Type II diabetes-related enzyme inhibition and molecular modeling study of a novel series of pyrazolone derivatives

Article abstract of DOI:10.1007/s00044-013-0846-3

Inhibitors of alpha-Amylase are targets for the development of novel drugs for the treatment of diabetes and obesity. Alpha amylase is an enzyme which increases the bio availability of glucose in the blood. Hence, the inhibition effects of alpha amylase of 2-[1-(4-isobutylphenyl)ethyl]-5-methyl-4-[2-(aryl- substituted)hydrazinylidene]-2,4-dihydro-3H-pyrazol-3-one (4a-l) were investigated, among them compounds 4d, 4f, 4a, and 4g have displayed good inhibitory activity. The compounds with significant results were further evaluated for their molecular modeling study using in silico method. The new series of compounds were synthesized by solvent-free microwave irradiation method and were characterized by spectral and analytical data.

Full text of DOI:10.1007/s00044-013-0846-3

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2014) 23:2834–2846
DOI 10.1007/s00044-013-0846-3
ORIGINAL RESEARCH
Type II diabetes-related enzyme inhibition and molecular
modeling study of a novel series of pyrazolone derivatives
•
Shobhitha Shetty Balakrishna Kalluraya
•
•
Nithinchandra S. K. Peethambar
•
Sandeep B. Telkar
Received: 21 June 2013 / Accepted: 14 October 2013 / Published online: 17 November 2013
Ó Springer Science+Business Media New York 2013
Abstract Inhibitors of alpha-amylase are targets for the
development of novel drugs for the treatment of diabetes and
obesity. Alpha amylase is an enzyme which increases the bio
availability of glucose in the blood. Hence, the inhibition
effects of alpha amylase of 2-[1-(4-isobutylphenyl)ethyl]-5-
methyl-4-[2-(aryl-substituted)hydrazinylidene]-2,4-dihy-
dro-3H-pyrazol-3-one (4a–l) were investigated, among
them compounds 4d, 4f, 4a, and 4g have displayed good
inhibitory activity. The compounds with significant results
were further evaluated for their molecular modeling study
using in silico method. The new series of compounds were
synthesized by solvent-free microwave irradiation method
and were characterized by spectral and analytical data.
Introduction
Pyrazolone derivatives form an important class of com-
pounds having significant pharmacological activities such
as anti-inflammatory, antimicrobial (Bekhit et al., 2010),
analgesic (Hall et al., 2008), angiotensin antagonists
(Sharma and Kohli, 2013), cytotoxic agents (Xu et al.,
2013). Aryl pyrazoles are reported to have non-nucleoside
HIV-I reverse transcriptase inhibitory activity (Genin et al.,
2000). Furthermore, pyrazoles with a wide array of
substituted groups were reported to be selective inhibitors
of cyclooxygenase (Reddy et al., 2008) and also exhibit
antidiabetic (Hassan et al., 2011; Eduardo et al., 2013),
herbicidal (Wu et al., 2012) properties. Coupling products
of diazonium salts with compounds having active hydrogen
are widely used as intermediates in the synthesis of variety
of heterocyclic compounds (Oruc¸ et al., 2006).
Keywords Pyrazolone ꢀ Green technique ꢀ Antidiabetic ꢀ
a-Amylase inhibition ꢀ In silico molecular modeling study
Type 2 diabetes mellitus (T2DM) is a complex meta-
bolic disorder results from the body’s ineffective use of
insulin (American Diabetes Association, 2012), leading to
abnormal metabolism of not only glucose but also lipids
and amino acids. The pharmacophoric moiety 3,5-disub-
stituted pyrazoles as antidiabetic agents was first reported
by Wright et al. (1964). Based on this pharmacophore
numerous pyrazole derivatives were synthesized and
evaluated for antidiabetic activity. A number of pyrazole
compounds have been cited in the literature that elicit anti-
hyperglycemic effects, including 1,3-disubstituted pyra-
zoles, 1,3,5-trisubstituted pyrazoles, and pyrazolones
(Bertrand et al., 2002; Hassan et al., 2011).
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-013-0846-3) contains supplementary
material, which is available to authorized users.
S. Shetty ꢀ B. Kalluraya (&) ꢀ Nithinchandra
Department of Studies in Chemistry, Mangalore University,
Mangalagangothri 574 199, Karnataka, India
e-mail: kallurayab@yahoo.com; bkalluraya@gmail.com
S. K. Peethambar
Department of Bio-Chemistry, Kuvempu University,
Jnanasahyadri, Shankaraghatta 577 451, Karnataka, India
a-Amylase is an important key enzyme responsible for
carbohydrate digestion. So inhibitors of a-amylase can
effectively retard the digestion and assimilation at the early
steps of starch digestion, and thus succeed in a significant
delay of postprandial hyperglycemia and have a beneficial
S. B. Telkar
Department of Biotechnology, Kuvempu University, Bioscience
Complex, Jnanasahyadri, Shankaraghatta 577 451, Karnataka,
India
123

Med Chem Res (2014) 23:2834–2846
2835
effect on insulin resistance (Michelle de Sales et al., 2012).
So a-amylase catalyze the hydrolysis of a-(1,4)-glycosidic
linkages in starch and are considered to be one of the best
targets for the development of type II diabetes therapeutic
agents (Qin et al., 2011). For this purpose inhibitors such as
acarbose and voglibose are clinically used. However, they
often cause severe gastrointestinal side effects such as
abdominal pain, flatulence, and diarrhea (van de Laar et al.,
2005). Therefore, a number of studies have been conducted
in the search for new a-amylase inhibitors (Matsui et al.,
2007).
either on a Waters UPLC mass spectrometer or API3000
LCMS instrument operating at 70 eV.
The synthesis of hitherto unreported title compounds
was as outlined in Scheme 1. Synthesis of new pyrazolone
derivatives using 2-[4-isobutylphenyl]propanoyl hydrazine
and ethyl-2-[2-(aryl substituted)hydrazinylidene]-3-oxo-
butanoate by both conventional and microwave-assisted
methods were carried out. Diazotisation of appropriately
substituted anilines (1a–l) with nitrous acid gave the diazo
compound. The in situ coupling of this diazo compound
with ethyl aceto acetate in the presence of sodium acetate
as catalyst gave the required ethyl-2-arylhydrazono-3-
oxobutyrates (3a–l). The 2-[4-isobutylphenyl]propanoyl
hydrazine were obtained from the esterification of 2-[4-
isobutylphenyl]propanoic acid followed by hydrazinolysis
with hydrazine hydrate as per the literature procedure
(Nithinchandra et al., 2012).
Computational biology and bioinformatics play a major
role in designing the drug molecules and have the potential
of speeding up the drug discovery process. Molecular
docking methods are commonly used for predicting bind-
ing modes and energies of ligands to proteins. Molecular
docking of the drug molecule with the receptor gives
important information about drug receptor interactions and
is commonly used to find out the binding orientation of
drug candidates to their target sites in order to predict the
affinity and activity.
Characterization data of 2-[4-isobutylphenyl]propanoyl
hydrazine is given below:
It was obtained as a white solid with mp 71–72 °C; Yield
:80 %, IR (KBr) c/cm-1: 3,270.3 (N–H), 2,954.4 (C–H
stretching), 1,686.2 (C=O stretching); 1H NMR (DMSO-d6),
d in ppm: 0.84 (d, 6H, J = 6.56 Hz, (CH3)2), 1.30 (d, 3H,
J = 7 Hz, CH3), 1.81–1.74 (m, 1H, CH–(CH3)2), 2.38 (d, 2H,
J = 7.04 Hz, CH2), 3.48 (q, 1H, CH), 4.16 (s, 2H, NH2), 7.06
(d, 2H, J = 7.72 Hz, 30,50-Ib–Ar–H), 7.20 (d, 2H,
J = 7.72 Hz, 20,60-Ib–Ar–H), 9.13 (s, 1H, NH); 13C NMR
(DMSO-d6 100 MHz): d 14.11 (isobutyl-(CH3)2), 19.15
(CH3), 30.14 (isobutyl CH), 42.01 (isobutyl-CH2), 43.06
(CH), 127.20 (C-4 of isobutylphenyl), 127.84 (C-3 and C-5 of
isobutylphenyl), 131.16 (C-2 and C-6 of isobutylphenyl),
137.23 (C-1 of isobutylphenyl), 161.9 (C=O); LC–MS (m/z):
220.8 (M?). Anal. Calcd. For C13H20N2O: C, 70.87; H, 9.15;
N, 12.72, Found: C, 70.85; H, 9.17; N, 12.70.
The driving force for microwave developments in
organic synthesis by green techniques has many benefits
because of their simplicity in operation, enhanced reaction
rates, and greater selectivity (Jyothi et al., 2007). The
increase in requirement for environmentally clean tech-
nology that minimizes the production of waste (Escalante
´
and Dıaz-Coutin˜o, 2009) is another important criterion of
green technology. Keeping in view of the above observa-
tions and in continuation of our search for biologically
active heterocycles (Girisha et al., 2012; Nithinchandra
et al., 2012), we herein report a new series of pyrazolone
derivative synthesized by microwave irradiation technique.
These new molecules were tested for a-amylase inhibition.
Molecular docking simulations of compounds with signif-
icant amylase inhibition effects and acarbose to a-amylase
were performed in order to gain functional and structural
insight into the mechanism of inhibition.
General procedure for the preparation of ethyl-2-
arylhydrazono-3-oxobutyrate (3a–l)
Appropriately substituted amine 1 (0.01 mol) was dis-
solved in dilute hydrochloric acid (10 ml) and cooled to
0 °C in an ice bath. To this, a cold solution of sodium
nitrite (0.02 mol) was added. The diazonium salt solution
was filtered into a cold solution of ethyl acetoacetate
(0.02 mol) and sodium acetate (0.02 mol) in 15 ml of
aqueous ethanol. The separated solid was filtered, washed
with water, and recrystallized from ethanol. Compounds
prepared according to this procedure and their character-
ization data are given below:
Experimental
Microwave-assisted synthesis was carried out using Catalyst
system microwave oven, CATA R ranging from power levels
1–9 at 140–700 W. Melting points were determined using the
apparatus Innovative DTC-967A and are uncorrected. The IR
spectra (in KBr pellets) were recorded on a Shimadzu FT-IR
157 spectrophotometer. The 1H NMR spectra were recorded
on a Bruker AMX-400 (400 MHz) NMR spectrometer using
TMS as an internal standard. The chemical shifts are
expressed in d scale downfield from TMS and proton signals
are indicated as s = singlet, d = doublet, t = triplet,
q = quartet, m = multiplet. The mass spectra were recorded
Ethyl-2-(4-chlorophenyl)hydrazono-3-oxobutyrate (3a)
It was obtained as a yellowish solid with melting point 92–
93 °C; Yield : 76 %; IR (KBr) c/cm-1: 3,315.7 (NH
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Med Chem Res (2014) 23:2834–2846
O
O
N2+}Cl-
NH2
CH3
O
CH3
O
N
H3C
NaNO2/HCl
0 -5 °C
O
NH
O
NaOAc, EtOH
R
CH3
R
3
R
2
CH
O
1
3
H C
NH
NH
3
2
CH
3
MW irradiation,
3-5minutes, 450W
AcOH, Δ,
8- 12 hrs
H3C
R
NH
CH3
N
N
N
CH3
CH3
O
O
4
R = 4-Cl, 3-NO2, 4-F, 2,4-difluoro, 4-carboxyl, 2-carboxyl, 3-Cl, 4-NO2, 2-NO2, 2-OCH3,
4-OCH3, 3-chloro-4-fluoro
Scheme 1 Synthetic protocols for the target compounds (4a–l)
1
stretch), 1,720 (C=O ester carbonyl), 1,680.1 (C=O
stretching of keto group), 1,558.1(NH–N=C), 2,978.8 (CH
stretch); H NMR (DMSO-d6 400 MHz) d in ppm: 1.84 (t,
3H, CH3), 2.46 (S, 3H, CH3–C=O), 4.45 (q, 2H, CH2),
7.50–7.28 (m, 4H, 20, 40, 50, and 60 protons of 3-nitro-
phenyl), 12.16 (1H, NH); 13C NMR (DMSO-d6 100 MHz)
d in ppm : 23.41 (ester CH3), 25.75 (acetyl CH3), 59.11
(CH2), 175.28 (C=O of acetyl), 167.17 (C=O of ester),
117.81 (C-5 of 3-nitrophenyl), 120.75 (C-6 of 3-nitro-
phenyl), 128.92 (C-4 of 3-nitrophenyl), 130.35 (C-2 of 3-
nitrophenyl), 134.42 (C-1 of 3-nitrophenyl), 135.98 (C-3 of
3-nitrophenyl) 139.93 (C=N–); LC–MS (m/z): 280.3
(M??1). Anal. Calcd. for C12H13N3O5: C, 51.61; H, 4.69;
N, 15.05. Found: C, 51.63; H, 4.70; N, 15.03.
1
stretch); H NMR (DMSO-d6 400 MHz) d in ppm: 1.78 (t,
3H, CH3), 2.42 (s, 3H, CH3–C=O), 4.8 (q, 2H, CH2),
7.23(d, 2H, ortho protons of 4-chlorophenyl), 7.50 (d, 2H,
meta protons of 4-chlorophenyl), 12.06 (s, 1H, NH); 13C
NMR (DMSO-d6 100 MHz) d in ppm : 22.50 (ester CH3),
24.91 (acetyl CH3), 60.01 (CH2), 165.21 (C=O of ester),
169.14 (C=O of acetyl), 120.21 (C-2 and C-6 of 4-chlo-
rophenyl), 129.80 (C-3 and C-5 of 4-chlorophenyl), 132.61
(C-1 of 4-chlorophenyl), 136.21 (C-4 of 4-chlorophenyl),
140.31(C=N–); LC–MS (m/z): 269 (M??1); Anal. Calcd.
for C12H13ClN2O3: C, 53.64; H, 4.88; N, 10.43. Found: C,
53.66; H, 4.89; N, 10.41.
Ethyl-2-(4-fluorophenyl)hydrazono-3-oxobutyrate (3c)
Ethyl-2-(3-nitrophenyl)hydrazono-3-oxobutyrate (3b)
It was obtained as a yellowish solid with melting point
99 °C; Yield : 75 %; IR (KBr) c/cm-1: 3,328.5 (NH
stretch), 1,716.8 (C=O ester carbonyl), 1,677 (C=O
stretching of keto group), 1,541.5 (NH–N=C), 2,988.7 (CH
Reddish brown solid with melting point 111–112 °C,
Yield: 68 %; IR (KBr) c/cm-1: 3,325.4 (NH stretch),
1,730.5 (C=O ester carbonyl), 1,670.8 (C=O stretching of
keto group), 1,554.8 (NH–N=C), 2,977.8 (CH stretch);
1,560 (NO2 asymmetric stretch), 1,346 (NO2 symmetric
1
stretch); H NMR (DMSO-d6 400 MHz) d in ppm: 1.88 (t,
3H, CH3), 2.38 (s, 3H, CH3–C=O), 4.76 (q, 2H, CH2), 7.20
(d, 2H, ortho protons of 4-fluorophenyl), 7.69 (d, 2H, meta
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Med Chem Res (2014) 23:2834–2846
2837
protons of 4-fluorophenyl), 12.11 (1H, NH); 13C NMR
(DMSO-d6 100 MHz) d in ppm : 21.62 (ester CH3), 25.22
(acetyl CH3), 63.04 (CH2), 170.25 (C=O of acetyl), 168.90
(C=O of ester), 116.67 (C-2 and C-6 of 4-fluorophenyl),
120.91 (C-3 and C-5 of 4-fluorophenyl), 128.25 (C-1 of 4-
fluorophenyl), 135.91 (C-4 of 4-fluorophenyl), 139.96
(C=N–); LC–MS (m/z): 253.3 (M??1); Anal. Calcd. for
C12H13FN2O3: C, 57.14; H, 5.19; N, 11.11. Found: C,
57.16; H, 5.16; N, 11.09.
(NH–N=C), 2,990.9 (CH stretch); 1H NMR (DMSO-d6
400 MHz) d in ppm: 1.79 (t, 3H, CH3), 2.61 (s, 3H, CH3–
C=O), 4.70 (q, 2H, CH2), 7.70–7.59 (m, 3H, 20, 50 and 60
protons of 3-chloro-4-fluorophenyl), 12.06 (1H, NH); 13C
NMR (DMSO-d6 100 MHz) d in ppm: 22.4 (ester CH3),
26.2 (acetyl CH3), 51.22 (CH2), 171.61 (C=O of acetyl),
169.22 (C=O of ester), 120.32 (C-6 of 3-chloro-4-fluoro-
phenyl), 122.51 (C-2 of 3-chloro-4-fluorophenyl), 129.32
(C-5 of 3-chloro-4-fluorophenyl), 132.27 (C-1 of 3-chloro-
4-fluorophenyl), 137.80 (C-3 of 3-chloro-4-fluorophenyl),
141.44 (C-4 of 3-chloro-4-fluorophenyl), 142.28 (C=N–);
LC–MS (m/z): 287 (M??1); Anal. Calcd. for
C12H12ClFN2O3: C, 50.27; H, 4.22; N, 9.77. Found: C,
50.29; H, 4.25; N, 9.79.
Ethyl-2-(2,4-difluorophenyl)hydrazono-3-oxobutyrate (3d)
Yellowish solid with melting point 109 °C. IR (KBr) c/
cm-1: 3,309.4 (NH stretch), 1,735.5 (C=O ester carbonyl),
1,689.5 (C=O stretching of keto group), 1,561.5 (NH–
N=C), 2,988.98 (CH stretch); 1H NMR (DMSO-d6 400
MHZ) d in ppm: 1.98 (t, 3H, CH3), 2.58 (s, 3H, CH3–
C=O), 4.86 (q, 2H, CH2), 7.68–7.57 (m, 3H, 30, 50 and 60
protons of 2,4-difluorophenyl), 12.81 (1H, NH); 13C NMR
(DMSO-d6 100 MHz) d in ppm: 24.41 (ester CH3), 28.21
(acetyl CH3), 59.63 (CH2), 162.6 (C=O of acetyl), 160.25
(C=O of ester), 116.2 (C-6 of 2,4-difluorophenyl), 120.2
(C-5 of 2,4-difluorophenyl), 124.23 (C-3 of 2,4-difluoro-
phenyl), 128.87 (C-1 of 2,4-difluorophenyl), 138.91 (C-4 of
2,4-difluorophenyl), 139.65 (C-2 of 2,4-difluorophenyl),
140.2 (C=N–); LC–MS (m/z): 271.3 (M??1); Anal. Calcd.
for C12H12F2N2O3: C, 53.34; H, 4.48; N, 10.37. Found: C,
53.30; H, 4.46; N, 10.39.
Ethyl-2-(2-carboxyphenyl)hydrazono-3-oxobutyrate (3g)
Yellow solid with melting point 84 °C, Yield 70 %; IR (KBr)
c/cm-1: 3,311.3 (NH stretch), 1,719.8 (C=O ester carbonyl),
1,701.5 (acid C=O), 1,681.5 (C=O stretching of keto group),
1,564.2 (NH–N=C), 2,980.9 (CH stretch); 1H NMR (DMSO-
d6 400 MHz) d in ppm: 1.83 (t, 3H, CH3), 2.58 (s, 3H, CH3–
C=O), 4.76 (q, 2H, CH2), 7.78–7.71 (m, 4H, 30, 40, 50, and 60
protons of 2-carboxyphenyl), 12.08 (1H, NH), 13.16 (1H,
OH); 13C NMR (DMSO-d6 100 MHz) d in ppm: 24.10 (ester
CH3), 26.34 (acetyl CH3), 54.91 (CH2), 169.62 (C=O of
acetyl), 165.27 (C=O of ester), 168.23 (C=O of carboxyl),
133.26 (C-5 of 2-carboxyphenyl), 134.24 (C-4 of 2-car-
boxyphenyl), 137.23 (C-6 of 2-carboxyphenyl), 138.21 (C-3
of 2-carboxyphenyl), 138.65 (C-1 of 2-carboxyphenyl),
139.89 (C-2 of 2-carboxyphenyl), 142.21 (C=N–); LC–MS
(m/z): 279 (M??1); Anal. Calcd. for C13H14N2O5: C, 56.11;
H, 5.07; N, 10.07. Found: C, 56.13; H, 5.05; N, 10.05.
Ethyl-2-(2-anisyl)hydrazono-3-oxobutyrate (3e)
Brownish yellow solid with melting point 114 °C; Yield:
70 %. IR (KBr) c/cm-1: 3,299.4 (NH stretch), 1,729.8
(C=O ester carbonyl), 1,689.1 (C=O stretching of keto
group), 1,567.5 (NH–N=C), 2,992.9 (CH stretch); 1H NMR
(DMSO-d6 400 MHz) d in ppm: 1.88 (t, 3H, CH3), 2.68 (s,
3H, CH3–C=O), 4.77 (q, 2H, CH2), 7.60–7.54 (m, 4H, 30,
40, 50 and 60 protons of 2-anisyl), 12.11 (s, 1H, NH); 13C
NMR (DMSO-d6 100 MHz) d in ppm: 22.1 (ester CH3),
25.91 (acetyl CH3), 50.15 (CH2), 64.20 (OCH3), 168.61
(C=O of acetyl), 158.20 (C=O of ester), 128.21 (C-4 of 2-
anisyl), 132.63 (C-3 of 2-anisyl), 133.26 (C-5 of 2-anisyl),
134.42 (C-6 of 2-anisyl), 136.20 (C-1 of 2-anisyl), 138.20
(C-2 of 2-anisyl), 140.21 (C=N–); LC–MS (m/z): 265
(M??1); Anal. Calcd. for C13H16N2O4: C, 59.08; H, 6.10;
N, 10.60. Found: C, 59.10; H, 6.13; N, 10.62.
Ethyl-2-(4-carboxyphenyl)hydrazono-3-oxobutyrate (3h)
Reddish solid with melting point 102 °C, Yield: 71 %; IR
(KBr) c/cm-1: 3,302.3 (NH stretch), 1,724.9 (C=O ester
carbonyl), 1,718.1 (C=O acid carbonyl), 1,679.2 (C=O
stretching of keto group), 1,564.6 (NH–N=C), 2,990.6 (CH
1
stretch); H NMR (DMSO-d6 400 MHz) d in ppm: 1.79 (t,
3H, CH3), 2.49 (s, 3H, CH3–C=O), 4.69 (q, 2H, CH2), 7.48
(d, 2H, ortho protons of 4-carboxyphenyl), 7.64 (d, 2H,
meta protons of 4-carboxyphenyl), 12.24 (1H, NH), 13.08
(1H, OH); 13C NMR (DMSO-d6 100 MHz) d in ppm:
24.52 (ester CH3), 26.91 (acetyl CH3), 56.22 (CH2), 169.29
(C=O of acetyl), 166.13 (C=O of ester), 167.11 (C=O of
acid), 129.66 (C-2 and C-6 of 4-carboxylphenyl), 133.84
(C-3 and C-5 and 4-carboxylphenyl), 136.23 (C-1 of 4-
carboxylphenyl), 138.35 (C-4 of 4-carboxyphenyl), 143.91
(C=N–); LC–MS (m/z): 279 (M??1); Anal. Calcd. for
C13H14N2O5: C, 56.11; H, 5.07; N, 10.07. Found: C, 56.09;
H, 5.05; N, 10.09.
Ethyl-2-(3-chloro-4-fluorophenyl)hydrazono-3-oxobutyrate
(3f)
Yellow solid with melting point 108 °C; Yield: 69 %. IR
(KBr) c/cm-1: 3,301.4 (NH stretch), 1,722.6 (C=O ester
carbonyl), 1,678.5 (C=O stretching of keto group), 1,560.5
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Med Chem Res (2014) 23:2834–2846
Ethyl-2-(4-nitrophenyl)hydrazono-3-oxobutyrate (3i)
nitrophenyl), 135.44 (C-3 of 2-nitrophenyl), 136.93 (C-1 of
2 -nitrophenyl), 137.92 (C-2 of 2-nitrophenyl), 141.02
(C=N–); LC–MS (m/z): 280 (M??1); Anal. Calcd. for
C12H13N3O5: C, 51.61; H, 4.69; N, 15.05. Found: C, 51.63;
H, 4.67; N, 15.07.
Brown solid with melting point 124–125 °C, [literature
(Ferguson et al., 2008): 127.5 °C]; Yield : 68 %. IR (KBr)
c/cm-1: 3,203.8 (NH stretch), 1,726.7 (C=O ester car-
bonyl), 1,679.1 (C=O stretching of keto group), 1,566.7
(NH–N=C), 1,532 (NO2 asymmetric stretch), 1,328.6 (NO2
Ethyl-2-(4-anisyl)hydrazono-3-oxobutyrate (3l)
1
symmetric stretch); H NMR (DMSO-d6 400 MHz): 1.78
(t, 3H, CH3), 2.42 (s, 3H, CH3–C=O), 4.46 (q, 2H, CH2),
7.23(d, 2H, ortho protons of 4-nitrophenyl), 7.50 (d, 2H,
meta protons of 4-nitrophenyl), 12.10 (1H, NH); 13C NMR
(DMSO-d6 100 MHz) d in ppm: 20.5 (ester CH3), 23.9
(acetyl CH3), 50.06 (CH2), 168.24 (C=O of acetyl), 161.42
(C=O of ester), 132.22 (C-2 and C-6 of 4-nitrophenyl),
135.61 (C-3 and C-5 of 4-nitrophenyl), 136.81 (C-1 of 4-
nitrophenyl), 137.12 (C-4 of 4-nitrophenyl), 148.31 (C=N–
); LC–MS (m/z): 279 (M?); Anal. Calcd. for C12H13N3O5:
C, 51.61; H, 4.69; N, 15.05. Found: C, 51.59; H, 4.72; N,
15.07.
Yellow solid with melting point 126–127 °C; Yield : 70 %.
IR (KBr) c/cm-1: 3,317.3 (NH stretch), 1,730.7 (C=O ester
carbonyl), 1,687.3 (C=O stretching of keto group), 1,557.8
(NH–N=C); 1H NMR (DMSO-d6 400 MHz) d in ppm: 1.72
(t, 3H, CH3), 2.44 (s, 3H, CH3–C=O), 3.06 (s, 3H, OCH3),
4.48 (q, 2H, CH2), 7.44 (d, 2H, ortho protons of 4-anisyl),
7.66 (d, 2H, meta protons of 4-anisyl), 12.28 (s, 1H, NH);
13C NMR (DMSO-d6 100 MHz) d in ppm : 21.98 (ester
CH3), 24.03 (acetyl CH3), 51.09 (CH2), 167.92 (C=O of
acetyl), 163.14 (C=O of ester), 67.44 (OCH3), 131.12 (C-3
and C-5 of 4-anisyl), 132.39 (C-2 and C-6 of 4-anisyl),
136.18 (C-4 of 4-anisyl), 136.72 (C-1 of 4-anisyl), 145.3
(C=N–); LC–MS (m/z): 265 (M??1); Anal. Calcd. for
C13H16N2O4: C, 59.08; H, 6.10; N, 10.60. Found: C, 59.07;
H, 6.12; N, 10.61.
Ethyl-2-(3-chlorophenyl)hydrazono-3-oxobutyrate (3j)
Brownish yellow solid with melting point 86–88 °C, [lit-
erature (Ferguson et al., 2008): 85 °C]; Yield : 72 %. IR
(KBr) c/cm-1: 3,213.8 (NH stretch), 1,723.4 (C=O ester
carbonyl), 1,677.3 (C=O stretching of keto group), 1,560.9
(NH–N=C); 1H NMR (DMSO-d6 400 MHz) d in ppm: 1.69
(t, 3H, CH3), 2.49 (s, 3H, CH3–C=O), 4.44 (q, 2H, CH2),
7.38–7.20 (m, 4H, 20, 40, 50, and 60 protons of 3-chloro-
phenyl); 12.70 (1H, NH); 13C NMR (DMSO-d6 100 MHz)
d in ppm: 23.0 (ester CH3), 25.82 (acetyl CH3), 50.31
(CH2), 169.28 (C=O of acetyl), 165.13 (C=O of ester),
121.91 (C-5 of 3-chlorophenyl), 125.96 (C-6 of 3-chloro-
phenyl), 126.82 (C-4 of 3-chlorophenyl), 130.22 (C-2 of 3-
chlorophenyl), 130.98 (C-1 of 3-chlorophenyl), 134.42 (C-
3 of 3-chlorophenyl), 141.31 (C=N–); LC–MS (m/z): 269
(M??1). Anal. Calcd. for C12H13ClN2O3: C, 53.64; H,
4.88; N, 10.43. Found: C, 53.66; H, 4.90; N, 10.45.
Procedure for the preparation of 2-[1-(4-
isobutylphenyl)ethyl]-5-methyl-4-[2-(aryl
substituted)hydrazinylidene]-2,4-dihydro-3H-pyrazol-
3-one (4a–l)
(i) Microwave method
An equimolar mixture of ethyl-2-arylhydrazono-3-oxobuty-
rate (0.01 mol) and 2-[4-isobutylphenyl]propanoyl hydrazine
(0.01 mol) were groundtogether to get a uniform mixture. For
this 5–6 drops of glacial acetic acid was added and mixed well
and it was kept inside a 100 ml round-bottom flask and sub-
jected to microwave irradiation on a catalyst systems micro-
wave oven operating at 490 W for about 3–5.5 min. Progress
ofthereactionwasmonitoredbyTLC.Afterthecompletionof
reaction, the reaction mass was poured into crushed ice, solid
obtained was filtered and recrystallized from ethanol–DMF
mixture.
Ethyl-2-(2-nitrophenyl)hydrazono-3-oxobutyrate (3k)
Brownish yellow solid with melting point 111–112 °C;
Yield: 71 %; IR (KBr) c/cm-1: 3,323.1 (NH stretch),
1,728.6 (C=O ester carbonyl), 1,673.7 (C=O stretching of
keto group), 1.557.8 (NH–N=C), 1,544.4 (NO2 asymmetric
stretch), 1,346.5 (NO2 symmetric stretch); 1H NMR
(DMSO-d6 400 MHz) d in ppm: 1.72 (t, 3H, CH3), 2.44 (s,
3H, CH3–C=O), 4.38 (q, 2H, CH2), 7.44–7.30 (m, 4H, 30,
40, 50, and 60 protons of 2-nitrophenyl); 12.23 (s, 1H, NH);
13C NMR (DMSO-d6 100 MHz) d in ppm: 21.94 (ester
CH3), 25.09 (acetyl CH3), 51.84 (CH2), 168.8 (C=O of
acetyl), 162.2 (C=O of ester), 122.61 (C-5 of 2-nitro-
phenyl), 129.28 (C-4 of 2-nitrophenyl), 134.02 (C-6 of 2-
(ii) Conventional method
Ethyl-2-arylhydrazono-3-oxobutyrate (0.01 mol) was dis-
solved in 25 ml glacial acetic acid. To this a solution of 2-
[4-isobutylphenyl]propanoyl hydrazine (0.01 mol) in gla-
cial acetic acid was added and the mixture was refluxed for
8 h. Progress of the reaction was monitored by TLC. After
the completion of the reaction it was cooled and allowed to
stand overnight and the separated solid was filtered, dried
and recrystallized from ethanol–DMF mixture.
123

Med Chem Res (2014) 23:2834–2846
2839
Characterization data of the title compounds are given
below:
isobutylphenyl), 128.91 (C-1 of isobutylphenyl), 129.98
(C-5 of 3-nitrophenyl), 131.16 (C-6 of 3-nitrophenyl), 134.23
(C-4 of 3-nitrophenyl), 134.98 (C-2 of 3-nitrophenyl), 133.88
(C-1 of 3-nitrophenyl), 138.22 (C-3 of 3-nitrophenyl), 140.62
(C-5 of pyrazolone), 142.4 (C-4 of pyrazolone), 159.04
(C=O of pyrazolone), 161.9 (C=O between pyrazolone and
isobutylphenyl); UPLC mass (m/z, %) 436.4 (M??1, 88).
Anal. Calcd. For C23H25N5O4: C, 63.44; H, 5.79; N, 16.08,
Found: C, 63.47; H, 5.81; N, 16.06.
2-[1-(4-Isobutylphenyl)ethyl]-5-methyl-4-[2-(4-
chlorophenyl)hydrazinylidene]-2,4-dihydro-3H-pyrazol-3-
one (4a)
Yellow solid, mp 270 °C, IR (KBr) c/cm-1: 3,153.3 (N–
H), 2,957.6 (C–H), 1,708.7 (pyrazolone carbonyl), 1,690
1
(C=O), 1,542.2 (C=N); H NMR (DMSO-d6 400 MHz), d
in ppm: 0.83 (d, 6H, J = 6.56 Hz, (CH3)2), 1.43 (d, 3H,
J = 7 Hz, CH3), 1.83–1.73 (m, 1H, CH–(CH3)2), 2.21 (s,
3H, pyrazolone CH3), 2.39 (d, 2H, J = 7.16 Hz, CH2),
4.86 (q, 1H, CH), 7.10 (d, 2H, J = 8.04 Hz, 30,50-Ib–Ar–
H), 7.24 (d, 2H, J = 8.04 Hz, 20,60-Ib–Ar–H), 7.49 (d, 2H,
J = 8.88 Hz, ortho protons of 4-chlorophenyl), 7.68 (d,
2H, J = 8.88 Hz, meta protons of 4-chlorophenyl), 13.0 (s,
1H, NH); 13C NMR (CDCl3-d6 100 MHz) d in ppm: 12.01
(isobutyl-(CH3)2), 19.15 (CH3), 22.40 (pyrazolone CH3),
30.14 (isobutyl CH), 44.01 (isobutyl-CH2), 45.06 (CH
between pyrazolone and isobutylphenyl), 117.18 (C-4 of
isobutylphenyl), 127.20 (C-3 and C-5 of isobutylphenyl),
127.84 (C-2 and C-6 of isobutylphenyl), 129.35 (C-1 of
isobutylphenyl), 129.88 (C-2 and C-6 of 4-chlorophenyl),
131.68 (C-3 and C-5 of 4-chlorophenyl), 137.23 (C-1 of 4-
chlorophenyl), 139.35 (C-4 of 4-chlorophenyl), 140.64 (C-
5 of pyrazolone), 143.83 (C-4 of pyrazolone), 159.04 (C=O
of pyrazolone), 161.35 (C=O between pyrazolone and
isobutylphenyl); UPLC mass (m/z, %) 425.4 (M??1, 100).
Anal. Calcd. For C23H25ClN4O2: C, 65.01; H, 5.93; N,
13.19, Found: C, 65.03; H, 5.95; N, 13.17.
2-[1-(4-Isobutylphenyl)ethyl]-5-methyl-4-[2-(4-
fluorophenyl)hydrazinylidene]-2,4-dihydro-3H-pyrazol-3-
one (4c)
Yellow solid, mp 277 °C, IR (KBr) c/cm-1: 3,251.3 (N–
H), 2,868.1 (C–H), 1,726.7 (pyrazolone carbonyl), 1,685.7
1
(C=O), 1,541.9 (C=N); H NMR (DMSO-d6), d in ppm:
0.83 (d, 6H, J = 6.56 Hz, (CH3)2), 1.43 (d, 3H, J = 7 Hz,
CH3), 1.73–1.83 (m, 1H, CH–(CH3)2), 2.21 (s, 3H, pyr-
azolone CH3), 2.39 (d, 2H, J = 7.16 Hz, CH2), 4.86 (q,
1H, CH), 7.10 (d, 2H, J = 8.04 Hz, 30,50-Ib–Ar–H), 7.24
(d, 2H, J = 8.04 Hz, 20,60-Ib–Ar–H), 7.17 (d, 2H,
J = 9.2 Hz, ortho protons of 4-fluorophenyl), 7.48 (d, 2H,
J = 9.2 Hz, meta protons of 4-fluorophenyl), 13.0 (S, 1H,
NH);13C NMR (DMSO-d6) d in ppm: 14.11 (isobutyl-
(CH3)2), 20.15 (CH3), 22.50 (pyrazolone CH3), 33.14
(isobutyl CH), 43.51 (isobutyl-CH2), 45.66 (CH between
pyrazolone and isobutylphenyl), 120.33 18 (C-4 of isobu-
tylphenyl), 129.60 (C-3 and C-5 of isobutylphenyl), 130.84
(C-2 and C-6 of isobutylphenyl), 131.35 (C-1 of isobutyl-
phenyl), 133.88 (C-2 and C-6 of 4-fluorophenyl), 134.16
(C-3 and C-5 of 4-fluorophenyl), 136.73 (C-1 of 4-fluoro-
phenyl), 137.31 (C-4 of 4-fluorophenyl), 141.62 (C-5 of
pyrazolone), 143.4 (C-4 of pyrazolone), 160.04 (C=O of
pyrazolone), 163.9 (C=O between pyrazolone and isobu-
tylphenyl); LC–MS (m/z): 409 (M??1). Anal. Calcd. For
C23H25FN4O2: C, 67.63; H, 6.17; N, 13.72, Found: C,
67.65; H, 6.19; N, 13.74.
2-[1-(4-Isobutylphenyl)ethyl]-5-methyl-4-[2-(3-
nitrophenyl)hydrazinylidene]-2,4-dihydro-3H-pyrazol-3-
one (4b)
Yellow solid, mp 269 °C, IR (KBr) c/cm-1: 3,080.7 (N–H),
2,954.5 (C–H), 1,734.5 (pyrazolone carbonyl), 1,669.7 (C=O),
1,561.2 (C=N), 1,350.1 (NO2 sym.), 1532.5 (NO2 asym.); 1H
NMR (DMSO-d6) d in ppm: 0.84 (d, 6H, J = 6.6 Hz, (CH3)2),
1.44 (d, 3H, J = 7 Hz, CH3), 1.80–1.75 (m, 1H, CH–(CH3)2),
2.23 (s, 3H, pyrazolone CH3), 4.83 (q, 1H, CH), 2.39 (d, 2H,
J = 7.08 Hz, CH2), 7.11 (d, 2H, J = 8.04 Hz, 30,50-Ib–Ar–H),
7.25 (d, 2H, J = 8.04 Hz, 20,60-Ib–Ar–H), 7.71 (t, 1H,
50-proton of 3-nitrophenyl), 8.02 (doublet of doublet, 1H,
J = 1.68 and 8.12 Hz, 60-proton of 3-nitrophenyl), 8.09
(doublet of doublet, 1H, J = 1.44 and 8.2 Hz, 40-proton of
3-nitrophenyl), 8.51 (s, 1H, 20-proton of 3-nitrophenyl), 13.08
(s, 1H, NH); 13C NMR (DMSO-d6) d in ppm: 13.09 (isobutyl-
(CH3)2), 19.91 (CH3), 21.40 (pyrazolone CH3), 31.44 (isobutyl
CH), 41.31 (isobutyl-CH2), 46.01 (CH between pyrazolone
and isobutylphenyl), 121.18 (C-4 of isobutylphenyl), 126.20
(C-3 and C-5 of isobutylphenyl), 127.84 (C-2 and C-6 of
2-[1-(4-Isobutylphenyl)ethyl]-5-methyl-4-[2-(2,4-
difluorophenyl)hydrazinylidene]-2,4-dihydro-3H-pyrazol-
3-one (4d)
Yellow solid, mp 267 °C, IR (KBr) c/cm-1: 3,153.3 (N–H),
2,984.6 (C–H), 1,728.7 (pyrazolone carbonyl), 1,668.7
(C=O), 1,541.2 (C=N); 1H NMR (DMSO-d6), d in ppm: 0.86
(d, 6H, J = 6.56 Hz, (CH3)2), 1.46 (d, 3H, J = 7 Hz, CH3),
1.81–1.71 (m, 1H, CH–(CH3)2), 2.26 (s, 3H, pyrazole CH3),
2.43 (d, 2H, J = 7.18 Hz, CH2), 4.80 (q, 1H, CH), 7.12 (d,
2H, J = 8.04 Hz, 30,50-Ib–Ar–H), 7.26 (d, 2H, J = 8.04 Hz,
20,60-Ib–Ar–H), 7.05–7.00 (m, 2H, 50 and 60-protons of 2,4-
difluorophenyl), 7.83 (m, 1H, 30-proton of 2,4-difluoro-
phenyl), 13.0 (s, 1H, NH); 13C NMR (DMSO-d6) d in ppm:
123

2840
Med Chem Res (2014) 23:2834–2846
13.64 (isobutyl-(CH3)2), 19.01 (CH3), 20.30 (pyrazolone
CH3), 32.46 (isobutyl CH), 40.96 (isobutyl-CH2), 45.31 (CH
between pyrazolone and isobutylphenyl), 119.28 (C-4 of
isobutylphenyl), 125.58 (C-3 and C-5 of isobutylphenyl),
126.20 (C-2 and C-6 of isobutylphenyl), 126.56 (C-1 of iso-
butylphenyl), 127.84 (C-6 of 2,4-difluorophenyl), 128.8 (C-5
of 2,4-difluorophenyl), 131.16 (C-3 of 2,4-difluorophenyl),
134.23 (C-1 of 2,4-difluorophenyl), 137.19 (C-4 of 2,4-
difluorophenyl), 138.91 (C-2 of 2,4-difluorophenyl), 141.82
(C-5 of pyrazolone), 143.24 (C-4 of pyrazolone), 162.04
(C=O of pyrazolone), 164.93 (C=O between pyrazolone and
isobutylphenyl); LC–MS (m/z): 426 (M?). Anal. Calcd. For
C23H24F2N4O2: C, 64.78; H, 5.67; N, 13.14, Found: C, 64.76;
H, 5.69; N, 13.17.
2H, J = 8.04 Hz, 20,60-Ib–Ar–H), 7.70–7.59 (m, 3H, 20, 50
and 60-protons of 3-chloro-4-fluorophenyl), 13.06 (s, 1H,
NH); 13C NMR (DMSO-d6) d in ppm: 14.11. (isobutyl-
(CH3)2), 19.99 (CH3), 21.10 (pyrazolone CH3), 31.36 (iso-
butyl CH), 39.26 (isobutyl-CH2), 44.87 (CH between pyraz-
olone and isobutylphenyl), 122.31 (C-4 of isobutylphenyl),
123.51 (C-3 and C-5 of isobutylphenyl), 124.81 (C-2 and C-6
of isobutylphenyl), 126.14 (C-1 of isobutylphenyl), 130.93
(C-6 of 3-chloro-4-fluorophenyl), 131.11 (C-2 of 3-chloro-4-
fluorophenyl), 131.76 (C-5 of 3-chloro-4-fluorophenyl),
132.91 (C-1 of 3-chloro-4-fluorophenyl), 134.23 (C-3 of 3-
chloro-4-fluorophenyl), 136.98 (C-4 of 3-chloro-4-fluoro-
phenyl), 141.82 (C-5 of pyrazolone), 143.24 (C-4 of pyrazo-
lone), 162.04 (C=O of pyrazolone), 164.93 (C=O between
pyrazolone and isobutylphenyl); UPLC-mass (m/z, %): 443.2
(M?, 89 %). Anal. Calcd. For C23H24ClFN4O2: C, 62.37; H,
5.46; N, 12.65, Found: C, 62.39; H, 5.44; N, 12.68.
2-[1-(4-Isobutylphenyl)ethyl]-5-methyl-4-[2-(2-
anisyl)hydrazinylidene]-2,4-dihydro-3H-pyrazol-3-one
(4e)
2-[1-(4-Isobutylphenyl)ethyl]-5-methyl-4-[2-(2-
carboxyphenyl)hydrazinylidene]-2,4-dihydro-3H-pyrazol-
3-one (4g)
It was obtained as a reddish brown solid, mp 248 °C, IR (KBr)
c/cm-1: 3,173.3 (N–H), 2,957.6 (C–H), 1,719.7 (pyrazolone
carbonyl), 1,668.7 (C=O), 1,542.2 (C=N); 1H NMR (DMSO-
d6), d in ppm: 0.83 (d, 6H, J = 6.56 Hz, (CH3)2), 1.43 (d, 3H,
J = 7 Hz, CH3), 1.83–1.73 (m, 1H, CH–(CH3)2), 2.21 (s, 3H,
pyrazolone CH3), 2.39 (d, 2H, J = 7.16 Hz, CH2), 4.86 (q,
1H, CH), 3.91 (s, 3H, OCH3), 7.10 (d, 2H, J = 8.04 Hz, 30,50-
Ib–Ar–H), 7.24 (d, 2H, J = 8.04 Hz, 20,60-Ib–Ar–H), 7.79–
6.99 (m, 4H, aromatic protons of 2-anisyl), 13.0 (s, 1H, NH);
13C NMR (DMSO-d6) d in ppm: 13.04. (isobutyl-(CH3)2),
18.99 (CH3), 21.40 (pyrazolone CH3), 33.56 (isobutyl CH),
40.06 (isobutyl-CH2), 44.68 (CH between pyrazolone and
isobutylphenyl), 56.88 (OCH3), 126.21 (C-4 of isobutyl-
phenyl), 127.11 (C-3 and C-5 of isobutylphenyl), 128.14 (C-2
and C-6 of isobutylphenyl), 128.84 (C-1 of isobutylphenyl),
129.16 (C-4 of 2-anisyl), 132.11 (C-5 of 2-anisyl), 133.13 (C-
3 of 2-anisyl), 133.91 (C-6 of 2-anisyl), 136.36 (C-2 of 2-
anisyl), 136.78 (C-1 of 2-anisyl), 144.82 (C-5 of pyrazolone),
145.21 (C-4 of pyrazolone), 162.91 (C=O of pyrazolone),
164.93 (C=O between pyrazolone and isobutylphenyl); LC–
MS (m/z): 421 (M??1).Anal. Calcd. For C24H28N4O3: C,
68.55; H, 6.71; N, 13.32, Found: C, 68.58; H, 6.73; N, 13.35.
Yellow solid, mp 272 °C, IR (KBr) c/cm-1: 3,158.3 (N–H),
2,947.6 (C–H), 1,729.3 (pyrazolone carbonyl), 1,719.2 (car-
boxyl C=O), 1,678.2 (C=O), 1,540.5 (C=N); 1H NMR
(DMSO-d6), d in ppm: 0.84 (d, 6H, J = 6.58 Hz, (CH3)2),
1.44 (d, 3H, J = 7 Hz, CH3), 1.79–1.67 (m, 1H, CH–(CH3)2),
2.31 (s, 3H, pyrazolone CH3), 2.43 (d, 2H, J = 7.16 Hz,
CH2), 4.80 (q, 1H, CH), 7.12 (d, 2H, J = 8.04 Hz, 30,50-Ib–
Ar–H), 7.22 (d, 2H, J = 8.04 Hz, 20,60-Ib–Ar–H), 6.98–7.79
(m, 4H, aromatic protons of 2-carboxyphenyl), 12.81 (s, 1H,
NH), 13.10 (s, broad, 1H, OH); 13C NMR (DMSO-d6) d in
ppm: 13.94. (isobutyl-(CH3)2), 19.09 (CH3), 22.40 (pyrazo-
lone CH3), 33.86 (isobutyl CH), 40.96 (isobutyl-CH2), 44.78
(CH between pyrazolone and isobutylphenyl), 119.91 (C-4 of
isobutylphenyl), 120.46 (C-3 and C-5 of isobutylphenyl),
122.11 (C-2 and C-6 of isobutylphenyl), 127.31 (C-1 of iso-
butylphenyl), 128.11 (C-5 of 2-carboxyphenyl), 129.21 (C-4
of 2-carboxyphenyl), 129.86 (C-6 of 2-carboxyphenyl),
130.11 (C-3 of 2-carboxyphenyl), 132.93 (C-1 of 2-car-
boxyphenyl), 134.84 (C-2 of 2-carboxyphenyl), 143.82 (C-5
of pyrazolone), 146.21 (C-4 of pyrazolone), 161.91 (C=O of
pyrazolone), 164.34 (C=O of carboxyl), 165.93 (C=O
between pyrazolone and isobutylphenyl); LC–MS (m/z): 435
(M??1). Anal. Calcd. For C24H26N4O4: C, 66.34; H, 6.03; N,
12.89, Found: C, 66.37; H, 6.00; N, 12.91.
2-[1-(4-Isobutylphenyl)ethyl]-5-methyl-4-[2-(3-chloro-4-
fluorophenyl)hydrazinylidene]-2,4-dihydro-3H-pyrazol-3-
one (4f)
Yellow solid, mp 270 °C, IR (KBr) c/cm-1: 3,200.9 (N–H),
2,955.9 (C–H), 1,795.9 (pyrazolone carbonyl), 1,657.5
(C=O), 1,560.2 (C=N); 1H NMR (DMSO-d6 300 MHz), d in
ppm: 0.84 (d, 6H, J = 6.6 Hz, (CH3)2), 1.51 (d, 3H,
J = 7.26 Hz, CH3), 1.81–1.70 (m, 1H, CH–(CH3)2), 2.36 (s,
3H, pyrazolone CH3), 2.39 (d, 2H, J = 7.29 Hz, CH2), 4.31
(q, 1H, CH), 7.08(d, 2H, J = 8.1 Hz, 30,50-Ib–Ar–H), 7.16 (d,
2-[1-(4-Isobutylphenyl)ethyl]-5-methyl-4-[2-(4-
carboxyphenyl)hydrazinylidene]-2,4-dihydro-3H-pyrazol-
3-one (4h)
Yellow solid, mp 258 °C, IR (KBr) c/cm-1: 3,178.7 (N–H),
3,317.56 (O–H), 2,922.16 (C–H), 1,757.2 (carboxyl C=O),
123

Med Chem Res (2014) 23:2834–2846
2841
2-[1-(4-Isobutylphenyl)ethyl]-5-methyl-4-[2-(3-
1,720 (pyrazolone carbonyl), 1,614.42 (C=O), 1,543.05
1
(C=N); H NMR (DMSO-d6): 0.85 (d, 6H, J = 6.56 Hz,
chlorophenyl)hydrazinylidene]-2,4-dihydro-3H-pyrazol-3-
one (4j)
(CH3)2), 1.32 (d, 3H, J = 7 Hz, CH3), 2.51–2.50 (m, 1H,
CH–(CH3)2), 2.16 (s, 3H, pyrazolone CH3), 2.89 (d, 2H,
J = 7.16 Hz, CH2), 4.86 (q, 1H, CH), 7.08 (d, 2H,
J = 8.08 Hz, 30,50-Ib–Ar–H), 7.24 (d, 2H, J = 8.00 Hz,
20,60-Ib–Ar–H), 7.62 (d, 2H, J = 8.64 Hz, ortho protons of 4-
carboxyphenyl), 7.98 (d, 2H, J = 8.68 Hz, meta protons of 4-
carboxyphenyl), 12.87, (s, 1H, NH), 13.20 (s, broad, 1H, OH);
13C NMR (DMSO-d6) d in ppm: 12.11 (isobutyl-(CH3)2),
19.69 (CH3), 22.49 (pyrazolone CH3), 30.54 (isobutyl CH),
44.61 (isobutyl-CH2), 45.36 (CH between pyrazolone and
isobutylphenyl), 120.18 (C-4 of isobutylphenyl), 128.75 (C-3
and C-5 of isobutylphenyl), 129.20 (C-2 and C-6 of isobu-
tylphenyl), 129.62 (C-1 of isobutylphenyl), 130.84 (C-2 and
C-6 of 4-carboxyphenyl), 131.16 (C-3 and C-5 of 4-car-
boxyphenyl), 136.23 (C-1 of 4-carboxyphenyl), 138.95 (C-4
of4-carboxyphenyl), 140.62(C-5ofpyrazolone), 142.11(C-4
of pyrazolone), 163.04 (C=O of pyrazolone), 165.11 (C=O of
carboxyl), 168.91 (C=O between pyrazolone and isobutyl-
phenyl); LC–MS (m/z): 433.3 (M?-1). Anal. Calcd. For
C24H26N4O4: C, 66.34; H, 6.03; N, 12.89, Found: C, 66.32; H,
6.05; N, 12.87.
Yellow solid, mp 276 °C, IR (KBr) c/cm-1: 3,253.3 (N–
H), 2,867.6 (C–H), 1,715.2 (pyrazolone carbonyl), 1,667.7
1
(C=O), 1,536.2 (C=N); H NMR (DMSO-d6), d in ppm:
0.84 (d, 6H, J = 6.56 Hz, (CH3)2), 1.68 (d, 3H, J = 7 Hz,
CH3), 1.78–1.82 (m, 1H, CH–(CH3)2), 2.47 (s, 3H, pyr-
azolone CH3), 2.36 (d, 2H, J = 7.14 Hz, CH2), 4.80 (q,
1H, CH), 7.16 (d, 2H, J = 8.06 Hz, 30,50-Ib–Ar–H), 7.36
(d, 2H, J = 8.08 Hz, 20,60-Ib–Ar–H), 7.69 (m, 3H, 40, 50
and 60-protons of 3-chlorophenyl), 8.36 (s, 1H, 20-proton of
3-chlorophenyl), 13.0 (s, 1H, NH);13C NMR (DMSO-d6) d
in ppm: 13.19. (isobutyl-(CH3)2), 19.41 (CH3), 21.44
(pyrazolone CH3), 32.44 (isobutyl CH), 42.01 (isobutyl-
CH2), 45.01 (CH between pyrazolone and isobutylphenyl),
120.18 (C-4 of isobutylphenyl), 128.20 (C-3 and C-5 of
isobutylphenyl), 128.92 (C-2 and C-6 of isobutylphenyl),
129.84 (C-1 of isobutylphenyl), 131.88 (C-5 of 3-chloro-
phenyl), 133.26 (C-6 of 3-chlorophenyl), 133.53 (C-4 of 3-
chlorophenyl), 133.98 (C-2 of 3-chlorophenyl), 135.88 (C-
1 of 3-chlorophenyl), 136.15 (C-3 of 3-chlorophenyl),
141.12 (C-5 of pyrazolone), 143.2 (C-4 of pyrazolone),
161.04 (C=O of pyrazolone), 163.95 (C=O between pyr-
azolone and isobutylphenyl) LC–MS (m/z): 425 (M??1).
Anal. Calcd. For C23H25ClN4O2: C, 65.01; H, 5.93; N,
13.19, Found: C, 64.99; H, 5.96; N, 13.21.
2-[1-(4-Isobutylphenyl)ethyl]-5-methyl-4-[2-(4-
nitrophenyl)hydrazinylidene]-2,4-dihydro-3H-pyrazol-3-
one (4i)
Yellow solid, mp 269 °C, IR (KBr) c/cm-1: 3,268.1 (N–H),
2,950.9 (C–H), 1,736.2 (pyrazolone carbonyl), 1,641.0
(C=O), 1,539.6 (C=N), 1,373.6 (NO2 sym.), 1,506.2 (NO2
asym.); 1H NMR (DMSO-d6), d in ppm: 0.85 (d, 6H,
J = 6.6 Hz, (CH3)2), 1.34 (d, 3H, J = 7.12 Hz, CH3), 1.81–
1.70 (m, 1H, CH–(CH3)2), 2.66 (s, 3H, pyrazolone CH3), 2.41
(d, 2H, J = 7.04 Hz, CH2), 4.64 (q, 1H, CH), 7.19 (d, 2H,
J = 7.76 Hz, 30,50-Ib–Ar–H), 7.27 (d, 2H, J = 8.04 Hz,
20,60-Ib–Ar–H), 8.03 (doublet of doublet 2H, J = 1.96 and
7.04 Hz, ortho protons of 4-nitrophenyl), 8.51 (doublet of
doublet, 2H, J = 2 and 7 Hz, meta protons of 4-nitrophenyl),
13.01 (s, 1H, NH); 13C NMR (DMSO-d6) d in ppm: 12.01
(isobutyl-(CH3)2), 19.21 (CH3), 22.43 (pyrazolone CH3),
31.14 (isobutyl CH), 43.91 (isobutyl-CH2), 45.86 (CH
between pyrazolone and isobutylphenyl), 119.97 (C-4 of
isobutylphenyl), 127.84 (C-3 and C-5 of isobutylphenyl),
128.20 (C-2 and C-6 of isobutylphenyl), 129.85 (C-1 of iso-
butylphenyl), 130.08 (C-2 and C-6 of 4-nitrophenyl), 132.11
(C-3 and C-5 of 4-nitrophenyl), 136.35 (C-1 of 4-nitro-
phenyl), 137.23 (C-4 of 4-nitrophenyl), 141.62 (C-5 of pyr-
azolone), 142.98 (C-4 of pyrazolone), 159.04 (C=O of
pyrazolone), 164.9 (C=O between pyrazolone and isobutyl-
phenyl);LC–MS (m/z): 436 (M??1).Anal. Calcd. For
C23H25N5O4: C, 63.44; H, 5.79; N, 16.08, Found: C, 63.47; H,
5.82; N, 16.06.
2-[1-(4-Isobutylphenyl)ethyl]-5-methyl-4-[2-(2-
nitrophenyl)hydrazinylidene]-2,4-dihydro-3H-pyrazol-3-
one (4k)
It was obtained as a yellow solid, mp 272 °C, IR (KBr) c/
cm-1: 3,203.9 (N–H), 2,950.9 (C–H), 1,712.2 (pyrazolone
carbonyl), 1,669.3 (C=O), 1,539.2 (C=N), 1,344.8 (NO2
sym.), 1,539 (NO2 asym.); 1H NMR (DMSO-d6), d in ppm:
0.85 (d, 6H, J = 6.56 Hz, (CH3)2), 1.41 (d, 3H, J = 7 Hz,
CH3), 1.66–1.79 (m, 1H, CH–(CH3)2), 2.40 (s, 3H, pyr-
azolone CH3), 2.37 (d, 2H, J = 7.16 Hz, CH2), 4.78 (q,
1H, CH), 7.14 (d, 2H, J = 8.04 Hz, 30,50-Ib–Ar–H), 7.22
(d, 2H, J = 8.04 Hz, 20,60-Ib–Ar–H), 8.12–7.69 (m, 4H,
aromatic protons of 2-nitrophenyl), 13.0 (s, 1H, NH); 13C
NMR (DMSO-d6) d in ppm : 12.31. (isobutyl-(CH3)2),
18.99 (CH3), 22.89 (pyrazolone CH3), 31.12(isobutyl CH),
43.61 (isobutyl-CH2), 46.36 (CH between pyrazolone and
isobutyl pheny), 120.96 (C-4 of isobutylphenyl), 121.91
(C-3 and C-5 of isobutylphenyl), 125.19 (C-2 and C-6 of
isobutylphenyl), 126.31 (C-1 of isobutylphenyl), 128.11
(C-5 of 2-nitrophenyl), 129.31 (C-4 of 2-nitrophenyl),
130.16 (C-6 of 2-nitrophenyl), 131.01 (C-3 of 2-nitro-
phenyl), 131.93 (C-1 of 2-nitrophenyl), 132.11 (C-2 of 2-
nitrophenyl), 142.82 (C-5 of pyrazolone), 145.21 (C-4 of
123

2842
Med Chem Res (2014) 23:2834–2846
pyrazolone), 162.01 (C=O of pyrazolone), 165.93 (C=O
between pyrazolone and isobutylphenyl); LC–MS (m/z):
436 (M??1). Anal. Calcd. For C23H25N5O4: C, 63.44; H,
5.79; N, 16.08, Found: C, 63.46; H, 5.81; N, 16.11.
of enzyme. Then, the enzyme was incubated with starch
solution without adding any inhibitors which act as positive
control. The inhibitory effect of the synthesized compound
was compared with standard a-inhibitor Acarbose solution
of same concentration. The inhibition percentage of a-
amylase was assessed by the following formula:
2-[1-(4-Isobutylphenyl)ethyl]-5-methyl-4-[2-(4-
À
Á
anisyl)hydrazinylidene]-2,4-dihydro-3H-pyrazol-3-one (4l)
% of Iaꢁamylase ¼ DAcontrol ꢁ DAsample =DAcontrol ꢂ 100;
Yellow solid, mp 279 °C, IR (KBr) c/cm-1: 3,201.3 (N–
H), 2,954.8 (C–H), 1,795.4 (pyrazolone carbonyl), 1,655.6
where DAcontrol ¼ ATest ꢁ ABlank
;
DAsample ¼ ATest ꢁ ABlank
:
1
(C=O), 1,560.7 (C=N); H NMR (DMSO-d6), d in ppm:
0.83 (d, 6H, J = 6.56 Hz, (CH3)2), 1.58 (d, 3H,
J = 7.08 Hz, CH3), 1.80–1.74 (m, 1H, CH–(CH3)2), 2.65
(s, 3H, pyrazolone CH3), 3.90 (s, 3H, OCH3), 2.39 (d, 2H,
J = 7.08 Hz, CH2), 4.27 (q, 1H, CH), 7.08 (d, 2H,
J = 7.92 Hz, 30,50-Ib–Ar–H), 7.22 (d, 2H, J = 7.96 Hz,
20,60-Ib–Ar–H), 7.33 (d, 2H, J = 8.96 Hz, ortho protons of
4-anisyl), 7.80 (d, 2H, J = 8.92 Hz meta protons of 4-
anisyl), 12.90 (s, 1H, NH); 13C NMR (DMSO-d6) in ppm:
13.11 (isobutyl-(CH3)2), 20.01 (CH3), 22.93 (pyrazolone
CH3), 32.11(isobutyl CH), 42.21 (isobutyl-CH2), 44.96
(CH between pyrazolone and isobutylphenyl), 55.87
(OCH3), 120.87 (C-4 of isobutylphenyl), 129.16 (C-3 and
C-5 of isobutylphenyl), 129.64 (C-2 and C-6 of isobutyl-
phenyl), 130.55 (C-1 of isobutylphenyl), 132.41 (C-2 and
C-6 of 4-anisyl), 133.08 (C-3 and C-5 of 4-anisyl), 136.25
(C-1 of 4-anisyl), 137.02 (C-4 of 2-anisyl), 142.62 (C-5 of
pyrazolone), 143.92 (C-4 of pyrazolone), 161.04 (C=O of
pyrazolone), 164.56 (C=O between pyrazolone and isobu-
tylphenyl); LC–MS (m/z): 421.5 (M??1). Anal. Calcd. For
C24H28N4O3: C, 68.55; H, 6.71; N, 13.32, Found: C, 68.57;
H, 6.74; N, 13.35.
Acute toxicity and gross behavioral studies
The acute oral toxicity study for the organic compound 4a–
l was carried out following the OECD guidelines No. 420.
Swiss albino male mice weighing 25–30 g were used for
the evaluation. Each group consisting of 6 male mice
(overnight fasted) was kept in the colony cage at
25 2 °C with 55 % relative humidity and 12-h light/dark
cycle was maintained. A specified fixed dose of 250, 500,
750, 1000, 1500, 2000, and 3000 mg/kg was selected and
administered orally as a single dose as fine suspension
prepared in saline using gum acacia powder. The acute
toxic symptoms and the behavioral changes produced by
the test compounds were observed continuously for 4th,
8th, 12th, and 24th h onset of toxic symptoms and gross
behavioral changes were also recorded (Jaouhari et al.,
1999).
Simulation studies
˚
X-ray crystallographic structure resolved at 2.0 A of
Aspergillus oryzae alpha-amylase complex with inhibitor
acarbose (PDB: 7TAA) was obtained from protein data-
bank. The structure 7TAA was simplified by removing
HETATOMS, water molecules, and co-crystallized com-
pounds. For all ligands (4a, 4d, 4f, 4g, and acarbose)
topology file and other force field parameters were gener-
ated using PRODRG server (van Aalten et al., 1996).
Flexible torsions for ligands were defined using AutoTors
provided with AutoDock suite. Acarbose was redocked
together with 4a, 4d, 4f, and 4g in the inhibitor binding
site. The docking site for all ligands in 7TAA was con-
sidered at co-crystallized position of acarbose. PyRx 0.8
interface was used (Wolf, 2009) with grid box size
89 9 85 9 74, spacing 0.375, grid center 41.0450,
39.9533, 27.6100, and assigning 3 degrees of Freedom.
The Lamarckian genetic algorithm was employed (Morris
and Goodsell, 1998) with 10 conformations, for each ligand
and best pose with the lowest binding energy was consid-
ered. For all the ligands binding energy and inhibition
constant (Ki) were tabulated. Python molecular viewer
Biological evaluation
In vitro a-amylase inhibition
The a-amylase inhibitory activity of newly synthesized
compounds was carried out using the method of Giancarlo
et al. (2006), with slight modifications. It was performed by
using 1 ml of enzyme solution (1 U/ml in 20 mM PBS of
pH 6.9), 1 ml of different concentration (25, 50, 75 and
100 lg) of synthesized compounds in dimethyl sulfoxide
(DMSO) was incubated at 25 °C, for 30 min, added 1 ml
of 0.5 % of starch solution and tubes were further incu-
bated for 3 min at 25 °C. To this, 1 ml of 96 mM of 3,5-
dinitro salicylic acid solution (prepared by using 5.31 M
sodium potassium tartrate in 2 M sodium hydroxide) was
added. The reaction mixture was shaken well and closed
tube was placed in a water bath at 85 °C for 15 min. The
reaction mixture was diluted with 9 ml of distilled water
and resulting solution was measured at 540 nm by spec-
trophotometer (Shimadzu-1800) to determine the inhibition
123

Med Chem Res (2014) 23:2834–2846
2843
Table 1 Comparative data
Comp. no.
Molecular formula
(mol. wt.)
Conventional method
Microwave method
showing the yield and reaction
time by conventional and
microwave irradiation methods
for the synthesis of pyrazolones
(4a–l)
Yield (%)
Reaction
time (h)
Yield (%)
Reaction
time (min)
4a
4b
4c
4d
4e
4f
C23H25ClN4O2 (424.92)
56
57
55
58
59
61
60
62
60
59
60
61
8
8
69
71
69
70
71
74
75
77
78
74
75
77
5.0
4.5
5.5
4.6
4.5
5.5
5.0
5.0
4.5
5.3
4.8
4.4
C23H25N5O4 (435.47)
C23H25FN4O2 (408.47)
C23H24F2N4O2 (426.46)
C24H28N4O3 (420.50)
C23H24ClFN4O2 (442.91)
C24H26N4O4 (434.48)
C24H26N4O4 (434.48)
C23H25N5O4 (435.47)
C23H25ClN4O2 (424.92)
C23H25N5O4 (435.47)
C24H28N4O3 (420.50)
10
9
11
10
9
4g
4h
4i
13
10
10
13
14
4j
4k
4l
(Michel, 1999) was used for docking confirmation
representation.
Table 2 In vitro alpha amylase inhibition activity of pyrazolone
derivatives (4a–l)
Compounds
% of inhibition
4a
46.2 1.3
10.6 0.9
26.7 1.5
61.6 1.3
40.0 0.8
60.4 1.6
47.3 1.5
32.0 2.1
–
Results and discussion
4b
4c
Chemistry
4d
4e
After optimization of the experimental conditions, 2-[1-(4-
isobutylphenyl)ethyl]-5-methyl-4-[2-(aryl substituted)hydraz-
inylidene]-2,4-dihydro-3H-pyrazol-3-one was synthesized by
the reaction of equimolar mixture of 2-[4-isobutylphenyl]
propanoyl hydrazine and ethyl-2-arylhydrazono-3-oxobutyrate
by both conventional heating and microwave irradiation
method. The main advantage of microwave-mediated method
is reduced time interval. In microwave irradiation method, the
yields were high as compared to the yield obtained by con-
ventional method. Microwave irradiation method facilitates the
polarization of the reacting molecule causing reactions to occur
at higher rate. A comparative study in terms of yield and
reaction time is shown in Table 1. It is noteworthy that the
reaction, which required 8–14 h in conventional method, was
completed within 3.50–5.50 min in microwave system at
power level of 490 W. Also yields have been remarkably
improved from 55–62 to 69–78 %.
4f
4g
4h
4i
4j
10.5 0.6
40.7 1.4
16.3 1.5
87.7 1.2
4k
4l
Acarbose
100
80
60
40
20
0
Pharmacology
Organic Compounds
In this study, we have evaluated the inhibitory effects of title
compounds (4a–l) against a-amylase to elucidate the pos-
sible use of title compounds as anti-hyperglycemic agents.
The inhibitory effect of the title compounds are shown in
Table 2. The bar diagram representation of comparative
activities of title compounds (4a–l) is shown in Fig. 1.
Fig. 1 Bar diagram showing a-amylase inhibition activity of com-
pounds 4a–l
Molecular docking simulation study of the synthesized
compounds and acarbose was carried out in order to gain
functional and structural insight into the mechanism of
123

2844
Med Chem Res (2014) 23:2834–2846
Table 3 The binding energy (kJ mol-1) and inhibition constant of
4a, 4d, 4f, 4g, and acarbose
Compound Binding energy (kJ mol-1
)
Inhibition constant (lM)
4a
-5.97
-2.09
-3.65
-5.38
-1.6
37.04
41.98
40.69
37.52
66.63
4d
4f
4g
Acarbose
Fig. 3 Acarbose docked in best of its conformation with a-amylase
(i.e., 7TAA). Ligand acarbose shown in ball and stick model as blue
color, amino acid residues in active pocket shown in ball and stick
model as colored by rasmol view and remaining residues of the
protein as lines colored by rasmol view. Hydrogen bonds represented
as green cylinders. Acarbose forms three hydrogen bonds in total, 2
˚
bound in common with GLU230 with bond lengths 2.171 and 2.11 A,
˚
and third bound with Asp206 with the bond length 2.183 A (Color
figure online)
compounds, which showed depression on the first day and
death on second day.
Structure–activity relationship
Fig. 2 4d docked in best of its conformation with a-amylase (i.e.,
7TAA). Ligand 4d shown in ball and stick model in blue color, amino
acid residues in active pocket shown in ball and stick model as
colored by rasmol view and remaining residues of the protein as lines
colored by rasmol view. Hydrogen bonds represented as green
cylinders. 4d forms two hydrogen bonds in total, 1 bound with
Based on the nature of substituents each of the synthesized
individual compounds showed sensitivity toward alpha
amylase inhibition. With regard to SAR we observed that
the substituent’s present on phenyl ring remarkably results
in the increase or decrease in the inhibition. Compounds
4d, 4f, 4a, and 4g exhibited good inhibitory activity against
a-amylase as compared to the control enzyme inhibitor,
whereas rest of the compounds showed weak inhibitory
activity. Introduction of disubstituted halogen derivatives
caused good inhibitory activity as observed in the case of
2,4-difluoro (4d) and 3-chloro-4-fluoro (4f) substituents.
This may be due to the high electronegativity effect of the
halogens. Also it is noticeable that difluoro derivative
showed slight higher inhibition than chloro–fluoro sub-
stituent. Whereas, the compounds carrying 4-chloro (4a)
and 2-carboxyl (4g) substituents showed moderate activity.
But when the carboxyl group is present at para position it
diminishes the activity compared to ortho position. Com-
pounds with meta substituents like 3-nitro and 3-chloro
groups showed very weak inhibition. Also the para-
substituted groups such as 4-carboxyl, 4-methoxy, and 4-
fluoro groups showed weak inhibition. In case of nitro
group, ortho-substituted group showed moderate activity,
but the para-substituted compound did not showed any
activity.
˚
GLU230 with bond length 2.041 A, and another with HIS210 with the
˚
bond length 1.875 A (Color figure online)
inhibition. Auto Dock 4.2 suite was used as molecular
docking tool. The docking of receptor a-amylase with
newly synthesized candidate ligands exhibited well estab-
lished binding energy in the receptor active pocket. The
binding energy, inhibition constant of the compound 4a,
4d, 4f, 4g, and acarbose is shown in Table 3. It is observed
that the compounds with good amylase inhibitory activity
docked in best of its conformation with a-amylase (i.e.,
7TAA) as shown in Fig. 2; and also acarbose docked in
best of its conformation with a-amylase (i.e., 7TAA) is
shown in Fig. 3.
The acute toxic symptoms and the behavioral changes
produced by the test compounds were observed. The
experimental studies revealed that these compounds were
quite safe up to 250, 500, 750, 1000, and 2000 mg/kg, and
no death of animals were recorded. Further, no significant
gross behavioral changes were observed in experimental
animals except in the 3,000 mg/kg of 4a, 4b, 4d, 4j, and 4l
123

Med Chem Res (2014) 23:2834–2846
2845
identification and SAR of 3- and 4-substituted derivatives. J Med
Chem 43(5):1034–1040
Conclusion
Giancarlo S, Rosa LM, Nadjafi F, Francesco M (2006) Hypoglycae-
mic activity of two spices extracts: Rhus coriaria L. and Bunium
persicum Boiss. Nat Prod Res 20:882–886
Pyrazolones are highly versatile members of the hetero-
cyclic compounds. They possess an array of interesting
chemical and variety of biological activities. From the
above studies it can be concluded that microwave tech-
nique is one of the promising technique of synthesis with
better yield, lower reaction time, and provides green
chemical pathway. Alpha amylase inhibition results of
pyrazolone derivatives revealed that compounds with high
electron withdrawing substituents showed good inhibitory
activity, i.e., compounds 4d and 4f with dihalo sub-
stituent’s showed good inhibition. In silico studies revealed
that synthesized molecules 4a, 4d, 4f, and 4g showed good
binding energy toward the target protein ranging from
-2.09 to -5.97 kJ mol-1 and 4d being the best among
them. All the docking results showed the best RMS value,
i.e., 0.0.
Girisha KS, Kalluraya B, Jois VHSS (2012) Synthesis, characteriza-
tion and antimicrobial activity of 3-methyl-6-(aryl)-1-phenyl-
1H-pyrazolo[3,4-b]pyridine. Indian J Chem 51B(12):1767
Hall A, Billinton A, Brown SH, Clayton NM, Chowdhury A, Gerald
MP, Goldsmith GP, Hayhow TG, Hurst DN, Kilford IR, Naylor
A, Passingham B (2008) Non-acidic pyrazole EP1 receptor
antagonists with in vivo analgesic efficacy. Bioorg Med Chem
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Hassan MF, Khalid AK, Abdullah MA (2011) Synthesis and
biological evaluation of new 3-trifluoromethylpyrazolesulfonyl-
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agents. J Fluor Chem 132:131–137
Jaouhari JT, Lazrek HB, Seddik A, Jana M (1999) Hypoglycaemic
response to Zygophyllum gaetulum extracts in patients with non-
insulin-dependent diabetes mellitus. J Ethnopharmacol 64:211–
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Jyothi CH, Rai SN, Kalluraya B (2007) Environmentally benign
synthesis of sydnone containing 1,3,4-thiadiazines under micro-
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The preliminary in vitro anti-diabetic activity of these
series of pyrazolone derivatives has shown that compounds
(4a, 4d, 4f, and 4g) have exhibited good a-amylase inhi-
bition. The in silico molecular docking results are matching
with the in vitro studies and they may be considered as
good inhibitor of a-amylase enzyme.
Michel F Sanner (1999) Python: a programming language for
software integration and development. J Mol Graph Mod
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Michelle de Sales P, de Souza PM, Simeoni LA, Magalha˜es PO,
Silveira D (2012) a-Amylase inhibitors: a review of raw material
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Morris GM, Goodsell DS (1998) Automated docking using a
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energy function. J Comput Chem 19(14):1639–1662
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selective reaction: synthesis, characterization and pharmacolog-
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sydnone. Eur J Med Chem 54:597–604
Acknowledgments The authors are thankful to Head, I.I.Sc., Ban-
galore and C.D.R.I., Lucknow for providing the spectral analysis and
Head, Department of Chemistry, Mangalore University for CHNS
analysis, and also to DST-Purse for providing fellowship to one of the
author Shobhitha Shetty.
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