Design, Synthesis, Drug-Likeness Studies and Bio-Evaluation of Some New Chalconeimines

Article abstract of DOI:10.1007/s11094-019-02084-y

Newly designed chalconeimines were synthesized, characterized and evaluated for their in vitro antioxidant and antibacterial effectiveness. Results of antioxidant activity assay reveal that all the tested compounds possess good to moderate antioxidant activity which is lower in comparison to that of a standard drug (gallic acid). On the other hand, all the synthesized compounds were found to exhibit a considerably wider spectrum of antibacterial activity, but it was also narrower in comparison to that of a standard drug (ciprofloxacin). Elucidation of structure—activity relationships revealed that electron donating groups (-OH, -OCH₃) contribute more to antioxidant potency, whereas electron withdrawing groups (-Cl) impart better antibacterial effectiveness. Moreover, results of drug-likeness studies indicate that a reasonable correlation exists between the drug-like properties and antioxidant activity of the synthesized chalconeimines.

Full text of DOI:10.1007/s11094-019-02084-y

DOI 10.1007/s11094-019-02084-y
Pharmaceutical Chemistry Journal, Vol. 53, No. 9, December, 2019 (Russian Original Vol. 53, No. 9, October, 2019)
DESIGN, SYNTHESIS, DRUG-LIKENESS STUDIES
AND BIO-EVALUATION OF SOME NEW CHALCONEIMINES
1
,*
1
Mithun Rudrapal and Mullapudi P. K. Sowmya
Original article submitted July 2, 2019.
Newly designed chalconeimines were synthesized, characterized and evaluated for their in vitro antioxidant
and antibacterial effectiveness. Results of antioxidant activity assay reveal that all the tested compounds pos-
sess good to moderate antioxidant activity which is lower in comparison to that of a standard drug (gallic
acid). On the other hand, all the synthesized compounds were found to exhibit a considerably wider spectrum
of antibacterial activity, but it was also narrower in comparison to that of a standard drug (ciprofloxacin). Elu-
cidation of structure—activity relationships revealed that electron donating groups (-OH, -OCH ) contribute
3
more to antioxidant potency, whereas electron withdrawing groups (-Cl) impart better antibacterial effective-
ness. Moreover, results of drug-likeness studies indicate that a reasonable correlation exists between the
drug-like properties and antioxidant activity of the synthesized chalconeimines.
Keywords: chalconeimines; enone azomethines; antioxidant activity; antibacterial activity; drug-likeness.
1
. INTRODUCTION
In recent years, chalcones (1,3-diaryl-2-propen-1-ones)
with increased oxidative stress (OS) in humans [16, 17].
However, the bio-efficacy of existing natural (for example,
plant polyphenols such as catechins, quercetin) and synthetic
antioxidants (for example, BHA and BHT) is negligible as
compared to the enzymatic (SOD, CAT and GPx) antioxidant
defense system of human body [16, 18 – 20]. It is therefore
required to develop new antioxidant molecules that would be
effective yet safe in preventing and/or treating OS induced
human disorders. The increasing incidence of bacterial infec-
tion caused by the rapid emergence of multi-drug resistant
bacterial strains against majority of the currently available
antibiotics has become a serious medical problem all over
the world [21]. Therefore, the discovery and development of
new antibacterial agents that would fight resistant bacterial
infection with some new mechanisms of action is a topical
task.
belonging to the class of natural flavonoids and related prod-
ucts have gained significant interest among medicinal chem-
ists owing to their diverse biologic activities [1 – 7]. Struc-
ture – activity relationship (SAR) study indicated the biolog-
ical potential of chalcone functional moiety (Ar-CO-CH=
CH-Ar¢), where the a,b-unsaturated keto group (enone func-
tion, -CO-CH=CH-) is considered to be the key pharmaco-
phore of chalcones [1, 8]. On the other hand, imines, also
known as Schiff¢s bases, are widely found in various natural
and synthetic compounds of medicinal interest and possess a
wide array of biological activities [9, 10]. Literature reveals
that the pharmacodynamic potential of bio-active imines is
mainly due to the presence of pharmacophoric azomethine
(
>C=N) moiety [11, 12]. The conjugated chalcone-imine de-
rivatives such as chalconeimines [13] and iminoflavones
14, 15] having enone-azomethine (-CH=CH-C=N-) phar-
In view of the above facts, it was assumed that the ratio-
nal design of new chalcone derivatives as chalconeimines
would be an attractive approach to developing biologically
active molecules. Accordingly, a series of structural analogs
based on the conjugated scaffold of various chalcones and
substituted chalcones coupled with a heteroaryl amine was
designed with modifications in the aryl rings (ring B, Fig. 1)
of the chalcone core. Newly designed chalconeimines were
synthesized, characterized, and evaluated in vitro for their
antioxidant and antibacterial properties. In the present work,
the synthesized compounds were evaluated in silico for mo-
lecular properties assessment and drug-likeness prediction
[
macophoric moiety have been reported as pharmacologically
potent compounds.
Literature reports suggest that many chronic and degen-
erative diseases such as cancer, diabetes, cardiovascular dis-
eases, neurodegenerative disorders, certain inflammatory
diseases and bacterial infections are commonly associated
1
Aditya Institute of Pharmaceutical Sciences and Research, Surampalem,
E.G. Dist., Andhra Pradesh, 533437 India.
*
e-mail: rudrapal.m03@gmail.com
814
0
091-150X/19/5309-0814 © 2019 Springer Science+Business Media, LLC

Design, Synthesis, Drug-Likeness Studies and Bio-Evaluation
815
1
13
was made on the basis of UV, FT-IR, H NMR, C NMR,
and mass spectroscopy, and elemental analysis. Procedures
used for the synthesis of target compounds and the related
physicochemical, spectroscopic, and analytical data are pre-
sented below.
Fig. 1. Chemical structure of chalcone.
2.2. Synthesis of Target Compounds 5a – 5f
General procedure. Chalcone and substituted chalcones
[
23] 3a – 3f (0.01 mol) and 2-amino-4-phenylthiazole 4 [24]
0.01 mol) were dissolved in absolute ethanol (30 mL), 2 – 3
drops of conc. H SO was added, and the mixture was
with the aim to assess their drug-likeness based on Lipinski¢s
(
rule of five with additional parameters for antioxidant activity.
2
4
refluxed for 6 – 12 h and kept in refrigerator overnight. The
separated solid was collected by filtration, dried in air, and
re-crystallized from absolute ethanol [14].
2
. EXPERIMENTAL
2
.1. Chemicals, Instrumentation and Analytical Methods
N-(1,3-Diphenylallylidene)-4-phenylthiazol-2-amine
O
(5a): %Yield: 60.65%, color: yellow, mp: 178 – 180 C, R :
All chemicals were obtained commercially from Merck
f
Specialists Pvt. Ltd., Mumbai; S.D. Fine Chemicals Ltd.,
Mumbai; and Qualigens Fine Chemicals, Mumbai, and were
used without further purification, unless otherwise stated.
The solvents and reagents used in the biological study were
of analytical grade. All reactions were performed in oven
dried glass ware using synthetic grade chemicals. The prog-
ress of reactions was monitored by the silica gel-G thin layer
chromatography (TLC) and the spots were visualized by io-
dine vapors.
0
.78 (EtOAc/CHCl = 1:1); UV spectrum (acetone), lmax, nm
3
-1
(
abs): 370 (0.498); IR (KBr; n, cm ): 3083.58, 3052.94 (aro-
matic C-H str.), 3032.85, 3015.19 (vinylic C-H str.,
1
-
(
-
CH=CH-), 1664.36 (>C=N- str., imine);
H
NMR
= 15.82 Hz,
trans
400 MHz, CDCl ; d, ppm): 8.03 (d, 1H, J
3
CH=CH-), 7.95 (d, 1H, J
= 15.80 Hz, =CH-Ar), 7.62 (s,
trans
1H, C5-thiazolyl-H), 7.58 – 7.52 (m, 5H, Ar-H), 7.46 – 7.42
1
3
m, 5H, Ar-H), 7.32 – 7.26 (m, 5H, Ar-H); C NMR
(
(
100 MHz, CDCl ; d, ppm): 198.66 (>C=N), 168.26, 152.62,
Melting points (mp) were measured in open capillaries
on an electrically heated melting point apparatus. UV-Vis
spectra were recorded on Labindia UV-3000 UV-Vis
spectrophotometer and the absorption band positions are re-
ported as maximum wavelength (lmax, nm). Infrared (IR) ab-
sorption spectra were obtained on a Bruker Alpha FT-IR
spectrometer using KBR disks and reported in terms of vi-
3
1
48.20 (thiazolyl-C); 138.26, 134.86, 132.89, 128.52,
(
vinylic- & aromatic-CH); MS (API-ES), m/z (%): 367.12
+
100), [M+H] , 368.12 (28), 368.90 (6), 369.38 (4); CHN
(
anal. for C H N S (366.48), calc. (%): C, 78.66, H, 4.95,
24 18
2
N, 7.64, found (%): C, 79.00, H, 4.90, N, 7.78.
N-(3-(4-Chlorophenyl)-1-phenylallylidene)-4-phenyl-
thiazol-2-amine (5b): %Yield: 82.20%, color: yellow, mp:
-1
1
13
brational frequencies (n, cm ). The H and C NMR spectra
were recorded on a Bruker AC-F 400 FT-NMR spectrometer
at 400 and 100 MHz, respectively, with tetramethylsilane
1
18 – 120°C, R : 0.72 (EtOAc/CHCl = 1:1); UV spectrum
f
3
-1
(
acetone), l , nm (abs): 370 (0.301); IR (KBr; n, cm ):
max
3
126.22, 3059.38 (aromatic C-H str.), 3024.85, 2922.19
(
TMS) as an internal standard and CDCl as a solvent. Chem-
3
(vinylic C-H str., -CH=CH-), 1658.22 (>C=N- str., imine),
1
ical shifts (d) are expressed in parts per million (ppm) rela-
1
1092.03 (aromatic C-Cl str.); H NMR (400 MHz, CDCl ; d,
3
tive to TMS (d = 0.00 ppm). The H NMR data are presented
ppm): 8.04 (d, 1H, J
= 15.83 Hz, -CH=CH-), 7.94 (d, 1H,
trans
in the following order: peak multiplicity (b, broad; s, singlet;
d, doublet; t, triplet; q, quartet; m, multiplet), number of pro-
tons (numerical integral value), coupling constants (J, in
hertz). The mass spectra (MS) were recorded on an LC–MS
Waters 4000 ZQ instrument using atmospheric pressure ion-
ization (API-ES). The m/z values were recorded in the range
of m/z = 150 – 1400 (in both the negative and positive ion
modes), and the m/z values of the most intense quasimo-
Jtrans = 15.85 Hz, =CH-Ar), 7.66 (s, 1H, C5-thiazolyl-H),
7
7
.56 – 7.51 (m, 5H, Ar-H), 7.48 – 7.44 (m, 4H, Ar-H),
1
3
.36 – 7.32 (m, 5H, Ar-H); C NMR (100 MHz, CDCl ; d,
3
ppm): 198.64 (>C=N), 168.40, 155.20, 149.52 (thiazolyl-C);
138.45, 134.39, 132.40, 126.06 (vinylic- & aromatic-CH);
+
MS (API-ES), m/z (%): 401.01 (100), [M+H] , 402.06 (29),
4
03.34 (9), 404.60 (2); CHN anal. for C H N SCl (400.92),
24 17
2
+
lecular ion [M+H] peaks (with relative intensities) in paren-
calc. (%): C, 71.90, H, 4.27, N, 6.99, found (%): C, 70.62, H,
.55, N, 7.82.
N-(3-(4-Methoxyphenyl)-1-phenylallylidene)-4-phenyl-
thiazol-2-amine (5c): %Yield: 82.32%, color: yellow, mp:
00 – 104°C, R : 0.79 (EtOAc/CHCl = 1:1); UV spectrum
4
theses, are given followed by peaks corresponding to major
fragment ions. Elemental analysis was performed on a Perkin
Elmer 2400 Series II CHNS/O analyzer. In the study of anti-
oxidant activity the absorbance readings were obtained using
an ELICO CL157 colorimeter.
1
f
3
-1
(acetone), l , nm (abs): 370 (0.490); IR (KBr; n, cm ):
max
Chemical methods involving standard reaction proce-
dures were employed for the synthesis of target compounds
3152.00, 3067.46 (aromatic C-H str.), 3035.29, 3015.46
(vinylic C-H str., -CH=CH-), 2964, 2889 (C-H str., CH3),
1657.45 (>C=N- str., imine), 1170.43, 1180.67 (-C-O-C str.);
5
a – 5f. The structural assignment of obtained compounds

8
16
Mithun Rudrapal and Mullapudi P. K. Sowmya
1
1
H NMR (400 MHz, CDCl ; d, ppm): 8.08 (d, 1H,
H NMR (400 MHz, CDCl ; d, ppm): 8.22 (d, 1H,
3
3
J
= 15.57 Hz, -CH=CH-), 7.89 (d, 1H, J
= 15.37 Hz,
J
= 16.42 Hz, -CH=CH-), 8.14 (t, 1H, J = 16.32 Hz,
trans
trans
trans
trans
=
CH-Ar), 7.62 (s, 1H, C5-thiazolyl-H), 7.58 – 7.54 (m, 5H,
-CH=CH-CH=),
=CH-CH=CH-), 7.78 (d, 1H, Jtrans = 16.86 Hz, =CH-Ar),
.62 (s, 1H, C5-thiazolyl-H), 7.56 – 7.52 (m, 5H, Ar-H),
7.89
(t,
1H,
Jtrans = 16.24 Hz,
Ar-H), 7.46 – 7.42 (m, 4H, Ar-H), 7.38 – 7.33 (m, 5H,
13
7
Ar-H), 3.86 (s, 3H, OCH3); C NMR (100 MHz, CDCl ; d,
3
13
7.48 – 7.44 (m, 5H, Ar-H), 7.40 – 7.36 (m, 5H, Ar-H);
C
ppm): 194.69 (>C=N), 168.20, 156.26, 149.22 (thiazolyl-C);
NMR (100 MHz, CDCl ; d, ppm): 192.47 (>C=N), 168.67,
1
38.45, 134.40, 132.52, 128.36, 126.23 (vinylic- & aro-
3
1
56.45, 149.33 (thiazolyl-C); 138.25, 134.53, 132.60, 126.50
matic-CH), 52.58 (OCH ); MS (API-ES), m/z (%): 397.96
3
+
100), [M+H] , 398.58 (28), 399.36 (12), 399.24 (8); CHN
(
vinylic- & aromatic-CH); MS (API-ES), m/z (%): 393.89
+
100), [M+H] , 393.07 (56), 394.48 (34), 395.02 (20); CHN
(
(
anal. for C H N OS (396.50), calc. (%): C, 71.90, H, 4.27,
2
5
20
2
anal. for C H N S (392.52), calc. (%):C, 79.56, H, 5.14, N,
N, 6.99, found (%): C, 70.62, H, 4.55, N, 7.82.
26 20
2
7
.14, found (%):C, 79.02, H, 6.27, N, 7.48.
N-(3-(2-Hydroxyphenyl)-1-phenylallylidene)-4-phenyl-
thiazol-2-amine (5d): %Yield: 89.52%, color: pale yellow,
O
2.3. Studying Antioxidant Activity
mp: 186 – 190 C, R : 0.94 (EtOAc/CHCl = 1:1); UV spec-
f
3
The in vitro antioxidant activity of the synthesized com-
trum (acetone), l , nm (abs): 380 (0.397); IR (KBr; n,
max
-1
pounds 5a – 5e was determined by two methods: 2,2-diphen-
yl-1-picrylhydrazyl (DPPH) radical scavenging assay and
ferric reducing antioxidant power (FRAP) assay [12].
DPPH free radical scavenging assay. Methanolic DPPH
solution (0.1 mmol) was added to 3.0 mL of test drug solu-
tions (in methanol) of three different concentrations (50, 100
and 200 mg/mL). The mixture was shaken vigorously and al-
lowed to stand at room temperature for 30 min. The
absorbance was then measured at 517 nm in a colorimeter.
Methanol was used as the blank, and the reaction mixture of
DPPH and methanol served as control. Gallic acid (1.0, 2.5
and 5.0 mg/mL) was used as the reference standard drug. The
percentage scavenging activity was calculated using the fol-
lowing formula:
cm ): 3430 (Phenolic O-H str.); 3158.34, 3080.71 (aromatic
C-H str.), 3034.36, 3012.38 (vinylic C-H str., -CH=CH-),
1
664.82 (>C=N- str., imine), 1280.96 (C-O str.); H NMR
1
(
400 MHz, CDCl ; d, ppm): 8.12 (d, 1H, J
= 15.68 Hz,
trans
3
-
CH=CH-), 7.88 (d, 1H, Jtrans = 15.25 Hz, =CH-Ar), 7.64 (s,
1
H, C5-thiazolyl-H), 7.58 – 7.55 (m, 5H, Ar-H), 7.46 – 7.43
(
m, 4H, Ar-H), 7.39 – 7.36 (m, 5H, Ar-H), 5.32 (bs, 1H,
1
3
OH); C NMR (100 MHz, CDCl ; d, ppm): 192.47 (>C=N),
3
1
68.67, 156.45, 149.33 (thiazolyl-C); 138.38, 134.73,
1
32.55, 128.63, 126.34 (vinylic- & aromatic-CH); MS
+
API-ES), m/z (%): 383.27 (100), [M+H] , 384.60 (40),
(
3
85.56 (30), 386.27 (12); CHN anal. for C H N OS
24 18 2
(
382.48), calc. (%): C, 75.37, H, 4.74, N, 7.32, found (%): C,
7.28, H, 4.78, N, 8.20.
N-(3-(4-Hydroxy-3-methoxyphenyl)-1-phenylallylide-
7
DPPH scavenging (%) = [(A – A )/A ] ´ 100,
0
1
0
ne)-4-phenylthiazol-2-amine (5e): %Yield: 87.86%, color:
O
buff, mp: 178 – 182 C, R : 0.96 (EtOAc/CHCl = 1:1); UV
f
3
where A is the absorbance of control (without test drug) and
0
spectrum (acetone), l , nm (abs): 370 (0.99); IR (KBr; n,
max
A1 is the absorbance of the test drug solution. All the tests
were carried out in triplicate, and results were expressed as
mean % inhibition ± SEM. The percentage scavenging activ-
ity was determined at various solution concentrations, and
the results of test samples were compared with that of the
standard drug, gallic acid. Data were subjected to statistical
analysis by Student¢s t-test using the SPSS Ver. 19.0 statisti-
cal software package. Results were considered as statistically
significant for p £ 0.05.
-1
cm ): 3423 (Phenolic O-H str.); 3147.30, 3058.20 (aromatic
C-H str.), 3033.20, 3011.10 (vinylic C-H str., -CH=CH-),
2
1
8
966, 2862 (C-H str., CH ), 1665.37 (>C=N- str., imine),
1
3
283.40 (>C-O str.); H NMR (400 MHz, CDCl ; d, ppm):
3
.26 (d, 1H, J
= 16.23 Hz, -CH=CH-), 7.92 (d, 1H,
trans
Jtrans = 16.26 Hz, =CH-Ar), 7.64 (s, 1H, C5-thiazolyl-H),
7
7
.59 – 7.54 (m, 5H, Ar-H), 7.48 – 7.42 (m, 3H, Ar-H),
.38 – 7.33 (m, 5H, Ar-H), 5.38 (bs, 1H, OH), 3.75 (s, 3H,
1
3
OCH3);
C NMR (100 MHz, CDCl ; d, ppm): 188.60
3
Ferric reducing antioxidant power (FRAP) assay. 1.0 mL
of test drug solution (in ethanol) of different concentrations
(
>C=N), 167.52, 154.28, 148.49 (thiazolyl-C); 138.27,
1
34.36, 132.44, 128.08 (vinylic- & aromatic-CH), 52.60
(
50, 100 and 200 mg/mL) was mixed with 2.5 mL of phos-
+
OCH ); MS (API-ES), m/z (%):413.14 (100), [M+H] ,
(
3
phate buffer (0.2M, pH 6.6) and 2.5 mL of 1% potassium
ferricyanide. The mixture was then incubated at 50°C for
20 min. 2.5 mL of 1% trichloroacetic acid (10%) was added
to the reaction mixture, which was then centrifuged at 3000
rpm for 10 min. The upper layer of the solution (2.5 mL) was
4
14.29 (38), 415.27 (26), 416.36 (8); CHN anal. for
C H N O S (412.50), calc. (%): C, 72.79 H, 4.89, N, 6.79,
2
5
20
2
2
found (%): C, 73.30 H, 5.46, N, 6.88.
N-(1,5-Diphenylpenta-2,4-dienylidene)-4-phenylthia-
zol-2-amine (5f): %Yield: 86.98%, color: dark yellow, mp:
O
mixed with 2.5 mL of distilled water and 0.5 mL of FeCl so-
3
1
90 – 194 C, R : 0.88 (EtOAc/CHCl = 1:1); UV spectrum
lution (0.1%). The absorbance was measured at 700 nm us-
ing a colorimeter. Gallic acid was used as the reference stan-
dard. All the tests were performed in triplicate, and the re-
sults are expressed as mean ± SEM.
f
3
-
1
(
acetone), l , nm (abs): 370 (0.160); IR (KBr; n, cm ):
max
3
123.45, 3079.80 (aromatic C-H str.), 3032.54, 3009.43
(
vinylic C-H str., -CH=CH-), 1658.40 (>C=N- str., imine);

Design, Synthesis, Drug-Likeness Studies and Bio-Evaluation
817
Scheme 1. Synthesis of target compounds (chalconeimines) 5a – 5f. Reagents and conditions: (a) 1, 2a – 2f, aq. NaOH, EtOH, 25 – 3O°C,
2
– 6 h; (b) 3a – 3f, 4, conc. H SO , EtOH, reflux, 8 – 12 h.
2 4
2
.4. Studying Antibacterial Activity
100 mL of test and standard solutions and vehicle were trans-
ferred to cups aseptically and labeled accordingly. The plates
were left undisturbed for 1 h at room temperature to allow
pre-incubation diffusion of the solution into the medium in
order to minimize the effects of variation in time between the
application of different solutions. After incubation of the
plates at 37 ± 1(C for 24 h, diameters of the zones of com-
plete inhibition surrounding each well were measured on a
millimeter scale. The studies were performed in triplicate
and mean ± SEM values (in mm) were determined. Data
were subjected to statistical analysis by Student¢s t-test using
the SPSS Ver. 19 statistical software package, and differ-
Test strain and bacterial inoculums. Three strains of
Gram positive bacteria, viz. Bacillus subtilis, Bacillus
pumilus, Staphylococcus aureus and three strains of Gram
negative bacteria, viz. Eschericia coli, Pseudomonas aerugi-
nosa, Pseudomonas vulgaris were used in this study. The
cultures of bacteria were maintained in their appropriate agar
o
slants at 4 C throughout the study and used as stock cultures.
Bacterial inoculums for each strain were prepared by trans-
ferring a loop full (0.005 mL) of stock culture into a clean
and sterilized tube containing 4 – 5 mL nutrient broth me-
dium. The broth cultures were then incubated at 37 ± 1(C for
1
8 h.
Drug dilution. Test drug solutions were prepared in ace-
ences were considered as statistically significant for p £ 0.05.
Drug-likeness prediction. The drug-likeness of synthe-
tone. Ciprofloxacin Injection IP (200 mg/100 mL; Cadila
Health care Ltd., Bhiwadi) was diluted with distilled water to
obtain a concentration of 200 mg/mL and used as positive
control. Acetone was used as negative control. The stock so-
lution (1 mg/mL) of each test compound 5a – 5f was used to
prepare a test solution of 200 mg/mL concentration.
sized compounds 5a – 5f was evaluated in silico using web
based Molsoft (molsoft.com/molprop/) and Molinspiration
Cheminformatics (www.molinspiration.com) academic soft-
ware packages. The drug-likeness prediction score was cal-
culated based on the following molecular descriptors: molec-
ular weight, aqueous solubility (logS), octanol – water parti-
tion coefficient (logP), acceptor and donor hydrogen-bond
count, polar surface area, and rotatable bonds. The drug-like-
ness score was calculated for all test compounds.
Agar disk diffusion assay [21, 25]. Nutrient agar me-
dium was sterilized by autoclaving at 121(C (15 lb/sq. inch.)
for 15 min. The flat-bottomed petri dishes (100 mm in diam-
eter) were sterilized previously in hot-air oven at 160(C for
o
about 1 h. Freshly prepared and cooled (45 – 50 C) molten
agar medium of about 25 mL volume was inoculated with a
standard inoculum (0.5 – 1.0 mL) aseptically in laminar air
flow unit. The medium was then transferred aseptically into
sterilized petri-plates to occupy a depth of about 4 mm. The
plates were then left at room temperature to allow solidifica-
tion. Under aseptic condition, four wells of 5 mm diameter
were made in each plate using a sterile borer. Accurately
3
. RESULTS AND DISCUSSION
In the present study, a series of new chalconeimines were
synthesized, characterized, and screened for their antioxidant
and antibacterial effectiveness.

8
18
Mithun Rudrapal and Mullapudi P. K. Sowmya
1
3
3
.1. Design and Synthesis
The C NMR data confirmed the presence of various
skeletal carbons in the conjugated chalcone-arylthiazole sys-
tem. The chemical shift for the carbon atom of azomethine
group (>C=N) was observed in the region of
d = 188 – 198 ppm. Three thiazolyl carbons, for example, in
compound 5b, appeared around d = 168, 155 and 149 ppm.
The methoxy carbon present in 5c and 5e appeared at
d = 52.58 and 52.60 ppm, respectively. Aromatic (quater-
nary, ArC and tertiary, ArCH) and vinylic carbons were con-
firmed by d values in the region of 138 – 126 ppm.
The design of target chalconeimines involved substitu-
tions on two terminal phenyl rings connected via conjugated
enone-imine moiety with different functional groups particu-
larly at 2¢-ortho and/or 4¢-para positions of ring B of the par-
ent chalcone moiety. The electronic character of substituents
is the main motif in this design strategy. The synthesis route
used to obtain the target compounds is depicted in Scheme 1.
Chalconeimines 5a – 5f were synthesized by condensation
reaction between chalcone/substituted chalcones 3a – 3f and
The mass spectra of compounds 5a – 5f exhibited promi-
2
-amino-4-phenylthiazole 4 in absolute ethanol as solvent in
O
the presence of conc. H SO as catalyst at about 100 C by
+
nent peaks of molecular ions [M+H] in accordance with the
2
4
anticipated masses corresponding to their molecular formu-
one-step reaction mechanism involving nucleophilic addi-
tion.
las. The results of elemental analyses were within the accept-
.
able limits of calculated values for all synthesized com-
pounds.
3
.2. Chemistry and Analysis
The purity of the synthesized compounds was confirmed
3.3. Antioxidant Activity
by melting point determination and TLC measurements on
silica gel G plates. All synthesized compounds 5a – 5f were
obtained in good yields with high purity. The spectral data
considered above are in close agreement with the proposed
structures of synthesized compounds 5a – 5f.
The results of antioxidant activity screening (Table 1)
showed that all tested compounds 5a – 5e exhibited moder-
ate to good antioxidant activity. From tabulated data, it is ev-
All compounds dissolved in acetone exhibited prominent
UV absorption maxima (with l
in the region of 370 –
TABLE 1. Data on the Antioxidant Activity of Synthesized Com-
pounds 5a – 5e
max
3
80 nm indicating the presence of strong chromophoric con-
jugated chalcone-imine (CH=CH-CO-)- (>C=N-) functional
moiety.
Concentration
-1
DPPH
(% Inhibition)*
Compd.
FRAP (Absorbance)*
(
mg mL )
50
IR spectroscopy showed characteristic absorption bands
of specific functional groups, which supported the proposed
structures of synthesized compounds. The presence of two
vinylic C-H bonds in 5b was manifested by weak bands at
$
$
5
a
28.634 ± 0.000
31.130 ± 0.000
0.305 ± 0.00033
$
$
$
1
00
0.360 ± 0.000
$
$
200
50
37.591 ± 0.000
0.404 ± 0.0010
-
1
3
024.85 and 2922.19 cm . The characteristic imine moiety
$$
$$
5
b
35.829 ± 0.0848
0.373 ± 0.0146
(
>C=N-) was manifested by a sharp peak of medium inten-
$
$
1
00
42.731 ± 0.000
0.415 ± 0.000
-1
sity at 1657.45 cm for compound 5c. The appearance of
$
$$
-1
200
50
4
5.668 ± 0.000
0.433 ± 0.0033
broad absorption peaks at 3430 and 3423 cm confirmed the
presence of phenolic –OH group in the structures of com-
pounds 5d and 5e, respectively. The presence of >C=N- and
C-O linkages in the structure of 5d was confirmed by the ap-
$
$
$
5c
37.004 ± 0.1697
0.307 ± 0.000
$
$$
1
2
00
00
41.703 ± 0.000
0.347 ± 0.0011
$
$
44.199 ± 0.000
0.370 ± 0.000
-1
pearance of peaks at 1664.82 and 1280.96 cm , respectively.
1
The HNMR spectra displayed various signals with char-
$
$
5d
50
93.098 ± 0.1697
0.392 ± 0.0006
$
$
1
00
200
50
93.538 ± 0.1695
0.398 ± 0.0012
acteristic chemical shift (d, ppm) values corresponding to
various protons present in the structure of synthesized com-
pounds. Two vinylic protons (-CH=CH-) of the chalcone
moiety in 5a were observed as two doublets at d = 8.03 and
$
$
9
3.832 ± 0.000
0.482 ± 0.000
$
$
$$
5
e
89.720 ± 0.000
90.455 ± 0.000
0.354 ± 0.0013
$
1
2
00
00
0.390 ± 0.000
7
.95 ppm. The geometry of each proton was assigned to be
$
$
$
93.147 ± 0.1296
18.20 ± 0.206
0.401 ± 0.0003
trans (E) as the coupling constant (J) values for these protons
were 15.82 and 15.80 Hz, respectively. A singlet at d = 7.62,
Gallic
acid
50.0
0.0028 ± 0.001
0.0221 ± 0.001
0.0033 ± 0.002
7
.64 or 7.66 ppm was attributed to C5 proton of thiazolyl
100.5
0
9.40 ± 0.265
moiety present in the structure of synthesized compounds. A
shift for the hydroxyl proton on the ring B of chalcone moi-
ety in compounds 5d and 5e was manifested by a broad sin-
glet peak at d = 5.32 and 5.38 ppm, respectively. Sharp
singlets at d = 3.86 and 3.75 were assigned to methoxy pro-
tons (ring B) for compounds 5d and 5e respectively [26].
2
00.0
02.37 ± 0.629
*
Values are expressed as mean ± SEM of three replicate observa-
tions; Gallic acid: standard drug; values are statistically significant
$
at p < 0.05 and
$$
p < 0.01 as compared to standard drug (gallic
acid).

Design, Synthesis, Drug-Likeness Studies and Bio-Evaluation
819
ident that the activity (% scavenging or reducing power) in-
creases with the concentration of test compound. All these
compounds were equally active within some degree of varia-
tion at all the three test concentrations. However, the overall
activity of compounds was comparatively lower than that of
the standard drug, gallic acid. Among the synthesized drugs,
compound 5d with hydroxy substituent and compound 5e
with both substituted hydroxy and methoxy groups showed
good activity, while the rest of synthesized analogs (com-
pound 5a without any substituent or compound 5b with
chloro group or compound 5c with only methoxy substi-
tuent) exhibited moderate radical scavenging activity.
actions in cellular components [18, 30]. The presence of
electron withdrawing groups (-Cl) seems to decrease the an-
tioxidant activity. Unsubstituted (5a) compound showed the
least activity in this series.
Results of the present investigation are in accordance
with previous reports [8] that various structural substitutions
with electron donating/releasing groups (in A and B aryl
rings) greatly influence the antioxidant potency of chalcone
derivatives by activating/deactivating the molecule (via
mesomeric effect) through delocalization of electrons over
these rings for target (DNA, proteins, etc.) specific interac-
tion of chalcone pharmacophoric moiety with the corre-
sponding receptor. The lipophilicity of a molecule (due to the
presence of bulky aryl fragments) is also important for
lipoidal membrane permeation in order to get access to cellu-
lar targets. Finally, it can be concluded that the chalcone-
arylthiazole conjugate having enone-azomethine pharmaco-
phoric moiety (-CH=CH-C=N-) is a structural feature impor-
tant for the antioxidant activity.
It is clear from the obtained results that the variation in
activity among the synthesized analogs might be related to
the nature of substituents or substitution pattern in ring B of
the parent chalcone moiety. The presence of para-OH group
(
5d) or ortho-3,4-(OH) (OCH ) groups (5e) is probably more
3
important for increasing antioxidant activity, whereas the
presence of para-OCH (5c) or para-Cl (5b) groups is likely
3
to be less important for the activity. The SAR study clearly
3.4. Antibacterial Activity
indicates that electron donating groups (-OH, -OCH ) are
3
more contributing toward the antioxidant activity than does
the electron withdrawing groups (-Cl). The antioxidant abil-
ity decreases in the following order: OH > OCH3 > Cl > H.
The presence of only methoxy group does not infer high ac-
tivity, but it may potentiate the antioxidant activity when
present in the hydroxylated ring, preferably in the ortho posi-
tion with respect to hydroxy (para) substitution. The –OH
group (at para position) alone significantly contributes to an-
tioxidant activity (5d, 5e) since it is converted into reactive
phenoxy radical after accepting electron from a reactive radi-
cal (ROS). A stable phenoxy radical imparts antioxidant ac-
tivity by disrupting free radical chain mediated oxidation re-
All synthesized compounds 5a – 5f were found active
against selected strains of Gram- positive and Gram-negative
bacteria at the test concentration. There was no inhibition of
growth by vehicle control (acetone). Results presented in Ta-
ble 2 clearly indicate that all compounds were equally active
with little variation, but were less potent in comparison to the
standard drug, ciprofloxacin. Although the antibacterial effi-
cacy of these compounds was less significant, they possess
considerably wider spectrum of activity. The results demon-
strate that compounds 5b (4¢-chloro) and 5c (4¢-methoxy) ex-
hibited somewhat higher inhibitory activity than compounds,
5a (unsubstituted), 5d (2¢-hydroxy) and 5e (3¢-methoxy,
TABLE 2. Data on the Antibacterial Activity of Synthesized Compounds 5a – 5f against Six Selected Bacterial Strains
*
Zone of inhibition ± SEM (mm)
Compd.
B. s
B. p
S. a
E. c
P. a
P. v
$
$
$
$
$$
$
$
$
$
$
5
5
5
5
5
5
a
b
c
d
e
f
3.0 ± 0.68
4.0 ± 0.47
3.0 ± 0.58
4.0 ± 0.55
2.0 ± 0.32
2.0 ± 0.40
3.0 ± 0.46
2.0 ± 0.36
2.0 ± 0.32
$$
3.0 ± 0.28
-
4.0 ± 0.33
$
$
$$
$$
$$
$$
$
$
3.0 ± 0.36
3.0 ± 0.24
3.0 ± 0.27
-
4.0 ± 0.34
3.0 ± 0.36
3.0 ± 0.26
3.0 ± 0.29
-
3.0 ± 0.49
3.0 ± 0.55
-
3.0 ± 0.36
$
$
$
$
$
$
$
$$
-
-
-
2.0 ± 0.46
2.0 ± 0.51
2.0 ± 0.48
3.0 ± 0.46
3.0 ± 0.42
$$
$
2.0 ± 0.30
$
$
2.0 ± 0.32
3.0 ± 0.46
#
Standard
16.0 ± 0.23
16.0 ± 0.22
12.0 ± 0.24
16.0 ± 0.20
16.0 ± 0.20
16.0 ± 0.22
#
#
Control
-
-
-
-
-
-
*
**
-1
#
##
Mean ± SD of triplicate test; Concentration, 200 mg mL ; Ciprofloxacin was used as positive control; Acetone was used as negative
$
$$
control; (–) no bacterial growth; values are statistically significant at p < 0.05 and p < 0.01 as compared to positive control. Bacterial strains:
B. s, Bacillus subtilis; B. p, Bacillus pumilus; S. a, Staphylococcus aureus; E. c, Eschericia Coli; P. a, Pseudomonas aeruginosa; P. v, Pseudomo-
nas vulgaris.

8
20
Mithun Rudrapal and Mullapudi P. K. Sowmya
4
¢-hydroxy), particularly against Gram-positive B. subtilis
ble 3. All these compounds showed moderate drug-like char-
acteristics based on Lipinski¢s rule of five with additional
parameters such as logS and PSA. Drug-likeness prediction
evaluates the acceptability of derivatives as drug molecules
based on Lipinski¢s rule of five [32]. Poor absorption or per-
meation of a ligand is more likely in the presence of molecu-
lar weight >500, logPo/w > 5, HBA > 10, HBD > 5, and RotB
and B. pumilus and Gram-negative P. vulgaris bacterial
strains. It was found that compound 5b was slightly more po-
tent than compound 5c, and compound, 5f showed lowest ac-
tivity in the series studied.
Variation in antibacterial activity among the synthesized
compounds seems to be related to different substitution pat-
terns in aryl components of the core chalcone moiety. The
nature of substitution is an additional contributing factor,
which further modulates the activity of parent chalcone scaf-
fold. The elucidated SAR suggests that electron-withdrawing
and electron-donating groups impart different electronic en-
vironments to the molecules, depending upon the number
and position of substituent(s) present in the aryl ring of the
chalcone moiety, which in turn influences the biological
properties of compounds. The presence of electron with-
drawing group like 4¢-chloro (5b) in ring B of the chalcone
moiety has greater contributing effect to the antibacterial ac-
tivity than do the electron donating groups like 4¢-methoxy
>
5. The assessment of drug-likeness score indicates the suit-
ability of derivatives as drug-like molecules.
The values of all calculated drug-like properties are
within the adopted range except for logP parameter. The
octanol-water partition coefficient (logP) is greater than 5
(within the range of 6.27 – 7.36) for all compounds 5a – 5f.
None of their molecular weights exceeds 500 Da. They pos-
sess HBAs less than or equal to 5 and have HBD values not
exceeding 1. The number of rotatable bonds and the PSA val-
ues of all compounds are also in the permissible range. Fur-
thermore, drug-likeness scores of compounds 5a – 5f ranges
from –0.53 to +0.06. However, since all the synthesized
compounds showed favorable drug-like properties, a reason-
able correlation can be drawn between the calculated
drug-like properties and their in vitro antioxidant activity
profile. LogPo/w is a direct indicator of lipophilicity of drug
substances. The higher the value of logP, the better the mem-
brane permeability. It is essential for a molecule to have suf-
ficient lipophilicity for optimal bioavailability and drug ac-
tion. Sufficient aqueous solubility (logS) is important for op-
timal bioavailability of drugs. Hydrogen bond acceptor and
donor groups are also of paramount importance for transport
property and bioavailability of drug molecules. Molecular
PSA is a useful parameter for drug transport properties. Prac-
tically, a compound with all these drug-like properties in the
desired range appears to exhibit high level of therapeutic po-
tency [22].
(
5c) and 4¢-hydroxy (5d). The activity of the unsubstituted
compound (5a) was almost equal to that of compound 5e
containing two electron releasing groups (4¢-hydroxy and
3
¢-methoxy). The results are consistent with previous reports
[
8] in that electron withdrawing groups in chalcones contrib-
ute more activity than do electron donating group. The elec-
tron withdrawing or donating ability of groups causes
delocalization of electrons over bulky aromatic rings of the
molecule either by inductive or mesomeric effect [31], which
in turn increases the lipophilicity of the molecule thus favor-
ing permeation through the lipoid layer of bacterial mem-
branes [12]. From the above considerations, it can be con-
cluded that conjugated chalcone-arylthiazole scaffold having
pharmacophoric enone-azomethine (-CH=CH-C=N-) moiety
is an important structural requirement for the antibacterial
activity of synthesized chalconeimines.
Higher antioxidant activity of compounds 5d and 5e
might be because of their favorable hydrophilic behavior. Po-
lar properties such as HBD and HBA groups and PSA have
therefore more activity contributing effect than lipophilic
property (logP). It can now be attributed that compounds 5d
3
.5. Drug-Likeness Assessment
The predicted drug-like properties and drug-likeness
score of synthesized compounds 5a – 5f are presented in Ta-
TABLE 3. Predicted Drug-Likeness Parameters for Antioxidant Activity
a
b
c
d
e
f
PSA (A )
2
Violation of the Drug-likeness
Comp.
Mol. wt.
log P
log S
HBA
HBD
Rot B
rule of 5
score
-0.53
-0.13
-0.24
-0.51
0.06
5
5
5
5
5
5
a
b
c
d
e
f
366.12
400.08
396.13
382.11
462.16
392.13
6.65
7.36
6.74
6.27
6.36
7.28
-7.80
-8.76
-8.06
-7.37
-7.64
-8.30
3
3
4
4
5
3
0
0
0
1
1
0
5
5
6
5
6
6
25.26
25.26
34.49
45.48
54.72
25.26
1
1
1
1
1
1
-0.53
a
b
Octanol/water partition coefficient, calculated lipophilicity; aqueous solubility in log(moles L ) ; acceptor hydrogen bond number;
-1
c
d
f
donor hydrogen bond number; polar surface area.

Design, Synthesis, Drug-Likeness Studies and Bio-Evaluation
821
5
. A. Detsi, M. Majdalani, C. A. Kontogiorgis, and D.
and 5e having PSA of 45.48 and 54.72, respectively, readily
dissociate to give reactive ion species (phenoxy radical),
which in turn is responsible for the antioxidant action.
Drug-likeness score of compound 5e (0.06) supports the
above considerations. Finally, it can be concluded that the
conjugated chalcone-arylthiazole scaffold possess antioxi-
dant as well as antibacterial potential. Different structural
substitutions (electron withdrawing or releasing groups) in
the aryl ring (ring B) of chalcone moiety further modulate
the bio-efficacy of chalconeimines.
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6
7
2
3
9
1
The newly synthesized chalconeimines can be used as
lead molecules for further structural modifications in order to
achieve various series of structural analogs with a spectrum
of pharmacological activity. Modifications can be carried out
in the aryl rings (rings A and B) of the chalcone moiety
and/or in the heteroaryl system with a multitude of substitu-
tions preferably with electron donating or withdrawing
groups at different positions. From another point of view, ra-
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and/or flavonoid analogs with diverse substitution pattern
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1
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(
ACKNOWLEDGEMENTS
2
1. M. Rudrapal, T. Mariya Babu, R. Ravi Chandra, et al., Anti-In-
fect. Agents, 12 (2), 198 – 205 (2014).
The authors express their sincere thanks to Dr. K.
Ravishankar, Principal, Sri Sai Aditya Institute of Pharma-
ceutical Sciences and Research, Surampalem, E. G. Dist.,
Andhra Pradesh (India) for his generous help and co-opera-
tion in studying out the antioxidant activity of synthesized
compounds. The authors also wish to thank Mr. N. Sathish
Reddy, Vice-Chairman, Aditya Educational Institutions,
Surampalem, E. G. Dist., Andhra Pradesh (India) for provid-
ing necessary laboratory facilities to carry out the work.
2
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ture Design and Methods, Academic Press, New York (2008),
pp. 7 – 9.
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Tatchell, Vogel¢s Text Book of Practical Organic Chemistry,
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CONFLICT OF INTEREST
2
6. R. M. Silverstein and F. X. Webster, Spectrometric Identifica-
tion of Organic Compounds, John Wiley and Sons, Inc., New
York (1963), pp. 203, 213.
The authors declare that they have no conflict of interest.
2
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26, 1001 – 1043 (2009).
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