
4-OCH3 190
33
34
25
26
42
76
77
72
73
71
4-CH3
4-OH
3-OH
3-NO2
185
225
230
190
Scheme I: Synthesis of 3-(substituted phenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde

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H
H
N
N
i
H2N
N
MW
R
O
3a-h
R
1a-h
2
Reflux
ii
a: R = -H
e: R = -4-CH3
f: R = -4-OH
g: R = -3-OH
h: R = -3-NO2
b: R = -4-Cl
c: R = -4-Br
d: R = -4-OCH3
N
R
N
i: Et-OH, HCl, MW 10-20 s
ii: POCl3/DMF, 70-80oC, Reflux 1 h
OHC
4a-h
Scheme II: Synthesis of 4-[3-(substituted phenyl)-1-phenyl-1H-pyrazole-4-ylmethylene]-3-methyl-4H-isoxaol-5-
one
O
O
O
O
O
C6H5COONa
1 h
NH2OH.HCl
N
5
6
7
O
O
N
R
N
C6H5COONa
2.5 hour
N
N
N
R
O
N
4a-h
O
O
8a-h
Scheme III-Proposed mechanism for synthesis of 8a–h

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Formation of 3-Methyl-4H-isoxazol-5-one
Step I-
O
O
H3C
O
C2H5
NH2OH.HCl
Sodium benzoate
O
O
O
N
N
N
-EtOH
HO
O
O
O
O
CH3
5
5a
5b
7
Step II- Formation of Knoevenagel product
O
X
N
OH
Sodium
Benzoate
X
N
N
H
N
O
O
O
O
HO
O
O
O
7c
7a
7b
X
Where X-
N
R
N
N
O
O
All compounds 8a–h show singlet for three protons of CH3 group in the range of 2.14 to 2.15 ppm. Also, multiplet
for one of the phenyl rings is observed from 7.45 to 7.60 ppm. Whereas the other phenyl ring aroused out of
phenylhydrazine fraction shows a downfield shift with 7.55 to 7.85 ppm. Singlet for one hydrogen corresponding to
pyrazole ring is observed at around 9.94 ppm. The singlet for azomethine (CAr=CH) is covered with aromatic
protons.
Strong peaks are observed in the IR spectra of all compounds 8a–h for aromatic C–H bending in the range of 660 to
860 cm-1. Strong peak around 1737 cm–1 can be attributed to cyclic keto group (C=O) and decrease in frequency
may be due to conjugation on one side due to azomethine double bond and ring oxygen on other side. Strong peak
for azomethine (CH=N) is observed at 1603 cm–1 in IR spectra of all compounds. Also, all compounds show strong
peak at around 1500 cm–1 for C=C–C asymmetric stretching. Strong C–O stretching peak is observed in all

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compounds at around 1226 to 1229 cm–1. Thus, except for substituents, the general IR spectrum for all compounds is
of a typical signature type as seen in Figure 1.
Figure 1: Infrared spectra of 8a–h.
We were able to obtain crystals suitable for X-ray structural analysis of five out of eight synthesized compounds 8a–
h, even though several attempts were made to study the whole series. X-ray crystal structures of 4-[1,3-diphenyl-1H-
pyrazole-4-ylmethylene]-3-methyl-4H-isoxazol-5-one (8a) and four other derivatives (8b, 8d, 8f–g) (Figure 2)
shows that all the bond lengths of compounds are within normal ranges. The isoxazole and pyrazole rings only
slightly deviate from coplanarity, the dihedral angle between both rings are in the range 3.49(10)–14.8(2)° for the
studied compounds. The 1-phenyl-1H-pyrazolone moiety is in 8d and 8f–g almost coplanar with the dihedral angles
in the range 1.24(12)–13.95(9)°, while in 8a and 8b this angle is 21.7(2)° and 20.25(12)°, respectively. In all
compounds the phenyl moiety at the position 3 of the central pyrazole ring is inclined to the pyrazole ring with
systematically larger dihedral angles being in the range 38.15(19)–47.00(9)° (Table 3).
Table 3: Selected torsion angles for 8a–b, 8d and 8f–g (°).
Torsion angle
ring 1 – ring 2
ring 2 – ring 3
8a
8b
8d
8f
8g
14.8(2)
21.7(2)
3.49(10)
20.25(12)
8.39(11)
1.24(12)
4.29(9)
13.95(9)
7.6(3)
8.6(2)

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ring 2 – ring 4
38.15(19)
47.00(9)
41.35(11)
43.27(8)
40.4(2)
Rings 1–4 are O1/N3/C17–C19, N1–C2/C1–C3, C4–C9 and C10–C15 rings, respectively.
Figure 2: Molecular structures and atom numbering schemes for 8a–b, 8d and 8f–g. Probability ellipsoids are
drawn at the 50% level.
In the crystal structure of 8a there are no significant hydrogen-bonding, CH···π or π∙∙∙π interactions. In chloro
derivative 8b π∙∙∙π stacking interactions are present between pyrazole and unsubstituted phenyl rings with centroid-
to-centroid distance of 3.6703(11) Å , and dihedral angle between the rings of 12.01(9) and between chlorophenyl
ring and unsubstituted phenyl rings with centroid-to-centroid distances of 3.9186(14) Å , and dihedral angle between
the rings of 12.38(12)° (Figure 3, Table 4). Although chlorine atom is present in the molecule, there were not
observed any halogen-halogen or C–H···Cl interactions. In 8d infinite chain is formed through C21–H21A···π
interactions between the methoxy group and the methoxyphenyl moiety of the adjacent molecule (Figure 4, Table
5).

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Figure 3: π∙∙∙π interactions in 8b (dashed lines). Hydrogen atoms have been omitted for clarity.
Table 4: Geometrical parameters (Å, °) for π···π stacking interactions in 8b and 8f–g.
CgI···CgJ
CgI···CgJ
α
β
CgI-Perp
Ring slippage
Symmetry code
8b
/
/
Cg2···Cg4
Cg3···Cg4
8f
3.6703(11)
3.9186(14
12.01(9)
24.62
20.56
3.5808(7)
½ – x, –½ + y, 3/2 – z
½ – x, ½ + y, 3/2 – z
12.35(12)
–3.6890(11)
1.762
1.870
1.800
Cg2···Cg2
Cg3···Cg3
Cg4···Cg4
8g
3.9439(9)
3.9439(11)
3.9439(9)
0
0
0
26.54
28.30
27.16
3.5284(6)
3.4724(8)
3.5090(6)
x, –1+ y, z
x, –1+ y, z
x, –1+ y, z
/
/
Cg2···Cg4
3.611(3)
3.863(3)
2.1(2)
2.1(2)
14.47
25.96
3.4955(18)
2 – x, –½+ y,1 – z
2 – x, ½ + y,1 – z
Cg2···Cg4
–3.4442(18)
CgI···CgJ, α, β and CgI-Perp are, respectively, the centroid-to-centroid distance between rings I and J, the inter-ring dihedral angle, slip angle and
the perpendicular distance of CgI from ring J. Cg2–Cg4 are N1–N2/C1–C3, C4–C9 and C10–C15 ring centroids, respectively.
Figure 4: Infinite chain formation in 8d generated by C21–H21A∙∙∙π interactions indicated by black dashed lines.
Hydrogen atoms except on methoxy group have been omitted for clarity.

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Table 5: Hydrogen bond geometry of 8d and 8f–g (Å and °).
D–H···A
D–H (Å)
H···A (Å)
D···A (Å)
D–H···A (°)
132
Symmetry code
8d
C21–H21A···Cg4
0.96
2.93
3.646(3)
3/2 – x, –½ + y, z
8f
O3–H3···N3
8g
0.82
2.15
2.9494(19)
165.6
½ + x, 2 – y, z
O3–H3···O2
0.82
0.93
1.98
2.59
2.758(4)
3.303(5)
158.6
133.9
x, y, 1 + z
C9–H9···O1
2 – x, –½ + y, –z
Cg4 is C10–C15 ring centroid.
In the crystal structure of 4-hydroxyphenyl derivative 8f molecules are linked via O3–H3∙∙∙N3 hydrogen bonding
involving the hydroxyl group as a hydrogen bond donor and the nitrogen atom of the isoxazole ring of the adjacent
molecule as a hydrogen bond acceptor thus forming infinite chain along a-axis (Figure 5, Table 5). Furthermore,
π∙∙∙π stacking interactions between parallel phenyl rings, pyrazole rings and 4-hydroxyphenyl rings are present with
centroid-to-centroid distances of 3.9439(9) Å (Figure 6, Table 4). The change of the position of hydroxyl group
from position 4 in 8f to position 3 in 8g causes also the change in the hydrogen-bonding pattern. In 8g the hydrogen-
bond acceptor is not the nitrogen N3 atom but instead the carbonyl O2 atom of the isoxazole ring and thus the O3–
H3∙∙∙O2 hydrogen bonding facilitates the formation of infinite chain along a-axis (Figure 5, Table 5). Furthermore,
chains in 8g are linked into layers via C9–H9···O1 interactions between the phenyl moiety and the oxygen atom of
isoxazole moiety of the adjacent molecule and π∙∙∙π stacking interactions between pyrazole rings and 3-
hydroxyphenyl rings are present with centroid-to-centroid distances of 3.611(3) and 3.863(3) Å, and dihedral angle
between the rings of 2.1(2)° (Figure 7, Table 4).

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Figure 5: Infinite chain formation in 8f generated by O3–H3∙∙∙N3 hydrogen bonding (top) and in 8g generated by
O3–H3∙∙∙O2 hydrogen bonding (bottom). Hydrogen-bonding is indicated by blue dashed lines.
Figure 6: π∙∙∙π interactions in 8f (dashed lines). Hydrogen atoms have been omitted for clarity.
Figure 7: 2D Layer formation in 8g generated by O3–H3∙∙∙O2 and C9–H9···O1 interactions (left) and π∙∙∙π stacking
interactions (right). Hydrogen-bonding and π∙∙∙π interactions are indicated by blue and black dashed lines,
respectively. Hydrogen atoms not involved in the motif shown have been omitted for clarity.

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Antibacterial and Antifungal activity: All synthesized compounds 8a–h were screened for antibacterial and
antifungal study against E. coli, P. aeruginosa, S. aureus and S. pyogenus as bacterial strain and C. albicans, A.
niger, A. clavatus as fungal strain. Compounds 8a and 8e show antibacterial activity against E. coli. 8a–h show
antibacterial activity against S. aureus. 8d, 8e and 8h show antibacterial activity against S. pyogenus close to
standard ampicillin (Table 6). It was found that compounds 8b and 8f show antifungal activity against C. albicans
close to standard greseofulvin (Table 7).
Table 6: Antibacterial activity study
Antibacterial activity study of 8a–h with standard drugs
Minimal inhibition concentration (μg/mL)
Sr.No. Code given
E. coli
(MTCC443)
62.5
200
250
62.5
100
200
125
P. aeruginosa S. aureus
(MTCC1688) (MTCC96)
S. pyogenus
(MTCC442)
200
250
250
100
62.5
200
250
100
0.5
1
8a
200
200
200
100
200
250
250
250
1
125
125
250
200
100
125
200
125
0.25
250
50
2
8b
3
8c
4
8d
5
8e
6
8f
7
8g
8
8h
200
0.05
100
9
Gentamycin
Ampicillin
Chloramphenicol 50
Ciprofloxacin
Norfloxain
10
11
12
13
-
50
100
50
50
25
10
25
50
10
10
10
Table 7: Antifungal activity study of 8a–h along with standard drugs
Antifungal activity study of 8a–h with standard drugs
Minimal inhibition concentration (μg/mL)
Sr.No.
Code given
C. albicans
(MTCC227)
1000
500
1000
>1000
>1000
500
>1000
>1000
100
A. niger
(MTCC282)
>1000
>1000
500
500
500
500
250
A. clavatus
(MTCC1323)
>1000
>1000
500
500
1000
500
250
1
2
3
4
5
6
7
8
9
10
8a
8b
8c
8d
8e
8f
8g
8h
>1000
100
100
>1000
100
100
Nystatin
Greseofulvin 500
Conclusion:
The method of synthesis of 8a–h using sodium benzoate is ecofriendly, cheaper and gives products in high yield.
Spectral analysis and crystallographic study proves the formation of desired compounds. Change in the position of

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hydroxyl group in 8f and 8g shows change in intermolecular hydrogen bonding pattern. The compound 8g shows
highest melting point (230°C) due to presence of cross linked hydrogen bonding whereas 8f shows slightly lower
melting point (225°C) due to linear hydrogen bonding. All the compounds show antibacterial action against S.
aureus and few (8b and 8f) show antifungal activity against C. albicans.
Acknowledgment: The authors are thankful to Microcare laboratory and tuberculosis centre, Surat for providing
antibacterial and antifungal activity. Financial support from the Slovenian Research Agency (ARRS) through project
P1-0175 is gratefully acknowledged. We thank the EN-FIST Centre of Excellence, Ljubljana, Slovenia, for using
SuperNova diffractometer.
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