Design, synthesis, characterization and anti-inflammatory evaluation of novel pyrazole amalgamated flavones

Article abstract of DOI:10.1016/j.bmcl.2012.12.094

A series of novel pyrazole amalgamated flavones has been designed and synthesized from 1-methyl-5-(2,4,6-trimethoxy-phenyl)-1H-pyrazole 6. The structures of regioisomers 6 and 7 were resolved by 2D ¹H- ¹H COSY, ¹H-¹³C HSQC and ¹H- ¹³C HMBC experiments. The newly synthesized compounds were tested for their in vitro COX inhibition and in vivo carrageenan induced hind paw edema in rats and acetic acid induced vascular permeability in mice. Although the compounds have inhibitory profile against both COX-1 and COX-2, some of the compounds are found to be selective against COX-2, supported by inhibition of paw edema and vascular permeability. Docking studies were also carried out to determine the structural features which sway the anti-inflammatory activity of the tested compounds. The keto and phenolic -OH are major factors that are prominently involved in interaction with COX-2 active site.

Full text of DOI:10.1016/j.bmcl.2012.12.094

Accepted Manuscript
Design, synthesis, characterization and anti-inflammatory evaluation of novel
pyrazole amalgamated flavones
Hemant V. Chavan, Babasaheb P. Bandgar, Laxman K. Adsul, Valmik D.
Dhakane, Pravin S. Bhale, Vishnu N. Thakare, Vijay Masand
PII:
S0960-894X(13)00004-8
DOI:
http://dx.doi.org/10.1016/j.bmcl.2012.12.094
BMCL 19979
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
7 October 2012
19 December 2012
28 December 2012
Please cite this article as: Chavan, H.V., Bandgar, B.P., Adsul, L.K., Dhakane, V.D., Bhale, P.S., Thakare, V.N.,
Masand, V., Design, synthesis, characterization and anti-inflammatory evaluation of novel pyrazole amalgamated
flavones, Bioorganic & Medicinal Chemistry Letters (2013), doi: http://dx.doi.org/10.1016/j.bmcl.2012.12.094
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Graphical Abstract
Design, synthesis, characterization and anti-inflammatory
evaluation of novel pyrazole amalgamated flavones
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A series of novel pyrazole amalgamated flavones have been designed synthesized and
evaluated for their anti-inflammatory potential.

Design, synthesis, characterization and anti-inflammatory
evaluation of novel pyrazole amalgamated flavones
Hemant V. Chavan,a Babasaheb P. Bandgar,a,* Laxman K. Adsul,a Valmik D.
Dhakane,a Pravin S. Bhale,a Vishnu N. Thakare,b Vijay Masandc
a
Medicinal Chemistry Research Laboratory, School of Chemical Sciences, Solapur
University, Solapur-413 255, Maharashtra, India.
b Department of Pharmacology, Sinhgad Institute of Pharmaceutical Sciences, Kusgaon (Bk),
Lonavala-410 401, Maharashtra, India.
c
Department of Chemistry, Vidyabharati Mahavidyalaya, Camp, Amravati-444 602,
Maharashtra, India.
*Corresponding author: Tel. /Fax: 0091-217-2351300; E-Mail: bandgar_bp@yahoo.com
A B S T R A C T
A series of novel pyrazole amalgamated flavones has been designed and synthesized from 1-
methyl-5-(2,4,6-trimethoxy-phenyl)-1H-pyrazole 6. The structures of regioisomers 6 and 7
1
1
1
were resolved by 2D H-1H COSY, H-13C HSQC and H-13C HMBC experiments. The
newly synthesized compounds were tested for their in vitro COX inhibition and in vivo
carrageenan induced hind paw edema in rats and acetic acid induced vascular permeability in
mice. Although the compounds have inhibitory profile against both COX-1 and COX-2, some
of the compounds are found to be selective against COX-2, supported by inhibition of paw
edema and vascular permeability. Docking studies were also carried out to determine the
structural features which sway the anti-inflammatory activity of the tested compounds. The
keto and phenolic –OH are major factors that are prominently involved in interaction with
COX-2 active site.
Keywords: Pyrazole, Flavones, HMBC, HSQC, COSY, Anti-inflammatory activity,
Molecular docking.
1

Flavonoids are natural phytoconstituents having various biological and pharmacological
activities.1 The flavonoid family includes chalcone, flavan-3-ol, flavanone, flavone, flavonol
and various biflavonoids and found to have elicited anti-inflammatory activity in vitro and in
vivo.2,3 The anti-inflammatory activity of flavonoids is due to its multiple cellular
mechanisms of action. In addition to their well-known anti-oxidative action, some flavonoids
exert inhibitory activity against eicosanoid and nitric oxide (NO) metabolism. For example,
some compounds inhibit phospholipase A2s (PLA2), cyclooxygenase (COX), lipoxygenase
(LOX) and nitric oxide synthase (NOS).4-6 Furthermore, flavonoids also found to inhibit and
down-regulate expression of other pro-inflammatory mediators viz. matrix metalloproteinase
(MMP).7 The flavones are characterized by the planar structure because of the double bond in
the central aromatic ring. Specifically, these flavonoids inhibit the expression of the inducible
forms of COX and NOS, interleukins, tumor necrosis factor-α (TNF-α) and adhesion
molecules in inflammatory cells and tissues. Many flavonoids and biflavonoids affect
multiple points in the aforementioned pathways, which suggests that they are anti-
inflammatory agents with multiple action mechanisms.8 One of the best described flavonoids,
quercetin, is a member of this group.
Pyrazoles are an important class of compounds for new drug development that attracted
much attention due to their broad spectrum of biological activities, such as anti-
inflammatory,9-12 antifungal,13 anticancer,14-16 and antiviral activities.17,18. Pyrazole
derivatives also act as antiangiogenic agents,19 kinase inhibitor for treatment of type-2
diabetes, hyperlipidemia, obesity,20 and thrombopiotinmimetics.21 Recently urea derivatives
of pyrazoles have been reported as potent inhibitors of p38 kinase.22 Among the highly
marketed COX-2 inhibitors that comprise the pyrazole nucleus, celecoxib is the one which is
treated as a safe anti-inflammatory and analgesic agent. It is considered as a typical model of
the diaryl heterocyclic template that is known to selectively inhibit the COX-2 enzyme. Some
other examples of pyrazole derivatives such as deracoxib, SC-558, ramifenazone,
famprofazone, rimonbant, fipronil, pyraclonil (Figure 1) have been reported as potent
NSAIDs.23-27
In continuation of our ongoing research programme on the development of novel anti-
inflammatory agents,28-30 and based on the diverse biological activities of flavonoids and
pyrazoles, in the present study we have synthesized some new synthetic flavones
amalgamated with pyrazole scaffold, as potential anti-inflammatory agents.
Synthesis of title compounds 10a-n was achieved by the oxidation of chalcones 9a-n
with iodine in DMSO. Claisen-Schmidt condensation of 1-(2-hydroxy-4,6-dimethoxy-3-(1-
2

methyl-1H-pyrazol-5-yl)phenyl)ethanone 8 with various aromatic / heteroaromatic aldehydes
in presence of NaOH in ethanol at room temperature furnished chalcones 9a-n in good to
excellent yields. 1-(2-Hydroxy-4,6-dimethoxy-3-(1-methyl-1H-pyrazol-5-yl)phenyl)ethanone
8 was readily prepared by the Friedel-Craft acetylation of key intermediate 1-methyl-5-
(2,4,6-trimethoxyphenyl)-1H-pyrazole 6 using acetic anhydride in presence of boron
trifluoride etherate under nitrogen atmosphere (Scheme 1).
The regioselective synthesis of key intermediate 1-methyl-5-(2, 4, 6-trimethoxyphenyl)-
1H-pyrazole 6 was carried out using the synthetic strategies illustrated in Scheme 2.
Xanthoxyline 2 was synthesized by the Friedel-Craft acetylation of 1,3,5-trimethoxybenzene
1 by adopting the literature precedent, and successive O-methylation of the resulting
xanthoxyline 2 using dimethyl sulphate and fused potassium carbonate in acetone under
reflux condition gave 1-(2,4,6-trimethoxyphenyl)ethanone 3 in excellent yield.31 2-
Formylation of acetophenone 3 with ethyl formate and sodium hydride gave 3-oxo-3-(2,4,6-
trimethoxyphenyl)propanal 4, and without further purification subsequent treatment of 4 with
hydrazine hydrate afforded the corresponding 5-(2,4,6-trimethoxyphenyl)-1H-pyrazole 5 in
96% yield. Treatment of compound 5 with methyl iodide and sodium hydride in dry DMF
under nitrogen atmosphere afforded a mixture of 1-methyl-5-(2,4,6-trimethoxyphenyl)-1H-
pyrazole 6 and 1-methyl-3-(2,4,6-trimethoxyphenyl)-1H-pyrazole 7, which were easily
separated by silica gel coloumn chromatography.
1
The structures of the regioisomers 6 and 7 were determined using 1D H, 13C NMR
1
1
spectra, 135-DEPT and 2D NMR experiments (1H-1H COSY, H-13C HSQC and H-13C
HMBC and NOESY), which were scanned on Bruker AV-400 and 500 MHz instrument.
These two regioisomers differ only in the position of methyl group on pyrazole nucleus,
1
13
therefore, the H and C NMR chemical shifts were found to be almost similar. Thus, the
structures of these two regioisomers were only assigned with the help of HMBC, HSQC and
COSY correlations. The most important HMBC and COSY correlations are summarized in
Figure 2 and the complete chemical shift assignment is shown in Table 4 and Table 5
(Supporting information). According to a Heteronuclear Multiple Bond Correlation (HMBC)
experiment performed with regioisomer 6, the HMBC correlation observed between H-13 (δH
3.65) and C-3 (δC 135.5) and with regioisomer 7, the HMBC correlation observed between
H-13 (δH 3.97) and C-1 (δC 130.12) allowing to unambiguous identification of the
regioisomer 6 and 7. Additionally, Heteronuclear single Quantum Coherence (HSQC)
experiment was used to assign proton signals to the corresponding carbon signals. There is a
strong 1H-1H COSY correlation between H-1 and H-2.
3

All the synthesized compounds (10a-n) were screened for their inhibitory potential
against the COX-1 and COX-2 enzymes at 100 μM by using Colorimetric COX (ovine)
Inhibitor Screening assay kit (Catalogue No. 760111, Cayman Chemicals Co, USA).
Indomethacin was used as a reference compound. The results are listed in Table 1. The
results showed that most of the synthesized compounds have an inhibitory profile against
both COX-1 and COX-2, some are found to be more selective towards COX-2 (10h, 10e, and
10i) by a small percentage of inhibition. The entire series of flavones exhibited significant
inhibitory profile against COX-2 (38-51%). Compounds carrying methoxy groups on phenyl
ring (10h and 10e) appeared as the most active compounds against COX-2 enzyme.
Moreover, compounds containing furan and thiophene heterocycles instead of phenyl ring
(10i and 10j respectively) showed significant inhibitory activity on COX-2 enzyme with
inhibiting COX-1 enzyme less than indomethacin (Table 1). In addition, substitution of
fluorine to the second / fourth position of phenyl ring (10n, 10b, and 10f) resulted in a
remarkable decrease in COX-2 inhibitory activity.
The synthesized compounds were also tested for their in vivo anti-inflammatory
potential by using carrageenan hind paw and acetic acid vascular permeability methods. From
the experimental findings, significant (p < 0.01) increase in paw volume was observed in rats
administered with carrageenan alone when compared with normal rats (Table 2). Increase in
paw edema indicates the inflammation due to release of inflammatory mediators
(prostaglandins, serotonin, histamine, etc.) at various time intervals. The synthesized
compounds viz. 10h, 10e and 10i are found to significantly inhibit (p<0.01) the edema
formation (Table 2). Inhibition of paw edema formation at 6 h is greater that might be due to
the inhibition of COX-II, which is known to be released at 4-6 h after the carrageenan
injection. In acetic acid induced vascular permeability in mice, significant inhibition of dye
leakage into peritoneal cavity indicated the anti-inflammatory effects of 10h, 10e and 10i
(Table 3). Since vascular permeability is one of the sign of inflammation, increased vascular
permeability in control group indicated the inflammatory response. Prevention and/or
inhibition of vascular permeability with 10h, 10e and 10i certainly elicited the potent anti-
inflammatory activity. Therefore, the compounds showing significant COX-2 inhibitory
potential (10h, 10e and 10i) also display potent anti-inflammatory activity in vivo.
The docking study was performed with MOE software program to understand the
orientations of the compounds in the active site of COX-2. We docked all the synthesised
compounds 10a-n and indomethcin in the active site of COX-2. In COX-2, the binding site is
comparatively long, slender, hydrophobic channel extending from the membrane-binding
4

region of the protein. A second cavity branched off from the main channel that lead to the
cyclooxygenase active site is observed in COX-2. A similar pocket also exists in COX-1 but
it is relatively small and less accessible due to presence of bulkier isoleucine at position
number 523. The well known anti-inflammatory agent Indomethacin binds to Val102,
Arg106, Val335, Leu338, Tyr341, Leu345, Trp373, Met508, Val509, Gly512, and Ala513
residues of COX-2. To get clear idea, we docked all the flavone derivatives in the pocket of
COX-2.
Hydrophobic as well as H-bonding interactions are involved in binding of most active
compound 10h with COX-2 (Figure 3). It appears that the water present inside the active site
of COX-2 plays crucial role.45, 46 The two oxygen atoms, one from ketone group of ring 2 and
-OMe from ring 1 adjacent to it, act as H-bond acceptor by interacting with TyrA 341 (a
neutral amino acid with polar but non-ionized side chain) with a distance of 3.34Ao (48%)
and 2.69 Ao (99%), respectively. Neutral amino acids with nonpolar side chain ValA335,
MetA99, LeuA517, AlaA513 surronds the ring 3 that indicates the presence of lipophilic
groups on this ring is important for binding with receptor. This fact is confirmed when we
compare efficiency of compound 10h and CLogP values (Table 1) with other derivatives in
which lipophilic groups are absent on ring 3. From Figure 3, it is clear that the binding
pocket is large and compound 10h is small, this means new larger groups can be introduced
on ring 3 to increase the interaction between drug and receptor. This suggests that compound
10h is attractive target as “ lead compound”. The docking and structure activity relationship
(SAR) analysis reveals that pyrazole play crucial role due to its lipophilic character and the
nitrogens are unable to act as H-bond acceptor. The summary of SAR study is shown in
Figure 4.
In conclusion, a series of new pyrazole amalgamated flavones were synthesized in order
to evaluate their inhibitory activities on COX-1 and COX-2 enzymes. Among the synthesized
compounds, 10h, 10e, and 10i showed significant inhibitory profiles against COX-2,
indicating that they are selective inhibitors for COX-2. Moreover, inhibitory effects of 10h,
10e, and 10i are also supported by their effects in inhibition of carrageenan induced paw
edema and prevention of dye leakage into peritoneal cavity due to the reduction of vascular
permeability which might be due to release of prostaglandins and other inflammatory
mediators. The binding mode of the tested compounds inside the active site of COX-2
enzyme was predicted using docking technique. Neutral amino acids with nonpolar side chain
ValA335, MetA99, LeuA517, AlaA513 surronds ring 3 of compound 10h, indicating the
presence of lipophilic groups on this ring is important for binding with receptor. It is also
5

clear that the binding pocket of COX-2 is large and compounds are small, this means
introduction of new larger groups on ring 3 may be useful to propose new molecules with
enhanced COX-2 selectivity.
Acknowledgements
Author H.V.C. thankful to Council of Scientific and Industrial Research (CSIR), New Delhi,
Govt. of India for financial support in the form of SRF. Authors are also grateful to Dr. S. R.
Naik and Dr. S. B. Bhise, Sinhgad Institute of Pharmaceutical Sciences, Lonavala, and Pune
for providing necessary facility for conducting pharmacological experiments.
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32. Preparation of 5-(2,4,6-Trimethoxy-phenyl)-1H-pyrazole (5): Ethyl formate (2.96
gm, 3.21 ml, 30 mmol) was added to a suspension of sodiun hydride (60 % in oil, 0.8
gm, 20 mmol) in tetrahydrofuran (3 ml) at room temperature. After stirring for 10 min,
a solution of 1-(2,4,6-trimethoxy-phenyl)-ethanone 3 (2.1 gm, 10 mmol) in THF (15
ml) was added, and the mixture was stirred for 1 h. To the reaction mixture was then
added 1M HCl (20 ml), and extracted with diethyl ether (3 x 25 ml). Organic layer was
concentrated under reduced pressure. To this hydrazine hydrate (5 gm, 4.84 ml, 100
mmol) was added, and the mixture was stirred for 30 min. The reaction mixture was
made alkaline by adding of 6 N NaOH (20 ml) and extracted with ethyl acetate. The
organic layer was washed with brine, dried over anhydrous magnesium sulphate,
evaporated in vacuo, and recrystallized from ethanol to obtain colorless crystals of title
compound 5. Yield: 85%; MP: 138-140oC; IR (KBr, cm-1): 3293, 3018, 2969, 2939,
1
2344, 1752, 1607, 1586, 1451, 1204; H NMR (400 MHz, CDCl3): δ 3.85 (s, 3H,
OCH3), 3.92 (s, 6H, OCH3), 6.22 (s, 2H, ArH), 6.82 (d, 1H, J = 1.6 Hz, C=CH), 7.6 (d,
1H, J = 1.6 Hz, N=CH), 11.38 (bs, 1H, NH); MS (ESI): m/e 235 (M+1).
33. Preparation of 1-methyl-5-(2,4,6-trimethoxy-phenyl)-1H-pyrazole (6): 5-(2,4,6-
Trimethoxy-phenyl)-1H-pyrazole 5 (2.34 gm, 10 mmol) was dissolved in dry DMF (15
ml) under N2 atmosphere. Cooled the flask in an ice bath and methyl iodide (2.84 gm,
1.25 ml, 20 mmol) was added to it. To this solution, sodium hydride (60 % in oil, 0.48
gm, 12 mmol) was added in portions and the resulted solution was then allowed to stir
at 0oC for 15 min. The reaction mixture was poured over crushed ice and the resulted
solid was filtered off, recrystallized from ethanol to afford the title compound 6 in pure
form. The filtrate was then extracted three times with ethyl acetate. The combined
extracts were washed with water. After drying over anhydrous MgSO4, the solvent was
distilled off under reduced pressure. The resulting residue was purified by silica gel
8

column chromatography using petroleum ether: ethyl acetate as eluent (9:1) to obtain
compound 7 in pure form.
1-methyl-5-(2,4,6-trimethoxy-phenyl)-1H-pyrazole (6): Yield: 82%; MP: 147-149oC;
IR (KBr, cm-1): 3064, 2962, 2943, 2841, 1612, 1584, 1549, 1473, 1458, 1234, 1161; 1H
NMR (500 MHz, CDCl3): δ 3.65 (s, 3H, NCH3), 3.75 (s, 6H, 2xOCH3), 3.87 (s, 3H,
OCH3), 6.20 (m, 3H, 2xArH, Pyr-H), 7.55 (s, 1H, Pyr-H); 13C NMR (125 MHz,
CDCl3): 36.66 (m, CH3), 55.40 (m, CH3), 55.75 (s, CH3), 90.54 (s, CH), 100.69 (w, C),
107.44 (m, CH), 135.52 (w, C), 137.91 (m, CH), 159.32 (m, C), 162.25 (w, C); 135-
DEPT: 36.65 (+), 55.40 (+), 55.75 (+), 90.53 (+), 107.44 (+), 137.89 (+); MS (ESI):
m/e 249 (M+1).
1-methyl-3-(2,4,6-trimethoxy-phenyl)-1H-pyrazole (7): Yield: 18%; MP: 104-106oC;
IR (KBr, cm-1): 3130, 2992, 2956, 2834, 1613, 1586, 1505, 1474, 1224, 1161; 1H NMR
(400 MHz, CDCl3): δ 3.76 (s, 6H, 2xOCH3), 3.85 (s, 3H, OCH3), 3.97 (s, 3H, NCH3),
6.20 (s, 2H, 2xArH), 6.31 (d, J = 2.8 Hz, 1H, Pyr-H), 7.41 (d, J = 2.8 Hz, 1H, Pyr-H);
13C NMR (100 MHz, CDCl3): 38.92 (m, CH3), 55.27 (m, CH3), 55.91 (s, CH3), 90.55
(s, CH), 104.51 (w, C), 107.81 (m, CH), 130.13 (m, CH), 144.40 (w, C), 159.37 (m, C),
161.07 (w, C); 135-DEPT: 38.94 (+), 55.28 (+), 55.93 (+), 90.56 (+), 107.81 (+),
130.11 (+); MS (ESI): m/e 249 (M+1).
34. Preparation
of
1-[2-Hydroxy-4,6-dimethoxy-3-(2-methyl-2H-pyrazol-3-yl)-
phenyl]-ethanone (8): Compound 6 (2.48 gm, 10 mmol) was dissolved in 25 ml of dry
CH2Cl2 under N2 atmosphere and flask was cooled in an ice bath. To this solution,
BF3.OEt2 (11.36 gm, 10.05 ml, 80 mmol) was added followed by a drop wise addition
of acetic anhydride (5.1 gm, 4.72 ml, 50 mmol). The resulted solution was then allowed
to stir for 24 h at room temperature. The reaction mixture was diluted with water,
rendered alkaline with Na2CO3, and extracted with CH2Cl2. Removal of solvent in
vacuo gave white solid that was recrystallized from methanol to obtain compound 8 in
pure form.
o
Yield: 95%; MF / FWt: C14H16N2O4 / 276; MP: 221-223 C; IR (KBr, cm-1): 3461,
2940, 2370, 1625, 1597, 1416, 1277, 1133; 1H NMR (400 MHz, CDCl3): δ 2.69 (s, 3H,
COCH3), 3.71 (s, 3H, NCH3), 3.88 (s, 3H, OCH3), 4.02 (s, 3H, OCH3), 6.08 (s, 1H,
ArH), 6.26 (d, 1H, J = 1.6 Hz, C=CH), 7.59 (d, 1H, J = 1.6 Hz, N=CH); MS (ESI): m/e
277 (M+1)
35. General procedure for the preparation of pyrazole-based chalcones (9a-n): 1-(2-
Hydroxy-4,6-dimethoxy-3-(1-methyl-1H-pyrazol-5-yl)phenyl)ethanone 8 (0.276 gm, 1
9

mmol) was dissolved in ethanol (10 ml) under stirring. To this was added NaOH (0.12
gm; 3 mmol, with a minimum of water) and stirred for 5 minutes. To this reaction
mixture, the aromatic aldehyde 9a-n (1.2 mmol) was then added and stirring continued
at room temperature for 24 h. Reaction was monitored by TLC. After completion of
reaction, the mixture was poured over crushed ice and acidified with acetic acid. The
separated solid was filtered and washed well with water. Crude product was dried and
recrystallized from ethanol to obtain the desired product in pure form.
(E)-1-(2-hydroxy-4,6-dimethoxy-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-
phenylprop-2-en-1-one (9a): Yield: 98%; MF / FWt: C21H20N2O4 / 364; MP: 178-180
1
oC; IR (KBr, cm-1): 3439, 2986, 2944, 2344, 1628, 1578, 1556, 1335, 799, 758; H
NMR (400 MHz, DMSO-d6): δ 3.55 (s, 3H, NCH3), 3.89 (s, 3H, OCH3), 4.08 (s, 3H,
OCH3), 6.15 (s, 1H, ArH), 6.43 (d, 1H, J = 1.6 Hz, Pyr-H), 7.41 (d, 1H, J = 1.6 Hz, Pyr-
H), 7.48 (m, 3H, ArH), 7.77 (m, 3H, 2xArH, CH=CHCO), 7.91 (d, 1H, J = 15.6 Hz,
CH=CHCO), 14.O3 (bs, 1H, OH); MS (ESI): m/e 365 (M+1)
36. General procedure for the synthesis of pyrazole amalgamated flavones (10a-n): To
the solution of (E)-1-(2-hydroxy-4,6-dimethoxy-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-
3-phenylprop-2-en-1-one 9a-n (1 mmol) in DMSO (10 ml), I2 (0.025 g, 0.1 mmol) was
added. The mixture was heated at 150oC for 3h. The completion of reaction was
monitored by TLC. After completion of reaction, the reaction mixture was poured into
cold water. Product precipitated was filtered off, washed with sodium thiosulphate,
dried and recrystallized from methanol to obtain the title compounds 10a-n in pure
form.
5,7-dimethoxy-8-(1-methyl-1H-pyrazol-5-yl)-2-phenyl-4H-chromen-4-one (10a):
o
Yield: 96 %; MF / FWt: C21H18N2O4 / 362; MP: 194-196 C; IR (KBr, cm-1): 3019,
1
2945, 2848, 2400, 1644, 1586, 1536, 1488, 1216, 769, 689; H NMR (400 MHz,
CDCl3): δ 3.69 (s, 3H, NCH3), 3.95 (s, 3H, OCH3), 4.10 (s, 3H, OCH3), 6.38 (d, J =
2Hz, 1H, Pyr-H), 6.49 (s, 1H, ArH), 6.69 (s, 1H, C=CHCO), 7.40 (m, 3H, ArH), 7.52
(d, J = 8 Hz, 2H, ArH), 7.65 (d, J = 2 Hz, 1H, Pyr-H); MS (ESI): m/e 363 (M+1).
37. In vitro COX inhibition assay: The assay was performed by using colorimetric COX
(ovine) inhibitor screening assay kit.38 Briefly, the reaction mixture contains, 150 µl of
assay buffer, 10 µl of heme, 10 µl of enzyme (either COX-1 or COX-2), and 50µl of
sample (0.1mM). The assay utilizes the peroxidase component of the COX catalytic
domain. The peroxidase activity can be assayed colorimetrically by monitoring the
appearance of oxidized N, N, N, N’-tetramethyl-p-phenylenediamine (TMPD) at
10

590nm. Indomethacin (0.1 mM) was used as a standard drug. The percent COX
inhibition was calculated using following equation,
T
____
C
X 100
1 -
COX inhibition activity (%) =
Where T= Absorbance of the inhibitor well at 590 nm
C= Absorbance of the 100% initial activity without inhibitor well at 590 nm
38. Maurias, M. Bioorg. Med. Chem. 2004, 12, 5571-5578.
39. Carrageenan induced hind paw edema in rats: Rats were divided into various
groups (n=6) and allowed to free access to water ad libitum. Different groups of rats
administered with indomethacin (100 mg/kg, b.w.) and various synthesized compounds
10a-10n (50 mg/kg,) orally. One group of rats served as a control and administered
with gum acacia (1 %, w/v; 10 ml/kg, b.w., p.o.). One hour after the drug
administration, to all groups of rats, hind paw edema was induced by the method of
winter et.al.40 by injecting 0.1 ml of 1 % (w/v) solution of carrageenan subcutaneously
into the subplanter region of hind paw. The hind paw edema volume was measured by
volume displacement method using plethysmometer (UGO, Besile 7140, Italy ) by
immersing the paw till the level lateral malleolus at various time intervals ( 1, 3 and 6
h) after carrageenan injection.
40. Winter, C.A.; Risley, E.A.; Nuss, G. W. Proc. Soc. Exp. Biol. Med. 1962, 111, 544-547.
41. Acetic acid induced vascular permeability in mice: Mice were divided in to various
groups (n=6) and allowed to free access to water ad libitum. Different groups of mice
administered with indomethacin (10 mg/kg) and selected test compounds from
carrageenan group (compounds eliciting significant paw edema inhibition) at dose of
100 mg/kg orally. Control group received gum acacia (2 %, 10 mL/kg, p.o.). One hr of
drug treatment mice were injected with 0.25 mL (0.6 %, v/v) solution of acetic acid by
intraperitoneally (i.p.). Immediately, 10 mL/kg (10 %, w/v) solution of Evan’s blue was
injected intravenously through tail vain. After 30 minutes of Evan’s blue injection,
mice were sacrificed with excess of ether anaesthesia. The abdomen was cut open and
exposed the viscera. The mice were held by a flap of abdominal wall and the viscera
irrigated with 5 mL normal saline over a Petri dish. The perfusate was collected and
then filtered through Whatmann filter paper, and the final volume of filtrate was made
up to 10 mL. The dye leaking out into peritoneal cavity in diluted fluid was measured
spectrophotomeically at 620 nm.42
42. Whittle, B. A.; Br J Pharmacol. 1964, 22, 246-253.
11

43. Experimental animals: Wistar rats of either sex were obtained from National
Toxicology Center, Pune, India. The rats were housed and maintained in clean
polypropylene cages under standard conditions: humidity (50 + 5 %), temperature (25 ±
20 C) and (12 h light/12 h dark cycle), fed with standard diet (Amrut laboratory animal
feed, Sangli, Maharashtra, India) and water ad libitum. Experimental protocol was
reviewed and approved by the Institutional Animal Ethics Committee
(SIPS/IAEC/2011-12/17) and conform to the Indian National Science Academy
Guidelines for the use and care of experimental animals in research. The animal house
registration number with Government of India is 962/c/06/CPCSEA.
44. Molecular modeling: In present work, we have used MOE 2008, which has improved
flexible docking as well as integration with a graphical interface along with other
modules, such as analysis, molecular mechanics, and molecular dynamics. To get
fruitful results, we docked the ligands into active site of receptor COX-2. A good
number of X-ray crystallographically resolved 3D structures are available for COX-2,
PDB ID-3NTG was chosen on the basis of following two reasons: (1) X-ray diffraction
resolution, 2.14 in present case. (2) The pdb 3NTG contains crystal structure of COX-
2 with selective compound 23d-(R). The experimental conformation of the "template"
ligand 23d-(R) was analysed to get idea about active site and then deleted, leaving
space for docking of candidate ligand to be docked into the protein. Before the actual
docking, protein structure was first repaired followed by appropriate protonation in
absence of ligand using the Protonate 3D module. The health of protein was checked by
plotting Ramchandran plot (Figure 5). Protein prepared in this manner were applied
directly for docking. During docking, the default parameters and settings using triangle
match placement method with London dG scoring were used for the docking except
that the number of retain were 20 instead of 30.
45. Niu Huang, N.; Shoichet, B. K. J. Med. Chem. 2008, 51, 4862–4865.
46. Kroemer, R. T. Curr. Protein Pept. Sci. 2007, 8, 312-328.
12

Table 1. In vitro COX-1 and COX-2 enzyme inhibitory activities of flavones bearing
pyrazole scaffold (10a-n)
Compound
% inhibition of COX (100 μM)a
CLogP
COX-1
54.1
COX-2
41.75
43.33
38.06
42.88
50.12
40.60
42.80
51.20
46.48
46.28
42.27
43.33
41.75
37.95
43.33
3.284
3.427
3.570
4.460
2.944
3.500
3.251
2.165
2.670
3.142
4.147
3.027
4.210
3.570
--
10a
10b
54.1
51.58
46.08
48.42
52.76
51.58
45.67
20.38
59.99
54.1
10c
10d
10e
10f
10g
10h
10i
10j
10k
47.38
48.86
52.18
70.07
10l
10m
10n
Indomethacin
aThe determination was performed in duplicate for two independent experiments;
13

Table 2. Effects of various synthesized compounds on carrageenan-induced paw edema in
rats with different time intervals
Paw volume (ml) Mean± SEM at various time intervals
Treatment and dose
(n=6)
(100 mg/kg.p.o.)
1 h
3 h
6 h
Normal group
0.40±0.04
0.94±0.05#
0.86±0.05
0.77±0.1
0.76±0.1
0.78±0.3
0.89±0.4
0.79±0.5
0.79±0.5
0.86±0.1
0.83±0.4
0.87±0.6
0.91±0.2
0.81±0.2
1.04±0.06
0.79±0.4
0.88±0.3
0.35±0.04
1.17±0.1#
0.95±0.07*
0.91±0.1
1.05±0.2*
1.01±0.4
1.11±0.6
0.96±0.7*
0.99±0.7
0.92±0.1
0.95±0.6*
0.94±0.4*
1.18±0.6
0.99±0.03*
1.00±0.04
1.46±0.08
1.24±0.2
0.38±0.06
1.69±0.08##
0.78±0.05**
1.22±0.1
Carrageenan control
Indomethacin
10a
1.17±0.1*
1.44±0.9
10b
10c
1.32±0.5
10d
0.99±0.5**
1.20±0.5
10e
10f
1.07±0.3*
0.97±0.6**
0.88±0.7**
1.55±0.6
10g
10h
10i
10j
10k
10l
1.11±0.1*
1.02±0.04*
1.01±0.6*
1.44±0.3
10m
10n
##p < 0.01, #p < 0.05 when compared with normal control group; **p < 0.01, *p < 0.05 when
compared with carrageenan group
14

Table 3. Effects of various prodrugs on acetic acid induced vascular permeability in mice
Amount of dye
leakage (µg / mL)
Mean±SEM
0.42±0.02
Inhibition of dye leakage
(%)
Treatment and dose
(100 mg / kg.p.o.)
Normal control
--
Vehicle treated control
22.03 ±1.42##
6.52±0.47**
10.59±1.7**
7.01±0.8**
16.44±2.6*
6.11 ±1.0**
8.7±0.9**
--
indomethacin
10b
70.40
51.92
68.17
25.32
72.26
60.50
35.08
33.27
57.78
10e
10g
10h
10i
14.3±2.5*
10k
14.7±1.4*
10l
9.3±2.1**
10m
##p < 0.01 when compared with normal control group; **p < 0.01, *p < 0.05 when compared
with vehicle treated control group
15

H2NO2S
H2NO2S
CF3
H2NO2S
N
N
N
N
N
N
CF3
CHF2
F
H3C
Br
O
Deracoxib
Celecoxib
N
SC-558
Cl
Cl
H3C HN
N
Cl
O
N
N
H3C
O
N
N
N
HN N
O
Famprofazone
Ramifenazone
Rimonabant
O
O
Cl
F3C
Cl
O
NH2
N
O
S
CF3
N
N
N
N
O
O
N
CN
Cl
N
O
O
N
N
CN
Fipronil
10h
Pyraclonil
Figure 1. Structures of some known pyrazole NSAIDs and compound 10h
16

11
11
O
7
O
7
4
8
9
8
6
5
6
5
9
10
12
12
10
4
O
O
O
O
Symbol Correlations
HMBC
13
3
3
N
N
2
2
1
1
N
N
COSY
13
6
7
Figure 2. HMBC (H to C) and 1H-1H COSY correlations
17

(a)
(b)
Figure 3. (a) 3D (b) 2D representation for docking pose of 10h in active site of COX-2.
18

Figure 4. Summary of SAR study of flavone derivatives for COX-2 selectivity
19

Figure 5. Ramchandran plot for 3NTG after optimization
20

O
O
O
O
O
a
b
O
O
O
OH
R
O
OH
N
N
N
N
N
N
6
8
9a-n
c
O
O
O
O
R
N
N
10a-n
F
Cl
OMe
OMe
F
O
R
=
O
F
F
Cl
Br
F
(a)
(b)
(d)
(e)
(f)
(g)
(c)
Cl
F
OMe
NO2
Cl
O
S
F
MeO
OMe
(l)
(m)
(n)
(h)
(i)
(j)
(k)
Scheme 1. Reagents and conditions: (a) Ac2O, BF3.OEt2, 0oC-rt, N2, 3 h ; (b) RCHO, NaOH,
EtOH, rt, 24 h; (c) I2, DMSO, reflux, 12 h.
21

O
O
O
a
b
c
O
OH
O
O
O
O
O
O
1
2
3
O
O
O
O
d
e
+
O
H
O
O
O
NH
O
O
O
O
O
N
N
N
N
N
O
4
5
7
6
(18%)
(82%)
o
Scheme 2. Reagents and conditions: (a) CH3COCl, AlCl3 (anh.), Et2O, N2, 0 C - rt; (b)
Me2SO4, K2CO3, acetone, reflux, 12 h; (c) HCOOEt, NaH, THF, N2, rt; (d) NH2NH2.H2O, rt;
(e) MeI, NaH, DMF, N2, 0 oC
22