MEDICINAL CHEMISTRY RESEARCH
Med Chem Res (2012) 21:2292–2299 DOI 10.1007/s00044-011-9746-6
ORIGINAL RESEARCH
Synthesis and biological evaluation of novel series of chalcone derivatives as inhibitors of cyclooxygenase and LPS-induced TNF-a with potent antioxidant properties Babasaheb P. Bandgar • Baliram S. Hote Nagesh A. Dhole • Rajesh N. Gacche
•
Received: 26 January 2011 / Accepted: 8 July 2011 / Published online: 28 July 2011 Ó Springer Science+Business Media, LLC 2011
Abstract Novel series of chalcones were synthesized and were evaluated as possible anti-inflammatory agents targeting the cyclooxygenase-1 and 2 (COX-1 and 2), b-glucuronidase, trypsin, and TNF-a. Amongst the tested chalcones the compound 4k was found to be most effective inhibitor of TNF-a exhibiting 85% inhibition activity (IC50 = 0.1 lM). The compounds 4a, 4f, 4l, and 4m were found to inhibit the COX-1 activity in as a range of 79.95–68.47% and COX-2 inhibition ranging 84.45–74.77%. The compounds 4l (81.71%) and 4f (72.10%) were found to be excellent inhibitors of trypsin and b-glucuronidase, respectively. Keywords WBCs TNF-a COX-1 COX-2 Antioxidant Enzyme inhibition
Introduction Chalcone is one of the major classes of natural product with wide spread distribution in fruit, vegetables, spices, tea, and soya-based foodstuff and it has been recently subjects of great interest for their interesting pharmacological activities (Di Carlo et al., 1999). Chalcones, or 1,3-
B. P. Bandgar (&) Medicinal Chemistry Research Laboratory, School of Chemical Sciences, Solapur University, Solapur, Maharashtra, India e-mail:
[email protected] B. P. Bandgar B. S. Hote Organic Chemistry Research Laboratory, School of Chemical Sciences, SRTM University, Nanded, Maharashtra, India N. A. Dhole R. N. Gacche Biochemistry Research Laboratory, School of Life Sciences, SRTM University, Nanded, Maharashtra, India
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diaryl-2-propen-1-ones, belong to the flavonoid family. Chemically they consist of open chain flavonoids wherein the two aromatic rings are joined by a three-carbon a, b-unsaturated carbonyl system. Varieties of naturally occurring chalcones are polyhydroxylated in the aryl rings. The radical quenching properties of the phenolic groups present in many chalcones have raised interest in using the compounds or chalcones rich plant extract as drugs or food preservatives (Dhar, 1981). A vast body of literature has accumulated in the recent past linking the importance of chalcones as anti-inflammatory agents involved in inhibition of cell migration and inhibition of TNF-a synthesis in mouse (Herencia et al., 2001). Many citations describe the diverse biological activities of chalcones and related derivatives as anti-cancer (Xia et al., 2000; Bois et al., 1998), anti-inflammatory (Hsieh et al., 1998, 2000; Herencia et al.,1998), antimitotic (Ducki et al., 1998), antitubercular (Linn et al., 2002), cardiovascular (Furman et al., 2001), and hyperglycemic agents (Satyanarayana et al., 2004). Cyclooxygenase (COX) is the rate-limiting enzyme of the prostanoid biosynthetic pathway and it catalyzes the conversion of arachidonic acid to important inflammatory mediators such as prostaglandins (PGs), prostacyclins, and thromboxanes (Vane and Botting, 1996). The existence of enzyme cyclooxygenase in its two distinct isoforms and thus nonselective action of classical non-steroidal antiinflammatory drugs (NSAIDs) results in certain mechanism-based side effects including dyspepsia, gastrointestinal ulcerations, bleeding, and nephrotoxicity (Pairet and Engelhardt, 1996). Both the isoforms differ in their regulation and expression. The constitutive COX-1 is responsible for the biosynthesis of PGs, which involves the cytoprotection of gastrointestinal tract and platelet aggregation (Kujubu et al., 1991). COX-2 is induced by pro-
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inflammatory molecules such as interleukin-1 (IL-1), tumor necrosis factor-a (TNF-a), lipopolysaccharide (LPS), carrageenan, etc. that leads to inflammation (Marnett, 2000). COX-2 levels are undetectable in most tissues under normal physiological conditions, but are significantly up regulated in acute and chronic inflammations. Inhibition of both isoforms by classical NSAIDs with preferential binding affinity for enzyme COX-1 causes serious side effects. The association of COX-2 with induced inflammation has led to the hypothesis that selective inhibition of COX-2 over COX-1 might provide good anti-inflammatory agents with reduced side effects than classical NSAIDs. Searching the alternatives to currently used NSAIDs has remained a major thrust area of the anti-inflammatory research. Tumor necrosis factor-a, an important cytokine produced by activated monocytes/macrophages, has been identified as an endotoxin-induced serum factor that causes hemorrhagic necrosis of transplanted solid tumors (Miyachi et al., 1996). TNF-a plays a critical role in several physiological immune systems, and can cause severe damage when it is produced in excess. Therefore, TNF-a can be regarded as possessing both favorable and unfavorable effects. The favorable effects include direct tumor killing action, stimulation of the host’s immune system, and acting as a growth factor for normal B cells. The unfavorable effects include induction of tissue inflammation, a tumor-promoting action, stimulation of human immunodeficiency virus (HIV) replication, and induction of insulin resistance. These pleiotropic effects associated with TNF-a clearly demonstrate that the cytokine production enhancers and inhibitors could be of use as biological response modifiers under various circumstances (Shibata et al., 1994; Miyachi et al., 1997). Trypsin is a member of the serine proteases family. These proteases are involved in initiation of inflammation; moreover, serine protease inhibition has been considered as one of the targets for design of anti-inflammatory drugs (Bilfinger and George, 2002). 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-glucuronidase. This enzyme is attributed as one of the mediators for initiating the process of inflammation. The enzyme inhibition assay was performed as per the method described (Nicolaides et al., 1998). According to recent studies, reactive oxygen species (ROS) are important mediators that initiate and propagate inflammatory responses by stimulating release of proinflammatory cytokines such as interleukin-1b (IL-1b) and tumor necrosis factor-a (Geronikaki and Gavalas, 2006). It was also found that ROS induced by activated neutrophils, eosinophils, monocytes, and macrophages during the
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inflammation process leads to tissue injury by damaging macromolecules and effecting the lipid peroxidation of membranes (Gutteridge, 1995). This indicates that free radical scavengers might be a useful means of attenuating inflammatory effects. In this study, series of novel chalcones were synthesized and evaluated as possible inhibitors of cyclooxygenase, LPS-stimulated TNF-a and pro-inflammatory enzymes such as trypsin and b-glucuronidase (Bandgar et al., 2010). Free radical scavenging assays were also performed so as determine the antioxidant potential.
Result and discussion The novel chalcone derivatives (4a–p) were synthesized (Scheme 1). In this study, 1-(2-hydroxyl-4,6-dimethoxyphenyl)ethanone (2) was prepared by reacting commercially available 1,3,5-trimethoxy benzene (1) with acetyl chloride in dry ether using anhydrous AlCl3 (Ahluwalia, 2005). The 1-(2-hydroxyl-4,6-dimethoxyphenyl)ethanone (2) on bromination with Br2 in glacial acetic acid with constant stirring at 20°C for 1 h obtained 1-(3-bromo-2-hydroxyl-4,6-dimethoxyphenyl)ethanone (3) (Cechinel-Filho et al., 1996). Finally the different substituted benzaldehydes treated with 1-(3-bromo-2-hydroxyl-4,6-dimethoxyphenyl)ethanone (3) by Claisen–Schmidt condensation (Cechinel-Filho et al., 1996). The desired derivatives were obtained in 46–83% yield after purification. All synthesized compounds were characterized by using IR, 1H NMR, mass spectra, and elemental analysis. Series of chalcone derivatives synthesized (Table 1) were biologically evaluated as antioxidant and possible inhibitors of pro-inflammatory cytokine TNF-a and enzymes such as COX, trypsin, and b-glucuronidase. The results of the TNF-a inhibition are shown in (Table 2). It was observed that the compounds 4k (85.0%), 4o (78.0%), 4g (72.0%), and 4d (63.0%) were found to inhibit the LPSinduced release of TNF-a in WBCs significantly as compared to other compounds which showed the TNF-a inhibition in a range of (37–57%) at 10 lM. Nevertheless, the minimum IC50 values were calculated for 4k (0.1 lM) and 4o (0.6 lM). From the results, it can be stated that the chalcones (4k and 4o) containing heterocyclic B-rings seems to be an important configuration for the demonstration of effective TNF-a inhibition. Moreover, the chalcones containing electron withdrawing substituents (4e, 4d, and 4i) were also observed to be effective inhibitors of TNF-a. The results of the COX-1 inhibition summarized in (Table 3) clearly indicated that the compounds 4a (79.95%), 4f (71.39%), 4l (69.47%), and 4b (62.61%) were effective, however, the compounds 4a (84.45%), 4f
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O
O
O
O
O
O
b
a
H
+ O
O
O
OH
O
R
OH Br
2
1
3 c
4 a = 4 - OMe
4 i = 4 - Cl
4 b = 2, 5 - (OMe)2
4 j = 3 - CF3
4 c = 2, 4 - (OMe)2
4 k = 3 - Pyridine
4d=3-F
4l=2-F
4 e = 3 - CN
4 m = 3 - NO2
4 f = 3 - Br
4 n = 3, 5 - (OMe)2
4 g = 2 - Cl
4 o = 2 - Pyrrole
4 h = 2, 4 - Cl2
4 p = 4 - Cl, 3 - NO2
O
O
R O
OH Br
4a-p
Scheme 1 Synthesis of novel series of chalcones
(78.41%), 4l (74.77%), and 4b (68.19%) were found to be most active COX-2 inhibitors. It is interesting to note that all the tested compounds are more selective toward COX-2 as compared to COX-1. The results obtained were compared with standard COX-1 and 2 inhibitors such as aspirin (34.90%) and SC 560 (38.54%). The results of the trypsin and b-glucuronidase inhibition are summarized in (Table 3). The compounds 4l (81.71%), 4j (71.84%), and 4i (67.81%) were observed to be effective inhibitors of trypsin, while all other compounds demonstrated the trypsin inhibition in a range of (33.67–65.92%) as compared to salicylic acid (86.64%). It was found that the compounds 4f (72.10%), 4l (68.46%), and 4j (62.11%) inhibited b-glucuronidase significantly as compared to other compounds having inhibition activity in a range of (38.45–61.32%). The results of the enzyme inhibition studies shows that the majority of compounds possessing electron withdrawing substituted chalcones (4f, 4l, and 4m) showed considerable pro-inflammatory enzyme inhibition activity. However, 4a was exceptionally found to be more active as compared to other compounds. The results of the free radical scavenging activities are depicted in (Table 4). With few exceptions, all the synthesized chalcones exhibited moderate to good free radical scavenging and reducing activities. Amongst the tested chalcones, the compound 4a was found to possess considerable antioxidant potential which demonstrated (38.37%), (49.41%), and (34.32%) DPPH, OH radical
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scavenging, and reducing activity, respectively. All other compounds were found to possess moderate to good antioxidant activities. The free radicals are closely implicated in the initiation and progression of inflammatory disorders. It has been also reported that the compounds possessing significant reducing abilities can be considered as a potential candidate for the design and development of novel COX inhibitors (Gacche et al., 2008).
Experimental General methods Chemicals were purchased from Aldrich Chemical Co., USA. Melting points were determined with a digital thermometer were uncorrected. IR spectra were recorded on FT–IR Shimadzu 8300 spectrophotometer and 1H NMR spectra were recorded on a Bruker 300 MHz spectrometer in CDCl3 using tetramethylsilane as an internal standard and chemical shifts are reported in d units and the coupling constants (J) are reported in hertz. Mass spectra were obtained with a Shimadzu LCMS-2010 EV. Chromatographic purification was performed with silica gel (100–200 mesh). Thin layer chromatography (TLC) was performed on pre-coated silica plates (Merck Kiesegel 60F254, 0.2 mm thickness) sheets. The spots could be visualized easily under ultraviolet light.
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Table 1 Analytical data of chalcone derivatives
Table 2 Profile of the inhibition of LPS-induced TNP-a production by selected chalcones
O
Entry
1'
1
B
A 3
5
3'
5'
4
4'
Entry A ring 1 4a 4b 4c
B ring 2 3
4
5
10 20
30
OMe H OMe Br OH H H H OMe H OMe Br OH H OMe H OMe H OMe Br OH H OMe H
40
50
Yield (%)
OMe H 40 H OMe 38 OMe H
47
4d
OMe H OMe Br OH H H
F
H
H
55
4e
OMe H OMe Br OH H Cl
CN
H
H
55
4f 4g 4h
OMe H OMe Br OH H H OMe H OMe Br OH H Cl OMe H OMe Br OH H Cl
Br H H
H H Cl
H H H
60 83 62
4i
OMe H OMe Br OH H H
H
Cl
II
80
4j
OMe H OMe Br OH H H
CF3
H
II
70
4k
OMe H OMe Br OH H H
H
N
H
52
4l
OMe H OMe Br OH H F
H
H
H
62
4m
OMe H OMe Br OH H H
NO2 H
H
60
4n
OMe H OMe Br OH H H
OMe H
OMe 67
4o
OMe H OMe Br OH H H
H
N
–
43
4p
OMe H OMe Br OH H H
NO2 CI
H
36
General Procedure for the synthesis of (4a–p) To a mixture of 1-(3-bromo-2-hydroxy-4,6-dimethoxyphenyl)ethanone 3 (1 mmol) in ethanol (15 ml) was added NaOH (2.5 mmol, 2–3 drops of water) and stirred for 5 min. Then, added substituted benzaldehyde (1 mmol) and stirred the reaction mixture at room temperature. The progress of reaction was checked by TLC and the reaction mixture was poured over crushed ice and acidified with acetic acid. The precipitated solid was filtered, washed with water, and oven dried. It was purified by column chromatography using silica gel mesh size (100–200 mesh) and elution with petroleum ether and ethyl acetate. (E)-1-(3-bromo-2-hydroxyl-4,6-dimethoxyphenyl)-3-(4methoxyphenyl)prop-2-en-1-one (4a) Recrystallized from ethanol; (yield 40%); mp 154–156°C; IR tmax cm-1 (KBr): 3420, 3130, 2974, 1685, 1615, 1587, 1538, 1478, 1420, 1011, 920, 748; 1H-NMR (CDCl3) d; 14.59 (1H, s, –OH), 7.89 (1H, d, J = 11.2 Hz), 7.62 (1H,
(IC50 lM)
1 lM
10 lM
4a
38
53
7.4
4b
22
57
10.9
4c 4d
21 36
59 63
10.2 1.2
4e
26
57
0.7
4f
13
57
8.2
4g
40
72
15.9
4h
46
53
8.9
4i
43
51
1.4
4j
27
52
3.2
4k
45
85
0.1
4l
24
38
N.A.
4m
31
50
10.1
4n
25
37
11.4
4o
42
78
0.6
4p
34
49
14.2
DMS
72
87
1
2'
2
Inhibition of TNF-a (%)
N.A. not active, DMS betamethasone
d, J = 11.2 Hz), 7.78 (2H, d, J = 1.7 Hz), 7.52 (2H, d, J = 1.9 Hz), 6.12 (1H, s, Ar-H), 4.04 (3H, s, –OCH3), 3.94 (6H, s, –OCH3 X 2). MS: m/e 394 (M ? 1). Anal.: C18H17BrO5/393. (E)-1-(3-bromo-2-hydroxyl-4,6-dimethoxyphenyl)-3-(2,5dimethoxyphenyl)prop-2-en-1-one (4b) Recrystallized from ethanol; (yield 38%); mp 134–136°C; IR tmax cm-1 (KBr): 3430, 3140, 3008, 2964, 1678, 1638, 1560, 1465, 1420, 1021, 962, 765; 1H-NMR (CDCl3) d; 14.52 (1H, s, –OH), 7.83 (2H, d, J = 1.2 Hz), 7.82 (2H, d, J = 12.2 Hz), 7.59 (1H, d, J = 11.4 Hz), 6.06 (1H, s, ArH), 4.02 (3H, s, –OCH3), 3.94 (9H, s, –OCH3 X 3). MS: m/ e 424 (M ? 1). Anal.: C19H19BrO6/423. (E)-1-(3-bromo-2-hydroxyl-4,6-dimethoxyphenyl)-3-(2,4dimethoxyphenyl)prop-2-en-1-one (4c) Recrystallized from ethanol; (yield 47%); mp 152–154°C; IR tmax cm-1 (KBr): 3620, 3110, 2984, 1686, 1635, 1590, 1438, 1420, 1009, 865, 728; 1H-NMR (CDCl3) d; 14.79 (1H, s, –OH), 8.04 (1H, d, J = 4.3 Hz), 7.36 (1H, d, J = 1.4 Hz), 7.21 (1H, d, J = 9.2 Hz), 7.94 (2H, d, J = 12.3 Hz), 7.72 (1H, d, J = 13.2 Hz), 6.09 (1H, s, ArH), 4.06 (6H, s, –OCH3 X 2), 3.93 (6H, s, –OCH3 X 2). MS: m/e 424 (M ? 1). Anal.: C19H19BrO6/423.
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Table 3 Profile of COX, trypsin, and b-glucuronidase inhibitory activity of (4a–p) chale one derivatives at 1 mM concentration COX-1 (%)
COX-2 (%)
Trypsin (%)
b-glucluronidase (%)
4a
79.95
84.45
65.14
4b
62.61
68.19
42.85
4c 4d
52.42 21.84
57.82 33.54
4e
62.42
4f
71.39
4g
Entry
Table 4 Summary of the results of DPPH, OH radical scavenging activity (%), and reducing activity (%) of (4a–p) chalcone derivatives at concentration (1 mM) Entry
DPPH (%)
OH (%)
Reducing activity (%)
52.91
4a
38.37
49.41
34.32
48.45
4b
22.98
31.58
22.90
39.47 55.42
41.29 61.32
4c 4d
20.24 12.00
28.76 NR
18.40 12.20
58.21
48.20
47.29
4e
27.42
25.62
17.27
78.41
51.90
72.10
4f
24.12
38.23
19.60
32.78
34.74
57.61
48.21
4g
15.75
32.27
14.29
4h
37.47
39.57
54.24
44.72
4h
17.29
30.18
17.38
4i
44.00
51.80
67.81
54.59
4i
18.41
38.23
15.70
4j
11.78
12.62
71.84
62.11
4j
13.67
47.05
10.00
4k
21.72
35.57
48.00
44.27
4k
21.11
44.11
16.56
4l
69.47
74.77
81.71
68.46
4l
29.95
16.76
29.70
4m
68.47
76.41
65.92
61.85
4m
23.67
18.82
26.22
4n
57.23
78.42
65.92
61.85
4n
NR
23.52
18.40
4o
35.18
61.38
33.67
38.45
4o
26.69
NR
26.00
4p
18.62
38.76
49.73
38.95
4p
14.27
NR
26.00
ASA
–
38.54
–
–
GA (1 mM)
91.02
–
–
– –
– 86.64
– 26.48
AA (1 mM)
–
41.17
76.60
SC 560 34.90 SA –
The results summarized are the mean values of n = 2 ASA acetyl salicylic acid, SA salicylic acid, SC560 a standard COX-1 inhibitor
1 mM is the stock concentration, from which only 10 lM was pipetted. Therefore, the actual concentration in the reaction mixture is lM only GA gallic acid, AA ascorbic acid, NR no reaction
(E)-1-(3-bromo-2-hydroxyl-4,6-dimethoxyphenyl)-3-(3fluorophenyl)prop-2-en-1-one (4d)
(E)-1-(3-bromo-2-hydroxyl-4,6-dimethoxyphenyl)-3-(3bromophenyl)prop-2-en-1-one (4f)
Recrystallized from ethanol; (yield 55%); mp 165–167°C; IR tmax cm-1 (KBr): 3450, 3131, 3010, 2986, 1667, 1623, 1530, 1454, 1423, 1012, 945, 815, 724; 1H-NMR (CDCl3) d; 14.52 (1H, s, –OH), 7.85 (1H, d, J = 8.6 Hz), 7.82 (1H, d, J = 1.6 Hz), 7.60 (1H, d, J = 1.6 Hz), 7.52 (1H, d, J = 8.7 Hz), 7.11 (2H, d, J = 8.8 Hz), 6.05 (1H, s, Ar-H), 4.02 (3H, s, –OCH3), 3.92 (3H, s, –OCH3). MS: m/e 382 (M ? 1). Anal.: C17H14BrFO4/381.
Recrystallized from ethanol; (yield 60%); mp 151–153°C; IR tmax cm-1 (KBr): 3320, 3110, 3008, 2987, 1678, 1624, 1510, 1468, 1410, 1010, 968, 818, 726; 1H-NMR (CDCl3) d; 14.61 (,1H, s, –OH), 7.80 (1H, d, J = 16 Hz), 7.72 (1H, d, J = 16 Hz), 7.71 (2H, d, J = 1.7 Hz), 7.55 (2H, d, J = 11.4 Hz), 7.28 (1H, d, J = 8 Hz), 6.05 (1H, s, Ar-H), 4.01 (3H, s, –OCH3), 3.95 (3H, s, –OCH3). MS: m/e 443 (M ? 2). Anal.: C17H14Br2O4/441.
3-((E)-3-(3-bromo-2-hydroxy-4,6-dimethoxyphenyl)-3oxoprop-1-enyl)benzonitrile (4e) Recrystallized from ethanol; (yield 55%); mp 170–172°C; IR tmax cm-1 (KBr): 3456, 3110, 3009, 2991, 1678, 1633, 1561, 1464, 1410, 1019, 933, 817, 720; 1H-NMR (CDCl3) d; 14.61 (1H, s, –OH), 7.86 (2H, d, J = 12.3 Hz), 7.51 (2H, d, J = 0.4 Hz), 7.35 (1H, d, J = 0.6 Hz), 7.13 (1H, d, J = 3.5 Hz), 6.10 (1H, s, Ar-H), 3.52 (6H, s, –OCH3 X 2). MS: m/e 389 (M ? 1). Anal.: C18H14BrNO4/388.
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(E)-3-(3-bromo-2-hydroxyl-4,6-dimethoxyphenyl)-3-(2chlorophenyl)prop-2-en-1-one (4g) Recrystallized from ethanol; (yield 83%); mp 154–156°C; IR tmax cm-1 (KBr): 3310, 3080, 3010, 2981, 1682, 1622, 1525, 1422, 1403, 1010, 936, 810, 712; 1H-NMR (CDCl3) d; 14.61 (1H, s, OH), 7.89 (1H, d, J = 9.6 Hz), 7.82 (1H, d, J = 9.6 Hz), 7.80 (1H, d, J = 0.4 Hz), 7.72 (1H, t, ArH), 7.67 (1H, t, Ar-H), 7.53 (1H, d, J = 0.3 Hz), 6.12 (1H, s, Ar-H), 4.01 (3H, s, –OCH3), 3.92 (3H, s, –OCH3). MS: m/e 398 (M ? 1). Anal.: C17H14BrClO4/397.
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(E)-3-(3-bromo-2-hydroxyl-4,6-dimethoxyphenyl)-3-(2,4dichlorophenyl)prop-2-en-1-one (4h) Recrystallized from ethanol; (yield 62%); mp 152–154°C; IR tmax cm-1 (KBr): 3430, 3129, 3010, 2964, 1678, 1635, 1590, 1520, 1470, 1410, 1010, 928, 825, 728; 1H-NMR (CDCl3) d; 14.60 (1H, s, –OH), 8.24 (1H, d, J = 12.1 Hz), 7.82 (1H, d, J = 12.1 Hz), 7.74 (1H, d, J = 0.3 Hz), 7.62 (1H, d, J = 0.3 Hz), 6.14 (1H, s, Ar-H), 4.03 (3H, s, – OCH3), 3.92 (3H, s, –OCH3). MS: m/e 433 (M ? 1). Anal.: C17H13BrCl2O4/432. (E)-1-(3-bromo-2-hydroxyl-4,6-dimethoxyphenyl)-3-(4chlorophenyl)prop-2-en-1-one (4i) Recrystallized from ethanol; (yield 80%); mp 217–219°C; IR tmax cm-1 (KBr): 3343, 3164, 3110, 2995, 1681, 1612, 1577, 1494, 1425, 1010, 943, 813, 736; 1H-NMR (CDCl3) d; 14.24 (1H, s, –OH), 7.90 (1H, d, J = 15.6 Hz), 7.81 (1H, d, J = 19.6 Hz), 7.66 (2H, d, J = 8.6 Hz), 7.45 (2H, d, J = 8.5 Hz), 6.21 (1H, s, Ar-H), 3.95 (3H, s, –OCH3), 3.92 (3H, s, –OCH3). MS: m/e 398 (M ? 1). Anal.: C17H14BrClO4/397. (E)-3-(3-bromo-2-hydroxyl-4,6-dimethoxyphenyl)-3-(3(trifluoromethyl)phenyl)prop-2-en-1-one(4j) Recrystallized from ethanol; (yield 70%); mp 139–141°C; IR tmax cm-1 (KBr): 3420, 3120, 3024, 2978, 1678, 1656, 1516, 1458, 1413, 1009, 928, 824, 721; 1H-NMR (CDCl3) d; 14.62 (1H, s, –OH), 7.88 (1H, d, J = 7.6 Hz), 7.81 (1H, d, J = 9.5 Hz), 7.73 (2H, t, Ar-H), 7.62 (1H, d, J = 1.7 Hz), 7.53 (1H, d, J = 6.1 Hz), 6.13 (1H, s, Ar-H), 4.01 (3H, s, –OCH3), 3.92 (3H, s, –OCH3). MS: m/e 432 (M ? 1). Anal.: C18H14BrF3O4/431. (E)-1-(3-bromo-2-hydroxyl-4,6-dimethoxyphenyl)-3(pyridine-3-yl)prop-2-en-1-one (4k) Recrystallized from ethanol; (yield 52%); mp 166–168°C; IR tmax cm-1 (KBr): 3310, 3144, 3110, 2987, 1681, 1632, 1570, 1456, 1415, 1014, 934, 813, 751; 1H-NMR (CDCl3) d; 14.72 (1H, s, –OH), 8.84 (1H, d, J = 2 Hz), 8.61 (1H, d, J = 3 Hz), 7.43 (2H, d, J = 11 Hz), 7.35 (1H, d, J = 12 Hz), 6.11 (1H, s, Ar-H), 3.97 (6H, s, –OCH3 X 2). MS: m/e 366 (M ? 2). Anal.: C16H14BrNO4/364. (E)-3-(3-bromo-2-hydroxyl-4,6-dimethoxyphenyl)-3-(2fluorophenyl)prop-2-en-1-one (4l) Recrystallized from ethanol; (yield 62%); mp 152–154°C; IR tmax cm-1 (KBr): 3400, 3165, 3021, 2974, 1678, 1665, 1545, 1526, 1470, 1010, 978, 824, 765; 1H-NMR (CDCl3)
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d; 14.82 (1H, s, –OH), 7.95 (1H, d, J = 14.6 Hz), 7.88 (1H, d, J = 14.6 Hz), 7.53 (1H, d, J = 2 Hz), 7.34 (1H, d, J = 3 Hz), 7.15(2H, m, Ar-H), 6.11 (1H, s, Ar-H), 3.98 (6H, s, –OCH3 X 2). MS: m/e 383 (M ? 2). Anal.: C17H14BrFO4/381. (E)-1-(3-bromo-2-hydroxyl-4,6-dimethoxyphenyl)-3-(3nitrophenyl)prop-2-en-1-one (4m) Recrystallized from ethanol; (yield 60%); mp 141–143°C; IR tmax cm-1 (KBr): 3410, 3122, 3124, 2987, 1684, 1662, 1575, 1468, 1425, 1012, 931, 825, 748; 1H-NMR (CDCl3) d; 14.59 (1H, s, -OH), 8.47 (1H, d, J = 2 Hz), 8.25 (1H, d, J = 3 Hz), 7.94 (2H, d, J = 15.5 Hz), 7.62 (1H, d, J = 15.5 Hz), 7.24 (1H, d, J = 3 Hz), 7.15 (1H, d, J = 2.1 Hz), 6.02 (1H, s, ArH), 3.97 (3H, s, –OCH3), 3.94 (3H, s,–OCH3). MS: m/e 410 (M ? 2). Anal.: C17H14BrNO6/408. (E)-1-(3-bromo-2-hydroxyl-4,6-dimethoxyphenyl)-3-(3,5dimethoxyphenyl)prop-2-en-1-one (4n) Recrystallized from ethanol; (yield 67%); mp 170–172°C; IR tmax cm-1 (KBr): 3470, 3168, 3008, 2989, 2858, 1678, 1618, 1567, 1590, 1410, 1021, 945, 822; 1H-NMR (CDCl3) d; 14.64 (1H, s, –OH), 7.82 (1H, d, J = 2.4 Hz), 7.64 (1H, d, J = 2.4 Hz), 7.62 (1H, d, J = 2.4 Hz), 7.61 (2H, d, J = 12 Hz), 6.04 (1H, s, Ar-H), 4.03 (6H, s, –OCH3 X 2), 3.92 (6H, s, –OCH3 X 2). MS: m/e 424 (M ? 1). Anal.: C19H19BrO6/423. (E)-1-(3-bromo-2-hydroxyl-4,6-dimethoxyphenyl)-3-(1Hpyrrol-3-yl)prop-2-en-1-one (4o) Recrystallized from ethanol; (yield 43%); mp 156–158°C; IR tmax cm-1 (KBr): 3410, 3109, 3144, 2968, 1681, 1638, 1510, 1456, 1420, 1012, 933, 822, 751; 1H-NMR (CDCl3) d; 14.21 (1H, s, –OH), 7.90 (1H, s, Ar-H), 7.82 (1H, d, J = 10.2 Hz), 7.60 (2H, d, J = 10.2 Hz), 7.44 (1H, d, J = 0.5 Hz), 7.42 (1H, s), 6.02 (1H, s, Ar-H), 4.02 (3H, s, –OCH3), 3.94 (3H, s, –OCH3). MS: m/e 352 (M ? 1). Anal.: C15H14BrNO4/351. (E)-1-(3-bromo-2-hydroxyl-4,6-dimethoxyphenyl)-3-(4chloro-3-nitrophenyl)prop-2-en-1-one (4p) Recrystallized from ethanol; (yield 36%); mp 176–178°C; IR tmax cm-1 (KBr): 3420, 3145, 3109, 2987, 2842, 1685, 1616, 1598, 1547, 1422, 1012, 972, 815, 725; 1H-NMR (CDCl3) d; 14.21 (1H, s, –OH), 7.92 (2H, d, J = 14.6 Hz), 7.72 (1H, s, Ar-H), 7.69 (1H, d, J = 12.2 Hz), 7.62 (1H, d, J = 1.7 Hz), 6.04 (1H, s, Ar-H), 4.09 (3H, s, –OCH3), 3.96 (3H,–s, OCH3). MS: m/e 443 (M ? 1). Anal.: C17H13BrClNO6/442.
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Whole blood cell culture (WBCs) TNF-a production by lipopolysaccharide (LPS) in whole blood was measured according to the method described by Wilson et. al. In brief, K3EDTA blood from healthy donors were diluted 1:5 in RPMI 1640 culture medium containing 100 U/ml penicillin and 100 mg/ml streptomycin with no added serum. 100 ll/well of diluted blood was transferred into 96 well culture plates. Following cell plating, 80 ll of medium and 1 ll of the test compounds or vehicle (0.5% DMSO) were added to each well and the plate was incubated at 37°C for 30 min. Finally 20 ll (10 lg/ml) per well of LPS (Escherichia coli 0127:B8) was added, for a final concentration of 1 lg/ml. Plates were incubated at 37°C up to 5 h in 5% carbon dioxide humidifier. Supernatants were harvested and assayed for TNF-a release compared to the control (Wilson et al., 1997). COX-1 and 2 inhibition micro-titer assay The COX-1 and 2 inhibition assay was performed as per the assay protocol instructions of ‘‘Colorimetric COX (ovine) inhibitor Screening Assay Kit,’’ Cayman Chemical Company, MI, USA. The reaction mixture of 100% initial activity wells contained 160 ll of assay buffer, 150 ll of heme, and 10 ll of either COX-1 or 2 enzyme solutions. While the reaction mixture of inhibitor wells was comprised of 150 ll of assay buffer, 10 ll of heme, and 10 ll of either enzyme COX-1 or 2, 10 ll 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 ll of the colorimetric substrate solution was added to all the wells, followed by the addition of 20 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 inhibition activity (%) was calculated using following formula. COX inhibition activity ð%Þ ¼ 1
T 100 C
where T is the absorbance of the inhibitor well at 590 nm, C is the absorbance of the 100% initial activity without inhibitor well at 590 nm (Bandgar et al., 2010).
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 5,000 RPM for 15 min. The amount of protein in the acid soluble fractions was estimated by this method (Tandon et al., 1982; Lowry et al., 1951). b-glucuronidase inhibition assay The effect of the selected compounds on activity of bglucuronidase was studied using a method described by (Nicolaides et al., 1998). One millimolar concentration of test sample (0.1 ml) in 0.1 M acetate buffer pH 7.4 for 5 min at 37°C were preincubated with 0.8 mL of 2.5 mM p-nitrophenyl-b-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 spectrophotometrically at 410 nm (Gacche and Dhole, 2006). DPPH radical scavenging assay The DPPH radical scavenging assay was performed as described by Bartolome et al. (2004). The reaction mixture contained 1 mM concentrations of individual test sample (in absolute ethanol) and DPPH radical (10-4 M in absolute ethanol) solution. The contents of the reaction mixture were observed spectrophotometrically at 517 nm after 20 min (Bartolome et al., 2004). OH radical scavenging activity OH radicals were generated by using the Ferric ion (Fe3?)/ ascorbic acid reaction system. The detection of OH radicals was carried out by measuring the amount of formaldehyde generated from the oxidation of dimethyl sulfoxide. The reaction cocktail contained 0.1 mM EDTA, 167 mM Fe3-, 33 mM DMSO in phosphate buffer of 50 mM pH 7.4, and 0.1 ml individual compound (1 mM) solution. Ascorbic acid (150 ll, 10 mM in phosphate buffer) was added finally to initiate the reaction. Trichloroacetic acid (17%, w/v) was used to terminate the reaction. The contents were observed spectrophotomertically at 412 nm for the detection of formaldehyde (Nash, 1953).
Trypsin inhibition assay Reducing activity assay The method is based on the measurement of inhibition of trypsin induced hydrolysis of bovine serum albumin (BSA). Trypsin (0.075 mg/ml) was initially incubated with 1 mM individual concentrations of test sample of 0.1 ml for 20 min. The substrate BSA (6 g/100 ml, in 0.1 M
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Reduction of Fe3? of K3Fe(CN)6 to Fe2? by antioxidants is the underlying mechanism of the reducing activity assay. The reducing potential of selected compounds was calculated by the fall in extinction of K3Fe(CN)6 at 420 nm
Med Chem Res (2012) 21:2292–2299
against appropriate blank. The reaction mixture contained 500 ml solution of individual sample (1 mM in 0.5% v/v dimethyl sulfoxide) in 3 ml of 1 mM potassium ferricynide solution and the absorbance was recorded at 420 nm after 10 min reaction time (Sasaki et al., 1991). Acknowledgment Authors are thankful to the Council of Scientific and Industrial Research (CSIR), New Delhi for the award of SRF to Mr. B.S.Hote, as well as to Mr. Mahesh Nambiar and Mrs. Asha Almeida, Piramal life Sciences Ltd., Mumbai for TNF-screening of the compounds.
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