Synthesis, docking studies and anti-inflammatory activity of 4,5,6,7-tetrahydro-2H-indazole derivatives

Article abstract of DOI:10.1016/j.bmc.2007.03.006

A regioselective synthesis of 2,3-disubstituted tetrahydro-2H-indazols, mediated by α-zirconium sulfophenylphosphonate-methanephosphonate, was reported. Docking studies into the catalytic site of COX-2 were used to identify potential anti-inflammatory lea

Full text of DOI:10.1016/j.bmc.2007.03.006

Bioorganic & Medicinal Chemistry 15 (2007) 3463–3473
Synthesis, docking studies and anti-inflammatory activity
of 4,5,6,7-tetrahydro-2H-indazole derivatives
Ornelio Rosati,a,* Massimo Curini,a Maria Carla Marcotullio,a Antonio Macchiarulo,a
Marina Perfumi,b Laura Mattioli,b Francesco Rismondob and Giancarlo Cravottoc
aDipartimento di Chimica e Tecnologia del Farmaco, Via del liceo 1, University of Perugia, 06123 Perugia (PG), Italy
b
`
`
Dipartimento di Medicina Sperimentale e Sanita Pubblica, Universita di Camerino Via Scalzino 3, 62032 Camerino (MC), Italy
cDipartimento di Scienza e Tecnologia del Farmaco, Via P. Giuria 9, University of Torino, 10125 Torino (TO), Italy
Received 12 December 2006; revised 28 February 2007; accepted 2 March 2007
Available online 6 March 2007
Abstract—A regioselective synthesis of 2,3-disubstituted tetrahydro-2H-indazols, mediated by a-zirconium sulfophenylphospho-
nate-methanephosphonate, was reported. Docking studies into the catalytic site of COX-2 were used to identify potential anti-
inflammatory lead compounds. Two lead derivatives were chosen endowed with good binding energies and good ADME profiling.
The biological in vivo evaluation of these compounds in two different experimental models (Freund’s adjuvant-induced arthritis and
carrageenan-induced oedema) proved the presence of anti-inflammatory activity. Noteworthy, both compounds evidenced the lack
of any gastric injury even at high doses in gastric ulcerogenic assays.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
COX-1.10 Thus, COX-2 is a validated molecular target
whose selective inhibition is sought in the development
of anti-inflammatory therapies (Fig. 1).
The pyrazole skeleton constitutes an important central
template for a wide variety of biologically active com-
pounds,1 such as anti-microbial (1),2 antiviral (2),3
anti-inflammatory (3),4 antidepressant (4),5 anti-hyper-
glycaemic (5)6 and pesticidal activity (6).7
The presence of a pyrazole nucleus is a common feature
in the chemical structure of several COX-2 inhibitors. In
particular, it consists in five- or six-membered carbocy-
clic or heterocyclic central template which is 1,2-disub-
stituted by aryl moieties. Two of the most known
COX-2 selective inhibitors containing a pyrazole moiety
are celecoxib (7, Celebrex)11 and SC-558 (8).11
In particular some of pyrazole derivatives were in depth
investigated as nonsteroidal anti-inflammatory drugs
(NSAIDs). The mechanism of action of this class of
compounds is linked to the nonselective or selective inhi-
bition of two cyclooxygenase isoforms, namely COX-1
and COX-2.8 While COX-1 is a constitutive enzyme
and is necessary for the proper function of the kidney
and stomach through the synthesis of prostaglandins,
COX-2 is an inducible form of the enzyme that mediates
the inflammatory processes.9
Recently, it has been reported that the cycloalkanopy-
razole nucleus with a 1,3-diaryl substituted pattern
(9a–c) is also a suitable scaffold for the development of
selective COX-2 inhibitors.4b
The synthesis of this class of compounds relies on syn-
thetic methodologies that use the pyrazole nucleus as
starting building block.4b,12,13 In searching for more
general and versatile synthetic methodologies to prepare
cycloalkanopyrazole derivatives, we recently reported
that the use of a heterogeneous catalyst, such as layered
zirconium sulfophenylphosphonate methanephospho-
nate [a-Zr(CH3PO3)1,2(O3PC6H4SO3H)0,8],14 can be
very useful for the synthesis of both pyrazole and
The selective inhibition of COX-2 avoids the presence of
gastrointestinal and renal side effects associated with the
inhibition of the production of prostaglandins by
Keywords: Tetrahydro-2H-indazoles; Anti-inflammatory activity;
Docking studies.
*
Corresponding author. Tel.: +39 075 5855103; fax: +39 075
5855116; e-mail: ornros@unipg.it
4,5,6,7-tetrahydro-1(2)H-indazole
derivatives
(1,2-
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.03.006

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O. Rosati et al. / Bioorg. Med. Chem. 15 (2007) 3463–3473
R
O
N
N
H
N
N
N
N
N
N
H
N
HS
OMe
O
N
N
NC
1
2
3
H
N
N
N
N
N
HO
CF3
N
N
6
R
S
4
5
Figure 1. Some representative examples of active compounds containing a pyrazole moiety.
Y
H2NO2S
H2NO2S
N
N
N
N
N
N
X
CF3
CF3
Br
9a X=H, Y=H
9b X=Cl, Y=H
8
7
9c X=Cl, Y=OCH3
disubstituted cycloalkanopyrazole), starting from
hydrazine and 1,3-diketones (Scheme 1).15 In particular,
this methodology has proven to be particularly effective
towards the use of less reactive hydrazine such as 2,4-
dinitrophenylhydrazine.
mixture formation,4a low/medium reaction yield.13a,3c or
expensive reagents.13b
We found that a-zirconium sulfophenylphosphonate-
methanephosphonate [a-Zr(CH3PO3)1,2(O3PC6H4SO3H)0,8]
is an effective catalyst for the regioselective synthesis
of 2,3-diaryl-4,5,6,7-tetrahydro-2H-indazole derivatives
(Scheme 2).
With the aim of developing novel anti-inflammatory
lead compounds based on the selective inhibition of
COX-2 enzyme and as a continuation of our ongoing
effort in the field of pyrazole derivatives, we afforded
the regio-selective synthesis of 2,3-disubstituted-4,5,6,7-
tetrahydro-2H-indazoles (1,2-diarylcycloalkanopyrazoles).
The preparation, molecular modelling studies and
in vivo biological assays of this class of compounds
are herein reported.
The 2,3-diaryl-4,5,6,7-tetrahydro-2H-indazoles 10–15
were synthesized starting from 2-benzoylcyclohexanone
and substituted hydrazines in the presence of
a-Zr(CH3PO3)1,2(O3PC6H4SO3H)0,8 as the catalyst, in
solvent-free and mild conditions. The adducts 10–15
were obtained with high yield as single isomer (Table
1). 1H NMR experiments, such as NOESY, were carried
out to establish the 1,2-disubstitution of the synthesized
compounds.
2. Chemistry
The synthesis of 2,3-diaryl-4,5,6,7-tetrahydro-2H-indaz-
oles somewhat demanding and can be affected by isomer
For this purpose, we considered the possibility to observe
NOE correlation between the aromatic substituents
R
R
O
O
neat, 40°C, RNHNH
α-Zr(CH3PO3)1.2(O3PC6H4SO3H)0.8
2
N
N
N
N
e/o
R1
R2
R1
R2
R1
R2
Scheme 1. Synthesis of Pyrazole derivatives mediated by zirconium sulfophenylphosphonate methanephosphonate.

O. Rosati et al. / Bioorg. Med. Chem. 15 (2007) 3463–3473
3465
R
1
2
O
O
N
N
3
7a
5
neat, 40°C, RNHNH
2
7
6
3a
α-Zr(CH3PO3)1.2(O3PC6H4SO3H)0.8
4
10-15
Scheme 2. Synthesis of 2,3-disubstituted tetrahydro-2H-indazole derivatives mediated by zirconium sulfophenylphosphonate methanephosphonate.
free energies with inhibitory activities of compounds.18
Table 1. Reaction of 2-benzoylcyclohexanones with hydrazines in the
presence of a-Zr(CH3PO3)1,2(O3PC6H4SO3H)0,8
Briefly, we carried out comparative docking experiments
of compounds 10–15 with known selective inhibitors of
COX-2 such as SC-558 (8)17 and a representative set of
1,3-diarylcycloalkanopyrazole derivatives (9a–c).4b The
obtained results were evaluated in terms of binding
energy and docking pose into the catalytic site of
COX-2. Docking calculations predicted the binding con-
formation of SC-558 (8) with a root mean square devia-
Compound Hydrazines
Yield Time
(%) (h)
10
11
12
13
14
15
Phenylhydrazine 96
2-Hydrazino-4-(trifluoromethyl)pyrimidine 95
2
5
4-(Trifluoromethyl)phenylhydrazine
Methyl hydrazinecarboxylate
2-Hydrazinopyridine
92
95
81
86
6
18
12
5
˚
tion (RMSD) of 1.56 A from the conformation obtained
2,5-Difluorophenylhydrazine
with X-ray crystallographic studies (Fig. 2). The high
RMSD value observed between the predicted and the
experimental bioactive conformation of SC-558 is
ascribed to the different orientation that the sulfonamide
group adopts in the docked conformation. However,
several studies pinpoint that the sulfonamide group
could bind at the enzyme in several conformations than
suggested by the crystal structure.17–19 In particular, Liu
et al. quantified the energetic preference of the sulfon-
amide group of SC-558 binding at COX-2 as resulting
from Autodock prediction.18 The authors indicated that
the predicted conformation was lower in energy than the
observed conformation in the crystal structure of COX-2.
and the protons of the cyclohexane ring system (H-4 and
H-7).
While it is possible to see the crosspeak of NOE correla-
tion between the protons of the phenyl group on C-3
and the H-4, the lack of crosspeak of NOE correlation
between the protons of the aromatic substituent on
nitrogen and the H-7 is of a fundamental meaning for
the assignment of 1,2-disubstitution in the synthesized
compounds.
3. Results and discussion
Table 2 shows the energetic scores of the top solutions
found during docking experiments of compounds
8–15. SC-558 (8) is endowed with the best binding
energy among the studied compounds (À11.53 kcal/
A number of molecular docking experiments were car-
ried out to identify potential COX-2 inhibitors among
the class of 2,3-diaryl-4,5,6,7-tetrahydro-2H-indazoles
(10–15). The resulting lead compounds were tested for
their anti-inflammatory property in male Wistar rats
(Harlan SRC, Milan, Italy), weighting 250–300 g, fol-
lowing two different experimental models: Freund’s
adjuvant-induced arthritis and carrageenan-induced
oedema, both causing articular inflammation, frequently
associated with erythema, swelling of periarticular tis-
sues, enlargement and distortion of the joints, and
involving limbs hardening and loss of limbs functional-
ity. In addition, the selected lead compounds were eval-
uated for their anti-nociceptive activity and gastric
ulcerogenic response.
3.1. Molecular docking experiments
To identify potential anti-inflammatory lead compounds
among 10–15 endowed with COX-2 inhibition, docking
calculations were performed using Autodock v3.016 into
the 3D model of the catalytic site of COX-2 enzyme
(pdb code: 1cx2).17
It should be mentioned that the Lamarckian genetic
algorithm implemented in Autodock has been success-
fully employed to dock inhibitors into the catalytic site
of the COX-2 and to well correlate the obtained binding
Figure 2. Superposition between the predicted conformation of SC-
558 (8) from docking experiments (carbon atoms in cyan colour) and
the crystallographic conformation (carbon atoms in grey colour).

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O. Rosati et al. / Bioorg. Med. Chem. 15 (2007) 3463–3473
Table 2. Binding energies of top scoring solutions as resulting from
docking experiments
Compound
Binding energy (kcal/mol)
IC50 (lM)
8
À11.53
À9.50
À9.96
À9.32
À9.67
À10.59
À10.88
À9.06
À9.76
À10.26
0.0093a
0.57b
0.62b
1.56b
—
9a
9b
9c
10
11
12
13
14
15
—
—
—
—
—
a Activity data from Ref. 17.
b Activity data from Ref. 4b.
mol). Two compounds (11 and 12) out of six 2,3-disub-
stituted-cycloalkanopyrazoles display improved binding
energies compared to the active 1,3-diarylcycloalkano-
pyrazole derivatives (9a–c) with a minimum difference
of 0.63 kcal/mol between compounds 11 and 9b.
The inspection of the obtained COX-2 complexes with
SC-558 (8) and compounds 11 and 12 reveals binding
poses located in the centre of the binding pocket of
COX-2. In this binding mode, three anchor sites (P1,
P2 and P3) can clearly be identified (Fig. 3).
The site P1 contains the recognition elements of the sul-
fonamide group of SC-558 (8) and the trifluoromethyl
moiety of compounds 11 and 12. In this pocket the ami-
do group of sulfonamide moiety acts as an hydrogen
bond donor with the backbone oxygen of Leu352 and
the side-chain oxygen of Gln192, while one oxygen of
the moiety acts as an hydrogen bond acceptor with the
guanidine group of Arg513 (Fig. 4a).
The trifluoromethyl moiety of compounds 11 and 12
forms hydrogen bond interactions with the backbone
of Ile517 and the amido group of Gln192 (Fig. 4b and c).
These hydrogen-bonding interactions play a key role
Figure 4. Residues involved in the interactions with compounds 8 (a),
11 (b) and 12 (c) in the binding site of COX-2 as resulting from
docking experiments.
in determining the 3D space position of the cycloalkano-
pyrazole moieties in the binding pocket of COX-2.
Thus, the difference observed between the binding mode
of the cycloalkanopyrazole moiety of compounds 11
and 12 is ascribed to the strong hydrogen-bonding inter-
Figure 3. Anchor sites (P1, P2 and P3) of compounds SC-558 (8), 11
and 12 in the binding pocket of COX-2.

O. Rosati et al. / Bioorg. Med. Chem. 15 (2007) 3463–3473
3467
action that the trifluoromethyl group of 11 forms in site
P1. This interaction is more energetically favoured than
the one resulting with the trifluoromethyl group occupy-
ing site P3. The occupancy of site P1 by the trifluorom-
ethyl group hampers that the cycloalkanopyrazole
moiety of compound 11 could assume a similar docking
pose as observed in compound 12.
terms of binding energy with a difference of 1.0 kcal/
mol from SC-558 (8). Compound 12 violates the role
of five once with its logP value. However, since this vio-
lation is subtle, 0.49 U above the cut-off value of 5, and
given its better binding energy within the series of 2,3-
disubstituted-cycloalkanopyrazoles, we took this com-
pound into account for in vivo biological assays.
The site P2 binds the trifluoromethyl group of SC-558,
the phenyl group of compound 11 and the cycloalkano-
pyrazole moiety of 12. While the trifluoromethyl group
of SC-558 (8) acts as an hydrogen bond acceptor with
the guanidine group of Arg120, the phenyl ring of com-
pound 11 forms p–cation interaction with the positively
charged side chain of Arg120. Conversely, hydrophobic
interactions with the side-chain carbons of Arg120 stabi-
lize the binding of the cycloalkanopyrazole moiety of
compound 12 at site P2.
3.2. Anti-inflammatory activity: Freund’s adjuvant-
induced arthritis
Freund’s adjuvant-induced arthritis has been used as a
model of sub-chronic or chronic inflammation in rats
and it is of considerable relevance for the study of path-
ophysiological and pharmacological control of inflam-
matory processes, as well as the evaluation of potential
analgesic or anti-inflammatory effects of drugs.23,24
One of the reasons for the wide utilization of this model
is due to the strong correlation between the efficiency of
therapeutic agents in this model and in rheumatoid
arthritis in humans. The arthritis is induced by a sub-
plantar injection of 0.1 ml of Freund’s adjuvant (com-
plete fraction of Mycobacterium butyricum suspended
in mineral oil; Sigma Chemical Co., USA) in the right
paw’s plantar surface. The adjuvant elicits arthritis pre-
dominantly in the joints of hind limbs, promoting signif-
icant reduction of motor activity and increased itching
and scratching behaviours.25
The anchor site P3 interacts with the p-bromo-phenyl
moiety of SC-558, the cycloalkanopyrazole moiety of
11 and the phenyl ring of compound 12 through hydro-
phobic interactions that involve residues Met522,
Val523 and Ala527.
The binding pose of SC-558 (8) and compound 12 is also
enforced through an hydrogen bond interaction between
the central pyrazole moiety and the hydroxyl group of
Tyr355.
All animals were subjected to assessment of body weight
and measurements of the right paw (the width and
height of the paw and width of the joints were measured
with a caliper ruler) (baseline). Subsequently, Freund’s
adjuvant was injected in the right paw: 3 days after the
administration (this period was established with preli-
minary tests and it is necessary for achieving a relevant
inflammatory disease), the animals were subjected again
to the measures to verify the arthritis development (day
0), and then they were randomly assigned to one of the
experimental groups.
The bioavailability of compounds 10–15 was assessed
using ADME (adsorption, distribution, metabolism,
elimination) prediction methods. In particular, we calcu-
lated the compliance of compounds to the Lipinski’s
role of five.20 Briefly, this simple role is based on the
observation that most orally administered drugs have
a molecular weight (MW) of 500 or less, a logP no high-
er than 5, five or fewer hydrogen bond donor sites and
10 or fewer hydrogen bond acceptor sites (N and O
atoms). In addition, we calculated the polar surface area
(PSA) since it is another key property that has been
linked to drug bioavailability.21 Thus, passively
Starting from this day (day 0), rats were orally injected
once daily for further 3 days (day 1, day 2, day 3) with
compound 11 (10, 50, 100, 200 mg/kg/10 ml), compound
12 (10, 50, 100 mg/kg/10 ml) or vehicle (10% DMSO,
10% TWEEN 80, 80% distilled water).
2
˚
absorbed molecules with a PSA > 140 A are thought
to have low oral bioavailabilities.22
On the basis of docking results (Table 2) and bioavail-
ability scores (Table 3) we choose compounds 11 and
12for in vivo evaluation of their anti-inflammatory
activity. Compound 11 is the third ranked molecule in
Evaluation of the anti-inflammatory effects of tested
compounds was performed on day 1, day 2, day 3 and
on day 4 (the day after the last injection), by monitoring
the width and height of the paw and the width of the
joints in the right paw.
Table 3. Compliance of compounds to computational parameters of
bioavailability
2
PSA (A )
˚
Compound
No. of role of five violations
The same experimental procedure was carried out to test
the anti-inflammatory activity of compound 11 (10, 50,
100, 200 mg/kg/2 ml), compound 12 (10, 50, 100 mg/
kg/2 ml) or vehicle (10% DMSO, 10% TWEEN 80,
80% distilled water), after intraperitoneal injection.
8
0
86.36
17.82
17.82
27.05
17.82
43.60
17.82
44.12
30.71
17.82
9a
9b
9c
10
11
12
13
14
15
0
1 (logP = 5.45)
1 (logP = 5.19)
0
0
1 (logP = 5.49)
The obtained results confirmed the ability of Freund’s
adjuvant administration to induce a severe inflamma-
tory status in rat’s hind paw. After a 4-day course of
treatment, progression of inflammation was reversed,
0
0
0

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O. Rosati et al. / Bioorg. Med. Chem. 15 (2007) 3463–3473
depending on the dose and on the route of compounds
administered.
oral or ip injection of compound 11 was ineffective to
decrease the joint size compared to vehicle group
[F(3,20) = 0.579; p > 0.05] and [F(3,20) = 1.400; p > 0.05],
respectively (Fig. 5c–f).
Specifically, both oral and ip administration of higher
doses of compound 11 reduced paw height
[F(3,20) = 3.898;
p < 0.05]
and
[F(3,20) = 4.052;
All doses of compound 12 were ineffective to reduce
paw-oedema when orally administered (paw height,
[F(3,20) = 1.073; p > 0.05]; paw width, [F(3,20) = 0.081;
p > 0.05]; joint width, [F(3,20) = 1.456; p > 0.05])
(Fig. 6a–c, respectively). Instead ip administration of
compound 12 (50, 100 mg/kg) significantly decreased
p < 0.05], respectively, from the 3rd or 2nd treatment
on (Fig. 5a–d), whereas paw width was significantly re-
duced by ip administration [F(3,20) = 7.486; p < 0.01]
(Fig. 5e) but not by oral administration [F(3,20) =
1.743; p > 0.05] (Fig. 5b). Regarding joint width, either
Figure 5. Anti-inflammatory activity of compound 11 on Freund’s adjuvant-induced arthritis in rats. (a–c) and (d–f) represent the effect of,
respectively, oral and ip administration of compound 11 (10, 50, 100 mg/kg) or vehicle on the different measurements of paw height, paw width and
joint width, respectively, before (baseline) and after (day 0) arthritis-induction and throughout 4-day course of treatment (days 1–4). Values are
*
expressed as means SEM. Difference from vehicle group: p < 0.05, **p < 0.01, where not indicated, the difference was not statistically significant.

O. Rosati et al. / Bioorg. Med. Chem. 15 (2007) 3463–3473
3469
Figure 6. Anti-inflammatory activity of compound 12 on Freund’s adjuvant-induced arthritis in rats. (a–c) and (d–f) represent the effect of,
respectively, oral and ip administration of compound 12 (10, 50, 100 mg/kg) or vehicle on the different measurements of paw height, paw width and
joint width, respectively, before (baseline) and after (day 0) arthritis-induction and throughout 4-day course of treatment (days 1–4). Values are
*
expressed as means SEM. Difference from vehicle group: p < 0.05, **p < 0.01, where not indicated, the difference was not statistically significant.
either paw height [F(3,20) = 15.709; p < 0.001], paw
width [F(3,20) = 8.770; p < 0.001] and joint width
[F(3,20) = 13.725; p < 0.001] from the 1st treatment on
(Fig. 6d–f, respectively).
inflammatory response on injection into tissues. Injected
into the footpad of rats it is known to cause physiological
and biochemical changes leading to oedema limited to the
affected limb,27,28 over a short time course (maximal effect
after4–6 h).29 Rats, fasted overnight, received orally com-
pound 11 (10, 50, 100 mg/kg/10 ml), compound 12 (10, 50,
100 mg/kg/10 ml) or vehicle.
3.3. Anti-inflammatory activity: carrageenan-induced
paw-oedema
Anti-inflammatory activity of lead compounds was also
determined by carrageenan-induced paw-oedema.26 Car-
rageenan is a polymeric irritant that induces an acute
One hour later, paw-oedema was induced by injection
of 0.1 ml of 1.0% k carrageenan suspension in the
sub-plantar region of the right hindpaw. An equivalent

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O. Rosati et al. / Bioorg. Med. Chem. 15 (2007) 3463–3473
Figure 7. Anti-inflammatory activity of compound 11 (a) and compound 12 (b), orally administered, on carrageenan-induced paw-oedema in rats,
expressed as weight difference of carrageenan-treated vs. vehicle-treated paw. Values are expressed as means SEM. Difference from vehicle group:
*p < 0.05, where not indicated, the difference was not statistically significant.
volume of saline solution was injected in the same region
of the left hindpaw. Four hours later, animals were
sacrificed and their hind paws cut and weighed.30 The
anti-inflammatory activity was evaluated by the weight
difference of right vs. left paw. Results confirmed the
ability of carrageenan injection to induce a marked
inflammation on hindpaw of treated rats. All doses of
compound 12 exhibited antiinflammatory properties,
since this compound dose-dependently reduced carra-
geenan-induced paw-oedema compared with vehicle-
treated rats [F(3,20) = 3.750; p < 0.05] (Fig. 7b), whereas
compound 11 was ineffective to reduce paw-oedema at
all doses tested [F(3,20) = 0.956; p > 0.05] (Fig. 7a).
received orallyfast indomethacin (10 mg/kg/5 ml) as po-
sitive controls.31 Four hours later, rats were killed and
their stomachs removed and opened along the greater
curvature. The ulcerative index (UI) was expressed as
the sum of the lengths (mm) of lesions found in the
mucosal membrane.33
Oral administration of indomethacin (10 mg/kg) caused
severe haemorrhagic lesions in the gastric mucosa of
arthritic rats (UI = 40.833 1.6). In contrast, neither
compound 11 [F(3,20) = 614.290; p < 0.01] nor com-
pound 12 [F(3,20) = 618.814; p < 0.01] induced any
damage in the stomach of arthritic rats, even at the high-
est dose of 100 mg/kg.
3.4. Gastric ulcerogenic response
3.5. Anti-nociceptive activity
Gastroenteropathy is the most common side effect
among patients taking NSAIDs for inflammatory disor-
ders, especially rheumatoid arthritis. It has been
reported that gastric mucosal lesions induced by conven-
tional NSAIDs, such as indomethacin, naproxen and
aspirin, were significantly aggravated in arthritis-
affected rats when compared with normal rats.31,32
In arthritis-affected rats the anti-nociceptive activity of
compound 12 was also evaluated using the hot plate
test.34 The apparatus consists of a 20-cm diameter
metal hot-plate surface (Basile, Milan, Italy) set at
55 0.5 ꢁC to give a latency-time of 15–18 s in control
group.
It has also been reported that the selective COX-2 inhib-
itors such as rofecoxib and celecoxib, even at a higher
dose (100 mg/kg), did not induce any damage in normal
rat stomachs but caused gross lesions in the stomach of
arthritic rats.32
Pain threshold was determined by the latency for noci-
ceptive response (licking of any hind paw or jump) with
a maximum cut-off time of 60 s. Arthritis-affected rats
(see above) were ip administered once daily for 4 days
with compound 12 (10, 50, 100 mg/kg/10 ml) or vehicle.
The day after the last injection, rats were tested twice,
30 min apart. A group of normal rats was added as
positive controls.
Taking into consideration this side effect, we investi-
gated the influence of our compounds on the integrity
of gastric mucosa in rats with adjuvant-induced arthritis
and compared the effects with those of indomethacin, a
conventional NSAID.
As shown in Figure. 8, arthritis-affected rats exhibited a
latency time for nociceptive response lower than that of
normal rats [F(1,10) = 24.694; p < 0.01], corresponding
to augmented sensitivity to pain. Intraperitoneal adminis-
tration of compound 12 at doses of 50, 100 mg/kg/10 ml
increased the latency time for nociceptive response com-
pared to vehicle-treated arthritis rats, but the difference
was not statistically significant [F(3,20) = 2.298; p > 0.05].
Therefore, arthritis was induced in rats as reported
above. Two days later, they were fasted overnight and,
the day after, orally administered with compound 11
(10, 50, 100 mg/kg/10 ml), compound 12 (10, 50,
100 mg/kg/10 ml) or vehicle. Another group of rats

O. Rosati et al. / Bioorg. Med. Chem. 15 (2007) 3463–3473
3471
Indeed both compounds show a lack of gastric injury
even at high doses.
5. Experimental methods
5.1. General remarks
All chemicals were purchased from the major chemical
suppliers as highest purity grade and used without any
further purification. Column chromatography was per-
formed with Merk silica gel 60 (70–230 mesh ASTM),
1
with hexane/ethyl acetate mixture. H and 13C NMR
were recorded in CDCl3 with a Brucker Avance DRX
200 spectrometer at a frequency of 200.1 and 50 MHz,
respectively, or Brucker Avance DPX 400 spectrometer
at a frequency of 400.13 and 100.62 MHz, respectively.
Chemical shifts (d) are reported in ppm relative to
TMS; J values are given in Hz. GC-MS analysis was
performed with an HP-6890 gas chromatograph (di-
methyl silicone column, 12.5 m) equipped with an HP-
5973 mass-selective detector at an ionizing voltage of
70 eV. Melting points are uncorrected. Reported yields
are for isolated compounds judged pure by NMR
analysis.
Figure 8. Anti-nociceptive activity of compound 12 (10, 50, 100 mg/kg
ip) in hot-plate test in rats, expressed as latency time for nociceptive
response of arthritis-affected rats compared to normal rats. Values are
expressed as means SEM. Difference from normal-group: +p < 0.05,
where not indicated, the difference was not statistically significant.
5.2. Typical reaction procedure
4. Conclusion
To the mixture of 2-benzoylcyclohexanone (1 mmol)
and a-Zr(CH3PO3)1. 2(O3PC6H4SO3H)0.8 (25 mg, ꢀ6%
molar), under stirring, under nitrogen and in neat,
hydrazine derivative (1 mmol) was added. The mixture
was left to react at 50 ꢁC and monitored by TLC. The
reaction mixture was diluted with dichloromethane,
filtered on buckner and then concentrated under vacuum.
The structures of 2,3-diaryl-4,5,6,7-tetrahydro-2H-
indazoles were determined on the basis of the typical
1H NMR chemical shifts and NOESY experiments.
Molecular docking calculations followed by a number of
in vivo biological assays were used to identify novel anti-
inflammatory agents among the class of 2,3-diaryl-
4,5,6,7-tetrahydro-2H-indazoles (10–15) and acting
through a COX-2 inhibition mechanism.
The obtained results indicate that both compounds 11
and compound 12 possess significant anti-inflammatory
activity both after oral and intraperitoneal administration.
5.3. 2,3-Diphenyl-4,5,6,7-tetrahydro-2H-indazole (10).13c
In Freund’s adjuvant-induced arthritis model, both
compounds are able to reduce the progression of inflam-
mation, but their anti-inflammatory activity is better
after ip than oral administration, probably due to a low-
er bioavailability following a reduced absorption or to
an increased metabolism of tested-compounds when or-
ally administered.
1
Yield, 96%; white powder; mp 107–108 ꢁC; H NMR
(400 MHz, CDCl3) d 1.81 (m, 2H, CH2), 1.91 (m, 2H,
CH2), 2.62 (t, J = 6.1 Hz, 2H, CH2), 2.84 (t,
J = 6.1 Hz, 2H, CH2), 7.29 (m, 10H, ArH); 13C NMR
(50 MHz, CDCl3) d 21.5, 23.3, 23.4, 23.5, 116.1, 124.8,
126.6, 127.7, 128.4, 128.7, 129.2, 130.8, 138.4, 140.4,
150.3; GC–MS m/z 274 (M+), 257, 246, 231, 218, 180,
153, 128, 115, 77.
After ip injection, compound 12 showed a dose-depen-
dent ready and steady effect, whereas at the same
doses, compound 11 exhibited a late and less effective
effect.
5.4. 3-Phenyl-2-[4-(trifluoromethyl)pyrimidin-2-yl]-
4,5,6,7- tetrahydro-2H-indazole (11)
Moreover, the difference in the anti-inflammatory activ-
ity of the two compounds is highlighted by the carra-
geenan-induced oedema test, where only the
compound 12 reduced paw-oedema at all doses tested
contrary to compound 11 that was ineffective.
Yield, 95%; white powder; mp 97–98 ꢁC; 1H NMR
(400 MHz, CDCl3) d 1.78 (m, 2H, CH2), 1.90 (m, 2H,
CH2), 2.53 (t, J = 6.3 Hz, 2H, CH2), 2.90 (t,
J = 6.3 Hz, 2H, CH2), 7.26 (m, 3H, ArH), 7.37 (m,
3H, ArH), 8.92 (d, J = 4.8 Hz, 1H, ArH); 13C NMR
(100 MHz, CDCl3) d 21.3, 23.3, 23.4, 24.1, 113.4,
120.0 (q, JCF = 274.2 Hz), 120.7, 128.19, 128.23, 129.3,
131.9, 141.3, 154.3, 156.7 (q, JCF = 37.2 Hz), 157.7,
161.5; GC–MS m/z 344 (M+), 316, 290, 275, 197, 147,
77.
In conclusion, the results of this study show that
compound 12, more than compound 11, exhibits anti-
inflammatory activity against adjuvants-induced arthri-
tis, and against early phase of inflammation (acute
paw-oedema), without any deleterious side effects.

3472
O. Rosati et al. / Bioorg. Med. Chem. 15 (2007) 3463–3473
5.5. 3-Phenyl-2-[4-(trifluoromethyl)phenyl]-4,5,6,7-tetra-
hydro-2H- indazole (12)
charges of residues were calculated using the Kollman
united charge model of the Amber force field as imple-
mented in Autodock tools package.37 Each docking
experiment consisted in 100 docking runs using the ge-
netic algorithm with a population size of 50 individuals
and 2,500,000 energy evaluations. Other parameters
were left to their respective default values. The search
was conducted in a grid of 60 points per dimension
and a step size of 0.375 centred on the binding site of
SC-558 as resulting from the crystallized complex with
COX-2 (pdb code 1cx2).17 Role of five violations and
polar surface area (PSA) were calculated using
JChem.13,38 All calculations were carried out on SGI
O2 R5000 and R10000 workstations.
1
Yield, 92%; white powder; mp 104–105 ꢁC; H NMR
(400 MHz, CDCl3) d 1.81 (m, 2H, CH2), 1.92 (m, 2H,
CH2), 2.60 (t, J = 6.1 Hz, 2H, CH2), 2.84 (t,
J = 6.1 Hz, 2H, CH2), 7.21 (d, J = 6 Hz, 2H, PhH),
7.37 (m, 5H, PhH), 7.54 (d, J = 6 Hz, 2H, PhH); 13C
NMR (100 MHz, CDCl3) d 21.6, 23.5, 23.6, 23.8,
118.3, 126.9 (q, JCF = 270.4 Hz), 124.4, 126.2 (q,
JCF = 4.0 Hz), 128.5, 129.0, 129.5, 130.8, 138.9, 143.4,
151.6; GC–MS m/z 342 (M+), 314, 265, 248, 197, 145,
77.
5.6. Methyl-3-phenyl-4,5,6,7-tetrahydro-2H-indazole-2-
carboxylate (13)
Acknowledgements
Yield, 95%; white powder; mp 85–86 ꢁC; 1H NMR
(400 MHz, CDCl3) d 1.73 (m, 2H, CH2), 1.84 (m, 2H,
CH2), 2.42 (t, J = 6.2 Hz, 2H, CH2), 2.79 (t,
J = 6.2 Hz, 2H, CH2), 3.92 (s, 3H, OCH3), 7.29–7.47
(m, 5H, PhH); 13C NMR (50 MHz, CDCl3) d 20.7,
22.7, 22.8, 23.7, 54.2, 120.4, 127.9, 128.4, 129.1, 130.6,
141.9, 150.5, 154.1; GC–MS m/z 256 (M+), 241, 265,
228, 197, 169, 141, 77.
Financial support from MIUR, National Project
‘‘Sviluppo di processi ecocompatibili nella sintesi orga-
nica’’ and CEMIN (Centro di Eccellenza Materiali
Innovativi Nanostrutturati per applicazioni chimiche,
fisiche e biomediche) is gratefully acknowledged.
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