Certain 4-iminoflavones derivatives: Synthesis, docking studies, antiasthmatic and antimicrobial agents

Article abstract of DOI:10.14233/ajchem.2016.19789

In this present study, certain substituted 4-iminoflavone derivatives 5(a-j) were synthesized. All the synthesized compounds were evaluated for antiasthmatic and antimicrobial activity. Among the synthesized compounds, 5f was found to be the most promising against asthmatic models, whilst, 5h was found active against all the microbial strains. Antiasthmatic activity was correlated and evaluated with various models, e.g., citric acid induced cough model, OVA induced asthma model (biochemical estimation for cell infiltrations, estimation of lipid peroxidation and glutathione and estimation of TNF-α and IL-6 and histamine induced response). Compound 5f was found to show 2.20 ± 0.047 number of cough whilst codeine showed 1.40 ± 0.548; compound 5f produced considerable reduction in neutrophils, lymphocytes, eosinophils and leukocytes; decrease in LPO level (lung and BALF) and increment in GSH level (lungand BALF); and decreased the level of TNF-α and IL-6, respectively. On the other hand, 5h showed MIC 6.25 μg/mL against B. subtilis and S. aureus, 3.1 μg/mL against E. coli, 1.55 μg/mL against S. typhi and 6.25 μg/mL against C. albicans, respectively. In addition to gain better understanding on the biological activities of synthesized compounds, molecular docking study was performed within the binding site of human histamine H₁ receptor and Glc-6-P synthase revealing compound 5f and 5h as best fit within the respective receptor pockets.

Full text of DOI:10.14233/ajchem.2016.19789

Asian Journal of Chemistry; Vol. 28, No. 8 (2016), 1687-1696
A
SIAN
J
OURNAL OF HEMISTRY
C
http://dx.doi.org/10.14233/ajchem.2016.19789
Certain 4-Iminoflavones Derivatives: Synthesis, Docking Studies,
Antiasthmatic and Antimicrobial Agents
1
1
1
2
4
SHAILA KHAH , ROHIT GOYAL , ANKUSH CHABBA , VARUN JAISWAL , GAURAV SHARMA3,* and MU. NAUSHAD
1School of Pharmaceutical Sciences, Shoolini University, Solan-173 212, India
2School of Computer Science and Bioinformatics, Shoolini University, Solan-173 212, India
3School of Chemistry, Shoolini University, Solan-173 212, India
4Department of Chemistry, College of Science, Building 5, King Saud University, Riyadh, Saudi Arabia
*Corresponding author: E-mail: gaurav8777@gmail.com
Received: 21 December 2015;
Accepted: 8 March 2016;
Published online: 30 April 2016;
AJC-17874
In this present study, certain substituted 4-iminoflavone derivatives 5(a-j) were synthesized.All the synthesized compounds were evaluated
for antiasthmatic and antimicrobial activity. Among the synthesized compounds, 5f was found to be the most promising against asthmatic
models, whilst, 5h was found active against all the microbial strains. Antiasthmatic activity was correlated and evaluated with various
models, e.g., citric acid induced cough model, OVA induced asthma model (biochemical estimation for cell infiltrations, estimation of
lipid peroxidation and glutathione and estimation of TNF-α and IL-6 and histamine induced response). Compound 5f was found to show
2.20 0.047 number of cough whilst codeine showed 1.40 0.548; compound 5f produced considerable reduction in neutrophils,
lymphocytes, eosinophils and leukocytes; decrease in LPO level (lung and BALF) and increment in GSH level (lungand BALF); and
decreased the level of TNF-α and IL-6, respectively. On the other hand, 5h showed MIC 6.25 µg/mL against B. subtilis and S. aureus, 3.1
µg/mL against E. coli, 1.55 µg/mL against S. typhi and 6.25 µg/mL against C. albicans, respectively. In addition to gain better understanding
on the biological activities of synthesized compounds, molecular docking study was performed within the binding site of human histamine
H1 receptor and Glc-6-P synthase revealing compound 5f and 5h as best fit within the respective receptor pockets.
Keywords: 4-Iminoflavone, Antiasthmatic, Antimicrobial activity.
ovipositor stimulant of phytoalexins, anti-HIV, vasodilator,
bactericidal, DNA cleavage, anti-inflammatory, antimutagenic,
anti-asthmatic and anticancer. Especially, flavones (2-phenyl-
chromones) exhibit a wide variety of activities [3-6]. Therefore,
they exhibit diverse type of properties that are beneficial for
human health via interacting with a number of cellular targets
involved in critical cell signaling pathways in the body. Espe-
cially, flavone derivatives have been reported to possess anti-
asthmatic activity via targeting FcεRI receptors as well as
exhibiting leukotriene antagonism [7]. Histamine is implicated
as a mediator of some of the symptoms of allergic rhinitis and
other allergic diseases, but its importance in asthma is much
less well understood. However, some studies also suggests the
potential value of H1 antihistamines in the treatment of asthma
and number of groups have been reported compounds of poten-
tial therapeutic benefit that were claimed to possess the combined
properties of both histamine H1 antagonism and mast cell
stabilization, likely, BM 15100 [8,9].
INTRODUCTION
Flavonoids are a group of chemical moieties of the compounds
whose structure is based on C6-C3-C6 i.e. two phenyl rings are
attached through a propane bridge. Flavonoids are a family of
plant compounds with a similar flavone backbone composed
of two aromatic rings and an oxygen heterocycle attached [1].
Flavonoids can be classified into various classes i.e. flavonols
(quercetin, kaempferol), flavones (luteolin, apigenin), flavanones
(hesperetin), flavonoidglycosides(rutin), flavonolignans(silibinin),
flavans (catechin, epicatechin), isoflavones (genistein, daidzein),
anthocyanidins (cyanidin, delphinidin), aurones (leptosidin,
aureusidin), leucoanthocyanidins (teracacidin), neoflavonoids
(coutareagenin, dalbergin), chalcones [2].All classes of flavonoids
exhibits variety of biological activities, but among them, the
flavones have been considerably explored. In recent years,
substituted synthetic flavonoids have been dragging continuous
attention due to their broad range of biological activities.
Flavonoids are very well know and documented to possess
antioxidant effects, antiviral and leishmanicidal activity,
Substituted flavones are also known to show potential
pharmacological profile but no natural flavonoids have been

1688 Khah et al.
Asian J. Chem.
reported with halogens as substituent. Substitution of the B
ring is known to enhance antibacterial activity, with 3′-chloro,
4′-chloro and 4′-bromo analogues each being approximately
twice as effective as their parent compound against S. aureus
and four times more active against Enterococcus faecalis.Also,
the 2′,4′-dichloro derivative exhibited a 4-8 folds improvement
in activity against S. aureus [10]. However, a little work has
been carried out on iminoavones revealing antiasthmatic and
antimicrobial activities. Additionally, we were also interested
to evaluate the antiasthmatic and antimicrobial activity of
flavones derivatives possessing nitrogen atom at 4-position.
Therefore, in order to search for new compounds that have
potential activity for asthma and microbial infections, this
paper describes the synthesis of variably substituted 4-
iminoavones. Moreover, to gain much understanding, the
docking studies were performed on specific targets in order to
examine the binding affinity of respective molecules within
the pocket.
tion of the initial adduct followed by intramolecular cyclization
resulting in its hemiacetal species (a) which gets converted to
more reactive flavylium ion, (b) furthermore, water molecules
attach on the more reactive position to form adduct and finally,
(c) on oxidation in presence of iodine yields final products i.e.
flavones 4(a-j). The final product series 5(a-j), 4-iminoflavones
was synthesized by refluxing the flavone 4(a-j) with phenyl-
hydrazine in presence of concentrated H2SO4 in ethanol absolute.
Antiasthmatic evaluation: Male Hartley Guinea pigs
(500-600 g of either sex) were procured from the animal house
Shoolini University Solan, India.Animals were maintained in
the animal house within ordinary cages and exposed to normal
day-night cycles under standard conditions with the temperature
at 25 5 °C and the relative humidity of 55-60 %, moreover,
food and water ad libitum.All the protocols were in accordance
with guidelines of committee for the purpose of control and
supervision of experiments on animals (CPCSEA) in India, in
addition, approved by Institutional Animal Ethics Committee
(IAEC).
EXPERIMENTAL
Spectral characterization of synthesized compounds
1-(2-Hydroxy-4-methoxyphenyl)-3-phenylprop-2-en-
1-one (3a): Yield 60 %; m.p.: 148-150 °C; IR (KBr, νmax, cm-1):
3396.32, 2989.54, 2978.74, 1687.35; 1H NMR (CDCl3) δ ppm:
10.1 (bs, 1H, OH), 8.43 (d, 1H, Ar-H), 7.93 (s, 1H, Ar-H),
7.16 -6.97 (m, 4H, Ar-H), 6.89 (s, 2H, CH=CH), 6.66 (s, 1H,
Ar-H), 3.78 (s, 3H, OCH3); 13C NMR (CDCl3) δ ppm: 180.3,
156.2, 154.5, 144.7, 135.6, 128.9, 127.6, 127.2, 124.3, 122.1,
120.2, 118.8, 116.4, 55.3, 23.6; MS (m/z %) C16H14O3: 254.09
[M+].
All chemicals and solvents were supplied by Merck and
S.D. Fine Chemical Limited, Mumbai. The reactions were
monitored with the help of thin-layer chromatography using
pre-coated aluminum sheets with GF254 silica gel, 0.2 mm layer
thickness (E. Merck). Solvent systems of benzene-acetone
(9:1) and chloroform-methanol (9:1), (9.5:0.5) were employed.
Melting points of the synthesized compounds were recorded
on theVeego (VMP-MP) melting point apparatus. IR spectrum
was obtained onAgilent technologies, Microlab-PC, Cary 630
1
3-(4-Bromophenyl)-1-(2-hydroxy-4-methoxyphenyl)-
Infrared spectrometer, (model FTIR-630). Both H NMR
(CDCl3/DMSO-d6) and 13C NMR (CDCl3/DMSO-d6) spectra
of the synthesized compounds were performed with Bruker
Avance-II 400 NMR Spectrometer operating at 400 MHz in
SAIF, Panjab University, Chandigarh, India. Chemical shifts
are reported in parts per million (ppm) using tetramethylsilane
(TMS) as an internal standard. Mass spectra of the synthesized
compounds were recorded at MAT 120 in SAIF, Panjab
University, Chandigarh, India.
In view of the importance of pharmacological active
flavones, 4-iminoflavones were aimed to synthesize. The
replacement of oxygen atom of the keto group was carried
out with an amino group containing phenyl hydrazine. Our
synthetic approach was initiated with the synthesis of various
intermediate chalcones 3(a-j) which was achieved through
Claisen-Schmidt condensation. Targets compounds were
synthesized via initial preparation of chalcones 3(a-j) using
easily accessible starting materials, 2-hydroxy-4-methoxy-
acetophenone and substituted aldehydes in presence of 10 %
KOH and ethanol (Scheme-I). Thereafter the chalcones were
then oxidative cyclized in the presence of iodine to furnish
the flavone derivatives 4(a-j) in good yields. Notably, the
reaction was conducted under 1,4-dioxane solvent system. It
has been reported that the method followed for preparation of
flavones corresponds to final products with satisfactory yields
with both electron withdrawing and electron donating moieties.
The plausible mechanism for the formation of flavone ring
system 4(a-j), using iodine mediated oxidative cyclization is
presented in Scheme-I. The mechanism involves isomeriza-
prop-2-en-1-one (3b):Yield 54 %; m.p.: 152-156 °C; IR (KBr,
ν
1
max, cm-1): 3402.28, 2999.04, 2984.25, 1695.59, 634.48; H
NMR (CDCl3) δ ppm: 10.7 (bs, 1H, OH), 8.59 (d, 1H, Ar-H),
7.98 (s, 1H, Ar-H), 7.48-7.02 (m, 4H, Ar-H), 7.12 (s, 2H,
CH=CH), 3.81 (s, 3H, OCH3); 13C NMR (CDCl3) δ ppm: 184.6,
158.5, 156.9, 149.4, 132.5, 129.3, 128.8, 126.5, 120.9, 117.2,
52.1, 23.6, 15.6, 11.2; MS (m/z %) C16H13O3Br: 332.2 [M+].
3-(4-Fluorophenyl)-1-(2-hydroxy-4-methoxyphenyl)-
prop-2-en-1-one (3c):Yield 54 %; m.p.: 158-160 °C; IR (KBr,
ν
max, cm-1): 3413.51, 3002.35, 2991.56, 1697.48, 1035.20; 1H
NMR (CDCl3) δ ppm: 12.01 (bs, 1H, OH), 9.11 (d, 1H, Ar-
H), 8.12 (s, 1H, Ar-H), 7.76-7.34 (m, 4H, Ar-H), 7.32 (s, 2H,
CH=CH), 3.98 (s, 3H, OCH3); 13C NMR (CDCl3) δ ppm: 188.7,
168.3, 160.5, 153.8, 145.2, 136.1, 130.5, 129.5, 128.2, 126.6,
125.4, 120.1, 118.5, 55.3; MS (m/z %) C16H13O3F: 272.12
[M+].
3-(4-Chlorophenyl)-1-(2-hydroxy-4-methoxyphenyl)-
prop-2-en-1-one (3d):Yield 48 %; m.p.: 152-154 °C; IR (KBr,
1
ν
max, cm-1): 3405.87, 2998.18, 2956.84, 1718.04, 640.49; H
NMR (CDCl3) δ ppm: 11.78 (bs, 1H, OH), 8.85 (d, 1H, Ar-
H), 8.58-7.53 (m, 5H, Ar-H), 7.22 (s, 2H, CH=CH), 3.81 (s,
3H, OCH3); C NMR (CDCl3) δ ppm: 186.3, 165.6, 159.2,
13
150.5, 144.1, 134.6, 128.2, 126.9, 125.4, 123.8, 120.4, 118.7,
114.6, 54.6; MS (m/z %) C16H13O3Cl: 288.70 [M+].
1-(2-Hydroxy-4-methoxyphenyl)-3-(4-methoxyphenyl)-
prop-2-en-1-one (3e): Yield 57 %; m.p.: 150-152 °C; IR (KBr,
1
ν
max, cm-1): 3388.92, 2984.42, 2943.17, 1710.37; H NMR
(CDCl3) δ ppm: 10.12 (bs, 1H, OH), 8.30-7.22 (m, 6H,Ar-H),

Vol. 28, No. 8 (2016)
Synthesis, Docking Studies, Antiasthmatic and Antimicrobial Activities of 4-Iminoflavones Derivatives 1689
O
H CO
3
OH
H CO
3
OH
OCH
i
R
+
R
3
O
O
3(a-j)
1
2
a) 4-H
b) 4-Br
c) 4-F
d) 4-Cl
e) 4-OCH
ii
O
HN
N
3
iii
f) 4-CH
OCH
OC H
3
3
3
g) 2-CH
3
h) 4-NO
i) 4-CN
j) 2-F
2
O
O
R
R
5(a-j)
4(a-j)
H O
2
H CO
3
OH
O
OCH
OCH
3
3
R
HO
O
+
O
R
-H O
R
2
I2
(I)
(II)
Isomerization followed by cyclization
O
OH
OCH
OC H
3
3
O
O
R
R
(III)
Scheme-I: Synthesis of substituted targeted compounds 5(a-j). Reactants used; (i) aldehydes, 10 % KOH, ethanol, (ii) I2, 1,4-dioxane, (iii)
reflux, phenyhydrazine, ethanol, conc. H2SO4 and, plausible mechanism of synthesized flavone using iodine
7.02 (s, 2H, CH=CH), 3.75 (s, 6H, OCH3); 13C NMR (CDCl3)
δ ppm: 183.8, 162.4, 155.8, 148.6, 142.5, 132.5, 126.1, 125.7,
123.5, 122.5, 120.8, 117.1, 115.5, 55.2; MS (m/z %) C17H16O4:
284.38 [M+].
1-(2-Hydroxy-4-methoxyphenyl)-3-p-tolylprop-2-en-1-
one (3f):Yield 61 %; m.p.: 144-146 °C; IR (KBr, νmax, cm-1):
3382.65, 2988.74, 2968.40, 1701.02; 1H NMR (CDCl3) δ ppm:
9.8 (bs, 1H, OH), 8.40 (s, 1H, Ar-H), 7.90 (s, 1H, Ar-H), 7.19
-6.86 (m, 4H, Ar-H), 6.94 (s, 2H, CH=CH), 6.72 (s, 1H, Ar-
H), 3.74 (s, 3H, OCH3), 2.67 (s, 3H, CH3); 13C NMR (CDCl3)
δ ppm: 178.4, 159.7, 152.6, 142.1, 136.4, 132.9, 128.2, 127.3,
126.8, 122.6, 120.7, 117.3, 112.7, 55.7, 23.3; MS (m/z %)
C17H16O3: 268.33 [M+].
H), 6.97 (s, 2H, CH=CH), 6.76 (s, 1H, Ar-H), 3.84 (s, 3H,
OCH3), 2.73 (s, 3H, CH3); 13C NMR (CDCl3) δ ppm: 175.3,
157.5, 150.8, 144.6, 136.7, 136.2, 130.5, 129.6, 127.8, 126.4,
124.2, 122.7, 120.5, 118.5, 114.7, 55.4, 23.6; MS (m/z %)
C17H16O3: 269.40 [M+1].
1-(2-Hydroxy-4-methoxyphenyl)-3-(4-nitrophenyl)-
prop-2-en-1-one (3h):Yield 63 %; m.p.: 162-164 °C; IR (KBr,
ν
max, cm-1): 3380.84, 3002.44, 2992.71, 2964.25, 1714.64; 1H
NMR (CDCl3) δ ppm: 10.06 (bs, 1H, OH), 8.28-7.15 (m, 6H,
Ar-H), 7.07 (s, 2H, CH=CH), 3.78 (s, 3H, OCH3); 13C NMR
(CDCl3) δ ppm: 185.7, 160.6, 154.1, 140.2, 136.6, 126.8,
126.2, 124.5, 123.3, 122.9, 120.1, 116.6, 114.3, 55.1; MS (m/z
%) C16H13NO5: 299.29 [M+].
1-(2-Hydroxy-4-methoxyphenyl)-3-o-tolylprop-2-en-1-
one (3g):Yield 54 %; m.p.: 140-142 °C; IR (KBr, νmax, cm-1):
3395.29, 2993.19, 2971.31, 1698.85; 1H NMR (CDCl3) δ ppm:
9.3 (bs, 1H, OH), 8.21 (s, 1H, Ar-H), 7.32 -6.70 (m, 5H, Ar-
4-[3-(2-Hydroxy-4-methoxyphenyl)-3-oxoprop-1-enyl]-
benzonitrile (3i): Yield 50 %; m.p.: 164-166 °C; IR (KBr,
1
ν
max, cm-1): 3392.18, 2974.69, 2857.83, 1718.73; H NMR
(CDCl3) δ ppm: 10.25 (bs, 1H, OH), 8.42 (s, 1H, Ar-H), 8.36-

1690 Khah et al.
Asian J. Chem.
7.29 (m, 5H,Ar-H), 7.19 (s, 2H, CH=CH), 3.81 (s, 3H, OCH3);
13C NMR (CDCl3) δ ppm: 179.2, 159.8, 152.1, 145.6, 142.9,
136.7, 132.5, 126.7, 125.2, 124.8, 122.0, 120.5, 115.2, 55.6,
23.2; MS (m/z %) C17H13NO3: 279.30 [M+].
125.1, 120.8, 118.5, 115.0, 104.5, 55.6, 23.8; MS (m/z %)
C17H14 O3: 266.30 [M+].
6-Methoxy-2-o-tolyl-4H-chromen-4-one (4g):Yield 75
%; m.p.: 160-162 °C; IR (KBr, νmax, cm-1): 2971.89, 1640.75,
1601.58; 1H NMR (CDCl3) δ ppm: 7.29 (d, 2H, Ar-H), 7.23
(d, 2H, Ar-H), 7.16 (s, 1H, Ar-H), 6.76-7.08 (m, 4H, Ar-H),
3-(2-Fluorophenyl)-1-(2-hydroxy-4-methoxyphenyl)-
prop-2-en-1-one (3j):Yield 48 %; m.p.: 152-156 °C; IR (KBr,
13
ν
max, cm-1): 3410.04, 3000.23, 2988.63, 1704.61, 1030.15; 1H
3.73 (s, 3H, OCH3), 2.81 (s, 3H, CH3); C NMR (CDCl3) δ
NMR (CDCl3) δ ppm: 11.78 (bs, 1H, OH), 9.04 (d, 1H, Ar-
H), 8.03 (s, 1H, Ar-H), 7.72-7.30 (m, 4H, Ar-H), 7.26 (s, 2H,
CH=CH), 3.88 (s, 3H, OCH3); 13C NMR (CDCl3) δ ppm: 189.4,
160.4, 158.7, 152.6, 144.6, 135.5, 130.5, 129.2, 128.8, 125.9,
125.2, 124.2, 121.6, 120.5, 117.9, 114.7, 55.8; MS (m/z %)
C16H13O3F: 272.25 [M+].
ppm: 182.2, 163.2, 155.8, 149.1, 137.4, 130.7, 128.9, 127.7,
126.4, 125.9, 125.3, 120.2, 118.2, 115.5, 105.7, 56.2, 23.5;
MS (m/z %) C17H14O3: 266.29 [M+].
6-Methoxy-2-(4-nitrophenyl)-4H-chromen-4-one (4h):
Yield 77 %; m.p.: 174-176 °C; IR (KBr, νmax, cm-1): 3002.53,
2986.71, 1654.03, 1612.65; 1H NMR (CDCl3) δ ppm: 7.28 (d,
2H, Ar-H), 7.19 (d, 2H, Ar-H), 7.09 (s, 1H, Ar-H), 6.71-7.06
(m, 4H, Ar-H), 3.77 (s, 3H, OCH3); 13C NMR (CDCl3) δ ppm:
183.8, 163.8, 155.7, 149.2, 147.5, 136.6, 127.3, 125.8, 121.5,
120.6, 118.9, 114.3, 104.7, 55.5; MS (m/z %) C16H11NO5:
297.26 [M+].
4-(6-Methoxy-4-oxo-4H-chromen-2-yl)benzonitrile
(4i): Yield 58 %; m.p.: 178-180 °C; IR (KBr, νmax, cm-1):
2974.29, 2848.52, 2215.35, 1658.76; 1H NMR (CDCl3) δ ppm:
7.32-6.96 (m, 8H, Ar-H), 3.78 (s, 3H, OCH3); 13C NMR
(CDCl3) δ ppm: 182.6, 162.5, 155.1, 149.6, 146.8, 136.9,
126.8, 125.3, 122.6, 120.2, 118.4, 115.7, 114.9, 104.5, 56.3;
MS (m/z %) C17H11NO3: 277.28 [M+].
2-(2-Fluorophenyl)-6-methoxy-4H-chromen-4-one
(4j): Yield 69 %; m.p.: 184-186 °C; IR (KBr, νmax, cm-1):
2989.06, 1659.38, 1612.33; 1H NMR (CDCl3) δ ppm: 7.42 (d,
2H, Ar-H), 7.35 (d, 2H, Ar-H), 7.20 (s, 1H, Ar-H), 6.84-7.23
(m, 4H, Ar-H), 3.93 (s, 3H, OCH3); 13C NMR (CDCl3) δ ppm:
182.4, 163.8, 156.4, 149.7, 137.8, 131.3, 128.5, 126.8, 125.6,
124.2, 120.5, 118.6, 114.1, 104.0, 56.5, 23.8; MS (m/z %)
C16H11O3F: 270.27 [M+].
6-Methoxy-2-phenyl-4H-chromen-4-one (4a):Yield 70
%; m.p.: 168-170 °C; IR (KBr, νmax, cm-1): 2978.49, 1652.16,
1601.03; 1H NMR (CDCl3) δ ppm: 7.30 (d, 2H, Ar-H), 7.21
(d, 2H, Ar-H), 7.15 (s, 1H, Ar-H), 6.71-7.02 (m, 4H, Ar-H),
3.71 (s, 3H, OCH3); 13C NMR (CDCl3) δ ppm: 180.5, 163.7,
155.2, 148.1, 130.6, 128.3, 128.0, 126.9, 124.8, 120.4, 118.2,
115.6, 105.3, 56.2; MS (m/z %) C16H12O3: 252.25 [M+].
2-(4-Bromophenyl)-6-methoxy-4H-chromen-4-one
(4b): Yield 71 %; m.p.: 164-166 °C; IR (KBr, νmax, cm-1):
2980.69, 1657.25, 1610.44, 630.28; 1H NMR (CDCl3) δ ppm:
7.43 (d, 2H, Ar-H), 7.30 (d, 2H, Ar-H), 7.23 (s, 1H, Ar-H),
6.82-7.14 (m, 4H, Ar-H), 3.82 (s, 3H, OCH3); 13C NMR
(CDCl3) δ ppm: 183.8, 165.1, 156.1, 149.2, 130.9, 129.3,
128.1, 126.3, 125.0, 120.8, 117.5, 114.8, 104.6, 55.8; MS (m/z
%) C16H11 O3Br: 331.18 [M+].
2-(4-Fluorophenyl)-6-methoxy-4H-chromen-4-one
(4c): Yield 68 %; m.p.: 172-174 °C; IR (KBr, νmax, cm-1):
2996.35, 1660.28, 1616.12, 1015.54; 1H NMR (CDCl3) δ ppm:
7.54 (d, 2H, Ar-H), 7.41 (d, 2H, Ar-H), 7.28 (s, 1H, Ar-H),
6.87-7.22 (m, 4H, Ar-H), 3.91 (s, 3H, OCH3); 13C NMR
(CDCl3) δ ppm: 185.1, 166.6, 155.7, 149.6, 131.4, 129.5,
128.4, 126.7, 125.3, 121.1, 118.8, 114.9, 106.2, 56.4; MS (m/z
%) C16H11 O3F: 270.26 [M+].
1-(6-Methoxy-2-phenyl-4H-chromen-4-ylidene)-2-
phenylhyrazine (5a): Yield 54 %; m.p.: 254-256 °C; IR (KBr,
1
ν
max, cm-1): 3312.65, 2993.58, 1672.67, 1624.42; H NMR
2-(4-Chlorophenyl)-6-methoxy-4H-chromen-4-one
(4d): Yield 65 %; m.p.: 166-168 °C; IR (KBr, νmax, cm-1):
2985.03, 1658.75, 1614.33, 654.15; 1H NMR (CDCl3) δ ppm:
7.47 (d, 2H, Ar-H), 7.36 (d, 2H, Ar-H), 7.25 (s, 1H, Ar-H),
6.84-7.15 (m, 4H, Ar-H), 3.84 (s, 3H, OCH3); 13C NMR
(CDCl3) δ ppm: 184.6, 165.4, 156.2, 149.8, 131.1, 129.4,
128.2, 126.6, 125.2, 120.9, 117.8, 114.8, 105.5, 56.1; MS (m/z
%) C16H11O3Cl: 286.72 [M+].
6-Methoxy-2-(4-methoxyphenyl)-4H-chromen-4-one
(4e): Yield 74 %; m.p.: 158-160 °C; IR (KBr, νmax, cm-1):
2971.47, 1648.35, 1605.24; 1H NMR (CDCl3) δ ppm: 7.26 (d,
2H, Ar-H), 7.15 (d, 2H, Ar-H), 7.04 (s, 1H, Ar-H), 6.66-6.95
(m, 4H, Ar-H), 3.74 (s, 6H, OCH3); 13C NMR (CDCl3) δ ppm:
182.5, 162.5, 154.6, 148.6, 130.2, 128.5, 128.1, 126.6, 124.7,
120.8, 118.5, 114.9, 105.5, 56.6; MS (m/z %) C17H14 O4: 282.29
[M+].
(DMSO-d6) δ ppm: 10.0 (s, 1H, NH), 7.72 (d, 1H, Ar-H), 7.58
(d, 1H, Ar-H), 7.48-7.02 (m, 5H, Ar-H), 6.84 (s, 1H, Ar-H),
6.74-6.42 (m, 7H, Ar-H), 3.62 (s, 3H, OCH3); 13C NMR
(DMSO-d6) δ ppm: 157.5, 155.2, 154.6, 148.4, 143.2, 130.7,
129.6, 128.5, 127.9, 126.3, 121.1, 120.7, 120.5, 118.4, 114.0,
113.8, 90.6, 55.8; MS (m/z %) C22H18N2O2: 343.39 [M+1].
1-(2-(4-Bromophenyl)-6-methoxy-4H-chromen-4-
yidene)-2-phenylhydrazine (5b): Yield 62 %; m.p.: 258-260
°C; IR (KBr, νmax, cm-1): 3322.49, 2998.62, 1684.37, 1620.08,
664; 1H NMR (DMSO-d6) δ ppm: 10.2 (s, 1H, NH), 7.79 (d,
1H, Ar-H), 7.68-7.09 (m, 5H,Ar-H), 6.89 (s, 1H, Ar-H), 6.74-
6.41 (m, 7H, Ar-H), 3.72 (s, 3H, OCH3); 13C NMR (DMSO-
d6) δ ppm: 157.8, 154.3, 153.9, 148.7, 143.5, 130.4, 129.5,
128.7, 127.2, 126.5, 121.1, 120.7, 118.7, 116.1, 114.9, 113.2,
90.8, 55.2; MS (m/z %) C22H17N2O2Br: 421.29 [M+].
1-[2-(4-Fluorophenyl)-6-methoxy-4H-chromen-4-
ylidene]-2-phenylhydrazine (5c): Yield 58 %; m.p.: 266-268
°C; IR (KBr, νmax, cm-1): 3328.60, 2999.84, 1685.42, 1624.24,
1010.51; 1H NMR (DMSO-d6) δ ppm: 10.32 (s, 1H, NH), 7.86
(d, 1H, Ar-H), 7.80-7.15 (m, 5H, Ar-H), 6.91 (s, 1H, Ar-H),
6.79-6.50 (m, 7H, Ar-H), 3.84 (s, 3H, OCH3); 13C NMR
(DMSO-d6) δ ppm: 158.5, 155.9, 153.5, 148.5, 142.8, 130.8,
6-Methoxy-2-p-tolyl-4H-chromen-4-one (4f): Yield 71
%; m.p.: 164-166 °C; IR (KBr, νmax, cm-1): 2976.24, 1644.06,
1602.45; 1H NMR (CDCl3) δ ppm: 7.25 (d, 2H, Ar-H), 7.19
(d, 2H, Ar-H), 7.11 (s, 1H, Ar-H), 6.69-6.98 (m, 4H, Ar-H),
13
3.78 (s, 3H, OCH3), 2.85 (s, 3H, CH3); C NMR (CDCl3) δ
ppm: 182.8, 163.6, 155.4, 149.7, 136.2, 129.5, 128.3, 126.6,

Vol. 28, No. 8 (2016)
Synthesis, Docking Studies, Antiasthmatic and Antimicrobial Activities of 4-Iminoflavones Derivatives 1691
129.2, 128.8, 127.5, 125.3, 121.8, 120.6, 118.6, 115.8, 114.1,
113.7, 91.6, 55.7; MS (m/z %) C22H17N2O2F: 360.4 [M+].
1-[2-(4-Chlorophenyl)-6-methoxy-4H-chromen-4-
ylidene]-2-phenylhydrazine (5d): Yield 63 %; m.p.: 262-264
°C; IR (KBr, νmax, cm-1): 3325.56, 2986.18, 1680.38, 1619.64,
590.28; 1H NMR (DMSO-d6) δ ppm: 10.22 (s, 1H, NH), 7.86-
7.12 (m, 6H, Ar-H), 6.86 (s, 1H, Ar-H), 6.73-6.45 (m, 7H, Ar-
H), 3.80 (s, 3H, OCH3); 13C NMR (DMSO-d6) δ ppm: 157.6,
155.4, 153.3, 148.8, 143.5, 130.4, 129.6, 128.7, 127.2, 125.5,
121.4, 120.2, 118.5, 116.8, 114.8, 113.2, 90.9, 55.5; MS (m/z
%) C22H17N2O2Cl: 377.8 [M+1].
1-[6-Methoxy-2-(4-methoxyphenyl)-4H-chromen-4-
ylidene]-2-phenylhydrazine (5e):Yield 60 %; m.p.: 258-260
°C; IR (KBr, νmax, cm-1): 3319.38, 2980.54, 1676.61, 1616.09;
1H NMR (DMSO-d6) δ ppm: 10.08 (s, 1H, NH), 7.75-6.92
(m, 6H, Ar-H), 6.78 (s, 1H, Ar-H), 6.64-6.28 (m, 7H, Ar-H),
3.71 (s, 6H, OCH3); 13C NMR (DMSO-d6) δ ppm: 156.9, 155.3,
153.5, 147.7, 143.9, 130.4, 129.4, 128.2, 127.6, 125.5, 121.9,
120.6, 118.9, 115.6, 114.5, 113.0, 91.2, 55.9; MS (m/z %)
C23H20N2O2: 372.4 [M+].
1006.76; 1H NMR (DMSO-d6) δ ppm: 10.24 (s, 1H, NH), 7.82
(d, 1H, Ar-H), 7.76-7.10 (m, 5H, Ar-H), 6.88 (s, 1H, Ar-H),
6.72-6.42 (m, 7H, Ar-H), 3.80 (s, 3H, OCH3); 13C NMR
(DMSO-d6) δ ppm: 158.9, 155.5, 153.3, 148.4, 143.6, 130.5,
129.2, 128.5, 127.6, 126.8, 125.8, 121.6, 120.7, 118.8, 116.6,
114.0, 113.1, 90.3, 52.2; MS (m/z %) C22H17N2O2F: 361.4
[M+1].
Citric acid-induced cough model: Animals were divided
into different groups. Each group included 5 animals (n = 5).
Citric acid induced cough model was used to perform the
experiment as reported by Laude et al. [11]. On 1st day, after a
3 min acclimatization period, the animals were initially
exposed to normal saline and 5 min later to aerosolized 7.5 %
w/v citric acid for a period of 10 min. Animal selection was
conducted on the basis of number of coughs (< 7 or15 >) on
exposure to aerosolized 7.5 % citric acid or their tendency to
cough on exposure to aerosolized saline was excluded as this
taken as an indication of infection or hyper-reaction. Whilst
on the same day, cough challenge was given for each animal
and a minimum interval of 24 h was allowed between challenges
to eliminate any short-term prophylaxis. Each animal will be
placed in a perspex chamber (30 cm × 20 cm × 20 cm) and
exposed to an aerosolized aqueous solution of 7.5 % w/v citric
acid for a period of 10 min. The animals were observed conti-
nuously and number of coughs and latency time to initial cough
response was documented. The samples were intraperitoneally
administered at 30 min before the second challenge of
aerosolized citric acid (75 % w/v) solution for 10 min. All the
synthesized compounds 5(a-j) were administered (i.p.) at dose
of 30 mg/kg, dissolved in normal saline. Codeine 10 mg/kg
was administered as standard.
1-[6-Methoxy-2-(p-tolyl)-4H-chromen-4-ylidene]-2-
phenylhydrazine (5f): Yield 55 %; m.p.: 250-252 °C; IR (KBr,
1
ν
max, cm-1): 3318.09, 2991.39, 1679.56, 1618.35; H NMR
(DMSO-d6) δ ppm: 10.03 (s, 1H, NH), 7.68 (d, 1H, Ar-H),
7.54-6.98 (m, 5H, Ar-H), 6.82 (s, 1H, Ar-H), 6.66-6.34 (m,
7H, Ar-H), 3.74 (s, 3H, OCH3), 2.75 (s, 3H, CH3); 13C NMR
(DMSO-d6) δ ppm: 158.2, 155.6, 153.6, 148.9, 143.2, 130.8,
129.1, 128.3, 127.8, 126.8, 121.9, 120.4, 118.6, 116.5, 114.5,
113.8, 91.2, 56.4, 23.8; MS (m/z %) C23H20N2O2: 357.2 [M+1].
1-[6-Methoxy-2-(o-tolyl)-4H-chromen-4-ylidene]-2-
phenylhydrazine (5g):Yield 58 %; m.p.: 248-250 °C; IR (KBr,
1
ν
max, cm-1): 3316.39, 2994.37, 1680.07, 1621.46; H NMR
OVA induced asthma model: According to the method
of Andersson and Bergstrand [12] animals were actively
sensitized. The intraperitoneal injection of 20 µg ovalbumin
and 100 mg Al(OH)3 dissolved in normal saline (0.2 mL) was
given twice in a gap of 7 days. After 2 weeks, the animals
were placed in perspex chamber equipped with an ultrasonic
nebulizer and challenged with 0.5 % ovalbumin inhalation in
order to verify the occurrence of sensitization. The animals
showing airway hyper-responsiveness, to the inhaled antigen,
were referred to as sensitized animals. Afterward, the animals
were divided into different groups, each comprising 5 animals
(n = 5): SC (sensitized control), AMP (aminophylline 50 mg/
kg) and all the test compounds 5(a-j) 30 mg/kg were given
intraperitoneally for 7 days dissolved in pyrogen free water.
Biochemical estimation for cell infiltrations: After final
ovalbumin challenge of 24 h, animals were sacrificed using
sodium pentobarbitone (200 mg/kg, i.p.). The trachea was
immediately cannulated and the lungs were lavaged with 5 ×
4 mL aliquots of Ca2+ and Mg2+ free 0.1 M phosphate buffered
saline solution of pH 7.4. The collected BAL fluid was centri-
fuges and used for further biochemical estimations. The
obtained lung tissue was dissected out, washed in cold saline,
dried and homogenated (10 %) in 10 mL phosphate buffer saline
pH 7.4. The homogenate was centrifuged and supernatant was
used for biochemical estimation [13].
(DMSO-d6) δ ppm: 10.05 (s, 1H, NH), 7.64-6.94 (m, 6H, Ar-
H), 6.78 (s, 1H, Ar-H), 6.62-6.30 (m, 7H, Ar-H), 3.76 (s, 3H,
OCH3), 2.72 (s, 3H, CH3); 13C NMR (DMSO-d6) δ ppm: 157.5,
155.6, 153.9, 148.3, 143.8, 130.5, 129.8, 128.2, 127.6, 126.4,
121.3, 120.7, 118.5, 116.4, 114.8, 113.2, 90.6, 55.8, 23.9; MS
(m/z %) C23H20N2O2: 356.2 [M+].
1-[6-Methoxy-2-(4-nitrophenyl)-4H-chromen-4-
ylidene]-2-phenylhydrazine (5h): Yield 60 %; m.p.: 272-274
°C; IR (KBr, νmax, cm-1): 3332.29, 3003.85, 2986.59, 1680.19,
1622.24; 1H NMR (DMSO-d6) δ ppm: 10.16 (s, 1H, NH), 7.92
(s, 1H, Ar-H), 7.86-6.98 (m, 5H, Ar-H), 6.76 (s, 1H, Ar-H),
6.70-6.30 (m, 7H, Ar-H), 3.77 (s, 3H, OCH3); 13C NMR
(DMSO-d6) δ ppm: 156.5, 155.7, 154.0, 147.6, 143.5, 130.6,
129.8, 128.7, 127.2, 126.1, 121.7, 120.6, 118.0, 115.8, 114.6,
113.8, 91.6, 55.2; MS (m/z %) C22H17N3O4: 387.4 [M+]
4-[6-Methoxy-4-(2-phenylhydrazono)-4H-chromen-2-
yl]benzonitrile (5i): Yield 48 %; m.p.: 268-270 °C; IR (KBr,
ν
max, cm-1): 3305.95, 2986.40, 2218.53, 1679.62, 1620.18; 1H
NMR (DMSO-d6) δ ppm: 10.06 (s, 1H, NH), 7.79-6.88 (m,
6H, Ar-H), 6.81 (s, 1H, Ar-H), 6.72-6.30 (m, 7H, Ar-H), 3.74
(s, 3H, OCH3); 13C NMR (DMSO-d6) δ ppm: 157.2, 155.6,
153.2, 146.6, 143.9, 130.8, 129.2, 128.9, 127.0, 125.2, 121.5,
120.4, 118.8, 116.4, 114.1, 113.8, 90.6, 55.6; MS (m/z %)
C23H17N3O2: 367.4 [M+].
1-[2-(2-Fluorophenyl)-6-methoxy-4H-chromen-4-
ylidene]-2-phenylhydrazine (5j): Yield 66 %; m.p.: 268-270
°C; IR (KBr, νmax, cm-1): 3326.27, 2998.09, 1683.48, 1622.46,
Estimation of lipid peroxidation (LPO) and glutathione
(GSH): The experiment was conducted according to the
method given by Ohkawa et al. [13]. Briefly, at first the organ

1692 Khah et al.
Asian J. Chem.
was rinsed with ice cold normal saline followed by 0.15 M
HCl (pH 7.4) for LPO estimation and 10 % w/v in 0.1 M phos-
phate buffer (pH 7.4) for GSH estimation. In case of LPO, the
conditions maintained were temperature (95 °C), pH (acidic),
absorbance (532 nm) and light path (1 cm) respectively.
Afterward, 0.2 mL of tissue homogenate was mixed with 0.2
mL of 8.1 % SDS, 1.5 mL of 20 % acetic acid, 1.5 mL of 8 %
TBA and the volume was made up to 4 mL with double distilled
water. The prepared sample was heated on water 95 °C for 60
min and allowed to cool. Afterward 5 mL of butanol:pyridine
(15:1) was added and vortexed for 2 min, followed by
centrifugation at 3000 rpm for 10 min. Upper organic layer
was collected and estimation was carried out spectrophoto-
metrically. Whilst, in case the conditions were maintained as
temperature (25 °C), pH (acidic), absorbance (412 nm) and
light path (1 cm) respectively. Homogenate sample was taken
and 20 % TCA and 1 µM EDTA was added. The sample was
centrifuged for 10 min at 2000 rpm. Supernatant (200 mL)
was taken and transferred to new tube followed by addition of
1.8 mL of Ellman/s reagent with volume make up to 2 mL
using double distilled water. The absorbance was recorded at
412 nm [14].
activity. The lowest concentration of the compound that
completely inhibits macroscopic growth was determined and
minimum inhibitory concentrations (MICs) were reported.
Docking study: All the synthesized derivatives were
examined for their binding affinity towards antiasthmatic and
antimicrobial activity. Crystal structure of human histamine
H1 receptor (PDB code 3RZE) [18] and Glc-6-P synthase (PDB
code 1MOQ) were obtained from RCSB protein data bank
and docking study was performed on each target. AutoDock
4.2 package software was used to examine the binding affinity
of newly synthesized derivatives within the binding pockets
[19]. Initially, ChemDraw Ultra 12.0 was used to draw chemical
structures and energy minimization was performed. Autogrid
program of AutoDock suit was utilized for generation of grid
around binding pocket within target protein. Finally, docking
simulation was carried out with AutoDock 4.2. Ligplot and
UCSF Chimera version 1.8.1 were used for analysis of docking
results i.e. protein ligand interaction and visualization of
docked proteins ligand complexes [20].
RESULTS AND DISCUSSION
Antiasthmatic activity
Estimation of TNF-α and IL-6 and histamine induced
response: For estimation of TNF-α and IL-6, RayBio® TNF-
α ELISA (Enzyme-linked immunosorbent assay) kit was used
and accordingly the assay was carried out. All standards and
samples were run using 96-well microliter plates. In addition,
response of synthesized best candidate was examined for
histamine induced response via goat tracheal method [15].
Briefly, goat trachea was procured and cut into individual ring.
Thereafter, it was suspended in bath containing kreb’s solution
maintained at 37 5 °C stream of CO2 and O2 was bubbled through
organ tube. As a result dose response effect was examined.
Antimicrobial activity: All the synthesized derivatives
5(a-j) were screened for in vitro antibacterial and antifungal
activity. Compounds were tested against the Bacillus subtilis
(ATCC-6633), Stapylococcus aureus (ATCC-25923),
Escherichia coli (ATCC-25922) and S. typhi (ATCC-6539).
However, to evaluate the antifungal activity of iminoflavones
Candida albicans (ATCC-24433) was considered [16].
Imipenem and amphotericin B were used as the standard drugs.
The antimicrobial testing was performed by the methods of
National Committee for Clinical Laboratory Standards [17].
Mirodilution method was employed and respective MICs were
obtained against specific strains. The solution of the newly
synthesized compounds and standard drugs was prepared at
200, 100, 50, 25, 12.5, 6.25, 3.1, 1.55, 0.78, 0.39, 0.19 µg/mL
concentrations in the wells of microplates by diluting in the
liquid double stranded nutrient broth. The bacterial suspensions
used for inoculation were prepared of 105 cfu/mL by diluting
fresh cultures at MacFarland 0.5 density (107 cfu/mL).
Suspensions of the bacteria at 105 cfu/mL concentration were
inoculated. There were 104 cfu/mL bacteria in the wells after
inoculations. Nutrient broth was used for diluting the bacterial
suspension. DMSO, pure microorganisms and pure media were
used as control wells. The trays were incubated at 37 °C in a
humid chamber and MIC endpoints were read after 24 h of
incubation. The same procedure was used for antifungal
Citric acid-induced cough model: It has been documented
that the guinea pigs and humans respond to similar concen-
tration of citric acid and responses are well correlated with
concentration-response relationship. Citric acid induction
resulted significant cough in Guinea pigs as evidenced by
decreased cough latency time and increased cough frequencies.
Pretreatment with codeine (standard) and compounds 5(a-j)
showed notable increase in cough latency as well as decrease in
cough frequencies per 10 min in comparison to control (Table-
1 and Fig. 1). Among the synthesized compounds, 5f showed
the highest antitussive effect at a dose of 30 mg/kg, i.p in
comparison to codeine (10 mg/kg). Compound 5f was found
to show 2.20 0.047 number of cough whilst codeine showed
1.40 0.548. However, compounds 5j, 5h and 5c also showed
substantial and considerable activity, likely, 3.60 0.548, 4.72
0.421 and 4.89 0.521, respectively. Interestingly, compound
having nitro at 4th position and methyl substitutions (position 4)
were highly active. In addition, fluoro containing moieties at
2nd and 4th position also showed prominent activity in comparison
to other halogen substituted compounds. However, most
electron withdrawing substituted groups showed promising
results among the synthesized derivatives.
OVA induced asthma model: This study presents the
respiratory hyper-reactivity induced by an exposure to ovalbumin
aerosol in sensitized guinea pigs. The interaperitoneal injection
TABLE-1
COUGH RESPONSE OF SYNTHESIZED COMPOUNDS 5(a-j)
Treatment
Control
Codeine
5a
No. of cough
18.80 0.447
1.40 0.548
10.80 0.837
10.92 0.721
4.89 0.521
13.40 1.517
Treatment
No. of cough
7.60 1.140
2.20 0.047
11.40 0.528
4.72 0.421
6.71 1.530
3.60 0.548
5e
5f
5g
5h
5i
5b
5c
5d
5j

Vol. 28, No. 8 (2016)
Synthesis, Docking Studies, Antiasthmatic and Antimicrobial Activities of 4-Iminoflavones Derivatives 1693
20
18
16
14
12
10
8
were also estimated, likely, neutrophils, lymphocytes and
leukocytes. It was found that the % count was quite high in
control animals, whereas, the synthesized compounds showed
promising results in comparison to standard drug i.e. codeine.
Control group did not receive any treatment and the cell infiltration
was estimated at a higher side. Among all the synthesized
compounds, 5f and 5j showed significant effect (Table-2).
Compound 5f produced considerable reduction in neutrophils
(7.68 0.811), lymphocytes (2.58 0.432), eosinophils (16.80
1.332) and leukocytes (10.38 0.870) whereas, compound
5j also showed notable effect on neutrophils (8.76 0.69),
lymphocytes (2.79 0.461), eosinophils (16.93 1.231) and
leukocytes (10.49 0.922) in comparison to codeine;
neutrophils (6.66 1.216), lymphocytes (2.40 0.412),
eosinophils (15.82 1.366) and leukocytes (9.96 0.602),
6
4
2
0
respectively. Therefore, the present study showed that the
compounds at an intended dose of 30 mg/kg were active in
comparison to codeine at a dose of 10 mg/kg. This study also
supports the previous experiment, as compound 5f was found to
be highly active among the synthesized ones and significantly
reduced the eosinophil accumulation in the sensitized guinea
pigs lungs and BALF.
Oxidative stress plays an important role in pathophysiology
of asthma leading various detrimental effects on airway
function, including airway smooth muscle contraction, mucus
hypersecretion and vascular exudation as evidenced by LPO
reaction and decrease in GSH levels in BALF and lung tissue,
as compared to control. Table-3 presents the LPO and GSH
Treatment
Fig. 1. Plot representing number of cough obtained with respect to the treatment
of 20 µg ovalbumin and 100 mg Al(OH)3 twice in a gap of 7
days caused allergic reactions in male Hartley guinea pigs and
the success rate of sensitization method was 89.79 %, which
showed allergic reactions to inhaled antigen. This exposure
resulted pulmonary asthma like symptoms in antigen induced
sensitized animals which was supported by the increase in
eosinophilic accumulation in bronchial tissue and BAL fluid
associated with chronic airway inflammation.
Eosinophilic inflammation is known to be a paramount
parameter of bronchial asthma. In addition, other cell infiltrates
TABLE-2
EFFECT OF TEST DRUGS ON ANTIGEN INDUCES CELL INFILTRATION
Treatment
Neutrophils (%)
14.40 1.840
6.66 1.216
10.22 0.210
11.61 0.761
9.99 0.731
10.21 1.230
12.08 1.076
7.68 0.811
11.82 0.841
9.96 0.532
10 0.231
Lymphocytes (%)
5.64 0.9840
2.40 0.4120
4.42 0.3770
4.67 0.4219
3.06 0.5320
4.20 0.6600
4.89 0.4810
2.58 0.4320
4.78 0.4970
2.99 0.5220
3.27 0.5620
2.79 0.4610
Eosinophils (%)
40.70 0.401
15.82 1.366
19.23 2.060
22.60 2.493
17.99 1.071
18.94 1.011
30.76 2.640
16.80 1.332
27.04 2.131
17.26 1.128
18.82 1.016
16.93 1.231
Leukocytes (%)
18.18 3.356
9.96 0.602
Control
AMN
5a
5b
5c
5d
5e
5f
5g
11.54 0.970
12.86 0.527
10.99 0.960
11.26 0.986
16.18 1.001
10.38 0.870
14.88 0.482
10.63 0.995
11.01 0.931
10.49 0.922
5h
5i
5j
8.76 0.690
TABLE-3
FFECT OF COMPOUNDS 5(a-j) TREATMENT ON OXIDATIVE STRESS IN LUNG AND BALF HOMOGENATES
Oxidative stress in lung homogenates
LPO (nM MDA/g wet tissue) GSH (nmol/g wet tissue)
Oxidative stress in BALF homogenates
LPO (nM MDA/g wet tissue) GSH (nmol/g wet tissue)
54.50 10.501 0.27 0.063
Treatment
Control
AMN
5a
5b
5c
5d
5e
5f
5g
51.16 6.811
17.17 5.349
25.50 8.131
32.23 8.231
19.26 2.23
0.35 0.054
0.71 0.113
0.52 0.072
0.46 0.064
0.62 0.123
0.56 0.079
0.39 0.065
0.7 0.123
0.43 0.081
0.65 0.086
0.59 0.089
0.69 0.121
18.50 7.583
25.23 6.79
0.67 0.144
0.51 0.029
0.45 0.018
0.55 0.231
0.53 0.035
0.38 0.071
0.66 0.113
0.41 0.101
0.59 0.129
0.51 0.230
0.63 0.105
31.17 8.772
22.79 5.233
24.50 6.604
41.83 8.028
19.61 7.831
39.17 10.448
21.27 5.431
23.98 5.692
20.16 5.578
23.29 6.275
39.17 6.625
18.83 2.236
33.17 11.844
19.01 2.261
20.61 6.121
18.99 2.241
5h
5i
5j

1694 Khah et al.
Asian J. Chem.
TABLE-4
levels expressed in BALF and lung tissue homogenate samples.
The control showed significant increase in LPO (lung; 51.16
6.811 and BALF; 54.50 10.501) and decrease in GSH
(lung; 0.35 0.054 and BALF; 0.27 0.063). The synthesized
compounds 5(a-j) were tested (30 mg/kg) in order to analyze
their impact over LPO and GSH. Compounds 5f, 5j, 5h, 5c
and 5i showed substantial and considerable effects over LPO
as well as GSH. Among these, 5f having methyl substitution
at para position was found to be the most active one. Compound
5f showed significant decrease in LPO level (lung; 18.83
2.236 and BALF; 19.61 7.831) and increment in GSH level
(lung; 0.7 0.123 and BALF; 0.66 0.113), respectively. The
present experiment showed that iminoflavones have a significant
impact on LPO and GSH levels.
ESTIMATION OF TNF-α AND IL-6 AFTER
TREATMENT WITH COMPOUND 5(a-j)
Treatment
IL-6 (ng/mL)
TNF-α (ng/mL)
19.98 1.764
4.05 0.896
6.86 1.102
8.31 1.118
5.14 0.901
5.51 0.931
13.49 0.504
4.33 0.520
12.81 0.739
4.00 0.731
5.20 0.921
4.10 0.611
Control
AMN
5a
5b
5c
5d
5e
5f
5g
10.97 1.034
2.82 0.214
5.62 0.421
6.79 0.409
3.76 0.577
4.69 0.171
8.99 0.610
3.03 0.281
8.23 0.521
3.59 0.461
4.52 0.161
3.23 0.321
5h
5i
5j
TNF-αand IL-6 have been identified as important pathogenic
mediators in asthma/inflammation associated asthma. TNF-α
and IL-6 were estimated in comparison to standard i.e. codeine
(TNF-α 4.05 0.896 and IL-6 10.97 1.034), whilst, in control
the level was found at higher side (TNF-α 19.98 1.764 and
IL-6 10.97 1.034) as shown in Table-4 and Fig. 2. All the
compounds were tested and 5f showed significant activity via
decreasing the level of TNF-α (4.33 0.520) and IL-6 (3.03
0.281).All the experiments were found to support each other as
compound 5f was found to be the most active one. Therefore,
the compound 5f was examined for response against histaminic
induced contraction. The compound at optimized doses showed
significant relaxation. Interestingly, the relaxation was found
to increase with increment in dose, whilst, control (at different
dose)werefoundtoshowcontraction due to induction of histamine.
This revealed that 5f considerably inhibited the contractile
effect of histamine thus produces bronchodilation. Therefore,
compound 5f was found active in all the experiments, suppor-
ting each other.
data revealed that compounds inhibited the growth of the
selected microorganisms in vitro showing MIC values between
3.1 to 100 µg/mL as shown in Table-5. Newly synthesized
derivatives 5(a-k) showed satisfactory to good potential against
gram negative bacteria (E. coli and S. typhi) and C. albicans.
Compounds 5h was found to the most active candidate among
the synthesized derivatives showing significant activity against
all the strains. Compound 5h showed MIC 6.25 µg/mL against
B. subtilis and S. aureus, 3.1 µg/mL against E. coli, 1.55 µg/mL
against S. typhi and 6.25 µg/mL against C. albicans, respectively.
Moreover, other compounds of the series likely, 5c, 5d, 5f and
5j were also found active against all strains.All the compounds
against C. albicans showed inhibition within a range of MIC
3.1-50 µg/mL. Compounds 5c, 5d and 5h were found to be the
most active candidate against C. albicans with MIC 3.1, 6.25
and 6.25 µg/mL, respectively (Table-6). Interestingly, compound
having halogen substitution were found to show better inhibi-
tion against C. albicans in comparison to other compounds.
However, very few of the synthesized compounds were found
inactive against specific strains.
Antimicrobial activity: The antimicrobial activity was
determined in terms of minimum inhibitory concentration
(MIC) of the synthesized compounds 5(a-j). The activity was
determined against the Gram-positive Bacillus subtilis,
Stapylococcus aureus, the Gram-negative Escherichia coli,
Salmonella typhi and the fungi Candida albicans. The obtained
Docking study: Most employed and popular software was
used in the present study in order to visualize and optimize
the binding affinity of synthesized new molecules on specific
20
15
10
5
10
8
6
4
2
0
0
Treatment
Treatment
Fig. 2. Plots presenting estimated TNF-α (ng/mL) and IL-6 (ng/mL) with respect to the treatment

Vol. 28, No. 8 (2016)
Synthesis, Docking Studies, Antiasthmatic and Antimicrobial Activities of 4-Iminoflavones Derivatives 1695
TABLE-5
198(A), Thr 112(A), Thr 194(A), Phe 424(A), Phe 435(A)
were taken to define binding pocket within target protein,
whereas, active site residues of glucosamine-6-phosphate
synthase according to co-crystal structure with inhibitors (Cys
300, Gly 301, Thr 302, Ser 303, Ser 347, Gln 348, Ser 349,
Thr 352, Val 399, Ser 401,Ala 602 and Lys 603) were taken to
define binding pocket within target protein. Ligands were
ranked according to docking score i.e. estimated free energy
of binding and free energy of binding of ligand was in the
range -8.63 to -10.48 (Histamine H1 receptor, Fig. 3) and -7.53
to -8.45 (glucosamine-6-phosphate synthase, Fig. 4). Free
energy of binding of ligands was in good agreement with wet
lab biological activities. Top ranked compounds were also
found active in lab experiments. Protein ligand analysis also
showed strong interactions with target protein and had four
hydrogen bond interaction with different residues (Tyr 108,
Ser 111, Tyr 431 and Tyr 458) in H1 receptor site, whereas,
six hydrogen bond interaction with different residues (Ser 303,
Ser 347, Thr 352, Thr 355 and Glu 488) in active site of
antimicrobial and antifungal activity.
RESPONSE OF 5f TO HISTAMINE (10 µg/mL)
INDUCED CONTRACTION AT DIFFERENT DOSES
Response
(mm)
Relaxation
Dose (mL)
Response
(%)
–
–
–
–
–
–
–
–
Histamine (0.05)
Histamine (0.1)
Histamine (0.2)
Histamine (0.4)
Histamine (0.8)
Histamine (1.6)
Histamine (3.2)
Histamine (6.4)
5f (5 mg/mL) +
Histamine (3.2)
5f (10 mg/mL) +
Histamine (3.2)
5f (20 mg/mL) +
Histamine (3.2)
13
20
28
37
46
51
56
48
22
Contraction
Contraction
Contraction
Contraction
Contraction
Contraction
Contraction
Contraction
Relaxation
60.71
18
15
Relaxation
Relaxation
67.85
73.21
drug targets. The significant result of antiasthmatic and
antimicrobial activity prompted is to investigate the docking
of molecules on well-known targets. Active site residues of
human histamine H1 receptor according to co-crystal structure
with inhibitors; Trp 428(A), Phe 432(A), Asp 107(A), Tyr
108(A), Ser 111(A), Tyr 431(A), Trp 158(A), Tyr 458(A),Asn
Conclusion
It is concluded that synthesized 4-iminoflavones 5(a-j)
showed good potential against asthma and microbial strains.
TABLE-6
ANTIMICROBIAL ACTIVITY AND DOCK SCORE OF THE SYNTHESIZED COMPOUNDS 5(a-j)
Compound
Dock score
B. subtilis
S. aureus
E. coli
S. typhi
C. albicans
5a
5b
5c
5d
5e
5f
5g
5h
-7.57
-7.86
-7.53
-7.81
-7.62
-7.86
-7.90
-8.47
-7.84
-7.96
–
–
–
–
100.00
25.00
12.50
50.00
50.00
12.50
6.25
3.10
50.00
3.10
50.00
50.00
6.25
25.00
50.00
25.00
12.50
1.55
–
–
50.00
6.25
25.00
50.00
25.00
25.00
6.25
–
12.50
25.00
–
25.00
–
6.25
–
6.25
1.55
–
3.10
6.25
25.00
50.00
–
6.25
–
12.50
–
5i
5j
100.00
6.25
0.78
12.50
3.10
–
Imipenem
Amphoterecin B
0.19
–
–
–
1.55
(–) denotes no activity
(a)
(b)
Fig. 3. Docking images (a) overlapping of all compounds 5(a-j) within binding pocket of histamine receptor and (b) binding of the high
ranking generated conformers for compound 5f

1696 Khah et al.
Asian J. Chem.
(a)
(b)
Fig. 4. Docking images (a) overlapping of all compounds 5(a-j) within 3D structure of GlcN-6-P synthase and (b) binding of the high
ranking generated conformers for compound 5h
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ACKNOWLEDGEMENTS
The authors thank Shoolini University for providing
necessary facilities and support.
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