Synthesis and biological evaluation of isoxazolo[4,5-d]pyridazin-4-(5H)-one analogues as potent anti-inflammatory agents

Article abstract of DOI:10.1016/j.bmc.2012.03.021

In this study, eighteen new isoxazolo[4,5-d]pyridazin-4(5H)-one derivatives possessing either a 1,3,4-thiadiazole or a 1,2,4-triazole-5-thione moiety were synthesized and tested for anti-inflammatory activity in vitro (COX-1/COX-2, 5-LOX) and in vivo (rat paw edema assay). Compounds 15, 16, 25, 26 and 28-30 showed dual COX-2 (IC₅₀'s in the 2.1-10.9 μM range), and 5-LOX (IC₅₀'s in the 6.3-63.5 μM range) inhibitory activity. When administered orally to rats, dual COX-2/5-LOX inhibitors showed higher anti-inflammatory activity in vivo (30-45% reduction of the inflammatory response) than the reference drug ibuprofen (18%). Among dual COX-2/5-LOX inhibitors, the most potent compound (28) exhibited the best anti-inflammatory profile by inhibiting both COX-2 (IC₅₀ = 2.1 μM) and 5-LOX (IC₅₀ = 6.3 μM) enzymes. We investigated the binding interactions of compound 28 by an enzyme-ligand molecular modeling (docking) studies, which showed favorable binding interactions in both COX-2 and 5-LOX active sites. Furthermore, the dual acting COX-2/5-LOX compound 28 exhibited a superior gastrointestinal safety profile (ulcer index = 0.25) compared to the reference drug ibuprofen (UI = 7.0) when administered orally at the same molar dose. These observations suggest that isoxazolo[4,5-d]pyridazin-4(5H)-one analogs represent a new scaffold to design potent, effective, and safe anti-inflammatory agents possessing dual COX-2/5-LOX inhibitory activity.

Full text of DOI:10.1016/j.bmc.2012.03.021

Bioorganic & Medicinal Chemistry 20 (2012) 2912–2922
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of isoxazolo[4,5-d]pyridazin-4-(5H)-one
analogues as potent anti-inflammatory agents
Keriman Özadalı a,b, Fügen Özkanlı a, Sarthak Jain b, Praveen P.N. Rao c, Carlos A. Velázquez-Martínez b,
⇑
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Hacettepe, 06100 Sihhiye, Ankara, Turkey
b Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, 2142-L Katz Group Centre for Pharmacy and Health Research, Edmonton, Alberta, Canada T6G 2E9
c School of Pharmacy, Health Sciences Campus, 200 University Ave. W., University of Waterloo, Waterloo, ON, Canada
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study, eighteen new isoxazolo[4,5-d]pyridazin-4(5H)-one derivatives possessing either a 1,3,4-thia-
diazole or a 1,2,4-triazole-5-thione moiety were synthesized and tested for anti-inflammatory activity
in vitro (COX-1/COX-2, 5-LOX) and in vivo (rat paw edema assay). Compounds 15, 16, 25, 26 and 28–30
Received 13 December 2011
Revised 29 February 2012
Accepted 7 March 2012
Available online 15 March 2012
showed dual COX-2 (IC50’s in the 2.1–10.9 lM range), and 5-LOX (IC50’s in the 6.3–63.5 lM range)
inhibitory activity. When administered orally to rats, dual COX-2/5-LOX inhibitors showed higher anti-
inflammatory activity in vivo (30–45% reduction of the inflammatory response) than the reference drug
ibuprofen (18%). Among dual COX-2/5-LOX inhibitors, the most potent compound (28) exhibited the best
Keywords:
Cyclooxygenase
Lipoxygenase
anti-inflammatory profile by inhibiting both COX-2 (IC50 = 2.1 lM) and 5-LOX (IC50 = 6.3 lM) enzymes.
We investigated the binding interactions of compound 28 by an enzyme–ligand molecular modeling
(docking) studies, which showed favorable binding interactions in both COX-2 and 5-LOX active sites. Fur-
thermore, the dual acting COX-2/5-LOX compound 28 exhibited a superior gastrointestinal safety profile
(ulcer index = 0.25) compared to the reference drug ibuprofen (UI = 7.0) when administered orally at the
same molar dose. These observations suggest that isoxazolo[4,5-d]pyridazin-4(5H)-one analogs represent
a new scaffold to design potent, effective, and safe anti-inflammatory agents possessing dual COX-2/5-LOX
inhibitory activity.
Isoxazolo[4,5-d]pyridazin-4-(5H)-one
NSAID
Docking
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
even when administered at low prophylactic doses (i.e., aspirin
81 mg/day).14 Furthermore, recent literature reports describe
The major pharmacological mechanism of action of non-steroi-
dal anti-inflammatory drugs (NSAIDs) is the enzymatic inhibition
of cyclooxygenase (COX)-mediated production of pro-inflamma-
tory prostaglandins (PGs) and thromboxanes (TXs). There are two
types of COX enzymes, namely COX-1 and COX-2; COX-1 is gener-
ally regarded as a constitutive enzyme involved in the physiologi-
cal production of PGs and provides maintenance functions such as
cytoprotection in the stomach. In contrast, COX-2 has been
regarded as an inducible enzyme expressed in inflammatory cells.1
However, recent reports have challenged these ‘traditional’ roles
for COX-1 and COX-2 enzymes, emphasizing the importance of
re-evaluating their roles not only in the inflammatory process,
but also in their mechanism-based toxicity.2
how the relatively high incidence of side-effects associated with
the chronic use of NSAIDs has led some clinicians (and patients)
to reduce their use.15
The development and clinical use of highly selective COX-2
inhibitors (coxibs) arrived with bold promises of improving gastro-
intestinal safety that have not been fully realized.2,16 Even though
these drugs produce less ulceration than conventional NSAIDs, cox-
ibs still produce significant gastric and cardiovascular adverse
events in susceptible individuals, especially if they are administered
concomitantly with aspirin.17,18 There is evidence in the literature
to be concerned about the dose-dependent hypertensive effect of
some NSAIDs, which is not easily predicted by their COX selectivity
alone.19 For this reason, Wallace et al. recently proposed that there
is still a ‘strong clinical need for effective anti-inflammatory drugs
with improved safety profiles over existing NSAIDs’.20 In this regard,
dual inhibitors of COX/LOX enzymatic pathways constitute a ratio-
nale approach for the design of more effective anti-inflammatory
agents with an improved safety profile, because of their ability to
synergistically block both pathways of the arachidonic acid cas-
cade.21,22 This supposition is based on the premise that inhibiting
only one of the COX/LOX pathways may shift the metabolism of
Epidemiological studies have shown a significant risk of gastro-
intestinal,3–6 renal,7 and hepatic8 side-effects associated with the
inhibition of COX-mediated PG synthesis.9–13 In this regard, gastro-
intestinal erosions and bleeding are two of the most common toxic
effects associated with the long-term administration of NSAIDs,
⇑
Corresponding author.
E-mail address: cvelazquez@pharmacy.ualberta.ca (C.A. Velázquez-Martínez).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.03.021

K. Özadalı et al. / Bioorg. Med. Chem. 20 (2012) 2912–2922
2913
O
arachidonic acid to the other uninhibited pathway, which may pro-
duce abnormally high concentrations of the corresponding COX-de-
rived prostaglandins (if only the LOX pathway is inhibited) or the
LOX-derived leukotrienes (if only the COX pathway is blocked).23
To improve the efficacy/safety profile of new NSAIDs, extensive
structure–activity relationship (SAR) studies have been carried out
using a wide variety of scaffolds exerting COX inhibitory profile,
including (but not limited to) pyridazinones,24–27 1,3,4-thiadiaz-
oles,28,29 and 1,2,4-triazol-5-thione rings.30 Michaux et al. reported
the isoxazolo[4,5-d]pyridazinone ring template (compound I,
Fig. 1) as a potent and selective COX inhibitor equipotent to rofec-
oxib31; Unsal-Tan et al. reported a similar isoxazolo[4,5-d]pyrida-
O
H3C
N
H3C
N
N
N
N
N
N
N
H
N
N
N
S
S
N
O
O
R2
R2
H
(13-21)
(22-30)
R1
R1
Figure 2. Chemical structure of 7-benzyl-3-methylisoxazolo[4,5-d]pyridazin-
4(5H)-one derivatives, bearing a 1,3,4-thiadiazole (13–21) or a 1,2,4-triazol-5-
thione moiety (22–30). R1 and R2 groups are described in Table 1.
zin-4(5H)-one (II) derivative as
a suitable scaffold for the
inhibition of COX enzymes,26 and Varandas et al. showed that hy-
brid drugs formed by known anti-inflammatory agents with a
1,3,4-thiadiazole moiety resulted in COX-2 selective molecules.28
Additionally, Navidpour et al. observed that compounds carrying
a 1,2,4-triazol-5-thione group were more selective than celecoxib
toward COX-2.30
Based on these observations, we were interested in evaluating
the pharmacological properties of a new series of 7-benzyl-3-
methylisoxazolo[4,5-d]pyridazin-4(5H)-one derivatives, bearing
either a 1,3,4-thiadiazole (13–21) or a 1,2,4-triazol-5-thione (22–
30) moiety (Fig. 2).
In this report we describe the chemical synthesis and biological
evaluation of 7-benzyl-3-methylisoxazolo[4,5-d]pyridazin-4(5H)-
one derivatives as anti-inflammatory agents. We also investigated
the potential (unwanted) gastric side-effects exerted by an oral
dose of these compounds, and finally, we conducted two molecular
modeling (docking) experiments to support the observed in vitro
and in vivo dual COX-2/5-LOX inhibitory profile exerted by these
compounds. The results of our investigation provide evidence sug-
gesting that 7-benzyl-3-methylisoxazolo[4,5-d]pyridazin-4(5H)-
one derivatives represent a new scaffold to develop potent and safe
anti-inflammatory agents.
of intermediates 3a–c were refluxed with an appropriately substi-
tuted isothiocyanate (R2NCS) to obtain 1-(2-(7-benzyl-3-methyl-4-
oxoisoxazolo[4,5-d]pyridazin-5(4H)-yl)acetyl)-4-thiosemicarbazides
(4–12).
The acid-catalyzed cyclization of thiosemicarbazides 4–12 using
concentrated sulfuric acid, afforded the corresponding 7-benzyl-3-
methyl-5-((5-alkylamino-1,3,4-thiadiazol-2-yl)methyl)isoxazolo[4,5-
d]pyridazin-4(5H)-ones (13–21), whereas the base-catalyzed
cyclization of thiosemicarbazides 4–12 using sodium bicarbonate
resulted in the formation of the corresponding 7-benzyl-5-((4-substi-
tuted-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)methyl)-3-methy-
lisoxazolo[4,5-d]pyridazin-4(5H)-ones (22–30). Both acid- and
base-catalyzed procedures were based on the work published by
Onkol et al.33
2.2. Determination of physicochemical properties
Considering that the COX-1 active site consists of a long narrow
channel about 8 Â 25 Å (total volume 316 Å2), and the COX-2 bind-
ing site is about 25% larger (394 Å3),34 bulkier molecules normally
fit better into the bigger active site of COX-2, and consequently,
larger compounds generally show a higher selectivity for this en-
zyme.35 This is a well established concept reported in the literature
which has been employed in the development of potent and selec-
tive COX-2 inhibitors by several groups.36–38 Consequently, we
determined the molecular length (Å), surface (Å2), and volume
(Å3) of experimental drugs to assess if they would possess the min-
imum essential structural requirements to enter the COX binding
site. In this regard, after a standard energy minimization procedure
using Alchemy 2000 (Version 2.0, 1997, Tripos Inc.), we deter-
mined these parameters for compounds 13–30 and the results
are summarized in Table 1.The majority of test compounds (except
13 and 22) showed a molecular volume higher than that required
to fit into the active site of COX-1, which suggested that they
would probably show a weak binding affinity for this enzyme.
However, the calculated molecular volumes (309–390 Å3) for com-
pounds 13–30 suggested that these molecules would be able to ac-
cess the long narrow channel of the COX-2 enzyme active site
(394 Å3), which in turn, would likely produce a higher binding
2. Results and discussion
2.1. Chemistry
The synthesis of 7-benzyl-3-methyl-5-((5-alkylamino-1,3,4-thia-
diazol-2-yl)methyl)isoxazolo[4,5-d]pyridazin-4(5H)-ones
13–21
and 7-benzyl-5-((4-substituted-5-thioxo-4,5-dihydro-1H-1,2,4-tria-
zol-3-yl)methyl)-3-methylisoxazolo[4,5-d]pyridazin-4(5H)-ones
22–30 is outlined in Scheme 1.
7-Benzyl-3-methylisoxazolo[4,5-d]pyridazin-5(4H)-ones (1a–c)
were prepared as described in the literature.32 Briefly, reaction of
intermediates 1a–c with ethyl bromoacetate in the presence of
potassium carbonate in acetone yielded 2-(7-benzyl-3-methyl-4-
oxoisoxazolo[4,5-d]pyridazin-5(4H)-yl)acetates (2a–c), which
were subsequently reacted with hydrazine hydrate under reflux
to obtain a series of 2-(7-benzyl-3-methyl-4-oxoisoxazolo[4,5-
d]pyridazin-5(4H)-yl)acetohydrazides (3a–c). Ethanolic solutions
H
N
OCH3
O
N CH
O
H3C
N
H3C
N
O
N
N
R
N
N
O
O
(I)
II
( )
CH3
Figure 1. Chemical structure of reported isoxazolo[4,5-d]pyridazin-4(5H)-ones SPB04674 (I), and (II).

2914
K. Özadalı et al. / Bioorg. Med. Chem. 20 (2012) 2912–2922
O
O
O
H3C
N
H3C
N
H3C
N
NHNH2
H
O
N
N
N
N
N
N
a
b
O
O
O
O
O
1a-c
2a-c
3a-c
R1
S
R1
R1
c
O
H3C
O
H
N
H3C
N
N
H
H
N
N
d
N
N
N
H
N
N
N
S
O
O
R2
O
N
R2
13-21
4-12
R1
R1
e
O
H3C
N
N
N
N
H
N
N
O
R2
S
22-30
R1
Scheme 1. Synthesis of target compounds 13–30. Reagents and conditions: (a) K2CO3, acetone, reflux 4 h, then EtOCOCH2Br and reflux 2 h; (b) NH2NH2, EtOH, reflux 1 h; (c)
R2NCS, EtOH, reflux 4 h; (d) H2SO4, H2O, 25 °C,1 h; (e) NaHCO3, EtOH, 25 °C, 1 h.
Table 1
two different linear distances between the two atoms farthest apart
from each other in the corresponding energy-minimized structures
(Table 1). Based on these parameters, we observed that all test com-
pounds (10.6–16.2 Å long) could theoretically fit into the active site
of COX (25 Å long). These preliminary parameters, along with the
corresponding molecular volumes, suggested that pyridazin-
4(5H)-ones had appropriate physicochemical parameters to access
the COX channel. Nevertheless, we recognized that even though
pyridazin-4(5H)-ones possessed proper volumes and lengths, they
may or may not interact favorably with essential residues within
the binding site of either enzyme (this would be better assessed
by specific molecular modeling—docking—studies, see discussion
below).
Prediction of physicochemical parameters for compounds 13–30: molecular volume
(expressed in cubic Angstroms, Å3); Length (expressed in Angstroms, Å); negative
logarithm of the distribution coefficient of each compound between n-octanol/water
(theoretical value, expressed as LogP)
a
a
Compd
R1
R2
Volumeb
Lengthc
LogPd
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
H
H
H
CH3
309.6
325.8
364.1
335.7
341.5
388.6
334.7
351.0
389.1
309.2
325.9
363.6
335.3
351.6
390.4
334.1
350.3
388.3
11.0, 13.2
11.8, 14.3
11.8, 15.2
13.0, 13.2
12.3, 14.1
13.1, 16.2
13.2, 13.6
13.7, 14.1
13.4, 14.7
10.7, 11.5
11.6, 11.9
11.8, 12.7
10.6, 13.9
11.4, 14.0
11.2, 11.5
11.4, 13.0
11.7, 13.9
13.2, 15.6
0.67 1.0
C2H5
C6H5
CH3
C2H5
C6H5
CH3
C2H5
C6H5
CH3
C2H5
C6H5
CH3
C2H5
C6H5
CH3
C2H5
C6H5
1.20 1.0
1.68 1.1
0.40 1.0
0.93 1.0
1.41 1.1
0.59 1.0
1.12 1.0
1.59 1.1
À0.68 1.0
À0.15 1.0
1.07 1.0
À0.95 1.1
À0.42 1.1
0.80 1.1
À0.76 1.1
À0.23 1.1
0.99 1.1
NO2
NO2
NO2
OCH3
OCH3
OCH3
H
2.3. Inhibition of cyclooxygenase enzymes
H
H
NO2
NO2
NO2
OCH3
OCH3
OCH3
The target compounds 13–30 were evaluated for their ability to
inhibit COX-2 (human recombinant) and COX-1 (ovine) enzymatic
activity using a fluorescent inhibitor screening assay kit (Cayman
Chemicals, Cat. No. 700100).The potency (IC50 values) of test com-
pounds was determined and compared to that of the reference
molecules DuP-697 (selective COX-2 inhibitor) and SC-560 (selec-
tive COX-1 inhibitor); these results are summarized in Table 2.
In general, 7-benzyl-3-methyl-5-((5-alkylamino-1,3,4-thia-
diazol-2-yl) methyl)isoxazolo[4,5-d]pyridazin-4(5H)-ones (13–
21), showed a modest cyclooxygenase inhibition in vitro (COX-1
a
See Scheme 1.
b
Calculated using Alchemy 2000 (Version 2.0, 1997, Tripos Inc.) after energy
minimization.
c
From the energy-minimized structures, different lengths were calculated by
measuring the linear distance between atoms farthest apart from each other.
d
Calculated using ACD/ChemSketch (Version 12.01 freeware, 2010, ACD Labs
IC50’s = 19–99.1 lM; COX-2 IC50’s = 2.1–39.6 lM) compared to ref-
Inc.).
erence compounds SC-560 (IC50 = 8.5 nM) and DuP-697
(IC50 = 41.6 nM). The most potent COX inhibitors in this series were
compounds 19 (COX-1 IC50 = 19.0
l
M; COX-2 IC50 = 14.7
M).
lM) and
affinity compared to COX-1. Therefore, it was expected that com-
pounds 13–30 would possess a selective COX-2 inhibitory profile.
In addition to molecular volumes, we also determined molecular
lengths (Å) for all test compounds. Considering the three dimen-
sional structure of pyridazin-4(5H)-ones (13–30), we determined
16 (COX-1 IC50 = 67.0 M; COX-2 IC50 = 6.0
l
l
Structure–activity relationship studies showed that the nature
of substituents (R2) on the 1,3,4-thiadiazol-2-yl ring was not a ma-
jor determinant on COX-1 inhibition, whereas substitution at the

K. Özadalı et al. / Bioorg. Med. Chem. 20 (2012) 2912–2922
2915
Table 2
Table 3
In vitro COX-1 and COX-2 enzyme inhibition for compounds 13–30
In vitro 5-LOX enzyme inhibition, in vivo anti-inflammatory and ulcerogenic activity
for compounds 15, 16, 25, 26, 28, 30
Compd
COX-1 IC50
(
l
M)a
COX-2 IC50
(l
M)a
SIb
Compd
5-LOX IC50
(
l
M)a
AIb
Ulcer indexc
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
99.1
>100
>100
67.0
46.7
77.4
19.0
>100
>100
6.4
21.3
>100
10.9
6.0
39.6
>100
14.7
>100
>100
5.5
6.1
>100
4.6
9.2
>100
2.1
4.6
n.d.c
>10
11.1
1.1
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
79.2
72.8
63.5
41.2
52.8
58.9
48.7
39.5
46.2
34.7
40.3
45.1
13.5
14.8
16.5
6.3
n.d.
n.d.
40.3 6.2
30.2 3.5
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
0.50 0.2
0.75 0.3
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
0.25 0.2
0
n.d.
0.25 0.2
n.d.
0
—
n.d.c
1.3
n.d.c
n.d.c
1.1
0.9
0.1
n.d.
n.d.
n.d.
>100
47.7
>100
>100
26.5
20.5
>100
0.0085
n.d.c
n.d.c
10.3
>10
n.d.c
12.6
9.15
>10
—
35.8 3.5
38.0 4.8
n.d.
45.0 2.8
n.d.
45.0 3.9
—
—
29
30
SC-560
DuP-697
2.2
10.8
n.d.c
0.04
29
30
8.6
7.7
—
Caffeic acid
Zileuton
Ibuprofen
4.0
1.1d
—
—
a
The in vitro test compound concentration required to produce 50% inhibition of
enzymatic activity. The result (IC50 M) is the mean of three determinations
acquired using the COX Fluorescent Inhibitor Screening Assay Kit (Catalog No.
700100, Cayman Chemicals Inc., Ann Arbor, MI, U.S.A.) and the deviation from the
mean is <10% of the mean value.
18.5 3.0
7.00 0.8
,
l
a
The in vitro test compound concentration required to produce 50% inhibition of
potato 5-LOX (Cayman Chemicals Inc. Catalog No. 60401). The result (IC50 M) is
the mean of two determinations acquired using a LOX assay kit (Catalog No.
760700, Cayman Chemicals Inc., Ann Arbor, MI, U.S.A.) and the deviation from the
mean is <10% of the mean value.
,
l
b
In vitro COX-2 selectivity index (COX-1 IC50/COX-2 IC50).
Not determined.
c
b
Inhibitory activity in a carrageenan-induced rat paw edema assay. The results
are expressed as the % inhibition of inflammation at 3 h following an oral dose
(0.14 mmol) of the corresponding test compounds equivalent to 30 mg/kg of ibu-
profen. Results are expressed as the mean SEM (n = 4).
The average overall length (in mm) of individual ulcers in each stomach SEM,
n = 4, 6 h after oral administration of the test compounds (0.14 mmol equivalent to
7-benzyl ring (R1) modulated the potency of compounds in this
series; molecules possessing an electronegative nitro group
(16–18) were more active than unsubstituted compounds
(R1 = H, 13–15), and molecules having an electron donating meth-
oxy group (20, 21) were the least potent COX-1 inhibitors, with the
notable exception of compound 19, which was the most potent in
this group.
c
30 mg/kg of ibuprofen).
d
Ref. value.43
A similar analysis conducted on 7-benzyl-5-((4-substituted-5-
thioxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)methyl)-3-methylisoxaz-
olo[4,5-d]pyridazin-4(5H)-ones (22–30) revealed that inhibition of
COX-1 and COX-2 was modulated mostly by the size of substitu-
ents (R2) located on the 1,2,4-triazol-3-yl ring, since compounds
possessing small alkyl groups (22, 25, 28 R2 = methyl; 23, 26, 27
R2 = ethyl) showed increased potency on both COX-1 (IC50’s in
reference drug ibuprofen (18.5% decrease) on a molar basis
(0.14 mmol/kg, equivalent to 30 mg/kg of ibuprofen), exerting a
1.6–2.4 fold-increase in anti-inflammatory activity. These results
are in agreement with a previous report by Amir et al. describing
the in vivo anti-inflammatory profile of 2,5-disubstituted 1,3,4-
oxadiazoles and 1,2,4-triazoles as ‘arylacetic acid derivatives’ with
significant analgesic and anti-inflammatory activity.40
the 0.9–47.7
l
M range) and COX-2 (IC50 = 2.1–9.2
l
M) enzymes,
There are several plausible explanations for the increased anti-
inflammatory profile exerted by these compounds (especially 28
and 30) compared to ibuprofen. One of them is the modest increase
in their selectivity towards COX-2 enzyme (COX-1/COX-2 selectiv-
ity index = 10 or higher). However, we recognize the limitations of
correlating in vitro potency and selectivity with in vivo results, be-
cause the incubation of test compounds in the presence of purified
enzymes does not account for all the pharmacokinetic and phar-
macodynamic factors evaluated in the animal model. Another pos-
sible explanation may be that compounds 15, 16, 25, 26, 28, and 30
are absorbed at a higher extent compared to ibuprofen, which con-
tains an ionizable COOH group; however, without a proper phar-
macokinetic study we cannot conclude this is the case. In this
regard, one of the main factors controlling the passive diffusion
of molecules across cell membranes is their lipophilic character,
and therefore, we determined the theoretical LogP values for com-
pounds 13–30 (Table 1). However, the result of these calculations
showed a lower lipophilic character for the test compounds (LogP
values in the À0.95–1.68 range), compared to the reference drug
ibuprofen (LogP = 3.72 0.23). Based on these results, we could
not correlate theoretical LogP values to the observed in vivo anti-
inflammatory activity; for example, compounds 15 (LogP = 1.68)
and 25 (LogP = À0.95) showed similar in vivo anti-inflammatory
potencies (40.3% and 35.8% decrease in inflammatory response
compared to analogs possessing a larger phenyl group (24, 27,
30) at the same position, which did not inhibit cyclooxygenase
activity at the highest test compound concentration (100
The only exception was compound 30, which displayed a relatively
strong COX-2 enzyme inhibition (IC50 = 10.8 M). In this regard,
we also observed that compounds 22–30 showed an increased
selectivity toward COX-2 (selectivity indices = 0.15–12.65) com-
pared to analogs possessing a 5-substituted-1,3,4-thiadiazol-2-yl
moiety (13–21). Compounds 26 and 30 were selective COX-2
inhibitors (SI >10), but their potency (COX-2 IC50 = 9.2 and
lM).
l
10.8
erence compound DuP-697 (IC50 = 41.6 nM). The most potent COX-
2 inhibitor (28, IC50 = 2.1 M) was about 52 fold-less potent than
lM) was not nearly as good as that of the corresponding ref-
l
the reference drug DuP-697 (IC50 = 0.04 lM).
2.4. Anti-inflammatory activity
Based on the inhibitory potencies obtained in vitro (COX-2
selectivity), we screened a group of compounds (15, 16, 25, 26,
28, and 30) for in vivo anti-inflammatory activity using the tradi-
tional carageenan-induced rat paw edema assay39 (Table 3). We
observed that these compounds were significantly more active
(30.2–45.0% decrease in the inflammatory response) than the

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K. Özadalı et al. / Bioorg. Med. Chem. 20 (2012) 2912–2922
respectively). This wide range of LogP values suggested that the
in vivo anti-inflammatory response exerted by 7-benzyl-3-methy-
lisoxazolo[4,5-d]pyridazin-4(5H)-one derivatives is (probably) not
modulated by their lipophilic character.
A third possible explanation for the improved in vivo anti-
inflammatory profile observed for the test compounds, is the inhi-
bition of other enzymes involved in the inflammatory process. One
of these enzymes is 5-lipoxygenase (5-LOX), which mediates the
production of leukotrienes (LTs), species implicated not only in
inflammation but also in fever, arthritis, and bronchospasm.21,22
2.5. Inhibition of 5-lipoxygenase (5-LOX)
It has been suggested that inhibition of only one of the COX/LOX
pathways could shift the metabolism of arachidonic acid towards
the other pathway, thereby inducing potential side-effects.23
Therefore, to support the hypothesis of dual COX/5-LOX inhibition
by the test compounds, we evaluated the in vitro inhibitory profile
of drugs 15, 16, 25, 26, 28, and 30 (Table 3). We observed that the
5-LOX inhibitory potency of the test compounds was moderate to
relatively high (IC50’s in the 6.3–79.2
compounds 28 (IC50 = 6.3 M) and 30 (IC50 = 7.7
the most active, showing an inhibitory potency similar to that of
the reference compound caffeic acid (IC50 = 4.0 M). However,
lM range), especially for
l
lM) which were
l
these inhibitory potencies represent a 5 to 7-fold decrease in po-
tency compared to Zileuton (ZyfloÒ), an orally active inhibitor of
5-LOX.
The dual inhibition of COX and LOX enzymatic pathways has
been extensively reported in the literature, and constitutes a ratio-
nale method for the design of more effective anti-inflammatory
agents, because of their ability to synergistically block both path-
ways of the AA cascade. In this regard, considering that the re-
ported mechanisms of enzymatic inhibition of 5-LOX involve
either chelation of the iron atom present in the active site
(hydroxamic acids and N-hydroxyureas),41 or direct inhibition by
reversible binding interactions (classical enzyme inhibition),42 we
can assume that 7-benzyl-3-methyl-5-((5-alkylamino-1,3,4-thia-
diazol-2-yl)methyl)isoxazolo[4,5-d]pyridazin-4(5H)-ones (15, 16)
and 7-benzyl-5-((4-substituted-5-thioxo-4,5-dihydro-1H-1,2,4-tria-
zol-3-yl)methyl)-3-methylisoxazolo[4,5-d]pyridazin-4(5H)-ones (25,
26, 28, and 30) inhibited 5-LOX by classical enzyme (reversible
binding) interactions. At this point we do not have evidence to sup-
port a possible chelation of iron as the potential mechanism of LOX
inhibition. Nevertheless, these compounds represent a new scaffold
to design relatively potent and selective dual COX/5-LOX inhibitors.
Figure 3. The orientation of 28 (ball-and-stick) in the active site of mammalian
COX-2. Red dotted line represents hydrogen bonding. Hydrogen atoms are not
shown for clarity.
2.6. Molecular modeling
To support the in vitro and in vivo anti-inflammatory profile ex-
erted by isoxazolo[4,5-d]pyridazin-4(5H)-one (compound 28), we
also carried out enzyme–ligand molecular modeling (docking)
studies, which showed that the test compounds exerted favorable
interactions with both COX-2 (Fig. 3) and 5-LOX (Fig. 4).
The pyridazin-4(5H)-one ring was oriented in the central region
of the COX-2 active site and the fused isoxazole ring was within the
vicinity of COX-2 secondary pocket, surrounded by His90, Ser353,
Tyr355, Arg513 and Val523. The isoxazole nitrogen atom formed
a hydrogen bond with NH of His90 (distance = 2.30 Å) and the oxy-
gen atom of isoxazole was about 3.55 Å away from OH of Tyr355,
which suggests favorable binding interactions allowing this ring
to enter the mouth of the COX-2 active site. The substituted
1,2,4-triazole-5-thione ring was oriented toward the apex of the
COX-2 active site in a region comprised of Phe381, Leu384,
Tyr385, Trp387, Met522, Gly526 and Ala527. The C@S moiety
underwent nonpolar interactions with the side chain of Met522
(distance <5 Å) and was about 4.28 Å away from OH of Tyr385,
Figure 4. The orientation of 28 (ball-and-stick) in the active site of mammalian 5-
LOX. Red dotted line represents hydrogen bonding. Hydrogen atoms are not shown
for clarity.
whereas the N-CH3 group was oriented toward Tyr348, Ala527
and Ser530. The para-methoxy-benzyl substituent of isoxazol-
o[4,5-d]pyridazin-4(5H)-one was oriented in a hydrophobic region
comprised of Met113, Leu117, Ile345, Leu359, and Leu531 closer to
the mouth of the COX-2 active site (distance <5 Å). This orientation
differs from that reported by Unsal-Tan et al.,23 who reported the
benzyl ring, and not the 1,2,4-triazole-5-thione ring, to be the

K. Özadalı et al. / Bioorg. Med. Chem. 20 (2012) 2912–2922
2917
group orienting into the cavity occupied by Phe381, Leu384,
Tyr385, Trp387, Phe513 and Ser530.
We also investigated the binding interactions of compound 28
(5-LOX IC50 = 6.3 lM), which was the most potent LOX inhibitor
injuries to the stomachs of rats.44 Darling et al. found that NSAIDs
can induce gastric injury in transgenic mice deficient in either
COX-1 or COX-2, suggesting that simple inhibition of gastric
COX-1 activity cannot provide a suitable explanation for the
NSAID-induced gastric pathogenesis.45 Orally administered NSAIDs
associated with phosphatidylcholine (PC) inhibit gastric PGE2 to
the same extent as NSAIDs administered alone; however, PC-NSA-
IDs do not produce significant GI toxicity.46
It has been proposed that NSAIDs possessing carboxylic acid
groups produce epithelial damage at sites of contact. The re-ioniza-
tion of the uncharged, lipid-soluble drug within the gastric cells can
lead to osmotic swelling and lysis of the cells, and this process can
uncouple mitochondrial respiration leading to cell death.47 The the-
oretical LogP values calculated for isoxazolo[4,5-d]pyridazin-4(5H)-
one derivatives (LogP values in the À0.9–1.6 range) suggests low
lipophilicity for test compounds, especially for 7-benzyl-3-methyl-
5-((5-(substituted amino)-1,3,4-thiadiazol-2-yl)-methyl)isoxazol-
o[4,5-d]pyridazin-4(5H)-ones (13–21). However, despite their low
lipophilic character, compounds 15, 16, 25, 26, 28, and 30 showed
an improved anti-inflammatory activity compared to the reference
drug ibuprofen (calculated LogP value = 3.72 0.23), but were de-
void of gastric significant gastric toxicity at a 0.14 mmol dose
(equivalent to 30 mg/kg of ibuprofen).
in vitro (Fig. 4). This study showed that the fused isoxazolopyrida-
zin-4(5H)-one ring present in compound 28 was at the center of
the active site, and the pyridazin-4-one C@O group was oriented
toward the entrance of 5-LOX active site (Lys409) and formed a
hydrogen bond with the NH group of Phe402 (distance = 2.52 Å).
The fused isoxazole ring was oriented in a region comprised of
Ile167, Tyr383, Phe402 and Arg401. The isoxazole oxygen formed
a hydrogen bond with OH of Tyr383 (distance = 2.39 Å), whereas
the methyl substituent underwent non-polar interactions with
Val400, Ile404 and Ala405 (distance <5 Å). The substituted 1,2,4-
triazole-5-thione ring was oriented toward a polar pocket com-
prised of Gln15, Tyr81, Trp102, Asp170 and Glu622 (distance
<5 Å). Interestingly, the C@S of the triazole ring also formed a
hydrogen bond with the NH2 group of Gln15 (distance = 2.44 Å),
and additionally, the triazole nitrogens underwent electrostatic
interactions with the polar side chain of Arg401 (distance ꢀ3 Å).
The para-methoxy-benzyl substituent of isoxazolo[4,5-d]pyrida-
zin-4(5H)-one was oriented toward the catalytic site of 5-LOX in
a region comprised of Tyr383, Phe393, Val397 and His624 (dis-
tance <5 Å). These studies indicate that compound 28 (molecular
volume of 334.1 Å3) fits well within the large boot-shaped active
site of 5-LOX (volume = 663 Å3).51 The results obtained in these
docking studies suggest that compound 28 is able to form several
binding interactions, which allow it to inhibit the catalytic activity
of 5-LOX, establishing a correlation with its relatively high in vitro
inhibitory potency.
3. Conclusion
We propose the 3-methylisoxazolo[4,5-d]pyridazin-4(5H)-one
ring with a central 4-substituted benzyl ring at C-7, and a 1,3,4-
thiadiazole (13–21) or 1,2,4-triazole (22–30) moiety at N-5 as a
new scaffold for dual COX/5-LOX inhibition. Structure–activity
relationship studies showed that the nature of substituents (R2)
on the 1,3,4-thiadiazol-2-yl ring was not a major determinant on
COX inhibition, whereas substitution at the 7-benzyl ring (R1)
modulated their potency in vitro. The addition of a 5-alkylamino-
1,3,4-thiadiazol-2-yl, or a 5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-yl
moiety to the central 3-methylisoxazolo[4,5-d]pyridazin-4(5H)-
one ring provides a new class of anti-inflammatory agents with
negligible ulcerogenicity in vivo.
2.7. Ulcer index assay (UI)
Gastrointestinal erosions and ulcers are two of the most com-
mon side-effects associated with the chronic administration of
NSAIDs in subpopulations of patients classified at high risk. There-
fore, we were interested in determining if isoxazolo[4,5-d]pyrida-
zin-5(4H)-ones produce toxicity when administered orally at
their anti-inflammatory dose. To evaluate the potential (un-
wanted) ulcerogenic side-effects of dual COX/5-LOX inhibitors 15,
16, 25, 26, 28, and 30, we conducted a toxicity assay by administer-
ing the corresponding test compounds (0.14 mmol/kg), and the ref-
erence compound ibuprofen (0.14 mmol/kg, equivalent to 30 mg/
kg) po to rats. All drugs were suspended in 1% methylcellulose
solution.
Interestingly, the test compounds showed negligible toxicity
(UI = 0–0.75) compared to the reference drug ibuprofen (UI = 7.0,
Table 3). These results showed that the test compounds were sig-
nificantly less ulcerogenic (p <0.001) than the reference drug ibu-
profen when administered orally at equimolar doses, suggesting
that the 3-methylisoxazolo[4,5-d]pyridazin-4(5H)-one ring pos-
sessing either a 1,3,4-thiadiazole (15, 16) or a 1,2,4-triazole (25,
26, 28, and 30) moiety at N-5 have an improved efficacy/safety pro-
file compared to a classical NSAID such as ibuprofen.
A plausible explanation for the lack of acute gastric toxicity ex-
erted by the test compounds is the lack of acidic carboxylic acid
groups in isoxazolo[4,5-d]pyridazin-4(5H)-one derivatives. These
results showed that in spite of exerting a relatively high COX inhib-
itory profile in vitro and a potent anti-inflammatory activity
in vivo, the test compounds did not exert significant gastric toxic-
ity, and the inhibition of COX activity seems to be unrelated to the
extent of gastric toxicity. This observation is in agreement with
several reports described previously. For example, Ligumsky et al.
demonstrated that intragastrically- and parenterally-administered
aspirin exerted the same degree of gastric prostaglandin biosyn-
thesis inhibition, but only the former induced significant gastric
4. Experimental
4.1. Chemistry
Melting points were determined with a Thomas–Hoover capil-
lary melting point apparatus (Philadelphia, PA, U.S.A.) and are
uncorrected. IR spectra (KBr) were recorded on a Jasco FTIR420
Fourier spectrometer. The 1H NMR spectra (DMSO-d6) were re-
corded on a Varian Mercury 400 FT NMR spectrometer using
TMS as internal reference (Chemical shift represented in d ppm).
The ESI-MS spectra were determined using a micromass ZQ-4000
single quadruple mass spectrometer. Elemental analyses (C, H, N)
were performed on a Leco CHNS 932 analyzer (University of
Hacettepe). 7-benzyl-3-methylisoxazolo[4,5-d]pyridazin-5(4H)-ones
(1a–c),32 ethyl 2-(7-benzyl-3-methyl-4-oxoisoxazolo[4,5-d]pyri-
dazin-5(4H)-yl)acetate (2a),26 and 7-benzyl-3-methyl-4-oxoisox-
azolo[4,5-d]pyridazin-5(4H)-yl)acetohydrazide
(3a)26
were
synthesized following previously reported methods.
4.1.1. General procedure for the preparation of ethyl 2-(7-
benzyl-3-methyl-4-oxoisoxazolo[4,5-d]pyridazin-5(4H)-
yl)acetates (2b–c)
Anhydrous potassium carbonate (0.075 mol) was added to a
solution of 0.05 mol of 1a–c in acetone (100 mL). This reaction
mixture was stirred under reflux for 4 h before adding a solution
of ethyl bromoacetate (0.05 mol) in acetone (25 mL) dropwise.

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K. Özadalı et al. / Bioorg. Med. Chem. 20 (2012) 2912–2922
After 2 h of continuous reflux, the mixture was poured into a bea-
ker containing about 250 mL of ice. The solid was filtered out and
purified by recrystallization from ethanol/water.
(4).
Obtained as a white solid (66% yield) by reacting 3a with
methyl isothiocyanate; mp 169–171 °C. IR; 3288, 3163 (N–H),
1693 (C@O, pyridazinone and hydrazide), 1205 (C@S). 1H NMR
(DMSO-d6); d 2.53 (3H, s, –CH3), 2.88 (3H, d, –NH–CH3, J: 4.4 Hz),
4.22 (2H, s, –CH2–C6H5), 4.91 (2H, s, –CH2–CONH–), 7.23–7.34 (5H,
m, –CH2–C6H5), 7.94 (1H, q, –NH–CH3, J: 4.4 Hz), 9.38 (1H, br s, –
NH–NH–CS–), 10.15 (1H, br s, –CO–NH–NH–). ESI-MS (m/z); 409
[M+Na]+ (100%), 387 [M+H]+. Anal. calcd for C17H18N6O3S: C,
52.84; H, 4.70; N, 21.75; S, 8.30. Found: C, 52.44; H, 4.59; N, 21.41;
S, 8.21.
4.1.1.1. Ethyl 2-(7-(4-nitrobenzyl)-3-methyl-4-oxoisoxazolo-[4,
5-d]pyridazin-5(4H)-yl)acetate (2b).
60% yield; mp 128–
129 °C. IR: 1733 (C@O, ester), 1689 (C@O, pyridazinone).1H NMR
(DMSO-d6); d 1.16 (3H, t, –CH2–CH3, J: 6.8 Hz), 2.52 (3H, s, –CH3),
4.13 (2H, q, –CH2–CH3, J: 6.8 Hz), 4.41 (2H, s, –CH2–C6H5), 4.93
(2H, s, –CH2COO–), 7.57 (2H, d, phenyl-H2 and H6, J: 8.8 Hz), 8.17
(2H, d, phenyl-H3 and H5, J: 8.8 Hz). ESI-MS (m/z); 395 [M+Na]+
(100%), 373 [M+H]+. Anal. calcd for C17H16N4O6: C, 54.84; H, 4.33;
N, 15.05. Found: C, 54.78; H, 4.28; N, 15.04.
4.1.3.2. 1-(2-(7-Benzyl-3-methyl-4-oxoisoxazolo[4,5-d]pyridazin-
5(4H)-yl)acetyl)-4-ethylthiosemicarbazide (5).
Obtained as a
white solid (87% yield) by reacting 3a with ethyl isothiocyanate; mp
189–191 °C. IR; 3356, 3262 (N–H), 1684 (C@O, pyridazinone), 1670
(C@O, hydrazide), 1204 (C@S). 1H NMR (DMSO-d6, 400 MHz); d 1.11
(3H, t, –CH2CH3, J: 6.8 Hz), 2.54 (3H, s, –CH3), 3.46–3.49 (2H, m, –
CH2CH3), 4.24 (2H, s, –CH2–C6H5), 4.91 (2H, s, –CH2–CONH–), 7.23–
7.37 (5H, m, –CH2–C6H5), 7.87 (1H, t, –NH–CH2CH3, J: 5.2 Hz), 9.38
(1H, br, –NH–NH–CS–), 10.19 (1H, br, –CO–NH–NH–). ESI-MS (m/z);
423 [M+Na]+ (100%), 401 [M+H]+. Anal. calcd for C18H20N6O3S: C,
53.99; H, 5.03; N, 20.99; S, 8.01. Found: C, 53.76; H, 4.73; N, 20.74; S,
7.78.
4.1.1.2. Ethyl 2-(7-(4-methoxybenzyl)-3-methyl-4-oxoisox-azol-
o[4,5-d]pyridazin-5(4H)-yl)acetate (2c).
93% yield; mp 102–
104 °C. IR; 1742 (C@O, ester), 1689 (C@O, pyridazinone).1H NMR
(DMSO-d6); d 1.20 (3H, t, –CH2–CH3, J: 6.8 Hz), 2.51 (3H, s, –CH3),
3.68 (3H, s, –OCH3), 4.13–4.15 (4H, m, –CH2–CH3 and –CH2–
C6H5), 4.94 (2H, s, –CH2COO–), 6.85 (2H, d, phenyl-H3 and H5, J:
8.4 Hz), 7.19(2H, d, phenyl-H2 and H6, J: 8.4 Hz). ESI-MS (m/z);
380 [M+Na]+ (100%), 358 [M+H]+. Anal. calcd for C18H19N3O5: C,
60.50; H, 5.36; N, 11.76. Found: C, 60.39; H, 5.28; N, 11.75.
4.1.2. General procedure for the preparation of 2-(7-benzyl-3-
methyl-4-oxoisoxazolo[4,5-d]pyridazin-5(4H)-yl)acetohydraz
ides (3b–c)
A solution of hydrazine hydrate (0.2 mol) and the correspond-
ing ethyl 2-(7-benzyl-3-methyl-4-oxoisoxazolo[4,5-d]pyridazin-
5(4H)-yl)acetate (2a–c, 0.01 mol) in absolute ethanol (50 mL) was
refluxed for 1 h with vigorous stirring. The solvent was evaporated
under vacuum and the residue was purified by recrystallization
from ethanol.
4.1.3.3. 1-(2-(7-Benzyl-3-methyl-4-oxoisoxazolo[4,5-d]pyridazin-
5(4H)-yl)acetyl)-4-phenylthiosemicarbazide (6)
.
Obtained
as a white solid (70% yield) by reacting 3a with phenyl isothiocya-
nate; mp 186–188 °C. IR; 3496, 3173 (N–H), 1714 (C@O, pyridazi-
none), 1659 (C@O, hydrazide), 1205 (C@S). 1H NMR (DMSO-d6); d
2.52 (3H, s, –CH3), 4.24 (2H, s, –CH2–C6H5), 4.97 (2H, s, –CH2–
CONH–), 7.17–7.52 (10H, m, –CH2–C6H5 and –NH–C6H5), 9.53
(1H, br s, –NH–C6H5), 9.83 (1H, br s, –NH–NH–CS–), 10.47 (1H, br
s, –CO–NH –NH–). ESI-MS (m/z); 471 [M+Na]+ (100%), 449
[M+H]+. Anal. calcd for C22H20N6O3S: C, 58.92; H, 4.49; N, 18.74;
S, 7.15. Found: C, 58.41; H, 4.48; N, 18.84; S, 6.95.
4.1.2.1. 2-(7-(4-Nitrobenzyl)-3-methyl-4-oxoisoxazolo[4,5-d]py-
ridazin-5(4H)-yl)acetohydrazide (3b)
.
87% yield; mp 206–
208 °C. IR; 3321 (N–H), 1698 (C@O, pyridazinone), 1655 (C@O,
hydrazide). 1H NMR (DMSO-d6); d 2.54 (3H, s, –CH3), 4.29 (2H, br
s, –NH2), 4.41 (2H, s, –CH2–C6H5), 4.73 (2H, s, –CH2–CONH–),
7.61 (2H, d, phenyl-H2 and H6, J: 8.4 Hz), 8.20 (2H, d, phenyl-H3
and H5, J: 8.4 Hz), 9.25 (1H, br s, –NH–). ESI-MS (m/z); 381
[M+Na]+ (100%), 359 [M+H]+. Anal. calcd for C15H14N6O5: C,
50.28; H, 3.94; N, 23.45. Found: C, 50.42; H, 3.78; N, 23.25.
4.1.3.4. 1-(2-(7-(4-Nitrobenzyl)-3-methyl-4-oxoisoxazolo[4,5-d]pyrida-
zin-5(4H)-yl)acetyl)-4-methylthiosemicarbazide (7).
Obtained as a
white solid (63% yield) by reacting 3b with methyl isothiocyanate,
mp 187–189 °C. IR; 3476, 3143 (N–H), 1680 (C@O, pyridazinone
and hydrazide), 1209 (C@S). 1H NMR (DMSO-d6, 400 MHz); d 2.55
(3H, s, –CH3), 2.89 (3H, d, –NH–CH3, J: 4.4 Hz), 4.23 (2H, s, –CH2–
C6H5), 4.90 (2H, s, –CH2–CONH–), 7.61(2H, d, phenyl-H2 and H6, J:
8.8 Hz), 7.94 (1H, q, –NH–CH3, J: 4.4 Hz), 8.20 (2H, d, phenyl-H3 and
H5, J: 8.8 Hz), 9.40 (1H, br s, –NH–NH-CS–), 10.15 (1H, br s, –CO–
NH–NH–). ESI-MS (m/z); 454 [M+Na]+ (100%), 432 [M+H]+. Anal. calcd
for C17H17N7O5S: C, 47.33; H, 3.97; N, 22.73; S, 7.43. Found: C, 47.40;
H, 4.12; N, 22.51; S, 6.96.
4.1.2.2. 2-(7-(4-Methoxybenzyl)-3-methyl-4-oxoisoxazolo-[4,5-
d]pyridazin-5(4H)-yl)acetohydrazide (3c).
88% yield; mp
190–192 °C. IR; 3282 (N–H), 1694 (C@O, pyridazinone), 1656
(C@O, hydrazide). 1H NMR (DMSO-d6); d 2.50 (3H, s, –CH3), 3.68
(3H, s, –OCH3), 4.11 (2H, s, –CH2–C6H5), 4.26 (2H, br s, –NH2),
4.71 (2H, s, –CH2–CONH–), 6.85 (2H, d, phenyl-H3 and H5, J:
8.4 Hz), 7.20(2H, d, phenyl-H2 and H6, J: 8.4 Hz), 9.24 (1H, br s, –
NH–). ESI-MS (m/z); 366 [M+Na]+ (100%), 345 [M+H]+. Anal. calcd
for C16H17N5O4: C, 55.97; H, 4.99; N, 20.40. Found: C, 55.67; H,
5.02; N, 20.05.
4.1.3.5. 1-(2-(7-(4-Nitrobenzyl)-3-methyl-4-oxoisoxazolo[4,5-d]pyrida-
zin-5(4H)-yl)acetyl)-4-ethylthiosemicarbazide (8)
.
Obtained
as a white solid (89% yield) by reacting 3b with ethyl isothiocya-
nate; mp 203–205 °C. IR; 3256 (N–H), 1681 (C@O, pyridazinone
and hydrazide), 1208 (C@S). 1H NMR1H NMR (DMSO-d6); d 1.08
(3H, t, –CH2CH3, J: 6.8 Hz), 2.53 (3H, s, –CH3), 3.43–3.46 (2H, m, –
CH2CH3), 4.41 (2H, s, –CH2–C6H5), 4.86 (2H, s, –CH2–CONH–),
7.59 (2H, d, phenyl-H2 and H6, J: 8.8 Hz), 7.82 (1H, t, –NH–CH2CH3,
J: 5.2 Hz), 8.18 (2H, d, phenyl-H3 and H5, J: 8.8 Hz), 9.33 (1H, br s, –
NH–NH–CS–), 10.14 (1H, br s, –CO–NH–NH–). ESI-MS (m/z); 468
[M+Na]+ (100%), 446 [M+H]+. Anal. calcd for C18H19N7O5S: C,
48.53; H, 4.30; N, 22.01; S, 7.20. Found: C, 48.45; H, 4.46; N,
21.91; S, 6.94.
4.1.3. General procedure for the preparation of 1-(2-(7-benzyl-
3-methyl-4-oxoisoxazolo[4,5-d]pyridazin-5(4H)-yl)acetyl)-4-
substituted thiosemicarbazides (4–12)
An equimolar amount of the corresponding isothiocyanate was
added to a solution of hydrazide (3a–c) in ethanol (50 mL); this
reaction mixture was refluxed for 4 h and then allowed to reach
room temperature. The precipitated solid was filtered out, washed
with water, and purified by recrystallization from ethyl acetate.
4.1.3.6. 1-(2-(7-(4-Nitrobenzyl)-3-methyl-4-oxoisoxazolo[4,5-d]pyri-
4.1.3.1. 1-(2-(7-Benzyl-3-methyl-4-oxoisoxazolo[4,5-d]pyrida-
zin-5(4H)-yl)acetyl)-4-methylthiosemicarbazide
dazin-5(4H)-yl)acetyl)-4-phenylthiosemicarbazide (9).
Obtained

K. Özadalı et al. / Bioorg. Med. Chem. 20 (2012) 2912–2922
2919
as a white solid (90% yield) by reacting 3b with phenyl isothiocyanate; mp
195–197 °C. IR; 3324 (N–H), 1668 (C@O, pyridazinone and hydrazide),
1208 (C@S). 1H NMR (DMSO-d6); d 2.51 (3H, s, –CH3), 4.41 (2H, s, –CH2–
C6H5), 4.93 (2H, s, –CH2–CONH–), 7.14–8.18 (9H, m, –CH2–C6H4 and –
NH–C6H5), 9.47 (1H, br, –NH–C6H5), 9.75 (1H, br s, –NH–NH–CS–), 10.40
(1H, br, –CO–NH–NH–). ESI-MS (m/z); 516 [M+Na]+ (100%), 494 [M+H]+,
327, 299, 102. Anal. calcd for C22H19N7O5S: C, 53.54; H, 3.88; N, 19.87; S,
6.50. Found: C, 53.39; H, 3.65; N, 19.66; S, 6.33.
as a white solid (72% yield from 5); crystallized from acetone/water;
mp 180–182 °C.IR; 3384 (N–H), 1683 (C@O). 1H NMR (DMSO-d6); d
1.15 (3H, t, –CH2CH3, J: 6.8 Hz), 2.54 (3H, s, –CH3), 3.23–3.27 (2H, m,
–CH2CH3), 4.24 (2H, s, –CH2–C6H5), 5.49 (2H, s, –N–CH2–), 7.24–7.33
(5H, m, –CH2–C6H5), 7.75 (1H, t, –NH–CH2CH3, J: 5.6 Hz). ESI-MS (m/
z); 405 [M+Na]+ (100%), 383 [M+H]+, 325, 233, 102. Anal. calcd for
C18H18N6O2S: C, 56.53; H, 4.74; N, 21.97; S, 8.38. Found: C, 56.65; H,
4.64; N, 21.59; S, 8.32.
4.1.3.7. 1-(2-(7-(4-Methoxybenzyl)-3-methyl-4-oxoisoxazolo-[4, 5-d]pyri-
4.1.4.3. 7-Benzyl-3-methyl-5-((5-(phenylamino)-1,3,4-thiadiazol-2-
dazin-5(4H)-yl)acetyl)-4-methylthiosemicarbazide (10).
Obtained as a
yl)methyl)isoxazolo[4,5-d]pyridazin-4(5H)-one (15).
Obtained
white solid (83% yield) by reacting 3c with methyl isothiocyanate, mp 186–
as a white solid (83% yield from 6); crystallized from acetone; mp
207–210 °C (dec). IR; 3255 (N–H), 1686 (C@O). 1H NMR (DMSO-d6);
d 2.55 (3H, s, –CH3), 4.25 (2H, s, –CH2–C6H5), 5.60 (2H, s, –N–CH2–),
6.99–7.59 (10H, m, –CH2–C6H5 and –NH–C6H5), 10.40 (1H, s, NH–
C6H5). ESI-MS (m/z); 453 [M+Na]+(100%), 431 [M+H]+. Anal. calcd for
C22H18N6O2S: C, 61.38; H, 4.21; N, 19.52; S, 7.45. Found: C, 61.29; H,
4.03; N, 19.41; S, 7.55.
188 °C. IR; 3305, 3195 (N–H), 1685 (C@O, pyridazinone and hydrazide), 1209
1
(C@S). H NMR (DMSO-d6); d 2.51 (3H, s, –CH3), 2.88 (3H, d, –NH–CH3, J:
4.4 Hz), 3.68 (3H, s, –OCH3), 4.13 (2H, s, –CH2–C6H5), 4.88 (2H, s, –CH2–
CONH–), 6.86 (2H, d, phenyl-H3 and H5, J: 8.4 Hz), 7.20 (2H, d, phenyl-H2 and
H6, J: 8.4 Hz), 7.94 (1H, q, –NH–CH3, J: 4.4 Hz), 9.39 (1H, br s, –NH–NH–CS–),
10.15 (1H, br s, –CO–NH –NH–). ESI-MS (m/z); 439 [M+Na]+ (100%), 417
[M+H]+. Anal. calcd for C18H20N6O4S: C, 51.91; H, 4.84; N, 20.18; S, 7.70. Found:
C, 51.69; H, 4.98; N, 19.97; S, 7.69.
4.1.4.4. 7-(4-Nitrobenzyl)-3-methyl-5-((5-(methylamino)-1,3,4-thiadiazol-
2-yl)methyl)isoxazolo[4,5-d]pyridazin-4(5H)-one (16).
Obtained as a
4.1.3.8. 1-(2-(7-(4-Methoxybenzyl)-3-methyl-4-oxoisoxazolo-[4,5-d]pyri-
white solid (75% yield from 7); crystallized from ethanol; mp186–188 °C.
IR; 3216 (N–H), 1682 (C@O). 1H NMR (DMSO-d6); d 2.53 (3H, s, –CH3),
2.81 (3H, d, –NH–CH3, J: 4.8 Hz), 4.40 (2H, s, –CH2–C6H5), 5.44 (2H, s, –N–
CH2–), 7.58(2H, d, phenyl-H2 and H6, J: 8.8 Hz), 7.65 (1H, q, –NH–CH, J:
4.8 Hz), 8.16 (2H, d, phenyl-H3 and H5, J: 8.8 Hz). ESI-MS (m/z); 436
[M+Na]+ (100%), 414 [M+H]+, 325, 233, 149, 102. Anal. Calcd for C17H15N7O4S:
C, 49.39; H, 3.66; N, 23.72; S, 7.76. Found: C, 49.18; H, 3.69; N, 23.19; S, 7.71.
dazin-5(4H)-yl)acetyl)-4-ethylthiosemicarbazide (11).
Obtained as a
white solid (83% yield) by reacting 3c with ethyl isothiocyanate; mp 193–
194 °C. IR; 3364, 3161 (N–H), 1675 (C@O, pyridazinone and hydrazide),
1202 (C@S). 1H NMR (DMSO-d6); d 1.08 (3H, t, –CH2CH3, J: 6.8 Hz), 2.51 (3H,
s, –CH3), 3.43–3.45 (2H, m, –CH2CH3), 3.69 (3H, s, –OCH3), 4.14 (2H, s, –CH2–
C6H5), 4.87 (2H, s, –CH2–CONH–), 6.86 (2H, d, phenyl-H3 and H5, J: 8.8 Hz),
7.20 (2H, d, phenyl-H2 and H6, J: 8.8 Hz), 7.82 (1H, t, –NH–CH2CH3, J: 5.6 Hz),
9.33 (1H, br, –NH–NH–CS–), 10.15 (1H, br, –CO–NH–NH–). ESI-MS (m/z);
453 [M+Na]+ (100%), 431 [M+H]+. Anal. calcd for C19H22N6O4S: C, 53.01; H,
5.15; N, 19.52; S, 7.45. Found: C, 53.36; H, 4.91; N, 19.84; S, 7.18.
4.1.4.5. 7-(4-Nitrobenzyl)-3-methyl-5-((5-(ethylamino)-1,3,4-thiadiazol-
2-yl)methyl)isoxazolo[4,5-d]pyridazin-4(5H)-one (17).
Obtained as
a white solid from (65% yield from 8); crystallized from ethanol; mp
183–185 °C. IR; 3187 (N–H), 1686 (C@O). 1H NMR (DMSO-d6); d 1.12
(3H, t, –CH2CH3), 2.53 (3H, s, –CH3), 3.21 (2H, q, –CH2CH3, J: 6.8 Hz), 4.40
(2H, s, –CH2–C6H5), 5.44 (2H, s, –N–CH2–), 7.58 (2H, d, phenyl-H2 and
H6, J: 8.8 Hz), 7.71 (1H, t, –NH–CH2CH3, J: 5.6 Hz), 8.16 (2H, d, phenyl-H3
and H5, J: 8.8 Hz). ESI-MS (m/z); 450 [M+Na]+ (100%), 428 [M+H]+. Anal.
Calcd for C18H17N7O4S: C, 50.58; H, 4.01; N, 22.94; S, 7.50. Found: C,
50.30; H, 4.05; N, 22.63; S, 7.60.
4.1.3.9. 1-(2-(7-(4-Methoxybenzyl)-3-methyl-4-oxoisoxazolo-[4,5-d]pyri-
dazin-5(4H)-yl)acetyl)-4-phenylthiosemicarbazide (12).
Obtained as
a white solid (83% yield) by reacting 3c with phenyl isothiocyanate; mp
184–185 °C. IR; 3335, 3217 (N–H), 1673 (C@O, pyridazinone and hydrazide),
1204 (C@S). 1H NMR (DMSO-d6); d2.51 (3H, s, –CH3), 3.70 (3H, s, –OCH3), 4.16
(2H, s, –CH2–C6H5), 4.95 (2H, s, –CH2–CONH–), 6.85–7.51 (9H, m, –CH2–C6H4
and –NH–C6H5), 9.51 (1H, br s, –NH–C6H5), 9.80(1H, br s, –NH–NH–CS–), 10.45
(1H, br, –CO–NH–NH–). ESI-MS (m/z); 501 [M+Na]+ (100%), 479 [M+H]+.Anal.
calcd for C23H22N6O4S: C, 57.73; H, 4.63; N, 17.56; S, 6.70. Found: C, 57.69; H,
4.72; N, 17.41; S, 6.47.
4.1.4.6. 7-(4-Nitrobenzyl)-3-methyl-5-((5-(phenylamino)-1,3,4-
thiadiazol-2-yl)methyl)isoxazolo[4,5-d]pyridazin-4(5H)-one
(18).
Obtained as a white solid (90% yield from 9); crystallized
from ethyl acetate/acetone; mp 234–236 °C dec. IR; 3198 (N–H),
1698 (C@O). 1H NMR (DMSO-d6); dd 2.53 (3H, s, –CH3), 4.41 (2H,
s, –CH2–C6H5), 5.55 (2H, s, –N–CH2–), 6.96–8.16 (9H, m, –CH2–
C6H4 and –NH–C6H5), 10.33 (1H, s, NH–C6H5). ESI-MS (m/z); 498
[M+Na]+(100%), 476 [M+H]+. Anal. Calcd for C22H17N7O4S: C,
55.57; H, 3.60; N, 20.62; S, 6.74. Found: C, 55.66; H, 3.78; N,
20.48; S, 6.81.
4.1.4. General procedure for the preparation of 7-benzyl-3-
methyl-5-((5-(substituted amino)-1,3,4-thiadiazol-2-yl)-
methyl)isoxazolo[4,5-d]pyridazin-4(5H)-ones (13–21)
Concentrated sulfuric acid (1 mL) was added dropwise to the
corresponding thiosemicarbazide (4–12, 1 mmol), stirring at room
temperature for 1 hour. The reaction mixture was poured into a
beaker containing ice (50 mL), and the precipitated solid was fil-
tered out, washed with water, and purified by recrystallization.
4.1.4.7.
1,3,4-thiadiazol-2-yl)methyl)isoxazolo[4,5-d]pyridazin-4(5H)-
one (19). Obtained as a white solid (40% yield from 10); crys-
7-(4-Methoxybenzyl)-3-methyl-5-((5-(methylamino)-
4.1.4.1. 7-Benzyl-3-methyl-5-((5-(methylamino)-1,3,4-thiadiazol-2-
tallized from acetone/water; mp 186–188 °C. IR; 3220 (N–H), 1688
(C@O). 1H NMR (DMSO-d6, 400); d 2.51 (3H, s, –CH3), 2.81 (3H, d, –
NH–CH3, J: 4.8 Hz), 3.68 (3H, s, –OCH3), 4.13 (2H, s, –CH2–C6H5),
5.45 (2H, s, –N–CH2–), 6.85 (2H, d, phenyl-H3 and H5, J: 8.8 Hz),
7.20 (2H, d, phenyl-H2 and H6, J: 8.8 Hz), 7.65 (1H, q, –NH–CH3, J:
4.8 Hz). ESI-MS (m/z); 421 [M+Na]+ (100%), 399 [M+H]+. Anal. Calcd
for C18H18N6O3S: C, 54.26; H, 4.55; N, 21.09; S, 8.05. Found: C,
54.13; H, 4.59; N, 20.82; S, 8.18.
yl)methyl)isoxazolo[4,5-d]pyridazin-4(5H)-one (13).
Obtained
as a white solid (63% yield, from compound 4); crystallized from ace-
tone/water; mp 152–154 °C. IR; 3230 (N–H), 1686 (C@O). 1H NMR
(DMSO-d6); d 2.54 (3H, s, –CH3), 2.84 (3H, d, –NH–CH3, J: 4.4 Hz), 4.22
(2H, s, –CH2–C6H5), 5.50 (2H, s, –N–CH2–), 7.24–7.33 (5H, m, –CH2–
C6H5), 7.69 (1H, q, –NH–CH3, J: 4.4 Hz). ESI-MS (m/z); 391 [M+Na]+
(100%), 369 [M+H]+. Anal. calcd for C17H16N6O2S: C, 55.42; H, 4.38; N,
22.81; S, 8.70. Found: C, 55.67; H, 4.37; N, 22.28; S, 8.68.
4.1.4.8. 7-(4-Methoxybenzyl)-3-methyl-5-((5-(ethylamino)-1,3,4-thiadiazol-
4.1.4.2. 7-Benzyl-3-methyl-5-((5-(ethylamino)-1,3,4-thiadiazol-2-
2-yl)methyl)isoxazolo[4,5-d]pyridazin-4(5H)-one (20).
Obtained as a
yl)methyl)isoxazolo[4,5-d]pyridazin-4(5H)-one (14).
Obtained
white solid (35% yield from acetone/water); mp 183–185 °C. IR; 3206 (N–H),

2920
K. Özadalı et al. / Bioorg. Med. Chem. 20 (2012) 2912–2922
1687 (C@O). 1H NMR (DMSO-d6); d 1.12 (3H, t, –CH2CH3, J: 6.8 Hz), 2.51 (3H, s,
–CH3), 3.22 (2H, m, –CH2CH3), 3.68 (3H, s, –OCH3), 4.13 (2H, s, –CH2–C6H5), 5.45
(2H, s, –N–CH2–), 6.85 (2H, d, phenyl-H3 and H5, J: 8.8 Hz), 7.20(2H, d, phenyl-
H2 and H6, J: 8.8 Hz), 7.71 (1H, t, –NH–CH2CH3, J: 5.2 Hz). ESI-MS (m/z); 435
[M+Na]+(100%), 413 [M+H]+. Anal. Calcd for C19H20N6O3S: C, 55.33; H, 4.89;
N, 20.38; S, 7.77. Found: C, 54.85; H, 4.86; N, 19.82; S, 7.64.
(N–H), 1685 (C@O), 1330 (C@S). 1H NMR (DMSO-d6); d 2.56 (3H, s,
–CH3), 3.38 (3H, s, –N–CH3), 4.40 (2H, s, –CH2–C6H5), 5.42 (2H, s, –
N–CH2–), 7.55(2H, d, phenyl-H2 and H6, J: 8.8 Hz), 8.19 (2H, d, phe-
nyl-H3 and H5, J: 8.8 Hz), 13.71 (1H, br s, –NH). ESI-MS (m/z); 436
[M+Na]+ (100%), 414 [M+H]+. Anal. Calcd for C17H15N7O4S: C, 49.39;
H, 3.66; N, 23.72; S, 7.76. Found: C, 49.50; H, 4.03; N, 23.33; S, 7.43.
4.1.4.9.
7-(4-Methoxybenzyl)-3-methyl-5-((5-(phenylamino)-1,3,4-thia-
Obtained
4.1.5.5. 7-(4-Nitrobenzyl)-5-((4-ethyl-5-thioxo-4,5-dihydro-1H-
1,2,4-triazol-3-yl)methyl)-3-methylisoxazolo[4,5-d]pyridazin-
diazol-2-yl)methyl)isoxazolo[4,5-d]pyridazin-4(5H)-one (21).
as a white solid (90% yield from 12); crystallized from acetone/water; mp
172–174 °C (dec). IR; 3203 (N–H), 1692 (C@O). 1H NMR (DMSO-d6,
400 MHz); d 2.52 (3H, s, –CH3), 3.67 (3H, s, –OCH3), 4.14 (2H, s, –CH2–C6H5),
5.56 (2H, s, –N–CH2–), 6.83–7.56 (9H, m, –CH2–C6H4 and –NH–C6H5), 10.34
(1H, s, NH–C6H5). ESI-MS (m/z); 483 [M+Na]+ (100%), 461 [M+H]+. Anal. Calcd
for C23H20N6O3S: C, 59.99; H, 4.38; N, 18.25; S, 6.96. Found: C, 59.74; H, 4.34;
N, 18.02; S, 7.02.
4(5H)-one (26).
Obtained as a white solid (89% yield from
8); crystallized from ethanol/acetone; mp 180–182 °C (dec). IR;
3359 (N–H), 1686 (C@O), 1320 (C@S). 1H NMR (DMSO-d6); d
1.11 (3H, t, –CH2CH3, J: 7.2 Hz), 2.56 (3H, s, –CH3), 3.97 (2H, q,
–CH2CH3, J: 7.2 Hz), 4.42 (2H, s, –CH2–C6H5), 5.45 (2H, s, –N–
CH2–), 7.56(2H, d, phenyl-H2 and H6, J: 8.8 Hz), 8.20 (2H, d, phe-
nyl-H3 and H5, J: 8.8 Hz), 13.74 (1H, br s, –NH). ESI-MS (m/z); 450
[M+Na]+ (100%), 428 [M+H]+. Anal. Calcd for C18H17N7O4S: C,
50.58; H, 4.01; N, 22.94; S, 7.50. Found: C, 50.74; H, 3.71; N,
22.06; S, 7.18.
4.1.5. General procedure for the preparation of 7-benzyl-5-((4-
substituted-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)methyl)
-3-methylisoxazolo[4,5-d]pyridazin-4(5H)-ones (22–30)
12.5 mL of 1 M aqueous sodium bicarbonate solution was
added to a solution of appropriately substituted thiosemicarba-
zide (4–12, 0.001 mol) in ethanol (7.5 mL); this mixture was stir-
red at room temperature for about 1 h and then poured into a
beaker containing ice/water (100 mL). The solution was adjusted
to pH 5–6 with 1 M HCl. The precipitated solid was filtered out,
rinsed with water and purified by recrystallization from an appro-
priate solvent.
4.1.5.6. 7-(4-Nitrobenzyl)-5-((4-phenyl-5-thioxo-4,5-dihydro-
1H-1,2,4-triazol-3-yl)methyl)-3-methylisoxazolo[4,5-d]pyrida-
zin-4(5H)-one (27).
9); crystallized from ethanol/acetone; mp 202–204 °C (dec). IR;
3357 (N–H), 1698 (C@O), 1333 (C@S). 1H NMR (DMSO-d6); d 2.45
(3H, s, –CH3), 4.33 (2H, s, –CH2–C6H5), 5.28 (2H, s, –N–CH2–),
7.20–8.21 (9H, m, –CH2–C6H4 and –NH–C6H5), 13.95 (1H, br s, –
NH). ESI-MS (m/z); 498 [M+Na]+ (100%), 476 [M+H]+. Anal. Calcd
for C22H17N7O4S: C, 55.57; H, 3.60; N, 20.62; S, 6.74. Found: C,
55.54; H, 3.75; N, 20.85; S, 6.68.
Obtained as a white solid (84% yield from
4.1.5.1. 7-Benzyl-5-((4-methyl-5-thioxo-4,5-dihydro-1H-1,2,4-tria-
zol-3-yl)methyl)-3-methylisoxazolo[4,5-d]pyridazin-4(5H)-one
4.1.5.7. 7-(4-Methoxybenzyl)-5-((4-methyl-5-thioxo-4,5-dihy-
dro-1H-1,2,4-triazol-3-yl)methyl)-3-methylisoxazolo[4,5-
d]pyridazin-4(5H)-one (28).
(22).
Obtained as a white solid (77% yield from 4); crystallized
from ethanol; mp 161–163 °C. IR; 3343 (N–H), 1680 (C@O), 1332
(C@S). 1H NMR (DMSO-d6); d 2.54 (3H, s, –CH3), 3.43 (3H, s, –N–
CH3), 4.21 (2H, s, –CH2–C6H5), 5.45 (2H, s, –N–CH2–), 7.24–7.32
(5H, m, –CH2–C6H5), 13.71 (1H, br s, –NH). ESI-MS (m/z); 391
[M+Na]+ (100%), 369 [M+H]+. Anal. Calcd for C17H16N6O2S: C, 55.42;
H, 4.38; N, 22.81; S, 8.70. Found: C, 55.48; H, 4.24; N, 22.65; S, 8.77.
Obtained as a white solid (91%
yield from 10); crystallized from ethanol; mp 195–197 °C. IR;
3359 (N–H), 1684 (C@O), 1320 (C@S). 1H NMR (DMSO-d6); d 2.54
(3H, s, –CH3), 3.44 (3H, s, –N–CH3), 3.72 (3H, s, –OCH3), 4.13 (2H,
s, –CH2–C6H5), 5.45 (2H, s, –N–CH2–), 6.88(2H, d, phenyl-H3 and
H5, J: 8.8 Hz), 7.18 (2H, d, phenyl-H2 and H6, J: 8.8 Hz), 13.71
(1H, br, –NH). ESI-MS (m/z); 421 [M+Na]+(100%), 399 [M+H]+. Anal.
Calcd for C18H18N6O3S: C, 54.26; H, 4.55; N, 21.09; S, 8.05. Found:
C, 53.89; H, 4.33; N, 20.74; S, 7.93.
4.1.5.2. 7-Benzyl-5-((4-ethyl-5-thioxo-4,5-dihydro-1H-1,2,4-tria-
zol-3-yl)methyl)-3-methylisoxazolo[4,5-d]pyridazin-4(5H)-one
(23).
Obtained as a white solid (85% yield from 5); crystallized
from ethanol; mp 172–174 °C. IR; 3365 (N–H), 1688 (C@O), 1350
(C@S). 1H NMR (DMSO-d6); d 1.12 (3H, t, –CH2CH3, J: 7.2 Hz), 2.54
(3H, s, –CH3), 4.01 (2H, q, –CH2CH3, J: 7.2 Hz), 4.22 (2H, s, –CH2–
C6H5), 5.48 (2H, s, –N–CH2–), 7.27–7.32 (5H, m, –CH2–C6H5), 13.74
(1H, br s, –NH). ESI-MS (m/z); 405 [M+Na]+ (100%), 383 [M+H]+. Anal.
Calcd for C18H18N6O2S: C, 56.53; H, 4.74; N, 21.97; S, 8.38. Found: C,
56.12; H, 4.96; N, 21.01; S, 8.07.
4.1.5.8. 7-(4-Methoxybenzyl)-5-((4-ethyl-5-thioxo-4,5-dihydro-
1H-1,2,4-triazol-3-yl)methyl)-3-methylisoxazolo[4,5-d]pyrida-
zin-4(5H)-one (29).
Obtained as a white solid (82% yield
from 11); crystallized from ethanol; mp 190–192 °C. IR; 3365
(N–H), 1684 (C@O), 1326 (C@S). 1H NMR (DMSO-d6); d 1.12
(3H, t, –CH2CH3, J: 7.2 Hz), 2.54 (3H, s, –CH3), 3.72 (3H, s, –
OCH3), 4.01 (2H, q, –CH2CH3, J: 7.2 Hz), 4.14 (2H, s, –CH2–C6H5),
5.47 (2H, s, –N–CH2–), 6.87 (2H, d, phenyl-H3 and H5, J: 8.8 Hz),
7.19(2H, d, phenyl-H2 and H6, J: 8.8 Hz), 13.72 (1H, br s, –NH).
ESI-MS (m/z); 435 [M+Na]+ (100%), 413 [M+H]+. Anal. Calcd for
4.1.5.3. 7-Benzyl-5-((4-phenyl-5-thioxo-4,5-dihydro-1H-1,2,4-tria-
zol-3-yl)methyl)-3-methylisoxazolo[4,5-d]pyridazin-4(5H)-one
(24).
Obtained as a white solid (95% yield from 6); crystallized
C19H20N6O3S: C, 55.33; H, 4.89; N, 20.38; S, 7.77. Found: C,
from ethanol; mp 240–242 °C (dec). IR; 3361 (N–H), 1691 (C@O),
1307 (C@S). 1H NMR (DMSO-d6); d 2.42 (3H, s, –CH3), 4.10 (2H,
s, –CH2–C6H5), 5.29 (2H, s, –N–CH2–), 7.17–7.36 (10H, m, –CH2–
C6H5 and –NH–C6H5), 13.93 (1H, br s, –NH). ESI-MS (m/z); 453
[M+Na]+ (100%), 431 [M+H]+. Anal. Calcd for C22H18N6O2S: C,
61.38; H, 4.21; N, 19.52; S, 7.45. Found: C, 61.55; H, 3.92; N,
19.43; S, 7.23.
55.16; H, 4.75; N, 20.15; S, 7.72.
4.1.5.9. 7-(4-Methoxybenzyl)-5-((4-phenyl-5-thioxo-4,5-dihydro-
1H-1,2,4-triazol-3-yl)methyl)-3-methylisoxazolo[4,5-d]pyridazin-
4(5H)-one (30).
Obtained as a white solid (86% yield from 12);
crystallized from ethanol); mp 174–176 °C. IR; 3360 (N–H), 1689
(C@O), 1304 (C@S). 1H NMR (DMSO-d6); d 2.43 (3H, s, –CH3), 3.72
(3H, s, –OCH3), 4.04 (2H, s, –CH2–C6H5), 5.30 (2H, s, –N–CH2–),
6.87–7.35 (9H, m, –CH2–C6H4 and –NH–C6H5), 10.94 (1H, br s, –
NH). ESI-MS (m/z); 483 [M+Na]+ (100%), 461 [M+H]+. Anal. Calcd for
4.1.5.4. 7-(4-Nitrobenzyl)-5-((4-methyl-5-thioxo-4,5-dihydro-1H-
1,2,4-triazol-3-yl)methyl)-3-methylisoxazolo[4,5-d]pyridazin-
4(5H)-one (25).
Obtained as a white solid (75% yield from 7);
C23H20N6O3S: C, 59.99; H, 4.38; N, 18.25; S, 6.96. Found: C, 59.66;
crystallized from ethanol/acetone; mp 209–211 °C (dec). IR; 3360
H, 4.44; N, 18.07; S, 6.78.

K. Özadalı et al. / Bioorg. Med. Chem. 20 (2012) 2912–2922
2921
4.2. Biological assays
the receptor–ligand Interactions protocol of Discovery Studio after
defining subsets of the enzyme within 10 Å sphere radius of the li-
gand. The force field, Chemistry at HARvard Macromolecular
Mechanics (CHARMM) was employed for all docking purposes.
The ligandÀenzyme assembly was then subjected to a molecular
dynamics (MD) simulation using Simulation protocol at a constant
temperature of 300 K with a 100 step equilibration for over 1000
iterations and a time step of 1 fs using a distance dependent dielec-
tric constant 4r. The optimal binding orientation of the li-
gandÀenzyme assembly obtained after docking was further
minimized for 1000 iterations using the conjugate gradient method
until a convergence of 0.001 kcal/mol Å was reached after which
Eintermolecular (kcal/mol) of the ligand–enzyme assembly was
evaluated.
4.2.1. Cyclooxygenase inhibition assay (in vitro)
The synthesized compounds were evaluated for their ability to
inhibit human recombinant COX-2 and ovine COX-1 using a fluo-
rescent inhibitor screening assay in vitro (catalog number
700100, Cayman Chemical, Ann Arbor, MI, USA) following the pro-
cedure suggested by the manufacturer (excitation wave-
length = 530–540 nm, emission wavelength of 585–595 nm).
DuP-697 (selective COX-2 inhibitor) and SC-560 (selective COX-1
inhibitor) were used as reference compounds.
4.2.2. 5-lipoxygenase inhibition assay
The ability of the test compounds (listed in Table 2) to inhibit
potato 5-LOX (catalog number 60401, Cayman Chemical, Ann Ar-
bor, MI, USA) (IC50 values,
l
M) was determined using an enzyme
4.4. Statistical analysis
immuno assay kit (catalog number 760700, Cayman Chemical)
according to the experimental procedure described previously.48
Data for the ulcer index assay are presented as the mean SEM,
with sample sizes of 4 rats/group. Comparisons between groups
were performed using one-way analysis of variance (ANOVA) fol-
lowed by pairwise multiple comparison procedures (Holm–Sidak
method).
4.2.3. Anti-inflammatory assay
The test compounds, as well as the reference drug ibuprofen,
were evaluated using the in vivo carrageenan-induced rat foot
paw edema model reported previously.39 All compounds were
administered orally (30 mg/kg) suspended in methylcellulose (1%
aqueous solution) as vehicle. This assay was carried out using pro-
tocols approved by the Health Sciences Animal Welfare Committee
at the University of Alberta.
Acknowledgments
The authors gratefully acknowledge the financial support pro-
vided by the Scientific Research Fund of Hacettepe University
through Project 0801301003, the Faculty of Pharmacy and Pharma-
ceutical Sciences at the University of Alberta, and the School of
Pharmacy at the University of Waterloo for providing start up
funds. The research work carried out by Keriman Özadalı at the
University of Alberta was partially supported by TUBITAK (The
Scientific and Technological Research Council of Turkey).
4.2.4. Ulcer index assay
Ulcerogenic activity was evaluated after oral administration of
test compounds (0.14 mmol/kg) or ibuprofen (0.14 mmol/kg). All
drugs were suspended and administered in 1.0 mL of a 1% methyl-
cellulose solution. Control rats received oral administration of
vehicle (1.0 mL of 1% methylcellulose solution). Food, but not
water, was removed 24 h before administration of test compounds.
Six hours after oral administration of the drug, rats were eutha-
nized in a CO2 chamber and their stomachs were removed, cut
out along the greater curvature of the stomach, gently rinsed with
water and placed on ice. The number and the length of ulcers ob-
served in each stomach were determined using magnifier lenses.
The severity of each gastric lesion was measured along its greatest
length (1 mm = rating of 1, 1–2 mm = rating of 2, >2 mm = rating
according to their length in mm). The ‘ulcer index’ (UI) for each test
compound was calculated by adding the total length (L, in mm) of
individual ulcers in each stomach and averaging over the number
of animals in each group (n = 4): UI = (L1+L2+L3+L4)/4.
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