Synthesis of new chemical entities from paracetamol and NSAIDs with improved pharmacodynamic profile

Article abstract of DOI:10.1016/j.bmc.2006.08.017

It was envisaged to combine high antipyretic activity of paracetamol into commonly used NSAIDs. To achieve this goal new chemical entities were synthesized by chemically combining paracetamol and NSAIDs, and biologically evaluated for their antipyretic, a

Full text of DOI:10.1016/j.bmc.2006.08.017

Bioorganic & Medicinal Chemistry 14 (2006) 8701–8706
Synthesis of new chemical entities from paracetamol
and NSAIDs with improved pharmacodynamic profile
Mange Ram Yadav,* Datta M. Nimekar, A. Ananthakrishnan,
Pathik S. Brahmkshatriya, Shrikant T. Shirude, Rajani Giridhar,
Arvind Parmar and R. Balaraman
Pharmacy Department, Faculty of Technology and Engineering, Kalabhavan, The M.S. University of Baroda, Vadodara 390 001, India
Received 5 June 2006; revised 11 August 2006; accepted 12 August 2006
Available online 7 September 2006
Abstract—It was envisaged to combine high antipyretic activity of paracetamol into commonly used NSAIDs. To achieve this goal
new chemical entities were synthesized by chemically combining paracetamol and NSAIDs, and biologically evaluated for their anti-
pyretic, analgesic, anti-inflammatory and ulcerogenic potential. The acid chloride of parent NSAIDs was reacted with excess of p-
aminophenol to yield the desired p-amidophenol derivatives (1B–7B). Acetate derivatives (1C–7C) of these phenols (1B–7B) were
also prepared by their treatment with acetic anhydride, in order to see the impact of blocking the free phenolic group on the bio-
logical activity of the derivatives. All the synthesized p-amidophenol derivatives showed improved antipyretic activity than paracet-
amol with retention of anti-inflammatory activity of their parent NSAIDs. These compounds elicited no ulcerogenicity unlike their
parent drugs.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
undesired side effects mainly on the gastrointestinal (GI)
tract. NSAIDs are known to have inhibitory activity for
both the isoforms of the cyclooxygenase (COX) enzyme.
They vary considerably in their tendency to cause gastric
erosions and ulcers.1 Gastric damage by these agents is
caused by at least two distinct mechanisms. One is by
inhibiting the cytoprotective COX-1 in the stomach and
second by physical contact and ion-trapping mecha-
nism.5,6 The use of prodrugs to temporarily mask acidic
group of NSAIDs has been postulated as an approach
to decrease the GI toxicity due to direct contact effect.7,8
These prodrugs release the parent moieties after absorp-
tion by undergoing enzymatic/chemical hydrolysis. On
the other hand selective inhibition of the inducible
COX-2 enzyme, sparing the constitutive COX-1, formed
the basis for designing of COXIBs with minimum degree
of ulcerogenic risk. This new concept of treating inflam-
mation related disease came into effect with the consecu-
tive launch of celecoxib9 and rofecoxib.10,11
Paracetamol is a commonly used analgesic and antipyret-
ic drug. However, it has only weak anti-inflammatory
effects. The failure of paracetamol to exert anti-inflamma-
tory activity may be attributed to the fact that paraceta-
mol is only a weak inhibitor of COX in the presence of
high concentration of the peroxides that are found in
the inflammatory lesions.1 In contrast, its antipyretic ef-
fect may be explained by its ability to inhibit COX in
the brain, where peroxide tone is low.2 Single or repeated
therapeutic doses of paracetamol have no effect on the
cardiovascular and respiratory system nor does the drug
produce gastric irritation/erosion. Recent reports3,4
suggest that overdose of paracetamol is the most com-
mon cause of acute liver failure (ALF) in United States
and United Kingdom.
Nonsteroidal anti-inflammatory agents (NSAIDs) are the
most commonly prescribed drugs in the world but, their
use as anti-inflammatory, antipyretic, antithrombotic
and analgesic agents continues to be limited due to their
Willoughby and colleagues12 described the effect of some
selective COX-2 inhibitors and some dual (COX-1 and
COX-2) inhibitors on carrageenan pleurisy in the rat over
a time period ranging from 0 to 48 h after injection of the
irritant. This investigation gave surprising results. The
COX-2inhibitorsshowedanti-inflammatoryactivityearlyin
the inflammatory response, coincident with the expression
Keywords: Paracetamol; NSAIDs; Antipyretic activity; Analgesic
activity; Anti-inflammatory activity; Ulcerogenicity.
*
Corresponding author. Tel.: + 91 265 2434187; fax: +91 265 2423898/
2418927; e-mail: mryadav11@yahoo.co.in
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.08.017

8702
M. R. Yadav et al. / Bioorg. Med. Chem. 14 (2006) 8701–8706
of COX-2 protein as did the older dual inhibitors such as
indomethacin. However, by6 htheCOX-2inhibitorswere
without effect although the dual inhibitors still showed
efficacy. At this point the COX-2 protein as shown by
Western blotting was no longer present. This study
showed the supremacy of conventional NSAIDs over the
COXIBs for chronic use. Moreover, withdrawal of rofec-
oxib from market recently by its originators due to adverse
cardiovasculareffectsputsa bigquestion mark on thesafe-
ty profile of other COXIBs in the long-term therapy.
2. Results and discussion
2.1. Chemistry
Commonly used carboxylic group containing NSAIDs
like ibuprofen (1A) naproxen (2A), 6-methoxy-2-naph-
thylacetic acid (6-MNA) (3A) (active metabolite of nab-
umetone (8)), ketorolac (4A), ketoprofen (5A),
flurbiprofen (6A) and biphenylacetic acid (7A) (active
metabolite of fenbufen (9)) were chosen for this study.
These compounds were converted into the acid chlorides
by treatment with thionyl chloride under anhydrous con-
ditions. The acid chloride was reacted with excess of
p-aminophenol in dioxane to yield a mixture containing
the desired p-amidophenol derivatives (1B–7B) and the
undesired 4-aminophenyl esters. The unwanted 4-amino-
phenyl esters were removed by washing the chloroform
solution of the mixture continuously with dilute hydro-
chloric acid until it became free from it. The products so
obtained were purified by repeated recrystallizations.
All the synthesized p-amidophenol derivatives were con-
verted into their acetates in order to see the impact of
blocking the free phenolic group on the biological activity
of the derivatives. The acetate derivatives (1C–7C) were
prepared by treatment of the free phenolic compounds
(1B–7B) with acetic anhydride in pyridine. The synthe-
sized compounds conform to the assigned structures, as
deduced on the basis of their spectral and elemental data.
Biphenylacetic acid (7A) was prepared by Friedel–Crafts
acetylation of biphenyl followed by Kindler-modified
Willgerodt reaction of the 4-acetyl derivative.14 Similarly,
6-MNA (3A) was obtained by carrying out Friedel–Crafts
acetylation of nerolin15 and then submitting the product
so obtained to Kindler-modified Willgerodt reaction to
obtain the desired product (3A).16
It has been reported13 that conversion of the carboxylic
group containing NSAIDs to ester and amide functions
makes them more selective towards COX-2 enzyme. Tak-
ing the above said report and the potential cardiovascular
dangers posed by COXIBs into cognizance, and the cost
and time involved in the discovery of a new drug, it was
thought of converting some common NSAIDs into
p-amidophenol derivatives. p-Aminophenol has been
evaluated in the past1 as analgesic–antipyretic but its N-
acetylated derivative, that is, paracetamol (10) has been
found to be the most suitable, therapeutically. It was
planned to substitute the acetyl group in paracetamol
(10) with the carboxylic group containing NSAIDs as
the acyl groups. Threefold advantages were visualized
through such chemical conversion. First, the free carbox-
ylic group present in the NSAIDs would be blocked by a
nonhydrolysable (at physiological pH) amide linkage
thereby preventing the local contact mechanism, which
was partially responsible for GIT ulceration by these
NSAIDs. Second, these new derivatives are expected to
show more selectivity towards COX-2 enzyme as report-
ed13 for the amide derivatives for NSAIDs, resulting in
further reduction in ulcerogenicity of the parent NSAIDs.
Finally, due to the structural resemblance of these amides
with paracetamol they are expected to exhibit substantial
antipyretic effect like the parent drug paracetamol (10).
Y
2.2. Biological
The compounds (1B–7C) were synthesized with a view
to incorporate the antipyretic activity component of
paracetamol (10) into the NSAIDs with their normal
anti-inflammatory activity but without GIT ulceration.
So, all the synthesized derivatives (1B–7C) were evaluat-
ed for their antipyretic activity in animal model17 using
lipopolysaccharide (LPS) (from Escherichia coli) endo-
toxin for producing pyrexia. Percent reversal of the
body temperature (antipyretic activity) was determined
(Table 1) using paracetamol (10) as the standard antipy-
retic drug. Interesting results were obtained as shown in
Figure 1. All the compounds with free phenolic group
have shown superior antipyretic activity than paraceta-
mol (10). It may be noted that this activity was deter-
mined on equal weight bases with paracetamol for all
the compounds. That means the p-amidophenol compo-
nent in the derivatives (1B–7C) on molar basis is much
less than that present in paracetamol (10). But, free phe-
nolic group seems to be essential in these compounds
also, like paracetamol (10) for superior antipyretic activ-
ity as the acylation of the phenolic hydroxyl group has
led to decrease in antipyretic activity.
X
Me
O
Me
Me
X
MeO
O
(2) Y = Me
(3) Y = H
(1)
Me
O
O
N
X
X
O
O
(4)
(5)
Y
(A) X =
(B) X =
OH
X
N
H
OH
O
z
(6) Y = Me ;Z = F
(7) Y = H; Z = H
N
H
OAc
(C) X =
O
O
Me
OH
O
MeO
(9)
(8)
H
N
Me
OH
O
COX-2 inhibiting activity for the synthesized com-
pounds was determined using the Cayman COX-2
(10)

M. R. Yadav et al. / Bioorg. Med. Chem. 14 (2006) 8701–8706
Table 1. Biological activities of synthesized compounds
8703
Compound
% inhibition
of COX-2 at
22 lM concn
Anti-inflammatory
activity
Ulcerogenic potential
Analgesic activity
Antipyretic activity
Pyrexia % reversal
of body temperature
Dose
mg/kg
% inhibition
Dose
mg/kg
Ulcer index
Dose
mg/kg
% inhibition
Dose
mg/kg
1A
1B
8.4
10.4
20
28.8
60.0
34.8
200
290
0.5013 0.021
Nil
20
28.8
41.9
19.1
—
25
10
25
10
—
25
10
25
—
25
25
—
25
25
ND
87.4
49.3
75.0
43.5
ND
59.5
25.7
12.5
ND
75.0
68.7
ND
55.8
37.5
ND
50.0
ND
58.5
18.5
ND
82.4
67.5
35.5
63.8
39.9
1C
NI
32.9
38.7
330
Nil
32.9
36.5
2A
2B
81.8
12.7
20
27.9
81.1
39.4
200
280
0.981 0.038
Nil
20
27.9
43.0
20.7
2C
3A
3B
3C
4A
4B
4C
5A
5C
6A
6B
6C
7A
7B
7C
NI
31.6
20
27.6
55.4
44.6
41.5
75.0
86.4
83.5
86.7
72.3
84.0
68.9
58.1
60.9
57.9
59.6
316
200
284
323
75
Nil
31.6
20
21.8
42.2
30.8
28.5
54.1
52.6
58.2
74.8
68.4
54.9
45.8
52.3
51.7
47.8
45.2
10.5
8.9
0.630 0.052
28.4
32.3
10
Nil
Nil
28.4
32.3
20
NI
100
31.8
11.1
100
4.3
0.484 0.030
13.5
15.2
20
101.3
114
200
295
25
Nil
Nil
25
30.4
25
0.822 0.039
29.5
8
Nil
36.9
10
25
—
25
25
—
25
25
10
25
10
15.4
12.3
NI
0.80 0.048
11
34.4
38.8
500
715
815
Nil
Nil
13.1
15.5
25
12.4
10
10.5
8.9
0.564 0.069
14.3
16.3
Nil
Nil
35.8
40.8
NI
10
—
—
—
—
—
—
—
NI, no inhibition at the test dose; ND, not determined.
100
90
80
70
60
50
40
30
20
10
0
10 1B 1C 2B 2C 3B 3C 4B 4C 5C 6B 6C 7B 7C
Compound (25 mg/kg)
Figure 1. Antipyretic activity of synthesized compounds in rats.
colorimetric screening kit.18 No uniform conclusion
could be drawn on the basis of this study except that
all the acetylated derivatives showed no inhibition at
the test dose level. So, it was planned to perform anti-in-
flammatory activity in the in vivo model using carra-
geenan-induced paw edema.19 The activity was carried
out on equimolar basis to the parent drug. The results
of the study (Table 1) indicate that the conversion of
free carboxylic group in the NSAIDs to the amide link-
age caused reduction in the anti-inflammatory activity
of the parent drugs, in general. However, ketorolac
(derivative 4B) was found to be an exception where an
increase in inflammatory activity was noticed. Acetyla-
tion of free phenolic group was observed not to change
this activity drastically. Since NSAIDs do possess
peripheral analgesic activity, the synthesized derivatives
were evaluated for this activity using the writhing meth-
od20 in mice. No straightforward conclusion could be
drawn from this study except that all the synthesized
derivatives possessed analgesic activity with somewhat
less potency than their parent NSAIDs.
Ulcerogenic potential of the synthesized compounds was
determined in rat model.21 It was very encouraging to
note that none of the compounds showed any ulcer for-
mation in the test animals. This may be due to the dual
factors of blocking of the free acidic group and more
selectivity of these compounds to inhibit COX-2.

8704
M. R. Yadav et al. / Bioorg. Med. Chem. 14 (2006) 8701–8706
3. Conclusion
for C19H23NO2: C, 76.74; H, 7.80; N, 4.71. Found: C,
76.48; H, 7.53; N, 4.38.
Conversion of the conventional carboxylic group con-
taining NSAIDs into p-amidophenol derivatives has
resulted into new potential drugs having much improved
antipyretic activity. These compounds also possessed
sufficiently good potency to be used as anti-inflammato-
ry drugs with nil ulcerogenicity in acute model of this
biological testing. Keeping in view the therapeutic supe-
riority of the classical NSAIDs over COXIBs on longer
duration of usage, newer potential compounds have
been developed possessing high analgesic–antipyretic
activity with nil/minimum GIT ulceration. Acetylation
of free phenolic group in these synthesized compounds
was not found to be a suitable proposition as it
decreased the potency.
4.2.2. 4-[2-(6-Methoxy-2-naphthyl)propionamido]phenol
(2B). Yield 35%; mp 156–159 ꢁC; IR (KBr): 3327, 1652,
1
1539, 1512, 1226, 826 cmꢀ1; H NMR (CDCl3): d 1.60
(d, 3H), 3.86–3.89 (q, 1H), 3.91 (s, 3H), 8.90 (br s, 1H),
6.65–6.69 (m, 2H), 7.10–7.15 (m, 1H), 7.32–7.55 (m,
4H), 7.69–7.77 (m, 3H). Anal. Calcd for C20H19NO3: C,
74.75; H, 5.96; N, 4.36. Found: C, 74.94; H, 6.19; N, 4.52.
4.2.3. 4-[2-(6-Methoxy-2-naphthyl)acetamido]phenol (3B).
Yield 38%; mp 201–203 ꢁC; IR (KBr): 3285, 1657, 1539,
1
1512, 825 cmꢀ1; H NMR (DMSO-d6): d 3.68 (s, 2H),
3.85 (s, 3H), 6.64–6.67 (m, 2H), 7.10–7.14 (m, 1H),
7.33–7.43 (m, 4H), 7.70–7.77 (m, 3H), 9.15 (br s, 1H),
9.92 (br s, 1H). Anal. Calcd for C19H17NO3: C, 74.25;
H, 5.58; N, 4.56. Found: C, 73.91; H, 5.24; N, 4.28.
4. Experimental
4.1. Chemistry
4.2.4. 5-Benzoyl-N-(4-hydroxyphenyl)-2,3-dihydro-1H-
pyrrolizine-1-carboxamide (4B). Yield 43%; mp 228–
229 ꢁC; IR (KBr): 3300, 1661, 1651, 1513, 1240,
1
Melting points were determined using a heating block-
type melting point apparatus and are uncorrected. Puri-
ty of the compounds was ascertained by thin-layer chro-
matography (TLC) on silica gel plates (60 F254; Merck),
visualizing with ultraviolet light or iodine vapors. The
yields reported here are unoptimized. IR spectra were
recorded using KBr disc method on a Shimadzu FT-
IR Model 8300. 1H NMR spectra on a Brucker
300 MHz spectrometer were recorded in CDCl3 (chemi-
cal shifts in d ppm). Assignment of exchangeable pro-
tons (NH) was confirmed by D2O exchange studies.
Elemental analyses were obtained on Carlo Erba, Italy,
and Perkin-Elmer instruments. All the compounds were
purified by recrystallization from acetone–pet. ether.
725 cmꢀ1; H NMR (CDCl3): d 2.81 (m, 2H), 4.07 (m,
1H), 4.52 (m, 2H), 6.09 (br s, 1H), 6.64–6.68 (m, 2H),
6.89–6.90 (d, 1H), 7.44–7.57 (m, 6H), 7.81–7.85 (m, 2H).
Anal. Calcd for C21H18N2O3: C, 72.82; H, 5.24; N, 8.09.
Found: C, 72.99; H, 5.52; N, 8.27.
4.2.5. 4-[2-(3-Benzoylphenyl)propionamido]phenol (5B).
Sticky material. Yield 47%; IR (KBr): 3286, 1652,
1535, 1510, 834 cmꢀ1
.
4.2.6. 4-[2-(2-Fluoro-4-biphenyl)propionamido]phenol (6B).
Yield 40%; mp 170–173 ꢁC; IR (KBr): 3291, 1652, 1539,
1531, 1237, 827 cmꢀ1 1H NMR (CDCl3): d 1.61 (d,
;
3H), 3.68–3.71 (q, 1H), 6.97 (br s, 1H), 6.74–6.77 (m,
2H), 7.16–7.23 (m, 2H), 7.28–7.47 (m, 6H), 7.53–7.55
(m, 2H). Anal. Calcd for C21H18FNO2: C, 75.21; H,
5.41; N, 4.18. Found: C, 75.66; H, 5.22; N, 4.37.
4.2. General procedure for the preparation of compounds
1B–7B
4.2.1. Representative preparation of 4-[2-(4-isobutylphe-
nyl)propionamido]phenol (1B). Ibuprofen 1A (2 g) was
dissolved in dry toluene (25 ml) and thionyl chloride
(2 ml) was added dropwise with constant stirring. The
reaction mixture was heated at 80 ꢁC on a water bath
for 2 h, and excess thionyl chloride and toluene was re-
moved under vacuum. The residue so obtained was dis-
solved in dry dioxane (25 ml) and added dropwise to a
solution of p-aminophenol (5 g) in dioxane (50 ml) with
stirring. The reaction mixture was stirred for 1 h at room
temperature and heated on water bath for 3 h. Excess of
dioxane was removed under vacuum, the reaction mix-
ture acidified with dilute hydrochloric acid and extracted
with chloroform (3· 50 ml). The combined organic ex-
tract was washed successively with hydrochloric acid
(5%) until free from the basic impurities, dried over sodi-
um sulfate and solvent removed. The residue so ob-
tained was purified by crystallization from acetone–
pet. ether. Yield 50%; mp 112–115 ꢁC; IR (KBr): 3299,
4.2.7. 4-[2-(4-Biphenyl)acetamido]phenol (7B). Yield 15%;
mp 205–208 ꢁC; IR (KBr): 3246, 1651, 1546, 1514, 1245,
1
749 cmꢀ1; H NMR (CDCl3 + DMSO-d6): d 3.68 (s,
2H), 7.30 (br s, 1H), 6.72–6.75 (m, 2H), 732–7.45 (m,
7H), 7.53–7.58 (m, 4H), 9.24 (br s, 1H). Anal. Calcd for
C20H17NO2: C, 79.19; H, 5.65; N, 4.62. Found: C,
79.49; H, 5.83; N, 4.38.
4.3. General procedure for the preparation of compounds
1C–7C
4.3.1. Representative preparation of 4-[2-(4-isobutylphe-
nyl)propionamido]phenyl acetate (1C). Compound 1B
(0.4 g) was dissolved in dry pyridine (1 ml) and cooled
in ice bath. Acetic anhydride (1.0 ml) was added drop-
wise with stirring. Reaction mixture was heated on
water bath for 3 h and allowed to cool to room temper-
ature. It was poured over crushed ice containing conc
hydrochloric acid (3 ml). The solid was filtered, dried
and recrystallized from acetone–pet. ether. Yield 85%;
mp 145–147 ꢁC; IR (KBr): 3354, 1734, 1684, 1541,
1
1653, 1536, 1510, 1234, 827 cmꢀ1; H NMR (CDCl3):
d 0.90 (d, 6H), 1.59 (d, 3H), 1.85 (m, 1H), 2.45 (d,
2H), 3.68–3.71 (q, 1H), 6.95 (br s, 1H), 6.68–6.73 (m,
2H), 7.10–7.15 (m, 4H), 7.28–7.31 (m, 2H). Anal. Calcd
1510, 839 cmꢀ1 1H NMR (CDCl3): d 0.90 (d, 6H),
;
1.59 (d, 3H), 1.85 (m, 1H), 2.26 (s, 3H), 2.46 (d, 2H),

M. R. Yadav et al. / Bioorg. Med. Chem. 14 (2006) 8701–8706
8705
3.67–3.70 (q, 1H), 7.21 (br s, 1H), 6.96–7.01 (m, 2H),
7.10–7.16 (m, 4H), 7.39–7.44 (m, 2H). Anal. Calcd for
C21H25NO3: C, 74.31; H, 7.42; N, 4.13. Found: C,
74.68; H, 7.21; N, 4.01.
rat.17 Male Sprague–Dawley rats (150–200 g) were fast-
ed for 16–18 h before use and were divided into parallel
groups (n = 5). The animals were placed temporarily in
restrainer and the resting rectal temperature was record-
ed using a flexible temperature probe connected to Bio-
Pac data acquisition system. The same probe and system
were used for all animals to reduce experimental error.
The animals were returned to their respective cages after
the temperature measurement. Animals were injected
either normal saline or LPS (0.36 mg/kg, Sigma, USA)
intraperitoneally and the rectal temperature was mea-
sured at 0, 5, 6 and 7 h after LPS injection. At 5 h when
the increase in rectal temperature had reached a plateau,
the LPS injected rats were given orally either vehicle (1%
CMC) or test compound (suspended in 1% CMC) to
determine whether the rise in temperature could be
reversed. Percent reversal (Antipyretic activity) was
calculated using the rectal temperature obtained at 7 h
taking this value in the vehicle control group as zero
reversal.
4.3.2. 4-[2-(6-Methoxy-2-naphthyl)propionamido]phenyl
acetate (2C). Yield 90%; mp 212–216 ꢁC; IR (KBr):
3380, 1748, 1668, 1528, 1502, 857 cmꢀ1 1H NMR
;
(CDCl3): d 1.60 (d, 3H), 2.25 (s, 3H), 3.86–3.89 (q, 1H),
3.91 (s, 3H), 8.97 (br s, 1H), 6.95–7.00 (m, 2H), 7.09–
7.14 (m, 1H), 7.38–7.54 (m, 4H), 7.66–7.78 (m, 3H). Anal.
Calcd for C22H21NO4: C, 72.71; H, 5.82; N, 3.85. Found:
C, 72.52; H, 5.62; N, 3.67.
4.3.3. 4-[2-(6-Methoxy-2-naphthyl)acetamido]phenyl ace-
tate (3C). Yield 84%; mp 175–176 ꢁC; IR (KBr): 3305,
1
1750, 1651, 1539, 1506, 839 cmꢀ1; H NMR (DMSO-
d6): d 2.27 (s, 3H), 3.67 (s, 2H), 3.85 (s, 3H), 6.99–7.03
(m, 2H), 7.11–7.15 (m, 1H), 7.33–7.47 (m, 4H), 7.70–
7.77 (m, 3H) 9.06 (br s, 1H). Anal. Calcd for
C21H19NO4: C, 72.19; H, 5.48; N, 4.01. Found: C,
72.49; H, 5.64; N, 3.88.
4.5. In vitro COX inhibition assay
4.3.4. N-(4-Acetoxyphenyl)-5-benzoyl-2,3-dihydro-1H-
pyrrolizine-1-carboxamide (4C). Yield 89%; mp 197–
198 ꢁC; IR (KBr): 3260, 1755, 1661, 1539, 1508,
COX-2 inhibiting activity for the synthesized compound
and parent drugs was determined18 using the colorimetric
ovine cyclooxygenase (COX) assay kit (Cayman Chemi-
cal Company, MI, USA). This assay analyzes the peroxi-
dase activity of the enzymes using N,N,N0,N0-
tetramethylphenylenediamine (TMPD) as the reducing
co-substrate.22 Valdecoxib was used as the positive con-
trol for COX-2 inhibition. The compounds were dissolved
in DMSO and diluted in the assay buffer before use. The
assays were run according to the manufacturer’s
instructions.
1
720 cmꢀ1; H NMR (CDCl3): d 2.28 (s, 3H), 2.80 (m,
2H), 4.08 (m, 1H), 4.53 (m, 2H), 6.17 (br s, 1H), 6.89–
6.90 (d, 1H), 7.04–7.06 (m, 2H), 7.45–7.57 (m, 6H),
7.82–7.85 (m, 2H). Anal. Calcd for C23H20N2O4: C,
71.12; H, 5.19; N, 7.21. Found: C, 71.48; H, 5.41; N, 7.52.
4.3.5. 4-[2-(3-Benzoylphenyl)propionamido]phenyl acetate
(5C). Yield 49%; mp 87–88 ꢁC; IR (KBr): 3357, 1730,
1
1683, 1661, 1538, 1508, 838 cmꢀ1; H NMR (CDCl3):
d 1.62 (d, 3H), 2.27 (s, 3H), 3.72–3.76 (q, 1H), 7.18 (br
s, 1H), 6.98–7.02 (m, 2H), 7.44–7.52 (m, 5H), 7.57–
7.65 (m, 2H), 7.68–7.72 (m, 1H), 7.78–7.82 (m, 3H).
Anal. Calcd for C23H21NO4: C, 73.58; H, 5.64; N,
3.73. Found: C, 73.79; H, 5.38; N, 3.49.
4.6. In vivo carrageenan-induced rat paw edema assay
Anti-inflammatory activity was determined by using car-
rageenan-induced rat paw edema method described by
Winter et al.19 Fasted male Sprague–Dawley rats (150–
200 g) were divided in parallel groups (n = 5) and were
given orally either the vehicle (1% CMC) or test com-
pound as suspension in 1% CMC. The activity was carried
out on equimolar basis to the parent drug. A line was
drawn using permanent marker at the ankle of the left
hind paw to define the arc of the paw to be monitored.
The paw volume (V0h) was measured using a Plethys-
mometer (Ugo-Basile, Italy). The animals were then
injected subplantarly with 50 ll of 1% carrageenan (Sig-
ma, USA) in normal saline solution (i.e., 500 lg carra-
geenan per paw). Three hours after the carrageenan
injection, the paw volume (V3h) was measured, and the in-
crease in paw volume (V3h ꢀ V0h) was calculated. The in-
crease in paw volume was compared with that in the
vehicle control group, and percent inhibition was calcu-
lated taking the values in the control group as 0%
inhibition.
4.3.6. 4-[2-(2-Fluoro-4-biphenyl)propionamido]phenyl ace-
tate (6C). Yield 90%; mp 165–168 ꢁC; IR (KBr): 3357,
1
1732, 1684, 1539, 1509, 764 cmꢀ1; H NMR (CDCl3): d
1.62 (d, 3H), 2.27 (s, 3H), 3.68–3.71 (q, 1H), 7.09 (br s,
1H), 6.98–7.03 (m, 2H), 7.16–7.23 (m, 2H), 7.32–7.49
(m, 6H), 7.52–7.56 (m, 2H). Anal. Calcd for
C23H20FNO3: C, 73.20; H, 5.34; N, 3.71. Found: C,
72.77; H, 5.06; N, 3.95.
4.3.7. 4-[2-(4-Biphenyl)acetamido]phenyl acetate (7C).
Yield 90%; mp 185–188 ꢁC; IR (KBr): 3176, 1751, 1646,
1549, 1501, 746 cmꢀ1; 1H NMR (CDCl3): d 2.26 (s, 3H),
3.77 (s, 2H), 7.15 (br s, 1H), 6.98–7.03 (m, 2H), 733–
7.48 (m, 7H), 7.57–7.63 (m, 4H). Anal. Calcd for
C22H19NO3: C, 76.50; H, 5.54; N, 4.06. Found: C,
76.84; H, 5.81; N, 3.72.
4.4. Antipyretic activity
4.7. Analgesic activity
Antipyretic activity of synthesized compounds (1B–7C)
was determined on rats using lipopolysaccharide (LPS)
(from E. coli) endotoxin for producing pyrexia in
Analgesic activity of the derivatives was evaluated using
the acetic acid writhing model in mice as described
by Koster et al.20 Swiss albino mice (18–25) divided in

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drome was elicited by intraperitoneal administration of
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4.8. Ulcerogenic effect21
Sprague–Dawley rats (n = 6) of either sex were fasted for
36 h with water ad libitum prior to administration of the
derivative. The animals were further kept on fasting for
4 h after dosing. The derivatives as suspension in 1%
CMC or vehicle (1% CMC) were administered orally.
The animals were sacrificed by cervical dislocation and
their stomach was dissected out, cut along the greater
curvature, washed with normal saline and kept in for-
malin solution (5%) for 15 min and gastric mucosa
was observed for the lesions using a 2 · 2 binocular
magnifier and the ulcer index was determined using
the following formula as given below:
Ulcer index ¼ 10ðAu=AmÞ;
where Au = Al + Ac + Ap, Al is the area of linear lesions
(l b), Ac is the area of circular lesions (Pr2), Ap is the
total no. of petechiae/5, and Am is the total mucosal
*
area (PD2/8) (D = diameter of stomach).
Acknowledgments
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Authors are grateful to University Grants Commission
(UGC), New Delhi, for the grant of a major research
project and to All India Council for Technical Educa-
tion (AICTE) for the award of National Doctoral
Fellowship (NDF) to S.T.S.
20. Koster, R.; Anderson, M.; de Beer, E. J. Fed. Proc. 1959,
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References and notes
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