Synthesis and SAR/3D-QSAR studies on the COX-2 inhibitory activity of 1,5-diarylpyrazoles to validate the modified pharmacophore

Article abstract of DOI:10.1016/j.ejmech.2005.03.016

Diverse analogs of 1,5-diarylpyrazoles having 3-hydroxymethyl-4-sulfamoyl (SO₂NH₂)/methyl sulfonyl (SO₂Me)-pheny group at N¹ were synthesized and evaluated for their in vitro cyclooxygenase (COX-1/COX-2) inhibitory activity. The SAR study mainly involved the variations at positions C-3, C-5 and N¹ of the pyrazole ring. Several small hydrophobic groups at/around position-4 of C-5 phenyl, viz. 3,4-dimethylphenyl analog 9, 3-methyl-4-methylsulfanylphenyl analog 14 and 2,3-dihydrobenzo[b] thiophenyl analog 17, exhibited impressive COX-2 inhibitory potency. In general, the sulfonamide analogues with a CHF₂ at C-3 were found to be more potent than those having a CF₃ group. The three dimensional quantitative structure activity relationship comprising comparative molecular field analysis (3D-QSAR-CoMFA) afforded the models with high predictivity which further validated the acceptance of hydroxymethyl (CH₂OH) group in the hydrophilic pocket of the COX-2 enzyme.

Full text of DOI:10.1016/j.ejmech.2005.03.016

European Journal of Medicinal Chemistry 40 (2005) 977–990
www.elsevier.com/locate/ejmech
Original article
Synthesis and SAR/3D-QSAR studies on the COX-2 inhibitory activity
of 1,5-diarylpyrazoles to validate the modified pharmacophore
Sunil K. Singh a,*, V. Saibaba a, K. Srinivasa Rao a, P. Ganapati Reddy a, Pankaj R. Daga a,
S. Abdul Rajjak a, Parimal Misra b,Y. Koteswar Rao a
a Discovery Chemistry, Discovery Research-Dr. Reddy’s Laboratories Ltd., Bollaram Road, Miyapur, Hyderabad 500 049, India
b Discovery Biology, Discovery Research-Dr. Reddy’s Laboratories Ltd., Bollaram Road, Miyapur, Hyderabad 500 049, India
Received 10 October 2004; received in revised form 7 March 2005; accepted 23 March 2005
Available online 14 June 2005
Abstract
Diverse analogs of 1,5-diarylpyrazoles having 3-hydroxymethyl-4-sulfamoyl (SO2NH2)/methyl sulfonyl (SO2Me)-pheny group at N1
were synthesized and evaluated for their in vitro cyclooxygenase (COX-1/COX-2) inhibitory activity. The SAR study mainly involved the
variations at positions C-3, C-5 and N1 of the pyrazole ring. Several small hydrophobic groups at/around position-4 of C-5 phenyl, viz.
3,4-dimethylphenyl analog 9, 3-methyl-4-methylsulfanylphenyl analog 14 and 2,3-dihydrobenzo[b]thiophenyl analog 17, exhibited impres-
sive COX-2 inhibitory potency. In general, the sulfonamide analogues with a CHF2 at C-3 were found to be more potent than those having a
CF3 group. The three dimensional quantitative structure activity relationship comprising comparative molecular field analysis (3D-QSAR-
CoMFA) afforded the models with high predictivity which further validated the acceptance of hydroxymethyl (CH2OH) group in the hydro-
philic pocket of the COX-2 enzyme.
© 2005 Elsevier SAS. All rights reserved.
Keywords: COX-2 inhibitors; 1,5-Diarylpyrazoles; SAR; 3D-QSAR
1. Introduction
matory disorders with greater GI safety. This new concept
was proved in clinic with the subsequent launch of two drugs,
viz. celecoxib [11] and rofecoxib [12] for the treatment of
rheumatoid and osteoarthritis. The continued effort in this area
further introduced valdecoxib [13] and etoricoxib [14,15] as
second generation therapy for the chronic inflammation
(Fig. 1). Apart from inflammation, few COX-2 inhibitors are
also being studied for treating other ailments like cancer
[16,17] and Alzheimer’s disease [18]. Though few adverse
reports have also appeared about this treatment [19], by and
large the patients on COX-2 inhibitors enjoy a greater benefit
over the conventional NSAIDs therapy.
Unlike the diverse chemical classes of non-selective
NSAIDs, the COX-2 inhibitors have two major structural
motifs: the acidic methanesulfonamide (MeSO2NH) contain-
ing diphenyl ethers exemplified by nimesulide [20,21] and
sulfamoyl (SO2NH2)/methanesulfonyl (SO2Me) containing
vicinal diarylheterocycles represented by celecoxib, valde-
coxib, rofecoxib and etoricoxib (Fig. 1). The latter class
received more attention due to the known COX-2 enzyme-
ligand co-crystal structure [22], and various carbocycles
The cyclooxygenase (COX) exists in two isoforms [1–3].
The COX-1, a constitutive enzyme, primarily expressed in
gastrointestinal (GI) tract, is responsible for the biosynthesis
of prostaglandins (PGs) required for the cytoprotection and
platelet aggregation [4,5]. Hence, any disturbance in its rou-
tine function for long time leads to gastrointestinal ulcer-
ation, bleeding and perforation [6,7]. In contrast, the COX-2,
an inducible enzyme, produced during injury by the pro-
inflammatory cytokines, viz. tumor necrosis factor-a (TNF-
a), interleukines, mitogens and endotoxins, plays a major role
in the biosynthesis of PGs required by inflammatory cells such
as monocytes and macrophages to cause pain, inflammation
and fever [8–10]. Thus, the selective inhibition of COX-2,
sparing COX-1, emerged as the new way of treating inflam-
> DRL Publication No. 283 B.
* Corresponding author. Tel.: +91 40 2304 5439; fax: +91 40 2304
5438/5007.
E-mail address: sunilkumarsingh@drreddys.com (S.K. Singh).
0223-5234/$ - see front matter © 2005 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2005.03.016

978
S.K. Singh et al. / European Journal of Medicinal Chemistry 40 (2005) 977–990
2. Chemistry
Disconnection for the synthesis of desired 1,5-
diarylpyrazoles 4–46 for SAR study is depicted in Scheme 1.
The basic requirement for the synthesis of these pyrazoles
was the availability of corresponding phenyl hydrazine hydro-
chlorides 1–2, the multi-step synthesis of which will be dis-
cussed elsewhere. Simple coupling of the latter with appro-
priate 1,3-diketones 3 in absolute ethanol under heating
condition afforded the desired diarylpyrazoles 4–46 (Scheme
2) [29–31,33]. Though the regiomeric bias was normally in
favor of 1,5-diarylpyrazoles, the minor undesired regio iso-
mers were easily eliminated by triturating the product with a
mixture of ethyl acetate–toluene after column chromato-
graphic purification. The required 1,3-diketones 3 were syn-
thesized by Claisen condensation using appropriate etha-
nones and ethyl/methyl ester of trifluoroacetic acid,
difluoroacetic acid, fluoroacetic acid, acetic acid, formic acid,
propionic acid and oxalic acid. This reaction was carried out
under slightly modified condition involving sodium hydride
in dry DMF at a temperature of –5 to 30 °C to afford 1,3-
diketones in 95–98% yield. The commercially unavailable
ethanones (substituted acetophenones) were prepared accord-
ing to the standard literature procedures [11]. The sulfoxide
(SOMe) analog 16 was prepared by the controlled oxidation
of corresponding methylsulfanyl (SMe) analog 14 using
m-CPBA at 0–5 °C [34]. The 3-carboxylic acid analog 47
was synthesized by the saponification of corresponding
methyl ester of 1,5-diarylpyrazole (Scheme 3). The 3-cyano
analog 48 was prepared by converting this ester to correspond-
ing carboxamide by stirring with aqueous ammonia in a sealed
flask at room temperature and dehydrating the latter with
TFFA–pyridine [35].All the compounds reported herein were
characterized spectroscopically.
Fig. 1. COX-2 inhibitors.
[23,24] and heterocycles [25–27] were discovered on this
basis. Though many COXIBs (COX-2 inhibitors) have been
launched so far, there still remains a need to develop more
effective drugs as an alternative to the steroidal and narcotic
drugs used in severe pains.
While introduction of small hydrophobic groups (F, Me)
adjacent to sulfonamide in diarylheterocycles and their influ-
ence on binding with the hydrophilic region of COX-2 enzyme
have yielded encouraging results [28,29], the effect of hydro-
philic groups at this position has not yet been explored. Thus,
we contemplated to introduce few such groups, viz. hydroxy-
alkyl and acylaminoalkyl adjacent to sulfonamide to study
their binding as well as pharmacological impact on the COX-
2 enzyme. Few compounds with the smallest hydroxymethyl
(CH2OH) group have already shown effective binding with
the enzyme and impressive COX-2 selectivity [30,31]. With
a view to optimize and validate the effect of this change, we
synthesized its diverse analogues involving C-3, C-5 and N1,
and report herein the outcome of the SAR and 3D-QSAR-
CoMFA [32] study on the series.
Scheme 1
.
Scheme 2. (a) NaH/DMF, –5 → 30 °C, 4–5 h. (b) 2 N HCl. (c) Absolute ethanol, 50–60 °C, 12 h.

S.K. Singh et al. / European Journal of Medicinal Chemistry 40 (2005) 977–990
979
Scheme 3. (a) NaH/DMF, –5 → 30 °C, 4–5 h. (b) 2 N HCl. (c) Absolute ethanol, 50–60 °C, 12 h. (d) NaOH/MeOH, 5 → 35 °C, 3–4 h. (e) Aq. NH3/MeOH,
20–25 °C, 15 h. (f) TFAA/Pyridine, dioxane, room temperature, 14 h.
3. Pharmacology
halogenated analogs [30]. Combination of two methyl groups
such as 3,4-dimethyl analog 9 enhanced the COX-2 potency
further (0.411 µM) whereas 2,4-dimethyl analog 8 lost the
activity drastically (8.500 µM). Generally 3,4-disubstituted
analogs such as 3-methyl-4-fluoro analog 10 (0.830 µM),
3-chloro-4-methoxy analog 13 (1.300 µM), 3-methyl-4-
methylsulfanyl 14 (0.139 µM) and 3-methoxy-4-
methylsulfanyl 15 (1.390 µM) exhibited a far superior potency
than the 2,4-disubstituted analogs 6, 8 and 11. This observa-
tion can be attributed to the bulkier groups which when near
to the central heterocyclic ring (e.g. position-2 of the C-5 phe-
nyl ring) may be causing distortion to the cis-stilbene geom-
etry of the vicinal diaryl system required for COX-2 potency.
Electronic factor seems to be less important at position-2, as
fluorine even being highly electronegative shows better activ-
ity because of its small size (e.g. compound 4) causing less
geometrical distortion. On the other hand the electronic fac-
tor becomes very important at/around position-4 and fluorine
being highly electronegative decreases the activity of com-
pound 5. Such could be the reason for the most potent com-
pound 14 becoming inactive when converted to the corre-
sponding sulfoxide analog 16. However, suitable hydrophobic
groups such as in 3,4-disubstituted compounds 7, 9, 10, 13–15
being favorable, enhanced the potency dramatically. Observ-
ing the favorable effect of hydrophobic groups at positions-
3 and 4, a similar hydrophobic environment was created by
introducing partially saturated fused heterocycles and car-
bocycles such as 2,3-dihydrobezothiophenyl analog 17,
6-chromanyl analog 18 and 1,2,3,4-tetrahydronaphthyl ana-
log 19. While 17 being prototype of the most potent com-
pound 14, remained the best (0.227 µM) among these, 18 and
19 also showed COX-2 activity even after being reasonably
bulkier. But the cyclohexyl analog 20 completely turned out
to be non-selective. The fused aromatics, 6-methoxynaphthyl
analog 21 and 3-indolyl analog 22 also exhibited very good
COX-2 activity but the simple heterocycles such as 3-thienyl
23 and 3-pyridyl 24 were found neither active nor selective.
Possible reason for the poor activity of 23 and 24 could be
the lack of effective access of these C-5 substitutions to the
hydrophobic region of the COX-2 enzyme.
Initially, all the compounds were screened for their ability
to inhibit COX-2 (recombinant human enzyme, expressed in
sf-9 cells infected with baculovirus) and COX-1 (obtained
from microsomal fraction of Ram Seminal Vesicles) at
100 µM. Based on the initial in vitro efficacy, the promising
compounds were further tested at lower concentrations. The
enzyme activity was measured by TMPD method and IC50s
were calculated using non-linear regression analysis of per-
cent inhibitions [36,37]. The in vitro potent compounds were
preliminary screened in the carrageenan induced rat paw
edema model of inflammation [38]. Celecoxib and indometha-
cin were used as reference COX-2 selective and non-selective
inhibitors respectively.
3.1. Results and discussion
The analogs of the new series have broadly been divided
into three classes based on the site of variation. Compound
25 was selected from the previous study for the ease of com-
parison.
3.1.1. Variation at position C-5
Since we already observed a trend of COX-2 selectivity
due to monosubstituted phenyl group at this position [30],
we opted here the diverse variations like disubstituted phe-
nyls, heterocycles, fused carbocycles, fused heterocycles,
saturated carbocycles, partially saturated fused carbocycles
and partially saturated fused heterocycles for the study. The
in vitro COX-1/COX-2 inhibitory activity of these analogs is
depicted in Table 1. Among dihalogenated phenyls, 2,4-
difluoro analog 4 (1.680 µM) was found to be more potent
than the corresponding dichloro analog 6 (5.100 µM). While
3,4-difluoro analog 5 suffered a loss in activity (6.900 µM),
the combination of both, 3-chloro-4-fluoro analog 7 drasti-
cally improved the COX-2 potency (0.570 µM). Lesser
potency of compound 5 was not surprising because simple
4-fluoro analog also showed least potency among the mono

980
S.K. Singh et al. / European Journal of Medicinal Chemistry 40 (2005) 977–990
Table 1
fluorine atoms were gradually removed from one of the most
COX-2 selective compounds 25 containing a CF3 group [30].
The CHF2 analog 26 exhibited a reasonable improvement in
potency (0.236 µM) whereas CH2F analog 27 was found to
be almost inactive. In contrast, the CH3 analog 28, again
turned out to be selective inhibitor of COX-2 (0.660 µM).
But, complete removal of this methyl group, e.g. compound
29 and its replacement with an ethyl group e.g. compound
30, led to complete loss of activity. This was a unique obser-
vation where methyl group at C-3 was found to bring COX-
2 selectivity. Compound 31 was synthesized to further con-
firm this observation and was found to have similar effect.
The variation observed due to the above fluorinated methyl
substituents at C-3 in compounds 25–27 can mainly be attrib-
uted to the number of fluorine atoms causing deviation in the
net dipole moment and finally in the orientation of the mol-
ecules from the COX-2 active site. But, the activity of the
C-3 methyl analogs 28 and 31 could only be explained on the
basis of its suitable size for hydrophobic interaction with the
COX-2 enzyme. The corresponding hydrogen and ethyl
replacement of this methyl group was found to be inactive
probably due to the ineffective hydrophobic interaction with
In vitro data of C-5 substituted 1,5-diarylpyrazoles
Compound
Ar
IC50 (µM) a
COX-1 COX-2
4
5
6
7
8
9
10
11
12
13
14
15
16
17
2,4-F2-phenyl
>850.0
500.0
500.0
393.0
500.0
108.0
199.0
1.680
6.900
5.100
0.570
8.500
0.411
0.830
3,4-F2-phenyl
2,4-Cl2-phenyl
3-Cl,4-F-phenyl
2,4-Me2-phenyl
3,4-Me2-phenyl
3-Me,4-F-phenyl
2,4-OMe2-phenyl
3-F,4-OMe-phenyl
3-Cl,4-OMe-phenyl
3-Me,4-SMe-phenyl
3-OMe,4-SMe-phenyl
3-Me,4-SOMe-phenyl
500.0 11.600
500.0
>500.0
130.0
300.0
9 b
4.450
1.300
0.139
1.390
3 b
Table 2
64.0
0.227
In vitro data of C-3 and C-5 substituted 1,5-diarylpyrazoles
18
100.0
100.0
1.000
2.100
19
20
21
100 c,d 100 c,d
Compounds Ar
4-OMe-phenyl
R
IC50 (µM)a
COX-1 COX-2
>200.0
>200.0
0.930
1.546
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
CF3
63.0
39.4
82 b
0.365
0.236
58 b
4-OMe-phenyl
4-OMe-phenyl
4-OMe-phenyl
4-OMe-phenyl
4-OMe-phenyl
4-Cl-phenyl
CHF2
CH2F
Me
22
67.0
0.660
64 b
54 b
H
0 b
Et
10 b
23
24
0 c
52 c
Me
349.2
412.0
500.0
>500.0
>500.0
41.0
1.007
0.502
1.300
1.000
2.200
0.213
1.45
4-Me-phenyl
CHF2
CHF2
CHF2
CHF2
CHF2
CHF2
CHF2
CHF2
CHF2
CHF2
CHF2
CHF2
3-Me-phenyl
26 c
2 c
4-Et-phenyl
3-Cl-phenyl
4-SMe-phenyl
4-NHEt-phenyl
2,4-F2-phenyl
3,4-Cl2-phenyl
3,4-Me2-phenyl
3-Me,4-F-phenyl
3-Me,4-SMe-phenyl
Indomethacin
Celecoxib
–
–
0.067 7.810
10.750 0.076
93.0
156.0
59.0
1.000
0.371
1.140
1.000
a Mean of three determinations.
b % Inhibition at 100 µM.
c % Inhibition at 10 µM.
134.0
100.0
d IC50 not determined. > Precipitation observed beyond this concentration
(IC50s may be much higher than the reported values).
48 b, 0 c 99 b, 26 c
50 b,d
100 b,
23c
3.1.2. Variation at position C-3
Since the newly introduced 3-hydroxymethyl-4-sulfamoyl-
phenyl group at N1 has generated encouraging SAR with
respect to C-5 substitution, it became obvious to reassess the
suitability of C-3 substitution. The effect of the systematic
variation at this position is depicted in Table 2. Number of
a Mean of three determinations.
b % Inhibition at 100 µM.
c % Inhibition at 10 µM.
d IC50 not determined. > Precipitation observed beyond this concentration
(IC50s may be much higher than the reported values).

S.K. Singh et al. / European Journal of Medicinal Chemistry 40 (2005) 977–990
981
the COX-2 pocket. Since we observed a significant improve-
3.2. 3D-QSAR-CoMFA study
ment caused by CHF2 over CF3, more number of analogs
were studied for this change. But this improvement was found
to be limited to the smaller groups at/around position-4 of the
C-5 phenyl ring, e.g. 4-methyl analog 32 (0.502 µM),
4-methylsulfanyl analog 36 (0.213 µM) and 3,4-dichloro ana-
log 39 (0.371 µM) only exhibited better potency. Other CHF2
analogs of potent C-5 substituted compounds selected from
Table 1, such as 3,4-dimethyl analog 40 and 3-methyl-4-
fluoro analog 41 suffered a reasonable loss in potency, while
3-methyl-4-methylsulfanyl analog 42 and 2,3-dihydrobezo-
thiophenyl analog 43 even turned out to be inactive. The rea-
son for this adverse observation may be the imbalance caused
by deficiency of fluorine atoms in case of CHF2 from the opti-
mally balanced system due to hydrophobic groups at/around
position-4 of C-5 phenyl ring in conjugation with CF3. An
entirely different variation at C-3 was brought about by intro-
ducing a carboxylic acid group e.g. in compound 47 and a
cyano group in compound 48 which were, respectively, found
to be inactive and COX-1 selective (Table 3).
Since the percentage inhibition could not be converted to
IC50/MIC values, the compounds having experimental IC50s
were only considered for CoMFA study. Out of 34 such com-
pounds, 24 were incorporated in training set and the remain-
ing 10 in the test set. These sets were formed based on the
diversity in structure and enzyme activity. The statistical valid-
ity of the model was judged by the high values of q2 (more
than 0.5) and r2 (more than 0.9) with low standard error of
estimate (SEE). Additional descriptors such as ClogP, CMR
and dipole moment were used as independent variables to
obtain an improved 3D-QSAR model (Table 4) [39,40]. How-
ever, these descriptors led to the reduced values of r2 and q2.
Among significant models, the one with highest q2 value was
utilized for further analysis. While value of the cross vali-
dated q2 was found to be 0.571 with six components, the non-
cross validated r2 was found to be 0.986 (SEE = 0.071). The
CoMFA contour plots for the steric and electrostatic fields
are shown in Fig. 3a, b, respectively.
The actual and predicted pIC50 values of the training set
are shown in Table 5. The graph of predicted versus actual
activity for the training set revealed an excellent correlation
between the two. The model was validated by predicting the
activity of the test set compounds (Table 6). This model also
afforded a significant value of r2pred as 0.591, and the pre-
dicted activity was highly consistent with the experimental
data. A graph depicting the actual versus predicted activity of
training and test set compounds is shown in Fig. 4. All the
residual values for the prediction were less than 1 log order
of activity. The CoMFA analysis provided contour plots of
steric and electrostatic interactions (Fig. 3). The green con-
tour near position-3 of C-5 phenyl ring indicated that a
medium size substituent would enhance the activity, and it
was found that the 3-methyl group of the most active com-
pound 14 occupied the same contour. The 2,3-
dihydrobenzothiophenyl ring of compound 17 also extended
towards this green contour and this could be the reason for its
high potency. The 3-fluoro group of compound 5 keeping
away from this contour was found to be less potent. The yel-
3.1.3. Variation at N1
Different polar and non-polar groups were studied adja-
cent to sulfonamide/methylsulfonyl during the search of sec-
ond substitution in this benzene ring and a few best perform-
ing C-5 substitutions were selected here to conclude the SAR
for sulfonamide vs. methylsulfonyl group [30]. The results
are mentioned in Table 3. The compounds with methylsulfo-
nyl (SO2Me) group, such as 4-methoxyphenyl analog 44, and
4-methylsulfanylphenyl analogs 45 and 46 exhibited lesser
potency when compared to the corresponding sulfonamide
(SO2NH2) analogs. The poor in vitro potency exhibited by
methylsulfonyl group is a general observation, and is due to
its comparatively lesser tendency to form hydrogen bond
(donor) with the polar region of COX-2 enzyme.
Table 3
In vitro data of 4-methylsulfonyl and 4-sulfamoylphenyl containing 1,5-
diarylpyrazoles
Compounds R1
Ar
R2
IC50 (µM)a
COX-1 COX-2
>300.0 3.200
300.0 3.000
300.0 2.000
44
45
46
47
48
SO2Me
4-OMe-phenyl
4-SMe-phenyl
4-SMe-phenyl
CF3
SO2Me
SO2Me
CF3
CHF2
CO2H 0 b
SO2NH2 4-OMe-phenyl
SO2NH2 4-OMe-phenyl
61 b
0 b
CN
96 b
a Mean of three determinations.
b % Inhibition at 100 µM. > Precipitation observed beyond this concentra-
tion (IC50s may be much higher than the reported values).
Fig. 2. Alignment of 1,5-diarylpyrazoles.

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S.K. Singh et al. / European Journal of Medicinal Chemistry 40 (2005) 977–990
Table 4
PLS statistics of CoMFA studies
Fields
CoMFA
CoMFA with additional descriptors
ClogP
0.978
Dipole moment
0.973
CMR
0.976
r2a
0.986
6
Nb
6
6
6
q2c
0.571
0.071
197.865
0.530
0.089
126.147
0.519
0.517
0.093
114.527
SEEd
0.098
F-valuee
103.171
0.516 (S)
0.483 (E)
0.001 (DM)
Field contributionf
0.524(S)
0.476(E)
0.493 (S)
0.507 (S)
0.463 (E)
0.482 (E)
0.046 (ClogP)
0.011 (CMR)
a Non-cross validated correlation coefficient.
b Optimum number of components obtained form cross validated PLS analysis and used in final non-cross validated analysis.
c Cross validated correlation coefficient.
d SEE.
e F-test value.
f Field contributions: steric (S), electrostatic (E), and dipole moment (DM) fields from CoMFA.
Fig. 3
. CoMFA contour plots with compound 14 (a) steric: green-bulky groups favorable; yellow-bulky groups unfavorable (b) electrostatic: blue-
electropositive groups favorable; red-electronegative groups favorable.
ably the reason for compound 14 to be so potent. The pres-
ence of electronegative groups at this position, viz. 3-chloro
and 3-fluoro could be the reason for the less potency of com-
pounds 5, 12, 13 and 35. Red contour seen near position-4 of
C-5 phenyl ring indicated electronegative groups enhancing
the activity profile e.g. in compounds 7, 10 and 31 (Tables 1
and 2). The electropositive groups at this position in com-
pounds 8 and 11 led to poor activity. Red contour seen near
CF3 group indicated electronegative groups favorable at this
position. This could be the reason for most of the compounds
having CF3/CHF2 group at this position to be potent inhibi-
tors of COX-2. Blue contour surrounding NH2 (SO2NH2)
group indicates electropositive groups enhancing the activ-
ity. This could be the possible reason for sulfonamide
(SO2NH2) containing compounds to be more potent than those
having methylsulfonyl (SO2Me) group.
Fig. 4. Predicted values vs. experimental values for the training and test sets
of CoMFA.
low contour accommodated position-2 of C-5 phenyl ring,
thereby indicating a steric group to be less favorable, e.g. 2,4-
dimethylphenyl compound 8 and 2,4-dimethoxy analog 11.
Blue contour near position-3 of phenyl ring indicated elec-
tropositive groups enhancing the activity, and that was prob-
Superposition of the CoMFA coefficient contour maps with
the most active compound 14 in the active site of COX-2 is
shown in Fig. 5. These studies established a meaningful cor-

S.K. Singh et al. / European Journal of Medicinal Chemistry 40 (2005) 977–990
983
Fig. 5. Superimposition of (a) steric and (b) electrostatic CoMFA contour plots in the active site of COX-2. Most active compound 14 is shown in the bac-
kground.
Table 5
Table 6
Experimental pIC50, corresponding predicted values and residuals of mole-
cules used in training set for CoMFA
Experimental pIC50, corresponding predicted values and residuals of mole-
cules used in test set
Compounds
Actual
pIC50
5.29
6.24
5.07
6.39
6.08
4.94
5.35
5.89
6.86
5.86
6.64
6.00
5.81
6.44
6.63
6.00
5.89
6.00
6.67
6.43
6.00
5.49
5.52
5.70
Predicted
pIC50
5.20
6.14
5.21
6.37
6.13
4.92
5.43
5.94
6.90
5.83
6.68
5.98
5.74
6.38
6.69
6.04
5.89
6.08
6.61
6.37
5.99
5.52
5.46
5.71
Residuals
Compounds
Actual
pIC50
5.77
5.16
5.68
6.03
6.18
6.30
5.66
5.84
6.00
5.94
Predicted
pIC50
5.29
Residuals
6
7
8
9
0.09
4
5
0.48
0.10
5.73
–0.57
–0.52
–0.20
–0.29
0.56
–0.14
0.02
19
21
28
32
35
37
38
40
6.20
6.23
10
11
12
13
14
15
17
18
22
25
26
31
33
34
36
39
41
44
45
46
–0.05
0.02
6.47
5.74
–0.08
–0.05
–0.04
0.03
5.95
–0.29
–0.23
0.71
6.07
5.29
6.30
–0.36
–0.04
0.02
on the binding site of COX-2 enzyme is shown in Fig. 5b.
The blue contour seen at position-3 of the C-5 phenyl ring
indicated the electropositive groups causing increased inter-
actions with residuesVal-349, Leu-352 and Leu-384 whereas
red contour near position-4 of this ring indicated electrone-
gative groups showing favorable interactions with Tyr-
385 and Trp-387. Red contour above the CF3 group indicated
electronegative substituent forming more favorable electro-
static interactions with guanidyl group ofArg-120. Blue con-
tour above the amino group of sulfonamide indicated an elec-
tropositive group interacting better with hydrophilic region
formed by His-90, Gln-192 and Ile-517.
0.07
0.06
–0.06
–0.04
0.00
–0.08
0.06
0.06
0.01
–0.03
0.06
–0.01
relation between receptor–ligand binding and the biological
activity. Superposition of the steric fields on the binding site
of COX-2 enzyme is shown in Fig. 5a. The 3-methyl group
of C-5 phenyl ring in compound 14 nearing green contour
could be mapped to the hydrophobic residues such as Phe-
381, Leu-384 and Tyr-385 of the COX-2 pocket.Yellow con-
tour seen near position-2 of the C-5 phenyl ring indicated
that the groups with appropriate size were required to bind
this narrow pocket. Superposition of the electrostatic fields
3.3. In vivo results
The in vitro potent compounds were tested in the carrag-
eenan induced rat paw edema model at 30 mg kg–1 where
compounds 4, 10, 14, 17, 18, 21, 26, 28, 31, 32 and 36 showed
50–60% reduction in paw volume [38].

984
S.K. Singh et al. / European Journal of Medicinal Chemistry 40 (2005) 977–990
4. Conclusion
41.66 mmol) was injected and the reaction mixture was
allowed to stir at ambient temperature for 4–5 h. The reaction
mixture was poured into ice water, acidified with 2 N HCl
and extracted with ethyl acetate. The combined organic layer
was washed with water, dried and evaporated to get a residue
which was stirred with petroleum ether to afford a low melt-
ing solid of the title compound (7.37 g, 90%). This solid was
used in the next step without further purification [11]. IR
(Neat) 3330, 1614, 1405 cm–1. 1H NMR (CDCl3) d 7.80 (d,
J = 8.2 Hz, 2H), 7.35 (d, J = 8.0 Hz, 2H), 6.52 (s, 1H), 3.85
(s, 3H). MS (CI Method) 246 (M+).
Herein, we presented the synthesis and a systematic SAR
study on 1,5-diarylpyrazole class of COX-2 inhibitors with a
modified pharmacophore. The results of the variations at C-3,
C-5 and N1 afforded many potent inhibitors of COX-2. Of
many variations at the most sensitive C-5 phenyl ring, the
small hydrophobic groups at position-3 and 4, e.g. 3-methyl-
4-methylsulfanyl analog 14 and their saturated cyclic proto-
type 2,3-dihydrothiophenyl analog 17 exhibited excellent
COX-2 inhibitory potency. Generally, the sulfonamide ana-
logues with a CHF2 group at C-3 were found to be better than
those having CF3. Thus, the biological findings coupled with
a systematic 3D-QSAR-CoMFA study supported the accep-
tance of newly introduced hydroxymethyl (CH2OH) group
adjacent to sulfonamide in the hydrophilic region of the COX-
2 enzyme.
5.3. General procedure for 1,5-diarylpyrazoles.
Representative preparation of 2-hydroxymethyl-4-[5-(4-
methoxyphenyl)-3-trifluoromethyl-1H-1-pyrazolyl]-1-
benzenesulfonamide 25
4-Hydrazino-2-hydroxymethyl-1-benzenesulfonamide 1
(2.00 g, 9.21 mmol) was dissolved in MeOH (10 ml) under
argon atmosphere and acidified to pH 1–2 using IPA-HCl.
The reaction mixture was stirred at room temperature for 0.5 h
and solvent was completely removed under high vacuum at
40–50 °C to afford a solid. The mixture of this solid and above
prepared 4,4,4-trifluoro-1-(4-methoxyphenyl)-1,3-butane-
dione (2.04 g, 8.29 mmol) taken in absolute alcohol (30 ml),
was heated at 50–60 °C for 10–12 h under argon atmosphere.
The reaction mixture was cooled, poured over ice cold water
and extracted with ethyl acetate. The combined organic layer
after washing with brine and water, was dried over anhyd.
Na2SO4. The residue obtained after solvent evaporation was
purified by column chromatography using 30% ethyl acetate–
petroleum ether, and the product so obtained was finally tritu-
rated with ethyl acetate–toluene to get the colorless solid of
the title compound 25 (4.84 g, 79%). M.p. 188–189 °C. IR
5. Experimental section
5.1. Chemistry
Acetophenones and halo esters were either purchased from
Lancaster/Aldrich Co. or prepared according to the standard
procedures. While celecoxib was prepared according to the
literature procedure [11], indomethacin was extracted from
the capsules purchased from the medical store. The yields
reported are unoptimized. Melting points were determined
on Buchi melting point B-540 apparatus and are uncorrected.
IR spectra were recorded on Perkin–Elmer FT-IR 1650 spec-
trometer. 1H NMR and 13C NMR experiments were, respec-
tively, performed at 200 and 50 MHz on Varian Gemini
200 spectrometer and their chemical shifts are reported in d
units with respect to TMS as internal standard. Mass spectra
were recorded on HP-5989A spectrometer. Elemental analy-
ses were carried out for C, H, N using Perkin–Elmer 2400 se-
ries II CHN-O analyzer. All the analyses were performed by
the Analytical Research Group of Discovery Research-Dr.
Reddy’s Laboratories Ltd. The purity of the final compounds
were determined by HPLC using “System 1” which con-
sisted column Hichrom RPB (250 mm), mobile phase 0.01 M
KH2PO4/CH3CN (50:50) and “System 2” which comprised
column Intersil ODS 3V (250 mm), mobile phase
H2O/CH3CN (50:50), both running at 1.0 ml min–1 with UV
1
(Neat) 3311, 1609 cm–1. H NMR (CDCl3) d 7.97 (d,
J = 8.6 Hz, 1H), 7.62 (d, J = 1.8 Hz, 1H), 7.25 (d, J = 8.0 Hz,
1H), 7.15 (d, J = 8.4 Hz, 2H), 6.88 (d, J = 8.6 Hz, 2H), 6.71
(s, 1H), 5.43 (bs, 2H), 5.01 (d, J = 3.8 Hz, 2H), 3.83 (s, 3H),
2.65 (bs, 1H). 13C NMR (DMSO-d6) d 160.1, 145.1, 142.8,
142.0 (q, J = 35.5 Hz, 1C), 141.5, 139.9, 130.3 (2C), 128.1,
123.9, 123.3, 121.5 (q, J = 267.4 Hz, 1C), 120.6, 114.4 (2C),
105.9, 59.3, 55.3. MS (CI Method) 428 (M + H)+, 409 (100%),
392, 346. HPLC (System 1) 98.35%.Anal. (C18H16F3N3O4S)
C, H, N.
5.3.1. Compound 4
detection at respective kmax
.
Yield 76%. M.p. 142–144 °C. IR (KBr) 3425, 3260,
1625 cm–1. 1H NMR (CDCl3) d 8.00 (d, J = 8.6 Hz, 1H), 7.62
(d, J = 1.8 Hz, 1H), 7.26–7.19 (m, 2H), 6.97–6.88 (m, 2H),
6.87 (s, 1H), 5.41 (bs, 2H), 5.04 (d, J = 4.2 Hz, 2H), 2.64 (bs,
1H). MS (CI Method) 434 (M + H)+, 416, 398, 352. HPLC
(System 1) 99.62%. Anal. (C17H12F5N3O3S) C, H, N.
5.2. General procedure for 1,3-diketones 3. Representative
preparation of 4,4,4-trifluoro-1-(4-methoxyphenyl)-1,3-
butanedione 3a (Ar = 4-OMe-Ph; R2 = CF3)
4-Methoxyacetophenone (5.00 g, 33.33 mmol) was dis-
solved in 25 ml of dry DMF under argon atmosphere and
60% NaH (1.66 g, 41.66 mmol) was added in three lots main-
taining temperature between –5 and 0 °C. After stirring at
this temperature for 0.5 h, ethyl trifluoroacetate (4.97 ml,
5.3.2. Compound 5
Yield 67%. M.p. 120–122 °C. IR (KBr) 3423, 1606,
1486 cm–1. 1H NMR (CDCl3) d 8.05 (d, J = 8.6 Hz, 1H), 7.65

S.K. Singh et al. / European Journal of Medicinal Chemistry 40 (2005) 977–990
985
(s, 1H), 7.35–6.95 (m, 4H), 6.79 (s, 1H), 5.40 (bs, 2H), 5.08
(s, 2H), 2.60 (bs, 1H). MS (CI Method) 433 (M+), 414, 352.
HPLC (System 2) 97.32%. (C17H12F5N3O3S) C, H, N.
6.75–6.72 (m, 2H), 6.71 (s, 1H), 5.45 (bs, 2H), 5.05 (s, 2H),
3.92 (s, 3H), 3.80 (s, 3H), 2.65 (bs, 1H). MS (CI Method)
457 (M+) (440, 100%). HPLC (System 1) 97.97%. Anal
(C19H18F3N3O5S) C, H, N.
5.3.3. Compound 6
5.3.9. Compound 12
Yield 71%. M.p. 126–128 °C. IR (KBr) 3371, 3228,
1600 cm–1. 1H NMR (CDCl3) d 7.94 (d, J = 8.4 Hz, 1H), 7.60
(d, J = 2.4 Hz, 1H), 7.46 (d, J = 1.8 Hz, 1H), 7.35 (d,
J = 1.8 Hz, 1H), 7.23 (d, J = 7.2 Hz, 1H), 7.10 (dd, J = 6.2 &
2.2 Hz, 1H), 6.78 (s, 1H), 5.41 (bs, 2H), 5.00 (d, J = 3.2 Hz,
2H), 2.72 (bs, 1H). MS (CI Method) 466 (M+), 447, 384.
HPLC (System 1) 99.62%. Anal. (C17H12Cl2F3N3O3S) C, H,
N.
Yield 78%. M.p. 206–208 °C. IR (KBr) 3407, 3313, 1476,
1348 cm–1. 1H NMR (CDCl3 + DMSO-d6) d 7.98
(d, J = 8.3 Hz, 1H), 7.70 (s, 1H), 7.22–7.16 (m, 1H), 7.04–
6.90 (m, 3H), 6.74 (s, 1H), 5.00 (d, J = 4.0 Hz, 2H), 3.92 (s,
3H). MS (EI Method) 445 (M+), 426, 410, 364. HPLC (Sys-
tem 1) 98.25%. Anal. (C18H15F4N3O4S) C, H, N.
5.3.10. Compound 13
5.3.4. Compound 7
Yield 69%. M.p. 211–212 °C. IR (KBr) 3429, 1605,
1473 cm–1. 1H NMR (CDCl3 + DMSO-d6) d 7.95
(d, J = 8.3 Hz, 1H), 7.79 (s, 1H), 7.34 (s, 1H), 7.16 (dd,
J = 10.0 & 1.6 Hz, 1H), 7.08 (d, J = 8.6 Hz, 1H), 7.00 (s,
2H), 6.95 (d, J = 8.8 Hz, 1H), 6.77 (s, 1H), 5.28 (bs, 1H),
5.00 (d, J = 4.0 Hz, 2H), 3.92 (s, 3H). MS (EI Method) 461
(M+), 441, 426, 380. HPLC (System 1) 99.16%; (System 2)
99.20%. Anal. (C18H15Cl F3N3O4S) C, H; N: calcd, 9.10;
found, 8.54.
Yield 80%. M.p. 148–150 °C. IR (KBr) 3458, 3237, 1604,
1473 cm–1. 1H NMR (CDCl3) d 8.00 (d, J = 8.6 Hz, 1H), 7.63
(d, J = 1.8 Hz, 1H), 7.39 (d, J = 5.0 Hz, 1H), 7.26–7.01 (m,
3H), 6.78 (s, 1H), 5.02 (s, 2H). MS (CI Method) 450 (M+),
414, 368, 340, 319. HPLC (System 2) 98.62%. Anal.
(C17H12ClF4N3O3S) C, H, N.
5.3.5. Compound 8
Yield 74%. M.p. 96–98 °C. IR (KBr) 3420, 1604,
1470 cm–1. 1H NMR (CDCl3) d 7.86 (d, J = 8.2 Hz, 1H), 7.59
(s, 1H), 7.20–7.00 (m, 4H), 6.65 (s, 1H), 5.45 (bs, 2H), 4.95
(s, 2H), 2.80 (bs, 1H), 2.35 (s, 3H), 1.95 (s, 3H). MS (CI
Method) 425 (M+), 407, 344. HPLC (System 1) 96.52%; (Sys-
tem 2) 97.10%. Anal. (C19H18F3N3O3S) C, H, N.
5.3.11. Compound 14
Yield 59%. M.p. 115–117 °C. IR (KBr) 3422, 1604 cm–1.
1H NMR (CDCl3) d 8.00 (d, J = 8.8 Hz, 1H), 7.60 (s, 1H),
7.30–7.20 (m, 1H), 7.10–7.04 (m, 2H), 6.94 (d, J = 7.0 Hz,
1H), 6.75 (s, 1H), 5.40 (bs, 2H), 5.00 (d, J = 4.4 Hz, 2H),
2.65 (bs, 1H), 2.47 (s, 3H), 2.28 (s, 3H). MS (CI Method)
457 (M+), 443. HPLC (System 1) 98.28%. Anal.
(C19H18F3N3O3S2) C, H, N.
5.3.6. Compound 9
Yield 58%. M.p. 132–134 °C. IR (KBr) 3522, 1605,
1472 cm–1. 1H NMR (CDCl3) d 7.95 (d, J = 8.4 Hz, 1H), 7.66
(s, 1H), 7.25 (d, J = 7.8 Hz, 1H), 7.16 (m, 2H), 6.86 (d,
J = 7.4 Hz, 1H), 6.74 (s, 1H), 5.50 (bs, 2H), 5.03 (s, 2H),
2.80 (bs, 1H), 2.28 (s, 3H), 2.24 (s, 3H). 13C NMR
(DMSO-d6) d 145.4, 142.8, 142.2 (q, J = 37.5 Hz, 1C), 141.5,
140.0, 137.9, 137.1, 129.8 (2C), 128.0, 126.2, 125.8, 123.9,
123.3, 121.5 (q, J = 266.8 Hz, 1C), 106.1, 59.4, 19.3, 19.2.
MS (CI Method) 425 (M+), 407, 379, 327. HPLC (System 1)
97.55%; (System 2) 97.45%. Anal. (C19H18F3N3O3S) C, N;
H: calcd, 4.26; found, 4.68.
5.3.12. Compound 15
Yield 48%. M.p. 168–170 °C. IR (KBr) 3467, 1602,
1
1472 cm–1. H NMR (CD3OD) d 7.90 (d, J = 8.4 Hz, 1H),
7.73 (s, 1H), 7.20 (dd, J = 6.4 & 2.0 Hz, 1H), 7.05 (dd, J = 6.4
& 2.0 Hz, 1H), 6.85–6.75 (m, 2H), 6.73 (s, 1H), 4.90 (s, 2H),
3.75 (s, 3H), 2.02 (s, 3H). MS (CI Method) 473 (M+), 456,
392. HPLC (System 1) 98.01%. (System 2) 96.93%. Anal.
(C19H18F3N3O4S2) C, H, N.
5.3.13. Compound 17
5.3.7. Compound 10
Yield 82%. M.p. 158–159 °C. IR (Nujol) 3319, 1598,
1461 cm–1. 1H NMR (CDCl3) d 8.00 (d, J = 8.4 Hz, 1H), 7.67
(d, J = 2.2 Hz, 1H), 7.40–7.20 (m, 2H), 7.07 (s, 1H), 6.94 (d,
J = 7.8 Hz, 1H), 6.72 (s, 1H), 5.45 (bs, 2H), 5.04 (d,
J = 4.8 Hz, 2H), 3.43 (t, J = 7.4 Hz, 2H), 3.30 (t, J = 7.6 Hz,
2H), 2.75 (bs, 1H). MS (CI Method) 455 (M+), 437, 355.
HPLC (System 2) 98.25%. Anal. (C19H16F3N3O3S2) C, H,
N.
1
Yield 66%. IR (KBr) 3455, 3274, 1603, 1481 cm–1. H
NMR (CDCl3) d 7.98 (d, J = 8.4 Hz, 1H), 7.63 (d, J = 1.6 Hz,
1H), 7.30–7.15 (m, 2H), 7.05–6.96 (m, 2H), 6.74 (s, 1H),
5.54 (bs, 2H), 5.02 (s, 2H), 3.05 (bs, 1H), 2.27 (s, 3H). MS
(CI Method) 429 (M+), 410, 394, 348. HPLC (System 1)
95.35%; HPLC (System 2) 96.12%. Anal. (C18H15F4N3O3S)
C, N; H: calcd, 3.52; found, 3.95.
5.3.8. Compound 11
5.3.14. Compound 18
Yield 68%. M.p. 125–127 °C. IR (KBr) 3343, 1602,
1484 cm–1. 1H NMR (CDCl3) d 8.02 (d, J = 8.4 Hz, 1H), 7.65
(s, 1H), 7.30 (d, J = 1.8 Hz, 1H), 6.80 (d, J = 8.8 Hz, 1H),
Yield 64%. M.p. 92–94 °C. IR (KBr) 3386, 1603,
1475 cm–1. 1H NMR (CDCl3) d 7.98 (d, J = 8.6 Hz, 1H), 7.67
(d, J = 2.2 Hz, 1H), 7.26 (d, J = 8.2 Hz, 1H), 6.97 (s, 1H),

986
S.K. Singh et al. / European Journal of Medicinal Chemistry 40 (2005) 977–990
6.86 (d, J = 6.6 Hz, 1H), 6.74 (d, J = 6.8 Hz, 1H), 6.65 (s,
1H), 5.52 (bs, 2H), 5.04 (s, 2H), 4.25 (t, J = 5.0 Hz, 2H), 2.75
(t, J = 5.0 Hz, 2H), 2.06-1.97 (m, 2H). MS (EI Method)
453 (M+), 435. HPLC (System 1) 98.96%. Anal.
(C20H18F3N3O4S) C, H, N.
5.3.21. Compound 26
Yield 83%. M.p. 166–168 °C. IR (Nujol) 3300, 1608,
1460 cm–1. 1H NMR (CDCl3 + DMSO-d6) d 7.94 (dd, J = 6.0
& 2.6 Hz, 1H), 7.61 (s, 1H), 7.31 (s, 1H), 7.17 (d, J = 8.0 Hz,
2H), 6.90 (d, J = 7.0 Hz, 2H), 6.76 (t, J = 53.4 Hz, 1H), 6.67
(s, 1H), 6.28 (bs, 2H), 4.94 (s, 2H), 3.82 (s, 3H), 2.70 (bs,
1H). MS (EI Method) 409 (M+), 389, 374. HPLC (System 1)
99.50%; (System 2) 99.71%. Anal. (C18H17F2N3O4S) C, H;
N: calcd, 10.26; found, 9.58.
5.3.15. Compound 19
Yield 73%. M.p. 122–125 °C. IR (KBr) 3488, 1602,
1468 cm–1. 1H NMR (CDCl3) d 7.96 (d, J = 8.6 Hz, 1H), 7.70
(d, J = 2.0 Hz, 1H), 7.24–7.19 (dd, J = 6.4 & 2.0 Hz, 1H),
7.04–7.00 (m, 2H), 6.81 (dd, J = 6.6 & 1.4 Hz, 1H), 6.71 (s,
1H), 5.44 (bs, 2H), 5.04 (s, 2H), 2.78–2.71 (m, 4H), 2.65 (bs,
1H), 1.80–1.75 (m, 4H). MS (CI Method) 451 (M+), 433,
353. HPLC (System 1) 99.04%. Anal. (C21H20F3N3O3S) C,
H, N.
5.3.22. Compound 27
Yield 33%. M.p. 148–150 °C. IR (KBr) 3302, 1611,
1511 cm–1. 1H NMR (CDCl3 + DMSO-d6) d 7.87 (d,
J = 8.4 Hz, 1H), 7.60 (d, J = 2.0 Hz, 1H), 7.36 (s, 1H), 7.12
(d, J = 8.8 Hz, 2H), 6.84 (d, J = 8.8 Hz, 2H), 6.54 (s, 1H),
6.53 (bs, 2H), 5.40 (d, J = 48.2 Hz, 2H), 4.90 (s, 2H), 3.80 (s,
3H), 2.90 (bs, 1H). MS (CI Method) 391 (M+), 356, 310.
HPLC (System 1) 97.22%; HPLC (System 2) 96.20%. Anal.
(C18H18FN3O4S) C, H, N.
5.3.16. Compound 20
Yield 55%. M.p. 85–87 °C. IR (KBr) 3380, 3275, 1492,
1395 cm–1. 1H NMR (CDCl3) d 8.15 (d, J = 8.5 Hz, 1H), 7.60
(s, 1H), 7.45 (d, J = 8.3 Hz, 1H), 6.49 (s, 1H), 5.06 (s, 2H),
2.82–2.63 (m, 1H), 1.87–1.74 (m, 6H), 1.47–1.22 (m, 4H).
MS (EI Method) 403 (M+), 384, 368, 322. HPLC (System 1)
98.93%. Anal. (C17H20F3N3O3S) C, H, N.
5.3.23. Compound 28
Yield 75%. M.p. 177–179 °C. IR (KBr) 3320, 3196, 1610,
1578, 1511 cm–1. 1H NMR (CDCl3 + DMSO-d6) d 7.90 (d,
J = 8.6 Hz, 1H), 7.62 (d, J = 1.8 Hz, 1H), 7.12 (d, J = 8.8 Hz,
2H), 7.10 (m, 1H), 6.85 (t, J = 8.8 Hz, 2H), 6.31 (s, 1H), 5.40
(bs, 2H), 4.96 (s, 2H), 3.82 (s, 3H), 2.64 (bs, 1H), 2.38 (s,
3H). MS (CI Method) 374 (M + H)+, 356, 322, 295. HPLC
(System 1) 97.86%; HPLC (System 2) 97.65%. Anal.
(C18H19N3O4S) H; C: calcd, 57.90; found, 58.43; N: calcd,
11.25; found, 10.66.
5.3.17. Compound 21
Yield 82%. M.p. 207–208 °C. IR (KBr) 3427, 3329, 3252,
1630 cm–1. 1H NMR (CDCl3 + DMSO-d6) d 7.90 (d,
J = 8.4 Hz, 1H), 7.77–7.71 (m, 2H), 7.69–7.67 (m, 2H), 7.18
(d, J = 6.4 Hz, 1H), 7.16–7.14 (m, 3H), 6.85 (s, 1H), 6.45 (s,
2H), 4.96 (s, 2H), 3.94 (s, 3H), 2.46 (bs, 1H). MS (CI Method)
477 (M+), 396, 368. HPLC (System 2) 98.52%. Anal.
(C22H18F3N3O4S) C, H, N.
5.3.24. Compound 29
Obtained by the general method using hydrazine hydro-
chloride 1 and 3-(4-methoxyphenyl)-3-oxopropanal in 35%
yield. M.p. 166–167 °C. IR (KBr) 3320, 1608 cm–1. 1H NMR
(CDCl3 + CD3OD) d 7.90 (d, J = 8.4 Hz, 1H), 7.73 (d,
J = 2.0 Hz, 1H), 7.64 (d, J = 2.0 Hz, 1H), 7.15 (d, J = 8.8 Hz,
2H), 7.06 (d, J = 2.2 Hz, 1H), 6.88 (d, J = 8.8 Hz, 2H), 6.49
(d, J = 2.0 Hz, 1H), 4.96 (s, 2H), 3.82 (s, 3H). MS (CI Method)
359 (M+), 342, 324. HPLC (System 1) 98.47%; HPLC (Sys-
tem 2) 98.08%. Anal. (C17H17N3O4S) C, H, N.
5.3.18. Compound 22
Yield 38%. M.p. 230–232 °C. IR (KBr) 3403, 3298, 1592,
1459 cm–1. 1H NMR (CDCl3 + DMSO-d6) d 10.63 (bs, 1H),
7.91 (d, J = 4.4 Hz, 1H), 7.78 (d, J = 1.4 Hz, 1H), 7.51–7.33
(m, 3H), 7.25–7.08 (m, 2H), 6.93 (d, J = 2.4 Hz, 1H), 6.86 (s,
1H), 6.62 (s, 2H), 4.94 (s, 2H), 2.68 (bs, 1H). MS (CI Method)
437 (M+), 419. HPLC (System 1) 97.53%; (System 2)
96.82%. Anal. (C19H15F3N4O3S) C, H, N.
5.3.19. Compound 23
5.3.25. Compound 30
Yield 80%. M.p. 167–168 °C. IR (KBr) 3320, 3246, 1604,
1488, 1348 cm–1. 1H NMR (CDCl3 + DMSO-d6) d 8.00 (d,
J = 8.3 Hz, 1H), 7.74 (s, 1H), 7.39–7.27 (m, 3H), 6.92 (d,
J = 4.9 Hz, 1H), 6.81 (s, 1H), 5.00 (d, J = 4.5 Hz, 2H). MS
(CI Method) 403 (M+), 384, 368, 322. HPLC (System 2)
98.50%. Anal. (C15H12F3N3O3S2) C, H, N.
Yield 48%. M.p. 168–170 °C. IR (KBr) 3481, 1606,
1511 cm–1. 1H NMR (CDCl3) d 7.75 (d, J = 8.8 Hz, 1H), 7.45
(d, J = 2.0 Hz, 1H), 7.06 (d, J = 8.8 Hz, 2H), 7.00–6.90 (dd,
J = 8.4 & 2.4 Hz, 1H), 6.81 (d, J = 8.8 Hz, 2H), 6.27 (s, 1H),
5.88 (bs, 2H), 4.88 (s, 2H), 3.82 (s, 3H), 2.74 (q, J = 7.4 Hz,
2H), 2.65 (bs, 1H), 1.30 (t, J = 7.4 Hz, 3H). MS (CI Method)
387 (M+), 372, 352. HPLC (System 1) 99.01%; HPLC (Sys-
tem 2) 99.19%. Anal. (C19H21N3O4S) H, N; C: calcd, 58.90;
found, 59.41.
5.3.20. Compound 24
Yield 47%. M.p. 155–156 °C. IR (KBr) 3380, 3212, 1585,
1455 cm–1. 1H NMR (DMSO-d6) d 9.30 (s, 1H), 8.85–8.78
(m, 2H), 8.00–7.95 (m, 4H), 7.35 (s, 1H), 6.00 (bt, 1H), 4.95
(d, J = 4.4 Hz, 2H). MS (CI Method) 399 (M + H, 100%)+,
381. HPLC (System 2) 98.73%. Anal. (C16H13F3N4O3S) C,
H, N.
5.3.26. Compound 31
Yield 65%. M.p. 167–169 °C. IR (KBr) 3314, 3215, 1600,
1
1497 cm–1. H NMR (DMSO-d6) d 7.76 (s, 1H), 7.72 (d,

S.K. Singh et al. / European Journal of Medicinal Chemistry 40 (2005) 977–990
987
J = 8.2 Hz, 1H), 7.47 (bs, 2H), 7.45 (d, J = 8.4 Hz, 2H), 7.23
(d, J = 8.2 Hz, 2H), 7.08 (d, J = 8.8 Hz, 1H), 6.51 (s, 1H),
5.46 (bt, J = 5.4 Hz, 1H), 4.84 (d, J = 5.0 Hz, 2H), 2.27 (s,
3H). MS (EI Method) 378 (M + H)+, 360. HPLC (System 1)
97.21%; HPLC (System 2) 96.80%. Anal. (C17H16ClN3O3S)
H; C: calcd, 54.04; found, 54.66; N: calcd, 11.12; found,
10.41.
(s, 1H), 7.26 (s, 1H), 7.00 (d, J = 8.4 Hz, 2H), 6.63 (t,
J = 54.9 Hz, 1H), 6.63 (s, 1H), 6.53 (d, J = 8.2 Hz, 2H), 5.02
(s, 2H), 3.19 (q, J = 7.2 Hz, 2H), 1.25 (t, J = 7.4 Hz, 3H). MS
(CI Method) 423 (M + H)+, 389, 344, 242. HPLC (System 2)
98.31%. Anal. (C19H20F2N4O3S) C, H, N.
5.3.33. Compound 38
Yield 56%. M.p. 155–157 °C. IR (KBr) 3481, 1599, 1563,
1
1487, 1457 cm–1. H NMR (CDCl3) d 7.94 (d, J = 8.4 Hz,
5.3.27. Compound 32
Yield 71%. M.p. 138–139 °C. IR (KBr) 3333, 3211, 1607,
1492 cm–1. 1H NMR (CDCl3) d 7.95 (d, J = 8.3 Hz, 1H), 7.60
(d, J = 1.7 Hz, 1H), 7.26–7.09 (m, 5H), 6.77 (t, J = 54.8 Hz,
1H), 6.72 (s, 1H), 5.02 (d, J = 4.1 Hz, 2H), 2.38 (s, 3H). MS
(CI Method) 393 (M+), 375, 358, 312. HPLC (System 2)
98.21%. Anal. (C18H17F2N3O3S) C, H, N.
1H), 7.59 (d, J = 1.9 Hz, 1H), 7.30–7.17 (m, 2H), 7.06–6.87
(m, 2H), 6.79 (s, 1H), 6.79 (t, J = 54.6 Hz, 1H), 5.02 (d,
J = 4.1 Hz, 2H). MS (CI Method) 415 (M+), 396 (100%),
380, 334. HPLC (System 1) 98.90%.Anal. (C17H13F4N3O3S)
C, H, N.
5.3.34. Compound 39
5.3.28. Compound 33
Yield 68%. M.p. 160–162 °C. IR (KBr) 3441, 3210, 3094,
1
Yield 58%. M.p. 134–136 °C. IR (KBr) 3315, 1601, 1579,
1601, 1575 cm–1. H NMR (CDCl3) d 8.02 (d, J = 8.4 Hz,
1
1486, 1415 cm–1. H NMR (CDCl3) d 7.96 (d, J = 8.5 Hz,
1H), 7.61 (s, 1H), 7.45–7.41 (m, 2H), 7.30–7.19 (m, 2H),
7.00 (d, J = 6.6 Hz, 1H), 6.77 (t, J = 54.6 Hz, 1H), 5.04 (s,
2H). MS (CI Method) 448 (M+), 429, 412, 366. HPLC (Sys-
tem 2) 98.72%. Anal. (C17H13Cl2F2N3O3S) C, H, N.
1H), 7.59 (d, J = 1.9 Hz, 1H), 7.26–7.12 (m, 4H), 6.96–6.93
(m, 2H), 6.77 (t, J = 54.8 Hz, 1H), 5.00 (s, 2H), 2.34 (s, 3H).
MS (CI Method) 393 (M+), 374, 358, 347, 312. HPLC (Sys-
tem 1) 98.50%. Anal. (C18H17F2N3O3S) C, H, N.
5.3.35. Compound 40
Yield 70%. M.p. 140–142 °C. IR (KBr) 3403, 1603 cm–1.
1H NMR (CDCl3) d 7.93 (d, J = 8.6 Hz, 1H), 7.60 (s, 1H),
7.26–7.10 (m, 2H), 7.05 (s, 1H), 6.85 (d, J = 7.4 Hz, 1H),
6.77 (s, 1H), 6.69 (t, J = 54.8 Hz, 1H), 5.55 (s, 2H), 5.00 (s,
2H), 2.28 (s, 3H), 2.23 (s, 3H), 1.92 (bs, 1H). MS (CI Method)
407 (M+), 389, 361. HPLC (System 1) 96.41%; HPLC (Sys-
tem 2) 95.82%.
5.3.29. Compound 34
Yield 65%. M.p. 133–135 °C. IR (KBr) 3273, 2479, 1831,
1636 cm–1. 1H NMR (CDCl3) d 7.97 (d, J = 8.3 Hz, 1H), 7.60
(s, 1H), 7.28–7.12 (m, 5H), 6.79 (t, J = 54.8 Hz, 1H), 6.73 (s,
1H), 5.01 (s, 2H), 2.75 (q, J = 7.4 Hz, 2H), 1.27 (t, J = 8.0 Hz,
3H). MS (CI Method) 407 (M+), 389, 372, 354, 325. HPLC
(System 1) 98.82%. Anal. (C19H19F2N3O3S) C, H, N.
5.3.30. Compound 35
5.3.36. Compound 41
Yield 62%. M.p. 146–149 °C. IR (KBr) 3361, 1600, 1580,
Yield 72%. M.p. 134–136 °C. IR (KBr) 3474, 3329, 1741,
1604, 1553 cm–1. 1H NMR (CDCl3 + DMSO-d6) d 7.94 (d,
J = 8.4 Hz, 1H), 7.62 (s, 1H), 7.16 (d, J = 7.6 Hz, 2H), 6.97
(d, J = 7.2 Hz, 2H), 6.76 (t, J = 54.6 Hz, 1H), 6.69 (s, 1H),
4.95 (s, 2H), 2.26 (s, 3H). MS (CI Method) 411 (M+), 391,
376, 357. HPLC (System 1) 98.33%.Anal. (C18H16F3N3O3S)
C, H, N.
1
1485, 1413 cm–1. H NMR (CDCl3) d 8.00 (d, J = 8.3 Hz,
1H), 7.59 (d, J = 2.0 Hz, 1H), 7.42–7.19 (m, 4H), 7.06–7.02
(m, 1H), 6.79 (t, J = 54.9 Hz, 1H), 6.77 (s, 1H), 5.03 (s, 2H).
MS (EI Method) 413(M+), 394 (100%), 378, 332. HPLC (Sys-
tem 1) 99.21%. Anal. (C17H14ClF2N3O3S) C, H, N.
5.3.31. Compound 36
Yield 67%. M.p. 166–167 °C. IR (KBr) 3306, 1600 cm–1.
1H NMR (CDCl3) d 7.98 (d, J = 8.4 Hz, 1H), 7.61 (d,
J = 2.0 Hz, 1H), 7.25 (d, J = 5.4 Hz, 2H), 7.15 (d, J = 8.6 Hz,
2H), 7.12–7.05 (m, 1H), 6.77 (t, J = 54.8 Hz, 1H), 6.73 (s,
1H), 5.48 (bs, 2H), 5.03 (d, J = 3.8 Hz, 2H), 2.80 (bs, 1H),
2.51 (s, 3H). 13C NMR (DMSO-d6) d 147.3 (t, J = 28.8 Hz,
1C), 144.3, 142.7, 141.7, 140.1, 139.5, 129.1 (2C), 128.1,
125.6 (2C), 125.0, 123.6, 123.0, 111.4 (t, J = 231.7 Hz, 1C),
105.6, 59.4, 14.2. MS (CI Method) 425 (M+), 406, 390, 344.
HPLC (System 2) 98.64%. Anal. (C18H17F2N3O3S2) C, H,
N.
5.3.37. Compound 42
Yield 58%. M.p. 170–172 °C. IR (KBr) 3332, 1603 cm–1.
1H NMR (CDCl3) d 7.96 (d, J = 8.6 Hz, 1H), 7.64 (d,
J = 1.8 Hz, 1H), 7.27 (s, 1H), 7.23 (dd, J = 6.6 & 2.0 Hz,
1H), 7.08 (d, J = 8.6 Hz, 2H), 6.82 (t, J = 56.0 Hz, 1H), 6.66
(s, 1H), 5.50 (bs, 2H), 5.03 (s, 2H), 2.80 (bs, 1H), 2.49 (s,
3H), 2.30 (s, 3H). MS (CI Method) 439 (M+), 419, 358. HPLC
(System 1) 95.95%; HPLC (System 2) 96.20%.
5.3.38. Compound 43
Yield 55%. M.p. 160–162 °C. IR (KBr) 3313, 1602,
1452 cm–1. 1H NMR (CDCl3 + DMSO-d6) d 7.90 (d,
J = 8.4 Hz, 1H), 7.67 (d, J = 1.6 Hz, 1H), 7.20 (d, J = 6.8 Hz,
2H), 7.16 (d, J = 5.8 Hz, 1H), 6.98 (d, J = 5.8 Hz, 1H), 6.85
(s, 2H), 6.55 (s, 1H), 6.50 (t, J = 54.8 Hz, 1H), 4.99 (s, 2H),
5.3.32. Compound 37
Yield 65%. M.p. 165–166 °C. IR (KBr) 3344, 3302, 1613,
1453 cm–1. 1H NMR (CDCl3) d 7.96 (d, J = 8.3 Hz, 1H), 7.60

988
S.K. Singh et al. / European Journal of Medicinal Chemistry 40 (2005) 977–990
3.47 (t, J = 4.5 Hz, 2H), 3.22 (t, J = 4.8 Hz, 2H), 2.54 (bs,
1H). MS (CI Method) 437 (M+), 419, 338. HPLC (System 1)
98.92%; HPLC (System 2) 98.62%.Anal. (C19H17F2N3O3S2)
C, H, N.
5.3.43. 1-(3-Hydroxymethyl-4-sulfamoylphenyl)-5-(4-meth-
oxyphenyl)-1H-3-pyrazolecarboxylic acid 47
Sodium hydroxide (57 mg, 1.43 mmol) was dissolved in
methanol (5 ml) and methyl 1-(3-hydroxymethyl-4-sulfa-
moylphenyl)-5-(4-methoxyphenyl)-1H-3-pyrazolecarbo-
xylate (500 mg, 1.19 mmol), prepared by the general method
5.3.39. Compound 44
Prepared using hydrazine hydrochloride 2 and the corre-
sponding diketone. Yield 74%. M.p. 136–139 °C. IR (KBr)
3517, 1613, 1473 cm–1. 1H NMR (CDCl3) d 8.25 (d,
J = 1.4 Hz, 1H), 7.84 (dd, J = 8.2 & 2.0 Hz, 1H), 7.19 (d,
J = 8.2 Hz, 1H), 7.06 (d, J = 8.4 Hz, 2H), 6.85 (d, J = 8.8 Hz,
2H), 6.81 (s, 1H), 4.60 (s, 2H), 3.88 (s, 3H), 3.17 (s, 3H),
1.95 (bs, 1H). 13C NMR (DMSO-d6) d 160.0, 146.1, 142.3
(q, J = 37.5 Hz, 1C), 141.8, 141.0, 140.1, 129.8 (2C), 129.0,
126.5 (2C), 121.2 (q, J = 267.0 Hz, 1C), 120.0, 114.3 (2C),
104.4, 58.3, 55.2, 43.4. MS (CI Method) 426 (M+), 409, 397.
HPLC (System 1) 98.16%. Anal. (C19H17F3N2O4S) C, H, N.
using hydrazine hydrochloride
1 and methyl 4-(4-
methoxyphenyl)-2,4-dioxobutanoate 3b (Scheme 3), was
dumped at room temperature, and stirred for 5 h. The reac-
tion mixture was poured in ice water, acidified with 2 N HCl
and extracted with ethyl acetate. The combined organic layer
was washed with water and evaporated to get a solid which
on further trituration with ethyl acetate–petroleum ether
afforded a white solid of the title compound 47 (330 mg,
68%). M.p. 250–252 °C. IR (KBr) 3333, 1700, 1608,
1465 cm–1. 1H NMR (DMSO-d6) d 7.86–7.83 (m, 2H), 7.24–
7.19 (m, 3H), 7.01–6.93 (m, 2H), 6.87 (s, 1H), 4.89 (s, 2H),
3.76 (s, 3H). MS (CI Method) 403 (M+, 100%), 383, 322,
307. HPLC (System 2) 98.92%. Anal. (C18H17N3O6S) C, H,
N.
5.3.40. Compound 45
Yield 64%. M.p. 97–99 °C. IR (KBr) 3439, 1603 cm–1. 1H
NMR (CDCl3) d 8.27 (d, J = 1.80 Hz, 1H), 7.84 (dd, J = 7.4
& 1.8 Hz, 1H), 7.28–7.16 (m, 3H), 7.05 (d, J = 8.8 Hz, 2H),
6.85 (s, 1H), 4.60 (d, J = 7.0 Hz, 2H), 3.20 (bt, 1H), 3.11 (s,
3H), 2.54 (s, 3H). MS (CI Method) 442 (M+), 425, 413. HPLC
(System 1) 96.05%; HPLC (System 2) 95.20%. Anal.
(C19H17F3N2O3S2) C, H; N: calcd, 6.33; found, 6.74.
5.3.44. 4-[3-Cyano-5-(4-methoxyphenyl)-1H-1-pyrazolyl]-
2-hydroxymethyl-1-benzenesulfonamide 48 [35]
Methyl 1-(3-hydroxymethyl-4-sulfamoylphenyl)-5-(4-
methoxyphenyl)-1H-3-pyrazolecarboxylate
(500
mg,
1.19 mmol), prepared by the general method using hydrazine
hydrochloride 1 and methyl 4-(4-methoxyphenyl)-2,4-
dioxobutanoate 3b (Scheme 3), was taken in aq. ammonia
(25%, 5 ml) and stirred at ambient temperature in a closed
flask for 15 h. The reaction mixture was poured in ice water,
acidified with 2 N HCl and extracted with ethyl acetate. The
combined organic layer was washed with water and evapo-
rated to get a gummy mass which on trituration with dichlo-
romethane afforded an expected off white solid of carboxa-
mide (300 mg, 62%). The sample was used in the next step
without further purification. IR (KBr) 3432, 2926, 1680, 1600,
1328 cm–1. 1H NMR (DMSO-d6) d 7.85–7.80 (m, 2H), 7.78
(bs, 1H), 7.62 (bs, 2H), 7.42 (bs, 1H), 7.40–7.20 (m, 3H),
7.25–6.98 (m, 3H), 5.50 (bt, 1H), 4.89 (d, J = 4.2 Hz, 2H),
3.78 (s, 3H). MS (CI Method) 402 (M+, 100%), 384, 305.
The carboxamide (100 mg, 0.24 mmol) was dissolved in dry
dioxane (5 ml), treated with pyridine (20 mg, 0.25 mmol)
and cooled to 0–5 °C. Trifluoroacetic anhydride (100 mg,
0.47 mmol) was added at this temperature and reaction mix-
ture was left for stirring overnight at room temperature. The
reaction mixture was poured over ice water and extracted with
ethyl acetate. The combined organic layer was washed with
water and evaporated to get a gummy mass which was puri-
fied by column chromatography to get the title product 48 as
viscous liquid (50 mg, 52%). IR (Neat) 3252, 2244, 1612,
1498 cm–1. 1H NMR (CDCl3) d 8.00 (d, J = 8.2 Hz, 1H), 7.61
(s, 1H), 7.25–7.14 (m, 1H), 7.12 (d, J = 8.8 Hz, 2H), 6.90 (d,
J = 8.6 Hz, 2H), 6.82 (s, 1H), 5.42 (bs, 2H), 5.03 (d,
J = 4.2 Hz, 2H), 3.83 (s, 3H), 2.86 (bs, 1H). MS (CI Method)
384 (M+), 349, 303. HPLC (System 1) 96.28%; HPLC (Sys-
tem 2) 97.20%. Anal. (C18H16N4O4S) C, H, N.
5.3.41. Compound 46
Yield 58%. M.p. 86–88 °C. IR (KBr) 3434, 1602,
1451 cm–1. 1H NMR (CDCl3) d 8.24 (d, J = 1.8 Hz, 1H), 7.80
(dd, J = 5.8 & 2.4 Hz, 1H), 7.20–7.10 (m, 3H), 7.04 (d,
J = 8.4 Hz, 2H), 6.82 (s, 1H), 6.76 (t, J = 54.4 Hz, 1H), 4.61
(d, J = 6.4 Hz, 2H), 3.47 (bt, 1H), 3.11 (s, 3H), 2.49 (s, 3H).
MS (CI Method) 424 (M+), 407, 395. HPLC (System 1)
98.10%. HPLC (System 2) 97.71%.Anal. (C19H18F2N2O3S2)
C, H, N.
5.3.42. 2-Hydroxymethyl-4-[5-(3-methyl-4-methylsulfi-
nylphenyl)-3-trifluoromethyl-1H-1-pyrazolyl]-1-
benzenesulfonamide 16 [34]
m-Chloroperbenzoic acid (202 mg, 1.74 mmol) was slowly
added to a pre-cooled (0–5 °C) solution of compound 14
(400 mg, 0.87 mmol) in dichloromethane (10 ml) and stirred
the reaction mixture at room temperature for 2 h. The reac-
tion mixture was poured over ice water and extracted with
dichloromethane. The combined organic layer was sequen-
tially washed with 10% aqueous sodium bicarbonate and
water. The solvent was evaporated after drying to get a vis-
cous liquid which on further purification by column chroma-
tography using 20% ethyl acetate–petroleum ether afforded a
white solid of the title compound 16 (380 mg, 92%). M.p.
174–176 °C. IR (KBr) 3359, 1733, 1602, 1467 cm–1. 1H NMR
(DMSO-d6) d 7.86–7.75 (m, 3H), 7.42 (s, 1H), 7.31–7.19 (m,
3H), 4.91 (d, J = 3.8 Hz, 2H), 2.71 (s, 3H), 2.33 (s, 3H). MS
(CI Method) 474 (M + H)+, 457, 440, 392. HPLC (System 1)
98.57%. Anal. (C19H18F3N3O4S2) C, H, N.

S.K. Singh et al. / European Journal of Medicinal Chemistry 40 (2005) 977–990
989
5.4. In vitro biochemical assays.
activities of the compounds in the set. The predictive r2 for
the test set was calculated using the formula:
Spectrophotometric assay for COX-1 and COX-2 [36,37]
SD − PRESS
r2pred
=
Microsomal fraction of ram seminal vesicles were used as
a source of COX-1 enzyme, and microsomes from sf-9 cells
infected with baculovirus containing human COX-2 c-DNA
were used as a source of COX-2 enzyme. Enzyme activity
was measured using a chromogenic assay based on oxidation
of N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) dur-
ing the reduction of PGG2 to PGH2. The assay mixture
(1000 µl) contained Tris pH 8.0 (100 mM), EDTA (3 mM),
hematin (15 µM), enzyme (150 units) and DMSO (8%). The
mixture was pre-incubated at 25 °C for 15 min before initia-
tion of enzymatic reaction in presence of compound/vehicle.
The reaction was initiated by the addition of arachidonic acid
(100 µM) and TMPD (120 µM). The enzyme activity was
measured by the estimation of initial velocity of TMPD oxi-
dation over the first 25 s of the reaction followed by tracking
the increase in absorbance at 603 nM.
SD
where SD is the sum of the squared deviations between the
biological activities of the test set and mean activity of the
training set molecules, PRESS is sum of squared deviation
between predicted and actual activity values from each mol-
ecule in the test set.
Acknowledgements
The authors are thankful to Dr. K. Anji Reddy and Profes-
sor J. Iqbal for their constant support and encouragement,
and to Analytical Research group, DR-DRL, for spectral
analysis.
References
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with the IRIX 6.5 operating system. All the molecular struc-
tures were sketched using the coordinates of SC-558 (6COX)
[22] and their energies were minimized using MMFF94 force
field with a gradient convergence of 0.05 kcal mol–1. Further
geometrical optimizations were performed using AM1 [42]
method in order to obtain electrostatic potential charges. The
negative logarithm of the molar IC50 values (pIC50) was used
for the study in order to achieve a normal distribution of activ-
ity values. The most active compound 14 was used as the
template, and others were aligned to it by atom fit method
(Fig. 2).
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5.6. CoMFA
The steric and electrostatic potential fields for CoMFA
were calculated at each lattice intersection of a regularly
spaced grid of 2.0 Å. A sp3 carbon atom with + 1.0 charge
was selected as a probe for this calculation.Values of the steric
and electrostatic energies were truncated at 30 kcal mol–1.
The partial least-square (PLS) [43] method was used for fit-
ting the 3D structural features and their biological activities.
It was cross validated using the leave one out method with a
2 kcal mol–1 column filter to check the consistency and pre-
dictivity. The optimum number of components used to derive
the non-cross validated model was defined as the one leading
to the considerably higher value of q2 with the least standard
error of prediction (SEP). Statistical validity of the model was
assessed by the variance (r2), SEE and the F-value. Models
were further evaluated on the basis of their ability to predict
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