Synthesis and biological evaluation of nitrogen-containing chalcones as possible anti-inflammatory and antioxidant agents

Article abstract of DOI:10.1016/j.bmcl.2009.11.068

A novel series of nitrogen-containing chalcones were synthesized by Mannich reaction and were screened for anti-inflammatory related activities such as inhibition of cyclooxygenase-2 (COX-2), trypsin and β-glucuronidase. The antioxidant potential was demonstrated using 1,1-diphenyl-2-picryl hydrazine (DPPH) radical scavenging activity. The results of the above studies shows that the compounds synthesized were found to be effective inhibitors of above pro-inflammatory enzymes, and were found to be possess moderate radical scavenging potential. Overall, the results of the studies reveal that the chalcones with N-methyl piperazine methyl and piperidine methyl substitution (4c, 3b, 4d, 6b) seems to be important for inhibition of β-glucuronidase. Whereas the chalcones with piperidine methyl substitution (8b, 7b, 7c, 6c, 4b, 3c, 3b) were observed as effective inhibitors of COX-2, while the same compounds were found to be less reactive against COX-1 as compared to COX-2.

Full text of DOI:10.1016/j.bmcl.2009.11.068

Bioorganic & Medicinal Chemistry Letters 20 (2010) 730–733
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of nitrogen-containing chalcones
as possible anti-inflammatory and antioxidant agents
b
c
b
b
Babasaheb P. Bandgar a,b, , Sachin A. Patil , Rajesh N. Gacche , Balaji L. Korbad , Balwant S. Hote ,
*
Santosh N. Kinkar b, Shivkumar S. Jalde b
a Organic Chemistry Research Laboratory, School of Chemical Sciences, Solapur University, Solapur 413 255, India
b Organic Chemistry Research Laboratory, School of Chemical Sciences, Swami Ramanand Teerth Marathwada University, Nanded 431 606, India
c School of Life Sciences, Swami Ramanand Teerth Marathwada University, Nanded 431 606, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 17 June 2009
Revised 14 October 2009
Accepted 13 November 2009
Available online 11 December 2009
A novel series of nitrogen-containing chalcones were synthesized by Mannich reaction and were
screened for anti-inflammatory related activities such as inhibition of cyclooxygenase-2 (COX-2), trypsin
and b-glucuronidase. The antioxidant potential was demonstrated using 1,1-diphenyl-2-picryl hydrazine
(DPPH) radical scavenging activity. The results of the above studies shows that the compounds synthe-
sized were found to be effective inhibitors of above pro-inflammatory enzymes, and were found to be
possess moderate radical scavenging potential. Overall, the results of the studies reveal that the chal-
cones with N-methyl piperazine methyl and piperidine methyl substitution (4c, 3b, 4d, 6b) seems to
be important for inhibition of b-glucuronidase. Whereas the chalcones with piperidine methyl substitu-
tion (8b, 7b, 7c, 6c, 4b, 3c, 3b) were observed as effective inhibitors of COX-2, while the same compounds
were found to be less reactive against COX-1 as compared to COX-2.
Keywords:
Nitrogen-containing chalcones
Anti-inflammatory
Antioxidant activity
Ó 2009 Elsevier Ltd. All rights reserved.
Inflammation is a general name for reactions occurring in sev-
eral types of tissue injuries, infections, or immunologic stimulation
as a defense against foreign or altered endogenous substances. The
process of inflammation comprises of a series of changes of the ter-
minal tissues, which tend to eliminate the injurious agents and to
repair the damage tissue.1 In the treatment of rheumatic diseases,
two types of drugs, that is, non-steroidal anti-inflammatory drugs
(NSAIDs)2 and disease-modifying anti-rheumatic drugs (DMARDs)
are prescribed as anti-inflammatory agents.3 NSAIDs, the most
effective and widely used anti-inflammatory drugs are reputed
for inhibition of cyclooxygenase (COX-1 and 2) enzymes (a key
enzyme involved in recruiting inflammation). Worldwide in the
area of anti-inflammatory research there is a trend of searching
alternatives to NSAIDs. Although the treatment by NSAIDs is more
effective and dramatically ameliorates the inflammations, still
there is diversity in patient’s response towards NSAIDs. Moreover,
these drugs are suffering from many long lasting side effects espe-
cially the gastrointestinal ulcer.4–9 Therefore, to overcome the
problem of inflammation an alternatives to NSAIDs, new targets
are being identified and explored for the design and development
of new anti-inflammatory agents.10 As a part of this trend we at-
tempted the synthesis of novel N-alkylated chalcones as anti-
inflammatory and antioxidant agents.
Chalcones a group of naturally occurring compounds belongs to
a flavonoid family and are present in variety of plant species such
as fruits, vegetables, spices, tea, and soy based foodstuff. Chalcones
have been recently focused as a pharmacologically significant
group for their interesting biological activities. Chalcones isolated
from natural products are known to possess several important
activities including anti-fungal,11 leishmanicidal,12 and anti-malar-
ial.13 Recent reports indicates the importance of chalcones as anti-
inflammatory agents involved in inhibition of cell migration and
inhibition of TNF-
a
synthesis in mouse.2 Plethora of literature is
available describing the role of chalcones and related derivatives
such as anti-cancer,14 anti-inflammatory,15 antimitotic,16 anti-
tubercular,17 cardiovascular,18 and hyperglycemic agents.19
Compounds described in this study were prepared using a
straight forward chemistry (Scheme 1). Xanthoxyline 1 was syn-
thesized following the method described in the literature with
minor modifications.20 The starting compounds (chalcones) were
synthesized by Claisen–Schmidt condensation between substi-
tuted 2-hydroxy-4,6-dimethoxyacetophenone and various benzal-
dehydes in alkaline medium, according to previously described
method.21 Condensation of chalcones with the various secondary
amines and formaldehyde in the presence of hydrochloric acid in
isopropanol furnished desired derivatives.22
The single crystal X-ray analysis of compound 7a was reported
previously by Liu et al.23 The CCDC deposition number of
compound 7a is 287623.24 It was confirmed that the synthesized
* Corresponding author. Tel./fax: +91 217 2351300.
E-mail address: bandgar_bp@yahoo.com (B.P. Bandgar).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.11.068

B. P. Bandgar et al. / Bioorg. Med. Chem. Lett. 20 (2010) 730–733
731
O
O
O
O
O
a
b
R
O
OH
O
OH
O
O
1
2
c
O
O
R
O
OH
R1
3a-d
Where R = 3-Cl, 3-Br, 3-F, 2-F, 2-Cl, 2-Br.
R1
=
O
(d)
N
(b)
(a)
(c)
N
N
N
N
Scheme 1. Reagents and conditions: (a) AlCl3, CH3COCl, dry ether, 0 °C, 48 h rt; (b) benzaldehydes, NaOH, EtOH, H2O, rt, 88–91%; (c) secondary amines, 37% HCHO,
isopropanol, HCl, reflux, 51–65%.
chalcones of trans-configurations, which also proved by the data of
1H NMR spectra (J = 16 Hz).25
The synthesized nitrogen containing compounds were assayed
for their biological activities such as antioxidant, pro-inflammatory
(trypsin and b-glucuronidase), and COX-2 studies.26
curonidase. This enzyme is attributed as one of the mediators for ini-
tiating the process of inflammation. The enzyme inhibition assay
was performed as per the method described.33 The results of the test
compounds shows that with few exceptions (6c, 6d) the overall b-
glucuronidase inhibition profile of all the samples was found to be
significant. Whereas the sample 4c > 3b > 4d > 6b > 7a > 8c > 8b
> 7c showed significant inhibition of b-glucuronidase. N-Methyl-
morpholine methyl, piperidine methyl and pyrrolidine methyl sub-
stitution at 30 of chalcone (4c, 3b, 4d, 6b) were observed to be
important for inhibition of b-glucuronidase. However, the same can-
not be treated as the only responsible factor as the same substitution
at 20 of chalcone has also shown moderate activity against b-
glucuronidase.
‘Cyclooxygenase-2 enzyme inhibition: as a target for design of
anti-inflammatory agents’ has remained a critical issue in the
mainstream of anti-inflammatory pharmaceutical research. More-
over, majority of the traditional NSAIDs have been designed as
selective inhibitors of COX-2.2 The COX-2 inhibition assay was
performed as per the assay protocol instructions of ‘Colorimetric
COX (ovine) inhibitor Screening Assay Kit’, Cayman Chemical Com-
pany, MI, USA. All the samples under study were found to be good
to excellent inhibitors of COX-2. Interestingly majority of the sam-
ples showed COX-2 inhibition more effective than aspirin (a known
COX-2) inhibitor. The overall range of effective COX-2 inhibition
was observed to be 7b > 3b > 4b > 3c > 8b > 7c, while all other sam-
ples showed moderate to good to COX-2 inhibition. The com-
pounds showing promising COX-2 activity were also evaluated
for inhibition of COX-1: a housing keeping enzyme known for its
gastro-protective effects. The results summarized in Table 2,
clearly show that the compounds showing effective COX-2 inhibi-
tion are less reactive towards COX-1. The presence of piperidine
methyl and N-methylmorpholine methyl substitution at 20 and 30
of chalcones were found to be more favorable for COX-2 inhibition.
However, it is indeed difficult to underlay a common structure–
activity relationship with other samples which showed moderate
to good inhibition of COX-2.
The series of N-alkylated chalcones were evaluated as possible
anti-inflammatory and antioxidant agents by performing various
tests and assays. The results are compared with (E)-1-(2-hydroxy-
4,6-dimethoxyphenyl)-3-phenylprop-2-eb-1-one (NC): a analogue
of an active natural chalcone.27 The DPPH radical scavenging assay
was performed in order to determine the antioxidant potential.28
DPPH is a stable nitrogen-centered free radical. Its reaction rates cor-
relatedirectlywithantioxidantactivity, thehighertherate, themore
effective the antioxidant.29 The summary of reduction of DPPH (%)
has been shown in Table 1. The overall trend of DPPH reduction
wasobservedtobe4c > 4d > 6c > 4b > 6d, allothersampleswereob-
served as moderate radical stabilizers. As a part of structure–activity
relationship, the presence of electronegative environment (–OCH3
or –OH) on A-ring may be the electron donors for DPPH reduction.
However, with few exceptions it was noted that the N-methyl piper-
azine group substitution at 3 positions were found to be more suit-
able for reduction of DPPH.
As a part of evaluating anti-inflammatory related activities we
have carried out trypsin inhibition assay.30,31 Trypsin is a member
of the serine proteases family. These proteases are involved in initia-
tion of inflammation; moreover serine protease inhibition has been
considered as one of the targets for design of anti-inflammatory
drugs.32 With an exception of 5a, none of the sample was observed
to be significant in case of trypsin inhibition. The presence of fluoro
substitutionat3-positionofchalcone(5a) seems to betheonlyimpor-
tantsubstitutionmoreappreciablefortrypsininhibitionasthesimilar
group substitution at 2-position (6a) failed to inhibit the trypsin.
The enzyme b-glucuronidase has been considered as one of the
target in the design of anti-inflammatory agents as it plays pro-
inflammatory role in the initiation of inflammation reaction. The
lysosomes of the polymorphonuclear neutrophils are rich in b-glu-

732
B. P. Bandgar et al. / Bioorg. Med. Chem. Lett. 20 (2010) 730–733
Table 1
Profile of DPPH radical scavenging activity (%), inhibition of trypsin, b-glucuronidase and COX-2 activity (%) of N-alkylated chalcones at concentration* (1 mM)
Compound
R
R1
DPPH reduction (%)
21.87
Trypsin inhibition (%)
1.1
b-Glucuronidase inhibition (%)
COX-2 inhibition (%)
48.25
3a
3-Cl
NH
NH
NH
NH
13.87
O
3b
3c
3d
3-Cl
3-Cl
3-Cl
20.90
5.20
3.10
0.5
1.3
0.9
66.03
23.72
14.82
87.92
77.78
29.58
N
4a
4b
4c
3-Br
3-Br
3-Br
O
10.11
33.00
37.00
1.2
2.8
1.4
29.28
15.39
75.43
22.39
87.45
33.33
NH
NH
NH
NH
NH
N
4d
3-Br
35.67
1.3
59.81
19.87
5a
5b
3-F
3-F
24.00
14.6
19.50
2.4
10.82
7.06
38.91
21.67
O
NH
5c
3-F
18.4
NR
26.71
24.58
N
O
NH
NH
NH
NH
5d
6a
6b
3-F
2-F
2-F
11.72
19.2
NR
1.3
2.0
1.4
19.45
21.8
21.38
27.11
19.89
56.63
6c
2-F
35.30
NR
NR
44.89
N
O
NH
NH
NH
NH
6d
7a
7b
2-F
32.87
24.8
18.4
1.2
NR
0.7
NR
21.45
25.64
91.45
2-Cl
2-Cl
48.08
31.2
7c
2-Cl
2-Cl
10.8
7.89
0.1
0.8
35.48
32.42
68.84
42.52
N
NH
NH
7d
8a
8b
8c
2-Br
2-Br
2-Br
16.67
58.34
27.78
1.6
2.1
1.2
32.84
39.92
42.00
42.34
69.78
48.92
O
N
NH
NH
NH
NH
8d
2-Br
25.00
1.5
34.70
20.44
NC
QR
SA
16.67
86.30
ND
0.8
ND
89.24
ND
28.42
ND
23.30
ND
66.96
ND
ND
—
—
—
—
—
—
ASA
ND
35.11
The results summarized are the mean values of n = 2, QR = quercetin, SA = salicylic acid, ASA = acetyl salicylic acid, NC = natural chalcone, NR = no reaction, ND = not
determined, *1 mM is the stock concentration, from which only 10 lM was pipetted. Therefore the actual concentration in the reaction mixture is in lM only.
In summary, a novel series of nitrogen-containing chalcones
were synthesized, of the tested samples (4c, 3b, 4d, 6b) have
shown significant activity against trypsin and b-glucuronidase:
the pro-inflammatory enzymes. While the samples (8b, 7b, 7c,
6c, 4b, 3c, 3b) were observed to be excellent inhibitors of COX-2
and comparatively poor inhibitors of COX-1. The compounds hav-
ing selective COX-2 inhibition but less reactive towards COX-1 are
appreciated as novel anti-inflammatory agents in the mainstream
of anti-inflammatory research.34 The antioxidant potential of all
the compounds is not appreciable. The present investigations show
that the majority of the synthesized molecules can be considered
as lead molecules for optimization and explains them as a novel
and effective anti-inflammatory agents. Further studies in relation
Table 2
COX-1 inhibitory activity of selected compounds showing promising COX-2 inhibitory
activity at concentration 1 mM
Compounds
COX-2
COX-1
7b
7c
6c
4b
3c
3b
ASA
SC560
91.45
68.84
44.89
87.45
77.78
87.92
35.11
—
30.4
11.9
19.2
21.7
28.7
25.2
—
32.9
ASA: acetyl salicylic acid; SC 560: selective COX-1 inhibitor.

B. P. Bandgar et al. / Bioorg. Med. Chem. Lett. 20 (2010) 730–733
733
NMR (300 MHz, CDCl3, d): 7.72 (d, 1H, J = 16 Hz), 7.62 (s, 2H), 7.52–7.47 (d, 1H,
J = 16 Hz), 7.31 (t, 2H), 6.01 (s, 1H), 3.97 (s, 3H, OCH3), 3.93 (s, 3H, OCH3), 3.77
(br s, 6H), 2.46 (m, 4H, –CH2–CH2–), 1.45 (m, 2H, –CH2–); MS: m/z 416 [M+1]+.
Compound 3c: Yellow solid, mp 109–112 °C: IR 3742, 3050, 2926, 2795, 1620,
to toxicity and in vivo anti-inflammatory activities are needed for
better understanding.
1268, 1023, 793 cmꢀ1 1H NMR (300 MHz, CDCl3, d): 7.78 (d, 1H, J = 16 Hz),
;
Acknowledgements
7.60 (d, 1H, J = 16 Hz), 7.56 (dd, 1H, J = 2 Hz, 2 Hz), 7.34 (m, 1H), 7.17 (dd, 1H,
J = 2 Hz, 2 Hz), 7.13 (m, 1H), 6.00 (s, 1H), 3.90 (s, 3H, OCH3), 3.88 (s, 3H, OCH3),
3.70 (s, 2H, CH2), 2.62 (br s, 4H), 2.46 (br s, 4H), 2.27 (s, 3H); MS: m/z 431
[M+1]+.
The authors are thankful and acknowledge the financial support
by Council of Scientific and Industrial Research [CSIR, Scheme No.
01(2023)/05/EMR-II], New Delhi. S.A.P. thanks CSIR, New Delhi
for Senior Research fellowship.
26. DPPH radical scavenging assay: The DPPH radical scavenging assay was
performed as described by Bartolome (2004). The reaction mixture contained
1 mM concentrations of individual test sample (in absolute ethanol) and DPPH
radical (10ꢀ4 M in absolute ethanol) solution. The contents of the reaction
mixture were observed spectrophotometrically at 517 nm after 20 min.
Quercetin was used a reference drug (86.30%).
References and notes
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individual concentrations of test sample of 0.1 mL for 20 min. The substrate
BSA (6 g/100 mL, in 0.1 M phosphate buffer, pH 7.6) was added after 20 min.
The reaction mixture was incubated for 25 min at 37 °C. The reaction was
terminated by the addition 3 mL of CCl3COOH (5%, w/v). The acid soluble
fractions were obtained by centrifuging the contents at 5000 RPM for 15 min.
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of (Lowry 1951). Salicylic acid (10ꢀ6 M) was used as a reference drug (89.24%).
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glucuronidase was studied using a method described by Demetrios (1998). One
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D
-glucopyranosiduronic acid and 0.1 mL of b-glucuronidase was added. The
mixture was incubated for 30 min. Reaction was terminated by addition of
2 mL of 0.5 N NaOH. The reaction mixtures were observed spectro-
photometrically at 410 nm. Salicylic acid (1 mM) was used as a reference
compound (23.30%).
COX-2 inhibition micro-titer assay: The reaction mixture of 100% initial activity
wells contained 160
enzyme solution. While the reaction mixture of inhibitor wells was comprised
of 150 L of assay buffer, 10 L of heme, and 10 L of enzyme COX-2, 10 L of
the test samples (1 mM). The plates were carefully shaken for 5 s and were
incubated for 5 min at 25 °C. After 5 min incubation 20 L of the colorimetric
substrate solution was added to all the wells, followed by the addition of 20
lL of assay buffer, 150 lL of heme and 10 lL of COX-2
l
l
l
l
l
lL
of arachidonic acid to all the wells. The plates were shaken gently for few
seconds and again incubated for 5 min at 25 °C. The absorbance of all the wells
was read at 590 nm using Thermo make Automatic Ex-Microplate Reader (M
51118170). The COX-2 inhibition activity (%) was calculated using following
formula
T
C
COX-2 inhibition activity ð%Þ ¼ ꢁ 100
where T = absorbance of the inhibitor well at 590 nm, C = absorbance of the
100% initial activity without inhibitor well at 590 nm.
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25. Compound 3a: Yellow solid, mp 118–121 °C: IR 3742, 3060, 2930, 2845, 1623,
1375, 1271, 1043, 754 cmꢀ1
;
1H NMR (300 MHz, CDCl3, d): 7.60 (s, 2H), 7.56 (s,
1H), 7.46–7.43 (m, 1H), 7.35 (d, 1H, J = 16 Hz), 7.27 (d, 1H, J = 16 Hz), 6.02 (s,
1H), 3.94 (s, 3H, OCH3), 3.90 (s, OCH3), 3.71 (t, 4H, CH2–O–CH2), 3.66 (s, 2H),
2.58–2.54 (t, 4H, CH2–N–CH2); MS: m/z 418 [M+1]+. Compound 3b: Yellow
34. Anna, L. B.; Lawrence, J. M. J. Med. Chem. 2007, 50, 1425.
solid, mp 132–134 °C: IR 3555, 3010, 2976, 2847, 1622, 1217, 759 cmꢀ1 1H
;