Microwave-Assisted Synthesis, In Vivo Anti-Inflammatory and In Vitro Anti-Oxidant Activities, and Molecular Docking Study of New Substituted Schiff Base Derivatives

Article abstract of DOI:10.1007/s11094-018-1835-0

In view of considerable interest in the design and synthesis of new heterocyclic compounds with promising biological activities for medical and biological applications, a series of eight imine derivatives have been synthesized through microwave-assisted S

Full text of DOI:10.1007/s11094-018-1835-0

DOI 10.1007/s11094-018-1835-0
Pharmaceutical Chemistry Journal, Vol. 52, No. 5, August, 2018 (Russian Original Vol. 52, No. 5, May, 2018)
MICROWAVE-ASSISTED SYNTHESIS, IN VIVO ANTI-INFLAMMATORY
AND IN VITRO ANTI-OXIDANT ACTIVITIES, AND MOLECULAR
DOCKING STUDY OF NEW SUBSTITUTED SCHIFF BASE DERIVATIVES
Muhammad Hanif,2 Mubashir Hassan,1 Muhammad Rafiq,1,3 Qamar Abbas,4
Ansa Ishaq,5 Saba Shahzadi,6,7 Sung-Yum Seo,2 and Muhammad Saleem8,*
Original article submitted November 21, 2017.
In view of considerable interest in the design and synthesis of new heterocyclic compounds with promising bi-
ological activities for medical and biological applications, a series of eight imine derivatives have been syn-
thesized through microwave-assisted Schiff base formation by reacting 2-(4-methoxyphenyl)acetohydrazide
(3) and 4-amino-3-(4-methoxybenzyl)-1H-1,2,4-triazole-5(4H)-thione (6) with various substituted aldehydes.
Structures of the newly synthesized compounds were characterized by FT-IR, 1H NMR and 13C NMR spectral
analysis. All the synthesized derivatives were screened for their in vivo anti-inflammatory and in vitro anti-ox-
idant activities using carrageenan induced rat paw edema test and DPPH free radical scavenging assay, respec-
tively. In addition, molecular docking experiment was also performed to check the actual binding affinity of
ligand against target protein. Compounds 4a, 4c, 7a, and 7c screened as potent anti-inflammatory drugs signif-
icantly lowered the volume of rat paw edema (P < 0.05). In case of anti-oxidant assay, compound 7a with
ferrocenyl group as substituent R and 3,4-disubstitued 1,2,4-triazole as side coupled group exhibited IC50
value of 7.2 ± 2.7 mg/mL comparable with that of the reference ascorbic acid (2.61 ± 0.29 mg/mL) and was the
most active compound among the series. However, no prominent results were obtained in case of aralkanoic
acid hydrazide substituted Schiff base derivatives 4a – 4d. It is believed that the synthesized Schiff base deriv-
atives can be used for the development of potent anti-inflammatory and anti-oxidant drugs with considerable
advantages of convenient synthetic strategy possessing high product yield, short reaction time, and convenient
handling. The molecular docking results were found in good correlation with experimental IC50 values.
Keywords: microwave assisted synthesis; Schiff base derivatives; in vivo anti-inflammatory activity; in vitro
anti-oxidant activity.
1. INTRODUCTION
duced granuloma formation, peritoneal capillary permeabil-
ity test, and ethyl phenyl propionate or arachidonic acid
induced ear edema are usually employed. Among all the in-
flammatory tests, the most extensively used test for the
screening of new anti-inflammatory drugs is the carrageenan
induced paw edema in rat [2]. Inflammation is a part of com-
plex biological response for vascular tissues and mediators
involved in the inflammation reaction which can maintain,
induce, or aggravate many diseases. Steroidal or non-stero-
Inflammation is a physiological reaction to injury and in-
fectious, allergic, or chemical irritation. Inflammatory pro-
cesses are complex biochemical phenomena characterized by
tissue edema, pain, and leukocyte infiltration [1]. To evaluate
the anti-inflammatory activity of test samples, various types
of anti-inflammatory assays such as carrageenan induced
paw edema, serotonin induced paw edema, cotton pellet in-
1
Department of Chemistry, Government College University, Faisalabad (Sub-Campus Layyah), Pakistan.
College of Natural Sciences, Department of Biology, Kongju National University, Gongju, Chungcheongnam 32588, Republic of Korea.
Department of Biochemistry and Biotechnology (Baghdad-ul-Jadeed Campus), The Islamia University of Bahawalpur 63100, Pakistan.
Department of Physiology, University of Sindh, Jamshoro 76080, Pakistan.
2
3
4
5
6
7
8
Department of Chemistry, University of Sargodha (Sub-Campus Mianwali), Pakistan.
Department of Bioinformatics, Virtual University (Davis Road Campus), Lahore, Pakistan.
Institute of Molecular Science and Bioinformatics, 28 Nisbet Road, Opt. Dyal Singh Trust Library, Lahore 54000, Pakistan.
Department of Chemistry, University of Sargodha, sub-campus Bhakkar.
* e-mail: saleemqau@gmail.com
424
0091-150X/18/5205-0424 © 2018 Springer Science+Business Media, LLC

Microwave-Assisted Synthesis
425
idal chemical therapies are the current treatment for inflam-
matory disorders. Pain is the most prevalent inflammatory
symptom, and the two severe forms of inflammatory pain are
osteoarthritis (OA) and rheumatoid arthritis (RA). For treat-
ing pain and inflammation, nonsteroidal anti-inflammatory
drugs (NSAIDs) are used worldwide, which inhibit cyclo-
oxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) en-
zymes [3, 4].
the capability of such protective systems. In addition, ROS
induce lipid peroxidation causing the deterioration of foods,
which induces the oxidation of lipids and DNA resulting in
membrane damage, increased membrane fluidity, and
changes that lead to cancer via DNA mutation [14, 17]. Anti-
oxidant-based drug formulations are increasingly being rec-
ommended because they act directly upon oxidative pro-
cesses and are used for the prevention and treatment of com-
plex diseases such as stroke, atherosclerosis, Alzheimer’s
disease, diabetes, cancer, and health problems related to ag-
ing [18]. Hydroxy-substituted Schiff bases have been re-
ported as good free-radical scavenging agents [19]. Metal
complexes containing 2,6-di-tert-butylphenol group have
been reported as anti-oxidant, anti-inflammatory and ROS
scavenging agents [20]. Oxidative stress is thought to result
from imbalance between the free radicals and ROS and the
antioxidants that scavenge them [21].
Total phenolics, total anthocyanins, total flavonoids, to-
tal triterpenes, and monomeric compounds have been re-
ported as anti-oxidant and anti-inflammatory agents [5]. Re-
cently, Brazilian red propolis and its bioactive compounds
such as vestitol and neovestitol have been reported to possess
anti-inflammatory and antimicrobial activity [6]. Bleeding
and gastrointestinal tract (GIT) erosions are two major side
effects of NSAIDs, which have been observed even at low
doses of aspirin. In this regard, hybrid prodrugs NO-NSAIDs
have been reported [7]. Calcineurin (CN) inhibitors such as
tacrolimus and cyclosporine A (CsA) possess potent anti-in-
flammatory and immunosuppressive properties and have
been reported as beneficial treatment for autoimmune and in-
flammation diseases, but these drugs also exhibit severe nep-
hrotoxicity and hepatotoxicity side effects [8]. Norartocar-
petin and mornigrol D obtained from M. nigra root bark
inhibit the release of b-glucuronidase from rat polymor-
phonuclear leucocytes and show potent anti-inflammatory
activity [9, 10]. Several studies showed the effect of various
types of “berries” such as strawberry, blueberry and choke-
berry as anti-inflammatory agents [11]. Hence, over the last
century, inflammation remains the topic of numerous studies
at the cellular and molecular level. As of today, several medi-
ators that can modulate, initiate, and reduce inflammation
have been identified. More recently, several novel special-
ized pro-resolving mediators (SPMs) have identified [12].
The term antioxidant is applied in medicine and nutrition
to substances with low concentration levels that reduce reac-
tive nitrogen species (RNS) or reactive oxygen species
(ROS) before they can oxidize other molecules [13]. A grow-
ing amount of evidence has been accumulated indicating the
role of ROS such as singlet oxygen (1O2), superoxide anion
(O2·–), peroxyl radicals (ROO•), and hydroxyl radical (HO•)
in the pathophysiology of aging and various degenerative
diseases [14]. Among the cellular and extracellular compo-
nents, proteins, enzymes, and lipids, RNA and DNA are the
major targets for these reactive species. The antioxidative ef-
fect is mainly due to phenolic components, such as phenolic
acids, flavonoids and phenolic diterpenes [15]. In addition to
their role as antioxidants, these compounds exhibit a wide
spectrum of medicinal properties, such as anti-allergic,
anti-inflammatory, anti-microbial, anti-thrombotic, cardio-
protective, and vasodilatory effects [16]. Living cells possess
protective enzymatic and non-enzymatic defense systems of
antioxidants that suppress excess production and enables in-
activation of ROS. However, aging and various external fac-
tors such as alcohol, smoke, diet, and some drugs decrease
Herein, we report the microwave-assisted synthesis of
novel Schiff base derivatives and their in vivo anti-inflamma-
tory activity using carrageenan induced rat paw edema test as
well as in vitro anti-oxidant activity employing DPPH free-
radical scavenging assay. Compounds 4a, 4c, 7a, and 7c sig-
nificantly lower the volume of rat paw edema (P < 0.05)
when screened as potent anti-inflammatory drugs among the
series, while one of the synthesized compounds (7a) with
ferrocenyl group as substituent R and 3,4-disubstitued
1,2,4-triazole as side coupled group exhibited IC value of
50
7.2 ± 2.7 mg/mL comparable with that of reference ascorbic
acid (2.61 ± 0.29 mg/mL) and was the most active compound
among this series. In addition, computational experiment
was performed to check for significant binding of these com-
pounds with COX-1 and COX-2. The molecular docking re-
sults were found in good correlation with experimental IC
50
values.
Recently, Yehye, et al. [22] reported on triazole Schiff
base derivatives and evaluated their anti-oxidant activity.
Several aralkyl and alkoxy phenyl moieties were hybridized
to the triazole skeleton via imine linkage. Among the synthe-
sized materials, a Schiff base containing the phenyl group
substituted with tertiary butyl and hydroxyl unit was found to
possess highest biological activity with IC of 46.13 ±
50
0.31 mM [22]. Our synthesized compounds exhibited bicyc-
lo, ferocenyl, or bithiophenyl units connected to the central
triazole skeleton through imine linkage. The most potent
compound contained a ferocenyl unit hybridized to the tria-
zole skeleton with biological activity (IC = 7.2 ± 2.7 µM)
50
about six times better than that of reported material [22] and
much more close to the reference drug ascorbic acid. In addi-
tion, we have studied the synthesized compounds for molec-
ular docking in order to better evaluate the drug binding
modes. We believe that the presented data will be useful for
the future investigation and development of bioactive com-
pounds.

426
Muhammad Hanif et al.
Scheme 1. Synthesis of Schiff base derivatives 4a – 4d and 7a – 7d. Reagents and conditions: (i) POCl3, ClCH2CH2Cl, reflux 3 h; (ii)
hydrazine hydrate, TEA, MeCN, reflux 3 h; (iii) various substituted aldehydes, methanol, microwave pulses (10 min); (iv) CS2, KOH, metha-
nol, stirring at 0°C, 1 h; (v) hydrazine hydrate (80 %), water, reflux, 10 – 12 h; (vi) various substituted aldehydes, methanol, microwave pulses
(10 min).
2. EXPERIMENTAL
(ppm) measured downfield from the internal standard
tetramethylsilane in the indicated organic solution. Peak
multiplicities are denoted as follows: s, singlet; d, doublet;
m, multiplet. Other abbreviations: DMSO–d6, dimethyl
sulfoxide-d6; FT–IR spectroscopy, Fourier-transform infra-
red spectroscopy.
2.1. Materials and Methods
Substrate and reagents. 2-(4-Methoxyphenyl) acetic
acid, ferrocenecarboxaldehyde, 2,3-dihydrobenzo[b]dioxine-
6-carbaldehyde, benzo[b]thiophene-2-carbaldehyde, [2,2¢-bi-
thiophene]-5-carbaldehyde and phosphorous oxychloride
were purchased from Aldrich. Hydrazine hydrate (80%),
2.2. Chemical Synthesis
Schematic representation of the synthetic route adopted
to obtain target compounds 4a – 4d and 7a – 7d is shown in
Scheme 1.
TEA, CS , KOH, sodium hydrogen carbonate (Sigma-
2
Aldrich); ethanol, methanol, chloroform, water, acetonitrile,
dimethyl sulfoxide, petroleum ether, ethyl acetate, n-hexane
(Samchun Chemicals, Korea); and H SO , HCl (Jin Chemi-
2.2.1. Synthesis of 2-(4-methoxyphenyl) acetohydra-
zide (3). 2-(4-Methoxyphenyl)acetyl chloride (2) was syn-
thesized by reacting 2-(4-methoxyphenyl) acetic acid (1)
(1 mmol) in the presence of 1,2–dichloroethane (12 mL) sol-
vent with phosphorus oxychloride (0.4 mL) chlorinating
agent under reflux for 3 h. Then, the resulting solution was
cooled to room temperature and the solvent was removed un-
der reduced pressure to afford compound 2 that was directly
used in the next step without further purification. 2-(4-Me-
thoxyphenyl)acetyl chloride (2) was dissolved in acetonitrile
(80 mL), added dropwise to a solution containing hydrazine
hydrate (1 mmol), TEA (0.5 mL), acetonitrile (20 mL), and
allowed to reflux for 3 h with monitoring by TLC. After con-
sumption of the starting material, the reaction mixture was
cooled to room temperature. Evaporation of the solvent un-
der reduced pressure yielded crude 2-(4-methoxyphenyl)
2
4
cal & Pharmaceutical Co. Ltd., Korea) were used in experi-
ments.
Analytical instrumentation. The reaction progress was
monitored by thin layer chromatography (TLC) and the R
f
values were determined by employing pre–coated silica gel
aluminum plates Kieselgel 60 F from Merck (Germany).
254
TLC patterns were visualized under a UV lamp (VL–4 LC,
France). The melting points (MPs) were determined using a
Fisher Scientific (USA) melting point apparatus and re-
mained uncorrected. The IR spectra were recorded in KBr
pellets on a Shimadzu FTIR–8400S spectrophotometer
(Kyoto, Japan). Proton and carbon nuclear magnetic reso-
nance (1H NMR and 13C NMR) spectra were recorded on a
Bruker Avance 400 MHz spectrometer with TMS as the in-
ternal standard. The chemical shifts are reported as d values

Microwave-Assisted Synthesis
427
acetohydrazide (3) as a white solid upon cooling, which was
purified by column chromatography when necessary and
crystallized from methanol.
yellow solid; yield, 85%; R , 0.50 (chloroform – metha-
nol, 9:1); IR (n, cm-1): 3203 (NH), 3058, 2928 (CH), 1663
1
(C=O), 1603 (C=N), 1548, 1494 (C=C); H NMR (400 M
f
2-(4-Methoxyphenyl)acetohydrazide (3): white solid;
Hz, DMSO-d6; d, ppm): 10.13 (s, 1H, imine), 8.77 (s, 1H,
broad signal, NH), 7.84 – 7.79 (m, 2H, aromatic), 7.71 (s,
1H, thiophene), 7.67 – 7.51 (m, 2H, aromatic), 7.36 – 7.21
(m, 2H, aromatic), 6.97 – 6.85 (m, 2H, aromatic), 3.65 (s,
yield, 86%; MP, 134-136°C; R , 0.54 (n-hexane – ethyl ace-
tate, 1:1); FT-IR (n, cm-1): 3334, 3302 (NH ), 3205 (NH)
f
2
3037 (sp2 CH), 2958, 2837 (sp3 CH), 1618 (C=O), 1541,
2H, aliphatic CH ), 3.71 (s, 3H, methoxy); 13C NMR
1
1511, 1497 (C=C of phenyl ring); H NMR (400 MHz,
2
(100 MHz, DMSO-d6; d, ppm): 171.4, 159.1, 146.2, 138.4,
136.5, 135.4, 132.3, 130.1, 127.5, 127.2, 126.5, 122.7, 119.1,
114.9, 56.1, 38.8.
DMSO-d6; d, ppm): 9.21 (s, 1H, NH), 7.19 (aromatic, d, 2H,
J = 8.4 Hz), 6.86 (aromatic, d, 2H, J = 8.4 Hz), 4.23 (s, 2H,
broad, NH ), 3.71 (s, 3H, OCH ), 3.28 (s, 2H, CH ); 13C
2
3
2
(E)-N¢-[(2,2¢-Bithiophen-5-yl)-methylene]-2-(4-metho-
xyphenyl) acetohydrazide (4d): light-brown solid; yield,
NMR (100 MHz, DMSO-d6; d, ppm): 170.7, 159.6, 143.2,
129.7, 120.8, 114.6, 111.7, 55.3, 35.4.
88%; R , 0.50 (chloroform – methanol, 9:1); IR (n, cm-1):
2.2.2. General procedure for the synthesis of Schiff
base derivatives (4a – 4d). 2-(4-Methoxyphenyl) acetohyd-
razide 3 (0.125 mol, 1 eq) was dissolved in methanol
(20 mL) and various substituted aldehydes (0.125 mol, 1 eq)
were separately dissolved in methanol (20 mL). The two so-
lutions were mixed together prior to exposure under micro-
wave irradiation for 10 min with constant monitoring the re-
action progress by TLC after regular 2-min intervals. On
complete consumption of starting materials, the reaction
mixture was evaporated at reduced pressure and crystallized
from methanol. The product was purified when necessary by
column chromatography with n-hexane – ethyl acetate sol-
vent system.
f
3202 (NH), 3064, 2934 (CH), 1663 (C=O), 1592 (C=N),
1549, 1513, 1494 (C=C); 1H NMR (400 M Hz, DMSO-d6; d,
ppm): 10.10 (s, 1H, imine), 8.87 (s, 1H, broad signal, NH),
7.85 – 7.76 (m, 2H, aromatic), 7.73 – 7.71 (m, 2H, aro-
matic), 7.35 – 7.31 (m, 2H, aromatic), 7.24 – 7.18 (m, 2H,
aromatic), 7.14 – 7.09 (m, 2H, aromatic), 6.96 – 6.87 (m,
2H, aromatic), 3.65 (s, 2H, aliphatic CH ), 3.74 (s, 3H,
2
methoxy); 13C NMR (100 MHz, DMSO-d6; d, ppm): 171.8,
159.0, 143.9, 136.7, 131.4, 129.7, 127.4, 126.5, 126.1, 122.3,
121.4, 119.7, 115.2, 114.3, 55.4, 38.6.
2.2.3. Synthesis of 4-amino-3-(4-methoxybenzyl)-1H-
1,2,4-triazole-5(4H)thione (6). To potassium hydroxide
(0.125 mol) dissolved in dry methanol (50 mL) was added
2-(4-methoxyphenyl) acetohydrazide (3) (0.125 mol) and the
solution was cooled on ice. To this, solution was slowly
added carbon disulfide (0.125 mol) in small portions with
constant stirring for 2–3 h. The solid product of potassium
2-[2-(4-methoxyphenyl)acetyl]hydrazinecarbodithioate (5)
was filtered, washed with chilled diethyl ether, and dried. It
was directly used in the next step without purification. To po-
tassium 2-[2-(4-methoxyphenyl)acetyl]hydrazine carbodi-
thioate (5) in deionized water (20 mL) was added hydrazine
hydrate (0.250 mol) and the mixture was refluxed for
8 – 10 h. The reaction mixture turned yellowish-green with
evolution of hydrogen sulfide and finally it became homoge-
neous. Then, the mixture was poured in crushed ice and neu-
tralized with hydrochloric acid. The white precipitate of
4-amino-3-(4-methoxybenzyl)-1H-1,2,4-triazole-5(4H)thion
e (6) was filtered, washed with cold water, and crystallized
from aqueous methanol.
(E)-N¢-(Ferrocenylmethylene)-2-(4-methoxyphenyl)-
acetohydrazide (4a): dark-brown solid; yield, 86%; R , 0.52
f
(chloroform – methanol, 9:1); IR (n, cm-1): 3202 (NH), 3062,
2958 (CH), 1677 (C=O), 1602 (C=N), 1547, 1494 (C=C); 1H
NMR (400 M Hz, DMSO-d6; d, ppm): 10.16 (s, 1H, imine),
8.99 (s, 1H, broad signal, NH), 7.37 – 7.22 (m, 2H, aro-
matic), 6.90 – 6.89 (m, 2H, aromatic), 4.82 – 4.01 (m, 9H,
ferrocene), 3.65 (s, 2H, aliphatic CH ), 3.62 (s, 3H,
2
methoxy); 13C NMR (100 MHz, DMSO-d6; d, ppm): 171.1,
158.9, 145.2, 130.6, 127.3, 78.5, 77.4, 76.8, 73.2, 70.6, 69.3,
68.1, 67.7, 60.4, 55.2, 38.9.
(E)-N¢-[{2,3-Dihydrobenzo(b](1,4)dioxin-6-yl}methy-
lene]-2-(4-ethoxyphenyl) acetohydrazide (4b): off-white
solid; yield, 85%; R : 0.53 (chloroform – methanol, 9:1); IR
f
(n, cm-1): 3203 (NH), 3087, 3011 (CH), 1663 (C=O), 1601
(C=N), 1548, 1493 (C=C); 1H NMR (400 M Hz, DMSO-d6;
d, ppm): 10.03 (s, 1H, imine), 8.70 (s, 1H, broad signal, NH),
7.89 (s, 1H, aromatic), 7.69 (d, 1H, J = 7.5, aromatic), 7.43
(d, 1H, J = 7.5, aromatic), 7.32 – 7.22 (m, 1H, aromatic),
7.19 – 7.14 (m, 1H, aromatic), 6.92 – 6.85 (m, 2H, aro-
matic), 4.36 (t, 2H, aliphatic), 4.27 (t, 2H, aliphatic), 3.64 (s,
4-Amino-3-(4-methoxyphenyl)-1H-1,2,4-triazole-5(4H)-
thione (6): white solid; yield, 75%; MP, 215 – 217°C; R
f,
0.71 (petroleum ether – ethyl acetate, 7:3); IR (n, cm-1):
3294, 3130 (NH), 3350 – 3273 (NH ), 2937 (sp2 CH), 1585
2
(C=N), 1561 – 1506 (C=C), 1335 (C=S); 1H NMR
2H, aliphatic CH ), 3.65 (s, 3H, methoxy); 13C NMR
(400 MHz, CDCl ; d, ppm): 13.81 (s, 1H, NH), 7.98 – 7.95
2
3
(100 MHz, DMSO-d6; d, ppm): 174.2, 158.5, 147.8, 145.5,
143.8, 130.5, 127.5, 125.9, 121.4, 117.5, 115.6, 114.2, 64.5,
64.1, 55.3, 38.5.
(m, 2H, Ar-H), 7.07 – 6.97 (m, 2H, Ar-H), 5.76 (s, 2H, NH ),
2
3.72 (s, 3H, OCH ); 13C NMR (100 MHz, CDCl ; d, ppm):
3
3
167.12, 161.31, 158.22, 132.57, 130.11, 118.61, 56.32.
2.2.4. General procedure for the synthesis of Schiff
base derivatives (7a – 7d). 4-amino-3-(4-methoxybenzyl)-
(E)-N¢-[Benzo(b)thiophen-2-ylmethylene]-2-(4-metho-
xyphenyl) acetohydrazide (4c):

428
Muhammad Hanif et al.
1H-1,2,4-triazole-5(4H)thione (6) (0.125 mol, 1 eq) was dis-
solved in methanol (20 mL) and various substituted alde-
hydes (0.125 mol, 1 eq) were separately dissolved in metha-
nol (20 mL). The two solutions were mixed together prior to
exposure under microwave irradiation for 10 min with con-
stant monitoring of the reaction progress by TLC at regular
2-min intervals. Upon complete consumption of starting ma-
terials, the reaction mixture was evaporated at reduced pres-
sure and crystallized from methanol. The product was puri-
fied when necessary by column chromatography with n-hex-
ane – ethyl acetate solvent system on needed.
7.81 – 7.64 (m, 2H, aromatic), 7.34 – 7.11 (m, 5H, aromatic),
6.97 – 6.89 (m, 2H, aromatic), 3.58 (s, 2H, aliphatic CH ),
2
3.67 (s, 3H, methoxy); 13C NMR (100 MHz, DMSO-d6; d,
ppm): 164.5, 159.6, 157.8, 149.5, 144.5, 137.8, 136.4, 135.5,
131.1, 129.4, 128.3, 122.4, 119.8, 118.1, 114.5, 55.5, 38.4.
2.3. Biological Activity
2.3.1. In vivo anti-inflammatory activity protocol.
Anti-inflammatory activity of synthetic compounds was
evaluated using the carrageenan induced rat paw edema test
by following the reported procedure [23, 24].
(Z)-4-{(ferrocenylmethylene)amino}-3-(4-methoxyben-
zyl)-1H-1,2,4-triazole-5(4H)thione (7a): dark-brown solid;
Animals and maintenance. Healthy adult male Spra-
gue – Dawley rats (n = 50, average body weight 150 ± 10 g)
were obtained from the Daehan Biolink Co. (Chungcheong-
buk-Do, Korea). Five animals were housed per cage. They
were maintained at standard conditions of laboratory temper-
ature and photoperiod and had free access to standard rodent
diet.
yield, 86%; R , 0.54 (chloroform – methanol, 9:1); IR (n,
f
cm-1): 3079, 2977 (CH), 1588 (C=N), 1561, 1544, 1479
1
(C=C), 1329 (C=S); H NMR (400 M Hz, DMSO-d6); d,
ppm): 13.88 (s, 1H, broad, NH), 10.16 (s, 1H, imine),
7.24 – 7.18 (m, 2H, aromatic), 6.98 – 6.87 (m, 2H, aro-
matic), 4.84 – 4.06 (m, 9H, ferrocene), 3.56 (s, 2H, aliphatic
Dosage. 1% (w/v) solution of lambda carrageenan
(Sigma) was prepared in 0.9 % saline. The model edema was
induced by injecting 0.1 mL carrageenan solution. Standard
drug indomethacin (Sigma) was injected at 10 mg/kg only to
animals in the positive control group. Test compounds were
injected at a dose of 5 mg/animal.
CH ), 3.54 (s, 3H, methoxy); 13C NMR (100 MHz,
2
DMSO-d6; d, ppm): 162.1, 159.6, 157.7, 149.5, 132.9, 124.9,
122.3, 77.6, 77.3, 75.8, 73.2, 70.5, 68.9, 68.1, 60.3, 55.5,
38.6.
(Z)-4-[{(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methy-
lene}amino]-3-(4-methoxybenzyl)-1H-1,2,4-triazole-5(4H)-
Experimental design. Rats were weighed and marked
with individual numbers. Weighed rats were divided into ten
groups, each group containing five animals as follows:
(i) normal control group treated with standard drug indo-
methacin;
thione (7b): yellow solid; yield, 85%; R , 0.51 (chloro-
f
form – methanol, 9:1); IR (n, cm-1): 3094, 2981 (CH), 1597
1
(C=N), 1561, 1527, 1487 (C=C), 1329 (C=S); H NMR
(400 M Hz, DMSO-d6; d, ppm): 13.89 (s, 1H, broad, NH),
10.04 (s, 1H, imine), 7.94 – 7.91 (m, 1H, aromatic),
7.51 – 7.47 (m, 1H, aromatic), 7.27 – 7.18 (m, 2H, aro-
matic), 6.96 – 6.88 (m, 3H, aromatic), 4.29 (t, 2H, aliphatic),
(ii) positive control (carrageenan edema) group pretre-
ated with standard drug indomethacin; (iii – vi) carrageenan
edema test groups pretreated with compounds 4a, 4b, 4c, and
4d, respectively;
4.29 (t, 2H, aliphatic), 3.59 (s, 2H, aliphatic CH ), 3.55 (s,
2
(vii – x) carrageenan edema test groups pretreated with
compounds 7a, 7b, 7c, and 7d, respectively.
3H, methoxy); 13C NMR (100 MHz, DMSO-d6; d, ppm):
162.9, 159.4, 157.5, 154.6, 149.5, 149.2, 136.5, 132.0, 129.5,
128.6, 121.5, 118.2, 115.4, 65.3, 65.1, 55.6, 38.3.
One hour after injecting test compounds or standard
drug, the edema model was induced in the rat paw by inject-
ing 0.1 mL of freshly prepared 1% w/v carrageenan solution
into subplantar tissues of the left hind paw of each rat. The
paw volume of the rat was determined instantaneously after
the carrangeenan injection (0 h) and then at every 1 h inter-
val for 7 h by the volume displacement method using digital
plethysmometer (Ugo Basile, 21025 Comerio (VA), Italy).
The percentage inhibition of edema was calculated using the
following formula:
(Z)-4-{(benzo[b]thiophen-2-ylmethylene)amino}-3-(4-
methoxybenzyl)-1H-1,2,4-triazole-5(4H)thione (7c): beige
solid; yield, 85%; R , 0.52 (chloroform – methanol, 9:1); IR
f
(n, cm-1): 3070, 2961, 2833 (CH), 1587 (C=N), 1584, 1452
1
(C=C), 1331 (C=S); H NMR (400 M Hz, DMSO-d6; d,
ppm): 13.85 (s, 1H, broad, NH), 10.12 (s, 1H, imine),
7.88 – 7.77 (m, 2H, aromatic), 7.64 – 7.53 (m, 3H, aro-
matic), 7.34 – 7.23 (m, 2H, aromatic), 7.97 – 7.84 (m, 2H,
aromatic), 3.59 (s, 2H, aliphatic CH ), 3.67 (s, 3H, methoxy);
2
13C NMR (100 MHz, DMSO-d6; d, ppm): 165.5, 159.3,
158.2, 149.8, 137.1, 136.5, 134.4, 131.3, 129.2, 126.3, 122.4,
122.3, 118.7, 115.9, 55.8, 38.9.
N.C-C.E
Percentage edema inhibition =
´100,
N.C
(Z)-4-[{(2,2¢-bithiophen)-5-ylmethylene}amino]-3-(4-
methoxybenzyl)-1H-1,2,4-triazole-5(4H)thione (7d): mus-
where N. C is the mean increase in paw volume of negative
control and C. E is the mean increase in paw volume of com-
pound treated or positive group.
tard solid; yield, 87%; R , 0.54 (chloroform – methanol, 9:1);
f
IR (n, cm-1): 3078, 2954 (CH), 1592 (C=N), 1554, 1533,
1478 (C=C), 1334 (C=S); 1H NMR (400 M Hz, DMSO-d6; d,
ppm): 13.81 (s, 1H, broad, NH), 10.14 (s, 1H, imine),
Statistical Analysis. Experimental data were analyzed
through one-way analysis of variance (ANOVA) using the
Statistical Package for Social Sciences (SPSS version 16.0

Microwave-Assisted Synthesis
429
Inc. Chicago, Illinois, USA). P < 0.05 was considered statis-
tically significant difference.
mera 1.10.1. The Molsoft (http://www.molsoft.com/) and
Molinspiration (http://www.molinspiration.com/) online
ligand analysis tools were employed to predict the drug-like-
ness and basic biological properties of these designed struc-
tures. Moreover, Lipinski’s rule of five (RO5) was analyzed
using Molsoft and Molinspiraion tools. The numbers of ro-
tatable bonds, H-bond acceptors (HBA), and H-bond donors
(HBD) were also confirmed by PubChem (https://pubchem.
ncbi.nlm.nih.gov/). The pharmacokinetic properties like Ab-
sorption, Distribution, Metabolism, Excretion and Toxicity
(ADMET) were evaluated using pkCSM server [41].
2.3.2. Free radical scavenging assay protocol. Free
radical scavenging activity of the synthesized compounds
were determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH)
assay following the reported procedure [25 – 28]. Briefly, the
assay solution consisted of 100 mL of 150 mM DPPH and
20 mL of increasing concentration of test compound, with the
total volume adjusted to 200 mL with DMSO in each well.
The reaction mixtures were then incubated for 30 min at
room temperature. Ascorbic acid (vitamin C) was used as the
reference drug. The assay measurements were carried out for
2.4.3. Grid generation and molecular docking using
PyRx tool. Prior to molecular docking, the optimized
COX-1 and COX-2 structures were prepared. Bond orders
were assigned and hydrogen atoms were added to the pro-
tein. Grid preparation and molecular docking study was car-
ried out for the synthesized ligands 7a – 7d against COX-1
and COX-2 to obtain different docked complexes by PyRx
tool (Vina Wizard) [42]. The grid box parametric values of
X = 28.6432, Y = 28.6975, and Z = 9.4725 with 1.0 Å spac-
ing, and exhaustiveness values (8) were adjusted to attain the
finest binding conformational pose of ligands. Molecular
docking approach was employed for all ligands (7a – 7d)
against COX-1 and COX-2 to obtain various docked com-
plexes. Furthermore, the obtained COX1 and COX-2 docked
complexes were further evaluated on lowest binding energy
(kcal/mol) values and ligand – protein binding interaction
patterns (hydrogen/hydrophobic). Docking analysis was em-
ployed by Discovery Studio (2.1.0) and UCSF Chimera
1.10.1.
96-well plates using a microplate reader (OPTI
, Tunable
Max
Micro Plate Reader; wavelength range, 340 – 850 nm) at
517 nm. The reaction rates of test compounds were com-
pared to the reference drug and the percentage inhibition was
calculated as 100 – (Abs /Abs
) ´ 100, where Abs is
control
test
the peak absorbance [29]. Each concentration was analyzed
in three independent experimental runs in triplicate. The IC
50
values were determined by the data analysis and graphing
64-bit Origin 8.6 software.
2.4. Computational Methodology
2.4.1. Retrieval of protein structures from PDB.
COX-1 and COX-2 receptors were used to identify the bind-
ing affinity of synthesized ligands. In our experimental study
we analyzed the inflammatory activity, therefore, both
COX-1 and COX-2 were accessed and used as target mole-
cules. Preliminary results showed that both structures could
be used to predict anti-inflammatory activity [30]. Both tar-
get proteins (COX-1 and COX-2) were accessed through
homology based modeling and Protein Data Bank. The crys-
tal structure of human COX-1 is not present in PDB [31, 32].
Therefore, homology modeling approach was employed to
predict three-dimensional (3D) structure of COX-1 protein.
The amino acid sequences of COX-1 in FASTA format hav-
ing accession number (P23219) was obtained from Uniprot
Knowledge database (http://www.uniprot.org/). To build
COX-1 model, the Swiss modeling approach was employed
using sheep (Ovis aries) template having PDBID 401Z on
the basis of sequence identity 92.97% and query coverage
31 – 583 amino acids. The crystal structure of human COX-2
was retrieved from the PDB with PDBID 5F19. Furthermore,
the validity of predicted structure was confirmed using vari-
ous evaluation tools such as ERRAT, Anolea, ProCheck, and
Rampage [33 – 36]. Energy minimization of COX-1 was
performed through Vega-ZZ [37] while UCSF Chimera 1.6
was employed to visualize the predicted structures [38]. The
overall stereo-chemical properties, hydrophobicity, and
Ramachandran values of COX-1 and COX-2 were assessed
using Discovery Studio 4.0 and MolProbity server, respec-
tively [39, 40].
3. RESULTS AND DISCUSSION
3.1. Chemistry
The conversion of 2-(4-methoxyphenyl)acetic acid (1) to
2-(4-methoxyphenyl)acetyl chloride (2) was indicated by IR
spectral data as the disappearance of broad signal in the
range of 3400 – 2500 cm-1 related to acid hydroxyl group.
The formation of 2-(4-methoxyphenyl)acetohydrazide (3)
was indicated by IR spectral data as the appearance of new
peaks at 3334 and 3302 – 3205 cm-1 related to primary and
secondary amino group of acid hydrazide. In addition, there
was a slight shift in the carbonyl stretching vibrations from
1733 to 1618 cm-1 indicating the successful conversion of
2-(4-methoxyphenyl)acetyl chloride (2) into 2-(4-methoxy-
phenyl)acetohydrazide (3). The appearance of a broad singlet
at 9.21 ppm due to primary NH proton resolution as well as
4.23 ppm due to secondary NH proton resolution further
confirmed the formation of 2-(4-methoxyphenyl)
acetohydrazide (3). The transformation of 2-(4-methoxyphe-
nyl) acetohydrazide (3) into substituted Schiff base deriva-
tives 4a – 4d was initially indicated by the FT-IR spectral
data as the appearance of new signals in the range of
1603 – 1592 cm-1 related to C=N stretching vibrations and
2.4.2. Computational design of synthesized ligands.
The synthesized compounds 7a – 7d were sketched in draw-
ing ACD/ChemSketch tool and minimized by UCSF Chi-

430
Muhammad Hanif et al.
complete decline in NH peak of bare hydrazide moiety. Fur-
lowered the edema volume (P < 0.05; Fig. 1G). Compound
7d caused no any notable change in edema volume (Fig. 1H).
It appears from the present study that compounds 4a, 4c, 7a
and 7c exhibited almost similar activities in increasing per-
centage inhibition of rat paw edema. Compounds 4a and 7a
contain ferrocenyl group as substituent R, while in case of
compounds 4c and 7c there is 2-methylbenzo[b]thiophenyl
group as substituent R. Meanwhile, compounds 4b, 4d, 7b
and 7d did not show any significant anti-inflammatory activ-
ity. In case of 4b and 7b, there was 6-methyl-2,3-dihydro-
benzo[b][1, 4]dioxinyl group as substituent R, while com-
pounds 4d and 7d have 5-methyl-2,2¢-bithiophenyl group as
substituent R. From these results, it was concluded that iso-
lated thiophenyl group attached through Schiff base forma-
tion in the core skeleton of the target compounds caused sup-
pression of anti-inflammatory potential, while in bicyclic
system with phenyl ring, this group caused increase in
anti-inflammatory activity. Meantime, dioxane moiety in
connection to phenyl ring as bicyclic system reduced the
anti-inflammatory potential of Schiff base derivatives
(Fig. 1).
2
ther confirmation of target Schiff base derivatives 4a – 4d
was based on 1H NMR and 13C NMR spectral analysis. The
1
appearance of downfield singlet peak in case of H NMR
spectra in the range of 10.13 – 10.03 ppm due to imine pro-
ton and disappearance of NH signal at 4.23 ppm confirm the
2
formation of target compounds 4a – 4d. Further evidence
about product formation was obtained from 13C NMR spec-
tral analysis. The formation of 4-amino-3-(4-methoxyben-
zyl)-1H-1,2,4-triazole-5(4H)-thione (6) was confirmed by
IR, 1H NMR and 13C NMR spectral analysis. In the IR spec-
trum, a relatively broad peak in the range of 3294 –
3130 cm-1 with shoulder for NH stretching vibration and a
2
new signal at 1335 cm-1 for C=S stretching vibration were
observed, which indicated the transformation of 2-(4-metho-
xyphenyl)acetohydrazide (3) into 4-amino-3-(4-methoxy-
1
benzyl)-1H-1,2,4-triazole-5(4H)-thione (6). In the H NMR,
the presence of characteristic highly downfield signal at
13.81 ppm due to triazole ring secondary amino proton and
disappearance of secondary amino proton confirmed the for-
mation of 4-amino-3-(4-methoxybenzyl)-1H-1,2,4-triazole-
5(4H)-thione (6). The condensation of 4-amino-3-(4-metho-
xybenzyl)-1H-1,2,4-triazole-5(4H)-thione (6) with various
substituted aldehydes was confirmed by NMR (1H NMR and
13C NMR) spectral analysis and FT-IR spectroscopy. The
FT-IR spectrum indicated the formation of target compounds
7a – 7d by the disappearance of relatively broad peak in the
In conclusion, the results showed considerable variation
in biological activity of the synthesized compounds depend-
ing on changes in their structures. The aralkyl moiety in the
target compounds 4a – 4d and 7a – 7d did not exert much ef-
fect on the anti-inflammatory potential of the synthesized
Schiff base derivatives. On the other hand, a change in
substituent R led to considerable alteration in the biological
activity. The ferrocenyl group and 2-methylbenzo[b]thiophe-
nyl were found to be excellent candidates to incorporate the
anti-inflammatory potential in the synthesized Schiff base
derivatives 4a – 4d and 7a – 7d.
range of 3280 – 3169 cm-1 with shoulder for NH stretching
2
vibration. The NMR spectra provide further confirmation
about successful condensation of different substituted alde-
hydes with the 4-amino-3-(4-methoxybenzyl)-1H-1,2,4-
triazole-5(4H)-thione (6) through the disappearance of broad
signal in the range of 3.59 – 3.56 ppm and the appearance of
new downfield signal in the range of 10.16 – 10.04 ppm due
to imine proton resolution. Furthermore, there was a slight
shift in the triazole NH signal in the 1H NMR spectra of the
resulting compounds alongside with the additional signals in
both carbon and proton NMR spectra, which confirmed the
successful synthesis of target compounds 7a – 7d.
3.2.2. Anti-oxidant activity. In the DPPH free radical
scavenging assay, ascorbic acid with IC 2.61 ± 0.29 mg/mL
50
was used as the standard drug. Compound 7a with IC value
50
of 7.2 ± 2.7 mg/mL was the most active compound among
the series studied, while the IC values of other synthesized
50
compounds was in the range of 330.2 ± 10, 172.4 ± 4 and
403.4 ± 7 mg/mL for 7b, 7c and 7d, respectively. In case of
the synthesized Schiff base derivatives 4a – 4d, no signifi-
cant anti-oxidant potential was found. These results lead us
to conclude that the Schiff base derivatives containing
3,4-disubstitued 1,2,4-triazole nucleus exhibit considerable
anti-oxidant potential, while this anti-oxidant activity was
not triggered by only triazole subunit, as there were notice-
able fluctuations in the DPPH free-radical scavenging assay
results for varying side coupled R group. The most potent
compound with respect to DPPH free-radical scavenging as-
3.2. Pharmacology
3.2.1. In vivo anti-inflammatory activity. In this study,
eight compounds (4a – 4d and 7a – 7d) were tested on rats
for anti-inflammatory potential. In general, the animals re-
mained healthy and active, and no any deaths occurred in any
group. Compound 4a showed significantly lowered volume
of rat paw edema (P < 0.05; Fig. 1A). Compound 4b did not
show significant anti-inflammatory activity (Fig. 1B).
Highly significant anti-inflammatory activity was observed
for compound 4c (P < 0.05; Fig. 1C), whereas compound 4d
was not significantly active against inflammation (Fig. 1D).
Meanwhile, compound 7a showed highly momentous per-
centage inhibition of rat paw edema volume (P < 0.05;
Fig. 1E), while no significant change was observed for com-
pound 7b (Fig. 1F). Injection of compound 7c appreciably
say was 7a exhibiting IC values almost close to standard
50
drug (vitamin C), which was considered to be due to
3,4-disubstitued 1,2,4-triazole unit as well as substituted
ferrocenyl moiety. Meanwhile, the second and third most ac-
tive compounds among the series were 7c and 7b containing
2-methylbenzo[b]thiophenyl as well as 6-methyl-2,3-dihyd-
robenzo[b][1, 4]dioxine groups as substituent R along with

Microwave-Assisted Synthesis
431
Fig. 1. Percentage inhibition of rat paw edema treated with compounds 4a (A), 4b (B), 4c (C), 4d (D), 7a (E), 7b (F), 7c (G), 7d (H) and
indomethacin (reference drug, * P < 0.05).
side coupled groups. However, the least active compound
among synthesized compounds 7a – 7d was 7d, which con-
tained 5-methyl-2,2¢-bithiopheny as substituent R. Concur-
rently, there was no anti-oxidant potential in hydrazide sub-
stituted Schiff base derivatives 4a – 4d. The results of anti-
oxidant assay for the synthesized compounds 7a –7d along
with control are shown in Fig. 2.
In summary, from these results it was concluded that a
side coupled substituent alone exerts negligible effect on the
anti-oxidant potential, while in connection through Schiff
base formation with 3,4-disubstitued 1,2,4-triazole subunit, it
becomes more potent against scavenger during the time and
in connection to hydrazide through Schiff base formation it
does not implement any scavenging effect.

432
Muhammad Hanif et al.
Fig. 2. DPPH free-radical scavenging assay results for synthesized
Schiff base derivatives 7a – 7d (compounds 4a – 4d did not exhibit
any scavenging potential at concentrations up to 500 mg/mL).
3.3. Computational Analysis
3.3.1. COX-1 and COX-2 structure evaluation. The
COX-1 and COX-2 are functionally involved in prostaglan-
din (PG). COX-1 contains 599 amino acids, whereas
COX-2 consists of 604 residues. Available research data
showed that that the crystal structure of COX-1 was not re-
ported; therefore, in-silico approach was used to predict 3D
structure of COX-1 protein. The reliability and efficacy of
both COX-1 and COX-2 structures were analyzed on the ba-
sis of Ramachandran and hydrophobicity graphs. The
Ramachandran plots of both COX-1 and COX-2 indicate that
95.01% and 97.3% residues, respectively, are present in fa-
vorable regions. The generated Ramachandran graphs for
COX-1 and COX-2 protein also depicted the good accuracy
of phi (f) and psi (y) angles among the coordinates, and
most of the protein (COX-1 and COX-2) residues are plum-
meted in the acceptable region. The hydrophobicity graphs
for both domains showed residues within the favored region.
Moreover, the protein architectures including helices,
b-sheets, and coils were also keenly observed through
VADAR 1.8 tool. The predicted results show that
Fig. S1. Superimposed structures of COX-1 (blue) and COX-2
(yellow).
COX-1 contains 44% helices, 13% b-sheets, and 42% coils,
whereas COX-2 comprises 41% helices, 6% b-sheets, and
52% coils. Superimposed structures of both COX-1 and
COX-2 with binding pocket are presented in Fig. S1. The
hydrophobicity and Ramachandran graphs for COX-1 and
COX-2 are shown in Figs. S2 and S3, respectively.
3.3.2. Chemo-informatics evaluation and RO5 analy-
sis of synthesized ligands. Multiple computational servers
and tools were used to predict the biochemical properties of
synthesized compounds (Table 1). Research data referred to
TABLE 1. Chemo-Informatics Evaluation of Synthesized Compounds
Properties
Mol. Weight (g/mol)
7a
458.18
4
7b
382.11
6
7c
380.08
5
7d
412.05
6
Nunber of HBA
Number of HBD
Mol. LogP
1
1
1
1
5.95
0
3.59
0
4.97
0
5.45
0
Number of stereo centers
Mol. volume (A3)
Drug Likeness Score
436.13
0.27
388.19
0.06
383.02
0.07
402.18
0.14

Microwave-Assisted Synthesis
433
Fig. S2. (A) Hydrophobicity and (B) Ramachandran graphs of COX-1.
Fig. S3. (A) Hydrophobicity and (B) Ramachandran graphs of COX-2.

434
Muhammad Hanif et al.
standard values of molecular weight (MW) from 160 to
480 g/mol. Comparative analyses showed that the predicted
results for all compounds were comparable with standard
values. RO5 analysis also confirmed the therapeutic potential
of all ligands. Hydrogen-bonding affinity has also been con-
sidered as a significant parameter for evaluating the drug
permeability. Some studies showed that excessive values of
HBA (>10) and HBD (>5) in ligands resulted in their poor
permeation in the body [43, 44]. Our chemo-informatics
analyses show that all the designed compounds possess
HBA < 10 and HBD < 5, which may confirm their good pen-
etration within the body. Moreover, their logP values were
also comparable with standard values . However, there are
plenty of examples available for RO5 violation among the
existing drugs [45 – 47]. The drug score value describes the
significance of compounds as lead-like behavior. Results
showed that all the compounds may have strong therapeuti-
cal potential and potent drug-like behavior. Our in-silico de-
piction for all the compounds agrees with these results.
pharmacokinetic behavior of synthesized ligands (7a – 7d), a
freely accessible web server pkCSM was employed to pre-
dict their ADMET properties (Table 2). In ADMET evalua-
tion, the predicted values absorption-like aqueous solubility
(log mol/L), intestinal absorption (% Abs), and skin perme-
ability (logKp) indicated strong therapeutic potential of se-
lected compounds. It was found that compounds with good
absorption have potency to cross the gut barrier by passive
penetration to reach the target molecule. The aqueous solu-
bility data showed that compounds 7a – 7d had good log
mol/L values (–5.296, –3.657, –4.39, and –4.47, respec-
tively). Moreover, the intestinal solubility predictions also
justified their good efficacy compared to standard values.
The generated results showed that all compounds possess
good intestinal solubility values compared to the standard
(30% absorption). Compound 7b showed higher intestinal
solubility (92.475% Abs). Investigations showed that com-
pounds with less than 30% Abs value must be considered as
poorly absorbed. Furthermore, the skin permeability values
(logKp) of all compounds were also greater than standard
value (–2.5 logKp) which showed their significance as good
lead structures and justified their drug-like behavior. More-
over, blood – brain barrier (BBB) and central nervous system
(CNS) permeability values of compounds were also compa-
rable with the standard values (>0.3 and £1 logBB; >2 and
£3 logPS). It has been found that compounds with greater
than 0.3 logBB value have potential to cross BBB, while
those with less than –1 value are poorly distributed to the
brain. Similarly, the ligands having >2 logPS value are con-
3.3.3. Pharmacokinetic evaluations of designed com-
pounds. The development of novel drugs requires high at-
tention to their good pharmacokinetic properties. Mostly,
ADMET properties assessment is used to confirm the effi-
cacy of results for lead molecules. For calculation of the
TABLE 2. ADMET Assessment of Designed Compounds
ADMET properties*
7a
7b
-3.657
92.475
-2.753
0.02
7c
7d
-4.47
87.182
-2.736
-0.567
0.147
-1.958
Yes
Absorp-
WS
IS
-5.296
83.667
-2.884
0.306
0.211
-1.585
Yes
-4.39
88.717
-2.738
-0.374
0.222
-1.951
Yes
sidered to penetrate the CNS, while those with £3 logPS are
difficult to move into the CNS.
tion
SP
The computation generated values for all compounds
7a – 7d were comparable with the standard values. Our pre-
dicted results showed that selected compounds displayed a
significant potential to cross the barriers and may be targeted
directly to receptor molecule. Moreover, metabolic behavior
with inhibitory potential was confirmed for CYP1A2,
CYP2C19, CYP2C9, and CYP3A4. Positive values for all
compounds confirmed the strong inhibitory behavior against
target proteins with good metabolic behavior. The predicted
excretion and toxicity values also justified the drug-like be-
havior of these compounds on the basis of total clearance
(log mL/min/kg), AMES toxicity, maximum tolerated dose
Distribu-
tion
VDss
BBBP
CNSP
CYP1A2
CYP2C19
CYP2C9
CYP3A4
TC
-1.355
-2.449
Yes
Metabo-
lism
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Excretion
Toxicity
-0.032
No
0.031
No
0.102
Yes
0.055
Yes
AMES
toxicity
MTD
-0.308
0.106
2.476
Yes
0.243
2.727
Yes
0.287
2.767
No
(MTD), and LD values. The AMES toxicity prediction for
50
ORAT(LD50) 2.755
selected compounds confirmed that there is non-mutagenic
and non-toxic behavior. The hepatotoxicity and skin sensitiv-
ity negative behavior was showed their non-toxic and less
sensitive effects. Compounds 7b and 7c showed negative
hepatotoxic behavior as compared to other compounds. The
absence of skin sensitization effect was observed for all com-
pounds. Thus, hypothetical ADMET properties confirmed
that these ligands exhibited good lead-like potential for fur-
ther evaluation.
HT
SS
Yes
No
No
No
No
* Abbreviations: WS = water solubility (log mol/L); IS = intestinal
solubility (% abs); SP = skin permeability (log Kp); VDss = volumn
of distribution (log L/kg); BBBP = blood brain barrier permeability
(logBB); CNSP = CNS permeability (logPS); TC = total clearance
(log mL/min/kg); MTD = maximum tolerated dose; ORAT = oral
rat acute toxicity; HT = hepatotoxicity; SS = skin sensitization.

Microwave-Assisted Synthesis
435
3.4. Molecular Docking Study
3.4.1. Binding energy analysis. Docking experiments
were used only to estimate the binding potential of synthe-
sized ligands against molecular targets (COX1, COX2). The
docking (protein – ligand) complexes of all compounds
7a – 7d were analyzed individually and evaluated for mini-
mum energy (kcal/mol) values presented in Fig. 3. The en-
ergy values justified the binding potential of synthesized
compounds against the target proteins. Results showed that
the docking complexes of compound 7a were most active
against both receptors (COX-1 and COX-2) with lowest
binding energy value (–10.4 and –11.0 kcal/mol, respec-
tively) compared to others compounds. The docking energy
values of all docking complexes were calculated using the
following equation:
DG binding = DG gauss + DG repulsion + DG hbond +
+ DG hydrophobic + DG tors.
(1)
Here, DG gauss is the attractive term for dispersion of
two gaussian functions, DG repulsion is the square of the dis-
tance if closer than a threshold value, DG hbond is the ramp
function (also used for interactions with metal ions), DG hy-
Fig. 3. Docking energy values of synthesized compounds against
COX-1 and COX-2.
Fig. 4. Docking interactions of compounds 7a – 7d with COX-1 enzyme. COX-1 is highlighted in purple color in wire ribbon and surface for-
mat with all the four compounds embedded in the middle of diagrams. In detailed interaction patterns against COX-1, ligands 7a – 7d are shown
in red, yellow, green, and blue color, respectively.

436
Muhammad Hanif et al.
Fig. 5. Docking interactions of compounds 7a – 7d with COX-2 enzyme. COX-2 is highlighted in green color in wire ribbon and surface for-
mat with all the four compounds embedded in the middle of diagram. In detailed interactions patterns against COX-2, ligands 7a – 7d are shown
in red, yellow, green, and blue color, respectively.
residues were common in the interaction pattern, which may
confirm their significance in downstream signaling path-
ways. In COX-2 docking, compound 7a forms eight hydro-
phobic interactions at various residual positions including
Gly104, Pro122, Pro125, Cys4, Pro123, Arg12, Leu121, and
Val14. However, in 7b and 7c docking complexes, Cys15
was the commonly interacting residue involved in hydrogen
bonding with bond lengths presented in Fig. 5.
drophobic is the ramp function, and DG tors is proportional
to the number of rotatable bonds. Previous research showed
that the standard error for Autodock is about 2.5 kcal/mol.
However, the predicted energy values for all docking com-
plexes were less than the standard value. Although the basic
nucleus of all the synthesized compounds was similar, most
of ligands possessed good effective energy values and had no
big energy fluctuations.
3.4.2. Structure – activity relationship analysis of synthe-
sized ligands. Structure activity relationship analysis showed
that all compounds 7a – 7d possessed an active region for
target proteins (COX-1 and COX-2). Figure 4 shows that in
COX-1 docking a bonding interactions were observed in all
docking complexes of compounds 7a – 7d. Seven hydropho-
bic interactions against 7a were observed at different resi-
dues such as Leu151, Cys46, Pro152, Ile45, Arg60, Tyr129
and Gly44. However, 7b interacts with COX-1 with hydro-
gen and hydrophobic interactions at Asp134, Val47, Ile45,
Pro152, Cys46, Cys35 and Pro135, respectively having good
bonding distances. Five bonds were observed between 7c
and COX-1 protein. One hydrogen bond was observed at
Cys46 with bond length 3.92 Å and four hydrophobic inter-
actions were observed at Pro155, Cys35, Pro152 and Val47.
In 7d docking complex, seven hydrophobic interactions were
observed at Asp134, Cys35, Pro155, Pro152, Cys46, Val47
and Cys40. In comparative results, it was found that most of
4. CONCLUSION
A series of eight imine derivatives were synthesized
through microwave-assisted Schiff base formation and the
structures of target compounds were characterized by spec-
tral data. All the synthesized derivatives were screened for
their in vivo anti-inflammatory as well as in vitro anti-oxi-
dant activities using carrageenan induced rat paw edema test
and DPPH free-radical scavenging assay, respectively. Fur-
thermore, molecular docking experiments were also per-
formed to check the actual binding affinity of ligands against
target proteins. Compounds 4a, 4c, 7a, and 7c significantly
decreased the volume of rat paw edema (P < 0.05) and were
screened as potent anti-inflammatory drugs among the series.
Moreover, docking results of these compounds showed their
good binding affinity within the active region of target pro-
teins. It is believed that the synthesized Schiff base deriva-

Microwave-Assisted Synthesis
437
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of convenient synthetic strategy ensuring high product yield,
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DECLARATION OF INTERESTS
25. S. A. M. Reddya, J. Mudgala, P. Bansal, et al., Bioorg. Med.
Chem. 19(1) 384 – 392 (2011).
The authors declare no competing interests. M. Hanif
and M. Hassan contributed equally to this manuscript.
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