Selective COX-1 inhibition as a target of theranostic novel diarylisoxazoles

Article abstract of DOI:10.1016/j.ejmech.2013.12.023

Cyclooxygenase(COX)-1 role in some diseases is increasingly studied. 3-(5-Chlorofuran-2-yl)-5-methyl-4-phenylisoxazole (P6), a highly selective cyclooxygenase-1 inhibitor, was used as a lead to design new isoxazoles (2a-m), differently selective towards COX-1. Those isoxazoles might be useful as novel theranostic agents and also to better clarify COX-1 role in the human physiology and diseases. 2a-m were prepared in fair to good yields developing suitable synthetic strategies. They were evaluated in vitro for their COX-inhibitory activity and selectivity. Structure-activity relationship studies of the novel set of diarylisoxazoles allowed to identify new key determinants for COX-1 selectivity, and to uncover compounds appropriate for a deep pharmacokinetic and pharmacodynamic investigation. 3-(5-Chlorofuran-2yl)-4- phenylisoxazol-5-amine (2f) was the most active compound of the series, its inhibitory activity was assessed in purified enzyme (COX-1 IC₅₀ = 1.1 μM; COX-2 IC₅₀ > 50 μM) and in the ovarian cancer cell line (OVCAR-3) expressing only COX-1 (IC₅₀ = 0.58 μM). Furthermore, the high inhibitory potency of 2f was rationalized through docking simulations in terms of interactions with a crystallographic model of the COX-1 binding site. We found critical interactions between the inhibitor and constriction residues R120 and Y355 at the base of the active site, as well as with S530 at the top of the side pocket.

Full text of DOI:10.1016/j.ejmech.2013.12.023

European Journal of Medicinal Chemistry 74 (2014) 606e618
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Selective COX-1 inhibition as a target of theranostic novel
diarylisoxazoles
,
,
,
**
Paola Vitale a 1, Maria Grazia Perrone a 1, Paola Malerba a, Antonio Lavecchia b
,
Antonio Scilimati a
,
*
a Department of Pharmacy & Pharmaceutical Sciences, University of Bari “A. Moro”, Via Orabona 4, 70125 Bari, Italy
b Department of Pharmacy, “Drug Discovery” Laboratory, University of Naples Federico II, Via D. Montesano 49, 80131 Naples, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Cyclooxygenase(COX)-1 role in some diseases is increasingly studied. 3-(5-Chlorofuran-2-yl)-5-methyl-
4-phenylisoxazole (P6), a highly selective cyclooxygenase-1 inhibitor, was used as a “lead” to design new
isoxazoles (2aem), differently selective towards COX-1. Those isoxazoles might be useful as novel
theranostic agents and also to better clarify COX-1 role in the human physiology and diseases. 2aem
were prepared in fair to good yields developing suitable synthetic strategies. They were evaluated in vitro
for their COX-inhibitory activity and selectivity. Structureeactivity relationship studies of the novel set of
diarylisoxazoles allowed to identify new key determinants for COX-1 selectivity, and to uncover com-
pounds appropriate for a deep pharmacokinetic and pharmacodynamic investigation. 3-(5-Chlorofuran-
2yl)-4-phenylisoxazol-5-amine (2f) was the most active compound of the series, its inhibitory activity
Received 30 July 2013
Received in revised form
5 December 2013
Accepted 19 December 2013
Available online 2 January 2014
Keywords:
Cyclooxygenase(COX)-1 inhibitors
Diarylisoxazole scaffold
Docking
Diagnostic probes
OVCAR-3 cell line
Ovarian cancer
was assessed in purified enzyme (COX-1 IC50 ¼ 1.1
m
M; COX-2 IC50 > 50
mM) and in the ovarian cancer
cell line (OVCAR-3) expressing only COX-1 (IC50 ¼ 0.58
m
M). Furthermore, the high inhibitory potency of
2f was rationalized through docking simulations in terms of interactions with a crystallographic model of
the COX-1 binding site. We found critical interactions between the inhibitor and constriction residues
R120 and Y355 at the base of the active site, as well as with S530 at the top of the side pocket.
Ó 2014 Published by Elsevier Masson SAS.
1. Introduction
former was enlightened by the use of aspirin, which is a preferential
platelet COX-1 inhibitor when given at low-doses [4], whereas the
Cyclooxygenase (COX) is the target of nonsteroidal anti-
inflammatory drugs (NSAIDs) [1]. Two COX isoforms (COX-1 and
COX-2) are known as a product of different genes [2]. COX-1 is
considered a “housekeeping gene” by virtue of constitutive low-
levels of expression in most cell types. However, high levels of
constitutive COX-1 expression have been detected in the stomach
and platelets, where it catalyzes the arachidonic acid (AA) conversion
into prostaglandins capable to protect the gastrointestinal tract and
to modulate platelet function. COX-2, even if promotes the same AA
biseoxygenation reaction, is mainly involved in inflammation and
pain. The use of coxibs in humans allowed to unravel the protective
role of COX-2 in the cardiovascular system mostly through the
generation of prostacyclin [3]. COX-1 role in humans is quite vague
except for platelet activation and gastrointestinal protection. The
latter was demonstrated indirectly using coxibs which spare COX-1
[5,6]. The use of COX-1 knockout mice enlightened the potential
pathogenic contribution of prostanoids synthesized via COX-1 to
inflammatory arthritis [7], atherogenesis [8], intestinal polyposis [9],
carcinogenesis [10], neuroinflammation [11] and gastrointestinal
bleeding and ulceration [12]. In addition, although COX-2 is over-
expressed in most of cancer types, in human ovarian cancer the
major expressed isoform is COX-1 where it promotes angiogenic
growth factor production [13], as well as it is responsible of pain
processing and sensitization in spinal cord and gracile nucleus after
surgery [14]. Recently, a marked involvement of COX-1 in the very
early stages of neurodegenerative diseases, with a marked inflam-
matory component, has also been demonstrated [15,16].
Thus, the availability of highly specific COX-1 inhibitors which
better mirror pharmacological treatment in humans will be of valu-
ablehelptoprovetheroleofCOX-1inhumanhealthanddiseases[12].
Together with the classical NSAIDs aspirin and indomethacin,
few examples of selective COX-1 inhibitors (mofezolac, SC-560,
FR122047, P6, ABEX-3TF) have been reported so far as potential
new analgesic and antiproliferative agents (Fig. 1) [17e23].
* Corresponding author. Tel.: þ39 080 5442753; fax: þ39 080 5442427.
** Corresponding author. Tel.: þ39 081 678613; fax: þ39 081 678012.
E-mail addresses: lavecchi@unina.it (A. Lavecchia), antonio.scilimati@uniba.it
(A. Scilimati).
1
These authors contributed equally to the work.
0223-5234/$ e see front matter Ó 2014 Published by Elsevier Masson SAS.
http://dx.doi.org/10.1016/j.ejmech.2013.12.023

P. Vitale et al. / European Journal of Medicinal Chemistry 74 (2014) 606e618
607
Fig. 1. Selective COX-1 inhibitors.
In one of our previous studies, we reported the 3-(5-
chlorofuran-2-yl)-5-methyl-4-phenylisoxazole (P6, Fig. 1) as a
highly selective COX-1 inhibitor [24,25], which allowed us to clarify
and confirm that within the 3,4-diarylisoxazole class the p-sulfa-
moylphenyl group is essential for good COX-2 inhibitory potency,
and the lack of the sulfonamide moiety reverse the COX-2 selec-
tivity in favor of COX-1. Based on these findings, a novel set of se-
lective COX-1 inhibitors has been synthesized, in order to recognize
the molecular determinants necessary to have a selective diary-
lisoxazole COX-1 inhibitor, as well as to develop new compounds as
new antiplatelet agents, analgesics, anti-inflammatories and che-
mopreventive drugs [26]. Moreover, structureeactivity relationship
studies of a series of diarylpyrazoles analogs of P6 allowed us also
to clarify that the isoxazole core ring and furyl group are crucial
determinants for high COX-1 selectivity [27].
Herein, we report the synthesis of a set of novel P6 analogs,
designed collectively considering the known chemical and struc-
tural features to assure good COX-1 selectivity. New moieties are
also explored (i.e., amino- and amido-group) aimed at developing
both new pharmacological [26] and diagnostic tools [28]. The
newly synthesized target compounds were evaluated for in vitro
COX-1 and COX-2 inhibitory activity and selectivity. Furthermore,
the affinity data of the most active inhibitors were rationalized
through enzyme docking simulations in terms of interactions with
a crystallographic model of the COX-1 binding site.
domain of PGHS. The crystal structure of ovine PGHS complexed
with AA shows that, in the cyclooxygenase active site nineteen
aminoacidic residues determine fifty contacts with the substrate:
among them two are hydrophilic and forty-eight establish hydro-
phobic interactions [29].
This is the reason for which at least three different chemical
classes of COXs inhibitors (carboxylic acids, diarylheterocycles,
phenazones) exist. Coxibs development solved in part the selec-
tivity problem in favor of COX-2. Their identification was triggered
as an attempt to reduce or null the GI side effect associated to the
poor COXs selectivity of most of tNSAIDs [12].
During the last two decades, as above-mentioned, few efforts
were devoted to develop COX-1 inhibitors due to the scarce
knowledge of its tissue/cell distribution and role in some human
diseases onset and progression. On the contrary, a lot of work was
performed to identify a large number of COX-2 inhibitors belonging
to the chemical class of diarylheterocycles. The major difference
between compounds of this class is the nature of the central ring.
Thiazole, isoxazole, thiophene, cyclopentene structures and many
others were proposed.
Recently, we studied the effect of the substitution of the het-
erocycle core ring on the COX-1 inhibition by maintaining some P6
moieties and replacing the isoxazole central ring with pyrazole or
thiazole [27].
2. Result and discussion
2.1. Design of new 3,4-diarylisoxazoles
CH3
N
N
Our discovery of P6 [3-(5-chlorofuran-2-yl)-5-methyl-4-
CH3
O
O
phenylisoxazole],
a highly selective COX-1 inhibitor [24,25],
prompted us to attempt more specific structural modifications
without changing the isoxazole core ring, with the aim to investi-
gate by SAR studies, how chemical moieties replacement could
affect COX-1 selective inhibition.
The selectivity towards COXs is still an open target. The cyclo-
oxygenase active site is a long hydrophobic channel that protrudes
from the membrane binding domain into the core of the globular
P9
P10
COX-1 IC50 = 0.09 μM
COX-2 IC50 = 2.49μM
COX-1 IC50 = 0.05 μM
COX-2 IC50 = 1.49 μM
Fig. 2. 3,4-Diphenyl-5-ethylisoxazole (P9) and 3,4-diphenyl-5-methyl-isoxazole (P10)
analogs of P6.

608
P. Vitale et al. / European Journal of Medicinal Chemistry 74 (2014) 606e618
Structural features of P6 (Fig. 1) and its analogs 3,4-diphenyl-5-
at COX-1 active site. Furthermore, 2f, 2g, 2h and 2l were prepared
to verify the importance of acidity or basicity of the substituent at
C5-isoxazole ring position for COX-inhibitory activity and -selec-
tivity. 2e and 2m were prepared to study the influence of modifi-
cation of the substituent on the phenyl at isoxazole-C4 on COX-1
activity.
ethylisoxazole (P9, Fig. 2), 4-diphenyl-5-methyl-isoxazole (P10,
Fig. 2) [24,25] were used to project new COX-1 inhibitors, taking
into account that in the diarylheterocycles the size of the diaryls is
one of the COX selectivity determinants (compare P6, P9 and P10
IC50 values; Fig. 2 and Table 3).
Diarylisoxazoles 2bel (Table 1) were designed to evaluate the
effect of the C5-substituent modification on the COX-1/COX-2
inhibitory activity, with the aim to identify, through a SAR study,
further steric and electronic requirements for selective interactions
3-(5-Chlorofuran-2-yl)-4-phenylisoxazol-5-amine (2f) was
found to be highly potent and selective COX-1 inhibitor. In contrast,
its derivatives 2g and 2h did not inhibit COX activities. A similar
result was observed when the substituent was a carboxylic group,
Table 1
Molecular recognition in the 3-(5-chlorofuran-2-yl)-5-methyl-4-phenylisoxazole (P6) analogs.

P. Vitale et al. / European Journal of Medicinal Chemistry 74 (2014) 606e618
609
as for 2l, designed as a P6 derivative equivalent to mofezolac (Fig.
1). It was found scarcely active on COXs. To better rationalize
such results, we investigate also the electronic effect of the sub-
stituent on the methylene at C5, by evaluating in vitro COX-1 and
COX-2 inhibitory activity and selectivity of isoxazoles 2c,d and 2iel.
On the other hand, 2a and 2b were prepared to verify the role of
the oxygen on the substituent on isoxazole-C3, with the aim to
identify a functional group alternative to the furan, to be usefully
modified also in the preparation of radiolabelled compounds con-
taining the [11C] as a radionuclide in the place of [12C] in CH3O.
With the same objective we prepared the compound 2e,
designed as analog of P6 with EWG on para-position of C4 aromatic
ring as well as intermediate in the preparation of radiolabelled
compounds.
Then, 3-(5-chlorofuran-2-yl)-4-phenylisoxazol-5-amine (2f)
was used as starting material for the preparation of 2g and 2h, by
alkylation or acetylation reaction, respectively (Scheme 3).
Besides, we prepared 2-[3-(5-chlorofuran-2-yl)-4-phenyli
soxazol-5-yl]acetic acid (2l), starting from 5-(bromomethyl)-3-(5-
chlorofuran-2-yl)-4-phenylisoxazole (2i), obtained from a bromi-
nation of P6 (Scheme 4).
P6 was reacted with NBS/AIBN to afford 2i in 47% yield; it was
transformed by NaCN/(n-Bu)4NHSO4 into 2-[3-(5-chlorofuran-2-
yl)-4-phenylisoxazol-5-yl]acetonitrile 2j, further converted into
methyl 2-[3-(5-chlorofuran-2-yl)-4-phenylisoxazol-5-yl]acetate 2k
in the presence of p-TsOH and MeOH by microwave irradiation.
Finally, the methyl ester 2k was hydrolyzed with KOH/THF to afford
2-[3-(5-chlorofuran-2-yl)-4-phenylisoxazol-5-yl]acetic acid (2l) in
good yield (Scheme 4).
3-(5-Chlorofuran-2-yl)-5-methyl-4-(4-nitrophenyl)isoxazole
(2e) was reduced in the presence of stannous chloride and 37%
2.2. Chemistry
hydrochloric
acid
to
afford
4-[3-(5-chlorofuran-2-yl)-5-
We prepared a number of 3,4-diarylisoxazoles 2aee, by a one-
pot procedure, with fair to good yields, just reacting arylnitrile
oxides with sodium enolates of 3-aryl-2-propanones 1aee
(Scheme 1, Table 2) obtained by reacting the suitable propanone
with NaH at 0 ꢀC [25,30]. As previously found [24,25,31], we
often observed the complete dehydration-aromatization of 5-
hydroxyisoxazolines to the corresponding isoxazoles, and this oc-
curs “spontaneously” in basic conditions, possibly by an E1cB
mechanism [32], due to the presence of NaH in the reaction
mixture.
methylisoxazol-4-yl]benzenamine (2m) (Scheme 5).
2.3. Pharmacology
We have previously found [24,35] that P6 and its analogs P9 and
P10 inhibited platelet COX-1 and monocyte COX-2 activities with
IC50 values (95% confidence interval) equal to 0.5 and >100
mM for
P6, 0.05 and 1.49 M for P9 and 0.09 and 2.49 M for P10,
m
m
respectively, assessed by the in vitro human whole blood assay [36].
Structural features of P6 and its analogs were used to design a new
series of diarylisoxazoles (Table 1) with the aim to identify, through
a SAR study, steric and electronic requirements for selective in-
teractions between COX-1 active site and such compounds.
Initially, all compounds were screened at 50 mM on COX-1 and
COX-2, and their effects on COX inhibition are summarized in
Table 3. 2a, 2c, 2f and 2m showed a reasonable COX-1 inhibitory
activity at the final concentration of 50 mM, compared to the “lead”
Scheme 1. Reagents and conditions: (a) NaH/THF, 1 h, 0 ꢀC; (b) ArCNO/THF, 12 h, r.t.;
(c) sat. aq. NH4Cl.
compound P6, hence their IC50 values were determined (Table 3).
The obtained results showed that the substitution of the furan
ring with a methoxyphenyl group lead to the selective COX-1 in-
hibitor 2a (61% at 50 mM), that similarly to P6 was inactive towards
Table 2
COX-2. When a methoxyphenyl was present on C3 of the isoxazole
ring and a CF3 (2b) replaces a methyl group, a lower inhibition of
COX-1 isoenzyme was observed, but with a little improvement of
COX-2 inhibition (39% of inhibition for COX-1 and 10% for COX-2,
Table 3). The oxygen on the furan ring and/or on methoxyphenyl
Yields of 3,4-diarylisoxazoles 2aee obtained by 1,3-dipolar cycloaddition of aryl-
nitrile oxides to sodium enolates of 3-aryl-2-propanones 1aee and “in situ” sub-
sequent dehydration/aromatization under basic conditions.
Ar
R1
R2
2aef (yield (%))
2-Methoxyphenyl-
2-Methoxyphenyl-
5-Chlorofuran-2-yl-
5-Chlorofuran-2-yl-
5-Chlorofuran-2-yl-
5-Chlorofuran-2-yl-
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
4-NO2C6H4
CH3
CF3
CH3
CH2CH3
CH2CH2CH3
CH3
2a (43)
2b (43)
P6 (60)
2c (46)
2d (21)
2e (24)
Cl
Cl
Cl
O
O
O
Ph
Ph
NH2
Ph
b
a
COCH3
N
N
N
N
N
O
O
O
COCH3
A similar approach was used for the synthesis of the amino-
isoxazole 2f, obtained (40% yield) by using the lithiated phenyl-
acetonitrile as dipolarophile instead of enolates, (Scheme 2)
[33,34].
2h
2f
2g
Scheme 3. Reagents and conditions: (a) paraformaldehyde, AcOH, NaBH3CN, 25 ꢀC; (b)
Ac2O, Py, ambient temperature.
Scheme 2. Reagents and conditions: (a) LDA, ꢁ78 ꢀC, 2 h; (b) arylnitrile oxide; (c) sat. aq. NH4Cl.

610
P. Vitale et al. / European Journal of Medicinal Chemistry 74 (2014) 606e618
Cl
Cl
Cl
O
O
O
a
b
N
N
N
O
CH3
O
CH2Br
O
CH2CN
P6
2j
2i
c
Cl
Cl
d
O
O
N
N
CH2COOH
CH2COOCH3
O
O
2l
2k
Scheme 4. Reagents and conditions: (a) NBS/AIBN, CCl4; (b) NaCN/(n-Bu)4NHSO4; (c) MeOH/p-TsOH (MW); (d) KOH, THF.
seems to have a central role in the interaction with specific COX-1
catalytic site amino acids Trp 387 and Tyr 385 [26].
Although in previous structureeactivity/selectivity relation-
ships studies [24] the p-sulfamoyl moiety on the phenyl at iso-
xazole-C4 was found to be detrimental for COX-1 and not COX-2
inhibitory activity, we designed the compound 2e bearing the
NO2 group endowed with similar electronic effect (-M, -I).
When a CH2CH3 replaces a methyl group as in 2c, the COX-1
inhibitory activity was almost unchanged (COX-1 IC50 ¼ 30
mM)
compared to P6.
A further alkyl chain elongation (one more methylene, 2d in
Table 3) markedly reduced COX-1 inhibitor activity (only 17% at
2e was found to be not active, but it is suitable to be replaced by
19F as “cold” compound or 18F radionuclide, providing a radiotracer
50
mM), whereas a 20% COX-2 inhibition was observed.
Lower COX-1 inhibition was obtained also by linking to the
Table 3
methylene a bromine atom (2i), a cyano (2j), and a carboxy methyl
group (2k). They were found to be completely inactive towards
COX-2 (Table 3). Based on these data, both the group size and
electronic features of the substituent on the isoxazole-C5 seems to
play a crucial role for COX-1 inhibitory activity and selectivity.
A different result was achieved with an acidic substituent, as for
2l, designed combining P6 and mofezolac structural moieties (Fig. 1
and Table 1). It shown a selectivity shifted towards COX-2.
Since such different kind of substituents on C5 of the isoxazole
core ring were definitely detrimental for COX-1 inhibitory activity
and selectivity, we also investigated the presence of nitrogen atom-
containing groups, directly linked to the isoxazole or in any way
present in the molecule. Hence, the amines 2f, 2g, as well as the
amide 2h (Table 3) were prepared. Moreover, the influence on COX-
1 activity of the amine group on phenyl at isoxazole-C4 (2m) was
also studied.
Interestingly, 3-(5-chlorofuran-2-yl)-4-phenylisoxazol-5-amine
(2f) was found to inhibit COX-1 with a potency 20 fold higher
than P6, and this result seems particularly relevant in the exploi-
tation of the structural modifications that can also improve the
pharmacokinetic properties of such compounds (Fig. 3).
Moreover, by looking at the tertiary amine 2g and amide 2h
behavior, the basicity of the nitrogen atom seems to be an important
feature for COX selectivity and also for COX-1 inhibitory potency.
COX inhibitory activity of P6 and its analogs.
Compd Ar
R1
R2
COX-1 %
COX-2 %
inhibitiona inhibitiona
(IC50
61
)
(IC50
n.a.
)
2a
2-Methoxyphenyl- C6H5
CH3
(27
39
mM)
2b
P6
2-Methoxyphenyl- C6H5
5-Chlorofuran-2-yl- C6H5
CF3
CH3
10
10
69
(22
55
(30
m
m
M)
M)
2c
5-Chlorofuran-2-yl- C6H5
5-Chlorofuran-2-yl- C6H5
5-Chlorofuran-2-yl- 4-NO2C6H4 CH3
5-Chlorofuran-2-yl- C6H5
CH2CH3
n.a.
2d
2e
2f
CH2CH2CH3 17
20
n.a
n.a.
n.a.
94
NH2
(1.1
n.a.
mM)
2g
2h
2i
2j
2k
2l
5-Chlorofuran-2-yl- C6H5
5-Chlorofuran-2-yl- C6H5
5-Chlorofuran-2-yl- C6H5
5-Chlorofuran-2-yl- C6H5
5-Chlorofuran-2-yl- C6H5
5-Chlorofuran-2-yl- C6H5
N(CH3)2
N(COCH3)2 25
CH2Br
CH2CN
CH2COOCH3 n.a.
CH2COOH
n.a.
n.a.
n.a.
n.a.
n.a.
58
35
12
32
2m
5-Chlorofuran-2-yl- 4-NH2C6H4 CH3
78
38
(4.3
95
(0.077
81
(0.099
40
m
M)
NO2
NH2
SC-560
2
Cl
Cl
m
m
M)
a
Indomethacin
Celecoxib
93
O
O
M) (4.2
87
m
M)
N
N
(0.28
mM)
O
O
a
Compounds were screened at a concentration of 50
mM, by a Cayman colori-
2e
2m
metric COX (ovine) inhibitor screening assay kit. Values are the means of at least
three independent measurements; n.a. ¼ not active: no inhibitory activity was
Scheme 5. Reagents and conditions: (a) SnCl2/HCl, reflux, 4 h.
observed at the concentration of 50 mM.

P. Vitale et al. / European Journal of Medicinal Chemistry 74 (2014) 606e618
611
100
80
60
40
20
0
-9
-8
-7
-6
-5
-4
-3
Fig. 4. Concentrationeresponse curve and IC50 of 2f by OVCAR-3 cell (8 mM
14C-AA,
log [inhibitor] (M)
30 min at 37 ꢀC).
Fig. 3. Concentrationeresponse curves for the inhibition of COX-1 activity by P6 (
;
IC50 ¼ 22 ꢂ 2.4
m
M), 2f ( ; IC50 ¼ 1.1 ꢂ 0.26 M) and 2m ( ; IC50 ¼ 4.3 ꢂ 0.09 mm),
m
obtained by using a colorimetric COX (ovine) assay kit. IC50 values represent the
superior to the other scoring functions in GOLD for both pose
prediction and virtual screening [41].
mean ꢂ SEM from three separate experiments.
The ChemPLP-CS docking protocol was adopted in this study. In
this protocol, the poses obtained with the ChemPLP function are
rescored and reranked with the GOLD implementation of the
ChemScore function. Several examples of successful applications of
this protocol are known [27,39,42e46]. Alternative conformations
of R120, Y355, S353 and S530 side chains have been previously
observed in the COX crystal structures complexed with several
inhibitors, highlighting the innate plasticity of the active site [47e
49]. Accordingly, the side chain of R120, Y355, S353, and S530
was allowed to move during the docking experiments [50].
Docking of compounds 2f and 2m into COX-1 active site showed
an orientation similar to that observed by us for previously pub-
lished COX-1 selective isoxazoles [26]. As illustrated in Fig. 5, the
ligands lie on the internal side of the constriction, at the base of the
funnel-shaped entrance to the COX-1 active site. The constriction,
made up of residues R120, E524, and Y355, constitutes a gate that
must open and close for substrates and inhibitors to pass into or out
of the COX active site [51,52]. Both O1 furan oxygen and N2 iso-
xazole nitrogen of compounds 2f and 2m is within H-bonding
distance of the OH group of S353. The O1$$$O and N2$$$O atoms
for PET-CT imaging of physio-pathological organ in which COX-1 is
a biomarker (i. e. the ovarian cancer).
Further studies are in progress to deeply investigate other
substituents on this aromatic ring with quite different steric and
electronic properties to definitively list determinants responsible of
selective inhibition of COX-1 activity, at least for those compounds
belonging to the diarylheterocycle class.
The overall obtained results suggest that (i) the oxygenated
substituent on the aryl linked to the isoxazole-C3 is important for
COX-1 inhibition selectivity, as demonstrated by comparing the
activity of P6 and its two analogs 2a and 2b, lacking the furan ring
replaced by a methoxyphenyl; these new selective COX-1 inhibitors
could be also transformed into a PET-CT radiotracer by replacing
the O12CH3 with O11CH3; (ii) the ethyl group (instead of a methyl)
on isoxazole-C5 of 2c, still find enough space in the COX-1 active
site, however, the selectivity is lower than P6; the increase of the
substituent size reduces COX-1 activity and selectivity, as by
comparing P6 and propyl, bromomethyl, cyanomethyl and carboxy
methyl derivatives; (iii) the carboxylic acid derivative 2l exhibits
reversal selectivity toward COX isoforms, thus giving some impor-
tant indications on the different mechanism of mofezolac, P6 and
their derivate isoxazolic compounds; (iv) the introduction of a e
NH2 (2f) in place of a methyl on the isoxazole-C5 gives 2f, a new
selective COX-1 inhibitor, with a COX-1 IC50 20 fold higher than P6
(Table 3, Fig. 3); a similar result was obtained if the NH2 is in para
position of the phenyl on C4 of isoxazole (2m, Fig. 3).
ꢀ
ꢀ
are separated by 2.6 A and 2.8 A, respectively, and display a
favorable geometry for H-bonding. The furan O1 atom of the 2f also
ꢀ
accepts a weak H-bond from the OH group of Y355 (dO1$$$O ¼ 3.3 A).
The O1 isoxazole oxygen of 2f is engaged in a further H-bond with
ꢀ
the NH2 group of R120 (dO1$$$N ¼ 2.5 A), which plays a crucial role in
binding substrates and carboxylic acid containing inhibitors.
Finally, the NH2 group at position 5 of isoxazole ring of compound
ꢀ
2f establishes an H-bond with the S530 OH group (dN5$$$O ¼ 2.6 A),
Finally, the inhibitory activity of the most active compound 2f
was evaluated in the OVCAR-3 cells expressing only hCOX-1 (the
second isoform is completely absent). 2f was found to inhibit the
which adopts a “down” position during the flexible docking
simulation. Unexpectedly, the NH2 group at para position of phenyl
ꢀ
ring of 2m also interacts with S530 OH group (dN$$$O ¼ 2.8 A), since
hCOX-1 with high potency (IC50 ¼ 0.58
mM, Fig. 4).
this residue switches in an “up” conformation. In line with our
results, shifts in the position of the side chain of S530 have been
observed previously in crystal structures of COX-1 and COX-2 in
complex with fatty acid substrates [48,49]. It is worth noting that
the extensive H-bonding network between 2f and the COX-1 active
site provides a tight anchor for the ligand, explaining its higher
inhibitory potency compared to 2m.
2.4. Docking studies
To get a better comprehension of the high COX-1 inhibitory
potency of compounds 2f and 2m at a molecular level and guide
further SAR studies, docking experiments were conducted using
the ovine COX-1 in complex with celecoxib (PDB code: 3kk6) [37].
Docking investigations were carried out through the automated
docking program GOLD 5.0.1 [38], selecting ChemPLP as a fitness
function (rescoring with ChemScore) [39]. This scoring function,
which uses a piecewise linear potential for hydrophobic and non-
complementary interactions and Chemscore terms for H-bonding
and internal energy [40], has been recently demonstrated to be
The phenyl ring at position 4 of isoxazole of both ligands is
oriented towards the apex of the COX-1 active site and forms hy-
drophobic interactions with residues L352, F381, L384, Y385, W387,
F518, M522 and G526. Importantly, the 5-chlorofuryl moiety of 2f
and 2m is oriented towards the side pocket (residues 513e520) of
COX-1 and makes hydrophobic contacts with residues H90, L352,
I517, F518 and I523.

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P. Vitale et al. / European Journal of Medicinal Chemistry 74 (2014) 606e618
Fig. 5. Compounds 2f (a, yellow) and 2m (c, purple) docked into the COX-1 binding site. Docked position of compounds 2f (b, yellow) and 2m (d, purple) superimposed on the
ꢀ
crystal structure position of celecoxib (magenta). Only amino acids located within 5 A of the bound ligand are displayed and labeled. H-bonds discussed in the text are depicted as
dashed black lines. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Conformational superimposition of compounds 2f and 2m
(from the docking simulation) and celecoxib (from the X-ray crystal
structure of celecoxib/COX-1 complex), depicted in Fig. 5b and d,
shows that they bind to the COX-1 active site with position and
orientation very similar to that of the crystal structure of celecoxib
complexed with COX-1. Whether or not this binding results in
functional inhibition is more complicated to predict, as it may be
related to kinetic of dissociation rather than to the stability of the
complex. Given our biological results, it can be speculated that the
orientation observed for compounds 2f and 2m in COX-1 is able to
inhibit the access of arachidonic acid to the catalytic site and thus
ultimately inhibit the synthesis of prostaglandins, prostacyclin, and
thromboxanes.
Unfortunately, docking experiments into the binding site of
COX-2 (PDB code: 6COX) [47] were not able to explain the low
activity of compounds P6, 2f and 2m in inhibiting COX-2. As
depicted in Fig. 6, the best solution predicted by GOLD for the
representative compound 2f was very similar to the crystallo-
graphic disposition of the selective COX-2 inhibitor SC-558 in the
binding pocket of COX-2, although it did not show strong in-
teractions nor with the COX-2 active site residues nor with the
residues lining the side pocket such as H90, R513, and N192.
A mechanism proposed for diarylheterocyclic derivatives inhi-
Fig. 6. The binding mode found for compound 2f (yellow) in comparison with SC-558
bition of COX-2 consists of three sequential reversible steps [53].
(brown) from the X-ray crystal structure of SC-558/COX-2 complex (PDB code: 6COX).
(For interpretation of the references to color in this figure legend, the reader is referred
The first step represents the interaction of the inhibitor at the
surface near the membrane-binding region of the enzyme (lobby
to the web version of this article.)

P. Vitale et al. / European Journal of Medicinal Chemistry 74 (2014) 606e618
613
ꢀ
region), approximately 25 A away from the constriction formed by
fluorescent indicator, the spots on the TLC were observed under
ultraviolet light or were visualized by I2 vapor exposure. Column
chromatography was conducted by using silica gel 60 with a par-
R120, E524, and Y355. The second step represents the translocation
of the inhibitor from the lobby to the COX-2 active site. It can be
assumed that these two steps are similar for P6, 2f and 2m and
several selective COX-2 inhibitors such as valdecoxib or celecoxib.
However, the COX-2-selective diarylheterocycles are able to form, in
a third and final step, a tightly bound enzyme inhibitor complex in
the active site and a side pocket of COX-2 due to their phenyl-
sulfonamide or a phenylsulfamoyl group. We suppose that com-
pounds P6, 2f and 2m, lacking the sulfonamide or sulfamoyl group,
cannot form this tight complex due to the absence of any profitable
interaction, and are easily released from the binding pocket of COX-2.
ticle size distribution 40e63 mm and 230-400 ASTM. GCeMS ana-
lyses were performed on an HP 5995C model. MS-ESI analyses were
performed on Agilent 1100 LC/MSD trap system VL. All synthesized
compounds were analyzed by HPLC analysis, performed on an
Agilent 1260 Infinity instrument equipped with a 1260 DAD VLþ
detector on a column Poroshell 120 EC-18 3 ꢃ 50 mm 2.7Micron
(eluent CH3CN/H2O ¼ 70/30; ¼ 280 nm), and their purity is higher
l
than 95%.
4.1.2. Materials
3. Conclusions
Tetrahydrofuran from commercial source was purified by
distillation (twice) from sodium wire under nitrogen. Dichloro-
methane from commercial source was purified by distillation from
CaH2 under nitrogen atmosphere. Dimethylformamide from com-
mercial source was purified by distillation from CaH2 under
reduced pressure. Standardized (2.5 M) n-butyllithium in hexanes
was purchased from Aldrich Chemical Co. and its titration was
performed with N-pivaloyl-o-toluidine [54]. Diisopropylamine
from commercial source was purified by distillation from CaH2
under reduced pressure and nitrogen atmosphere. All other
chemicals and solvents were commercial grade further purified by
distillation or crystallization prior to use. Arylnitrile oxides were
prepared from aldehydes through their conversion into the corre-
sponding oximes and then into benzohydroximinoyl chlorides
[31,32,55,56]. These were finally converted into nitrile oxides by
treatment with Et3N, followed by vacuum filtration of Et3N.HCl from
the solution. Oximes [24,57e59], prepared from reaction of alde-
hydes/EtOH and NH2OH.HCl/aq.NaOH, had analytical and spectro-
scopic data identical to those previously reported or commercially
available.
All the new compounds have been prepared in good yields by
developing suitable, efficient and versatile synthetic methodologies
[34].
Many structural modifications have been proposed during this
investigation, that allowed us also to find out the rationale of the
molecular basis of some selective interactions between COX-1
enzyme and P6 and/or its analogs: SAR studies have contributed
to define the importance of the oxygen of substituent linked to
isoxazole-C3 for the inhibition of COX-1 activity and COX selectivity.
A number of these compounds are precursors of possible radio-
labelled selective COX-1 inhibitors to be used as PET-CT radiotracers
in ovarian cancer diagnosis. In addition, the substituent size on C5
of isoxazole ring seems to be determinant for potency and selec-
tivity in COX-1 inhibition. The substitution of methyl with an amino
group on that position increases COX-1 inhibitory activity and
selectivity (2f) compared to P6. Low or no activity at all was instead
observed for its N,N-dialkyl (2g) and N,N-diacetyl (2h) derivatives.
Molecular docking studies of the most active inhibitor 2f into the
active site of COX-1 suggest critical interactions between the in-
hibitor and the constriction residues R120 and Y355 at the base of
the active site, as well as an interaction with S530 at the top of the
pocket.
The data herein reported suggest some molecular determinants
necessary to design new diarylisoxazoles as inhibitors of cyclo-
oxygenases, mainly COX-1. A full pharmacological characterization
of some of the above compounds will be carried out in order to
prove their pharmacokinetic properties, metabolic stability and the
possibility of their use as diagnostic tools, analgesics, anti-
inflammatories, antiproliferative and chemopreventive drugs.
Further studies are in progress aimed to fine tune the isoxazole
structure to clarify at atomic level, including X-ray of the COX-1-
inhibitor complexes, the reasons of the marked inhibitory activity
and selectivity of the novel series of isoxazoles above described.
4.1.3. General procedure for the synthesis of 3,4-diarylisoxazoles
(2aee)
A solution of the 3-aryl-2-propanones 1aee (0.6 mmol) in THF
(3 mL) was dropwise added to a suspension of NaH (95% w/w,
1.2 mmol) in THF (6 mL) kept at 0 ꢀC under nitrogen atmosphere,
using a nitrogen-flushed, three necked flask equipped with a
magnetic stirrer, a nitrogen inlet and two dropping funnels. After
the yellow mixture had been stirred for 1 h, a solution of arylnitrile
oxide (0.6 mmol) in THF (3 mL) was added. The reaction mixture
was allowed to reach room temperature, stirred overnight and then
quenched by adding aqueous NH4Cl solution. The reaction products
were extracted three times with ethyl acetate. The combined
organic phases were dried over anhydrous Na2SO4 and then
evaporated under vacuum. Column chromatography (silica gel,
petroleum ether:ethyl acetate ¼ 19/1) of the residue affords the
3,4-diarylisoxazoles (2aee) in 21e60% yields (Table 2). 3-(5-
Chlorofuran-2-yl)-4-phenyl-5-methylisoxazole (P6) spectroscopic
and pharmacological data have been previously reported [24,57].
4. Experimental protocols
4.1. Chemistry
4.1.1. General methods
4.1.3.1. 3-(2-Methoxyphenyl)-5-methyl-4-phenylisoxazole
(2a).
Melting points taken on electrothermal apparatus were uncor-
rected. 1H NMR and 13C NMR spectra were recorded on a Varian-
Mercury 300 MHz, on a Varian-Inova-400 MHz spectrometer, on
a Bruker-Aspect 3000 console 500 MHz spectrometer and chemical
43% yield (30 mg). Mp 90e92 ꢀC (Et2O), white crystals. FT-IR (KBr):
3050, 2925, 1604, 1496, 1466, 1411, 1249, 1103, 1018, 899, 798, 755,
699 cmꢁ1. 1H NMR (400 MHz, CDCl3,
d): 7.47e7.45(m, 1H, aromatic
proton); 7.41e7.36 (m, 1H, aromatic proton); 7.31e7.22 (m, 3H, ar-
omatic protons); 7.10e7.07 (m, 2H, aromatic protons); 7.03e6.98
(m, 1H, aromatic proton); 6.80 (d, 1H, J ¼ 8.3 Hz, aromatic proton);
shifts are reported in parts per million (d
). 19F NMR spectra were
recorded by using CFCl3 as internal standard. Absolute values of the
coupling constant are reported. FT-IR spectra were recorded on a
PerkineElmer 681 spectrometer. GC analyses were performed by
3.29 (s, 3H), 2.52 (s, 3H). 13C NMR (100 MHz, CDCl3,
d): 165.1, 160.2,
157.0, 131.5, 131.1, 130.96, 128.24, 128.22, 126.9, 120.7, 118.5, 117.2,
111.2, 54.8, 11.7. GCeMS (70 eV) m/z (int.rel.): 265 (Mþ, 100), 250
(38), 223 (35), 222 (18), 208 (23), 207 (18), 152 (8), 115 (7), 104 (6),
103 (8), 89 (11), 77 (8), 63 (6).
using an HP1 column (methyl siloxane; 30 m ꢃ 0.32 mm ꢃ 0.25
mm
film thickness) on an HP 6890 model, Series II. Thin-layer chro-
matography (TLC) was performed on silica gel sheets with

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P. Vitale et al. / European Journal of Medicinal Chemistry 74 (2014) 606e618
4.1.3.2. 3-(2-Methoxyphenyl)-4-phenyl-5-(trifluoromethyl)isoxazole
(2b). 43 % yield. Mp 44e46 ꢀC (hexane), white powder. FT-IR (KBr):
3062, 2963, 2926, 2850, 1606, 1584, 1515, 1496, 1468, 1448, 1437,
1412, 1324, 1260, 1194, 1089, 1046, 1024, 953, 798 754, 698 cmꢁ1. 1H
reaction products were extracted three times with ethyl acetate.
The organic phase was dried over anhydrous Na2SO4 and then the
solvent evaporated under vacuum. Column chromatography (silica
gel, petroleum ether:ethyl acetate ¼ from 20/1 to 8/2) of the res-
idue affords the 5-(chlorofuran-2-yl)-4-phenylisoxazol-5-amine
(105 mg) in 40% yield.
NMR (400 MHz, CDCl3, d): 7.47e7.39 (m, 2H, aromatic protons);
7.32e7.28 (m, 3H, aromatic protons); 7.16e7.12 (m, 2H, aromatic
protons); 7.06e7.01 (m,1H, aromatic proton); 6.78 (d,1H, J ¼ 8.2 Hz,
aromatic proton); 3.26 (s, 3H). 13C NMR (100 MHz, CDCl3,
d
): 161.7,
4.1.4.1. 3-(5-Chlorofuran-2-yl)-4-phenylisoxazol-5-amine
(2f).
156.9, 131.9, 131.2, 128.6 (m), 128.4, 128.1 (2C), 127.7, 120.8, 118.6 (q,
Mp 138.5e141 ꢀC (EtOAc/hexane). FT-IR (KBr): 3460, 3402.6, 3115,
1JCeF] 271 Hz), 116.4, 111.2, 54.7. 19F NMR (376 MHz, CDCl3,
2927, 1643, 1518, 1506, 1474, 1413, 1318, 1208, 1148, 1020, 988, 940,
d
): ꢁ65.6 (s). GCeMS (70 eV) m/z (int.rel.): 319 (Mþ, 100), 251 (17),
896, 786, 699 cmꢁ1. 1H NMR (400 MHz, CDCl3,
d): 7.45e7.41 (m, 2H,
250 (94), 235 (10), 220 (9), 207 (18), 178 (8), 115 (13), 89 (19), 77 (8),
63 (8), 51 (33).
aromatic protons); 7.39e7.31(m, 3H, aromatic protons); 6.36 (d,
J ¼ 3.5 Hz, 1H, furyl proton); 6.14 (d, J ¼ 3.5 Hz, 1H, furyl proton);
4.56 (bs, 2H, NH2: exchange with D2O). 13C NMR (100 MHz, CDCl3,
4.1.3.3. 3-(5-Chlorofuran-2-yl)-5-ethyl-4-phenylisoxazole
(2c).
d
): 165.8, 153.1, 143.9, 138.4, 130.0, 129.8, 129.3, 128.1, 113.7, 108.1,
93.5. GCeMS (70 eV) m/z (rel.int.): 262 [M (37Cl)þ, 11], 260 [M
(
35Cl)þ, 31], 225 (6), 218 (20), 216 (54), 197 (8), 188 (13), 180 (20),
46% yield. Mp 82e85 ꢀC, yellow solid. FT-IR (KBr): 3137, 3063, 2964,
2940, 1628, 1594, 1518, 1427, 1412, 1261, 1204, 1094, 1017, 864, 798,
711 cmꢁ1. 1H NMR (400 MHz, CDCl3,
d): 7.45e7.41 (m, 3H, aromatic
154 (15),153 (16),152 (32),131 (31),129 (100),127 (16),115 (10),113
(21), 105 (28), 104 (27), 103 (13), 101 (26), 94 (42), 89 (14), 85 (18),
77 (54), 76 (11), 63 (15), 51 (14).
protons), 7.29e7.26 (m, 2H, aromatic protons); 6.20 (d, 1H,
J ¼ 3.3 Hz); 6.12 (d, 1H, J ¼ 3.3 Hz); 2.73 (q, 2H, J ¼ 7.5 Hz); 1.26 (t,
3H, J ¼ 7.5 Hz). 13C NMR (100 MHz, CDCl3,
d): 171.4, 152.2, 143.6,
138.3, 130.0, 129.5, 128.7, 128.39, 113.9, 113.5, 107.8, 19.1, 12.2. GCe
MS (70 eV) m/z (rel.int.): 275 [M(37Cl)þ, 15], 273 [M(35Cl)þ, 44], 219
(34), 218 (16), 217 (100), 188 (8), 154 (24), 153 (17), 152 (9), 129 (8),
127 (14), 89 (11), 77 (6), 57 (12).
4.1.5. Synthesis of 3-(5-chlorofuran-2-yl)-N,N-dimethyl-4-
phenylisoxazol-5-amine (2g)
To a stirred mixture of 3-(5-chlorofuran-2-yl)-4-phenylisoxazol-
5-amine (2f) (475 mg, 1.83 mmol) and paraformaldehyde (548 mg,
18.27 mmol) in AcOH (5 mL) at 25 ꢀC under nitrogen, sodium cya-
noborohydride was added (574 mg, 9.14 mmol). The reaction
mixture was kept under stirring for 3 days. EtOAc and saturated
aqueous NaHCO3 solution (10 mL) were added. The two phases were
separated and the aqueous layer was extracted three times with
EtOAc. The combined organic extracts were dried over anhydrous
Na2SO4 and the solvent removed under reduced pressure. Column
chromatography (silica gel, mobile phase: petroleum ether/ethyl
acetate ¼ 9:1) of the residue afforded the product with 25% yield
[60].
4.1.3.4. 3-(5-Chlorofuran-2-yl)-4-phenyl-5-propylisoxazole
(2d).
21% yield. Yellow oil. FT-IR (neat): 3058, 2964, 2933, 2874, 1627,
1595, 1520, 1497, 1436, 1416, 1261, 1207, 1135, 1093, 1018, 987, 942,
900, 794, 771, 702 cmꢁ1. 1H NMR (400 MHz, CDCl3,
d): 7.47e7.43 (m,
3H, aromatic protons), 7.29e7.26 (m, 2H, aromatic protons); 6.18 (d,
1H, J ¼ 3.3 Hz); 6.12 (d, 1H, J ¼ 3.3 Hz); 2.68 (t, 2H, J ¼ 7.5 Hz); 2.68
(sextet, 2H, J ¼ 7.5 Hz); 0.93 (t, 3H, J ¼ 7.5 Hz). 13C NMR (100 MHz,
CDCl3, d): 170.4, 152.2, 143.6, 138.3, 130.1, 129.5, 128.7, 128.3, 113.5,
107.8, 94.4, 27.4, 21.1, 13.6. GCeMS (70 eV) m/z (rel.int.): 289
[M(37Cl)þ, 11], 287 [M(35Cl)þ, 32], 219 (31), 218 (15), 217 (100), 188
(7), 154 (20), 153 (17), 127 (45), 89 (17), 77 (13), 71 (29), 43 (39).
4.1.5.1. 3-(5-Chlorofuran-2-yl)-N,N-dimethyl-4-phenylisoxazol-5-
amine (2g). Mp 89.8e91 ꢀC (EtOAc/hexane). FT-IR (KBr): 3109,
3054, 2927, 2875, 1626, 1600, 1515, 1484, 1423, 1409, 1324, 1247,
1206, 1026, 820, 772, 739, 701, 515 cmꢁ1. 1H NMR (400 MHz, CDCl3,
4.1.3.5. 3-(5-Chlorofuran-2-yl)-5-methyl-4-(4-nitrophenyl)isoxazole
(2e). 24 % yield. Mp 153e155 ꢀC (hexane), white powder. FT-IR
(KBr): 3144, 3103, 3071, 2929, 2851, 1628, 1601, 1559, 1519, 1441,
1419, 1345, 1239, 1204, 1136, 1105, 1019, 988, 896, 867, 853, 797, 761,
d
): 7.39e7.37 (m, 3H, aromatic protons); 7.32e7.30 (m, 2H, aromatic
protons); 6.04 (d, J ¼ 3.5 Hz, 1H, furyl proton); 6.86 (d, J ¼ 3.5 Hz,
1H, furyl proton); 2.84 (s, 6H, CH3). 13C NMR (100 MHz, CDCl3,
):
730, 709, 688, 560, 513 cmꢁ1. 1H NMR (400 MHz, CDCl3,
d
): 8.32 (d,
d
2H, J ¼ 8.3 Hz, aromatic protons); 7.49 (d, 2H, J ¼ 8.3 Hz, aromatic
162.9,150.5,140.5,134.0,128.2,127.4,124.6,124.2,109.3,103.8, 87.9,
protons); 6.49 (d, 1H, J ¼ 3.5 Hz); 6.21 (d, 1H, J ¼ 3.5 Hz); 2.45 (s,
35.6. GCeMS (70 eV) m/z (rel.int.): 290 [M (37Cl)þ, 18], 288 [M
3H). 13C NMR (100 MHz, CDCl3,
d
): 167.7, 151.8, 147.6, 142.7, 138.8,
(
35Cl)þ, 44], 253 (27), 188 (11),145 (100),139 (10),129 (13),127 (22),
136.5, 130.8, 123.9, 113.7, 113.3, 108.1, 11.4. GCeMS (70 eV) m/z
(int.rel.): 306 (M(37Cl)þ, (15), 304 (M(35Cl)þ, (45), 264 (33), 262
(100), 245 (7), 217 (13), 215 (15),199 (7),188 (9),187 (12),152 (9), 89
(16), 73 (8), 63 (6), 43 (52).
105 (10), 89 (22), 77 (12), 76 (11), 72 (31), 64 (18), 63 (12).
4.1.6. Synthesis of N-acetyl-N-(3-(5-chlorofuran-2-yl)-4-
phenylisoxazol-5-yl)acetamide (2h)
To
a solution of 3-(5-chlorofuran-2-yl)-4-phenylisoxazol-5-
4.1.4. General procedure for the synthesis of 3-(5-chlorofuran-2-yl)-
4-phenylisoxazol-5-amine (2f)
amine (2f) (458 mg, 1.76 mmol) and pyridine (0.264 mL,
3.28 mmol), Ac2O (0.41 mL, 4.40 mmol) was added. The reaction
mixture was kept under stirring for 20 h at r.t. EtOAc and saturated
aqueous NaHCO3 solution (10 mL) were added. The two phases were
separated and the aqueous layer was extracted three times with
EtOAc. The combined organic extracts were dried over anhydrous
Na2SO4 and the solvent removed under reduced pressure. Column
chromatography (silica gel, mobile phase: petroleum ether/ethyl
acetate ¼ 10:1) of the residue afforded the product with 30% yield.
A 2.5 M solution of n-butyllithium in hexane (1.068 mL,
2.67 mmol) was added to diisopropylamine (0.413 mL, 2.97 mmol)
in anhydrous THF (10 mL) kept at 0 ꢀC under nitrogen atmosphere,
using a nitrogen-flushed, three necked flask equipped with a
magnetic stirrer, a nitrogen inlet and two dropping funnels. After
the mixture had been stirred for 15 min, the reaction mixture was
kept at ꢁ78 ꢀC, then phenylacetonitrile (0.300 mL, 2.23 mmol) was
dropwise added. The yellow reaction mixture was stirred at 0 ꢀC for
1 h, then the solution of arylnitrile oxide (2.23 mmol) in anhydrous
THF (10 mL) was added [24]. The orange-colored reaction mixture
was allowed to reach room temperature and stirred overnight.
After quenching by addition of aqueous NH4Cl solution, the
4.1.6.1. N-Acetyl-N-[3-(5-chlorofuran-2-yl)-4-phenylisoxazol-5-yl]
acetamide (2h). Mp 98e100 ꢀC (EtOAc/hexane). FT-IR (KBr): 3142,
3021, 2942, 1740, 1651, 1600, 1519, 1498, 1429, 1367, 1256, 1208,
1146, 1074, 1036, 1003, 994, 984, 942, 897, 799, 769, 703, 678 cmꢁ1
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P. Vitale et al. / European Journal of Medicinal Chemistry 74 (2014) 606e618
615
1H NMR (400 MHz, CDCl3,
d
): 7.47e7.44 (m, 3H, aromatic protons);
2260, 1631, 1518, 1466, 1412, 1261, 1207, 1096, 1018, 942, 798, 729,
701 cmꢁ1. 1H NMR (400 MHz, CDCl3,
): 7.52e7.49 (m, 3H, aromatic
7.28e7.26 (m, 2H, aromatic protons); 6.34 (d, J ¼ 3.5 Hz, 1H, furyl
d
proton); 6.18 (d, J ¼ 3.5 Hz, 1H, furyl proton); 2.30 (s, 6H, CH3). 13
C
protons); 7.35e7.31 (m, 2H, aromatic protons); 6.29 (d, J ¼ 3.5 Hz,
NMR (100 MHz, CDCl3, d): 170.9, 158.4, 153.9, 142.4, 139.3, 129.5,
1H, furyl proton); 6.16 (d, J ¼ 3.5 Hz, 1H, furyl proton); 3.83 (s, 2H,
129.2, 129.0, 126.7, 114.8, 114.7, 108.2, 25.6. GCeMS (70 eV) m/z
(rel.int.): 346 [M (37Cl)þ, 4], 344 [M (35Cl)þ, 12], 304 (25), 303 (13),
302 (73), 267 (13), 262 (13), 261 (37), 260 (37), 259 (100), 231 (10),
225 (32), 224 (15), 218 (20), 197 (27), 196 (17), 190 (14), 179 (19), 175
(12), 168 (14), 164 (13), 162 (10), 155 (45), 154 (26), 145 (10), 144
(10), 139 (15), 127 (36), 126 (12), 119 (14), 117 (64), 105 (25), 89 (13),
77 (29), 64 (7), 63 (7), 51 (9), 43 (57). ESI-MS: m/z (%): 367
[M þ Na]þ (100); MSeMS (367): 325 (100), 282 (62), 284 (27), 248
(23), 226 (13), 225 (10).
CH2). 13C NMR (75 MHz, CDCl3,
d): 157.7, 153.0, 142.6, 139.4, 130.1,
129.7, 129.5, 127.4, 117.7, 114.8, 113.6, 108.4, 15.4. GCeMS (70 eV) m/z
(rel.int.): 286 [M (37Cl)þ, 34], 285 (18), 284 [M (35Cl)þ, 100], 256
(15), 249 (36), 246 (10), 244 (29), 222 (6), 221 (34), 218 (9), 216 (27),
215 (10), 193 (20), 190 (24), 189 (14), 188 (75), 187 (17), 180 (12), 154
(10), 153 (42), 152 (40), 129 (61), 128 (18), 127 (27), 126 (11), 115
(12), 102 (9), 101 (8), 91 (15), 89 (54), 77 (18), 75 (15), 73 (15), 63
(16), 51 (8).
4.1.9. Synthesis of methyl 2-[3-(5-chlorofuran-2-yl)-4-
4.1.7. Synthesis of 5-(bromomethyl)-3-(5-chlorofuran-2-yl)-4-
phenylisoxazol-5-yl]acetate (2k)
phenylisoxazole (2i)
2-[3-(5-Chlorofuran-2-yl)-4-phenylisoxazol-5-yl]acetonitrile
(2j) (0.057 g, 0.179 mmol) and p-toluenesulfonic acid (p-TsOH)
(0.035 g, 0.179 mmol) were dissolved in MeOH (4 mL) in a glass
vessel with Teflon stoppers, and heated during 20 min at 100 ꢀC,
using a microwave apparatus at 300 W. Then, to the resulting so-
lution, water (10 mL) and EtOAc (10 mL) were added. The two
phases were separated and aqueous layer was extracted three
times with EtOAc. The combined organic extracts were dried over
anhydrous Na2SO4 and the solvent evaporated under vacuum.
Column chromatography (silica gel, petroleum ether:ethyl
acetate ¼ 10/1) of the residue affords 0.040 g of methyl 2-(3-(5-
chlorofuran-2-yl)-4-phenylisoxazol-5-yl)acetate in 88% yield.
To
a solution of 3-(5-chlorofuran-2-yl)-4-phenyl-5-methyl-
isoxazole (0.500 g, 1.930 mmol) (P6) dissolved in anhydrous CCl4
(10 mL), contained in a argon-flushed, three necked flask equipped
with a magnetic stirrer N-bromosuccinimide (412 mg, 2.32 mmol)
was added. Then, AIBN (0.158 g, 0.965 mmol) was added, and the
obtained suspension was stirred overnight at room temperature.
The reaction mixture was kept under reflux for 4 h, and then water
and ethyl acetate were added. The two phases were separated and
the aqueous layer was extracted three times with ethyl acetate.
The combined organic extracts washed with brine and KOH (1 M),
were dried over anhydrous Na2SO4 and the solvent evaporated
under vacuum. Column chromatography (silica gel, petroleum
ether:ethyl acetate ¼ 10/1) of the residue affords the 5-(bromo-
methyl)-3-(5-chlorofuran-2-yl)-4-phenylisoxazole (2i) in 47%
yield as a solid.
4.1.9.1. Methyl 2-[3-(5-chlorofuran-2-yl)-4-phenylisoxazol-5-yl]ace-
tate (2k). Yellow oil. FT-IR (neat): 3140, 2955, 2923, 2852, 1743,
1635, 1597, 1518, 1497, 1436, 1414, 1332, 1295, 1260, 1206, 1177,
1131, 1017, 987, 942, 898, 796, 772, 701, 680 cmꢁ1 1H NMR
.
4.1.7.1. 5-(Bromomethyl)-3-(5-chlorofuran-2-yl)-4-phenylisoxazole
(2i). Mp 88.5e90.3 ꢀC (hexane). FT-IR (neat): 3137, 3065, 2924,
1628, 1515, 1435, 1410, 1266, 1206, 1136, 1096, 1017, 1009, 988, 948,
(400 MHz, CDCl3, d): 7.46e7.43 (m, 3H, aromatic protons); 7.33e
7.30 (m, 2H, aromatic protons); 6.26 (d, J ¼ 3.2 Hz, 1H, furyl pro-
ton); 6.14 (d, J ¼ 3.2 Hz, 1H, furyl proton); 3.75 (s, 2H, CH2); 3.71 (s,
942, 902, 789, 774, 704 cmꢁ1. 1H NMR (400 MHz, CDCl3,
d): 7.50e
3H, CH3). 13C NMR (100 MHz, CDCl3,
d): 167.7, 162.3, 152.4, 143.1,
7.46 (m, 3H, aromatic protons); 7.39e7.36 (m, 2H, aromatic pro-
tons); 6.31 (d, J ¼ 3.5 Hz, 1H, furyl proton); 6.15 (d, J ¼ 3.5 Hz, 1H,
138.6, 129.9, 128.8, 128.7, 116.9, 113.9, 107.9 52.6, 31.4. GCeMS
(70 eV) m/z (rel.int.): 319 [M (37Cl)þ, 26], 318 (13), 317 [M (35Cl)þ,
76], 277 (6), 275 (19), 247 (16), 244 (23), 219 (33), 218 (18), 217
(100), 216 (14), 190 (7), 188 (22), 154 (48), 153 (19), 152 (15), 129
(23), 128 (9), 127 (19), 115 (15), 103 (9), 102 (10), 101 (11), 91 (12),
77 (14), 59 (14), 51 (4).
furyl proton); 4.38 (s, 2H, CH2). 13C NMR (75 MHz, CDCl3,
d): 164.6,
152.8, 143.1, 139.1, 130.2, 129.9, 129.3, 129.2, 117.2, 114.4, 108.3, 17.8.
GCeMS (70 eV) m/z (rel. int.): 341 [M(81Br, 37Cl)þ, 26], 339 [M (79Br,
37Cl)þ, 100], 337 [M (79Br, 35Cl)þ, 78], 258 (22), 246 (11), 244 (33),
232 (12), 230 (35), 223 (14), 219 (17), 218 (13), 217 (54), 216 (19),
190 (16), 188 (46), 167 (25), 166 (16), 163 (12), 154 (18), 153 (29), 152
(28), 140 (10), 139 (18), 129 (66), 128 (11), 127 (24), 115 (34), 103
(19), 102 (13), 91 (28), 89 (38), 77 (18), 75 (13), 73 (17), 63 (12), 51
(9).
4.1.10. Synthesis of 2-[3-(5-chlorofuran-2-yl)-4-phenylisoxazol-5-
yl]acetic acid (2l)
KOH (1.340 mmol) in H2O (7 mL) was added to the methyl 2-[3-
(5-chlorofuran-2-yl)-4-phenylisoxazol-5-yl]acetate (2k) (211 mg,
0.670 mmol) in THF (8 mL). The reaction mixture was stirred
overnight at room temperature. Then, THF was distilled under
reduced pressure and ethyl ether was added to the residue. The two
phases were separated.10% HCl was added to the aqueous phase till
pH < 5, and the product extracted three times with ethyl ether. The
combined organic phases were dried over anhydrous Na2SO4 and
then the solvent was evaporated under reduced pressure. The
residue recrystallized from CHCl3/hexane afforded the pure prod-
ucts as a pale yellow solid (60% yield).
4.1.8. Synthesis of 2-[3-(5-chlorofuran-2-yl)-4-phenylisoxazol-5-yl]
acetonitrile (2j)
To
a solution of 5-(bromomethyl)-3-(5-chlorofuran-2-yl)-4-
phenylisoxazole (2i) (1.520 g, 4.51 mmol) in dichloromethane
(61 mL), tetrabutylammonium hydrogen sulfate (3.06 g, 9.02 mmol)
and a solution of NaCN (0.265 g, 5.41 mmol) in water (61 mL) were
added, and the suspension kept under stirring at room temperature
for 5 h. Then, the two phases were separated and aqueous layer was
extracted three times with dichloromethane. The combined organic
extracts, washed with brine, were dried over anhydrous Na2SO4 and
the solvent evaporated under vacuum. Column chromatography
(silica gel, petroleum ether:ethyl acetate ¼ 20/1) of the residue af-
fords 1.050 g of 2-[3-(5-chlorofuran-2-yl)-4-phenylisoxazol-5-yl]
acetonitrile in 80% yield.
4.1.10.1. 2-[3-(5-Chlorofuran-2-yl)-4-phenylisoxazol-5-yl]acetic acid
(2l). Mp 61e63 ꢀC (CHCl3/hexane). FT-IR (neat): 3600e3000, 2927,
2590, 1722, 1635, 1519, 1436, 1414, 1292, 1208, 1156, 1134, 1020, 989,
942, 898, 791, 771, 703 cmꢁ1. 1H NMR (400 MHz, CDCl3,
d): 9.10e
8.90 (bs, 1H, OH: exchanges with D2O); 7.47e7.41 (m, 3H, aromatic
protons); 7.34e7.31 (m, 2H, aromatic protons); 6.27 (d, J ¼ 3.4 Hz,
1H, furyl proton); 6.14 (d, J ¼ 3.4 Hz, 1H, furyl proton); 3.79 (s, 2H,
4.1.8.1. 2-[3-(5-Chlorofuran-2-yl)-4-phenylisoxazol-5-yl]acetonitrile
(2j). Dec 170.5 ꢀC (hexane) FT-IR (neat): 3050, 2959, 2918, 2850,
CH2). 13C NMR (100 MHz, CDCl3,
d): 173.0, 161.7, 152.4, 142.9, 138.8,

616
P. Vitale et al. / European Journal of Medicinal Chemistry 74 (2014) 606e618
129.9, 128.95, 128.91, 128.2, 117.3, 114.1, 108.0, 31.2. ESI-MS: m/z (%):
326 [M þ Na]þ (100); 304 [M þ H]þ (100); MSeMS (326): 284 (15),
282 (100), 246 (83).
(30:4:1). The organic phase was spotted on a 20 ꢃ 20 cm TLC plate
(EMD Kieselgel 60, VWR). The plate was developed in EtOAc/
CH2Cl2/glacial AcOH (75:25:1), and radiolabeled prostaglandins
were quantified with a radioactivity scanner (Bioscan, Inc., Wash-
ington, D.C.). The percentage of total products observed at different
inhibitor concentrations was divided by the percentage of products
observed for cells pre-incubated with DMSO.
4.1.11. Synthesis of 4-[3-(5-chlorofuran-2-yl)-5-methylisoxazol-4-
yl]benzenamine (2m)
To a stirred mixture of stannous chloride (298 mg, 1.32 mmol)
in hydrochloric acid 37% (1 mL) at 25 ꢀC, a solution of 3-(5-
chlorofuran-2-yl)-5-methyl-4-(4-nitrophenyl)isoxazole
(2e)
4.2.3. Statistical analysis
(100 mg, 0.33 mmol) dissolved in absolute EtOH (8 mL) was
added. The reaction mixture was kept under reflux for 4 h. 10%
NaOH (10 mL) was added to the reaction mixture till pH ¼ 12,
and the aqueous phase extracted three times with EtOAc. The
combined organic extracts were dried over anhydrous Na2SO4
and the solvent removed under reduced pressure. The residue
afforded the product with 98% yield.
The IC50 values of the compounds reported in Table 3 and Fig. 3
and 4 were determined by nonlinear curve fitting using the
GraphPad Prism program (GraphPad Prism Software (Windows
version), Graph-Pad Software, Inc., San Diego, CA, USA) and are the
mean ꢂ SEM from three separate experiments.
4.3. Computational chemistry
4.1.11.1. 4-[3-(5-Chlorofuran-2-yl)-5-methylisoxazol-4-yl]benzen-
amine (2m). Mp 132e134 ꢀC (EtOAc/hexane). FT-IR (KBr): 3486,
3386, 3143, 3032, 2924, 1626, 1520, 1441, 1411, 1298, 1234, 1204,
Molecular modeling and graphics manipulations were per-
formed using the molecular operating environment (MOE) [61] and
UCSF-CHIMERA [62] software packages, running on a Linux work-
station with an Intel Core i7 920 CPU and 12 GB of RAM.
Figures were generated using Pymol 1.0 [63].
1180, 1136, 1016, 988, 941, 899, 838, 799, 739, 565, 518 cmꢁ1 1H
.
NMR (300 MHz, CDCl3, d): 7.26 (bs, 2H, NH2: exchange with D2O);
7.04 (d, 2H, J ¼ 8.4 Hz, aromatic protons); 6.74 (d, 2H, J ¼ 8.4 Hz,
aromatic protons); 6.28 (d, 1H, J ¼ 3.5 Hz, furyl proton); 6.13 (d, 1H,
4.3.1. Ligand and receptor preparation
J ¼ 3.5 Hz, furyl proton); 2.35 (s, 3H). 13C NMR (100 MHz, CDCl3,
d
):
Model building and geometry optimizations of compounds 2f
and 2m were accomplished with the MMFF94X force field, avail-
able within MOE. The crystal structures of ovine COX-1 in complex
with celecoxib (PDB code: 3kk6) [37] and murine COX-2 complexed
with SC-558 (PDB code: 6COX) [47] were used in the docking ex-
periments. Bound ligands and water molecules were removed. A
correct atom assignment for Asn, Gln, and His residues was done,
and hydrogen atoms were added using standard MOE geometries.
Partial atomic charges were computed by MOE using the Amber99
force field. All heavy atoms were then fixed, and hydrogen atoms
166.8, 152.7, 146.8, 144.1, 138.4, 131.2, 119.1, 115.4, 114.9, 113.8, 108.1,
11.4. GCeMS (70 eV) m/z (rel.int.): 276 [M (37Cl)þ, 34], 274 [M
(
35Cl)þ, 100], 234 (13), 233 (19), 232 (40), 231 (48), 203 (15), 169
(19),168 (29),159 (26),144 (29),142 (12), 119 (29),118 (21),117 (10),
113 (10), 89 (11), 77 (7), 65 (6), 63 (6), 51 (4), 43 (14).
4.2. Biology
4.2.1. Inhibition studies of cyclooxygenase activity
The final compounds were evaluated for their ability to inhibit
were minimized using the AMBER99 force field and a constant
ꢀꢁ1
ovine COX-1/COX-2 enzyme (percent inhibition at 50
mM). The in-
dielectric of 1, terminating at a gradient of 0.001 kcal molꢁ1ꢄ A
.
hibition of the enzyme was determined using a colorimetric COX
(ovine) inhibitor screening assay kit (Catalog No. 760111, Cayman
Chemicals, Ann Arbor, MI, USA) following the manufacturer’s
instructions.
4.3.2. Docking simulations
Docking of 2f and 2m to COX-1 and COX-2 was performed with
GOLD version 5.0.1, which uses a genetic algorithm for determining
the docking modes of ligands and proteins [38]. The coordinates of
the cocrystallized ligand (celecoxib for COX-1 and SC-558 for COX-
2) were chosen as an active-site origin. The active-site radius was
COX is a bifunctional enzyme exhibiting both cyclooxygenase
and peroxidase activities. The cyclooxygenase component cata-
lyzes the conversion of arachidonic acid into a hydroperoxide
(PGG2), and the peroxidase component catalyzes the reduction of
endoperoxidase into the corresponding alcohol (PGH2), the pre-
cursor of PGs, thromboxanes, and prostacyclin. The Colorimetric
COX Inhibitor Screening Assay measures the peroxidase compo-
nent of the cyclooxygenases. The peroxidase activity is assayed
colorimetrically by monitoring the appearance of oxidized
N,N,N’,N’-tetramethyl-p-phenylenediamine (TMPD) at 590 nm.
Stock solutions of test compounds were dissolved in a minimum
volume of DMSO.
ꢀ
set equal to 8 A. The mobility of R120, S353, Y355 and S530 side
chains was set up using the flexible sidechains option in the GOLD
front end, which incorporates the Lovell rotamer library [64]. Each
GA run used the default parameters of 100,000 genetic operations
on an initial population of 100 members divided into five sub-
populations, with weights for crossover, mutation, and migration
being set to 95, 95, and 10, respectively. GOLD allows a user-
definable number of GA runs per ligand, each of which starts
from a different orientation. For these experiments, the number of
GA runs was set to 200, and scoring of the docked poses was per-
formed with the ChemPLP scoring function with ChemScore
rescore. The best output poses (orientations) of the ligands gener-
ated were analyzed on the basis of ChemPLP/ChemScore score and
H-bonding to the enzyme.
4.2.2. In vitro OVCAR-3 cell assay
Human ovarian adenocarcinoma cells, OVCAR-3, passage 13e17,
mycoplasma negative by PCR detection method (Sigma VenorGem)
were grown in RPMI 1640 (Invitrogen/Gibco) þ10% FBS (Atlas) to
70% confluence. Cells were plated in 6-well plates (Sarstedt) and
grown to w60% confluency. Warm HBSS/Tyrodes 1:1 (2 mL) was
replaced in each well, and the cells were treated with inhibitor
Acknowledgments
dissolved in DMSO (0.2e50
37 ꢀC followed by the addition of [14C]-arachidonic acid [8
w2035 MBq/mmol, Perkin Elmer] for 30 min at 37 ꢀC. Aliquots
(400 l) were removed and the reactions were terminated by sol-
vent extraction in 400 l ice-cold Et2O/CH3OH/1 M citrate, pH 4.0
m
M, final concentration) for 30 min at
This work has been supported by a grant from Ministero dell’Is-
truzione dell’Università e della Ricerca (Italy, PRIN 20097FJHPZ_001).
Thanks are due also to Public Research Laboratories Networks of
Regione Puglia-APQ Research Programme ‘Integrated renewable
energy generation from the regional agro-industrial sector. Project
mM,
m
m

P. Vitale et al. / European Journal of Medicinal Chemistry 74 (2014) 606e618
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Quadro in materia di Ricerca Scientifica e II Atto Integrativo e PO
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‘‘Investiamo nel vostro futuro’’). Thanks are due also MIUR (Rome e
Italy) for Progetti di Ricerca Industriale nell'ambito del Programma
Operativo Nazionale R&C 2007e2013 e Project "Reasearch, Appli-
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Appendix A. Supplementary data
Supplementary data associated with this article can be found in
the online version, at http://dx.doi.org/10.1016/j.ejmech.2013.12.
023. These data include MOL files and InChiKeys of the most
important compounds described in this article.
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