Synthesis and antioxidant, cytotoxicity and antimicrobial activities of novel curcumin mimics

Article abstract of DOI:10.3109/14756366.2011.587416

Claisen-Schmidt condensation of 3-(1,2,3,6-tetrahydro-1-methylpyridin-4-yl) -2,4,5-trimethoxybenzaldehyde 3 and various aromatic, heterocyclic and alicyclic amides of 3-aminoacetophenone 6(as) afforded novel curcumin mimics. All the synthesized compounds

Full text of DOI:10.3109/14756366.2011.587416

Journal of Enzyme Inhibition and Medicinal Chemistry, 2012; 27(2): 267–274
© 2012 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2011.587416
RESEARCH ARTICLE
Synthesis and antioxidant, cytotoxicity and antimicrobial
activities of novel curcumin mimics
Babasaheb P. Bandgar1,2, Shivkumar S. Jalde2, Balaji L. Korbad2, Sachin A. Patil2, Hemant V.
Chavan1, Santosh N. Kinkar2, Laxman K. Adsul1, Sadanand N. Shringare1, and Shivraj H. Nile3
1Medicinal Chemistry Research Laboratory, School of Chemical Sciences, Solapur University, Solapur, Maharashtra,
India, 2Organic Chemistry Research Laboratory, School of Chemical Sciences, SRTM University, Nanded, Maharashtra,
India, and 3Biochemistry Research Laboratory, School of Life Sciences, SRTM University, Nanded, Maharashtra, India
Abstract
Claisen-Schmidt condensation of 3-(1,2,3,6-tetrahydro-1-methylpyridin-4-yl)-2,4,5- trimethoxybenzaldehyde 3 and
various aromatic, heterocyclic and alicyclic amides of 3- aminoacetophenone 6(a–s) afforded novel curcumin mimics.
All the synthesized compounds were characterized by IR, 1H NMR, Mass spectroscopy and evaluated for antioxidant,
cytotoxicity and antimicrobial activity. Out of the 20 compounds screened, compounds 7i, 7l, 7q, and 7n have
shown excellent radical scavenging activity, compounds 7o, 7t, 7f, and 7r have shown significant xanthine oxidase
inhibition, and compounds 7a, 7k and 7l were found to be potent inhibitors of selected cancer cell lines. Compounds
7h, 7t, 7l, 7i, and 7e have shown good antibacterial activity, whereas compounds 7j, 7f, 7o, 7h, and 7t exhibited
significant antifungal activity.
Keywords: Curcumin mimics, antioxidant, xanthine oxidase, cytotoxicity, antimicrobial
Introduction
cardiovascular13, inflammation14, and neurodegenerative
disorders such as Alzheimer’s and Parkinson15,16
Reactiveoxygenspecies(ROS),includingthehydroxylrad-
ical, superoxide anion, hydrogen peroxide and peroxyni-
tric species are continuously generated in the process of
respiration as natural byproduct of oxygen metabolism1.
ese species are sufficiently reactive to cause injury
to cells through the destruction of components such as
proteins, lipids, sugars and nucleotides2,3. Under nor-
mal circumstances, cells are able to defend against ROS
damage through the use of enzymes such as superoxide
dismutase and catalase4–6. Small molecule antioxidants
such as ascorbic acid, uric acid and glutathione also play
an important role as cellular antioxidants7–9. erefore,
under normal condition, ROS are maintained at constant
level in the body by the antioxidant defence mechanism.
However, imbalances between the formation and detoxi-
fication of ROS can result in significant damage to cells, a
situation known as oxidative stress. Oxidative stress leads
to the initiation or progression of various diseases such as
cancer10, ischemia-reperfusion injury11, atherosclerosis12,
.
Xanthine oxidase (XO) is a key enzyme that catalyzes
the oxidation of xanthine and hypoxanthine into uric acid
and plays a vital role in causing hyperuricemia and gout17.
It is considered to be a main source of oxidative stress
and destructive free radicals in ischemia-reperfusion
injury associated with heart attack, stroke and spinal cord
injury, as well as being a destructive force myocardial or
renal hypoxia and infarction18–20. Because of this, there is
a growing interest in the discovery of natural and unnatu-
ral antioxidants that attenuate oxidative stress and as a
result serve as protective agents against these diseases21.
Curcumin is a β-diketone constituent of the turmeric
that is obtained from the powdered rhizome of Curcuma
longa. Curcumin has a wide range of interesting biologi-
cal activities such as anti-inflammatory, antioxidant, anti-
viral, cutaneous wound healing, hypocholesterolemic
effects in diabetic patients, anti-angiogenic and stimu-
latory response to stress-induced biological activity22,23
.
Address for Correspondence: Dr. Babasaheb P. Bandgar, Medicinal Chemistry Research Laboratory, School of Chemical Sciences,
Solapur University, Solapur, Maharashtra, India, India. Tel./Fax: 0091–217-2351300. E-mail: bandgar_bp@yahoo.co.in
(Received 04 February 2011; revised 04 May 2011; accepted 09 May 2011)
267

268 B. P. Bandgar et al.
Curcumin has been demonstrated to possess preven-
tative activity against Aβ-aggregation in Alzheimer’s
model24. Several studies have shown that curcumin man-
ifests anti-proliferative activity against various cancers,
including leukemia, colon, liver, breast and prostrate
cancers23,25–27. Although curcumin is non-toxic and has
promising biological activities, preclinical and clinical
studies have indicated that poor bioavailability and phar-
macokinetic profile are poor due to its instability under
physiological condition which has limited its applica-
tion in anticancer therapies28–30. Evidences from both
in vitro and in vivo studies show that β-diketone moiety
is responsible for instability and weak pharmacokinetic
profiles of curcumin. During last decade, synthetic modi-
fications of curcumin, which were aimed at enhancing
its bioactivities, suggested that the stability and meta-
bolic profile of curcumin could be enhanced by deleting
β-diketone moiety. Recent studies from several inde-
pendent groups demonstrated that curcumin analogues
without β-diketone either retained or increased various
biological activities such as anticancer31,32, antibacterial33
and anti-inflammatory34. In continuation of our studies
in synthesizing various biologically active compounds35,
in the present study we have synthesized and character-
ized the novel curcumin mimics and evaluated these for
antioxidant, cytotoxic and antimicrobial activity.
3 under basic media afforded a residue, which on puri-
fication by column chromatography with 1% ammonia
and 0.5–1% methanol in chloroform as eluting solvent
furnished title compounds in good yields (Table 1). All
the synthesized curcumin mimics were characterized by
1
IR, H NMR and Mass spectral analysis. All the synthe-
sized curcumin mimics 7(a–s) and synthetic intermedi-
ate 7t were evaluated for antioxidant, cytotoxicity and
antimicrobial activity.
Results and discussion
Taking into the account of multifactorial character of
oxidative stress which is involved in many pathological
states, we have evaluated antioxidant activity of cur-
cumin mimics against DPPH stable free radical. Free
radical scavenging activity was measured in terms of %
DPPH inhibition and results are presented in Table 2.
All the synthesized compounds have shown excellent
to moderate radical scavenging activity. Compounds 7i,
7l, 7q, 7f, 7r, 7j, 7e, 7o, 7t and 7c have shown excellent
radical scavenging activity (90–80%) as compared to
standard trolox (80%), compounds 7k, 7s have shown
good radical scavenging activity (77–75%), whereas the
rest of the compounds have shown moderate radical
scavenging activity. Structure activity relationship (SAR)
study of a curcumin mimics demonstrated that the pres-
ence of electron donating substituents on the amide ring
increases antioxidant activity, whereas the presence of
electron withdrawing substituents on the amide ring
decreases the antioxidant activity except in the case of
compound 7eand 7o. Butylated compounds are excellent
inhibitors of reactive oxygen species (ROS42); however, in
the present case, compound 7m has shown moderate
antioxidant activity. is may be ascribed to the absence
of hydroxyl group ortho to butyl group on aromatic ring.
Compound 7n containing cinnamide moiety shows good
antioxidant activity which is as expected since cinnamic
derivatives have been known to be excellent ROS scav-
enger43. A comparison of the antioxidant activity of com-
pounds 7a, 7k, 7l reveal that the replacement of aromatic
amide moiety (7a) with heterocyclic amide moiety such
as, thiphen (7k) or furan (7l) results in increased anti-
oxidant activity, particularly in the latter case. Similarly,
incorporation of alicyclic amide moiety (7r) also showed
increased activity compared to (7a) which contains aro-
matic amide moiety.
Chemistry
Although curcumin is non-toxic and has promising bio-
logical activities, clinical studies indicate its poor bio-
availability and pharmacokinetic profile. To overcome
these barriers several research groups have synthesized
curcumin analogues. Robinson et al31. have synthesized
enone and dienone analogues, and Woo et al36. have
synthesized the curcumin mimics which were shown to
possess increased anti-angiogenic activity. Flavopiridol
(NSC 649890) [cis-5,7-dihydroxy-2-(2-chlorophenyl)-8-
[4-(3-hydroxy-1-methyl)-piperidinyl]-1-benzopyran-4-
one] is a synthetic flavone that has been shown to pos-
sess anti-tumour activity against various tumour cell
lines such as human lung carcinoma and breast carci-
noma37. Olefin analogues of flavopiridol have been syn-
thesized and shown to be potent inhibitor of CDK38 and
glycogen phosphorylase39 Figure 1. We herein report on
the synthesis of the novel curcumin mimic incorporat-
ing olefin as well as aromatic, alicyclic or heteroaromatic
amide moities. e title compounds were prepared using
straight forward chemistry (Scheme 1). Compound 2
(olefin) was prepared by reacting 1, 3, 5-trimethoxy ben-
zene with 1-methyl-4-piperidone in presence of hydro-
gen chloride gas in glacial acetic acid40. Compound 2 on
Vilsmier-Hack formylation gave 3-(1,2,3,6-tetrahydro-
1-methylpyridine-4-yl)-2,4,6-trimethoxybenzaldehyde
3. Compounds 6a–s were prepared by acylation of
3-aminoacetophenone with different acylchlorides in
basic medium, except compound 6r and 6s which were
prepared usinganearlierreportedmethod41. Compounds
6a–s on Claisen-Schmidt condensation with compound
Xanthine Oxidase (XO) is a cytosolic enzyme that is an
important source of oxygen free radicals. Results of XO
inhibition by curcumin mimics are presented in Table 2.
ese results indicate that all the synthesized compounds
have excellent to moderate XO inhibition. Among all
synthesized compounds, 7o, 7t, 7f, 7r, 7l, 7i, 7k and 7q
were excellent inhibitors of XO (94–90%) as compared to
standard allopurinol (90%), compounds 7d, 7n, 7m, 7c,
and 7h were good inhibitors of XO (89–80%), whereas
the rest of the compounds showed moderate inhibition.
SAR study revealed that compounds containing electron
Journal of Enzyme Inhibition and Medicinal Chemistry

Antioxidant, cytotoxicity and antimicrobial activities of novel curcumin mimics 269
withdrawing substituents on amide ring are excellent
Panc 1 (human pancreatic carcinoma), Calu 1 (human
non-small cell lung carcinoma), H460 (human non cell
lung carcinoma) and HCT 116 (human colon carcinoma)
cancer cell lines (92–100%) at 10 μM as compared to stan-
dard flavopiridol (68–73%) and gemcitabine (78–84%) at
700 and 500 nM, respectively. Compounds 7e, 7g, and
7s have moderate cytotoxicity. Of the 20 compounds
synthesized, compound 7a and 7k are cytotoxic to all
selected five cancer cell lines studied. e cytotoxicity of
these compounds are in compliance with the literature36.
Compound 7l has preferential cytotoxicity against pan-
creas, lung and non-small cell lung carcinoma cancer
cell lines. Similarly, compound 7g shows cytotoxicity
against non-small cell lung carcinoma, human colon and
renal cancer cell lines. In general, the following trend was
observed: (i) electron withdrawing groups on the amide
ring are more cytotoxic than their counterparts contain-
ing electron donating groups, (ii) curcumin mimics con-
taining heteroaromatic amide moiety showed highest
cytotoxicity, (iii) the order of cytotoxicity with respect to
inhibitors of XO compared to their counterparts contain-
ing electron donating groups with the exception 7i and 7q.
Interestingly, compound 7t an intermediate of curcumin
mimics, has shown excellent XO inhibition. Changing the
position of fluorine on amide ring from para position (7f)
to meta position (7d) showed a decrease in activity. ere
is further decrease in the activity as the position of fluorine
is changed from meta (7d) to ortho (7b). is suggests that
a substituent at para or meta position is favourable for
enzyme inhibition. In addition it was found that heteroar-
omatic amides such as furan amide (7l), thiophen amide
(7k) are better inhibitors than the aromatic amide (7a).
Similarly, curcumin mimics containing alicyclic cyclopro-
pyl amide moiety (7r) shows excellent XO inhibition as
compared to one containing aromatic amide moiety (7a).
Results shown in Table 3 indicate that all synthesized
compounds have shown cytotoxicity against all selected
cancer cell lines. Compounds 7a, 7k, and 7l completely
inhibited selected ACHN (human renal carcinoma),
Scheme 1. Reagents and conditions: (a) N-methyl- 4-piperidone, AcOH, HCl, 85-90 0C, 3.5h; (b) DMF, POCl3, rt, 2 h; (C) NaOH (d) NaOH,
EtOH, rt, 24 h
Figure 1. Flavopiridol and curcumin mimics.
© 2012 Informa UK, Ltd.

270 B. P. Bandgar et al.
position of substituents on the amide moiety was found
to be meta > para > ortho.
nystatin at tested concentration. Some of the compounds
are inactive against fungal strains especially against C.
albicans. Structure activity relationship study indicates
that compounds containing electron withdrawing groups
on the amide moiety have shown higher antifungal activ-
ity than their counterparts containing electron donating
groups except 7j. Alicyclic cyclopropyl amide 7r showed
higher antifungal activity than the aromatic amide 7a.
Compounds having substituents at para and meta position
of amide ring have higher antifungal activity than the ortho
substituted counterparts.
All synthesized compounds were subjected to anti-
microbial study by in vitro disk diffusion method against
Salmonella typhi, Proteus vulgaris, Pseudomonas aerugi-
nosa, Staphylococcus aureus, Bacilus subtilis, Penicillium
chrysogenum, Trichoderma viridae, Candida albicans,
Microsporum cannis and Fusarium moniliformae. Nystatin
and tetracycline used as standards against fungi and bac-
teria, respectively. e results are reported as a mean of
triplicate measurements. Antibacterial and antifungal
activities of compounds are shown in Table 4. Results show
that all the synthesized compounds possess good antimi-
crobial activity. Compounds 7h, 7t, 7l, 7i, 7e, 7j, 7o, 7c, and
7r show antibacterial activity more or less equal to that of
standard at tested concentration; however, the rest of the
compounds displayed weak antibacterial activity. It is
observed that of the synthesized 20 compounds screened
against three gram negative strains S. typhi, P. vulgaris, P.
aeruginosa and two gram positive strain S. aureus, B. subti-
lis, almost all the compounds are active against gram posi-
tive strains, whereas some of the compounds are inactive
against gram negative strain. In addition, it was observed
that substituents on amide ring at meta and para posi-
tions showed higher antibacterial activity as compared to
the respective ortho derivatives with the exception of 7h.
Interestingly, compound 7t, an intermediate of curcumin
mimic, has shown equal antibacterial activity as that of
curcumin mimics. Curcumin mimics containing heterocy-
clic and alicyclic amides moieties have shown promising
antibacterial activity. Compounds 7j, 7f, 7o, 7h, 7t, 7c, 7e,
and 7r showed strong antifungal activity as compared to
Conclusion
In this study, we have synthesized a series of novel cur-
cumin mimics and evaluated for antioxidant, cytotoxicity
and antimicrobial activity. Compounds 7i, 7l, 7q, and 7n
showed excellent antioxidant activity. e compounds
7o, 7t, 7f, and 7r have shown promising xanthine oxidase
inhibition. e presence of substituents at meta and para
position of amide was found to be essential for antioxidant
activity. Compounds 7a, 7k, and 7l have shown potent
cytotoxicity against selected human cancer cell lines. e
presence of substituents at meta position was found to be
necessary for cytotoxicity. Compounds 7h, 7t, 7l, 7i, and
7e showed good antibacterial activity and compounds 7j,
7f, 7o, 7h, and 7t exhibited antifungal activity against all
the strains tested. Overall compounds 7l, 7t, 7i, 7o, and
7r need to be screened further for other cancer cell lines
and in vivo study and toxicology. ese molecules can be
considered as lead molecules for novel cytotoxic and anti-
oxidant agent.
Table 2. Antioxidant activity of curcumin mimics.
Table 1. Synthesis of curcumin mimics.
% inhibition by DPPH
Xanthine oxidase
inhibition (%) 100μg/mL
Entry
1
R
Product
7a
7b
7c
Yielda (%)
88
MP
Compound
50μg/mL
7a
50.20 1.8
65.23 2.6
80.42 1.2
60.85 2.3
82.13 3.2
85.12 3.5
60.45 1.2
56.12 1.5
90.12 2.1
82.90 1.8
77.52 1.9
87.16 1.6
68.85 2.2
85.43 4.3
81.19 2.8
58.12 1.6
86.15 2.4
83.20 1.8
75.20 1.6
80.45 1.4
80.20 2.5
—
62.13 1.2
56.23 1.3
85.46 3.2
89.22 1.6
70.52 4.2
92.50 2.1
75.10 1.8
80.95 3.1
92.00 1.8
85.21 4.2
90.15 1.5
92.11 2.5
86.45 1.3
88.15 2.6
94.50 2.8
65.42 1.6
90.12 2.3
92.45 1.4
92.10 3.2
94.11 1.2
—
C6H5
89–91°C
7b
2
o-F C6H4
79
86–88°C
7c
3
m-Cl C6H4
m-F C6H4
m-CF3 C6H4
p-F C6H4
85
108–110°C
75–77°C
7d
4
7d
7e
91
7e
5
86
120–122°C
118–120°C
190–192°C
105–107°C
102–104°C
125–127°C
108–110°C
104–106°C
155–157°C
81–83°C
7f
6
7f
90
7g
7
m-Br C6H4
o-CF3 C6H4
m-CH3 C6H4
p-OCH3 C6H4
C4H4S
7g
76
7h
8
7h
7i
70
7i
9
84
7j
7k
10
11
12
13
14
15
16
17
18
19
20
7j
93
7l
7k
7l
72
7m
C4H4O
81
7n
p-C(CH3)3 C6H4
P-C2H2 C6H4
p-Cl C6H4
o-Cl C6H4
p-CH3 C6H4
C3H5
7m
7n
7o
7p
7q
7r
75
7o
81
7p
94
100–102°C
92–94°C
7q
83
7r
89
95–97°C
7s
69
75–77°C
7t
Troloxa
Allopurinola
m-CN C6H4
RCO=H
7s
65
113–115°C
216–218°C
7t
85
90.12 1.6
aIsolated yield.
aStandard; data represent mean of three replicates.
Journal of Enzyme Inhibition and Medicinal Chemistry

Antioxidant, cytotoxicity and antimicrobial activities of novel curcumin mimics 271
6-trimethoxybenzaldehyde 3 (1 mmol) was added and stir-
ring continued for 24h at room temperature. After comple-
tion of reaction (TLC), reaction mixture was poured over
crushed ice, extracted with chloroform, organic extract was
washed with water, dried (anhydrous Na2SO4) and con-
centrated. Purification was carried using silica gel column
using mixture of 0.5–1% methanol + 1% liquor ammonia in
chloroform as an eluent to obtain the title compounds.
Experimental
Chemistry
All the chemicals were purchased from Aldrich Chemical
Co. (Nanded, Maharashtra, India). Melting points were
determined with digital thermometer and were uncor-
rected. IR spectra were recorded on FT-IR Shimadzu 8300
spectrophotometer and 1H NMR spectra were recorded on
a Bruker 400MHz spectrometer in CDCl3 usingtetramethyl
silane as an internal standard. Mass spectra were obtained
with a Shimadzu LCMS-2010 EV. Chromatographic purifi-
cation was performed with silica gel (100–200 Mesh). in
layer chromatography was performed on pre-coated silica
plates (Merks kiesegel 60F254, 0.2mm thickness) sheet.
e spots could be visualized easily under UV light.
N-(3-((2E)-3-(1,2,3,6-tetrahydro-1-methylpyridin-4-yl)-2,4,6-
trimethoxyphenyl)acryloyl)phenyl)benzamide (7a):
IR (KBr) cm−1: 3470, 3170, 2986, 2852, 1649, 1617, 1590,
1
1570, 1510, 1420, 1020, 945, 844, 763. H NMR (CDCl3,
400MHz) δ: 10.50 (s, 1H, -NH), 8.48 (s, 1H), 8.08 (d, 1H, J =
8 Hz), 7.95 (m, 2H), 7.85 (d, 1H, J= 15.6 Hz), 7.72 (d, 1H, J =
7.6 Hz), 7.45 (m, 4H), 7.40 (m, 1H), 6.30 (s, 1H), 5.58 (1H),
3.98 (s, 3H), 3.86 (s, 3H), 3.77 (s, 3H), 3.12 (s, 2H), 2.64 (s,
2H), 2.42 (s, 3H), 2.35 (s, 2H). MS: m/z 513 [M+1].
Synthesis of 3-(1,2,3,6-tetrahydro-1-methylpyridin-4-yl)-2,4,5-
trimethoxybenzaldehyde (3)
Olefin 2(1.0g, 4 mmol) was dissolved in dry DMF (13.3mL)
under anhydrous condition. It was cooled to 0°C, POCl3
(7.2mL) was added drop wise for 30min and stirring con-
tinued for 2h at 25°C. After completion of reaction (TLC),
reaction mass was poured over crushed ice (50g) and bas-
ified with Na2CO3, extracted with chloroform, dried over
anhydrous Na2SO4, purifiedthroughsilicagelcolumnusing
0.5–1% methanol + 1% liquor ammonia in chloroform as
eluting solvent afforded product 3 (0.6g) in 51% yield.
2-fluoro-N-(3-((2E)-3-(3-(1,2,3,6-tetrahydro-1-methylpyridin-
4-yl)-2,4,6-trimethoxyphenyl)acryloyl)phenyl)benzamide(7b):
IR (KBr) cm−1: 3410, 3115, 2984, 1660, 1603, 1429, 1139,
1016, 982, 828, 760. 1H NMR (CDCl3, 400MHz) δ: 10.67 (s,
1H, -NH), 8.44 (s, 1H), 8.04 (m, 3H), 7.95 (d, 1H, J= 16 Hz),
7.79 (d, 1H, J= 7.08 Hz), 7.53 (m, 4H), 6.30 (s, 1H), 5.53 (s,
1H), 3.99 (s, 3H), 3.80 (s, 3H), 3.70 (s, 3H), 3.27 (s, 2H), 2.88
(s, 2H), 2.53 (s, 3H), 2.46 (s, 2H). MS: m/z 531 [M+1].
General procedure for the synthesis of curcumin mimics (7a-s)
Amides 6a–s (1 mmol) were dissolved in ethanol
(15mL), NaOH (20%) was added to it and stirred for
5min. 3-(1,2,3,6-tetrahydro-1-methylpyridine-4-yl)-2,4,
Biological activities
DPPH antioxidant assay
e DPPH radical scavenging activity of curcumin
mimics were measured according to the procedure
Table 3. Cytotoxicity profile of curcumin mimics against selected cancer cell lines.
Compound
Conc in μM
Panc
97
8
H460
99
43
25
61
60
22
77
71
7
Calu
97
0
HCT 116
97
32
22
69
67
9
ACHN
92
26
16
65
80
15
82
0
7a
10
10
7b
7c
10
12
58
78
0
38
55
82
18
54
0
7d
10
7e
10
7f
10
7g
10
61
25
20
23
98
91
4
80
84
15
12
98
79
2
7h
10
7i
10
0
35
29
97
72
8
7j
10
23
100
51
0
11
98
89
10
13
28
9
7k
10
7l
10
7m
10
7n
10
20
40
0
15
32
2
7
0
7o
10
18
10
6
39
9
7p
10
7q
10
12
37
62
42
68
78
0
21
0
15
18
55
14
73
84
7r
10
24
78
37
73
79
13
59
21
83
86
7s
10
52
29
82
87
7t
10
Flavopiridol
Gemcitabine
700 nM
500 nM
Panc1: pancreas, H460: non-small cell lung carcinoma, Calu-1: Lung, hct116: Colon cancer, ACHN: Renal.
© 2012 Informa UK, Ltd.

272 B. P. Bandgar et al.
described by Bandgar et al. with minor modification44. A
purple-coloured (DPPH˙) is a stable free radical, which is
reduced to DPPH (yellow coloured) by reacting with an
antioxidant. e reaction mixture consisted of 100 μM
DPPH in absolute ethanol with different concentrations
(0–1000 μM) of test compounds. A negative control with
the same DPPH concentration in ethanol without sample
was used. e mixture was shaken vigorously and allowed
to stand in the dark at room temperature for 10 min. e
decrease in absorbance of resulting solution was then
measured spectrophotometrically at 517 nm against
ethanol. All measurements were made in triplicate and
averaged. Trolox was used as standard.
Cytotoxicity assay
e selected cancer cell lines such as ACHN (human
renal carcinoma), Panc 1 (human pancreatic carcinoma),
Calu-1 (human non-small cell lung carcinoma), H460
(human non cell lung carcinoma) and HCT 116 (human
colon carcinoma) were used for evaluation of cytotoxic-
ity of test compounds. e propidium iodide florescence
assay was performed for the demonstration of cytotoxic
effect of test compound. Seed cells of 3000–7500 cells/
well of individual cell line were placed in 2000 μL of tissue
culture grade 96-well plates and allowed them to recover
for 24h in humidified 5% CO2 incubator at 37°C. After cul-
turing for 24h, individual test compounds (10 μM in 0.3%
DMSO) were added onto triplicate wells. DMSO (0.3%)
alone was used as control. After 48h in humidified 5% CO2
incubator at 37°C condition, the medium was removed
and treated with 25 μL of propidium iodide (50μg/mL in
water/medium) per well. e plates were frozen at −80°C
for 24h then thawed and allowed it to room temperature,
and the plate absorbance was read on fluorometer (polar-
star BMG Tech), using 530nm excitation and 620nm
emission wavelengths. Percent cytotoxicity of the test
compounds were calculated by using following formula.
Xanthine oxidase inhibition
Xanthine oxidase inhibitory activity was assayed spectro-
photometrically at 295nm under aerobic condition45. e
reaction mixture contained 200mM sodium pyrophos-
phate buffer (pH 7.5), 100 μm xanthine, and 0.4U of XO.
e absorption rate at 295nm indicate the formation of uric
acid at 25°C. Samples were dissolved directly in the buffer
and incorporated with enzyme assay to assess the inhibi-
tory activity. e experiments were carried out with three
sets of apparatus testing XO inhibitory activity. XO activity
was expressed as % inhibition of XO, calculated as % inhibi-
tion=(1-B/A)×100 where A is the change in absorbance of
the assay without the curcumin mimics and B is the change
in absorbance of the assay with curcumin mimics. e
enzyme kinetics was similar to XO assay method.
Cytotoxicity (%) = 1−T/C×100
T = OD of treated cells
C = OD of control
Flavopiridol (700 nM) and Gemcitabine (500 nM) were
used as standard anticancer drugs for comparison46.
Table 4. Antimicrobial activity of curcumin mimics (zone of inhibition in mm).
Compound
Bacteria 50μg/mL ST PV PA SA BS
Fungi 100μg/mL PC TV CA MC FM
7a
ND
12
20
10
20
18
ND
24
22
20
20
22
12
19
22
14
18
20
14
22
18
—
ND
ND
22
10
15
ND
10
22
12
22
22
12
20
22
22
21
24
ND
20
22
12
20
22
2
10
15
20
ND
20
20
10
24
25
24
25
20
15
22
22
10
20
20
10
23
20
—
12
15
17
12
17
19
ND
18
17
20
18
17
ND
17
20
12
17
17
12
18
—
16
ND
ND
18
ND
ND
20
ND
ND
22
ND
13
7b
7c
20
20
7d
ND
22
ND
20
ND
18
ND
20
ND
22
ND
20
7e
7f
22
21
19
20
20
20
7g
ND
22
ND
24
10
ND
20
ND
20
ND
20
7h
18
7i
22
22
18
19
20
18
7j
22
20
22
20
20
20
7k
19
22
20
18
18
18
7l
22
24
18
19
19
20
7m
ND
19
ND
20
ND
18
ND
18
ND
18
ND
18
7n
7o
24
20
22
20
18
18
7p
ND
20
ND
20
10
ND
20
ND
21
ND
19
7q
20
7r
22
20
18
20
22
20
7s
ND
24
ND
24
ND
19
ND
20
ND
22
12
7t
21
20
—
20
Tetracyclin
Nystatin
20
20
—
—
—
—
—
—
17
18
20
20
Data represent mean of three replicates.
ND, not determined; ST, Salmonella typhi; PV, Proteus vulgaris; PA, Pseudomonas aeruginosa; SA, Staphylococcus aureus; BS, Bacillus
subtilis; PC, Penicillium chrysogenum; TV, Trichoderma viridae; CA, Candida albicans; MC, Microsporum cannis; FM, Fusarium
moniliformae.
Journal of Enzyme Inhibition and Medicinal Chemistry

Antioxidant, cytotoxicity and antimicrobial activities of novel curcumin mimics 273
5. Schiavone JR, Hassan HM. e role of redox in the regulation of
manganese-containing superoxide dismutase biosynthesis in
Antimicrobial activity
Antimicrobial activities of all the synthesized com-
pounds were determined by agar diffusion method35. All
the bacteria and fungal species used were procured from
Institute of Microbial Type Culture Collection (IMTCC),
Chandigarh, India, and National Collection of Industrial
Microorganisms (NCIM), Pune, India, namely, P. vul-
garis, P. aeruginosa, S. aureus, B. subtilis, P. chrysogenum,
T. viridae, C. albicans, M. cannis and F. moniliformae. All
the synthesized compounds were dissolved to prepare
a stock solution of 1 mg/mL using DMSO. Stock solu-
tion was aseptically transferred and suitably diluted to
have solutions of concentration ranging 50–100 μg. For
antifungal activity, different fungal spore suspension in
sterile distilled water was adjusted to give a final concen-
tration of 106 cfu/mL. Inoculum of 0.1 mL spore suspen-
sion of each fungus was spread on Sabouraud’s Dextrose
agar plates (HiMedia). For antibacterial activity, Muller
Hinton agar was used (HiMedia) seeded with 0.1mL
of respective bacterial strains suspension prepared in
sterile saline (0.85%) of 105 cfu/mL dilution. e wells
of 6 mm diameter were filled with 0.1 mL each test com-
pound separately for each fungi and bacterial strain. e
DMSO alone was used as control. e antibiotics tetracy-
cline (10 μg/mL) and nystatin (30 μg/mL) were used as
reference for antibacterial and antifungal, respectively.
Inoculated plates in duplicate were then incubated
at 37 0.5°C for antibacterial activity for 24 h and at
28 0.2°C for antifungal activity for 48 h. After incubation,
the antimicrobial activity was measured in terms of the
zone of inhibition in mm.
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HVC is thankful to the Council of Scientific and Industrial
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as to Mahesh Nambiar and Mrs Asha Almeida Piramal
Life Sciences Ltd., Mumbai, for anticancer activity.
hypoxia-reoxygenation injury. Am
2002;282:C227–C241.
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Declaration of interest
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