Synthesis of E-stilbene azomethines as potent antimicrobial and antioxidant agents

Article abstract of DOI:10.3906/kim-1801-104

A series of new extensively conjugated E -stilbene azomethines (5a–5h) were synthesized and screened for their antioxidant and antimicrobial activity. The compounds were tested against bacterial (Escherichia coli, Staphylococcus aureus, Klebsiella pneumon

Full text of DOI:10.3906/kim-1801-104

Turk J Chem
Turkish Journal of Chemistry
(2018) 42: 1518 – 1533
© TÜBİTAK
http://journals.tubitak.gov.tr/chem/
doi:10.3906/kim-1801-104
Research Article
Synthesis of E-stilbene azomethines as potent antimicrobial and antioxidant
agents
1
2
1
Ahsan IQBAL ,, Zulfiqar Ali KHAN1,∗,, Sohail Anjum SHAHZAD ,, Muhammad USMAN ,,
3
4
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1
Shakeel Ahmad KHAN ,, Abdul H. FAUQ ,, Ayesha BARI ,, Muhammad Aamir SAJID ,
1
Department of Chemistry, Faculty of Physical Sciences, Government College University Faisalabad,
Faisalabad, Pakistan
2
Department of Chemistry, COMSATS University Islamabad, Abbottabad Campus, Abbottabad, Pakistan
3
Department of Chemistry, School of Science, University of Management and Technology, Lahore, Pakistan
4
Department of Chemistry, University of North Florida, Jacksonville, FL, USA
Received: 31.01.2018
•
Accepted/Published Online: 29.06.2018
•
Final Version: 06.12.2018
Abstract: A series of new extensively conjugated E -stilbene azomethines (5a–5h) were synthesized and screened for
their antioxidant and antimicrobial activity. The compounds were tested against bacterial (Escherichia coli, Staphy-
lococcus aureus, Klebsiella pneumoniae, and Bacillus subtilis) and fungal strains (Aspergillus niger, A. flavus, and
Trichoderma harzianum) using the agar well diffusion method. Among the tested compounds, N ’-(4-nitrobenzylidene)-
2-((E)-styryl)benzohydrazide (5g) was found to possess potent antimicrobial activity higher than some drugs with
significant activity reported in the literature, e.g., cefradine and terbinafine hydrochloride. Additionally, compounds
5a–5h were also evaluated for antioxidant potential using DPPH free radical scavenging and ferric thiocyanate (FTC)
assays. Among these, N ’-(4-hydroxybenzylidene)-2-((E)-styryl)benzohydrazide (5e) exhibited significant antioxidant
potential by both assays. Compound 5e demonstrated higher DPPH free radical scavenging activity (IC50 = 22 ± 0.19
µ g/mL) than the standard, butylated hydroxytoluene (BHT; IC50 = 28 ± 0.10 µ g/mL). A similar trend was observed
for compound 5e in FTC assay, which exhibited 86 ± 0.19% inhibition, whereas the BHT control showed 81 ± 0.21%
inhibition of linoleic acid peroxidase. The structure elucidation of the synthesized compounds was carried out by UV-Vis,
1
13
FT-IR, H NMR, C NMR, and elemental analysis and mass spectrometry. These results suggest possible use of these
compounds for the rational design of new antimicrobial and antioxidant agents.
Key words: E -Stilbenes, azomethine analogues, Mizoroki–Heck reaction, antimicrobial activity, antioxidant activity
1. Introduction
1
Phytoalexins are naturally occurring stilbenes reported to act as secondary metabolites in numerous plants.
One example of such compounds is resveratrol (3,4,5-trihydroxystilbene), which was first isolated from the
2
3
Japanese plant Veratrum grandiflorum. This compound has demonstrated significant antineoplastic activity,
4
5
6
angiogenesis, action against certain microbes and inflammation, activity against acquired immunodeficiency
syndrome, radical scavenging, and platelet aggregation. Other reported biological activities of resveratrol
include antagonist action against aryl hydrocarbons receptors (AhR) , enhancement of estrogenic potency,
neuroprotective effects, cAMP phosphodiesterase inhibition, and slowed progression of atherosclerosis.
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∗Correspondence: zulfiqarchem@gmail.com
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This work is licensed under a Creative Commons Attribution 4.0 International License.

IQBAL et al./Turk J Chem
Another example of naturally occurring phytoalexins is pterostilbene, which has been shown to be effective
13
13
in treating resistant hematological malignancies and diabetes. Thus, the multifarious biological effects of
stilbene analogues of phytoalexins may have potential for expansion into other application domains.
¯
Reactive oxygen species (ROS) such as superoxide (O2), hydroxide (OH •), and neutral radicals such as
14
nitric monoxide (NO) are believed to be the key players in damaging lipids, proteins, and DNA in living bodies.
These free radicals may divert cells to abnormal physiological functions, leading to disease states such as
15
cardiovascular complications, diabetes, or cancer. Antioxidants work to scavenge the ROS in biological systems
16
through reductive mechanisms. Overproduction of ROS in humans resulting from excessive electromagnetic
radiation exposure, extensive muscular work, and overconsumption of certain foods suppresses the endogenous
antioxidant-mediated redress. The detrimental effects of oxidants can be cured by using exogenous natural and
14
synthetic antioxidants. Antioxidant activity has become a topic of growing interest.
According to a report, the third most significant causes of mortality and morbidity worldwide are
14
infectious diseases. Microbial strains such as Pseudomonas aeruginosa, Escherichia coli, Enterobacteriaceae,
17
Klebsiella pneumoniae, and Staphylococcus aureus have been recognized as pathogens. Multiresistant microbial
18
species are known to cause serious global health problems. Hydrazide derivatives are present in numerous
biologically active structures and display a wide diversity of biological activities. Among them, antimicrobial
19−21
activity is most frequently met in the literature.
Drug design by structural modifications of the known
antimicrobial agents is a feasible way to address these issues. Keeping these literature findings in view, a small
library of Schiff bases of E -stilbene hydrazide analogues was synthesized and screened for in vitro antimicrobial
and antioxidant potential.
2. Results and discussion
A small library of eight E -stilbene azomethines (5a–5h) was synthesized to evaluate its potential for antimi-
crobial and antioxidant activity. The chemical structures of the target compounds were analyzed by UV-Vis,
1
13
FT-IR, H NMR, C NMR, and mass spectroscopic techniques. The UV-Vis spectra of the E -compounds
(5a–5h) are shown in Figure 1. The λ (maximum absorption bands) values of compounds 5a–5h were in
max
the range of 361–387 nm (Figure 1). A literature search of the λ
values of related stilbene analogues revealed
max
18
a range of 290–360 nm. The bathochromic shifts observed in compounds 5a–5h can be explained on the basis
of extended π-conjugation and the presence of auxochromes (chloro, hydroxyl, nitro, and methoxy groups).
The FT-IR absorption peaks ranging from 960 to 982 cm−1 are assigned to the out-of-plane bending vibration
18
of the C-H bond of the E -ethylene bridge of the stilbene molecule. The FT-IR spectrum of compound 5a is
shown in Figure 2.
1
H NMR spectra also confirmed the identity of specific protons in the synthesized compounds. The
chemicals shift (δ) values (6.85–8.20 ppm) are assigned to the aromatic and olefinic protons of the synthesized
22,23
compounds.
The trans-geometry of olefinic protons in the stilbene structure is confirmed by the presence
of two doublets in spectra with large coupling constants (J = 16–17 Hz). A singlet observed in the range
1
of δ 9.14–9.35 ppm is assigned to the -CH proton of the azomethine moiety of compounds 5a–5h. The
H
13
NMR (400 MHz, CDCl3) spectrum of compound 5a is shown in Figure 3. C NMR and mass spectral studies
also confirmed the synthesis of targeted compounds. The C NMR (75 MHz, CDCl3) and mass spectra
13
(electrospray ionization mode) of compound 5a are shown in Figure 4 and Figure 5, respectively.
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IQBAL et al./Turk J Chem
Figure 1. The UV-Vis spectra of the compounds 5a–5h.
Figure 2. FT-IR spectrum of compound 5a.
2.1. Biological activities
2.1.1. DPPH radical scavenging assay
A variety of mechanisms have been proposed for the antioxidant action in the body. Antioxidants have attracted
24
special attention since the discovery of age-related oxidative stress. The 2,2-diphenyl-1-picrylhydrazyl (DPPH)
25
free radical scavenging activity of resveratrol was found to be IC50 = 33.79 µ M. The antioxidant activity
of resveratrol is thought to be linked to the presence of hydroxyl groups located at specified positions in its
26
structure.
The results for DPPH free radical scavenging activity (expressed in terms of IC50 values) of compounds
5a–5h are collected in Figure 6. These values range from 22 ± 0.19 to 75 ± 0.21 µ g/mL compared to the
standard butylated hydroxytoluene (BHT) with IC50 = 28 ± 0.10 µ g/mL. The tested compounds (5a–5h)
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IQBAL et al./Turk J Chem
1
Figure 3. H NMR spectrum of compound 5a.
showed decreasing order of DPPH activity as follows: 5e > BHT > 5d > 5h > 5a > 5c > 5b > 5f
> 5g. The highest DPPH activity was shown by compound 5e with IC50 = 22 ± 0.19 µ g/mL compared
to the standard BHT (IC50 = 28 ± 0.10 µ g/mL). The enhancement of DPPH activity may be due to the
increased conjugation mediated by the electron-donating 4-hydroxyphenyl moiety as well as the amide-iminol
tautomerism in compound 5e where a push-pull mechanism operates within the molecule. The notion of
the antioxidant activity being associated with the push-pull electronic effect in compound 5e is supported by
the observed decrease in antioxidant activity for compound 5g, where the electron-withdrawing 4-nitrophenyl
moiety exerts the opposite electronic effect (IC50 = 75 ± 0.21 µ g/mL). Similarly, compounds 5b and 5c
with 2-chlorophenyl and 4-chlorophenyl moieties, respectively, showed reduced antioxidant activity compared
to compound 5e (Figure 6). As expected, compound 5a, having unsubstituted phenyl moiety, exhibited slightly
higher antioxidant activity than compounds 5b and 5c that have 2-chlorophenyl and 4-chlorophenyl moieties,
respectively (Figure 6).
The proposed mechanism for the DPPH free radical scavenging activity of the most potent compound
27
(5e) is illustrated in Figure 7, where an amide-iminol tautomerization promoted by the solvent is shown.
The amide-iminol tautomerism in compound 5e helps to promote extensive delocalization of the single electron,
28
resulting in greater stability of the DPPH-generated radical. The enhanced stability caused by the electron-
enriched extended conjugation of the radical generated in the structure of 5e is likely the cause of its antioxidant
activity.
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IQBAL et al./Turk J Chem
13
Figure 4.
C NMR spectrum of compound 5a.
Figure 5. Mass spectrum (MS-ESI) of compound 5a.
2.1.2. Antioxidant activity via inhibition of linoleic acid oxidation
ROS may attack the polyunsaturated fatty acid chains of cell membranes and start a self-propagation chain
reaction, exerting a damaging influence on the cellular and tissue environments. Antioxidants are also effective
in scavenging ROS related to the lipid peroxides providing protective health benefits.
The results of the inhibitory effects of the synthesized compounds (5a–5h) on linoleic acid peroxidase
are summarized in Figure 8. Compounds 5a–5h exhibited linoleic acid peroxidase inhibition in the range of 46
± 0.20% to 86 ± 0.19%. Compounds 5e and 5d inhibited linoleic acid peroxidase most effectively (86 ± 0.19%
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IQBAL et al./Turk J Chem
80
60
40
20
0
5a
5b
5c
5d
5e
5f
5g
5h BHT
Stilbene derivatives (5a-h)
Figure 6. The DPPH free radical scavenging activity of compounds 5a–5h.
Figure 7. Proposed mechanism for DPPH free radical scavenging activity of compound 5e.
and 81.5 ± 0.11% inhibition) (Figure 8). Compounds 5e and 5d with 4-hydroxyphenyl and 2-hydroxyphenyl
moieties, respectively, fared slightly better than the standard BHT (81.0 ± 0.21%), whereas compound 5g,
having 4-nitrophenyl, showed the least inhibition (46 ± 0.20%). The inhibition of linoleic acid oxidation for the
remaining compounds (5h, 5a, 5c, 5b, and 5f) was found to be 73.11 ± 0.19%, 71 ± 0.20%, 65 ± 0.11%, 59
± 0.18%, and 51 ± 0.12%, respectively. The decreasing order of linoleic acid hydroperoxidase inhibition was as
follows: 5e > 5d > BHT > 5h > 5a > 5c > 5b > 5f > 5g.
The antioxidant activity seems to be related to the molecular structure, and more precisely to the presence
of both azomethine moiety and the auxochromes, particularly the hydroxyl moiety located at the ortho- and
para-positions of the aromatic ring as well as an extension of the conjugation into the stilbene molecule. These
structural features of compounds may contribute to the expected high radical scavenging effect resulting from
the higher stability of the generated free radicals.
2.1.3. Antimicrobial activity
The antimicrobial activity is linked to the presence of strongly electron-withdrawing substituents on the stilbene
29
skeleton. This may result in the formation of charge transfer complexes, which play an important role in
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100
80
60
40
20
0
5a
5b
5c
5d
5e
5f
5g
5h
BHT
Stilbene derivatives (5a-h)
Figure 8. The percentage inhibition of linoleic acid peroxidation of compounds 5a–5h.
30
enhancing affinity with the membrane proteins. A decrease in activity has been observed for the electron-rich
hydroxylated stilbenes, especially against gram-negative bacteria.
31
Resveratrol detoxifications by extracellular laccases have also been reported. Fungal pathogens may
31
avoid the inhibitory effect of natural stilbenes by oxidative degradation. The natural stilbenes are not suitable
for the chemical control of pathogenic fungi. In essence, the compounds that are not prone to oxidative
32
degradation may represent excellent lead structures for developing efficient antimicrobial agents.
Minimum inhibitory concentrations (MICs) of resveratrol were found to be 119.23 µ g/mL, 171 µ g/mL,
33,12
and 342 µ g/mL against bacterial strains such as E. faecalis, S. aureus, and P. aeruginosa, respectively.
The
simple stilbene structure displayed weak inhibition against B. subtilis and P. syringae, and against the fungal
34
strains B. cinerea, A. niger, C. herbarum, and M. aucupariae. With this in mind, synthesis of trans-stilbenes
with azomethine moiety (with electron-donating and -withdrawing substituents on the aryl moiety) was carried
out to probe their antimicrobial effects.
The results of antimicrobial activities of compounds 5a–5h are given in terms of the zone of inhibition
(ZOI) diameter in millimeters (Figures 9 and 10). The antimicrobial activity results in terms of MICs are
summarized in Tables 1 and 2. The highest antibacterial activity was shown by compound 5g with 4-nitrophenyl
substituent moiety. The MIC values of compound 5g against four bacterial strains, E. coli, S. aureus, Klebsiella
pneumoniae, and B. subtilis, were recorded to be 0.22 ± 0.03 mg/mL, 0.07 ± 0.05 mg/mL, 0.10 ± 0.03
mg/mL, and 0.06 ± 0.04 mg/mL, respectively (Table 1). Compound 5g also showed strong inhibition with
ZOI diameters of 32 ± 0.07 mm, 33 ± 0.04 mm, 32 ± 0.03 mm, and 33 ± 0.08 mm against E. coli, S. aureus,
Klebsiella pneumoniae, and B. subtilis, respectively (Figure 9). Similarly, compound 5f, with a nitro group
at the meta-position of the benzene ring, showed significant antibacterial activity against E. coli, S. aureus,
Klebsiella pneumoniae, and B. subtilis with ZOI diameters of 27 ± 0.01 mm, 29 ± 0.03 mm, 31 ± 0.03 mm,
and 30 ± 0.03 mm (Figure 9) and MICs of 0.29 ± 0.03 mg/mL, 0.09 ± 0.03 mg/mL, 0.12 ± 0.04 mg/mL,
and 0.07 ± 0.03 mg/mL, respectively (Table 1). Compound 5a, having simple phenyl moiety, exhibited only
moderate antibacterial activity. The literature reveals that Z -stilbenes show higher inhibition potential against
35
gram-negative bacteria as compared to E -stilbenes. However, these newly synthesized E -stilbene analogues
(5a–5h) were found to be more effective against bacterial strains E. coli, B. subtilis, Klebsiella pneumoniae, and
S. aureus in comparison to resveratrol.The standard drug (cefradine) showed good inhibition (ZOI diameters of
28 ± 0.04 mm, 29 ± 0.08 mm, 30 ± 0.07, and 31 ± 0.06 mm) against E. coli, S. aureus, Klebsiella pneumoniae,
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IQBAL et al./Turk J Chem
and B. subtilis, respectively (Figure 9). In comparison with the standard drug, compound 5g, having a 4-
nitrophenyl group, exhibited strong bactericidal activity.
35
40
30
25
20
15
10
5
35
30
25
20
15
10
5
0
0
E. coli
5a
S. aureus
5c 5d
Klebsiella
5f 5g 5h
B. subtilis
cefradine
A. niger
A. flavus
T. harzianum
5b
5e
5a 5b 5c 5d 5e 5f 5g 5h Terbinafine hydrochloride
Figure 9. The antibacterial activity of compounds 5a–5h
(ZOIs in mm).
Figure 10. The antifungal activity of compounds 5a–5h
against tested fungal strains (ZOIs in mm).
Stilbene azomethines 5a–5h displayed moderate to high antifungal activity against tested fungal strains
(A. niger, A. flavus, and T. harzianum) as shown in Figure 10. Compound 5a, having unsubstituted phenyl moi-
ety, showed similar antifungal activity against A. niger compared to the standard drug terbinafine hydrochloride
with ZOI diameters of 31 ± 0.06 mm and 31 ± 0.04 mm, respectively. 4-Nitrophenyl-containing compound 5g
demonstrated the highest antifungal activity against the tested fungal strains (Table 2). Compound 5f with
3-nitrophenyl moiety had reduced antifungal activity in comparison to the 4-substituted compound (5g) (Table
2). This could be explained by the powerful electron-withdrawing nature (negative mesomeric and inductive
effect) of the polar nitro group.
2.2. Conclusion
In this study, thermodynamically more stable E -stilbene azomethines (5a–5h) were synthesized by using the
Mizoroki–Heck reaction as an essential step. It is worth mentioning that most of the compounds showed signifi-
cant antioxidant and antimicrobial activity. Compounds 5g and 5f demonstrated potent antimicrobial activity,
whereas compound 5e with 4-hydroxyphenyl showed strong antioxidant activity. The potent antioxidant ac-
tivity is linked to the presence of an extended π-conjugated system, which is further enhanced by the presence
of auxochromes that promote amide-iminol tautomerism. On the other hand, the antimicrobial effect of the
synthesized compounds, e.g., compounds 5g and 5f, seems to be promoted by the presence of the electron-
withdrawing groups, such as the nitro group. These extended π-conjugated systems are proposed to generate
stable free radicals that may act on the ROS. In sum, the newly synthesized E -stilbene analogues may hold
significant potential for use in the food industry as putative detoxifying additives. The current study provides
some pointers for further investigation into many other (E)-stilbene azomethines containing various electron-
donating and -withdrawing groups that may serve as potent, safe, and cost-effective antimicrobial agents for
the pharmaceutical industry.
3. Experimental
3.1. Chemical reagents
All chemicals were procured from Sigma Aldrich or Alfa Aesar and were used without further purification.
The solvents were dried using standard procedures. Melting points were determined with a Büchi 434 melting
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IQBAL et al./Turk J Chem
point apparatus. FT-IR spectra (KBr dis ks) were recorded on a Bruker FT-IR IFS48 spectrophotometer.
1
13
H NMR and C NMR spectra were recorded in CDCl3 using a Bruker AC400 (400 MHz and 300 MHz)
spectrophotometer. Mass spectra were recorded on mass spectrometer ESI Micromass ZMD-2000 in electrospray
ionization mode. The ions were observed as quasimolecular ions [M–H]− . The chemical shift δ values are given
in ppm. The multiplicity is given as s = singlet, d = doublet, dd = doublet of doublet, t = triplet, etc. The
progression of reactions was monitored by thin-layer chromatography (TLC) performed on 2 × 5 cm aluminum
sheets preloaded with silica gel 60F254 to a thickness of 0.25 mm (Merck). The CHN analysis was performed
on a Carlo Erba Strumentazione-Mod-1106. The chromatograms were visualized under ultraviolet light.
3.2. Chemistry
The synthesis of E -stilbenes 5a–5h is described in the Scheme. 2-Iodobenzoic acid (1) was refluxed with
36
absolute ethanol in the presence of sulfuric acid as a catalyst to furnish ethyl-2-iodobenzoate (2) in 75% yield.
The resulting ester (2) was subjected to Mizoroki–Heck reaction to furnish E -stilbene (3) in good yield.
37,38
The ester functionality of compound 3 was converted to E -stilbene hydrazide (4) by treatment with hydrazine
39
hydrate (Scheme) in the presence of ethanol as a solvent. The stilbene azomethines (5a–5h) were synthesized
40
by condensation of E -stilbene hydrazide (4) with a variety of aromatic aldehydes (Scheme).
3.2.1. Synthesis of ethyl-2-iodobenzoate (2)
36
Ethyl-2-iodobenzoate (2) was prepared using the reported procedure. Yield: 75%; IR (υmax , KBr, cm−1):
3010 (C-H aromatic), 2890 (C-H aliphatic), 1660 (C=O), 1110 (C-O).
3.2.2. Synthesis of stilbene (3) via Mizoroki–Heck reaction
To a 100-mL round-bottom flask, ethyl-2-iodobenzoate (2) (5.1 g, 0.018 mol), styrene (2.59 mL, 0.0225 mol),
triethylamine (5 mL), Pd(OAc)2 (0.4 g, 0.0018 mol), and triphenylphosphine (0.94 g, 0.0036 mol) were added.
The reaction mixture was stirred at room temperature for 30 min. The reaction mixture was subjected to reflux
at 120 °C for 12–15 h under argon atmosphere. After completion of the reaction, the reaction mixture was
dissolved in ethyl acetate (10 mL). The reaction mixture was adsorbed on silica gel under reduced pressure
and purified by column chromatography. The hexane/ethyl acetate mixture (90:10) was used as an eluent. The
product was obtained as a colorless solid. Yield: 80%; mp: 92 °C; IR (υmax , KBr, cm−1): 3010 (C-H aromatic),
2860 (C-H aliphatic), 1660 (C=O), 1595 (C=C), 966 (CH=CH).
3.2.3. Synthesis of E-2-styrylbenzohydrazide (4)
To a 100-mL round-bottom flask E -ethyl 2-styrylbenzoate (3) (2.52 g, 0.01 mol) in absolute ethanol (10 mL)
and 80% hydrazine hydrate (2.42 mL, 0.04 mol) were added. The reaction mixture was refluxed until the
completion of the reaction. Reaction completion was confirmed with TLC. After the completion of reaction,
the remaining hydrazine hydrate and ethanol were distilled off under reduced pressure. The crude product was
washed with water and crystallized from 30% aqueous ethanol. Yield: 80%; mp: 150 °C; colorless crystals;
IR (υmax , KBr, cm−1): 3340 (N-H), 3010 (C-H aromatic), 2860 (C-H aliphatic), 1660 (C=O), 1595 (C=C),
1
970 (CH=CH); H NMR (CDCl3 , 400 MHz) δ: 9.12, (s, 1H, N-H), 7.21–7.71 (m, 9H, Ar-H), 7.38 (d, 1H,
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IQBAL et al./Turk J Chem
Scheme. Synthesis of compounds 5a–5h.
HC=CH, J = 16 Hz), 6.98 (d, 1H, HC=CH, J = 16 Hz), 4.23 (broad singlet, 2H, NH2); C15 H14 N2 O (MW:
238.28 g/mol): C, 75.61%; H, 5.92%; N, 11.76%; Found: C, 75.62%; H, 5.92%; N, 11.75%.
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IQBAL et al./Turk J Chem
3.2.4. General procedure (GP-1) for synthesis of compounds 5a–5h
To a 100-mL round-bottom flask E -2-styrylbenzohydrazide (4) (0.5 g, 0.002 mol) and aromatic aldehydes (1.2
eq., 0.0024 mol) were added in the presence of absolute ethanol followed by the addition of a few drops of glacial
acetic acid. The reaction mixture was refluxed until the completion of the reaction. Reaction completion was
monitored by TLC. After the completion of reaction, the reaction mixture was cooled in a refrigerator overnight.
The appeared solid was filtered. Finally, the crude product was crystallized from chloroform to furnish stilbene
azomethines 5a–5h in 45%–66% yields.
3.3. N’-Benzylidene-2-((E)-styryl)benzohydrazide (5a)
Compound 5a was synthesized from E -2-styrylbenzohydrazide (4) (0.5 g, 0.002 mol) and benzaldehyde (0.24
mL, 0.0024 mol) by following GP-1. Yield: 66%; light brown solid; mp: 240 °C; λ
in nm: 370; FT-IR
max
(υmax , KBr, cm−1): 3395 (N-H), 3035 (C-H aromatic), 2855 (C-H aliphatic), 1680 (C=O), 1590 (C=N), 1620
1
(C=C), 1195 (C-N), 960 (CH=CH); H NMR (CDCl3 , 400 MHz) δ: 9.14, (s, 1H, C-H), 7.07–7.98 (m, 14H,
13
Ar-H), 7.42 (d, 1H, HC=CH, J = 16.1 Hz), 7.04 (d, 1H, HC=CH, J = 16.1 Hz), 5.85 (s, 1H, -NH); C NMR
(CDCl3 , 75 MHz) δ: 170.1, 159.8, 140.7, 139.9, 136.2, 133.2, 133.1, 129.0, 128.9, 128.2, 128.1, 127.8, 126.8,
126.6, 125.1, 124.3, 123.9, 122.8; ESI-MS: 325.14 [M–H]− . Anal. Calcd. for C22 H18 N2 O (MW: 326.39 g/mol):
C, 80.96%; H, 5.56%; N, 8.58%; Found: C, 80.95%; H, 5.57%; N, 8.56%.
3.3.1. N’-(2-Chlorobenzylidene)-2-((E)-styryl)benzohydrazide (5b)
Compound 5b was synthesized from E -2-styrylbenzohydrazide (4) (0.5 g, 0.002 mol) and 2-chlorobenzaldehyde
(0.34 g, 0.0024 mol) by following GP-1. Yield: 48%; yellow solid; mp: 212 °C; λ
in nm: 373; FT-IR (υ
,
max
max
KBr, cm−1): 3350 (N-H), 3030 (C-H aromatic), 2855 (C-H aliphatic), 1680 (C=O), 1620 (C=N), 1610 (C=C),
1
1170 (C-N), 980 (CH=CH), 743 (C-Cl); H NMR (CDCl3 , 400 MHz) δ: 9.30, (s, 1H, C-H), 7.21–7.85 (m,
13
13H, Ar-H), 7.39 (d, 1H, HC=CH, J = 16.5 Hz), 7.01 (d, 1H, HC=CH, J = 16.5 Hz), 5.95 (s, 1H, NH);
C
NMR (CDCl3 , 75 MHz) δ: 169.1, 159.9, 145.8, 140.1, 139.0, 137.3, 135.3, 133.5, 131.8, 131.3, 129.6, 129.4,
128.9, 128.5, 128.2, 128.0, 127.6, 127.1, 126.4, 120.1; ESI-MS: 359.11 [M–H]− . Anal. Calcd. for C22 H17 ClN2 O
(MW: 360.84 g/mol): C, 73.23%; H, 4.75%; N, 7.76%; Found: C, 73.25%; H, 4.74%; N, 7.76%.
3.3.2. N’-(4-Chlorobenzylidene)-2-((E)-styryl)benzohydrazide (5c)
Compound 5c was synthesized from E -2-styrylbenzohydrazide (4) (0.5 g, 0.002 mol) and 4-chlorobenzaldehyde
(0.34 g, 0.0024 mol) by following GP-1. Yield: 45%; yellow solid; mp: 230 °C; λ
in nm: 375; FT-IR (υ
,
max
max
KBr, cm−1): 3340 (N-H), 3045 (C-H aromatic), 2890 (C-H aliphatic), 1690 (C=O), 1630 (C=N), 1605 (C=C),
1
1160 (C-N), 982 (CH=CH), 760 (C-Cl); H NMR (CDCl3 , 400 MHz) δ: 9.22, (s, 1H, -CH), 7.25–7.91 (m, 13H,
13
Ar-H), 7.30 (d, 1H, HC=CH, J = 17 Hz), 6.96 (d, 1H, HC=CH, J = 17 Hz), 5.90 (s, 1H, -NH); C NMR
(CDCl3 , 75 MHz) δ: 169.9, 158.9, 145.9, 140.5, 139.2, 135.6, 133.1, 132.8, 129.8, 129.5, 129.1, 128.7, 128.4,
127.5, 127.3, 127.1, 126.4, 123.6; ESI-MS: 359.12 [M–H]− . Anal. Calcd. for C22 H17 ClN2 O (MW: 360.84
g/mol): C, 73.23%; H, 4.76%; N, 7.77%; Found: C, 73.21%; H, 4.77%; N, 7.78%.
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3.3.3. N’-(2-Hydroxybenzylidene)-2-((E)-styryl)benzohydrazide (5d)
Compound 5d was synthesized from E -2-styrylbenzohydrazide (4) (0.5 g, 0.002 mol) and 2-hydroxybenzaldehyde
(0.25 mL, 0.0024 mol) by following GP-1. Yield: 55%; yellow solid; mp: 270 °C; λ
(nm): 380; FT-IR (υ
,
max
max
KBr, cm−1): 3370 (N-H), 3310 (O-H, broad), 3050 (C-H aromatic), 2905 (C-H aliphatic), 1690 (C=O), 1610
1
(C=N), 1590 (C=C), 1150 (C-N), 965 (CH=CH); H NMR (CDCl3 , 400 MHz) δ: 9.14, (s, 1H, -CH), 7.35–
8.20 (m, 13H, Ar-H), 7.29 (d, 1H, HC=CH, J = 16.7 Hz), 6.87 (d, 1H, HC=CH, J = 16.7 Hz), 5.85 (s, 1H,
13
-NH), 4.20 (s, 1H, Ar-OH); C NMR (CDCl3 , 75 MHz) δ: 172.5, 161.7, 146.3, 141.5, 139.6, 137.1, 134.8,
134.1, 131.6, 131.3, 130.7, 129.2, 128.7, 128.4, 128.2, 127.9, 127.5, 127.1, 126.8, 122.4; ESI-MS: 341.14 [M–H]−
.
Anal. Calcd. for C22 H18 N2 O2 (MW: 342.39 g/mol): C, 77.17%; H, 5.30%; N, 8.18%; Found: C, 77.15%; H,
5.32%; N, 8.17%.
3.3.4. N’-(4-Hydroxybenzylidene)-2-((E)-styryl)benzohydrazide (5e)
Compound 5e was synthesized from E -2-styrylbenzohydrazide (4) (0.5 g, 0.002 mol) and 4-hydroxybenzaldehyde
(0.3 g, 0.0024 mol) by following GP-1. Yield: 50%; yellow solid; mp: 285 °C; λ
(nm): 383; FT-IR (υ
,
max
max
KBr, cm−1): 3360 (N-H), 3300 (O-H, broad), 3040 (C-H aromatic), 2890 (C-H aliphatic), 1660 (C=O), 1615
1
(C=N), 1625 (C=C), 1140 (C-N), 970 (CH=CH); H NMR (CDCl3 , 400 MHz) δ: 9.25 (s, 1H, -CH), 7.40–8.12
(m, 13H, Ar-H), 7.34 (d, 1H, HC=CH, J = 16.5 Hz), 7.10 (d, 1H, HC=CH, J = 16.5 Hz), 5.95 (s, 1H, -NH),
13
4.25 (s, 1H, Ar-OH); C NMR (CDCl3 , 75 MHz) δ: 175.2, 164.5, 147.4, 142.4, 140.3, 136.8, 134.5, 133.8,
131.5, 130.5, 129.8, 129.5, 129.1, 128.7, 128.2, 127.8, 127.2, 119.7; ESI-MS: 341.14 [M–H]− . Anal. Calcd. for
C22 H18 N2 O2 (MW: 342.39 g/mol): C, 77.17%; H, 5.30%; N, 8.18%; Found: C, 77.15%; H, 5.32%; N, 8.18%.
3.3.5. N’-(3-Nitrobenzylidene)-2-((E)-styryl)benzohydrazide (5f)
Compound (5f) was synthesized from E -2-styrylbenzohydrazide (4) (0.5 g, 0.002 mol) and 3-nitrobenzaldehyde
(0.36 g, 0.0024 mol) by following GP-1. Yield: 60%; yellow solid; mp: 205 °C; λ
(nm): 365; FT-IR (υ
,
max
max
KBr, cm−1): 3320 (N-H), 3070 (C-H aromatic), 2850 (C-H aliphatic), 1650 (C=O), 1620 (C=C), 1610 (C=N),
1
1350 (Ar-NO2), 1130 (C-N), 960 (CH=CH); H NMR (CDCl3 , 400 MHz) δ: 9.35, (s, 1H, -CH), 7.30–8.22
(m, 13H, Ar-H), 7.41 (d, 1H, HC=CH, J = 16 Hz), 7.03 (d, 1H, HC=CH, J = 16 Hz), 5.92 (s, 1H, -NH);
13
C NMR (CDCl3 , 75 MHz) δ: 173.6, 160.4, 145.1, 141.5, 140.7, 138.2, 134.2, 133.5, 132.7, 131.5, 130.7, 129.8,
128.5, 128.3, 128.2, 127.6, 127.4, 127.1, 126.4, 118.9; ESI-MS: 370.13 [M–H]− . Anal. Calcd. for C22 H17 N3 O3
(MW: 371.39 g/mol): C, 71.15%; H, 4.61%; N, 11.31%; Found: C, 71.14%; H, 4.62%; N, 11.31%.
3.3.6. N’-(4-Nitrobenzylidene)-2-((E)-styryl)benzohydrazide (5g)
Compound 5g was synthesized from E -2-styrylbenzohydrazide (4) (0.5 g, 0.002 mol) and 4-nitrobenzaldehyde
(0.36 g, 0.0024 mol) by following GP-1. Yield: 58%; yellow solid; mp: 225 °C; λ
(nm): 361; FT-IR (υ
,
max
max
KBr, cm−1): 3330 (N-H), 3040 (C-H aromatic), 2870 (C-H aliphatic), 1680 (C=O), 1620 (C=N), 1615 (C=C);
1
1360 (Ar-NO2), 1120 (C-N), 980 (CH=CH); H NMR (CDCl3 , 400 MHz) δ: 9.30, (s, 1H, -CH), 7.35–8.14
(m, 13H, Ar-H), 7.28 (d, 1H, HC=CH, J = 16 Hz), 6.85 (d, 1H, HC=CH, J = 16 Hz), 5.95 (s, 1H, -NH);
13
C NMR (CDCl3 , 75 MHz) δ: 169.9, 158.5, 145.2, 139.3, 138.6, 133.5, 131.5, 131.2, 129.7, 129.2, 128.7, 128.6,
128.3, 127.7, 127.4, 127.2, 125.6, 118.6; ESI-MS: 370.13 [M–H]− . Anal. Calcd. for C22 H17 N3 O3 (MW: 371.39
g/mol): C, 71.15%; H, 4.61%; N, 11.31%; Found: C, 71.16%; H, 4.60%; N, 11.32%.
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3.3.7. N’-(4-Hydroxy-3-methoxybenzylidene)-2-((E)-styryl)benzohydrazide (5h)
Compound 5h was synthesized from E -2-styrylbenzohydrazide (4) (0.5 g, 0.002 mol) and 4-hydroxy-3-methoxy-
benzaldehyde (0.36 g, 0.0024 mol) by following GP-1. Yield: 64%; dark brown solid; mp: 310 °C; λ
(nm):
max
387; FT-IR (υmax , KBr, cm−1): 3380 (N-H), 3320 (O-H, broad), 3010 (C-H aromatic), 2900 (C-H aliphatic),
1
1660 (C=O), 1615 (C=N), 1605 (C=C); 1150 (-OCH3); 1110 (C-N), 970 (CH=CH); H NMR (CDCl3 , 400
MHz) δ: 9.20, (s, 1H, C-H), 7.21–7.70 (m, 12H, Ar-H), 7.31 (d, 1H, HC=CH, J = 16 Hz), 6.97 (d, 1H,
13
HC=CH, J = 16 Hz), 5.97 (s, 1H, N-H), 4.22 (s, 1H, Ar-OH), 3.85 (s, 3H, Ar-OCH3); C NMR (CDCl3 ,
75 MHz) δ: 169.8, 159.2, 144.9, 142.5, 141.7, 139.1, 135.5, 134.1, 133.5, 132.6, 131.7, 129.5, 128.8, 128.5, 128.2,
127.5, 127.4, 127.2, 126.7, 118.3, 55.5; ESI-MS: 371.13 [M–H]− . Anal. Calcd. for C23 H20 N2 O3 (MW: 372.42
g/mol): C, 74.18%; H, 5.41%; N, 7.52%; Found: C, 74.16%; H, 5.43%; N, 7.52%.
3.4. Determination of antioxidant potential of compounds 5a–5h
3.4.1. DPPH free radical scavenging activity
DPPH free radical scavenging activity of compounds 5a–5h was determined using BHT as a standard antioxi-
41
dant according to a reported method. Different concentrations (20, 40, 60, 80, and 100 µ g/mL) of compounds
5a–5h were prepared. To 1 mL of 0.1 mM DPPH ethanolic solution was added 3 mL of each compound con-
centration. The solutions were stirred at room temperature for 10 min and then permitted to stand for 1 h in
the dark. The absorbance was determined at 517 nm beside ethanol as a blank. Antioxidant activity (D) was
calculated by the equation given below:
D(%) = {(AC − AS)AC} × 100,
(1)
where AC and AS are the absorbance of the control and sample, respectively.
3.4.2. Ferric thiocyanate (FTC) assay
16
Inhibitory effects of compounds 5a–5h were assessed by using the FTC assay. To 2.5 mL of linoleic acid
emulsion (0.02 M, pH 7) and 2.0 mL of phosphate buffer (0.02 M, pH 7) was added 0.2 mL (100 µ g/mL)
of each compound solutions 5a–5h. Each resultant mixture was incubated at 40 °C for 5 days. The mixture
without sample was taken as a blank. The incubated mixture (0.5 mL) was mixed with 75% ethanol (5 mL),
30% ammonium thiocyanate (0.5 mL), and 20 mM ferrous chloride in 3.5% HCl (0.1 mL). Then absorbance
was taken at 500 nm after 3 min. The % inhibition of peroxidation (IP %) was measured by the equation given
below:
IP(%) = [1 − (abs.ofsample)/(abs.ofcontrol)] × 100.
(2)
3.5. Antibacterial activity
Antibacterial activity of synthesized stilbene hybrid azomethines 5a–5h was determined by measuring their
42
ZOIs against different bacterial strains by using the agar well diffusion method.
by the serial dilution technique.
The MICs were determined
21
Bacterial strains Bacillus subtilis (ATCC 9637), Staphylococcus aureus
(ATCC 25923), Escherichia coli (ATCC 25922), and Klebsiella pneumoniae (ATCC 10031) were obtained from
the PCSIR laboratories complex, Lahore, Pakistan. Bacterial cultures were grown in potato dextrose agar.
Cefradine served as the standard antibacterial drug (positive control).
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3.6. Antifungal activity
Antifungal potential of synthesized stilbene azomethines 5a–5h in terms of ZOIs was evaluated against different
42
21
fungal strains by using the reported method. The MICs were determined by the serial dilution technique.
The used fungal strains, i.e. Aspergillus niger (ATCC 16404), Trichoderma harzianum (ATCC 20846), and
Aspergillus flavus (ATCC 9643), were already cultured in the microbiology laboratory of the Industrial Biotech-
nology Division of NIBGE Faisalabad, Pakistan. Terbinafine hydrochloride was used as the standard drug
(positive control).
Acknowledgments
The second and third authors are thankful to the Higher Education Commission under the National Research
Program for Universities Pakistan (Project No. 5290/Federal/NRPU/R&D/HEC/2016) for financial support.
The second author is also thankful to the GCUF-RSP (61-CHM-1) research grant program.
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