Trimethyl-4-oxo-4,5,6,7-tetrahydroindazole-1-acetic acid: A new lead compound with selective COX-2 inhibitory activity

Article abstract of DOI:10.1002/ardp.201200193

A novel series of 3,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydroindazole-1-acetic acid derivatives was designed and synthesized by a new one-step pathway. Structure elucidation of the synthesized compounds was confirmed by various spectral and elemental analyses. The prepared compounds were evaluated for their ability to inhibit cyclooxygenase-2 (COX-2) and cyclooxygenase-1 (COX-1) enzymes in vitro. Among the synthesized compounds, the 2-(3,6,6-trimethyl-4-oxo- 4,5,6,7-tetrahydroindazol-1-yl)acetic acid 4 emerged as the most potent COX-2 inhibitor (IC₅₀ value: 150 nM) with the highest selectivity index (COX-1/COX-2 inhibition ratio: 570.6). Docking studies of compound 4 in the active site of COX-2 recognized its potential binding mode to the enzyme. Based on the preliminary results, compound 4 was considered as a lead compound for further optimization. Copyright

Full text of DOI:10.1002/ardp.201200193

Arch. Pharm. Chem. Life Sci. 2012, 000, 1–6
1
Full Paper
Trimethyl-4-oxo-4,5,6,7-tetrahydroindazole-1-acetic Acid:
A New Lead Compound with Selective COX-2
Inhibitory Activity
Hamdy M. Abdel-Rahman1 and Keriman Ozadali2,3
1 Medicinal Chemistry Department, Faculty of Pharmacy, Assiut University, Assiut, Egypt
2 Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, 2142-L Katz Group,
Centre for Pharmacy and Health Research, Edmonton, Alberta, Canada
3 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Hacettepe, Sihhiye,
Ankara, Turkey
A novel series of 3,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydroindazole-1-acetic acid derivatives was
designed and synthesized by a new one-step pathway. Structure elucidation of the synthesized
compounds was confirmed by various spectral and elemental analyses. The prepared compounds
were evaluated for their ability to inhibit cyclooxygenase-2 (COX-2) and cyclooxygenase-1 (COX-1)
enzymes in vitro. Among the synthesized compounds, the 2-(3,6,6-trimethyl-4-oxo-4,5,6,7-
tetrahydroindazol-1-yl)acetic acid 4 emerged as the most potent COX-2 inhibitor (IC50 value:
150 nM) with the highest selectivity index (COX-1/COX-2 inhibition ratio: 570.6). Docking studies
of compound 4 in the active site of COX-2 recognized its potential binding mode to the enzyme. Based
on the preliminary results, compound 4 was considered as a lead compound for further optimization.
Keywords: Selective COX-2 inhibitors / Structure elucidation / Tetrahydroindazoles
Received: May 10, 2012; Revised: July 1, 2012; Accepted: July 11, 2012
DOI 10.1002/ardp.201200193
Supporting information available online
:
Introduction
resulting in inflammatory actions [2]. Thus, selective inhibition
of COX-2 is useful for the treatment of inflammations with
reduced gastrointestinal toxicities [3, 4].
Non-steroidal anti-inflammatory drugs (NSAIDs) are among
the most widely prescribed therapeutics worldwide,
primarily for the treatment of pain and inflammation.
They exert their pharmacological effect by inhibiting the
cyclooxygenase (COX) enzymes, which catalyze the trans-
formation of arachidonic acid to prostaglandins. It is well
established that there are at least two COX isozymes (COX-1
and COX-2). COX-1 is a housekeeping enzyme responsible for
providing the physiological maintenance actions (vascular
and renal homeostasis, gastroprotection) [1]. On the other
hand, COX-2 isozyme is induced by proinflammatory stimuli
The pyrazole unit is one of the core structures in a number
of anti-inflammatory drugs including selective COX-2 inhibi-
tors such as celecoxib and SC-558 (Fig. 1) [5–7]. Additionally,
aryl and heteroaryl acetic acid derivatives [8] have made a
significant impact in NSAIDs research including pyrazole
acetic acid derivatives that have been previously reported [9].
Tetrahydroindazoles are a member of the fused-pyrazole
system that have recently attracted attention in the wide field
of medicinal chemistry due to their wide range of biological
actions such as anti-inflammatory [10], HSP-90 inhibitory [11],
and anticancer [12]. Despite their pharmacological potential,
they have not been investigated as COX inhibitors so far.
Herein, we reported the synthesis of novel tetrahydro-
indazole-1-acetic acid derivatives and the findings of their
COX-1/COX-2 inhibitory activities.
Correspondence: Hamdy M. Abdel-Rahman, Medicinal Chemistry
Department, Faculty of Pharmacy, Assiut University, Assiut 71526, Egypt.
E-mail: hamdym01@yahoo.com
Fax: þ2088 2332776
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

2
H. M. Abdel-Rahman and K. Ozadali
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–6
Figure 2. Expected isomeric structures of the ester 3.
The connectivity of key protons and carbons were deter-
mined by HMBC (Fig. 3). In the HMBC spectrum, a correlation
between the CH2 protons at the acetic acid side chain and the
carbon atoms at C-7a and C-7 was found, with no correlations
between these CH2 and the carbon at C-3. Other correlations
were found in the HMBC spectrum (see Fig. 3 and supple-
mentary data). Based on the above information, the structure
of the product was conclusively identified as ethyl 2-(3,6,6-
Figure 1. Examples of selective COX-2 and COX-1 inhibitors.
trimethyl-4-oxo-4,5,6,7-tetrahydroindazol-1-yl)acetate
3
(structure A, Fig. 2). Upon hydrolysis of the ester 3, the free
acetic acid derivative 4 was obtained, Scheme 1.
Reactions of the ester 3 with appropriate amines at room
temperature afforded the N-substituted amide derivatives
5a–h (Scheme 1). The structure elucidation of 5a–h was con-
firmed by spectroscopic (IR, 1H-NMR, and 13C-NMR) as well as
elemental analyses data.
Results and discussion
Chemistry
Condensation of 2-acyl dimedone with hydrazines is a gen-
eral method used for the preparation of tetrahydroind-
azolones [12–14]. In the present investigation, the starting
compound, ethyl 2-(3,6,6-trimethyl-4-oxo-4,5,6,7-tetra-
hydroindazol-1-yl)acetate 3 was prepared by reaction of 2-
acetyl-dimedone 2 with ethyl hydrazinoacetate (Scheme 1).
From a literature review, the reaction of 2-acetyl-dimedone
with the hydrazine derivative is expected to proceed straight-
forward to give only one product [15]. However some recent
reports showed that two possible isomeric structures (Fig. 2,
structures A and B) can be formed [16, 17]. To resolve this
disagreement, the structure elucidation of compound 3 was
studied in detail by NMR analysis. The chemical shift of each
proton and carbon was assigned by 1H, 13C, COSY, and HMQC.
Cyclooxygenase inhibitory activity
Compounds 3, 4, and 5a–h were tested for their ability to
inhibit COX-1 and COX-2 using ovine COX-1 and recombinant
human COX-2 by a COX fluorescence inhibitor assay in vitro
(catalog number 700100, Cayman Chemical, Ann Arbor, MI,
USA) according to the manufacturer’s assay protocol. SC-560
and DuP-697 (Fig. 1) were used as reference drugs for selective
COX-1 and COX-2 inhibition, respectively. Test compounds
and reference drugs were tested at different concentrations
and the IC50’s were then determined.
Scheme 1. Synthesis of the target compounds 3, 4 and 5a–h. Reagents and conditions: (a) (CH3CO)2O, Et3N, DCM (b)
NH2NHCH2COOC2H5ꢀHCl, Et3N, EtOH; (c) NaOH solution (10% w/v), reflux 30 min; (d) NH2–R, EtOH.
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–6
Tetrahydroindazoles COX-2 Inhibitors
3
From the biological activity studies, several conclusions
can be deduced: (i) Derivatization of the acetic acid side chain
is critically important for activity. (ii) Compounds carrying
the free carboxyl group (compound 4) or the primary amide
group (compound 5a) showed superior COX-2 inhibitory
activity than the N-substituted amide derivatives 5b–e.
(iii) Amides having smaller N-substituents showed better
activities than bulky groups. This short SAR study supports
that compound 4 is highly potent with good selective COX-2
inhibitory activity deserving further biological evaluation.
Figure 3. HMBC correlations of the ester 3 (structure A).
The results (Table 1) show that the presence of the acetic
acid side chain is critically important. Having a free carboxyl
group as the side chain, compound 4 showed strong COX-2
inhibition (IC50 value: 0.15 mM) with the highest selectivity
index (COX-1/COX-2 inhibition ratio: 570.6). Interestingly,
esterification of the free carboxyl group resulted in complete
loss of both COX-1 and COX-2 activity.
Docking studies
Molecular docking studies of compound 4 were performed
in order to rationalize the obtained biological results and
understand its likely interactions with the COX-2 enzyme
active site (Fig. 4).
Docking studies were performed by MOE (Molecular
Operating Environment) [18] using murine COX-2 co-crystal-
lized with SC-558 (PDB ID: 1CX2) as a template. We performed
100 docking iterations for the ligand in the enzyme active
site and the top scoring configuration of the ligand–enzyme
complexes was selected on energetic grounds. This molecular
modeling of compound 4 showed that the acetic acid side
chain (CH2COOH) was oriented within the COX-2 secondary
pocket in a region comprising His90, Gln192, Arg513, Val523,
and Leu352. The N-2 atom of the compound 4 formed a
strong hydrogen bond interaction with the NH group of
Replacement of the free carboxyl group by the amides
generally resulted in slightly reduced COX-1 inhibitory
activity and noticeably reduced COX-2 activity. The primary
amide (compound 5a) showed the best COX-2 inhibitory
activity among this series (IC50 value: 0.86 mM) with good
selectivity index (COX-1/COX-2 inhibition ratio: 80.5).
Increasing the bulkiness of the N-substituent or elongation
of the side chain of the amides (5b–h) generally reduce COX-2
inhibitory activity.
˚
His90 (distance ¼ 1.97 A). Furthermore, the carbonyl group
Table 1. Primary in vitro COX-1 and COX-2 inhibition assay results.
at C-4 formed a hydrogen bond interaction with the Arg513
˚
(distance ¼ 2.69 A). Finally a hydrogen bond interaction
O
between the carboxyl OH and the carbonyl group of the
˚
backbone Phe518 was also observed (distance ¼ 3.58 A).
N
N
The C-6 dimethyl moiety was located near Val523 forming
˚
hydrophobic interactions (distance ¼ 2.33 A).
COR
Compd. no.
R
IC50 (mM)
SIa)
COX-1
COX-2
3
4
5a
5b
5c
5d
5e
5f
5g
5h
SC-560
DuP-697
OC2H5
OH
NH2
>100
85.6
69.2
>100
0.15
0.86
–
570.6
80.5
2.3
–
>1.3
>1.9
–
NHCH3
74.1
32.4
NH(CH2)3CH3
NH–C6H11
NH(CH2)2OH
NHCH2C6H4(4-OCH3)
NH(CH2)2N(CH3)2
NH(CH2)2morpholine
–
>100
>100
>100
>100
>100
>100
0.0085
Nd
>100
76.0
52.7
>100
85.6
>100
Ndb)
0.0416
>1.2
–
–
–
–
a)
SI, selectivity index calculated as the IC50 COX-1/IC50 COX-2.
Nd, not determined.
b)
Figure 4. A view of the energy-minimized structure of the complex
of 4 bound to COX-2, viewed using the Molecular Operating
Environment (MOE) program.
SC-560 was used as selective COX-1 inhibitor while DuP-697 was
used as a selective COX-2 inhibitor.
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4
H. M. Abdel-Rahman and K. Ozadali
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–6
(80%), mp 64–658C. IR (KBr, cmꢁ1): 3530, 3410, 2950, 1740, 1647,
1180. 1H NMR (400 MHz, CDCl3) d 1.05 (s, 6H, 2CH3), 1.21
(t, J ¼ 7.2 Hz, 3H, OCH2CH3), 2.26 (s, 2H, CH2), 2.37 (s, 3H, CH3),
2.52 (s, 2H, CH2), 4.16 (q, J ¼ 7.2 Hz, 2H, OCH2CH3), 4.71 (s, 2H,
–NCH2CO). 13C NMR (100 MHz, CDCl3) d 13.28 (CH3 ester), 14.08
(CH3 on C-3), 28.44 (CH3)2, 35.18 (C-7), 50.24 (C-5), 52.25 (C-6), 53.48
(NCH2–), 62.13 (CH2 ester), 116.05 (C-3), 149.08 (C-3a), 150.63 (C-7a),
167.09 (CO ester), 193.12 (C-4). Anal. calcd for C14H20N2O3; C, 63.62;
H, 7.63; N, 10.60. Found: C, 63.70; H, 8.00; N, 10.62.
The above observations show that compound 4 is strongly
bound to the COX-2 active site, the same as that resulting
from the selective COX-2 inhibitor, SC-558 [19]. Consequently,
it provides guidelines to establish the SAR of this class of
compounds and to correlate COX-inhibitory activities. Thus,
short and hydrogen donor substituents at N-1 are essential
for activity while increasing steric bulkiness may reduce the
activity.
Therefore, molecular docking experiments will guide the
design and synthesis of new analogues with anticipated good
COX-2 inhibitory activities such as (i) the hydroxamate and
sulfonate analogues of the acid 4, (ii) acetic acid analogues
with modifications in the tetrahydroindazole C-3, (iii) other
derivatives with short chain at the N-1 nitrogen.
2-(3,6,6-Trimethyl-4-oxo-4,5,6,7-tetrahydroindazol-1-yl)-
acetic acid 4
A mixture of the ester 3 (4 mmol) and NaOH solution (10% w/v,
5 mL) was refluxed for 30 min. The reaction mixture was cooled
to room temperature, and washed with ether, acidified with
dilute HCl solution, extracted with ether (20 mL ꢂ 2), dried over
anhydrous MgSO4, and filtered. The solvent was evaporated
under reduced pressure, and the residue was crystallized from
ether. Yield 25% as white powder, mp 191–1938C. IR (KBr, cmꢁ1):
Conclusions
1
3430, 2910, 1730, 1654. H NMR (400 MHz, CDCl3) d 1.05 (s, 6H,
In conclusion, based on this preliminary results, we ident-
ified 4 as a lead compound for the novel class of trimethyl-
4-oxo-tetrahydroindazole-1-derivatives as selective COX-2
inhibitors. A docking pose of the energy-minimized structure
of the complex of 4 bound to the COX-2 showed a tight
binding mode and provided a guide for further intensive
SAR modifications to be performed and comprehensively
evaluated as COX-2 inhibitors.
2CH3), 2.29 (s, 2H, CH2), 2.38 (s, 3H, CH3), 2.54 (s, 2H, CH2), 4.82
(s, 2H, –NCH2CO), 8.29 (bs. 1H, COOH). 13C NMR (100 MHz, CDCl3)
d 12.85, 28.46, 34.97, 35.62, 49.47, 52.17, 115.81, 149.38, 150.93,
169.06, 193.38. MS (EI): m/z 235.96 [74.9%, Mþ], ESI positive ion
mode m/z 237 [Mþþ1], m/z 259 [MþþNa].
General procedure for synthesis of 2-(3,6,6-trimethyl-4-
oxo-4,5,6,7-tetrahydroindazol-1-yl)acetamides 5a–h
The appropriate amine (2 mL) was added to a solution of the ester
3 (0.05 mmol) in ethanol (2 mL) with constant stirring at room
temperature. The mixture was stirred at this temperature until
completion of reaction (about 24–48 h as monitored by TLC).
The mixture was then evaporated under vacuum, extracted by
dichloromethane, washed with water and brine. The organic
layer was dried over anhydrous MgSO4, filtered, evaporated till
dryness. The solid materials obtained after drying in a vacuum
desiccator was crystallized from ethanol.
Experimental
Melting points were determined using an electrothermal appar-
atus (Stuart Scientific, England) and were uncorrected. IR spectra
were recorded as KBr disks using a Shimadzu IR 200-91527
spectrophotometer (Shimadzu Corp., Kyoto, Japan) and the data
are given in ymax (cmꢁ1). 1H-NMR (400 MHz) spectra were carried
out on a Bruker 400 MHz, (Varian, Palo Alto, CA, USA) and the
chemical shifts are given in d (ppm). Mass spectra were performed
on a Jeol, JMS-600 spectrometer at an ionization voltage of 70 eV
(Jeol, Tokyo, Japan). Elemental analyses were performed on
an ‘‘Analytischer Funktionstest vario EL Fab.-Nr. 11982027’’
(Germany). All reactions were monitored by thin-layer chroma-
tography (TLC) using silica gel 60 GF245 precoated sheets
20 ꢂ 20 cm2, layer thickness 0.2 mm (E-Merck, Germany) and
were visualized by UV-lamp at a wavelength (l) of 254 nm. All
chemicals and solvents were of reagent grade, and the latter were
distilled and dried before use.
2-(3,6,6-Trimethyl-4-oxo-4,5,6,7-tetrahydroindazol-1-yl)-
acetamide 5a
As reported in Ref. [17].
N-Methyl-2-(3,6,6-trimethyl-4-oxo-4,5,6,7-
tetrahydroindazol-1-yl)acetamide 5b
Yield 78% as pale yellow powder; mp 130–1328C. IR (KBr, cmꢁ1):
3270, 2925, 1665, 1645. H NMR (400 MHz, CDCl3) d 1.05 (s, 6H,
1
2CH3), 2.27 (s, 2H, CH2), 2.41 (s, 3H, CH3), 2.56 (s, 2H, CH2), 2.75
(d, J ¼ 4.8 Hz, 3H, –NHCH3), 4.58 (s, 2H, –NCH2CO), 6.21 (bs, 1H,
CONH). 13C NMR (100 MHz, CDCl3) d 13.38, 26.35, 28.48, 35.21,
35.59, 51.87, 52.30, 116.11, 150.26, 150.61, 166.96, 193.08. Anal.
calcd for C13H19N3O2; C, 62.63; H, 7.68; N, 16.85. Found: C, 62.59;
H, 8.00; N, 16.76.
Ethyl 2-(3,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydroindazol-
1-yl)acetate 3
Equimolar amounts of ethyl hydrazinoacetate HCl, 2-acetyldi-
medone 2 and triethylamine were mixed in ethanol; then the
mixture was refluxed for 4 h. The reaction mixture was cooled
and the solvent was evaporated under reduced pressure. The
residue was dissolved in ether, washed with water and brine,
dried over anhydrous MgSO4, then filtered off. The filtrate was
concentrated under reduced pressure to an oily material that
was purified with a small silica column using 6% CH3OH/CH2Cl2
to give a yellow oily product that solidified on standing. Yield
N-Butyl-2-(3,6,6-trimethyl-4-oxo-4,5,6,7-
tetrahydroindazol-1-yl)acetamide 5c
Yield 90% as yellow powder; mp 80–828C. IR (KBr, cmꢁ1): 3240,
2925, 1667, 1645. 1H NMR (400 MHz, CDCl3) d 0.82 (t, J ¼ 7.3 Hz,
3H, –CH2CH2CH2CH3), 1.05 (s, 6H, 2CH3), 1.20 (m, 2H, CH2), 1.35
(m, 2H, CH2), 2.28 (s, 2H, CH2 of indazole), 2.41 (s, 3H, CH3), 2.56
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–6
Tetrahydroindazoles COX-2 Inhibitors
5
(s, 2H, CH2 of indazole), 3.17 (q, J ¼ 7.1 Hz, 2H, –NHCH2), 4.57
(s, 2H, –NCH2CO), 6.08 (s, 1H, CONH). 13C NMR (100 MHz, CDCl3)
d 12.29, 12.62, 18.90, 27.43, 30.31, 34.17, 34.55, 38.31, 51.00,
51.27, 115.05, 149.11, 149.56, 165.19, 192.07. Anal. calcd
for C16H25N3O2; C, 65.95; H, 8.65; N, 14.42. Found: C, 65.75;
H, 8.91; N, 14.37.
2CH3), 2.28 (s, 2H, CH2), 2.35 (t, J ¼ 4.0 Hz, 4H, –N(CH2)2 of
morpholine), 2.39 (t, J ¼ 6.0 Hz, 2H, –CH2N of morpholine)
2.43 (s, 3H, CH3), 2.57 (s, 2H, CH2), 3.29 (q, J ¼ 5.7 Hz, 2H,
–CONHCH2), 3.58 (t, J ¼ 4.4 Hz, 4H, –O(CH2)2 of morpholine),
4.60 (s, 2H, –NCH2CO), 6.64 (s, 1H, CONH). 13C NMR (100 MHz,
CDCl3) d 13.49, 28.43, 35.24, 35.53, 35.64, 51.99, 52.31, 53.09,
56.30, 66.71, 116.08, 149.95, 150.55, 166.39, 192.98. Anal. calcd
for C18H28N4O3; C, 62.05; H, 8.10; N, 16.08. Found: C, 61.67;
H, 7.92; N, 15.81.
N-Cyclohexyl-2-(3,6,6-trimethyl-4-oxo-4,5,6,7-
tetrahydroindazol-1-yl)acetamide 5d
Yield 50% as yellow powder; mp 118–1208C. IR (KBr, cmꢁ1): 3265,
2910, 1658, 1644. 1H NMR (400 MHz, CDCl3) d 1.05 (s, 6H, 2CH3),
1.28 (m, 3H, cyclohexyl), 1.55 (m, 4H, cyclohexyl), 1.75 (m, 3H,
cyclohexyl), 2.28 (s, 2H, CH2 of indazole), 2.41 (s, 3H, CH3), 2.56
(s, 2H, CH2 of indazole), 3.70 (m, 1H, –NHCH of cyclohexyl),
4.55 (s, 2H, –NCH2CO), 5.91 (d, J ¼ 7.4 Hz, 1H, CONH). 13C NMR
(100 MHz, CDCl3) d 13.33, 15.30, 24.52, 24.98, 25.32, 28.48, 32.70,
33.15, 35.24, 35.63, 48.40, 52.33, 65.88, 116.13, 150.08, 150.58,
165.35, 193.14. Anal. calcd for C18H27N3O2; C, 68.11; H, 8.57;
N, 13.24. Found: C, 67.91; H, 8.80; N, 12.99.
Cyclooxygenase inhibition assay
In vitro cyclooxygenase (COX) inhibition assay: The ability of
compounds 3, 4, and 5a–h to inhibit ovine COX-1 and recombi-
nant human COX-2 was determined using a COX fluorescence
inhibitor assay (catalog number 700100, Cayman Chemical, Ann
Arbor, MI, USA) according to the manufacturer’s assay protocol.
SC-560 and DuP-697 were used as reference drugs for selective
COX-1 and COX-2 inhibitors, respectively. Compounds were
assayed in concentrations ranging from 100 to 0.01 mM; then
the IC50 values were calculated.
N-(2-Hydroxyethyl)-2-(3,6,6-trimethyl-4-oxo-4,5,6,7-
tetrahydroindazol-1-yl)acetamide 5e
Docking studies
All the molecular modeling studies were carried out on an Intel
Core i3 processor, 3 GB memory with Windows 7 operating
system using Molecular Operating Environment (MOE 2008,
Chemical Computing Group, Canada) as the computational soft-
Yield 60% as pale yellow powder; mp 110–1118C. IR (KBr, cmꢁ1):
3350, 3260, 2920, 1655. H NMR (400 MHz, CDCl3) d 1.06 (s, 6H,
1
2CH3), 2.29 (s, 2H, CH2), 2.42 (s, 3H, CH3), 2.60 (s, 2H, CH2), 2.75
(br s, 1H, OH) 3.37 (q, J ¼ 5.6 Hz, 2H, –NHCH2), 3.64 (t, J ¼ 5.1 Hz,
2H, –CH2OH), 4.66 (s, 2H, –NCH2CO), 6.82 (s, 1H, CONH). 13C NMR
(100 MHz, CDCl3) d 12.16, 27.43, 34.13, 34.56, 41.31, 50.77, 51.25,
60.47, 116.00, 149.00, 149.99, 165.89, 191.99. Anal. calcd
for C14H21N3O3; C, 60.20; H, 7.58; N, 15.04. Found: C, 59.90; H,
7.92; N, 14.74.
ware. All the minimizations were performed with MOE until a
˚
RMSD gradient of 0.05 kcal molꢁ1 ꢁ1 with MMFF94X force-field
A
and the partial charges were automatically calculated.
The X-ray crystallographic structure of murine COX-2 com-
plexed with SC-558 (PDB ID: 1CX2) was obtained from the protein
data bank. The enzyme was prepared for docking studies where:
(i) Ligand molecule was removed from the enzyme active site.
(ii) Hydrogen atoms were added to the structure with their
standard geometry. (iii) MOE Alpha Site Finder was used for
the active sites search in the enzyme structure and dummy
atoms were created from the obtained alpha spheres. (iv) The
obtained model was then used in predicting the ligand–enzyme
interactions at the active site.
N-(4-Methoxybenzyl)-2-(3,6,6-trimethyl-4-oxo-4,5,6,7-
tetrahydroindazol-1-yl)acetamide 5f
Yield 70% as yellow powder; mp 114–1168C. IR (KBr, cmꢁ1): 3230,
2920, 1673, 1649, 1250. 1H NMR (400 MHz, CDCl3) d 1.00 (s, 6H,
2CH3), 2.25 (s, 2H, CH2), 2.37 (s, 3H, CH3), 2.54 (s, 2H, CH2), 3.71 (s, 3H,
–OCH3), 4.29 (d, J ¼ 5.8 Hz, 2H, –NHCH2), 4.61 (s, 2H, –NCH2CO), 6.41
(s, 1H, CONH), 6.61 (d, J ¼ 8.7 Hz, 2H, Ar–H), 7.05 (d, J ¼ 8.6 Hz, 2H,
Ar–H). 13C NMR (100 MHz, CDCl3) d 13.18, 28.42, 35.19, 35.54, 43.07,
52.05, 52.29, 55.30, 114.13, 116.14, 128.93, 129.45, 149.98, 150.71,
159.16, 165.97, 192.93. Anal. calcd for C20H25N3O3; C, 67.58; H, 7.09;
N, 11.82. Found: C, 67.32; H, 7.16; N, 11.54.
The authors are grateful to Dr. Carlos Velazquez, Faculty of Pharmacy and
Pharmaceutical Sciences, University of Alberta, Canada, for the biological
screening help.
The authors have declared no conflicts of interest.
N-(2-(Dimethylamino)ethyl)-2-(3,6,6-trimethyl-4-oxo-
4,5,6,7-tetrahydroindazol-1-yl)acetamide 5g
Yield 92% as yellow powder; mp 88–908C. IR (KBr, cmꢁ1): 3390,
3266, 2930, 1678, 1646. H NMR (400 MHz, CDCl3) d 1.05 (s, 6H,
References
1
2CH3), 2.14 (s, 6H, N(CH3)2), 2.27 (s, 2H, CH2), 2.34 (t, J ¼ 6.0 Hz,
2H, –CH2N(CH3)2) 2.40 (s, 3H, CH3), 2.57 (s, 2H, CH2), 3.26
(q, J ¼ 5.6 Hz, 2H, –CONHCH2), 4.60 (s, 2H, –NCH2CO), 6.64 (s, 1H,
CONH). 13C NMR (100 MHz, CDCl3) d 13.75, 28.91, 35.71, 36.08,
37.16, 45.37, 52.48, 52.80, 57.64, 116.51, 150.35, 151.15, 166.78,
193.65. Anal. calcd for C16H26N4O2; N, 18.29. Found: N, 17.95.
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