Synthesis and cyclooxygenase inhibitory activities of some N-acylhydrazone derivatives of isoxazolo[4,5-d]pyridazin-4(5H)-ones

Article abstract of DOI:10.1016/j.ejmech.2010.02.012

In this study, new isoxazolo[4,5-d]pyridazin-4(5H)-one derivatives having an N-acylhydrazone moiety were synthesized. The compounds were tested for their COX inhibitory activities using NS-398 and indomethacine as reference compounds. Although the compounds had an inhibitory profile against both COX-1 and COX-2, most were found to be more selective against COX-2 by a small percentage of inhibition, at the concentration of 50?μM. Docking studies were done to understand the interactions of the tested compounds with the active site of COX-2. It was observed that the compounds fit into, and interacted with, the hydrophobic parts which are common in the active pocket of COX-1 and COX-2 enzymes but could not fit to the area which is specific for COX-2 enzyme.

Full text of DOI:10.1016/j.ejmech.2010.02.012

European Journal of Medicinal Chemistry 45 (2010) 2345–2352
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and cyclooxygenase inhibitory activities of some N-acylhydrazone
derivatives of isoxazolo[4,5-d]pyridazin-4(5H)-ones
a,
b,
c
b
a
*
¨
¨
, Kevser Ozden , Arvi Rauk , Ayla Balkan
Oya Unsal-Tan
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Hacettepe, Ankara, Turkey
b Department of Chemistry, University of Calgary, Alberta, Canada T2N 1N4
c Department of Biochemistry, Faculty of Medicine, University of Hacettepe, Ankara, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study, new isoxazolo[4,5-d]pyridazin-4(5H)-one derivatives having an N-acylhydrazone moiety
were synthesized. The compounds were tested for their COX inhibitory activities using NS-398 and
indomethacine as reference compounds. Although the compounds had an inhibitory profile against both
COX-1 and COX-2, most were found to be more selective against COX-2 by a small percentage of inhi-
Received 3 January 2010
Received in revised form
3 February 2010
Accepted 4 February 2010
Available online 10 February 2010
bition, at the concentration of 50 mM. Docking studies were done to understand the interactions of the
tested compounds with the active site of COX-2. It was observed that the compounds fit into, and
interacted with, the hydrophobic parts which are common in the active pocket of COX-1 and COX-2
enzymes but could not fit to the area which is specific for COX-2 enzyme.
Keywords:
Cyclooxygenase enzymes
Isoxazolo[4,5-d]pyridazin-4(5H)-one
N-Acylhydrazone
Ó 2010 Elsevier Masson SAS. All rights reserved.
Docking
1. Introduction
rofecoxib was banned in 2004 because of cardiac toxicity. Subse-
quently, some of the other coxibs have been voluntarily withdrawn
Nonsteroidal anti-inflammatory drugs (NSAIDs) are among the
most widely used therapeutics for the treatment of pain and
inflammation. The first NSAID discovered was aspirin which has
now been used for more than 100 years. However, the molecular
mechanism of aspirin and other NSAIDs was elucidated only in the
1970s. Vane [1] proposed that NSAIDs produce their therapeutic and
also unwanted effects by inhibition of cyclooxygenase (COX)
enzyme in prostaglandin synthetic pathway. Until recently, all
prostaglandin synthesis was thought to result from only one form of
COX enzyme. In the early 1990s it was discovered that COX exists in
two isoforms, COX-1 (constitutive form) and COX-2 (inducible
form). Thus a new hypothesis has been suggested to explain the
effects of NSAIDs; inhibition of COX-1 accounts for the undesirable
side effects, whilst inhibition of COX-2 accounts for the therapeutic
benefits of NSAIDs. This proposal has led to drug discovery programs
aimed at identifying new anti-inflammatory agents that selectively
inhibit COX-2 activity, and a large number of compounds with
common structural features, i.e., the presence of two aryl rings on
adjacent carbons of a cyclic/acylic moiety, has been investigated for
COX-2 inhibition. Among these compounds, rofecoxib, celecoxib,
valdecoxib and etoricoxib were approved and marketed. However,
fromthe market [2]. On the otherhand, some studies havesuggested
that rofecoxib’s adverse cardiac events may not be a class effect but
rather an intrinsic chemical property related to its metabolism [3].
For this reason novel scaffolds with COX-2 selective inhibitory
activity needed to be found and evaluated for their anti-
inflammatory effects. A number of molecules based upon a mono-
cyclic (5 or 6 membered) or bicyclic heterocyclic template have
been investigated for their COX-2 inhibitory activity. Among them,
some compounds having a pyridazinone structure (Fig. 1) have
showed selective COX-2 inhibitory activity [4–6]. Furthermore, the
analgesic and anti-inflammatory effects of N-acylhydrazones
substituted with different structures have been described [7–14].
These observations prompted us to synthesize some new 7-
benzyl-3-methylisoxazolo[4,5-d]pyridazin-4(5H)-one derivatives,
bearing an N-acylhydrazone moiety at the fifth position, as
potential COX-2 inhibitors.
2. Results and discussion
2.1. Chemistry
Thesyntheticroute used to synthesizeN0-substitutedbenzylidene-
2-(7-benzyl-3-methyl-4-oxoisoxazolo[4,5-d]pyridazin-4(5H)-yl)ace-
tohydrazides (7–28) is outlined in Scheme 1. 7-Benzyl-3-
* Corresponding author at: Department of Pharmaceutical Chemistry, Faculty of
Pharmacy, University of Hacettepe, Ankara, Turkey. Tel./fax: þ90 3123051872.
¨
E-mail address: oyaunsal@hacettepe.edu.tr (O. Unsal-Tan).
0223-5234/$ – see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.02.012

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O. Unsal-Tan et al. / European Journal of Medicinal Chemistry 45 (2010) 2345–2352
2346
[17] showed that N-acylhydrazones derived from aromatic alde-
O
O
hydes in solution are in the E form because of the hindered rotation
on the imine bond.
O
N
N
R1
R3
N
N
H
Syakaev and co-workers [18], who investigated the 1H NMR
spectra of N-acylhydrazones derived from acetone and aromatic
aldehydes, mentioned that N-acylhydrazone derived from
acetone had two rotamers due to the N–C(O) bond because of
symmetry around the C]N bond. They also noticed that,
N-acylhydrazones derived from acetone and aromatic aldehydes
demonstrated a similar pattern of 1H NMR spectra. By regarding
these data, they claimed that N-acylhydrazones derived from
aromatic aldehydes have two rotamers (cis and trans) due to the
N–C(O) bond.
R
N
N
R'
O
OR
R2
R7
CH3SO2
R6
R4
R5
Fig. 1. Selective COX-2 inhibitors having pyridazinone structure.
methylisoxazolo[4,5-d]pyridazin-4(5H)-one 4 was prepared using
the method which was described earlier [15,16]. But we observed that
using methyl acetoacetate instead of ethyl acetoacetate increased the
reaction yield of 1.
Compound 4 was transformed into ethyl 2-(7-benzyl-3-methyl-
4-oxoisoxazolo[4,5-d]pyridazin-4(5H)yl)acetate 5 by reacting with
ethyl bromoacetate in the presence of potassium carbonate. By
refluxing this compound with excess hydrazine hydrate, 2-(7-
benzyl-3-methyl-4-oxoisoxazolo[4,5-d]pyridazin-4(5H)-yl)acetoh-
ydrazide 6 was obtained. Finally, the reaction of this hydrazide with
a number of benzaldehydes in the presence of hydrochloric acid
gave 7–28.
Structures of the target compounds 7–28, were elucidated by
using spectral methods (IR, 1H NMR, ESI-MS) and elemental anal-
ysis. In the IR spectra of 7–28, we observed the bands at around
1700 cmꢀ1 and 1670 cmꢀ1 due to C]O (pyridazinone) and C]O
(hydrazide) stretching respectively. In addition to these bands,
a broad band appeared between 3600 and 3270 cmꢀ1 due to
(CO)N–H stretching.
It is known that N-acylhydrazones may exist as geometrical
isomers (E/Z) in respect to the C]N double bonds and as rotamers
(cis/trans) about amide N–C(O) bond [17,18]. Palla and co-workers
In our study, to understand the possible isomers of 7–28, we
calculated the relative free energy of eight isomers of 7 derived
from 2-fold rotations about the C(O)–N, N–N and C]N bonds.
These calculations were performed at an ab initio level of theory
using the electronic structure code, Gaussian 03. According to the
optimization studies, it was observed that the two most stable
isomers are E/E/trans and E/E/cis conformers (Fig. 2), which is
consistent with the literature.
In the 1H NMR spectra of 7–28, we observed the signals
belonging to N]CH, –CH2–C(O) and N–H protons (also some
protons in benzylidene) as double singlets. By regarding both
literature data and geometric optimization results we assumed that
these double singlets appeared because of cis and trans isomers of
7–28. The signals belonging to methylidene and imine protons
were seen at around 4.80 (trans), 5.20 (cis) ppm and 8.30 (trans),
7.90 (cis) ppm, respectively. The amide protons also appeared at
around 11–12 ppm as double singlets with some exception.
In the ESI mass spectra of 7–28, in addition to [M þ Na]þ, the
fragment formed by the cleavage of N–N bond with simultaneous
hydrogen migration was seen at m/z 282, in accord with literature
data [19,20].
O
CH3OOC
Mg(OC2H5)2
ClCH2COCl
CHO
CH3COCH2COOCH3
O
CH3
1
O
COOCH3
CH3OOC
H
CH3
NH2NH2
H
C
NH2OH
O
N
CH3
O
OH
2
3
O
O
CH3
CH3
N
CH2COOC2H5
NH
N
N
NH2NH2
N
N
BrCH2COOC2H5
O
O
5
4
O
O
CH3
N
CH3
N
R
CHO
N
N
CH2CONHN
CH
N
N
CH2CONHNH2
R
O
O
7-28
6
Scheme 1. Synthetic route of 1–28.

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O. Unsal-Tan et al. / European Journal of Medicinal Chemistry 45 (2010) 2345–2352
2347
fluoro substituents at the second position of the phenyl ring (11 and
14 respectively) were the most notable ones because they showed
a remarkable inhibitory activity on COX-2 enzyme with inhibiting
COX-1 enzyme less than indomethacine (Table 1).
In addition to these data, we observed that the substitution of
triflouromethyl group to the third position of the phenyl ring (21)
resulted in a remarkable decrease in COX-2 inhibitory potency,
whereas a Cl, Br, or NO2 group showed good selectivity for COX-2
over COX-1.
Furthermore, we noticed that the substituents on the phenyl ring
(except 15 and 21) did not cause an important effect on the activity.
The docking study was performed with MOE software program
to understand the orientations of the ligands in COX-2. To compare
orientations of the ligands, we superimposed each compound’s best
pose which was obtained by regarding the S score in MOE. Except 21
(3-CF3), 24 (3-OCH3) and 27 (3-NO2), all the ligands showed similar
orientation in the COX-2 active site (Fig. 3A). It was observed that the
benzyl ring of the compounds fitted into the cavity formed by
Phe381, Leu384, Tyr385, Trp387, Phe513 and Ser530 while the
3-methylisoxazole moiety fitted into the other cavity formed by
Met113, Val116, Val349, Tyr355, Leu359 and Leu531 (Fig. 3B). As
Kurumbail and co-workers [21] mentioned, these features are
common in non-selective and selective COX-2 inhibitors such as
flurbiprofen, indomethacine and SC-558. Furthermore, the OH
group of Tyr355 formed a hydrogen bond (w2.1 Å) with the C]O
moiety of the pyridazinone, and the C]O moiety of Leu352 and N–H
moiety of His90 hydrogen bonded to the NH and C]N of the
hydrazone respectively (Fig. 3C).
On the other hand, it is known that the main difference between
the two COX active sites is the replacement of Ile523 in COX-1 by
Val, a less bulky amino acid. This replacement creates an adjunct
pocket in the COX-2 active site which may be responsible for
selectivity. Our docking study showed that 7–28 were oriented so
that their N-acylhydrazone moiety did not fill this adjunct pocket
(Fig. 3B). This may explain why the compounds did not show
significant selective activity against COX-2 enzyme.
Fig. 2. Structure of the E/E/trans (left) and E/E/cis (right) conformers of 7.
2.2. Pharmacology
The inhibitory effects of the target compounds (7–28) on
COX-1 and COX-2 enzymes were evaluated at 50 M using
m
a COX Inhibitor Screening Kit. NS-398 and indomethacine were
used as reference compounds. The results are shown in Table 1.
Although the target compounds showed remarkable inhibitory
activity on COX-2 enzyme, none of them was found as effective
as the standard compound, NS-398. Furthermore, when their
activities were compared with indomethacine, it was deter-
mined that all the compounds inhibited COX-2 enzyme more
than indomethacine while, with the exception of 15, they
inhibited COX-1 enzyme less than that.
The compound carrying a fluoro substituent at the third position
of the phenyl ring,15, appeared as the most active compound in this
series against COX-2 enzyme. In addition, its inhibitory activity on
the COX-1 enzyme was found to be higher even than that of indo-
methacine. Among the series, the compounds having chloro and
Additionally, 21, which showed the lowest inhibitor activity
against COX-2 enzyme, oriented in a different way altogether
(Fig. 3D). This suggests that the general orientation obtained for the
other compounds was correct.
Table 1
In vitro COX-1 and COX-2 enzyme inhibition data for 7–28.
Compound
R
% Inhibition (50
COX-1
m
M)a
3. Conclusions
COX-2
In the present study, a series of new N0-substitutedbenzylidene-
2-(7-benzyl-3-methyl-4-oxoisoxazolo[4,5-d]pyridazin-4(5H)-yl)a-
cetohydrazide derivatives were synthesized in order to evaluate
their inhibitory activities on COX-1 and COX-2 enzymes. The
binding mode of the tested compounds inside the COX-2 active site
was predicted using a docking technique. When the results of the
inhibitory activity and docking studies are considered together, it
can be suggested that: (i) the isoxazolo[4,5-d]pyridazin-4(5H)-one
moiety is a suitable scaffold for COX-2 enzyme; (ii) the N-acylhy-
drazone moiety, which does not fill the adjunct pocket in the COX-2
active site, does not make any contribution to the selectivity; (iii)
instead of N-acylhydrazone, appropriate substitutions which can
fill the adjunct pocket and interact with the relatively polar resi-
dues such as Gln192 and Arg513 may be useful to propose new
molecules with enhanced selectivity towards COX-2.
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
H
2-Br
3-Br
4-Br
2-Cl
3-Cl
4-Cl
2-F
50.6 ꢁ 2.9
52.8 ꢁ 4.0
42.9 ꢁ 1.3
50.6 ꢁ 6.2
52.8 ꢁ 4.0
36.3 ꢁ 3.7
39.6 ꢁ 3.1
41.8 ꢁ 4.8
83.0 ꢁ 1.5
54.0 ꢁ 2.1
46.9 ꢁ 3.6
61.0 ꢁ 1.4
62.1 ꢁ 5.0
51.0 ꢁ 4.3
47.1 ꢁ 1.9
64.0 ꢁ 5.2
65.0 ꢁ 2.4
55.2 ꢁ 4.2
46.3 ꢁ 3.1
59.0 ꢁ 2.7
39.6 ꢁ 3.9
40.7 ꢁ 2.2
78.0 ꢁ 2.4
–
52.0 ꢁ 4.5
50.1 ꢁ 2.7
53.9 ꢁ 3.5
53.9 ꢁ 1.7
65.5 ꢁ 1.6
57.8 ꢁ 4.3
55.6 ꢁ 3.6
64.8 ꢁ 3.4
71.7 ꢁ 2.6
59.7 ꢁ 5.2
56.0 ꢁ 3.8
54.3 ꢁ 2.8
57.8 ꢁ 4.1
45.5 ꢁ 3.7
35.0 ꢁ 4.4
49.0 ꢁ 3.9
49.0 ꢁ 5.3
59.5 ꢁ 2.6
59.4 ꢁ 2.1
57.8 ꢁ 4.7
55.8 ꢁ 3.3
49.0 ꢁ 4.1
29.6 ꢁ 2.3
91.1 ꢁ 3.0
3-F
4-F
2-CH3
3-CH3
4-CH3
2-CF3
3-CF3
4-CF3
2-OCH3
3-OCH3
4-OCH3
2-NO2
3-NO2
4-NO2
23
24
25
26
27
28
4. Experimental
4.1. Chemistry
Indomethazine
NS-398
a
Melting points were determined with a Thomas-Hoover Capil-
lary Melting Point Apparatus (Philadelphia, PA, USA) and are
The determination was performed in duplicate for two independent
experiments.

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O. Unsal-Tan et al. / European Journal of Medicinal Chemistry 45 (2010) 2345–2352
2348
Fig. 3. (A) Superimposition of best poses of 7–28 (21, 24 and 27 are shown as pink). (B) The orientation of 15 in COX-2 active pocket (SC-558 is shown as orange). (C) The orientation
of 15 in COX-2 active pocket (H bonds are shown as green). (D) The orientation of 21 in COX-2 active pocket (H bonds are shown as green). (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article.)
uncorrected. IR spectra (KBr) were recorded on a Jasco FTIR420
Fourier spectrometer and were reported in cmꢀ1. The 1H NMR
spectra (DMSO-d6) were recorded on a Varian Mercury 400 FT NMR
spectrophotometer using TMS as an internal reference (chemical
(C]O, ester), 1655 (C]O, ring). 1H NMR (DMSO-d6, 400 MHz);
2.75 (3H, s, –CH3), 3.76 (3H, s, –OCH3), 6.90 (1H, s, –CH]), 7.5–
d
7.52 (3H, m, phenyl-H3, H4, H5), 7.92–7.95 (2H, m, phenyl–H2, H6).
ESI-MS (m/z); 267 [M þ Na]þ (100%), 245 [M þ H]þ.
shift represented in
d ppm). The ESI-MS spectra were measured on
a
micromass ZQ-4000 single quadruple mass spectrometer.
4.1.3. Methyl 5-(1-hydroxy-2-phenylvinyl)-3-methylisoxazole-4-
carboxylate (3) [16]
Elemental analyses (C, H, N) were performed on Leco CHNS 932
analyzer.
A mixture of 2 (0.01 mol), sodium acetate (0.012 mol), hydrox-
ylamine hydrochloride (0.011 mol), water (10 mL) and ethanol
(50 mL) was refluxed for 2 h. After cooling, yellow crystals were
collected and recrystallized from ethanol to give pure compound
(66% yield). Mp 135–6 ꢂC. IR (KBr); 1672 (C]O). 1H NMR (DMSO-d6,
4.1.1. Methyl 2-methyl-4-oxo-4,5-dihydrofuran-3-carboxylate (1)
[15]
Methyl acetoacetate (0.020 mol) was added to the suspension of
magnesium ethoxide (0.022 mol) in 6 mL absolute ethanol and
25 mL toluene, then the mixture was stirred at room temperature
for 1 h. The mixture was cooled to ꢀ10 ꢂC followed by addition of
acetonitrile (25 mL). The chloroacetyl chloride (0.022 mol) was
added dropwise to the stirred solution which was then kept at 0 ꢂC
for 1 h. Then the mixture was poured into ice/water (65 mL) con-
taining sulfuric acid (4 mL) and organic phase extracted with ether,
separated and dried (Na2SO4). Triethylamine (0.020 mol) in dry
ether (5 mL) was added to cooled solution (0 ꢂC). The precipitated
salt was filtered off and the filtrate was evaporated. The residue was
recrystallized from petroleum ether (45% yield). Mp 110–1 ꢂC. IR:
1711 (C]0, ester and ring). 1H NMR: 2.50 (3H, s, CH3), 3.63 (3H, s,
–OCH3), 4.72 (2H, s, –CH2–). ESI-MS (m/z); 179 [M þ Na]þ (100%),
157 [M þ H]þ.
400 MHz);
d 2.40 (3H, s, –CH3), 3.85 (3H, s, –OCH3), 6.27 (1H, s,
–CH]), 7.33–7.39 (3H, m, phenyl–H3, H4, H5), 7.72–7.82 (2H, m,
phenyl–H2, H6), 10.53 (1H, s, –OH). ESI-MS (m/z); 282 [M þ Na]þ
(100%), 260 [M þ H]þ.
4.1.4. 7-Benzyl-3-methylisoxazolo[4,5-d]pyridazin-4(5H)-one (4)
[16]
This compound was prepared according to the literature
method [16].
4.1.5. Ethyl 2-(7-benzyl-3-methyl-4-oxoisoxazolo[4,5-d]pyridazin-
4(5H)-yl)acetate (5)
Anhydrous potassium carbonate (0.075 mol) was added to
a solution of 0.05 mol 4 in acetone (100 mL). The reaction mixture
was refluxed for 4 h and then to it was added drop by drop
a solution of 0.05 mol ethyl bromoacetate in acetone (25 mL). The
reaction mixture was refluxed for 2 h and poured into ice-cold
water (250 mL). The solid was filtered and purified by recrystalli-
zation from ethanol to give 5 (98% yield). Mp 135–6 ꢂC. IR (KBr);
1745 (C]O, ester), 1694 (C]O, pyridazinone). 1H NMR (DMSO-d6,
4.1.2. Methyl 5-benzylidene-2-methyl-4-oxo-4,5-dihydrofuran-3-
carboxylate (2) [15]
Compound 1 (3.25 mmol) and equimolar amount of benzalde-
hyde were dissolved in 25 mL of anhydrous benzene. p-Toluene-
sulfonic acid (0.08 g) was added as a catalytic reagent. The mixture
was then heated with a Dean–Stark separator during 2 h. Benzene
was evaporated and residue recrystallized from ethyl acetate and
n-hexane mixture (1:1) (64% yield). Mp 118–9 ꢂC. IR (KBr); 1707
400 MHz);
d 1.20 (3H, t, –CH2–CH3), 2.53 (3H, s, –CH3), 4.16 (2H, q,
–CH2–CH3), 4.23 (2H, s, –CH2–C6H5), 4.97 (2H, s, –CH2COO–), 7.24–
7.34 (5H, m, phenyl). ESI-MS (m/z); 350 [M þ Na]þ (100%), 271, 185.

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O. Unsal-Tan et al. / European Journal of Medicinal Chemistry 45 (2010) 2345–2352
2349
Anal. Calcd. for C17H17N3O4: C, 62.38; H, 5.23; N, 12.84. Found: C,
62.21; H, 5.09; N, 12.89.
[M þ Na]þ (100%), 282. Anal. Calcd. for C22H18BrN5O3: C, 55.01; H,
3.78; N, 14.58. Found: C, 54.89; H, 3.73; N, 14.50.
4.1.6. 2-(7-Benzyl-3-methyl-4-oxoisoxazolo[4,5-d]pyridazin-
4(5H)-yl)acetohydrazide (6)
4.1.7.4. N 0-(4-Bromobenzylidene)-2-(7-benzyl-3-methyl-4-oxoisoxa-
zolo[4,5-d]pyridazin-4(5H)-yl)acetohydrazide (10). The derivative
10 was obtained as a white solid by condensation of 6 with 4-
bromobenzaldehyde in 87% yield, mp 264 ꢂC. IR (KBr); 3670–3380
(N–H), 1699 (C]O, pyridazinone), 1670 (C]O, hydrazide), 1H NMR
A solution of 0.20 mol of hydrazine hydrate (100%) and 0.01 mol of 5
in 50 mL of absolute ethanol was refluxed with stirring for 1 h. The
solvent was evaporated under reduced pressure. The residue was
purified by recrystallization from ethanol to give 6 (90% yield). Mp
191–2 ꢂC. IR (KBr); 3341, 3263 (N–H),1687 (C]O, pyridazinone),1664
(DMSO-d6, 400 MHz); d 2.39 (3H; s; –CH3), 4.09 (2H; s; –CH2–C6H5),
4.79 and 5.18 (2H; s; –CH2–CONH–), 7.10–7.18 (5H; m; –CH2–C6H5),
7.48–7.55 (4H; m; –N]CH–C6H5, H2, H3, H5, H6), 7.87 and 8.05 (1H;
s; –N]CH–), 11.70 (1H; s; –CO–NH–N]). ESI-MS (m/z); 504
[M þ Na þ2 ]þ, 502 [M þ Na]þ (100%), 282, 239. Anal. Calcd. for
C22H18BrN5O3: C, 55.01; H, 3.78; N, 14.58. Found: C, 54.86; H, 3.77;
N, 14.46.
(C]O, hydrazide). 1H NMR (DMSO-d6, 400 MHz);
d 2.52 (3H, s, –CH3),
4.20(2H, s,–CH2–C6H5), 4.29(2H, br, –NH2), 4.74 (2H, s, –CH2–CONH–),
7.20–7.38 (5H, m, phenyl), 9.25 (1H, br, –NH–). ESI-MS (m/z); 336
[M þ Na]þ (100%), 286, 240. Anal. Calcd. for C15H15N5O3: C, 57.50; H,
4.83; N, 22.35. Found: C, 57.57; H, 4.86; N, 22.18.
4.1.7.5. N 0-(2-Chlorobenzylidene)-2-(7-benzyl-3-methyl-4-oxoisoxa-
zolo[4,5-d]pyridazin-4(5H)-yl)acetohydrazide (11). The derivative
11 was obtained as a white solid by condensation of 6 with 2-
chlorobenzaldehyde in 93% yield, mp 191–2 ꢂC. IR (KBr); 3620–
3320 (N–H), 1700 (C]O, pyridazinone), 1671 (C]O, hydrazide), 1H
4.1.7. General procedure for the preparation of N-acylhydrazones
(7–28)
An equimolar amount of appropriate aromatic aldehyde was
added to a solution of hydrazide (6) in 20 mL of ethanol, in presence
of catalytic amount of HCl (2 drops). Reaction mixture was stirred
for 0.5–2 h at room temperature and poured into cold water. After
neutralization with 10% aqueous sodium bicarbonate solution, the
precipitate formed was filtered, washed three times with 20 mL
water and purified by recrystallization from acetonitrile.
NMR (DMSO-d6, 400 MHz); d 2.40 (3H; s; –CH3), 4.10 (2H; s; –CH2–
C6H5), 4.80 and 5.21 (2H; s; –CH2–CONH–), 7.09–7.20 (8H; m;
–CH2–C6H5, –N]CH–C6H5, H3, H4, H5), 7.80 and 7.89 (1H; dd;
–N]CH–C6H5, H6, J: 7.6 Hz, J: 1.6 Hz), 8.28 and 8.47 (1H; s; –N]
CH–), 11.82 and 11.87 (1H; s; –CO–NH–N]). ESI-MS (m/z); 460
[M þ Na þ 2]þ, 458 [M þ Na]þ (100%), 282, 254. Anal. Calcd. for
C22H18ClN5O3: C, 60.62; H, 4.16; N,16.07. Found: C, 60.55; H, 4.32; N,
16.05.
4.1.7.1. N0-Benzylidene-2-(7-benzyl-3-methyl-4-oxoisoxazolo[4,5-d]-
pyridazin-4(5H)-yl)acetohydrazide (7). The derivative
7
was
obtained as a white solid by condensation of 6 with benzaldehyde
in 80% yield, mp 253–4 ꢂC. IR (KBr); 3550–3300 (N–H), 1700 (C]O,
pyridazinone), 1671 (C]O, hydrazide). 1H NMR (DMSO-d6,
4.1.7.6. N 0-(3-Chlorobenzylidene)-2-(7-benzyl-3-methyl-4-oxoisoxa-
zolo[4,5-d]pyridazin-4(5H)-yl)acetohydrazide (12). The derivative
12 was obtained as a white solid by condensation of 6 with 3-
chlorobenzaldehyde in 87% yield, mp 243 ꢂC. IR (KBr); 3640–3310
(N–H), 1698 (C]O, pyridazinone), 1673 (C]O, hydrazide). 1H NMR
400 MHz);
d 2.53 (3H; s; –CH3), 4.23 (2H; s; –CH2–C6H5), 4.92 and
5.32 (2H; s; –CH2–CONH–), 7.23–7.31 (5H; m; –CH2–C6H5), 7.42–
7.50 (3H; m; –N]CH–C6H5, H3, H4, H5), 7.69–7.73 (2H; m; –N]CH–
C6H5, H2, H6), 8.03 and 8.21 (1H; s; –N]CH–), 11.78 (1H; s; –CO–
NH–N]). ESI-MS (m/z); 424 [M þ Na]þ (100%), 282, 219, 132. Anal.
Calcd. for C22H19N5O3: C, 65.83; H, 4.77; N,17.45. Found: C, 65.76; H,
4.61; N, 17.36.
(DMSO-d6, 400 MHz); d 2.40 (3H; s; –CH3), 4.09 (2H; s; –CH2–C6H5),
4.80 and 5.20 (2H; s; –CH2–CONH–), 7.09–7.20 (5H; m; –CH2–C6H5),
7.31–7.35 (2H; m; –N]CH–C6H5, H4, H5), 7.53 (1H; d; –N]CH–
C6H5, H6, J: 4.8 Hz), 7.62 and 7.67 (1H; s; –N]CH–C6H5, H2), 7.87
and 8.06 (1H; s; –N]CH–), 11.75 and 11.77 (1H; s; –CO–NH–N]).
ESI-MS (m/z); 460 [M þ Na þ 2]þ, 458 [M þ Na]þ (100%), 282, 254.
Anal. Calcd. for C22H18ClN5O3: C, 60.62; H, 4.16; N, 16.07. Found: C,
60.76; H, 4.15; N, 16.05.
4.1.7.2. N0-(2-Bromobenzylidene)-2-(7-benzyl-3-methyl-4-oxoisoxa-
zolo[4,5-d]pyridazin-4(5H)-yl)acetohydrazide (8). The derivative 8
was obtained as a white solid by condensation of 6 with 2-bro-
mobenzaldehyde in 97% yield, mp 183–4 ꢂC. IR (KBr); 3620–3360
(N–H), 1690 (C]O, pyridazinone and hydrazide). 1H NMR (DMSO-
4.1.7.7. N 0-(4-Chlorobenzylidene)-2-(7-benzyl-3-methyl-4-oxoisoxa-
zolo[4,5-d]pyridazin-4(5H)-yl)acetohydrazide (13). The derivative
13 was obtained as a white solid by condensation of 6 with 4-
chlorobenzaldehyde in 62% yield, mp 266 ꢂC. IR (KBr); 3650–3310
(N–H), 1699 (C]O, pyridazinone), 1671 (C]O, hydrazide). 1H NMR
d6, 400 MHz); d 2.40 (3H; s; –CH3), 4.10 (2H; s; –CH2–C6H5) 4.80 and
5.21 (2H; s; –CH2–CONH–), 7.08–7.32 (7H; m; –CH2–C6H5, –N]CH–
C6H5, H4, H5), 7.55 (1H; d; –N]CH–C6H5, H3, J: 8.0 Hz), 7.78 and 7.87
(1H; d; –N]CH–C6H5, H6, J: 8.0 Hz), 8.24 and 8.42 (1H; s; –N]
CH–), 11.84 and 11.90 (1H; s; –CO–NH–N]). ESI-MS (m/z); 504
[M þ Na þ 2]þ, 502 [M þ Na]þ (100%), 282, 136. Anal. Calcd. for
C22H18BrN5O3: C, 55.01; H, 3.78; N, 14.58. Found: C, 54.92; H, 3.99;
N, 14.54.
(DMSO-d6, 400 MHz);
d 2.54 (3H; s; –CH3), 4.24 (2H; s; –CH2–
C6H5), 4.95 and 5.34 (2H; s; –CH2–CONH), 7.24–7.35 (5H; m;
–CH2–C6H5), 7.50–7.53 (2H; m; –N]CH–C6H5, H3, H5), 7.73–7.77
(2H; m; –N]CH–C6H5, H2, H6), 8.03 and 8.21 (1H; s; –N]CH–),
11.86 (1H; s; –CO–NH–N]). ESI-MS (m/z); 460 [M þ Na þ 2]þ, 458
[M þ Na]þ (100%), 282, 254. Anal. Calcd. for C22H18ClN5O3: C,
60.62; H, 4.16; N, 16.07. Found: C, 60.48; H, 3.95; N, 15.98.
4.1.7.3. N0-(3-Bromobenzylidene)-2-(7-benzyl-3-methyl-4-oxoisoxa-
zolo[4,5-d]pyridazin-4(5H)-yl)acetohydrazide (9). The derivative 9
was obtained as a white solid by condensation of 6 with 3-bromo-
benzaldehydein90% yield, mp 239–40 ꢂC. IR (KBr);3640–3360 (N–H),
1698 (C]O, pyridazinone), 1673 (C]O, hydrazide), 1H NMR (DMSO-
4.1.7.8. N 0-(2-Fluorobenzylidene)-2-(7-benzyl-3-methyl-4-oxoisoxa-
zolo[4,5-d]pyridazin-4(5H)-yl)acetohydrazide (14). The derivative
14 was obtained as a white solid by condensation of 6 with 2-
fluorobenzaldehyde in 93% yield, mp 212–3 ꢂC. IR (KBr);
3680–3330 (N–H), 1699 (C]O, pyridazinone), 1673 (C]O,
d6, 400 MHz);
d 2.40 (3H; s; –CH3), 4.09 (2H; s; –CH2–C6H5), 4.81 and
5.20 (2H; s; –CH2–CONH–), 7.09–7.18 (5H; m; –CH2–C6H5), 7.23–
7.27(1H; m; –N]CH–C6H5, H5), 7.46 (1H; d; –N]CH–C6H5, H4, J:
7.2 Hz), 7.57 (1H; d; –N]CH–C6H5, H6, J: 7.2 Hz), 7.76 and 7.80 (1H; s;
–N]CH–C6H5, H2), 7.86 and 8.05 (1H; s; –N]CH–), 11.75 and 11.78
(1H; s; –CO–NH–N]). ESI-MS (m/z); 504 [M þ Na þ 2]þ, 502
hydrazide). 1H NMR (DMSO-d6, 400 MHz);
d 2.40 (3H; s;
–CH3), 4.09 (2H; s; –CH2–C6H5), 4.79 and 5.20 (2H; s; –CH2–
CO–), 7.08–7.20 (7H; m; –CH2–C6H5, –N]CH–C6H5, H3, H5),

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7.31–7.36 (1H; m; –N]CH–C6H5, H4), 7.73–7.53 and 7.79–7.83
(1H; m; –N]CH–C6H5, H4), 8.10 and 8.30 (1H; s; –N]CH–).
ESI-MS (m/z); 442 [M þ Na]þ (100%), 282, 254. Anal. Calcd. for
C22H18FN5O3: C, 63.00; H, 4.33; N, 16.70. Found: C, 62.86; H,
4.66; N, 16.63.
MS (m/z); 438 [M þ Na]þ (100%), 282, 254. Anal. Calcd. for
C23H21N5O3: C, 66.49; H, 5.09; N, 16.86. Found: C, 66.31; H, 5.10; N,
16.75.
4.1.7.14. N 0-(2-Trifluoromethylbenzylidene)-2-(7-benzyl-3-methyl-4-
oxoisoxazolo[4,5-d]pyridazin-4(5H)-yl)acetohydrazide
(20). The
4.1.7.9. N 0-(3-Fluorobenzylidene)-2-(7-benzyl-3-methyl-4-oxoisoxa-
zolo[4,5-d]pyridazin-4(5H)-yl)acetohydrazide (15). The derivative
15 was obtained as a white solid by condensation of 6 with 3-flu-
orobenzaldehyde in 90% yield, mp 255 ꢂC. IR (KBr); 3640–3280 (N–
H), 1698 (C]O, pyridazinone), 1673 (C]O, hydrazide). 1H NMR
derivative 20 was obtained as a white solid by condensation of 6
with 2-trifluoromethylbenzaldehyde in 87% yield, mp 129 ꢂC. IR
(KBr); 3600–3380 (N–H), 1669 (C]O, pyridazinone and hydrazide).
1H NMR (DMSO-d6, 400 MHz);
d 2.40 (3H; s; –CH3), 4.10 (2H; s;
–CH2–C6H5), 4.82 and 5.23 (2H; s; –CH2–CONH–), 7.09–7.18 (5H; m;
–CH2–C6H5), 7.47–7.51 (1H; m; –N]CH–C6H5, H4), 7.58–7.67 (2H;
m; –N]CH–C6H5, H3, H5), 8.01 and 8.10 (1H; d; –N]CH–C6H5, H6, J:
7.6 Hz), 8.26 and 8.44 (1H; s; –N]CH–), 11.89 and 11.98 (1H; s;
–CO–NH–N]). ESI-MS (m/z); 492 [M þ Na]þ (100%), 413, 254. Anal.
Calcd. for C23H18F3N5O3: C, 58.85; H, 3.86; N, 14.92. Found: C, 58.80;
H, 3.89; N, 14.85.
(DMSO-d6, 400 MHz); d 2.40 (3H; s; –CH3), 4.10 (2H; s; –CH2–C6H5),
4.80 and 5.20 (2H; s; –CH2–CONH–), 7.10–7.20 (6H; m; –CH2–C6H5,
–N]CH–C6H5, H4), 7.33–7.46 (3H; m; –N]CH–C6H5, H2, H5, H6),
7.89 and 8.08 (1H; s; –N]CH–), 11.79 (1H; s; –CO–NH–N]). ESI-MS
(m/z); 442 [M
þ
Na]þ (100%), 282, 254. Anal. Calcd. for
C22H18FN5O3: C, 63.00; H, 4.33; N, 16.70. Found: C, 62.93; H, 4.21; N,
16.61.
4.1.7.15. N 0-(3-Trifluoromethylbenzylidene)-2-(7-benzyl-3-methyl-4-
oxoisoxazolo[4,5-d]pyridazin-4(5H)-yl)acetohydrazide
0
4.1.7.10. N -(4-Fluorobenzylidene)-2-(7-benzyl-3-methyl-4-oxoisox-
(21). The
azolo[4,5-d]pyridazin-4(5H)-yl)acetohydrazide (16). The derivative
16 was obtained as a white solid by condensation of 6 with 4-flu-
orobenzaldehyde in 89% yield, mp 252 ꢂC. IR (KBr); 3620–3330 (N–
H), 1700 (C]O, pyridazinone), 1670 (C]O, hydrazide). 1H NMR
derivative 21 was obtained as a white solid by condensation of 6
with 3-trifluoromethylbenzaldehyde in 92% yield, mp 229–30 ꢂC. IR
(KBr); 3620–3290 (N–H), 1700 (C]O, pyridazinone), 1673 (C]O,
hydrazide). 1H NMR (DMSO-d6, 400 MHz);
d 2.51 (3H; s; –CH3), 4.22
(DMSO-d6, 400 MHz);
d
2.40 (3H; s; –CH3), 4.09 (2H; s; –CH2–C6H5),
(2H; s; –CH2–C6H5), 4.94 and 5.35 (2H; s; –CH2–CONH–), 7.21–7.30
(5H; m; –CH2–C6H5), 7.63–7.67 (1H; m; –N]CH–C6H5, H5), 7.75
(1H; d; –N]CH–C6H5, H4, J: 6.8 Hz), 8.00–8.05 (2H; m; –N]CH–
C6H5, H2, H6), 8.10 and 8.29 (1H; s; –N]CH–), 11.95 (1H; s; –CO–
NH–N]). ESI-MS (m/z); 492 [M þ Na]þ (100%), 282, 254. Anal.
Calcd. for C23H18F3N5O3: C, 58.85; H, 3.86; N, 14.92. Found: C, 58.69;
H, 3.65; N, 14.81.
4.78 and 5.18 (2H; s; –CH2–CONH), 7.10–7.18 (7H; m; –CH2–C6H5,
–N]CH–C6H5, H3, H5), 7.44–7.47 (2H; m; –N]CH–C6H5, H2, H6), 7.89
and 8.07 (1H; s; –N]CH–), 11.63 (1H; s; –CO–NH–N]). ESI-MS (m/
z); 442 [M þ Na]þ (100%), 282, 254. Anal. Calcd. for C22H18FN5O3: C,
63.00; H, 4.33; N, 16.70. Found: C, 62.96; H, 4.26; N, 16.63.
0
4.1.7.11. N -(2-Methylbenzylidene)-2-(7-benzyl-3-methyl-4-oxoisox-
azolo[4,5-d]pyridazin-4(5H)-yl)acetohydrazide (17). The derivative
17 was obtained as a white solid by condensation of 6 with 2-
methylbenzaldehyde in 91% yield, mp 205 ꢂC. IR (KBr); 3680–3270
(N–H), 1701 (C]O, pyridazinone), 1670 (C]O, hydrazide). 1H NMR
4.1.7.16. N 0-(4-Trifluoromethylbenzylidene)-2-(7-benzyl-3-methyl-4-
oxoisoxazolo[4,5-d]pyridazin-4(5H)-yl)acetohydrazide
(22). The
derivative 22 was obtained as a white solid by condensation of 6
with 4-trifluoromethylbenzaldehyde in 81% yield, mp 219 ꢂC. IR
(KBr); 3670–3320 (N–H), 1703 (C]O, pyridazinone), 1670 (C]O,
(DMSO-d6, 400 MHz); d 2.43 (3H; s; –C6H4–CH3), 2.53 (3H; s; –CH3),
4.23 (2H; s; –CH2–C6H5), 4.92 and 5.31 (2H; s; –CH2–CONH–), 7.22–
7.31 (8H; m; –CH2–C6H5, –N]CH–C6H5, H3, H4, H5), 7.76 (1H; dd;
–N]CH–C6H5, H6, J: 8.4 Hz, J: 1.6 Hz), 8.29 and 8.47 (1H; s;
–N]CH–). ESI-MS (m/z); 438 [M þ Na]þ (100%), 282, 254. Anal.
Calcd. for C23H21N5O3: C, 66.49; H, 5.09; N, 16.86. Found: C, 66.47;
H, 5.15; N, 16.77.
hydrazide). 1H NMR (DMSO-d6, 400 MHz);
d 2.54 (3H; s; –CH3),
4.25 (2H; s; –CH2–C6H5), 4.96 and 5.37 (2H; s; –CH2–CONH–),
7.25–7.33 (5H; m; –CH2–C6H5), 7.79–7.83 (2H; m; –N]CH–C6H5,
H3, H5), 7.92–7.97 (2H; m; –N]CH–C6H5, H2, H6), 8.12 and 8.30
(1H; s; –N]CH–), 12.00 (1H; s; –CO–NH–N]). ESI-MS (m/z); 492
[M þ Na]þ (100%), 282, 254. Anal. Calcd. for C23H18F3N5O3: C,
58.85; H, 3.86; N, 14.92. Found: C, 58.68; H, 3.86; N, 14.85.
4.1.7.12. N 0-(3-Methylbenzylidene)-2-(7-benzyl-3-methyl-4-oxoisox-
azolo[4,5-d]pyridazin-4(5H)-yl)acetohydrazide (18). The derivative
18 was obtained as a white solid by condensation of 6 with 3-
methylbenzaldehyde in 94% yield, mp 227 ꢂC. IR (KBr); 3600–3280
(N–H), 1698 (C]O, pyridazinone), 1671 (C]O, hydrazide). 1H NMR
4.1.7.17. N 0-(2-Methoxybenzylidene)-2-(7-benzyl-3-methyl-4-oxois-
oxazolo[4,5-d]pyridazin-4(5H)-yl)acetohydrazide (23). The deriva-
tive 23 was obtained as a white solid by condensation of 6 with 2-
methoxybenzaldehyde in 87% yield, mp 210–1 ꢂC. IR (KBr); 3640–
3280 (N–H), 1695 (C]O, pyridazinone), 1680 (C]O, hydrazide). 1H
(DMSO-d6, 400 MHz); d 2.33 (3H; s; –C6H4–CH3), 2.53 (3H; s; –CH3),
4.23 (2H; s; –CH2–C6H5), 4.91 and 5.32 (2H; s; –CH2–CONH–), 7.22–
7.33 (7H; m; –CH2–C6H5, –N]CH–C6H5, H4, H5), 7.47–7.53 (2H; m;
–N]CH–C6H5, H2, H6), 7.99 and 8.17 (1H; s; –N]CH–). ESI-MS (m/
z); 438 [M þ Na]þ (100%), 282, 254. Anal. Calcd. for C23H21N5O3: C,
66.49; H, 5.09; N, 16.86. Found: C, 66.44; H, 4.88; N, 16.78.
NMR (DMSO-d6, 400 MHz); d 2.52 (3H; s; –CH3), 3.82 (3H; s;
–OCH3), 4.21 (2H; s; –CH2–C6H5), 4.88 and 5.29 (2H; s; –CH2–
CONH–), 6.95–7.00 (1H; m; –N]CH–C6H5, H5), 7.08 (1H; d;
–N]CH–C6H5, H3, J: 8.4 Hz), 7.21–7.32 (5H; m; –CH2–C6H5), 7.36–
7.41 (1H; m; –N]CH–C6H5, H4), 7.76 and 7.84 (1H; d; –N]CH–
C6H5, H6, J: 7.6 Hz), 8.35 and 8.53 (1H; s; –N]CH–), 11.75 (1H; s;
–CO–NH–N]). ESI-MS (m/z); 454 [M þ Na]þ (100%), 432 [M þ H]þ,
282. Anal. Calcd. for C23H21N5O4: C, 64.03; H, 4.91; N, 16.23. Found:
C, 64.01; H, 4.67; N, 16.15.
4.1.7.13. N 0-(4-Methylbenzylidene)-2-(7-benzyl-3-methyl-4-oxoisox-
azolo[4,5-d]pyridazin-4(5H)-yl)acetohydrazide (19). The derivative
19 was obtained as a white solid by condensation of 6 with 4-
methylbenzaldehyde in 96% yield, mp 230–1 ꢂC. IR (KBr); 3680–
3340 (N–H), 1703 (C]O, pyridazinone), 1672 (C]O, hydrazide). 1H
4.1.7.18. N 0-(3-Methoxybenzylidene)-2-(7-benzyl-3-methyl-4-oxois-
oxazolo[4,5-d]pyridazin-4(5H)-yl)acetohydrazide (24). The deriva-
tive 24 was obtained as a white solid by condensation of 6 with
3-methoxybenzaldehyde in 79% yield, mp 213 ꢂC. IR (KBr); 3600–
3310 (N–H), 1685 (C]O, pyridazinone and hydrazide). 1H NMR
NMR (DMSO-d6, 400 MHz);
d 2.19 (3H; s; –C6H4–CH3), 2.40 (3H; s;
–CH3), 4.09 (2H; s; –CH2–C6H5), 4.78 and 5.17 (2H; s; –CH2–CONH–
), 7.09–7.18 (7H; m; –CH2–C6H5, –N]CH–C6H5, H3, H5), 7.44–7.47
(2H; m; –N]CH–C6H5, H2, H6), 7.86 and 8.03 (1H; s; –N]CH–). ESI-

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O. Unsal-Tan et al. / European Journal of Medicinal Chemistry 45 (2010) 2345–2352
2351
(DMSO-d6, 400 MHz);
d
2.52 (3H; s; –CH3), 3.77 (3H; s; –OCH3), 4.21
prostaglandin E2 (PGE2) using a COX Inhibitor Screening Kit
(Catalog No. 560131) from Cayman Chemical, Ann Arbor, MI,
USA.
(2H; s; –CH2–C6H5), 4.91 and 5.31 (2H; s; –CH2–CONH–), 6.94–6.99
(1H; m; –N]CH–C6H5, H4), 7.21–7.35 (8H; m; –CH2–C6H5, –N]CH–
C6H5, H2, H5, H6), 7.98 and 8.16 (1H; s; –N]CH–), 11.78 (1H; s; –CO–
NH–N]). ESI-MS (m/z); 454 [M þ Na]þ (100%), 432 [M þ H]þ, 282.
Anal. Calcd. for C23H21N5O4: C, 64.03; H, 4.91; N, 16.23. Found: C,
63.86; H, 4.59; N, 16.25.
Reaction mixtures were prepared in 100 mM Tris–HCl buffer, pH
8.0 containing 1
for 10 min in a waterbath (37 ꢂC). The reaction was initiated by the
addition of 10 M arachinodic acid (final concentration in reaction
mixture 100 M) After 2 min the reaction was terminated by adding
mM heme and COX-1 or COX-2 and preincubated
m
m
0
4.1.7.19. N -(4-Methoxybenzylidene)-2-(7-benzyl-3-methyl-4-oxois-
1 M HCl and PGE2 was quantitated by an ELISA method. The test
compounds were dissolved in DMSO and diluted to the desire
concentration with 100 mM potassium phosphate buffer (pH 7.4).
Following transfer to a 96-well plate coated with mouse anti-rabbit
IgG, the tracer prostaglandin acetylcholine esterase and primary
antibody (mouse anti PGE2) were added. Plates were then incu-
bated at room temperature overnight, reaction mixtures were
removed and wells were washed with 10 mM potassium phosphate
oxazolo[4,5-d]pyridazin-4(5H)-yl)acetohydrazide (25). The deriva-
tive 25 was obtained as a white solid by condensation of 6 with 4-
methoxybenzaldehyde in 89% yield, mp 208 ꢂC. IR (KBr); 3640–
3360 (N–H), 1699 (C]O, pyridazinone). 1683 (C]O, hydrazide), 1H
NMR (DMSO-d6, 400 MHz);
d 2.52 (3H; s; –CH3), 3.77 (3H; s;
–OCH3), 4.21 (2H; s; –CH2–C6H5), 4.89 and 5.28 (2H; s; –CH2–
CONH–), 6.96–6.99 (2H; m; –N]CH–C6H5, H3, H5), 7.21–7.30 (5H;
m; –CH2–C6H5), 7.60–7.64 (2H; m; –N]CH–C6H5, H2, H6), 7.95 and
8.13 (1H; s; –N]CH–). ESI-MS (m/z); 454 [M þ Na]þ (100%), 432
[M þ H]þ, 282. Anal. Calcd. for C23H21N5O4: C, 64.03; H, 4.91; N,
16.23. Found: C, 63.70; H, 4.86; N, 16.17.
buffer containing 0.05% Tween 20. Ellman’s reagent (200 mM) was
added to each well and the plate was incubated at room temper-
ature (exclusion of light) for 60 min, until the control wells yielded
an OD ¼ 0.3–0.8 at 412 nm. A standard curve with PGE2 was
generated from the same plate, which was used to quantify the
PGE2 levels produced in the presence of test samples. Results were
expressed as a percentage relative to a control (solvent-treated
samples).
4.1.7.20. N 0-(2-Nitrobenzylidene)-2-(7-benzyl-3-methyl-4-oxoisoxa-
zolo[4,5-d]pyridazin-4(5H)-yl)acetohydrazide (26). The derivative
26 was obtained as a white solid by condensation of 6 with 2-
nitrobenzaldehyde in 83% yield, mp 186–7 ꢂC. IR (KBr); 3640–3360
(N–H), 1683 (C]O, pyridazinone and hydrazide). 1H NMR (DMSO-
4.3. Molecular modeling
d6, 400 MHz);
d 2.54 (3H; s; –CH3), 4.24 (2H; s; –CH2–C6H5), 4.95
and 5.31 (2H; s; –CH2–CONH–), 7.23–7.31 (5H; m; –CH2–C6H5),
7.66–7.80 (2H; m; –N]CH–C6H5, H4, H5), 8.04–8.13 (2H; m;
–N]CH–C6H5, H3, H6), 8.40 and 8.63 (1H; s; –N]CH–), 12.04 and
12.09 (1H; s; –CO–NH–N]). ESI-MS (m/z); 469 [M þ Na]þ (100%),
297, 282. Anal. Calcd. for C22H18N6O5: C, 59.19; H, 4.06; N, 18.83.
Found: C, 59.25; H, 3.96; N, 18.80.
Ligand preparation: The structures of eight different conforma-
tions of N0-benzylidene-2-(7-benzyl-3-methyl-4-oxoisoxazolo[4,5-
d]pyridazin-4(5H)-yl)acetohydrazide 7 were optimized using the
hybrid density functional, B3LYP and the 6-31G(d) basis set as
implemented in Gaussian 03 [22]. Harmonic frequency analysis at
the same level of theory was carried out to verify that each
conformation was a local minimum on the potential energy surface,
and permitted the evaluation of zero point energies, entropies at
298 K and thermal corrections to the enthalpies. From these, the
relative free energy of each conformer was calculated. The two
most stable conformations are shown in Fig. 2. Two conformers of
each ligand obtained from Gaussian calculations were used for
docking study.
Docking: Docking studies were performed with MOE (The
Molecular Operating Environment) Version 2009.10, software
available from Chemical Computing Group Inc., 1010 Sherbrooke
Street West, Suite 910, Montreal, Canada H3A2R7, http://www.
chemcomp.com. The X-ray crystallographic structure of COX-2
complexed with 1-phenylsulfonamide-3-trifluoromethyl-5-(4-
bromophenyl)pyrazole (SC-558) was obtained from the Protein
Data Bank (PDB: 1CX2) [21]. Then properly protonated in the
presence of its ligand using the Protonate 3D process in MOE. The
default Triangle Matcher placement method, followed by molec-
ular mechanics refinement and GBVI scoring, was used for the
docking runs. 100 docking iterations were performed for two
conformers of each ligand and the final refined poses were ranked
by the MM/GBVI binding free energy estimation, written in the S
field. The best score of each ligand–enzyme complexes was
selected.
4.1.7.21. N0-(3-Nitrobenzylidene)-2-(7-benzyl-3-methyl-4-oxoisoxaz-
olo[4,5-d]pyridazin-4(5H)-yl)acetohydrazide (27). The derivative 27
was obtained as a white solid by condensation of 6 with 3-nitro-
benzaldehyde in 83% yield, mp 263 ꢂC. IR (KBr); 3645–3360 (N–H),
1697 (C]O, pyridazinone), 1672 (C]O, hydrazide). 1H NMR
(DMSO-d6, 400 MHz); d 2.53 (3H; s; –CH3), 4.24 (2H; s; –CH2–C6H5),
4.96 and 5.37 (2H; s; –CH2–CONH–), 7.23–7.32 (5H; m; –CH2–C6H5),
7.70–7.74 (1H; m; –N]CH–C6H5, H5), 8.16 and 8.35 (1H; s; N]CH),
8.17–8.19 (1H; d; –N]CH–C6H5, H6, J: 8.0 Hz), 8.23–8.26 (1H; m;
–N]CH–C6H5, H4), 8.52 (1H; s; –N]CH–C6H5, H2), 11.99 and 12.01
(1H; s; –CO–NH–N]). ESI-MS (m/z); 469 [M þ Na]þ (100%), 267.
Anal. Calcd. for C22H18N6O5: C, 59.19; H, 4.06; N, 18.83. Found: C,
58.92; H, 3.83; N, 18.65.
4.1.7.22. N 0-(4-Nitrobenzylidene)-2-(7-benzyl-3-methyl-4-oxoisoxa-
zolo[4,5-d]pyridazin-4(5H)-yl)acetohydrazide (28). The derivative
28 was obtained as a white solid by condensation of 6 with 4-
nitrobenzaldehyde in 82% yield, mp 279 ꢂC. IR (KBr); 3640–3320
(N–H), 1696 (C]O, pyridazinone), 1670 (C]O, hydrazide). 1H NMR
(DMSO-d6, 400 MHz); d 2.53 (3H; s; –CH3), 4.23 (2H; s; –CH2–C6H5),
4.96 and 5.37 (2H; s; –CH2–CONH–), 7.23–7.31 (5H; m; –CH2–C6H5),
7.98–8.00 (2H; m; –N]CH–C6H5, H2, H6), 8.25–8.30 (2H; m;
–N]CH–C6H5, H3, H5), 8.14 and 8.32 (1H; s; N]CH), 12.06 (1H; s;
–CO–NH–N]). ESI-MS (m/z); 469 [M þ Na]þ (100%). Anal. Calcd. for
C22H18N6O5: C, 59.19; H, 4.06; N, 18.83. Found: C, 58.88; H, 4.09; N,
18.47.
Acknowledgment
The authors gratefully acknowledge the financial support
provided by Scientific Research Fund of Hacettepe University
though Project 0501301001. One of the authors (AR) thanks the
Natural Sciences and Engineering Council (NSERC) of Canada for
financial support.
4.2. Cyclooxygenase assay
The effect of the compounds on COX-1 (ovine) and COX-2
(human recombinant) enzymes was determined by measuring

¨
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O. Unsal-Tan et al. / European Journal of Medicinal Chemistry 45 (2010) 2345–2352
[14] A. Rineh, N. Mahmoodi, M. Abdollahi, A. Foroumadi, M. Sorkhi, A. Shafiee,
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