Novel synthesis of 3,4-diarylisoxazole analogues of valdecoxib: Reversal cyclooxygenase-2 selectivity by sulfonamide group removal

Article abstract of DOI:10.1021/jm040782x

3,4-Diarylisoxazole analogues of valdecoxib [4-(5-methyl-3-phenylisoxazol- 4-yl)-benzensulfonamide], a selective cyclooxygenase-2 (COX-2) inhibitor, were synthesized by 1,3-dipolar cycloaddition of arylnitrile oxides to the enolate ion of phenylacetone regioselectively prepared in situ with lithium diisopropylamide at 0 °C. The corresponding 3-aryl-5-methyl-4-phenyl- isoxazoles were easily generated by a dehydration/aromatization reaction under basic conditions of 3-aryl-5-hydroxy-5-methyl-4-phenyl-2-isoxazolines and further transformed into their benzenesulfonamide derivatives. The biochemical COX-1/COX-2 selectivity was evaluated in vitro by using the human whole blood assays of COX isozyme activity. Three compounds not bearing the sulfonamide group present in valdecoxib were selective COX-1 inhibitors.

Full text of DOI:10.1021/jm040782x

J . Med. Chem. 2004, 47, 4881
4881
Novel Syn th esis of 3,4-Dia r ylisoxa zole An a logu es of Va ld ecoxib: Rever sa l
Cyclooxygen a se-2 Selectivity by Su lfon a m id e Gr ou p Rem ova l
Leonardo Di Nunno,† Paola Vitale,† Antonio Scilimati,*,† Stefania Tacconelli,‡ and Paola Patrignani‡
Dipartimento Farmaco-Chimico, Universita` di Bari, Via Orabona no. 4, 70125 Bari, Italy, and Dipartimento di Medicina e
Scienze dell’Invecchiamento, Universita` degli Studi “G. D’Annunzio”, Via dei Vestini, 66100 Chieti, Italy
Received February 3, 2004
3,4-Diarylisoxazole analogues of valdecoxib [4-(5-methyl-3-phenylisoxazol-4-yl)-benzensulfon-
amide], a selective cyclooxygenase-2 (COX-2) inhibitor, were synthesized by 1,3-dipolar
cycloaddition of arylnitrile oxides to the enolate ion of phenylacetone regioselectively prepared
in situ with lithium diisopropylamide at 0 °C. The corresponding 3-aryl-5-methyl-4-phenyl-
isoxazoles were easily generated by a dehydration/aromatization reaction under basic conditions
of 3-aryl-5-hydroxy-5-methyl-4-phenyl-2-isoxazolines and further transformed into their
benzenesulfonamide derivatives. The biochemical COX-1/COX-2 selectivity was evaluated in
vitro by using the human whole blood assays of COX isozyme activity. Three compounds not
bearing the sulfonamide group present in valdecoxib were selective COX-1 inhibitors.
In tr od u ction
The enzyme cyclooxygenase (COX) catalyzes the rate-
limiting step in the formation of prostanoids from
arachidonic acid (AA).1-5 Two isoforms of the COX
enzyme (known as COX-1 and COX-2), encoded by two
different genes, have been identified.6 COX-1 displays
the characteristics of a “housekeeping gene” and is
constitutively expressed in virtually all tissues. High
expression of COX-1 has been detected in platelet and
gastric mucosa. Differently, COX-2 is expressed in
response to inflammatory and mitogenic stimuli.7 How-
ever, this simplified paradigm of constitutive COX-1 and
inducible COX-2 has many exceptions: COX-1 can be
regulated during development,1,8 whereas COX-2 is
constitutively expressed in the brain,9 reproductive
F igu r e 1.
tissues,10 and kidney.11-13 The inhibition of COX-2 is
thought to mediate the therapeutic actions of nonselec-
leaving the thromboxane action unopposed. The cardio-
tive nonsteroidal antiinflammatory drugs (NSAIDs),
vascular implications of these effects are currently
while the inhibition of COX-1 results in unwanted side
effects, particularly in the gastrointestinal (GI) tract.14,15
In fact, selective COX-2 inhibitors (denominated coxibs)
are efficacious as nonselective NSAIDs for the treatment
of acute pain and chronic inflammatory diseases and
show the advantage of reducing the GI toxicity associ-
ated with the administration of nonselective NSAIDs
by virtue of COX-1 sparing.14,15 Several selective COX-2
inhibitors such as rofecoxib,16 celecoxib,17 and valde-
coxib18 (Figure 1) have been approved for the treatment
of rheumatoid arthritis (RA) and osteoarthritis (OA) and
for the relief of acute pain associated with dental
surgery and primary dysmenorrhea.
These compounds are diaryl heterocyclic derivatives
containing a phenylsulfone or a phenylsulfonamide
moiety that interact with the COX-2 side pocket through
slow, tight-binding kinetics.19,20 However, selective COX-2
inhibition reduces the biosynthesis of prostacyclin, thus
debated on the basis of the results of the Vioxx Gas-
trointestinal Outcomes Research (VIGOR) trial showing
a statistically significant difference in acute myocardial
infarction (MI) rates between rofecoxib and naproxen,
a nonselective NSAID (0.4% vs 0.1%, respectively).21
Recently, experimental and clinical results have sug-
gested a possible involvement of COX-1 in pain and
cancer development, thus providing the rationale for the
development of selective COX-1 inhibitors.22-28 In fact,
animal studies have demonstrated that COX-1 plays a
role in intestinal polyposis and skin carcinogenesis,
consistent with epidemiological data demonstrating that
regular use of low-dose aspirin, which inhibits only
platelet COX-1 activity, can reduce colon cancer inci-
dence and mortality.29-31 In addition, in ovarian cancer,
the COX-1 enzyme is overexpressed and promotes
angiogenic growth factor production.32 Moreover, it has
been suggested that COX-1 may play an important role
in pain processing and sensitization in spinal cord and
gracile nucleus after surgery.33 Thus, selective COX-1
inhibitors might be useful analgesic and chemopreven-
tive agents.
* To whom correspondence should be addressed. Telephone:
+39 080 5442762. Fax: +39 080 5442231. E-mail: ascilimati@
farmchim.uniba.it.
† Universita` di Bari.
‡ Universita` degli Studi “G. D’Annunzio”.
10.1021/jm040782x CCC: $27.50 © 2004 American Chemical Society
Published on Web 08/26/2004

4882 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 20
Di Nunno et al.
Ta ble 1. Yields and Diastereoisomeric Ratios in 1,3-Dipolar
Cycloaddition of Arylnitrile Oxides with “Thermodynamic”
Lithium Enolate of Phenylacetone
compd
Ar
yield (%)a
drb
1a
1b
1c
1d
1e
phenyl
mesityl
5-chloro-2-furyl
2,4,6-trimethoxyphenyl
3-chloro-2,4,6-trimethoxyphenyl
61
85:15
70:30
80:20
70:30
70:30
29c
45c
52
60
a
b
Yields refer to the recrystallized products. Diastereoisomeric
ratios were determined by 1H NMR. c The apparent low yields are
due to a partial spontaneous dehydration to isoxazoles (see text
and Table 2).
F igu r e 2.
Sch em e 1
In this study, we report a novel synthesis of a series
of new 3,4-diarylisoxazole analogues of valdecoxib.
Using the well-known human blood assays for COX
isozyme selectivity,34-36 we found that sulfonamide
group removal was associated with reversal COX-2
selectivity.
F igu r e 3.
Ta ble 2. Yields of Isoxazolines (1b and 1c) and Isoxazoles (2b
and 2c) Formed in the Reaction of Mesitylnitrile Oxide and
5-Chloro-2-furylnitrile Oxide with Lithium Enolate of
Phenylacetone
Ch em istr y
yield (%)a
Recently, we found that a number of 5-alkyl-3-
arylisoxazoles structurally related to valdecoxib can be
prepared by reacting arylnitrile oxides with free enolate
ions regioselectively obtained by metallation of various
alkyl methyl ketones with lithium diisopropylamide
(LDA) at -78 °C, followed by a dehydration/aromati-
zation reaction.37 The same procedure was successfully
applied to the synthesis of valdecoxib with better results
compared to other reported valdecoxib syntheses.38,39 To
find more selective and more potent COX-2 inhibitors,
we prepared other valdecoxib-like isoxazoles by using
the same synthetic approach (Figure 2).
Ar
mesityl-
mesityl-
5-chloro-2-furyl
reaction time (h)
isoxazoline
isoxazole
3
29 (1b)
55 (2b)
73 (2b)
13 (2c)
overnight
overnight
45 (1c)
a
Yields refer to the products isolated by chromatography.
Sch em e 2
The key step in this methodology is the preparation
of the needed enolate from phenylacetone. In principle,
the phenylacetone can afford two different enolates in
the presence of a base. By use of thermodynamic control
conditions, namely, phenylacetone with LDA at 0 °C,
the needed enolate was generated. Then, such an
enolate was allowed to react with various arylnitrile
oxides to afford 3-aryl-5-hydroxy-5-methyl-4-phenyl-2-
isoxazolines (1a -e) (Scheme 1) with fair to good yields
and diasteroselectivity ratios (cis/trans) ranging be-
tween 70:30 and 85:15 (Table 1).
isoxazole by a dehydration/aromatization reaction under
basic (or acidic) conditions so that their relative distri-
bution has no synthetic importance with reference to
the isoxazole synthesis.
Furthermore, in some cases (reaction of mesitylnitrile
oxide and 5-chloro-2-furylnitrile oxide with the enolate
ion of phenylacetone and subsequent chromatographic
separation), partial or complete dehydration/aromati-
zation of 5-hydroxyisoxazolines to the corresponding
isoxazoles can also occur “spontaneously” (Table 2 and
footnote c of Table 1).
1
The diastereoisomeric ratios were determined by H
NMR spectra analysis of the reaction crudes by at-
tributing the lower (more shielded) δCH3 values to cis
1a -e (major isomers) by analogy with other already
known similar systems40 (see, for example, a comparison
of δ values of cis-1a and trans-1a with those of cis- and
trans-5-hydroxy-3,5-dimethyl-4-phenyl-2-isoxazoline A
and B in Figure 3). However, both the hydroxyisoxazo-
line diastereoisomers can be transformed into the same
Complete conversion of all 5-hydroxyisoxazolines
1a -e into the corresponding 3-aryl-5-methyl-4-phenyl-
isoxazoles 2a -e required, however, a separate treat-
ment under less basic conditions (Scheme 2 and Table
3).
As previously found,41 optimal conditions for basic
dehydration/aromatization of 5-hydroxyisoxazolines

Analogues of Valdecoxib
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 20 4883
Sch em e 3
Ta ble 3. Yields of Isoxazoles 2a -e Formed from
5-Hydroxyisoxazolines (Scheme 2)
Sch em e 4
reaction
time
compd
Ar
yield (%)a
2a
2b
2c
2d
2e
phenyl
mesityl
5-chloro-2-furyl
2,4,6-trimethoxyphenyl
3-chloro-2,4,6-trimethoxyphenyl 2 h
1 h
15 min
1 h
80
50
60
74
75
2 h
a
Yields refer to the products actually isolated by chromatog-
Ta ble 4. Yields of Valdecoxib Analogues from Compounds 2
raphy.
compd
Ar
yield (%)a
should require a not very strong base. This should
ensure, in fact, the incomplete conversion into the
corresponding alcoholates, thus allowing the existence
of the undissociated hydroxy group likely involved in
the dehydration reaction (possibly by an E1cB mecha-
nism).
As mentioned above, we applied such an approach
(1,3-dipolar cycloaddition of arylnitrile oxides with
lithium enolate of phenylacetone and dehydration of the
formed 5-hydroxyisoxazolines) to the synthesis of the
potent and selective COX-2 inhibitor 4-(5-methyl-3-
phenylisoxazol-4-yl)benzenesulfonamide, well-known as
valdecoxib (Scheme 3).42
The synthesis was accomplished by subjecting 5-meth-
yl-3,4-diphenylisoxazole 2a to reaction with ClSO3H/
NH4OH or even directly reacting hydroxyisoxazoline 1a
with ClSO3H/NH4OH. In the latter case, dehydration
to isoxazole occurs by means of ClSO3H (acid conditions)
and the conversion into valdecoxib is performed by a
“one-pot” procedure (just like already reported in the
previous synthetic approach).38,42
The actual overall yields (i.e., after separation of the
final product by chromatography and with reference to
the initial reagents) resulted in both cases being notably
higher (55% yield by performing the last conversion by
the “one-pot” procedure) compared to that (32%) re-
ported in the literature.38,39
3a
3c
3e
phenyl
5-chloro-2-furyl
3-chloro-2,4,6-trimethoxyphenyl
90
73
23
a
Yields refer to the products actually isolated by chromatog-
raphy.
effects of methoxy groups, compensating the deactiva-
tion toward the electrophiles associated with both the
preexisting chloro atom and the unfavorable position 3
of isoxazole. However, chlorination instead of sulfona-
tion of the ring is unusually observed in this case, which
could be explained by assuming that the electrophilic
species originated from ClSO3H still retains Cl (e.g.,
ClS2O6H)43,44 and, possibly due to the concomitant steric
effects of the methoxy groups, preferentially attack in
this case using the peripheral Cl rather than an internal
S atom.
Finally, preferential or exclusive reaction of ClSO3H/
NH4OH at the mesityl ring is observed in the case of
3-mesityl-5-methyl-4-phenylisoxazole 2b. In this case,
in fact, variable amounts of sulfonamides 3b and 4 were
isolated (Scheme 6 and Table 5). And this is once again
the result of the activation of such a ring by methyl
groups, associated, however, with lower steric effects
compared to 2e, as can be deduced by comparing some
reported steric parameters (v or V a constants) of Me
and OMe groups.45
On the other hand, following the same synthetic
approach, some valdecoxib analogues were also pre-
pared (Scheme 4). While 3a (valdecoxib) and 3c were
obtained in high yields (90% and 73% yield, respectively;
yields referred to the sole conversion of 2 to 3), 3e was
instead obtained in only 23% yield (Table 4). In this
case, some competition of trimethoxycholorophenyl ring
toward the reaction with ClSO3H is also observed
(Scheme 5). This is possibly due to the electron-donating
P h a r m a cology
To investigate the effect of valdecoxib molecular
structure modification on COX-1/COX-2 inhibitory ac-
tivity, we tested the synthesized isoxazoles by the
human whole blood assays, as summarized in the Table
6.
We assessed the inhibitory effects of test compounds
on lipopolysaccharide (LPS) stimulated whole blood

4884 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 20
Di Nunno et al.
Sch em e 5
Sch em e 6
F igu r e 4. Concentration-response curves for the inhibition
of whole blood COX-1 and COX-2 activities by 2a . Increasing
concentrations of 2a (0.01-100 µM) were incubated with 1 mL
of heparinized whole blood samples, drawn from healthy
volunteers pretreated with 300 mg of aspirin 48 h before
sampling, in the presence of LPS (10 µg/mL) for 24 h, and
plasma PGE2 levels were assayed as a reflection of monocyte
COX-2 activity (2). 2a (0.001-100 µM) was also incubated with
1 mL of whole blood samples (drawn from healthy subjects
when they had not taken any NSAID during the 2 weeks
preceding the study) allowed to clot for 1 h, and serum TXB2
levels were measured as a reflection of platelet COX-1 activity
(9). Results are depicted as percentage inhibition from three
to five separate experiments.
Ta ble 5. Yields of Compounds 3b and 4 Formed in the
Reaction Depicted in the Scheme 6
yield (%)
isoxazole/ClSO3H
3b
4
1:8
1:11
44
-
16
60
a
Yields refer to the products actually isolated by chromatog-
raphy.
PGE2 production and thrombin stimulated whole blood
TXB2 production, indexes of monocyte COX-2 and
platelet COX-1 activity, respectively.34-36 LPS-stimu-
lated whole blood samples, drawn from healthy subjects
treated with 300 mg of aspirin 48 h before sampling,
produced 33 ( 5.8 ng of PGE2 per mL of plasma (mean
( SEM, n ) 13). TXB2 production in clotting whole blood
samples, obtained from the same subjects in aspirin-
free periods, averaged 466 ( 53 ng/mL (mean ( SEM,
n ) 13).
sion (>90%) of platelet COX-1 activity occurred at
concentrations that did not affect monocyte COX-2
activity (Figure 5). Similar results were obtained with
2a and 2g (Figures 4 and 6). Interestingly, removal of
the sulfonamide moiety gave compounds 2g, 2a , and 2c,
which inhibited platelet COX-1 activity with IC50 values
that were 540-, 300-, and 54-fold lower than that of
valdecoxib (3a ) (used as active control), respectively,
while inhibiting monocyte COX-2 activity with IC50
values that were 2.6-, 4.3-, and >175-fold higher than
that of valdecoxib (Table 6); thus, they shared a reversal
COX-2 selectivity (2g, 2a , and 2c: COX-2/COX-1 IC50
ratios of 27.7, 30, and >200, respectively). The other
synthesized compounds showed IC50 values toward
monocyte COX-2 and platelet COX-1 activities that were
>100 µM.
These results confirm that (a) within the 3,4-diaryl-
isoxazole class (valdecoxib and its analogues) of COX-2
inhibitors, the p-sulfamoylphenyl group is essential for
good COX-2 inhibitory potency, (b) lacking the sulfona-
mide moiety reverses the COX-2 selectivity, (c) the
reduced side pocket volume of COX-1 is not the only
source of diarylheterocycle COX-2 inhibitors selectiv-
As shown in Figures 4, 5, 6, and 7, 2a ,46,47 2c, 2g,46,48
and valdecoxib (3a ) inhibited LPS-induced monocyte
COX-2 and thrombin-stimulated platelet COX-1 activi-
ties in a concentration-dependent fashion. The IC50
values for inhibition of platelet COX-1 and monocyte
COX-2 activities are detailed in Table 6.
Valdecoxib (3a ) inhibited monocyte COX-2 and plate-
let COX-1 activities with the following IC50 (95%
confidence interval) values, i.e., 27 (16.22-44.63) and
0.57 (0.4619-0.7056) µM, respectively, similar to previ-
ously published results.49
The analysis of the sigmoidal concentration-response
curves for inhibition of monocyte COX-2 and platelet
COX-1 by 2c showed that virtually complete suppres-

Analogues of Valdecoxib
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 20 4885
Ta ble 6. Inhibitory Activity of Valdecoxib Analogues by Human Whole Blood Assays in Vitroa
compd
2a
Ar
R1
R2
CH3
COX-1 IC50 (µM)
0.090b
COX-2 IC50 (µM)
2.49c
phenyl
phenyl
H
H
(0.05-0.144)
(1.577-3.954)
2g
CH3CH2
0.05b
1.49d
(0.028-0.097)
>100
(0.812-2.768)
>100
2b
2c
mesityl-
5-chloro-2-furyl
H
H
CH3
CH3
0.5b
>100b
(0.2641-0.9248)
2d
2e
2,4,6-trimethoxyphenyl
3-chloro-2,4,6-trimethoxyphenyl
phenyl
H
H
CH3
CH3
CH3
>100
>100
>100
>100
valdecoxib
SO2NH2
27e
0.57f
(3a )
3b
3c
3e
4
(16.22-44.63)
(0.4619-0.7056)
2,4,6-trimethyl-3-benzenesulfonamide
5-chloro-2-furyl
3-chloro-2,4,6-trimethoxyphenyl
2,4,6-trimethyl-3-benzenesulfonamide
H
CH3
CH3
CH3
CH3
>100
>100
SO2NH2
SO2NH2
SO2NH2
>100
>100
>100
>100
>100
>100
a
b
d
Values are the mean of at least two measurements. Values in parentheses are the IC50 confidence interval. n ) 3. c n ) 5. n ) 4.
e n ) 11. f n ) 13.
F igu r e 6. Concentration-response curves for the inhibition
of whole blood COX-1 and COX-2 activities by 2g. Increasing
concentrations of 2g (0.01-100 µM) were incubated with 1 mL
of heparinized whole blood samples, drawn from healthy
volunteers pretreated with 300 mg of aspirin 48 h before
sampling, in the presence of LPS (10 µg/mL) for 24 h, and
plasma PGE2 levels were assayed as a reflection of monocyte
COX-2 activity (2). 2g (0.001-100 µM) were also incubated
with 1 mL of whole blood samples (drawn from healthy
subjects when they had not taken any NSAID during the 2
weeks preceding the study) allowed to clot for 1 h, and serum
TXB2 levels were measured as a reflection of platelet COX-1
activity (9). Results are depicted as percentage inhibition from
three to four separate experiments.
F igu r e 5. Concentration-response curves for the inhibition
of whole blood COX-1 and COX-2 activities by 2c. Increasing
concentrations of 2c (0.1-100 µM) were incubated with 1 mL
of heparinized whole blood samples, drawn from healthy
volunteers pretreated with 300 mg of aspirin 48 h before
sampling, in the presence of LPS (10 µg/mL) for 24 h, and
plasma PGE2 levels were assayed as a reflection of monocyte
COX-2 activity (2). 2c (0.03-100 µM) was also incubated with
1 mL of whole blood samples (drawn from healthy subjects
when they had not taken any NSAID during the 2 weeks
preceding the study) allowed to clot for 1 h, and serum TXB2
levels were measured as a reflection of platelet COX-1 activity
(9). Results are depicted as percentage inhibition from 11 to
13 separate experiments.
recorded on a Mercury 300 MHz spectrometer, and chemical
shifts are reported in parts per million (δ). Absolute values of
the coupling constant are reported. IR spectra were recorded
ity,50 and (d) the striking feature of 2a , 2g, and in
particular 2c is their activity against COX-1.
In conclusion, a series of 3,4-diarylisoxazole analogues
of valdecoxib were synthesized in good overall yields.
Reversal COX-2 selectivity was obtained in 2a , 2c, and
2g compounds by removal of the sulfonamide group. The
valdecoxib analogue 2c characterized by a high selectiv-
ity toward COX-1 may represent an adequate tool to
study the involvement of COX-1 in cancer development
and pain.32,33,51-55
on
a Perkin-Elmer 681 spectrometer. GC analyses were
performed by using an HP1 column (methyl siloxane; 30 m ×
0.32 mm × 0.25 µm film thickness) on a HP 6890 model, series
II. Thin-layer chromatography (TLC) was performed on silica
gel sheets with fluorescent indicator, the spots on the TLC
were observed under ultraviolet light or were visualized with
I2 vapor. Chromatography was conducted by using silica gel
60 with a particle size distribution of 40-63 µm and 230-400
ASTM. GC-MS analyses were performed on an HP 5995C
model, and microanalyses were performed on an elemental
analyzer 1106 (Carlo Erba instrument). ESI-MS analyses were
performed on an Agilent 1100 LC/MSD trap system VL.
Ma ter ia ls. DMF from a commercial source was purified by
distillation from CaH2 under reduced pressure. Tetrahydro-
Exp er im en ta l Section
Gen er a l Meth od s. Melting points taken on an Electro-
thermal apparatus were uncorrected. 1H NMR spectra were
furan (THF) from a commercial source was purified by

4886 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 20
Di Nunno et al.
3H), 6.36 (s, 1H, aromatic proton), 8.40 (s, 1H), 10.00-10.60
(bs, 1H, OH, exchange with D2O). 13C NMR (75 MHz, CDCl3,
δ): 56.22, 56.58, 61.79, 92.68, 108.73, 109.33, 143.66, 156.99,
157.48, 158.01. GC-MS (70 eV) m/z (rel int): 247 [M(37Cl)+,
12], 245 [M(35Cl)+, 36], 230 (16), 229 (28), 228 (47), 227 (62),
215 (35), 214 (15), 213 (100), 212 (10), 186 (12), 185 (22), 184
(25), 179 (12), 178 (96), 171 (12), 170 (15), 169 (11), 163 (16),
155 (17), 143 (10), 69 (9).
2,4,6-Tr im et h oxyb en zon it r ile Oxid e a n d 3-Ch lor o-
2,4,6-tr im eth oxyben zon itr ile Oxid e. To a stirred and cooled
to 0 °C solution of 2,4,6-trimethoxybenzaldoxime (0.112 g, 0.53
mmol) dissolved in CHCl3 (5 mL), pyridine (0.027 mL) was
added. NCS (0.0706 g, 0.53 mmol) was then slowly added in
the round-bottom flask, and the turbid reaction mixture was
stirred for 2 h at room temperature. Then, water was added,
and the reaction mixture became a limpid yellow solution. The
two phases were separated, and the aqueous layer was
extracted three times with CH2Cl2. The combined organic
extracts were washed with water to remove succinimide, then
the solution was dried over anhydrous Na2SO4, and then the
solvent evaporated under vacuum. The reaction crude was
crystallized from hexane, affording the product in 70% yield.
3-Chloro-2,4,6-trimethoxybenzonitrile oxide was prepared in
quantitative yield from 2,4,6-trimethoxybenzaldoxime by using
2 equiv of NCS.
F igu r e 7. Concentration-response curves for the inhibition
of whole blood COX-1 and COX-2 activities by valdecoxib (3a ).
Increasing concentrations of valdecoxib (3a ) (0.01-100 µM)
were incubated with 1 mL of heparinized whole blood samples,
drawn from healthy volunteers pretreated with 300 mg of
aspirin 48 h before sampling, in the presence of LPS (10 µg/
mL) for 24 h, and plasma PGE2 levels were assayed as a
reflection of monocyte COX-2 activity (2). Valdecoxib (3a )
(0.01-300 µM) was also incubated with 1 mL of whole blood
samples (drawn from healthy subjects when they had not
taken any NSAID during the 2 weeks preceding the study)
allowed to clot for 1 h, and serum TXB2 levels were measured
as a reflection of platelet COX-1 activity (9). Results are
depicted as percentage inhibition from 11 to 13 separate
experiments.
2,4,6-Tr im eth oxyben zon itr ile Oxid e. Quantitative yield.
Mp 200-210 °C (hexane; dec 135 °C), yellow powder. FT-IR
(KBr): 3060, 2947, 2840, 2306, 1607, 1466, 1334, 1211, 1158,
1
1129, 1063, 1032, 962, 917, 847, 804, 769, 687 cm-1. H NMR
(CDCl3, δ): 3.83 (s, 6H), 3.84 (s, 3H), 6.05 (s, 2H). 13C NMR
(75 MHz, CDCl3, δ): 55.88, 56.14, 56.21, 84.58, 90.72, 160.25,
162.56, 163.65, 163.97. GC-MS (70 eV) m/z (rel int): 209 (100),
194 (11), 180 (8), 179(8), 166 (45), 151 (10), 123 (8), 69 (8). All
these data are identical to those of the commercially available
compound.
distillation (twice) from sodium wire under nitrogen. Stan-
dardized (2.5 M) n-butyllithium in hexane was purchased from
Aldrich Chemical Co., and its titration was performed by
N-pivaloyl-o-toluidine.56 Benzonitrile oxide41 and 5-chloro-2-
furylnitrile oxide were prepared from aldehydes through their
conversion into the corresponding oximes and then into
benzohydroximinoyl chlorides. These were finally converted
into nitrile oxides by treatment with NEt3. All other chemicals
and solvents were commercial grade further purified by
distillation or crystallization prior to use.
3-Ch lor o-2,4,6-tr im eth oxyben zon itr ile Oxid e. Quanti-
tative yield. Mp 124.6-129.8 °C (hexane, dec 117 °C), yellow
powder. FT-IR (KBr): 3023, 2986, 2949, 2847, 2305, 1609,
1583, 1468, 1454, 1436, 1414, 1334, 1231, 1210, 1162, 1131,
1
1043, 1023, 949, 915, 811 cm-1. H NMR (CDCl3, δ): 3.90 (s,
3H), 3.94 (s, 3H), 3.96 (s, 3H), 6.27 (s, 1H). 13C NMR (75 MHz,
CDCl3, δ): 56.25, 56.55, 61.78, 90.79, 91.69, 108.66, 158.80,
159.51, 161.48. GC-MS (70 eV) m/z (rel int): 245 [M(37Cl)+,
33], 243 [M(35Cl)+, 100], 228 (19), 202 (14), 200 (40), 187 (10),
185 (29), 69 (5). Anal. (C10H10ClNO4) C, H, N.
5-Ch lor o-2-fu r a n ca r boh yd r oxim oyl Ch lor id e. 2-Furan-
carbaldehydoxime57 (0.500 g, 4.505 mmol) dissolved in anhy-
drous DMF (5 mL), contained in a round-bottom flask equipped
with magnetic stirrer, was cooled to 0 °C. NCS (1.203 g, 9.009
mmol) was slowly added, and the obtained suspension was
stirred for 5 h to room temperature. Then, Et2O was added
and the solution was washed three times with water to remove
DMF. The combined organic extracts were dried over anhy-
drous Na2SO4, and then the solvent was evaporated under
vacuum. The residue was dissolved in ethyl acetate. A pale-
yellow solid formed in 75% yield by slow addition of petroleum
ether (ethyl acetate/petroleum ether ) 1:1). Mp 82-83 °C. FT-
IR (KBr): 3550-3200, 3130, 2924, 2851, 1622, 1498, 1433,
Gen er a l P r oced u r e for th e Syn th esis of 3-Ar yl-5-
h yd r oxy-5-m eth yl-4-p h en yl-2-isoxa zolin es 1a -e by Re-
a ction of Ar yln itr ile Oxid es w ith En ola te Ion of P h en -
yla ceton e (Ta ble 1). A 2.28 M solution of n-butyllithium in
hexane (1.86 mL, 4.26 mmol) was added to diisopropylamine
(0.596 mL, 4.26 mmol) in THF (20 mL) 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, phenylacetone (0.518 mL, 3.87 mmol) in THF (20 mL)
was dropwise added. The yellow reaction mixture was stirred
at 0 °C for 1 h, then a solution of arylnitrile oxide (3.87 mmol)
in THF (20 mL) was added. The reaction mixture was allowed
to reach room temperature and was stirred overnight, with
the exception of 1b that was stirred for 3 h. The reaction was
quenched by adding aqueous NH4Cl solution. The reaction
products were extracted three times with ethyl acetate. The
organic phase was dried over anhydrous Na2SO4 and then was
evaporated under vacuum. Column chromatography (silica gel,
petroleum ether/ethyl acetate ) 8:2; the ratio of the two
solvents was 6:4 for 1d and 1e) of the residue affords the
3-aryl-5-hydroxy-5-methyl-4-phenyl-2-isoxazolines in 29-61%
yield (Table 1).
1353, 1278, 1214, 1154, 1041, 1021, 1000, 939, 874, 799 cm-1
.
1H NMR (CDCl3, δ): 6.27-6.29 (d, 1H, J ) 3.50 Hz), 6.82-
6.84 (d, 1H, J ) 3.50 Hz), 8.40-10.30 (bs, 1H, OH, exchange
with D2O). 13C NMR (75 MHz, CDCl3, δ): 145.03, 139.95,
130.22, 116.07, 108.92.
3-Ch lor o-2,4,6-tr im eth oxyben za ld oxim e. In a round-
bottom flask equipped with magnetic stirrer the 2,4,6-tri-
methoxybenzaldoxime (0.300 g, 1.422 mmol) was dissolved in
anhydrous DMF (5 mL), then the suspension was cooled to 0
°C. NCS (0.189 g, 1.422 mmol) was slowly added, and the
suspension was stirred for 3 h to 0 °C. Then, water was added,
and the reaction mixture became a limpid yellow solution. The
two phases were separated, and the aqueous layer was
extracted three times with CH2Cl2. The combined organic
extracts were washed with water to remove succinimide, then
the solution was dried over anhydrous Na2SO4. A pale-yellow
solid was obtained in 88% yield after evaporation of the solvent
under vacuum. Mp 186-190 °C (lit.58 192 °C). FT-IR (KBr):
3500-3200, 3020, 2985, 2950, 1607, 1580, 1465, 1450, 1432,
1410, 1335, 1232, 1210, 1165, 1130, 1046, 1021, 948, 910, 812
5-Hyd r oxy-5-m eth yl-3,4-d ip h en yl-2-isoxa zolin e (1a ).38
61% yield. Diastereoisomeric ratio cis/trans ) 80:20. Mp 163-
165 °C, white powder. FT-IR (KBr): 3484 (very strong broad
band), 3064, 3029, 3005, 2992, 2941, 1561, 1496, 1444, 1385,
1
1356, 1235, 1147, 1094, 902, 797, 699 cm-1. H NMR (CDCl3,
1
cm-1. H NMR (CDCl3, δ): 3.84 (s, 3H), 3.92 (s, 3H), 3.94 (s,
δ): 1.28 (s, 3H, major isomer), 1.77 (s, 3H, minor stereoisomer),

Analogues of Valdecoxib
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 20 4887
2.66 (bs, 1H, OH, exchanges with D2O, minor stereoisomer),
3.26 (bs, 1H, OH, exchanges with D2O, major stereoisomer),
4.46 (s, 1H, minor stereoisomer), 4.52 (s, 1H, major stereoiso-
mer), 7.14-7.64 (m, 10H, aromatic protons, 5H for each
stereoisomer). 13C NMR (75 MHz, CDCl3, δ): 22.38, 63.35,
109.51, 127.26, 127.57, 128.34, 128.57, 128.85, 129.12, 129.46,
130.36, 134.85, 160.90. GC-MS (70 eV) m/z (rel int): 253 (M+,
1), 235 (24), 220 (7), 194 (31), 193 (100), 192 (26), 178 (7), 165
(27), 152 (3), 134 (6), 116 (9), 103 (6), 91 (10), 90 (15), 89 (21),
77 (13), 63 (5), 51(3), 43 (9). Anal. (C16H15NO2) C, H, N.
(KBr): 3466 (strong broad band), 3082, 2940, 2847, 1592, 1453,
1435, 1396, 1345, 1310, 1213, 1113, 917, 891, 807, 731 cm-1
.
1H NMR (CDCl3, δ): 1.25 (s, 3H, major isomer), 1.77 (s, 3H,
minor isomer), 2.95-3.08 (bs, 1H, OH, exchanges with D2O,
minor isomer), 3.45-3.55 (bs, 1H, OH, exchanges with D2O,
major isomer), 3.71 (s, 3H, minor isomer), 3.80 (s, 3H, major
isomer), 3.83 (s, 3H, minor isomer), 3.84 (s, 3H, major isomer),
3.92 (s, 3H, major isomer), 3.95 (s, 3H, minor isomer), 4.71 (s,
2H, one for each stereoisomer), 6.18 (s, 1H, aromatic proton
of the minor isomer), 6.25 (s, 1H, aromatic proton of the major
isomer), 7.13-7.28 (m, 10H, five aromatic protons for each
stereoisomer). 13C NMR (75 MHz, CDCl3, δ): 22.22 (major
isomer), 25.55 (minor isomer), 56.53, 62.79, 64.00 (minor
isomer), 65.65 (major isomer), 92.65, 92.91, 107.04, 107.17,
108.72, 109.36, 128.01, 128.13, 128.80 129.12, 129.49, 132.80,
134.53, 156.59, 156.77, 157.47, 157.67, 157.78. GC-MS (70 eV)
m/z (rel int): 379 [M(37Cl)+, 2], 377 [M(35Cl)+, 6], 361 (36), 360
(24), 359 (100), 346 (12), 344 (19), 319 (19), 318 (17), 317 (53),
316 (18), 304 (14), 302 (35), 287 (18), 272 (8), 227 (12), 178
(11), 134 (9), 115 (12), 103 (11), 91 (17), 77 (9), 43 (18). Anal.
(C19H20NO5Cl) C, H, N.
5-Hyd r oxy-3-(2,4,6-tr im eth ylp h en yl)-5-m eth yl-4-p h en -
yl-2-isoxa zolin e (1b). 29% yield. Diastereoisomeric ratio cis/
trans ) 75:25. Oil. FT-IR (neat): 3383 (very strong broad
band), 3020, 2922, 2852, 1612, 1495, 1455, 1383, 1312, 1264,
1216, 1136, 1095, 1034, 931, 851, 813, 758 cm-1 1H NMR
.
(CDCl3, δ): 1.45 (s, 3H, major isomer), 1.80 (s, 3H, minor
isomer), 2.20 (s, 3H for each stereoisomer), 2.25 (s, 3H for each
stereoisomer), 2.32 (s, 3H for each stereoisomer), 2.70-2.80
(bs, 1H, OH, exchanges with D2O, minor isomer), 3.00-3.10
(bs, 1H, OH, exchanges with D2O, major isomer), 4.35 (s, 1H,
major isomer), 4.65 (s, 1H, minor isomer), 6.77-6.84 (s, 2H
aromatic protons for each stereoisomer), 7.10-7.40 (m, 5H
aromatic protons for each stereoisomer). 13C NMR (75 MHz,
CDCl3, δ): 20.64, 21.12, 21.18, 22.05, 66.87, 108.66, 128.39,
128.64, 128.80, 129.05, 129.24, 129.65, 129.84, 137.56, 138.82,
162.32, 163.23. GC-MS (70 eV) m/z (rel int): 295 (M+, 4), 279
(6), 278 (27), 277 (44), 262 (11), 236 (9), 235 (38), 234 (100),
220 (13), 219 (11), 218 (13), 204 (10), 191 (4), 178 (3), 158 (15),
145 (12), 134 (19), 130 (19), 115 (11), 103 (8), 91 (29), 89 (9),
77 (12), 43 (10). Anal. (C19H21NO2) C, H, N.
Gen er a l P r oced u r e for th e Syn th esis of Isoxa zoles 2.
A solution of Na2CO3 (2.24 mmol) in water (10 mL) was added
to 3-aryl-5-hydroxy-5-methyl-4-phenyl-2-isoxazoline (1.12 mmol)
in THF (10 mL). The reaction mixture was then heated under
reflux (reaction time reported in Table 3). The two phases were
separated, and the aqueous layer was extracted three times
with ethyl acetate. The combined organic extracts were dried
over anhydrous Na2SO4 and the solvent was evaporated under
reduced pressure, affording the isoxazoles in 50-80% yield
(Table 3).
3-(5-Ch lor ofu r a n -2-yl)-5-h yd r oxy-5-m eth yl-4-p h en yl-2-
isoxa zolin e (1c). 45% yield. Diastereoisomeric ratio cis/trans
) 80:20. Mp 160.0-161.6 °C, white powder. FT-IR (KBr): 3495
(strong broad band), 3120, 3085, 3029, 3006, 2960, 1610, 1547,
1489, 1454, 1385, 1377, 1235, 1207, 1152, 1064, 1016, 955, 908,
889, 790, 752, 700 cm-1. 1H NMR (CDCl3, δ): 1.27 (s, 3H, major
isomer), 1.60-1.73 (bs, 1H, OH, exchanges with D2O, minor
isomer), 1.75 (s, 3H, minor isomer), 3.26-3.40 (bs, 1H, OH,
exchange with D2O, major isomer), 4.34 (s, 1H, minor isomer),
4.39 (s, 1H, major isomer), 6.08-6.13 (m, 3H, one furyl proton
of the major isomer and two furyl protons of the minor isomer),
6.43-6.44 (d, J ) 3.50 Hz, 1H, furyl proton of the major
isomer), 7.11-7.41 (m, 10H, five aromatic protons for each
stereoisomer). 13C NMR (75 MHz, CDCl3, δ): 22.10, 63.06,
108.69, 109.36, 115.39, 128.42, 128.55, 129.42, 134.35, 139.52,
144.09, 152.57. GC-MS (70 eV) m/z (rel int): 279 [M(37Cl)+,
7], 277 [M(35Cl)+, 20], 260 (13), 259 (9), 219 (14), 218 (22), 217
(29), 216 (14), 190 (7), 188 (16), 182 (12), 155 (19), 154 (100),
139 (11), 127 (17), 115 (10), 102 (12), 91 (11), 89 (13), 77 (10),
63 (7), 43 (18). Anal. (C14H12NO3Cl) C, H, N.
5-Meth yl-3,4-d ip h en ylisoxa zole (2a ).46,47 80% yield. Mp
97-98 °C (hexane), white crystals. FT-IR (KBr): 3051, 2928,
1619, 1597, 1573, 1497, 1464, 1436, 1414, 1376, 1304, 1239,
1074, 915, 769, 696 cm-1 1H NMR (CDCl3, δ): 2.45 (s, 3H),
.
7.17-7.47 (m, 10H). 13C NMR (75 MHz, CDCl3, δ): 11.82,
116.01, 127.90, 128.69, 128.95, 129.38, 129.60, 130.05, 130.61,
161.39, 166.85. GC-MS (70 eV) m/z (rel int): 235 (M+, 100),
220 (28), 194 (14), 193 (90), 192 (37), 165 (28), 103 (10), 90
(12), 89 (62), 78 (10), 77 (24), 63 (23), 51 (48), 43 (70). Anal.
(C16H13NO) C, H, N.
3-(2,4,6-T r im e t h y lp h e n y l)-5-m e t h y l-4-p h e n y lis o x -
a zole (2b).59 50% yield (Table 3), 73% yield if 2b is prepared
in one step, namely, directly from the mesitylnitrile oxide
without the isolation of 1b (Table 2). Mp 92-93 °C, yellow
crystals. FT-IR (KBr): 3061, 3038, 2974, 2919, 2856, 1614,
1495, 1440, 1396, 1235, 1015, 968, 883, 850, 774, 695 cm-1
.
1H NMR (CDCl3, δ): 2.03 (s, 6H), 2.28 (s, 3H), 2.58 (s, 3H),
6.86 (s, 2H), 7.01-7.04 (m, 2H, aromatic protons), 7.23-7.25
(m, 3H, aromatic protons). 13C NMR (75 MHz, CDCl3, δ):
12.39, 20.30, 21.40, 116.92, 125.88, 127.39, 128.31, 128.53,
128.81, 130.59, 137.48, 138.84, 161.69, 165,76. GC-MS (70 eV)
m/z (rel int): 277 (M+, 76), 262 (19), 235 (23), 234 (100), 219
(14), 218 (19), 204 (11), 158 (6), 115 (8), 103 (6), 91 (11), 77
(8), 43 (6). Anal. (C19H19NO) C, H, N.
5-Hydr oxy-3-(2,4,6-tr im eth oxyph en yl)-5-m eth yl-4-ph en -
yl-2-isoxa zolin e (1d ). 52% yield. Diastereomeric ratio cis/
trans ) 70:30. Mp 147-151 °C (dec 143 °C), white solid. FT-
IR (KBr): 3461 (strong broad band), 3100, 3084, 2940, 1592,
1435, 1396, 1345, 1213, 1113, 917, 891, 722 cm-1 1H NMR
.
(CDCl3, δ): 1.23 (s, 3H, major isomer), 1.76 (s, 3H, minor
isomer), 2.90-3.10 (bs, 1H, OH, exchanges with D2O, minor
isomer), 3.50-3.70 (bs, 1H, OH, exchanges with D2O, major
isomer), 3.72-3.75 (m, 18H, OCH3, nine protons for each
stereoisomer), 4.66 (s, 1H, major isomer), 4.74 (s, 1H, minor
isomer), 6.01 (s, 2H, aromatic protons, minor isomer), 6.04 (s,
2H, aromatic protons, major isomer), 7.12-7.27 (m, 10H, five
aromatic protons for each stereoisomer). 13C NMR (75 MHz,
CDCl3, δ): 21.71 (major isomer), 25.46 (minor isomer), 55.51
(minor isomer), 56.17 (major isomer), 64.24 (minor isomer),
66.23 (major isomer), 91.09, 100.49, 106.58, 108.57, 127.83,
128.58, 129.18, 129.47, 133.43, 134.86, 156.96, 157.15, 159.71,
159.88, 162.62. GC-MS (70 eV) m/z (rel int): 343 (M+, 7), 326
(24), 325 (100), 310 (20), 284 (11), 283 (48), 282 (27), 268 (19),
266 (9), 253 (8), 193 (25), 179 (8), 168 (8), 115 (13), 91 (17), 77
(12), 69 (11), 43 (17). Anal. (C19H21NO5) C, H, N.
3-(5-Ch lor o-2-fu r yl)-5-m et h yl-4-p h en ylisoxa zole (2c).
60% yield. Mp 71-73 °C, yellow crystals. FT-IR (KBr): 3147,
3051, 2927, 2848, 1633, 1520, 1435, 1412, 1236, 1204, 1134,
1020, 985, 940, 926, 897, 796, 775, 704 cm-1. 1H NMR (CDCl3,
δ): 2.36 (s, 3H), 6.11-6.12 (d, 1H, J ) 3.57 Hz), 6.25-6.27 (d,
1H, J ) 3.57 Hz), 7.25-7.30 (m, 2H, aromatic protons), 7.40-
7.47 (m, 3H, aromatic protons). 13C NMR (75 MHz, CDCl3, δ):
11.41, 108.14, 113.87, 114.99, 128.58, 129.01, 129.63, 130.20,
138.59, 143.76, 152.42, 167.10. GC-MS (70 eV) m/z (rel int):
261 [M(37Cl)+, 5], 259 [M(35Cl)+, 15], 219 (11), 217 (36), 154
(17), 127 (10), 115 (5), 102 (5), 89 (14), 77 (9), 63 (10), 51 (12),
43 (100). Anal. (C14H10NO2Cl) C, H, N.
3-(2,4,6-Tr im e t h oxyp h e n yl)-5-m e t h yl-4-p h e n ylisox-
a zole (2d ). 74% yield. Mp 210-217 °C (dec 137 °C), yellow
powder. FT-IR (KBr): 3060, 2923, 2851, 1610, 1590, 1461,
1413, 1342, 1230, 1207, 1132, 893, 812, 779, 701 cm-1. 1H NMR
(CDCl3, δ): 2.51 (s, 3H), 3.56 (s, 6H), 3.81 (s, 3H), 6.09 (s, 2H),
7.06-7.30 (m, 5H). 13C NMR (75 MHz, CDCl3, δ): 12.15, 55.55,
55.95, 90.99, 100.06, 118.08, 126.99, 128.41, 128.46, 131.57,
5-Hydr oxy-3-(2,4,6-tr im eth oxy-3-ch lor oph en yl)-5-m eth -
yl-4-p h en yl-2-isoxa zolin e (1e). 60% yield. Diastereomeric
ratio cis/trans ) 70:30. Mp 139-147 °C, white solid. FT-IR

4888 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 20
Di Nunno et al.
157.00, 159.76, 162.66, 164.93. GC-MS (70 eV) m/z (int rel):
325 (M+, 100), 310 (27), 284 (5), 283 (29), 282 (32), 268 (14),
266 (11), 253 (6), 115 (10), 103 (4), 89 (5), 77 (4), 69 (5), 43 (5).
Anal. (C19H19NO4) C, H, N.
1595, 1546, 1488, 1448, 1424, 1373, 1337, 1234, 1176, 1118,
1
1019, 886, 875, 782, 699, 665 cm-1. H NMR (CDCl3, δ): 2.01
(s, 3H), 2.42 (s, 3H), 2.58 (s, 3H), 2.65 (s, 3H), 4.93 (bs, 2H,
NH2, exchange with D2O), 6.97-7.00 (m, 3H), 7.23-7.28 (m,
3H). 13C NMR (75 MHz, CDCl3, δ) 12.37, 19.61, 21.27, 23.67,
117.08, 127.85, 128.24, 129.07, 129.76, 129.85, 132.94, 137.74,
138.11, 139.44, 142.14, 161.15, 166.59. GC-MS (70 eV) m/z
(rel int): 356 (M+, 100), 341 (4), 313 (10), 275 (20), 274 (10),
260 (13), 248 (10), 234 (15), 232 (70), 231 (13), 230 (10), 218
(21), 217 (19), 216 (9), 204 (5), 189 (6), 115 (13), 103 (8), 91
(10), 77 (9), 43 (10). MS-ESI m/z (%): 355 [M - H]- (100). Anal.
(C19H20N2O3S) C, H, N, S.
4-[3-(5-Ch lor ofu r a n -2-yl)-5-m et h ylisoxa zol-4-yl]b en -
zen su lfon a m id e (3c). 73% yield. Mp 145-147 °C (dec 114
°C), yellow crystals. FT-IR (KBr): 3330, 3258, 3005, 2924,
2848, 1632, 1564, 1520, 1442, 1336, 1165, 1020, 987, 896, 794,
572 cm-1. 1H NMR (CDCl3 , δ): 2.40 (s, 3H), 5.25 (bs, 2H, NH2,
exchange with D2O), 6.17-6.18 (d, 1H, J ) 3.57 Hz), 6.40-
6,42 (d, 1H, J ) 3.57 Hz), 7.44-7.47 (m, 2H), 8.00-8.02 (m,
2H). 13C NMR (75 MHz, CDCl3, δ): 11.62, 108.36, 113.73,
114.08, 127.09, 130.89, 134.62, 138.99, 142.02, 143.09, 152.14,
167.83. GC-MS (70 eV) m/z (rel int): 340 [M(37Cl)+, 3], 338
[M(35Cl)+, 8], 296 (18), 215 (9), 152 (4), 89 (6), 43 (100). Anal.
(C14H11N2O4SCl) C, H, N, S.
3-(2,4,6-Tr im eth oxy-3-ch lor op h en yl)-5-m eth yl-4-p h en -
ylisoxa zole (2e). 75% yield. Mp 147-151 °C (dec 143 °C),
yellow powder. FT-IR (KBr): 3061, 3006, 2943, 2862, 1599,
1455, 1388, 1350, 1297, 1215, 1112, 1017, 920, 895, 785, 749,
1
703 cm-1. H NMR (CDCl3, δ): 2.52 (s, 3H), 3.52 (s, 3H), 3.75
(s, 3H), 3.90 (s, 3H), 6.26 (s, 1H), 7.08-7.30 (m, 5H). 13C NMR
(75 MHz, CDCl3, δ): 12.11, 56.10, 56.56, 62.39, 92.65, 106.93,
109.02, 118.06, 127.34, 128.57, 128.63, 130.88, 156.62, 157.72,
165.34. GC-MS (70 eV) m/z (rel int): 361 [M(37Cl)+, 40],
359 [M(35Cl)+, 100], 344 (23), 318 (10), 317 (24), 316 (18), 302
(21), 287 (11), 115 (11), 77 (5), 43 (8). Anal. (C19H18NO4Cl) C,
H, N.
3-(3,5-Dich lor o-2,4,6-t r im et h oxyp h en yl)-5-m et h yl-4-
p h en ylisoxa zole (2f). 5% yield. Mp 104-106 °C (dec 81 °C),
yellow powder. FT-IR (KBr): 3060, 2945, 2868, 1601, 1455,
1380, 1297, 1215, 1112, 1017, 920, 894, 783, 747 cm-1. 1H NMR
(CDCl3, δ): 2.56 (s, 3H), 3.67 (s, 6H), 3.93 (s, 3H), 7.13-7.34
(m, 5H). 13C NMR (75 MHz, CDCl3): 12.12, 61.11, 62.32,
117.37, 117.94, 119.83, 127.77, 128.78, 128.94, 130.01, 154.82,
155.19, 156.14, 165.93. GC-MS (70 eV) m/z (rel int): 397
[M(37Cl2)+, 12], 395 [M(37Cl)+, 66], 393 [M(35Cl)+, 100], 380 (12),
378 (18), 353 (10), 351 (15), 350 (10), 338 (15), 336 (26), 321
4-[3-(3-Ch lor o-2,4,6-t r im et h oxyp h en yl)-5-m et h ylisox-
a zol-4-yl]ben zen su lfon a m id e (3e). 23% yield. Mp 130 °C
dec, white powder. FT-IR (KBr): 3406, 3295, 3003, 2942, 2850,
1606, 1574, 1523, 1497, 1457, 1436, 1388, 1339, 1217, 1190,
(14), 115 (17), 103 (11), 89 (9), 77 (7), 43 (19). Anal. (C19H17
NO4Cl2) C, H, N.
-
1168, 1110, 1015, 920, 892, 839, 815, 778, 748, 616, 565 cm-1
.
5-Eth yl-3,4-d ip h en ylisoxa zole (2g).46,48 n-Butyllithium in
hexane (2.19 M, 0.213 mL, 0.4675 mmol) was added to
5-methyl-3,4-diphenylisoxazole (0.100 g, 0.425 mmol) in THF
(5 mL), and the mixture was kept at -78 °C under nitrogen
using a nitrogen-flushed, three-necked flask equipped with a
magnetic stirrer, a nitrogen inlet, and two dropping funnels.
The obtained red reaction mixture was stirred for 1 h at -78
°C before addition of CH3I (4.25 mmol). The reaction mixture
was allowed to reach room temperature, and then the reaction
was quenched by adding aqueous NH4Cl solution. The two
phases were separated, and the aqueous layer was extracted
three times with ethyl acetate. The combined organic extracts
were dried over anhydrous Na2SO4, and then the solvent was
evaporated under vacuum. Column chromatography (silica gel,
petroleum ether/ethyl acetate ) 10:1) of the residue afforded
the 3,4-diphenyl-5-ethylisoxazole in 75% yield. Mp 85-87 °C
(hexane), white crystals. FT-IR (KBr): 3029, 3005, 2923, 2848,
1625, 1596, 1493, 1467, 1437, 1410, 1327, 1282, 1210, 1011,
1H NMR (CDCl3, δ): 2.54 (s, 3H), 3.55 (s, 3H), 3.76 (s, 3H),
3.91 (s, 3H), 5.01 (s, 2H, NH2, exchange with D2O), 6.27 (s,
1H), 7.22-7.26 (m, 2H), 7.78-7.81 (m, 2H). 13C NMR (75 MHz,
CDCl3, δ): 12.22, 56.12, 56.60, 62.56, 92.68, 106.03, 109.13,
116.86, 126.85, 129.06, 135.84, 140.68, 156.34, 156.50, 157.54,
158.06, 166.23. GC-MS (70 eV) m/z (rel int): 440 (M+ + 2,
37), 438 (M+, 100), 423 (13), 396 (19), 395 (8), 301 (11), 103
(10), 77 (6), 43 (19). MS-ESI m/z (%): 437 [M - H]- (100). Anal.
(C19H19N2O6SCl) C, H, N, S.
4-[3,5-Dich lor o-2,4,6-tr im eth oxyp h en yl)-5-m eth ylisox-
a zol-4-yl]b en zen su lfon a m id e (3f). 10% yield. Mp 125 °C
dec, yellow powder. FT-IR (KBr): 3405, 3265, 3005, 2918, 2850,
1581, 1451, 1388, 1341, 1293, 1261, 1236, 1165, 1095, 996, 932,
1
798, 750, 658.1, 612, 562 cm-1. H NMR (CDCl3, δ): 2.57 (s,
3H), 3.70 (s, 6H), 3.94 (s, 3H), 4.84 (s, 2H, NH2, exchange with
D2O), 7.28-7.31 (d, 2H, J ) 8.32 Hz), 7.83-7.86 (d, 2H, J )
8.32 Hz). 13C NMR (CDCl3, δ): 12.18, 61.21, 62.51, 116.78,
120.15, 127.17, 129.40, 134.96, 141.10, 154.67, 155.52, 155.87,
166.79. GC-MS (70 eV) m/z (rel int): 476 (M+ + 4, 15), 475
(M+ + 3, 16), 474 (M+ + 2, 69), 473 (M+ + 1, 23), 472 (M+,
100), 459 (9), 457 (12), 432 (11), 430 (15), 415 (12), 335 (15),
334 (10), 320 (13), 77 (5), 43 (31). Anal. (C19H18N2O6Cl2S) C,
H, N, S.
905, 771, 702 cm-1 1H NMR (200 MHz, CDCl3, δ): 1.29 (t,
.
3H, J ) 7.60 Hz), 2.78 (q, 2H, J ) 7.60 Hz), 7.12-7.43 (m,
10H). 13C NMR (75 MHz, CDCl3, δ): 12.52, 19.67, 115.20,
127.90, 128.67, 128.93, 129.41, 129.53, 130.16, 130.66, 161.34,
171.43. GC-MS (70 eV) m/z (rel int): 249 [M+, 100], 234 (6),
221 (18), 220 (99), 194 (9), 193 (61), 192 (46), 165 (17), 115 (7),
103 (8), 89 (53), 77 (15), 63 (10), 51 (10). Anal. (C17H15NO) C,
H, N.
2,4,6-Tr im eth yl-3-[5-m eth yl-4-(4-su lfa m oylp h en yl)isox-
a zol-3-yl]ben zen esu lfon a m id e (4). 16% yield. Mp 123-125
°C, yellow crystal. FT-IR (KBr): 3369, 3263, 3095, 2980, 2926,
1622, 1594, 1556, 1429, 1371, 1330, 1239, 1163, 1098, 900, 841,
759, 615, 561 cm-1. 1H NMR (CD3CN, δ): 2.04 (s, 3H), 2.39 (s,
3H), 2.60 (s, 3H), 2.65 (s, 3H), 5.69 (bs, 4H, NH2, exchange
with D2O), 7.17 (s, 1H), 7.25-7.28 (d, 2H, J ) 8.27 Hz), 7.77-
7.80 (d, 2H, J ) 8.27 Hz). 13C NMR (75 MHz, CDCl3, δ): 11.73,
19.01, 19.74, 22.89, 115.69, 126.64, 129.01, 129.37, 132.91,
134.26, 137.68, 138.97, 139.56, 141.70, 142.48, 161.06, 168.29.
MS-ESI m/z (%): 434 [M - H]- (100). Anal. (C19H21N3O5S2)
C, H, N, S.
P h a r m a cologica l Meth od s. We tested the synthesized
isoxazoles by the human whole blood assays in vitro34-36 to
investigate the effect of substituents on COX-1/COX-2 inhibi-
tory activity with respect to valdecoxib.
Hu m a n Wh ole Blood Assa ys. Su bjects. Eleven healthy
volunteers (six females aged 25-29 years) were enrolled to
participate in the study after its approval by the ethical
committee of the University “G. D’Annunzio” in Chieti. In-
formed consent was obtained from each subject. The same
healthy volunteers were studied on different occasions.
Gen er a l P r oced u r e for th e Syn th esis of Ben zen e-
su lfon a m id es 3. To the isoxazole cooled to 0 °C in a round-
bottom flask equipped with magnetic stirrer chlorosulfonic acid
(1:11) was added. The brown reaction mixture was stirred at
0 °C for 2 h and then dropwise added to a stirring suspension
of ice (10 mL) and dichloromethane (10 mL). Then, the layers
were separated and the organic phase was added directly to a
solution of ammonium hydroxide (28% NH3 in water) kept at
0 °C. This biphasic mixture was vigorously stirred at 0 °C for
2 h. The two phases were separated, and the aqueous layer
was extracted three times with CH2Cl2. The combined organic
extracts were dried over anhydrous Na2SO4 and then evapo-
rated under vacuum. Column chromatography (silica gel,
petroleum ether/ethyl acetate ) 6:4) of the residue afforded
the product in the 10-90% yield (Tables 4 and 5). Analytical
and spectroscopic data of valdecoxib (3a ) were identical to
those reported.38
2,4,6-Tr im eth yl-3-(5-m eth yl-4-p h en ylisoxa zol-3-yl)ben -
zen esu lfon a m id e (3b). 44% yield. Mp 187-189 °C; white
crystals. FT-IR (KBr): 3341, 3236, 3066, 2921, 2853, 1617,

Analogues of Valdecoxib
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 20 4889
COX-2 Assa y. One milliliter aliquots of peripheral venous
blood samples containing 10 IU of sodium heparin were
incubated in the presence of LPS (10 µg/mL) or saline for 24
h at 37 °C as previously described. The contribution of platelet
COX-1 activity was suppressed by pretreating the subjects
with 300 mg of aspirin 48 h before sampling. Plasma was
separated by centrifugation (10 min at 2000 rpm) and kept at
-70 °C until assayed for prostaglandin (PG) E2 as an index of
LPS-induced monocyte COX-2 activity.34
COX-1 Assa y. Peripheral venous blood samples were drawn
from the same donors when they had not taken any NSAID
during the 2 weeks preceding the study. One milliliter aliquots
of whole blood were immediately transferred into glass tubes
and allowed to clot at 37 °C for 1 h. Serum was separated by
centrifugation (10 min at 3000 rpm) and kept at -70 °C until
assayed for thromboxane (TX) B2. Whole blood TXB2 produc-
tion was measured as a reflection of maximally stimulated
platelet COX-1 activity in response to endogenously formed
thrombin.35,36
Effects of th e Test Com p ou n d s on Wh ole Blood COX-2
a n d COX-1 Activities. Compounds (0.005-150 mM) were
dissolved in DMSO, and 2 µL aliquots of the solutions were
pipetted directly into test tubes to give final concentrations of
0.01-300 µM in blood. Four to nine different concentrations
of each compound were incubated with heparinized whole
blood samples in the presence of LPS (10 µg/mL) for 24 h or
with whole blood samples allowed to clot at 37 °C for 1 h to
examine the concentration dependence of COX-2 vs COX-1
inhibition.34,35 The actual concentrations of the compounds
used for each assay are reported in the figure captions.
An a lyses of P GE2 a n d TXB2. PGE2 and TXB2 concentra-
tions were measured by previously described and validated
radioimmunoassays.36,60 Unextracted plasma and serum
samples were diluted in the standard diluent of the assay (0.02
M phosphate buffer, pH 7.4) and assayed in a volume of 1.5
mL at a final dilution of 1:50 to 1:30000. We used 4000 dpm
of [3H]PGE2 or [3H]TXB2 and specific anti-PGE2 and anti-TXB2
sera diluted 1:100000 and 1:120000, respectively. The least
detectable concentration was 1-2 pg/mL for both prostanoids.
Ma ter ia ls. [3H]PGE2 and [3H]TXB2 (specific activity, >100
Ci/mmol) were from Perkin-Elmer Life Science Products
(Brussels, Belgium). Authentic PGE2 and TXB2 were from
Cayman Chemical Company (Ann Arbor, MI). Anti-PGE2 and
anti-TXB2 sera were obtained in our laboratory, and their
characteristics have been described previously. Heparin, LPS
derived from Escherichia coli 026:B6, and dimethyl sulfoxide
(DMSO) were purchased from Sigma Chemical Company
(St. Louis, MO).
Note Ad d ed a fter ASAP P ostin g. This paper was
published ASAP without final corrections. The corrected
version was reposted 09/08/2004.
Refer en ces
(1) Smith, W. L.; Langenbach, R. Why there are two cyclooxygenase
isozymes. J . Clin. Invest. 2001, 12, 1491-1495.
(2) Dennis, E. A. Phospholipase A2 in eicosanoid generation. Am.
J . Respir. Crit. Care. Med. 2000, 161, S32-S35.
(3) Marnett, L. J .; Rowlinson, S. W.; Goodwin, D. C.; Kalgutkar, A.
S.; Lanzo, C. A. Arachidonic acid oxygenation by COX-1 and
COX-2. Mechanisms of catalysis and inhibition. J . Biol. Chem.
1999, 274 (33), 22903-22906.
(4) J akobsson, P. J .; Thoren, S.; Morgenstern, R.; Samuelsson, B.
Identification of the human prostaglandin E synthase: A mi-
crosomal, glutathione-dependent, inducible enzyme, constituting
a potential novel drug target. Proc. Natl. Acad. Sci. U.S.A. 1999,
96 (13), 7220-7225.
(5) Tanioka, T.; Nakatani, Y.; Semmyo, N.; Murakami, M.; Kudo,
I. Molecular identification of cytosolic prostaglandin E2 synthase
that is functionally coupled with cyclooxygenase-1 in immediate
prostaglandin E2 biosynthesis. J . Biol. Chem. 2000, 275 (42),
32775-32782.
(6) Xie, W.; Chipman, J . G.; Robertson, D. L.; Erikson, R. L.;
Simmons, D. L. Expression of a mitogen-responsive gene encod-
ing prostaglandin synthase is regulated by mRNA splicing. Proc.
Natl. Acad. Sci. U.S.A. 1991, 88, 2692-2696.
(7) Fitzpatrick, F. A.; Soberman, R. Regulated formation of
eicosanoids. J . Clin. Invest. 2001, 107, 1347-1351.
(8) Rocca, B.; Spain, L. M.; Pure, E.; Langenbach, R.; Patrono, C.;
FitzGerald, G. A. Distinct roles of prostaglandin H synthases 1
and 2 in T-cell development. J . Clin. Invest. 1999, 103, 1469-
1477.
(9) Yamagata, K.; Andreasson, K. I.; Kaufmann, W. E.; Barnes, C.
A.; Worley, P. F. Expression of a mitogen-inducible cyclooxy-
genase in brain neurons: regulation by synaptic activity and
glucocorticoids. Neuron 1993, 11, 371-386.
(10) Kniss, D. A. Cyclooxygenases in reproductive medicine and
biology. J . Soc. Gynecol. Invest. 1999, 6, 285-292.
(11) Harris, R. C.; McKanna, J . A.; Akai, Y.; J acobson, H. R.; Dubois,
R. N.; Breyer, M. D. Cyclooxygenase-2 is associated with the
macula densa of rat kidney and increases with salt restriction.
J . Clin. Invest. 1994, 94, 2504-2510.
(12) Vio, P.; Cespedes, C.; Gallardo, P.; Masferrer, J . L. Renal
identification of cyclooxygenase-2 in a subset of thick ascending
limb cells. Hypertension 1997, 30, 687-692.
(13) Ferreri, N. R.; An, S. J .; McGiff, J . C. Cyclooxygenase-2 expres-
sion and function in the medullary thick ascending limb. Am.
J . Physiol. 1999, 277, F360-F368.
(14) Fitzgerald, G. A.; Patrono, C. The coxibs, selective inhibitors of
cyclooxygenase-2. N. Engl. J . Med. 2001, 345, 433-442.
(15) Patrono, C.; Patrignani, P.; Garc`ıa Rodriguez, L. A. Cyclooxy-
genase-selective inhibition of prostanoid formation: transducing
biochemical selectivity into clinical read-outs. J . Clin. Invest.
2001, 108, 7-13.
Sta tistica l An a lysis. For each experiment, the amount of
PGE2 produced in LPS-stimulated whole blood in the presence
of an inhibitor was subtracted from that produced in the
presence of saline and DMSO. The effects of the test com-
pounds were calculated and represented as percent inhibition
of prostanoid production assessed in the absence of the test
compounds (control). Concentration-response curves were fit-
ted, and IC50 values were analyzed with PRISM (GraphPad,
San Diego, CA). The IC50 values were reported as mean values,
and 95% confidence intervals were calculated.
(16) Clemett, D.; Goa, K. L. Celecoxib:
a review of its use in
osteoarthritis, rheumatoid arthritis and acute pain. Drugs 2000,
59 (4), 957-980.
(17) Matheson, A. J .; Figgitt, D. Rofecoxib: a review of its use in the
management of osteoarthritis, acute pain and rheumatoid
arthritis. Drugs 2001, 61 (6), 833-865.
(18) Ormrod, D.; Wellington, K.; Wagstaff, A. J . Valdecoxib. Drugs
2002, 62 (14), 2059-2071.
(19) Kurumbail, R. G.; Stevens, A. M.; Gierse, J . K.; McDonald, J .
J .; Stegeman, R. A.; Pak, J . Y.; Gildehaus, D.; Miyashiro, J . M.;
Penning, T. D.; Seibert, K.; Isakson, P. C.; Stallings, W. C.
Structural basis for selective inhibition of cyclooxygenase-2 by
anti-inflammatory agents. Nature 1996, 384, 644-648.
(20) Walker, M. C.; Kurumbail, R. G.; Kiefer, J . R.; Moreland, K. T.;
Koboldt, C. M.; Isakson, P. C.; Seibert, K.; Gierse, J . K. A three-
step kinetic mechanism for selective inhibition of cyclo-oxyge-
nase-2 by diarylheterocyclic inhibitors. Biochem. J . 2001, 357,
709-718.
Ack n ow led gm en t. This work was financially sup-
ported by the Ministero dell’Istruzione, dell’Universita`
e della Ricerca (MIUR, Rome), Ministero della Salute
(MinSan, Rome) under the framework of the National
Project “Progetto Strategico BS2, “PREVENZIONE,
DIAGNOSTICA PRECOCE E CONTROLLO DELLA
MALATTIA NEOPLASTICA”, the University of Bari,
the Istituto di Chimica dei Composti OrganoMetallici
(ICCOM-CNR, Bari), and the Center of Excellence on
Aging, “G. d’Annunzio” University of Chieti.
(21) Bombardier, C.; Laine, L.; Reicin, A.; Shapiro, D.; Burgos-Vargas,
R.; Davis, B.; Day, R.; Ferraz, M. B.; Hawkey, C. J .; Hochberg,
M. C.; Kvien, T. K.; Schnitzer, T. J . Comparison of upper
gastrointestinal toxicity of rofecoxib and naproxen in patients
with rheumatoid arthritis (VIGOR Study Group). N. Engl. J .
Med. 2000, 343, 1520-1528.
(22) Kitamura, T.; Itoh, M.; Noda, T.; Matsuura, M.; Wakabayashi,
K. Combined effects of cyclooxygenase-1 and cyclooxygenase-2
selective inhibitors on intestinal tumorigenesis in adenomatous
polyposis coli gene knockout mice. Int. J . Cancer 2004, 109 (4),
576-580.
Su p p or tin g In for m a tion Ava ila ble: Elemental analysis
data of the compounds in the manuscript. This material is
available free of charge via the Internet at http://pubs.acs.org.

4890 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 20
Di Nunno et al.
(23) Rotondo, S.; Krauze-Brzosko, K.; Manarini, S.; Evangelista, V.;
Cerletti, C. Licofelone, an inhibitor of cyclooxygenase and
5-lipoxygenase, specifically inhibits cyclooxygenase-1-dependent
platelet activation. Eur. J . Pharmacol. 2004, 488 (1-3), 79-83.
(24) Bottone, F. G., J r; Martinez, J . M.; Alston-Mills, B.; Eling, T. E.
Gene modulation by COX-1 and COX-2 specific inhibitors in
human colorectal carcinoma cancer cells. Carcinogenesis 2004,
25 (3), 349-357.
(41) Di Nunno, L.; Scilimati, A. Synthesis of 3-aryl-4,5-dihydro-5-
hydroxy-1,2-oxazoles by reaction of substituted benzonitrile
oxides with the enolate ion of acetaldehyde. Tetrahedron 1987,
43, 2181-2189.
(42) Sorbera, L. A.; Leeson, P. A.; Castan˜er, J .; Castan˜er, R. N.
Valdecoxib and Parecoxib Sodium. Drugs Future 2001, 26, 133-
140.
(43) van Albada, M. P.; Cerfontain, H. Aromatic Sulphonation. Part
60. Sulphonation in the Reaction of Aromatic Compounds with
Chlorosulphuric Acid in Nitromethane and in Dichloromethane.
J . Chem. Soc., Perkin Trans. 2 1977, 1548-1557.
(44) van Albada, M. P.; Cerfontain, H. Aromatic Sulphonation. Part
61. Sulphonylation in the Reaction of Aromatic Compounds with
Chlorosulphuric Acid in Nitromethane and in Dichloromethane.
J . Chem. Soc., Perkin Trans. 2 1977, 1557-1559.
(25) Krul, E. S. Methods of treating or preventing a cardiovascular
condition using
a cyclooxygenase-1 inhibitor. U.S. Patent
2003162824, 2003; CAN 139:191440, 2003.
(26) Teng, X. W.; Abu-Mellal, A. K. M.; Davies, N. M. Formulation
dependent pharmacokinetics, bioavailability and renal toxicity
of a selective cyclooxygenase-1 inhibitor SC-560 in the rat. J .
Pharm. Pharm. Sci. 2003, 6 (2), 205-210.
(27) Dooley, C. M.; Devocelle, M.; McLoughlin, B.; Nolan, K. B.;
Fitzgerald, D. J .; Sharkey, C. T. A novel family of hydroxamate-
based acylating inhibitors of cyclooxygenase. Mol. Pharmacol.
2003, 63 (2), 450-455.
(45) Smith, M. B.; March, J . Effects of Structure on Reactivity. In
March’s Advanced Organic Chemistry: Reactions, Mechanisms,
and Structure, 5th ed.; A Wiley-Interscience Publication, J ohn
Wiley & Sons: New York, 2001; pp 374-375.
(28) Penning, T. D.; Talley, J . J .; Bertenshaw, S. R.; Carter, J . S.;
Collins, P. W.; Docter, S.; Graneto, M. J .; Lee, L. F.; Malecha, J .
W.; Miyashiro, J . M.; Rogers, R. S.; Rogier, D. J .; Yu, S. S.;
Anderson, G. D.; Burton, E. G.; Cogburn, J . N.; Gregory, S. A.;
Koboldt, C. M.; Perkins, W. E.; Seibert, K.; Veenhuizen, A. W.;
Zhang, Y. Y.; Isakson, P. C. Synthesis and biological evaluation
of the 1,5-diarylpyrazole class of cycloxygenase-2 inhibitors:
identification of 4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1H-
pyrazol-1-yl]benzenesulfonamide (SC-58635, Celecoxib). J . Med.
Chem. 1997, 40, 1347-1365.
(29) Chulada, P. C.; Thompson, M. B.; Mahler, J . F.; Doyle, C. M.;
Gaul, B. W.; Lee, C.; Tiano, H. F.; Morham, S. G.; Smithies, O.;
Langenbach, R. Genetic disruption of Ptgs-1, as well as Ptgs-2,
reduces intestinal tumorigenesis in Min mice. Cancer Res. 2000,
60, 4705-4708
(30) Tiano, H. F.; Loftin, C. D.; Akunda, J .; Lee, C. A.; Spalding, J .;
Sessoms, A.; Dunson, D. B.; Rogan, E. G.; Morham, G.; Smart,
R. C.; Langenbach, R. Deficiency of either cyclooxygenase
(COX)-1 or COX-2 alters epidermal differentiation and reduces
mouse skin tumorigenesis. Cancer Res. 2002, 62 (12), 3395-
3401.
(31) Thun, M. J .; Henley, S. J .; Patrono, C. Nonsteroidal anti-
inflammatory drugs as anticancer agents: mechanistic, phar-
macologic, and clinical issues. J Natl. Cancer Inst. 2002, 94 (4),
252-266.
(32) Gupta, R. A.; Tejada, L. V.; Tong, B. J .; Das, S. K.; Morrow, J .
D.; Dey, S. K.; DuBois, R. N. Cyclooxygenase-1 is Overexpressed
and Promotes Angiogenic Growth Factor Production in Ovarian
Cancer. Cancer Res. 2003, 63, 906-911.
(33) Zhu, X.; Conklin, D.; Eisenach, J . C. Cyclooxygenase-1 in the
spinal cord plays an important role in postoperative pain. Pain
2003, 104, 15-23.
(34) Patrignani, P.; Panara, M. R.; Greco, A.; Fusco, O.; Natoli, C.;
Iacobelli, S.; Cipollone, F.; Ganci, A.; Creminon, C.; Maclouf, J .;
Patrono, C. Biochemical and pharmacological characterization
of the cyclooxygenase activity of human blood prostaglandin
endoperoxide synthases. J . Pharmacol. Exp. Ther. 1994, 271,
1705-1721.
(35) Patrignani, P.; Filabozzi, P.; Patrono, C. Selective cumulative
inhibition of platelet thromboxane production by low-dose aspirin
in healthy subjects. J . Clin. Invest. 1982, 69, 1366-1372.
(36) Patrono, C.; Ciabattoni, G.; Pinca, E.; Pugliese, F.; Castrucci,
G.; De Salvo, A.; Satta, M. A.; Peskar, B. A. Low dose aspirin
and inhibition of thromboxane B2 production in healthy subjects.
Thromb. Res. 1980, 17 (3-4), 317-327.
(37) Di Nunno, L.; Scilimati, A.; Vitale, P. Regioselective synthesis
and side-chain metallation and elaboration of 3-aryl-5-alkyl-
isoxazoles. Tetrahedron 2002, 58, 2659-2665.
(38) Talley, J . J .; Brown, D.; Nagarajan, S.; Carter, J . S.; Weier, R.
M.; Stealey, M. A.; Collins, P. W.; Rogers, R. S.; Seibert, K.
Substituted Isoxazoles for the treatment of Inflammation. U.S.
Patent 5,633,272, 1997; CAN 127:65756, 1997.
(39) Dileep Kumar, J . S.; Ho, M. M.; Leung, J . M.; Toyokuni, T.
Convenient Approach to 3,4-Diarylisoxazoles Based on the
Suzuki Cross-Coupling Reaction. Adv. Synth. Catal. 2002, 344,
1146-1151.
(46) Bellec, C.; Bertin, D.; Colau, R.; Deswarte, S.; Maitte, P.; Viel,
C. Pre´paration d’isoxazoles trisubstitue´s par re´duction chimique
et e´lectrochimique d’R-acyl â-nitrostilbe`nes (Preparation of
trisubstituted isoxazoles by chemical and electrochemical reduc-
tion of R-acyl-â-nitrostilbenes). J . Heterocycl. Chem. 1979, 16 (8),
1657-1759.
(47) Letendre, L. J .; Kunda, S. A.; Gallagher, D. J .; Seaney, L. M.
Method for preparing valdecoxib and related benzenesulfonyl
compounds. PCT Int. Appl. WO 2003029230, 2003; CAN 138:
304268, 2003.
(48) Ethyl-3,4-diphenylisoxazole was synthesized as reported in the
Experimental Section. See also the following: Scilimati, A.; Di
Nunno, L.; Vitale, P.; Patrignani, P.; Tacconelli, S. Italian Patent
MI2004A000019, 2004.
(49) Tacconelli, S.; Capone, M. L.; Sciulli, M. G.; Ricciotti, E.;
Patrignani, P. The selectivity of novel COX-2 inhibitors in
human whole blood assays of COX-isozyme activity. Curr. Med.
Res., Opin. 2002, 18 (8), 503-511.
(50) Hood, W. F.; Gierse, J . K.; Isakson, P. C.; Kiefer, J . R.;
Kurumbail, R. G.; Seibert, K.; Monahan, J . B. Characterization
of celecoxib and valdecoxib binding to cyclooxygenase. Mol.
Pharmacol. 2003, 63, 870-877.
(51) Chandrasekharan, N. V.; Dai, H.; Roos, L. T.; Evanson, N. K.;
Tomsik, J .; Elton, S. T.; Simmons, D. L. COX-3, a cyclooxyge-
nase-1 variant inhibited by acetaminophen and other analgesic/
antipyretic drugs: cloning, structure, and expression. Proc. Natl.
Acad. Sci. U.S.A. 2002, 99, 13926-13931.
(52) Schwab, J . M.; Schluesener, J .; Laufer, S. COX-3: just another
COX or the solitary elusive target of paracetamol? Lancet 2003,
361, 981-982.
(53) Schwab, J . M.; Beiter, T.; Linder, J . U.; Laufer, S.; Schulz, J .
E.; Meyermann, R.; Schluesener, H. J . COX-3: a virtual pain
target in humans? FASEB J . 2003, 17 (15), 2174-2175.
(54) Warner, T. D.; Mitchell, J . A. Cyclooxygenase-3 (COX-3): filling
in the gaps toward a COX continuum? Proc. Natl. Acad. Sci.
U.S.A. 2002, 99, 13371-13373.
(55) McAdam, B. F.; Mardini, I. A.; Habib, A.; Burke, A.; Lawson, J .
A.; Kapoor, S.; FitzGerald, G. A. Effect of regulated expression
of human cyclooxygenase isoforms on eicosanoid and isoe-
icosanoid production in inflammation. J . Clin. Invest 2000, 105
(10), 1473-1482.
(56) Suffert, J . Simple Direct Titration of Organolithium Reagents
Using N-Pivaloyl-o-toluidine and/or N-Pivaloyl-o-benzylaniline.
J . Org. Chem. 1989, 54, 509-510.
(57) Kanemasa, S.; Matsuda, H.; Kamimura, A. Kakinami, T. Syn-
thesis of Hydroximoyl Chlorides from Aldoximes and Benzylt-
rimethylammonium Tetrachloroiodate (BTMA ICl4). Tetrahedron
2000, 56, 1057-1064.
(58) Lloyd, G.; Whalley, W. B.; Travers, R. B. Some Chlorinated
Derivatives of Phloroglucinol. J . Chem. Soc. 1956, 3209-3212.
(59) Davies, M. W.; Wybrow, R. A. J .; J ohnson, C. N.; Harrity, J . P.
A. A regioselective cycloaddition route to isoxazoleboronic esters.
Chem. Commun. 2001, 1558-1559.
(60) Ciabattoni, G.; Pugliese, F.; Spaldi, M.; Cinotti, G. A.; Patrono,
C. Radioimmunoassay measurement of prostaglandins E2 and
F2alpha in human urine. J . Endocrinol. Invest. 1979, 2 (2),
173-182.
(40) Escale, R.; Petrus, F.; Verducci, J . Synthe`se et caracte´risation
d’hydroxy-5-isoxazolines-2 (Synthesis and characterization of
hydroxy-5-isoxazolines-2). Bull. Soc. Chim. Fr. 1974, 3-4 (part
2), 725-733.
J M040782X