Synthesis of some 5-phenylisoxazole-3-carboxylic acid derivatives as potent xanthine oxidase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2010.02.013

A number of 5-phenylisoxazole-3-carboxylic acid derivatives (5a-e, 11a-e) were synthesized and analyzed for their ability to inhibit xanthine oxidase. Most of the compounds exhibited potency levels in the micromolar/submicromolar range. The presence of a cyano group at the 3-position of phenyl moiety turned out to be the preferred substitution pattern, as its transformation into the nitro group determined a general reduction of the inhibitory potency. A molecular modeling study on compound 11a was performed to gain an insight into its binding mode with xanthine oxidase, and to provide the basis for further structure-guided design of new non-purine xanthine oxidase inhibitors related with 5-phenylisoxazole-3-carboxylic acid scaffold.

Full text of DOI:10.1016/j.ejmech.2010.02.013

European Journal of Medicinal Chemistry 45 (2010) 2663–2670
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis of some 5-phenylisoxazole-3-carboxylic acid derivatives as potent
xanthine oxidase inhibitors
,
b
a
a
a
a
a
Shaojie Wang a , Jufang Yan , Jian Wang , Jiarun Chen , Tingjian Zhang , Yong Zhao , Mingxing Xue
*
a Key Laboratory of New Drugs Design and Discovery of Liaoning Province, Shenyang Pharmaceutical University, 103 Culture Road, Shenhe District, Shenyang 110016, China
b Drug Screening Center, Di Ao Pharmaceutical Group, Chengdu 123388, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A number of 5-phenylisoxazole-3-carboxylic acid derivatives (5a–e, 11a–e) were synthesized and
analyzed for their ability to inhibit xanthine oxidase. Most of the compounds exhibited potency levels in
the micromolar/submicromolar range. The presence of a cyano group at the 3-position of phenyl moiety
turned out to be the preferred substitution pattern, as its transformation into the nitro group determined
a general reduction of the inhibitory potency. A molecular modeling study on compound 11a was per-
formed to gain an insight into its binding mode with xanthine oxidase, and to provide the basis for
further structure-guided design of new non-purine xanthine oxidase inhibitors related with 5-phenyl-
isoxazole-3-carboxylic acid scaffold.
Received 10 November 2009
Received in revised form
1 February 2010
Accepted 4 February 2010
Available online 10 February 2010
Keywords:
5-Phenylisoxazole-3-carboxylic acid
Synthesis
Ó 2010 Elsevier Masson SAS. All rights reserved.
Xanthine oxidase inhibitor
1. Introduction
Europe last year and in the US this year [7]. Encouraged by the
outstanding potency of these inhibitors, we attempt to adopt other
Gout and hyperuricemia, associated with an elevated uric acid
(UA) level in the blood, are common human metabolic disorders.
These lead to the accumulation of UA in joints and kidneys, which
in turn causes gouty arthritis and UA nephrolithiasis [1]. Xanthine
oxidase (XO) is a key enzyme in the last two steps of purine
metabolic pathway, catalyzing the oxidation of hypoxanthine to
xanthine, and then to UA [2]. Since reducing plasma levels of UA can
prevent gout, therapy is possible with XO inhibitors that block the
production of UA from purine [3]. Allopurinol (Fig. 1), a known XO
inhibitor and an analogue of hypoxanthine, has been widely
prescribed as a treatment for hyperuricemia and gout. In some
cases, however, severe life-threatening side effects have been
reported. These include a toxicity syndrome dramatized by eosin-
ophilia, vasculitis, rash hepatitis, and progressive renal failure [4].
Therefore, novel non-purine alternatives to allopurinol with potent
XO inhibitory activity, but possessing fewer side effects are in great
demand.
five-membered rings, such as isosteres of pyrazole and thiazole, to
design their analogues with the hope of extending the structure–
activity relationships of non-purine XO inhibitors and finding more
potent compounds.
In this paper, we describe the synthesis and preliminary struc-
ture–activity relationships of 5-phenylisoxazole-3-carboxylic acid
derivatives as potent XO inhibitors.
2. Results and discussion
2.1. Chemistry
The synthesis of the target 5-phenylisoxazole-3-carboxylic acid
derivatives 5a–e and 11a–e was performed as outlined in Scheme 1.
The commercially available 1-(4-hydroxyphenyl)ethanone was
nitrated with concentrated nitric acid in hot acetic acid to give 1-(4-
hydroxy-3-nitrophenyl)ethanone 1, which was then alkylated with
appropriate alkyl halide in DMF in the presence of potassium
carbonate to obtain 1-(4-alkyloxy-3-nitrophenyl)ethanones 2a–e
[8] (Scheme 1).
The 1-(4-alkyloxy-3-cyanophenyl)ethanones 8a–e, as the key
intermediates for the synthesis of target compounds 11a–e, were
prepared through a three-step sequence. In the process, 1-(4-
hydroxyphenyl)ethanone was treated with iodine and potassium
iodide in aqueous ammonia hydroxide to afford 1-(4-hydroxy-3-
Under efforts to find novel XO inhibitors without purine back-
bone, 2-phenylthiazoles [5] and 1-phenylpyrazoles [6] had been
designed and tested as xanthine oxidase inhibitors. Among them,
febuxostat and Y-700 (Fig. 1) were shown to be promising xanthine
oxidase inhibitors [5,6]. The former had already been approved in
* Corresponding author. Tel./fax: þ86 24 2398 6421.
E-mail address: sjwang_99@yahoo.com.cn (S. Wang).
0223-5234/$ – see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.02.013

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S. Wang et al. / European Journal of Medicinal Chemistry 45 (2010) 2663–2670
potency of 5-phenylisoxazole-3-carboxylic acid derivatives could
be inferred.
The presence of a cyano group at the 3-position of phenyl
moiety (11a–e) caused a significant improvement in the activity
with respect to the nitro group (5a–e). In particular, compounds
11a, 11b, and 11d showed submicromolar inhibitory activity. The
same effect also happened in XO inhibitors 1-phenypyrazoles,
represented by Y-700 [6].
Fig. 1. Chemical structures of allopurinol, febuxostat, and Y-700.
The different alkyloxy groups at the 4-position of phenyl moiety
were introduced to verify the influence of substitution pattern on
the inhibitory potency of these molecules. Within the series,
compounds 5a and 11a bearing isobutyl substituent proved to be
iodophenyl)ethanone 6 [9], which was alkylated as described for 2b
[8] and subsequently cyanided with cuprous cyanide in DMF [10]
(Scheme 1).
extremely potent, with an IC50 value of 1.00 and 0.36 mM, respec-
tively. The replacement of isobutyl group with benzyl group
decreased the inhibitory effectiveness, similar to the case of
compounds 5b and 11b. In order to explore the possibility of
improving the inhibitory activity of benzyl-substituted derivatives,
compounds 5c–e and 11c–e were synthesized. As shown in Tables 1
and 2, except for compounds 5c and 11c that proved to be inactive,
most of the compounds exhibited potency levels in the micro-
molar/submicromolar range, but they were all less active as
compared to the unsubstituted compounds 5b and 11b. The
insertion of a chlorine atom (5d and 11d) as an electron-
withdrawing group determined a general increase in the inhibi-
tory potency with respect to the insertion of the electron-releasing
groups (5c, 5e, 11c, and 11e). The presence of bulky tert-butyl group
(5e and 11e) improved the inhibitory activity when compared to
compounds 5c and 11c, which bear methyl group.
The diketo esters 3a–e and 9a–e, which exclusively exist in their
enol tautomeric forms, were obtained by the condensation of 2a–e
and 8a–e with diethyl oxalate in THF in the presence of sodium
ethoxide [11,12]. Finally, cyclocondensation of the appropriate
diketo ester with hydroxylamine hydrochloride in refluxing
ethanol provided isoxazoles 4a–e and 10a–e [13], which were then
hydrolyzed with sodium hydroxide to obtain (after acidification)
the target 5-phenylisoxazole-3-carboxylic acid derivatives 5a–e
and 11a–e [14].
The structures of the synthesized target compounds were
elucidated by TOF-HRMS, 1H NMR, and IR findings. All spectral data
were in accordance with assumed structures. In the TOF-HRMS
analysis, compound 11a showed M þ H ion peak, while the other
compounds showed M þ Na ion peaks. The IR spectra displayed the
nitro group stretching vibrations at 1531–1533 cmꢀ1 for
compounds 5a–e, and the cyano group stretching vibrations at
2223–2229 cmꢀ1 for compounds 11a–e. In the 1H NMR spectra, the
H-4 of isoxazole was observed around 7.50 ppm as a singlet. In the
case of compounds 5b, 5d, 5e, 11a, and 11b, it was overlapped with
the protons of benzene rings.
Compound 11a represented the most potent molecule among
the series. This indicates that the combination of a cyano group and
a bulky isobutyl group is favorable for the XO inhibitory activity of
5-phenylisoxazole-3-carboxylic acid derivatives, as previously
observed by Ishibuchi et al. for 1-phenylpyrazole inhibitors [6].
2.2. Biological activity
2.3. Molecular modeling
The in vitro bovine XO inhibitory activity of compounds 5a–e
and 11a–e was measured spectrophotometrically by following uric
acid levels at 295 nm [15,16]. Febuxostat was included as a refer-
ence compound. The results are shown in Tables 1 and 2. In general,
most of the 5-phenylisoxazole-3-carboxylic acid derivatives
exhibited potent inhibitory activity, but much lower than that of
febuxostat. Although the number of synthesized target compounds
is limited, some interesting characteristics for the XO inhibitory
The crystal structures of complexes of xanthine dehydrogenase
with febuxostat and Y-700 show a highly specific binding pocket,
presenting a long, narrow cavity leading toward the molybdenum–
pterin center [4,17]. The molybdenum–pterin sites of both xanthine
oxidase and xanthine dehydrogenase are structurally equivalent
[18].
To better understand the fairly good XO inhibitory potency of
compound 11a at the molecular level, and to gain an insight into its
Scheme 1. Reagents and conditions: (i) conc. HNO3, HOAc, 50–60 ꢁC; (ii) isobutyl bromide, K2CO3, PEG-400, DMF, 80 ꢁC or alkyl halide, K2CO3, KI, DMF, 65 ꢁC; (iii) conc. NH4OH, I2,
KI, H2O, r.t.; (iv) alkyl halide, K2CO3, KI, DMF, 65 ꢁC; (v) CuCN, DMF, 140 ꢁC; (vi) diethyl oxalate, EtONa, EtOH, –6 ꢁC then r.t.; (vii) NH2OH.HCl, EtOH, reflux; (viii) 4% NaOH solution,
THF, 50 ꢁC, then acidified with a 5% HCl solution.

S. Wang et al. / European Journal of Medicinal Chemistry 45 (2010) 2663–2670
2665
Table 1
However, the potency of compound 11a to inhibit XO was weaker
than that of febuxostat and Y-700 [5,6]. The difference in the
positions of nitrogen atom of heterocyclic rings may be one reason.
The N-3 of febuxostat and N-2 of Y-700 are presented at the similar
position relative to the phenyl portion, while the nitrogen atom of
isoxazole ring of compound 11a is far from the phenyl portion than
that of febuxostat and Y-700, making the nitrogen atom adjacent to
the phenyl portion as the preferred orientation. The difference in
the steric bulk of the heterocyclic rings may provide another
plausible explanation for the potency difference between
compound 11a and febuxostat, as suggested by Fukunari et al. [17].
The C-4 of compound 11a is smaller than the corresponding sulfur
atom of febuxostat, and the compound 11a also lacks a methyl
group, which could introduce additional bulk and hydrophobic
interaction. Thus, introducing some groups into the C-4 position to
increase its bulky and hydrophobic effects may be favorable for the
potency of 5-phenylisoxazole-3-carboxylic acid derivatives against
XO.
In vitro xanthine oxidase inhibitory activity of 5-phenylisoxazole-3-carboxylic acids
5a–e.
N
O
COOH
RO
NO2
a
Compound
R
IC50 (mM)
5a
5b
5c
5d
Isobutyl
Benzyl
4-Me-benzyl
4-Cl-benzyl
4-tert-Butyl-benzyl
1.00
0.97
n.a.b
2.83
12.75
0.003
5e
Febuxostat
a
IC50 values: the concentration of the inhibitor required to produce 50% inhibi-
tion of xanthine oxidase.
b
n.a.: not active (less than 50% inhibition at 10 mg/mL).
Lastly, the bovine and human sequences of this enzyme are
characterized by 90% identity, and almost all of the amino acids
interact with the compound 11a are conserved in the bovine and
human enzymes [20,21]. Therefore, the mechanism of the XO
inhibition by the compound 11a seems applicable to human XO as
well.
binding mode with XO, experimental docking into the binding
pocket of the bovine milk xanthine dehydrogenase/febuxostat
complex (PDB entry code 1N5X) [4] was performed. The docking
experiment was carried out using the Molegro Virtual Docker [19],
and the carboxyl group of compound 11a was calculated in disso-
ciated form.
It is known from the crystal structures of complexes of xanthine
dehydrogenase with febuxostat and Y-700 that they bind xanthine
dehydrogenase with the carboxylate moiety interacting with
Arg880 and Thr1010, the nitrogen atom of heterocycles forming
hydrogen bond with Glu802, and the nitrile group of phenyl unit
forming hydrogen bond with Asn768 [4,17]. Accordingly,
compound 11a was expected to bind with XO in a similar way. As
shown in Fig. 2, compound 11a indeed forms hydrogen bonds with
Arg880, Thr1010, Glu802, and Asn768. Moreover, additional
hydrogen binding between the carboxyl group of Glu802 and the
oxygen atom of isoxazole ring occurred.
The isoxazole ring as a whole was sandwiched between Phe914
and Phe1009. The benzonitrile portion was inserted between
Leu873 and Leu1014. The hydrophobic 4-isobutoxy tail surrounded
by several hydrophobic amino acid residues was positioned at the
entrance of the active site-directed channel. All these hydrophobic
interactions are very similar to those of febuxostat and Y-700 [4,17]
and may contribute to the binding of compound 11a to XO.
The binding mode of compound 11a is thus very similar to that
of febuxostat and Y-700 [4,17]. This explains the fairly good XO
inhibitory potency of compound 11a at the molecular level.
3. Conclusions
We report the synthesis and in vitro XO inhibitory activity of the
5-phenylisoxazole-3-carboxylic acid derivatives. Among the
derivatives synthesized, compounds 5b, 11a, 11b, and 11d with IC50
values lower than 1 mM were obtained. The presence of a cyano
group at the 3-position of phenyl moiety turned out to be the
preferred substitution pattern, as its transformation into the nitro
group determined a general reduction of the inhibitory potency.
The present study also points out that the combination of a cyano
group and a bulky isobutyl group is preferred for the XO inhibitory
activity of 5-phenylisoxazole-3-carboxylic acid derivatives.
Among the xanthine oxidase inhibitors used, allopurinol is the
most widely employed. However, adverse side effects associated
with the use of allopurinol are sometime very serious and a search
for substitutes is therefore highly warranted. The non-purine XO
inhibitors related with 5-phenylisoxazole-3-carboxylic acid scaf-
fold might circumvent such problems. Obviously, the toxicological
profile of 5-phenylisoxazole-3-carboxylic acid derivatives and their
in vivo hypouricemic effects have to be established before their
therapeutic potentials can be substantiated. However, these
compounds, especially compound 11a, constitute plausible lead
candidates for further investigations.
Table 2
In vitro xanthine oxidase inhibitory activity of 5-phenylisoxazole-3-carboxylic acids
11a–e.
4. Experimental protocols
N
O
COOH
4.1. Chemistry
RO
Melting points were recorded via a YRT-3 melting point appa-
ratus, and were uncorrected. 1H NMR spectra were recorded in
DMSO-d6 or CDCl3 on a Bruker ARX-300 spectrometer, and chem-
CN
a
Compound
R
IC50 (mM)
11a
11b
11c
11d
11e
Febuxostat
Isobutyl
Benzyl
4-Me-benzyl
4-Cl-benzyl
4-tert-Butyl-benzyl
0.36
0.59
n.a.b
0.63
1.01
ical shifts (d) were given in ppm downfield from TMS and used as
the internal standard. Coupling constant (J) values were in Hz. IR
spectra were determined as KBr pellets on Bruker IFS-55 spec-
trometer, and were expressed in cmꢀ1. The progress of the reactions
was monitored by TLC using several solvent systems of different
polarities. TOF-HRMS spectra were determined on an Agilent 1100
instrument. All solvents and reagents were of analytical grade and
used as received.
0.003
a
IC50 values: the concentration of the inhibitor required to produce 50% inhibi-
tion of xanthine oxidase.
b
n.a.: not active (less than 50% inhibition at 10 mg/mL).

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S. Wang et al. / European Journal of Medicinal Chemistry 45 (2010) 2663–2670
Fig. 2. Amino acid residues interacting with compound 11a (gray) in the structure of the enzyme inhibitor complex. Hydrogen bonds are represented by the dashed green lines.
4.1.1. Synthesis of 1-(4-hydroxy-3-nitrophenyl)ethanone (1)
A solution of 1-(4-hydroxyphenyl)ethanone (15 g, 0.11 mol) in
acetic acid (40 mL) was heated to 45 ꢁC, to which concentrated
nitric acid (10.5 mL, 0.11 mol) was gradually added to maintain the
temperature at 50–60 ꢁC. Then, the mixture was stirred at this
temperature for 0.5 h, and then poured into ice-cold water
(200 mL). The precipitate was filtered, washed with water, and
recrystallized from ethanol to give 1 (10.5 g, 52.6%) as a yellow
solid, m.p. 131.0–132.6 ꢁC (m.p. 128–130 ꢁC [8]).
4.1.4. Synthesis of 1-[4-(4-methylbenzyloxy)-
3-nitrophenyl]ethanone (2c)
Compound 2c was synthesized in the same manner as that for
2b, but using 4-methylbenzyl chloride instead of benzyl chloride as
a yellow solid with yield of 66%, m.p. 108.8–111.8 ꢁC; 1H NMR
(CDCl3):
d
8.40 (d, 1H, J ¼ 2.2 Hz, Ar–H), 8.20 (dd, 1H, J ¼ 2.2 and
8.8 Hz, Ar–H), 7.56 (d, 1H, J ¼ 8.9 Hz, Ar–H), 7.35 (d, 2H, J ¼ 8.0 Hz,
Ar–H), 7.22 (d, 2H, J ¼ 7.9 Hz, Ar–H), 5.37 (s, 2H, CH2), 2.58 (s, 3H,
CH3), 2.31(s, 3H, CH3).
4.1.2. Synthesis of 1-(4-isobutyloxy-3-nitrophenyl)ethanone (2a)
A mixture of compound 1 (15.8 g, 0.087 mol), isobutyl bromide
(41.6 g, 0.31 mol), anhydrous K2CO3 (42.2 g, 0.31 mol), PEG-400
(4 mL), and DMF (150 mL) was heated under stirring at 80 ꢁC for
7 h. After the reaction was completed, the mixture was poured into
water (600 mL), and extracted with methylene dichloride
(80 mL ꢂ 3). The combined organic layer was washed with a 3%
potassium hydroxide solution (100 mL ꢂ 2), dried over anhydrous
Na2SO4 and filtered. The filtrate was evaporated under vacuum to
obtain a residue. Water (100 mL) was then added to the residue.
The precipitate was collected and washed with water to yield 2a
(12.1 g, 58.4%) as a dark yellow solid, m.p. 64.4–66.4 ꢁC. 1H NMR
4.1.5. Synthesis of 1-[4-(4-chlorobenzyloxy)-
3-nitrophenyl]ethanone (2d)
Compound 2d was synthesized in the same manner as that for
2b, but using 4-chlorobenzyl chloride instead of benzyl chloride as
a light yellow solid with yield of 53.3%, m.p. 118.7–120.0 ꢁC. 1H NMR
(DMSO-d6):
d
8.42 (d, 1H, J ¼ 2.1 Hz, Ar–H), 8.22 (dd, 1H, J ¼ 2.2 and
8.8 Hz, Ar–H), 7.56 (d, 1H, J ¼ 8.9 Hz, Ar–H), 7.50 (s, 4H, Ar–H), 5.43
(s, 2H, CH2), 2.59 (s, 3H, CH3).
4.1.6. Synthesis of 1-[4-(4-tert-butylbenzyloxy)-
3-nitrophenyl]ethanone (2e)
Compound 2e was synthesized in the same manner as that for
2b, but using 4-tert-butylbenzyl chloride instead of benzyl chloride
as a light yellow solid with yield of 59.8%, m.p. 96.1–97.3 ꢁC (from
(DMSO-d6):
d
8.41 (d, 1H, J ¼ 2.2 Hz, Ar–H), 8.20 (dd, 1H, J ¼ 2.2 and
8.8 Hz, Ar–H), 7.48 (d, 1H, J ¼ 8.9 Hz, Ar–H), 4.04 (d, 2H, J ¼ 6.4 Hz,
CH2), 2.59 (s, 3H, CH3), 2.12–1.99 (m, 1H, CH), 0.98 (d, 6H, J ¼ 6.7 Hz,
2CH3).
ethanol). 1H NMR (DMSO-d6):
d
8.40 (d, 1H, J ¼ 2.1 Hz, Ar–H), 8.21
(dd, 1H, J ¼ 2.2 and 8.8 Hz, Ar–H), 7.58 (d, 1H, J ¼ 8.9 Hz, Ar–H), 7.42
(m, 4H, Ar–H), 5.38 (s, 2H, CH2), 2.58 (s, 3H, CH3), 1.28 (s, 9H, 3CH3).
4.1.3. Synthesis of 1-(4-benzyloxy-3-nitrophenyl)ethanone (2b)
A mixture of compound 1 (5 g, 0.028 mol), benzyl chloride
(6.4 mL, 0.055 mol), anhydrous K2CO3 (11.4 g, 0.083 mol), KI
(0.27 g), and DMF (50 mL) was heated under stirring at 65 ꢁC for
3 h. After the reaction was completed, the mixture was poured into
water (300 mL). The precipitate was collected by filtration and
recrystallized from ethyl acetate to yield 2b (5 g, 66.8%) as a light
yellow solid, m.p. 132.7–134.7 ꢁC (m.p. 134–136 ꢁC [8]).
4.1.7. General procedure for the synthesis of compounds 3a–e
Sodium metal (0.31 g, 13.4 mmol) was cautiously added to
absolute ethanol (15 mL) gave a solution of sodium ethoxide, to
which a solution of diethyl oxalate (1.78 g, 12.2 mmol) and the
corresponding 2 (12.2 mmol) in THF (25 mL) was added dropwise
under stirring at ꢀ6 ꢁC. The mixture was stirred at the same
temperature for 1 h, and was then stirred at room temperature for

S. Wang et al. / European Journal of Medicinal Chemistry 45 (2010) 2663–2670
2667
4 h. After the reaction was completed, the solvent was removed
under reduced pressure. Water (80 mL) was added to the residue,
which was acidified with a 5% HCl solution to pH 3. The resulting
precipitate was collected by filtration and recrystallized to yield the
desired product.
J ¼ 2.2 and 8.8 Hz, Ar–H), 7.65 (d, 1H, J ¼ 8.9 Hz, Ar–H), 7.58 (s, 1H,
4-H), 7.49–7.36 (m, 5H, Ar–H), 5.42 (s, 2H, CH2), 4.40 (q, 2H,
J ¼ 7.1 Hz, CH2CH3), 1.34 (t, 3H, J ¼ 7.1 Hz, CH2CH3).
4.1.8.3. Ethyl 5-[4-(4-methylbenzyloxy)-3-nitrophenyl]isoxazole-3-
carboxylate (4c). Yield of 72.0%, a white solid, m.p. 189.0–189.7 ꢁC
4.1.7.1. (Z)-Ethyl 2-hydroxy-4-(4-isobutoxy-3-nitrophenyl)-4-oxobut-
(from acetonitrile); 1H NMR (DMSO-d6):
d
8.49 (d, 1H, J ¼ 2.2 Hz,
2-enoate (3a). Yield of 55.8%, a white solid, m.p. 102.5–104.1 ꢁC
Ar–H), 8.21 (dd, 1H, J ¼ 2.2 and 8.8 Hz, Ar–H), 7.63 (d, 1H, J ¼ 9.0 Hz,
Ar–H), 7.56 (s, 1H, 4-H), 7.35 (d, 2H, J ¼ 8.0 Hz, Ar–H), 7.22 (d, 2H,
J ¼ 7.9 Hz, Ar–H), 5.36 (s, 2H, CH2), 4.40 (q, 2H, J ¼ 7.1 Hz, CH2CH3),
2.31 (s, 3H, CH3), 1.34 (t, 3H, J ¼ 7.1 Hz, CH2CH3).
(from ethanol); 1H NMR (CDCl3):
d 15.14 (br s, 1H, OH), 8.48 (d,
1H, J ¼ 2.2 Hz, Ar–H), 8.17 (dd, 1H, J ¼ 2.3 and 8.9 Hz, Ar–H), 7.16
(d, 1H, J ¼ 8.9 Hz, Ar–H), 7.02 (s, 1H, olefin-H), 4.42 (q, 2H,
J ¼ 7.1 Hz, CH2CH3), 3.96 (d, 2H, J ¼ 6.4 Hz, CH2), 2.24–2.15 (m,
1H, CH), 1.42 (t, 3H, J ¼ 7.1 Hz, CH2CH3), 1.08 (d, 6H, J ¼ 6.7 Hz,
2CH3).
4.1.8.4. Ethyl 5-[4-(4-chlorobenzyloxy)-3-nitrophenyl]isoxazole-3-
carboxylate (4d). Yield of 86.4%, a white solid, m.p. 174.3–175.2 ꢁC
(from ethanol); 1H NMR (DMSO-d6):
d
8.51 (d, 1H, J ¼ 2.1 Hz, Ar–H),
4.1.7.2. (Z)-Ethyl 4-(4-benzyloxy-3-nitrophenyl)-2-hydroxy-4-oxobut-
8.24 (dd, 1H, J ¼ 2.1 and 8.8 Hz, Ar–H), 7.63 (d, 1H, J ¼ 9.0 Hz, Ar–H),
7.58 (s, 1H, 4-H), 7.50 (s, 4H, Ar–H), 5.42 (s, 2H, CH2), 4.40 (q, 2H,
J ¼ 7.1 Hz, CH2CH3), 1.34 (t, 3H, J ¼ 7.1 Hz, CH2CH3).
2-enoate (3b). Yield of 36.5%, a light yellow solid, m.p. 116.4–117.4 ꢁC
(from ethanol); 1H NMR (CDCl3):
d 15.13 (br s, 1H, OH), 8.49 (d, 1H,
J ¼ 2.3 Hz, Ar–H), 8.15 (dd, 1H, J ¼ 2.3 and 8.9 Hz, Ar–H), 7.47–7.36
(m, 5H, Ar–H), 7.23 (d, 1H, J ¼ 8.9 Hz, Ar–H), 7.01 (s, 1H, olefin-H),
5.35 (s, 2H, CH2), 4.41 (q, 2H, J ¼ 7.1 Hz, CH2CH3), 1.42 (t, 3H,
J ¼ 7.1 Hz, CH2CH3).
4.1.8.5. Ethyl 5-[4-(4-tert-butylbenzyloxy)-3-nitrophenyl)]isoxazole-
3-carboxylate (4e). Yield of 71.4%, a white solid, m.p. 165.5–166.4 ꢁC
(from ethanol); 1H NMR (DMSO-d6):
d
8.50 (d, 1H, J ¼ 2.2 Hz, Ar–H),
8.23 (dd, 1H, J ¼ 2.2 and 8.9 Hz, Ar–H), 7.65 (d, 1H, J ¼ 9.0 Hz, Ar–H),
7.58 (s, 1H, 4-H), 7.42 (q, 4H, J ¼ 8.4 Hz, Ar–H), 5.37 (s, 2H, CH2), 4.40
(q, 2H, J ¼ 7.1 Hz, CH2CH3), 1.34 (t, 3H, J ¼ 7.1 Hz, CH2CH3), 1.28 (s,
9H, 3CH3).
4.1.7.3. (Z)-Ethyl 2-hydroxy-4-[4-(4-methylbenzyloxy)-3-nitrophenyl]-
4-oxobut-2-enoate (3c). Yield of 41.5%, a light yellow solid, m.p.
109.4–111.2 ꢁC (from ethyl acetate); 1H NMR (DMSO-d6):
d 8.24 (d,1H,
J ¼ 1.6 Hz, Ar–H), 8.03 (d, 1H, J ¼ 8.6 Hz, Ar–H), 7.44 (d, 1H, J ¼ 8.6 Hz,
Ar–H), 7.35 (d, 2H, J ¼ 7.7 Hz, Ar–H), 7.21 (d, 2H, J ¼ 7.8 Hz, Ar–H), 6.25
(s,1H, olefin-H), 5.29 (s, 2H, CH2), 4.10 (q, 2H, J ¼ 7.1 Hz, CH2CH3), 2.31
(s, 3H, CH3), 1.23 (t, 3H, J ¼ 7.1 Hz, CH2CH3).
4.1.9. General procedure for the synthesis of compounds 5a–e
A mixture of the corresponding 4 (2.33 mmol), 4% NaOH solu-
tion (23.3 mL), and THF (10 mL) was heated at 50 ꢁC for 1.5 h. After
the reaction was completed, the solvent was then removed under
reduced pressure. The resulting pasty residue was dissolved in
water (80 mL) and acidified with a 5% HCl solution to pH 1. The
suspension was stirred at room temperature for 20 min. The solid
was collected by filtration, washed with water, and recrystallized to
yield the desired product.
4.1.7.4. (Z)-Ethyl 4-[4-(4-chlorobenzyloxy)-3-nitrophenyl]-2-hydroxy-
4-oxobut-2-enoate (3d). Yield of 49.0%, a yellow solid, m.p. 130.9–
132.1 ꢁC (from acetone-ethanol); 1H NMR (DMSO-d6):
d 8.61 (s, 1H,
Ar–H), 8.38 (d,1H, J ¼ 8.4 Hz, Ar–H), 7.60 (d,1H, J ¼ 8.4 Hz, Ar–H), 7.50
(s, 4H, Ar–H), 7.18 (s, 1H, olefin-H), 5.45 (s, 2H, CH2), 4.32 (q, 2H,
J ¼ 7.1 Hz, CH2CH3), 1.32 (t, 3H, J ¼ 7.1 Hz, CH2CH3).
4.1.9.1. 5-(4-isobutoxy-3-nitrophenyl)isoxazole-3-carboxylic
(5a). Yield of 41.0%, a light yellow solid, m.p. 181.3–182.2 ꢁC (from
methanol); 1H NMR (DMSO-d6):
14.1 (br s, 1H, COOH), 8.46 (d, 1H,
acid
4.1.7.5. (Z)-Ethyl 4-[4-(4-tert-butylbenzyloxy)-3-nitrophenyl]-2-hydroxy-
4-oxobut-2-enoate (3e). Yield of 38.3%, a yellow solid, m.p. 128.3–
d
130.0 ꢁC (from ethyl acetate); 1H NMR (DMSO-d6):
d
8.49 (d, 1H,
J ¼ 2.1 Hz, Ar–H), 8.19 (dd, 1H, J ¼ 2.1 and 8.8 Hz, Ar–H), 7.54 (d, 1H,
J ¼ 8.9 Hz, Ar–H), 7.48 (s, 1H, 4-H), 4.04 (d, 2H, J ¼ 6.4 Hz, CH2),
2.12–1.99 (m, 1H, CH), 0.99 (d, 6H, J ¼ 6.7 Hz, 2CH3); IR (KBr, cmꢀ1):
3453, 2964,1720,1624, 1531, 1447,1397,1384,1286,1167,1001; TOF-
J ¼ 2.1 Hz, Ar–H), 8.23 (dd, 1H, J ¼ 2.1 and 8.8 Hz, Ar–H), 7.63 (d, 1H,
J ¼ 9.0 Hz, Ar–H), 7.50 (s, 5H, Ar–H, olefin–H), 5.31 (s, 2H, CH2), 4.39 (q,
2H, J ¼ 7.1 Hz, CH2CH3), 1.25 (m, 12H, 3CH3, CH2CH3).
HRMS
329.0750).
m/z ¼ 329.0739
[M þ Na]þ(C14H14N2NaO6
requires
4.1.8. General procedure for the synthesis of compounds 4a–e
A mixture of the corresponding 3 (5.93 mmol), hydroxylamine
hydrochloride (0.46 g, 5.93 mmol), and anhydrous ethanol (60 mL)
was heated under reflux for 6 h. After the reaction was completed,
about one-third of the solvent was evaporated, and the residue was
let to stand at room temperature for 2 h to complete the precipi-
tation. The precipitate was collected by filtration, washed with
ethanol, and recrystallized to yield the desired product.
4.1.9.2. 5-(4-benzyloxy-3-nitrophenyl)isoxazole-3-carboxylic
acid
(5b). Yield of 67%, a light yellow solid, m.p. 189.6–191.5 ꢁC (from
acetic acid); 1H NMR (DMSO-d6):
d
8.48 (d, 1H, J ¼ 2.0 Hz, Ar–H),
8.21 (dd, 1H, J ¼ 2.0 and 8.8 Hz, Ar–H), 7.64 (d, 1H, J ¼ 8.9 Hz, Ar–H),
7.49–7.36 (m, 6H, 4-H, Ar–H), 5.42 (s, 2H, CH2); IR (KBr, cmꢀ1):
3453, 2926, 1711, 1625, 1531, 1496, 1445, 1385, 1356, 1301, 1268;
TOF-HRMS m/z ¼ 363.0593 [M þ Na]þ(C17H12N2NaO6 requires
363.0593).
4.1.8.1. Ethyl 5-(4-isobutoxy-3-nitrophenyl)isoxazole-3-carboxylate
(4a). Yield of 80.7%, a white solid, m.p. 134.8–136.1 ꢁC (from
ethanol); 1H NMR (CDCl3):
d
8.27 (d, 1H, J ¼ 2.2 Hz, Ar–H), 7.95 (dd,
4.1.9.3. 5-[4-(4-methylbenzyloxy)-3-nitrophenyl]isoxazole-3-carbox-
ylic acid (5c). Yield of 61%, a light yellow solid, m.p. 173.2–174.7 ꢁC
1H, J ¼ 2.3 and 8.8 Hz, Ar–H), 7.18 (d, 1H, J ¼ 8.9 Hz, Ar–H), 6.91 (s,
1H, 4-H), 4.48 (q, 2H, J ¼ 7.1 Hz, CH2CH3), 3.94 (d, 2H, J ¼ 6.4 Hz,
CH2), 2.24–2.15 (m, 1H, CH), 1.45 (t, 3H, J ¼ 7.1 Hz, CH2CH3), 1.08 (d,
6H, J ¼ 6.7 Hz, 2CH3).
(from methanol); 1H NMR (DMSO-d6):
d
8.47 (d, 1H, J ¼ 2.1 Hz, Ar–
H), 8.20 (dd,1H, J ¼ 2.4 and 9.3 Hz, Ar–H), 7.63 (d, 1H, J ¼ 9.0 Hz, Ar–
H), 7.49 (s, 1H, 4-H), 7.35 (d, 2H, J ¼ 8.0 Hz, Ar–H), 7.22 (d, 2H,
J ¼ 8.1 Hz, Ar–H), 5.36 (s, 2H, CH2), 2.31 (s, 3H, CH3); IR (KBr, cmꢀ1):
3511, 3139, 2920, 1725, 1624, 1531, 1449, 1384, 1277, 1173, 996;
TOF-HRMS m/z ¼ 377.0738 [M þ Na]þ(C18H14N2NaO6 requires
377.0750).
4.1.8.2. Ethyl 5-(4-benzyloxy-3-nitrophenyl)isoxazole-3-carboxylate
(4b). Yield of 86.4%, a white solid, m.p. 171.8–173.0 ꢁC (from
ethanol); 1H NMR (DMSO-d6):
d 8.51 (s, 1H, Ar–H), 8.23 (dd, 1H,

2668
S. Wang et al. / European Journal of Medicinal Chemistry 45 (2010) 2663–2670
4.1.9.4. 5-[4-(4-chlorobenzyloxy)-3-nitrophenyl]isoxazole-3-carbox-
4.1.15. Synthesis of 1-[4-(4-tert-butylbenzyloxy)-
ylic acid (5d). Yield of 25.8%, a light yellow solid, m.p. 195.5–
3-iodophenyl]ethanone (7e)
196.9 ꢁC (from ethanol–THF); 1H NMR (DMSO-d6):
d
8.49 (d, 1H,
Similar to the procedure used to prepare 2b, reaction of 6 with
4-tert-butylbenzyl chloride provided 7e in a yield of 66.9% as
a yellow solid, m.p. 107.9–108.8 ꢁC (from ethanol). 1H NMR (CDCl3):
J ¼ 2.1 Hz, Ar–H), 8.23 (dd, 1H, J ¼ 2.1 and 8.8 Hz, Ar–H), 7.62 (d, 1H,
J ¼ 9.0 Hz, Ar–H), 7.50 (s, 5H, 4-H, Ar–H), 5.42 (s, 2H, CH2); IR (KBr,
cmꢀ1): 3487, 3145,1719, 1624, 1533, 1494, 1444, 1304, 1264, 1011;
TOF-HRMS m/z ¼ 397.0196 [M þ Na]þ(C17H11ClN2NaO6 requires
397.0204).
d
8.41 (d, 1H, J ¼ 2.1 Hz, Ar–H), 7.91 (dd, 1H, J ¼ 2.1 and 8.6 Hz, Ar–
H), 7.42 (s, 4H, Ar–H), 6.89 (d,1H, J ¼ 8.6 Hz, Ar–H), 5.20 (s, 2H, CH2),
2.53 (s, 3H, CH3), 1.33 (s, 9H, 3CH3).
4.1.9.5. 5-[4-(4-tert-butylbenzyloxy)-3-nitrophenyl]isoxazole-3-car-
4.1.16. General procedure for the synthesis of compounds 8a–e
A mixture of the corresponding 7 (18.3 mmol), cuprous cyanide
(2.63 g, 29.3 mmol), and DMF (50 mL) was heated at 140 ꢁC for 7 h.
After the reaction was completed, the mixture was cooled to room
temperature, poured into water (200 mL), and stirred at room
temperature for 0.5 h. The formed precipitate was collected by
filtration and dried at room temperature to give the crude product
as a solid. The solid was extracted with hot chloroform (100 mL)
and filtrated. The filtrate was evaporated under reduced pressure,
and the residue was then recrystallized to yield the desired
product.
boxylic acid (5e). Yield of 62.5%, a yellow solid, m.p. 158.5–159.8 ꢁC
(from ethanol); 1H NMR (DMSO-d6):
d
8.47 (d, 1H, J ¼ 2.2 Hz, Ar–H),
8.21 (dd, 1H, J ¼ 2.2 and 8.8 Hz, Ar–H), 7.65 (d, 1H, J ¼ 9.0 Hz, Ar–H),
7.49–7.38 (m, 5H, 4-H, Ar–H), 5.37 (s, 2H, CH2), 1.28 (s, 9H, 3CH3); IR
(KBr, cmꢀ1): 3458, 3143, 2962, 1732, 1625, 1596, 1532, 1449, 1384,
1284; TOF-HRMS m/z ¼ 419.1218 [M þ Na]þ(C21H20N2NaO4 requires
419.1219).
4.1.10. Synthesis of 1-(4-hydroxy-3-iodophenyl)ethanone (6)
To a solution of KI (60.1 g, 0.37 mol) and I2 (18.7 g, 0.074 mol) in
water (160 mL), 1-(4-hydroxyphenyl)ethanone (10 g, 0.074 mol) in
concentrated NH4OH (500 mL) was added rapidly with stirring. The
resulting mixture was stirred overnight at room temperature (color
changed from gray to yellow). After the reaction was completed, the
mixture was filtered. The filtrate was acidified with concentrated
HCl solution, and the yellow precipitate was collected by filtration.
The crude product was recrystallized from methanol–water (7:3) to
afford 6 (10.8 g, 56.1%) as a yellow solid, m.p. 153.4–156.0 ꢁC (m.p.
153–155 ꢁC [9]).
4.1.16.1. 1-(3-cyano-4-isobutoxyphenyl)ethanone
58.4%, a white solid, m.p. 97.1–97.9 ꢁC (from ethanol); 1H NMR
(DMSO-d6):
(8a). Yield
of
d
8.34 (d, 1H, J ¼ 2.2 Hz, Ar–H), 8.20 (dd, 1H, J ¼ 2.2 and
8.9 Hz, Ar–H), 7.37 (d, 1H, J ¼ 8.9 Hz, Ar–H), 4.03 (d, 2H, J ¼ 6.5 Hz,
CH2), 2.57 (s, 3H, CH3), 2.16–2.03 (m, 1H, CH), 1.02 (d, 6H, J ¼ 6.7 Hz,
2 CH3).
4.1.16.2. 1-(4-benzyloxy-3-cyanophenyl)ethanone (8b). Yield of 66.7%,
a light yellow solid, m.p. 126.5–127.3 ꢁC (from ethanol); 1H NMR
4.1.11. Synthesis of 1-(4-isobutoxy-3-iodophenyl)ethanone (7a)
Similar to the procedure used to prepare 2b, reaction of 6 with
isobutyl bromide provided 7a in a yield of 70.2% as a light yellow
(300 MHz, DMSO-d6): d 8.33 (s, 1H, Ar–H), 7.98 (d, 1H, Ar–H), 7.52–
7.32 (m, 6H, Ar–H), 5.40 (s, 2H, CH2), 2.57 (s, 3H, CH3).
solid, m.p. 50.6–51.7 ꢁC. 1H NMR (CDCl3):
d
8.38 (d, 1H, J ¼ 2.2 Hz,
4.1.16.3. 1-[3-cyano-4-(4-methylbenzyloxy)phenyl]ethanone
(8c).
Ar–H), 7.91 (dd, 1H, J ¼ 2.2 and 8.6 Hz, Ar–H), 6.79 (d, 1H, J ¼ 8.6 Hz,
Ar–H), 3.84 (d, 2H, J ¼ 6.3 Hz, CH2), 2.54 (s, 3H, CH3), 2.24–2.11 (m,
1H, CH), 1.10 (d, 6H, J ¼ 6.7 Hz, 2CH3).
Yield of 52.2%, a dark yellow solid, m.p. 102.9–103.8 ꢁC (from
ethanol); 1H NMR (CDCl3):
d
8.19 (d, 1H, J ¼ 2.2 Hz, Ar–H), 8.10 (dd,
1H, J ¼ 2.3 and 8.9 Hz, Ar–H), 7.33 (d, 2H, J ¼ 8.0 Hz, Ar–H), 7.20 (d,
2H, J ¼ 8.0 Hz, Ar–H), 7.06 (d, 1H, J ¼ 8.9 Hz, Ar–H), 5.26 (s, 2H, CH2),
2.56 (s, 3H, CH3), 2.36 (s, 3H, CH3).
4.1.12. Synthesis of 1-(4-benzyloxy-3-iodophenyl)ethanone (7b)
Similar to the procedure used to prepare 2b, reaction of 6 with
benzyl chloride provided 7b in a yield of 44.7% as a yellow solid,
m.p.126.0–127.3 ꢁC (from ethyl acetate). 1H NMR (300 MHz, DMSO-
4.1.16.4. 1-[4-(4-chlorobenzyloxy)-3-cyanophenyl]ethanone (8d).
Yield of 79.8%, a light yellow solid, m.p. 119.6–120.9 ꢁC; 1H NMR
d6):
d
8.33 (d, 1H, J ¼ 2.1 Hz, Ar–H), 7.98 (dd, 1H, J ¼ 2.1 and 8.6 Hz,
(CDCl3):
d
8.20 (d, 1H, J ¼ 2.2 Hz, Ar–H), 8.12 (dd, 1H, J ¼ 2.2 and
Ar–H), 7.52–7.32 (m, 5H, Ar–H), 7.19 (d, 1H, J ¼ 8.7 Hz, Ar–H), 5.32
8.9 Hz, Ar–H), 7.39 (s, 4H, Ar–H), 7.05 (d, 1H, J ¼ 8.9 Hz, Ar–H), 5.26
(s, 2H, CH2), 2.53 (s, 3H, CH3).
(s, 2H, CH2), 2.57 (s, 3H, CH3).
4.1.13. Synthesis of 1-[3-iodo-4-(4-methylbenzyloxy)-
phenyl]ethanone (7c)
Similar to the procedure used to prepare 2b, reaction of 6 with
4-methylbenzyl chloride provided 7c in a yield of 53.7% as a light
yellow solid, m.p. 117.3–118.9 ꢁC (from ethyl acetate). 1H NMR
4.1.16.5. 1-[4-(4-tert-butylbenzyloxy)-3-cyanophenyl]ethanone (8e).
Purification by flash silica gel column chromatography using ethyl
acetate as eluting solvent provided 8e in a yield of 70.9% as
a yellow solid, m.p. 106.9–108.3 ꢁC. 1H NMR (CDCl3):
d 8.19 (d, 1H,
J ¼ 2.2 Hz, Ar–H), 8.11(dd, 1H, J ¼ 2.2 and 8.9 Hz, Ar–H), 7.43
(d, 2H, J ¼ 8.5 Hz, Ar–H), 7.38 (d, 2H, J ¼ 8.4 Hz, Ar–H), 7.08 (d, 1H,
J ¼ 8.9 Hz, Ar–H), 5.26 (s, 2H, CH2), 2.56 (s, 3H, CH3), 1.33 (s, 9H,
3CH3).
(CDCl3):
d
8.40 (d, 1H, J ¼ 2.0 Hz, Ar–H), 7.90 (dd, 1H, J ¼ 2.0 and
8.6 Hz, Ar–H), 7.36 (d, 2H, J ¼ 7.9 Hz, Ar–H), 7.20 (d, 2H, J ¼ 7.8 Hz,
Ar–H), 6.86 (d, 1H, J ¼ 8.6 Hz, Ar–H), 5.18 (s, 2H, CH2), 2.53 (s, 3H,
CH3), 2.36 (s, 3H, CH3).
4.1.17. (Z)-Ethyl 4-(3-cyano-4-isobutoxyphenyl)-2-hydroxy-
4-oxobut-2-enoate (9a)
4.1.14. Synthesis of 1-[4-(4-chlorobenzyloxy)-
3-iodophenyl]ethanone (7d)
Similar to the procedure used to prepare 3, reaction of diethyl
oxalate with 8a provided 9a in a yield of 55.2% as a white solid, m.p.
106.7–107.3 ꢁC (from ethanol). 1H NMR (300 MHz, DMSO-d6):
Similar to the procedure used to prepare 2b, reaction of 6 with
4-chlorobenzyl chloride provided 7d in a yield of 73.4% as a light
yellow solid, m.p. 126.5–127.7 ꢁC (from ethanol–chloroform). 1H
d
15.06 (br s, 1H, OH), 8.23 (d, 1H, J ¼ 2.2 Hz, Ar–H), 8.18 (dd, 1H,
NMR (CDCl3):
d
8.40 (d, 1H, J ¼ 2.1 Hz, Ar–H), 7.92 (dd, 1H, J ¼ 2.1
J ¼ 2.3 and 8.9 Hz, Ar–H), 7.06 (d, 1H, J ¼ 8.9 Hz, Ar–H), 6.99 (s, 1H,
olefin-H), 4.41 (q, 2H, J ¼ 7.1 Hz, CH2CH3), 3.94 (d, 2H, J ¼ 6.5 Hz,
CH2), 2.29–2.16 (m, 1H, CH), 1.42 (t, 3H, J ¼ 7.1 Hz, CH2CH3), 1.10 (d,
6H, J ¼ 6.8 Hz, 2CH3).
and 8.6 Hz, Ar–H), 7.43 (d, 2H, J ¼ 8.6 Hz, Ar–H),7.38 (d, 2H,
J ¼ 8.6 Hz, Ar–H), 6.85 (d, 1H, J ¼ 8.6 Hz, Ar–H), 5.18 (s, 2H, CH2),
2.54 (s, 3H, CH3).

S. Wang et al. / European Journal of Medicinal Chemistry 45 (2010) 2663–2670
2669
4.1.18. (Z)-Ethyl 4-(4-benzyloxy-3-cyanophenyl)-2-hydroxy-
4-oxobut-2-enoate (9b)
Similar to the procedure used to prepare 3, reaction of diethyl
oxalate with 8b provided 9b in a yield of 52.3% as a white solid, m.p.
125.2–126.3 ꢁC (from ethanol–ethyl acetate). 1H NMR (300 MHz,
J ¼ 8.0 Hz, Ar–H), 7.15 (d,1H, J ¼ 8.9 Hz, Ar–H), 6.86 (s,1H, H-4), 5.26
(s, 2H, CH2), 4.47 (q, 2H, J ¼ 7.1 Hz, CH2CH3), 2.36 (s, 3H, CH3),1.45 (t,
3H, J ¼ 7.1 Hz, CH2CH3).
4.1.25. Ethyl 5-[4-(4-chlorobenzyloxy)-3-cyanophenyl]isoxazole-
3-carboxylate (10d)
DMSO-d6):
d
8.56 (s, 1H, Ar–H), 8.36 (d, 1H, J ¼ 8.2 Hz, Ar–H), 7.52–
7.35 (m, 6H, Ar–H), 7.17 (s, 1H, olefin-H), 5.42 (s, 2H, CH2), 4.31 (q,
Similar to the procedure used to prepare 4, reaction of hydrox-
ylamine hydrochloride with 9d provided 10d in a yield of 76.7% as
2H, J ¼ 7.1 Hz, CH2CH3), 1.31 (t, 3H, J ¼ 7.1 Hz, CH2CH3).
a white solid, m.p. 175.1–175.8 ꢁC. 1H NMR (300 MHz, CDCl3):
d 8.03
4.1.19. (Z)-Ethyl 4-[3-cyano-4-(4-methylbenzyloxy)phenyl]-
2-hydroxy-4-oxobut-2-enoate (9c)
Similar to the procedure used to prepare 3, reaction of diethyl
oxalate with 8c provided 9c in a yield of 66.6% as a dark yellow
solid, m.p. 154.2–155.4 ꢁC (from ethanol–ethyl acetate). 1H NMR
(d,1H, J ¼ 2.2 Hz, Ar–H), 7.95(dd,1H, J ¼ 2.2and8.9 Hz, Ar–H), 7.40 (s,
4H, Ar–H), 7.11 (d, 1H, J ¼ 8.9 Hz, Ar–H), 6.88 (s, 1H, H-4), 5.26 (s, 2H,
CH2), 4.47 (q, 2H, J ¼ 7.1 Hz, CH2CH3), 1.45 (t, 3H, J ¼ 7.1 Hz, CH2CH3).
4.1.26. Ethyl 5-[4-(4-tert-butylbenzyloxy)-3-cyanophenyl]-
isoxazole-3-carboxylate (10e)
(DMSO-d6):
d
8.56 (s, 1H, Ar–H), 8.35 (d, 1H, J ¼ 8.5 Hz, Ar–H), 7.50
(d, 1H, J ¼ 9.0 Hz, Ar–H), 7.39 (d, 2H, J ¼ 7.7 Hz, Ar–H), 7.24 (d, 2H,
J ¼ 7.7 Hz, Ar–H), 7.19 (s, 1H, olefin-H), 5.36 (s, 2H, CH2), 4.32 (q, 2H,
J ¼ 7.0 Hz, CH2CH3), 2.32 (s, 3H, CH3),1.32 (t, 3H, J ¼ 7.0 Hz, CH2CH3).
Similar to the procedure used to prepare 4, reaction of hydrox-
ylamine hydrochloride with 9e provided 10e in a yield of 83.2% as
a yellow solid, m.p. 181.3–182.2 ꢁC. 1H NMR (DMSO-d6):
d 8.42 (s,
1H, Ar–H), 8.24 (d, 1H, J ¼ 8.9 Hz, Ar–H), 7.54 (d, 2H, Ar–H, H-4),
7.48–7.41 (m, 4H, Ar–H), 5.35 (s, 2H, CH2), 4.40 (q, 2H, J ¼ 7.1 Hz,
CH2CH3), 1.37–1.29 (m, 12H, CH2CH3, 3CH3).
4.1.20. (Z)-Ethyl 4-[4-(4-chlorobenzyloxy)-3-cyanophenyl]-
2-hydroxy-4-oxobut-2-enoate (9d)
Similar to the procedure used to prepare 3, reaction of diethyl
oxalate with 8dprovided9dinayieldof59.7%as ayellowbrownsolid,
m.p. 132.2–133.1 ꢁC (ethanol–ethyl acetate). 1H NMR (300 MHz,
4.1.27. 5-(3-Cyano-4-isobutoxyphenyl)isoxazole-
3-carboxylic acid (11a)
DMSO-d6):
d
8.58 (s, 1H, Ar–H), 8.37 (d, 1H, J ¼ 8.9 Hz, Ar–H), 7.52 (s,
Similar to the procedure used to prepare 5, hydrolysis of 10a
provided 11a in a yield of 88.7% as a white solid, m.p.180.0–180.5 ꢁC
4H, Ar–H), 7.48 (s, 1H, Ar–H), 7.20 (s, 1H, olefin–H), 5.42 (s, 2H, CH2),
4.32 (q, 2H, J ¼ 7.1 Hz, CH2CH3), 1.32 (t, 3H, J ¼ 7.1 Hz, CH2CH3).
(from methanol). 1H NMR (DMSO-d6):
d 14.07 (br s, 1H, COOH), 8.37
(d, 1H, J ¼ 2.1 Hz, Ar–H), 8.20 (dd, 1H, J ¼ 2.1 and 8.9 Hz, Ar–H),
7.44–7.41 (m, 2H, Ar–H, H-4), 4.02 (d, 2H, J ¼ 6.5 Hz, CH2), 2.16–2.03
(m, 1H, CH), 1.02 (d, 6H, J ¼ 6.7 Hz, 2CH3); IR (KBr, cmꢀ1): 3543,
2228, 1730, 1618, 1495, 1451, 1385, 1302, 1264, 1177, 1123; TOF-
HRMS m/z ¼ 287.1018 [M þ H]þ(C15H15N2O4 requires 287.1033).
4.1.21. (Z)-Ethyl 4-[4-(4-tert-butylbenzyloxy)-3-cyanophenyl]-
2-hydroxy-4-oxobut-2-enoate (9e)
Similar to the procedure used to prepare 3, reaction of diethyl
oxalate with 8e provided 9e in a yield of 39.4% as a yellow brown
solid, m.p. 110.9–112.1 ꢁC (from acetic acid). 1H NMR (DMSO-d6):
d
8.57 (s, 1H, Ar–H), 8.37 (d, 1H, J ¼ 8.7 Hz, Ar–H), 7.52 (d, 1H,
4.1.28. 5-(4-Benzyloxy-3-cyanophenyl)isoxazole-
3-carboxylic acid (11b)
Similar to the procedure used to prepare 5, hydrolysis of 10b
provided 11b in a yield of 65.5% as a white solid, m.p.183.4–184.1 ꢁC
J ¼ 9.1 Hz, Ar–H), 7.46 (d, 2H, J ¼ 8.5 Hz, Ar–H), 7.42 (d, 2H,
J ¼ 8.5 Hz, Ar–H), 7.20 (s, 1H, olefin-H), 5.38 (s, 2H, CH2), 4.32 (q, 2H,
J ¼ 7.0 Hz, CH2CH3), 1.34–1.29 (m, 12H, CH2CH3, 3CH3).
(from acetic acid). 1H NMR (DMSO-d6):
d
8.40 (d, 1H, J ¼ 2.1 Hz, Ar–
4.1.22. Ethyl 5-(3-cyano-4-isobutoxyphenyl)isoxazole-
3-carboxylate (10a)
H), 8.22 (dd, 1H, J ¼ 2.1 and 8.9 Hz, Ar–H), 7.53(d, 1H, J ¼ 8.9 Hz, Ar–
H), 7.50–7.38 (m, 6H, Ar–H, H-4), 5.39 (s, 2H, CH2); IR (KBr, cmꢀ1):
3423, 2225, 1708, 1616, 1490, 1455, 1385, 1295, 1261, 1179, 1121;
TOF-HRMS m/z ¼ 343.0684 [M þ Na]þ(C18H12N2NaO4 requires
343.0695).
Similar to the procedure used to prepare 4, reaction of hydrox-
ylamine hydrochloride with 9a provided 10a in a yield of 85.4% as
a white solid, m.p.176.6–177.5 ꢁC. 1H NMR (300 MHz, DMSO):
d 8.39
(d, 1H, J ¼ 2.3 Hz, Ar–H), 8.21 (dd, 1H, J ¼ 2.3 and 8.9 Hz, Ar–H),
7.51(s, 1H, H-4), 7.42 (d, 1H, J ¼ 9.0 Hz, Ar–H), 4.39 (q, 2H, J ¼ 7.1 Hz,
CH2CH3), 4.02 (d, 2H, J ¼ 6.5 Hz, CH2), 2.16–2.03 (m, 1H, CH), 1.34 (t,
3H, J ¼ 7.1 Hz, CH2CH3), 1.02 (d, 6H, J ¼ 7.3 Hz, 2CH3).
4.1.29. 5-[3-Cyano-4-(4-methylbenzyloxy)phenyl]-
isoxazole-3-carboxylic acid (11c)
Similar to the procedure used to prepare 5, hydrolysis of 10c
provided 11c in a yield of 63.4% as a white solid, m.p. 194.7–195.1 ꢁC
4.1.23. Ethyl 5-[4-(benzyloxy)-3-cyanophenyl]isoxazole-
3-carboxylate (10b)
Similar to the procedure used to prepare 4, reaction of hydrox-
ylamine hydrochloride with 9b provided 10b in a yield of 86.5% as
a light yellow solid, m.p. 184.0–184.7 ꢁC. 1H NMR (300 MHz, CDCl3):
(from ethanol–THF). 1H NMR (DMSO-d6):
d 8.37 (s, 1H, Ar–H), 8.20
(d, 1H, J ¼ 8.8 Hz, Ar–H), 7.51 (d, 1H, J ¼ 8.9 Hz, Ar–H), 7.42 (s, 1H, H-
4), 7.39 (d, 2H, J ¼ 7.9 Hz, Ar–H), 7.23 (d, 2H, J ¼ 7.8 Hz, Ar–H), 5.33
(s, 2H, CH2), 2.31 (s, 3H, CH3); IR (KBr, cmꢀ1): 3423, 2225, 1729,
1616, 1490, 1453, 1385, 1295, 1270, 1179, 1121; TOF-HRMS
m/z ¼ 357.0838 [M þ Na]þ(C19H14N2NaO4 requires 357.0852).
d
8.02 (d, 1H, J ¼ 2.2 Hz, Ar–H), 7.92 (dd, 1H, J ¼ 2.2 and 8.9 Hz, Ar–
H), 7.47–7.35 (m, 5H, Ar–H), 7.13 (d, 1H, J ¼ 8.9 Hz, Ar–H), 6.87 (s,
1H, H-4), 5.30 (s, 2H, CH2), 4.47 (q, 2H, J ¼ 7.1 Hz, CH2CH3), 1.44 (t,
3H, J ¼ 7.1 Hz, CH2CH3).
4.1.30. 5-[4-(4-Chlorobenzyloxy)-3-cyanophenyl]isoxazole-
3-carboxylic acid (11d)
Similar to the procedure used to prepare 5, hydrolysis of 10d
provided 11d in a yield of 68.1% as a light yellow solid, m.p. 191.5–
4.1.24. Ethyl 5-[3-cyano-4-(4-methylbenzyloxy)phenyl]isoxazole-
3-carboxylate (10c)
192.3 ꢁC (from ethanol–THF). 1H NMR (DMSO-d6):
d 8.40 (d, 1H,
Similar to the procedure used to prepare 4, reaction of hydrox-
ylamine hydrochloride with 9c provided 10c in a yield of 63.7% as
a white solid, m.p. 190.3–191.5 ꢁC (from ethyl acetate). 1H NMR
J ¼ 2.1 Hz, Ar–H), 8.22 (dd, 1H, J ¼ 2.1 and 8.9 Hz, Ar–H), 7.55–7.50
(m, 5H, Ar–H), 7.43 (s, 1H, H-4), 5.39 (s, 2H, CH2); IR (KBr, cmꢀ1):
3474, 2223, 1731, 1617, 1496, 1454, 1385, 1291, 1269, 1242, 1124;
TOF-HRMS m/z ¼ 377.0300 [M þ Na]þ(C18H11ClN2NaO4 requires
377.0305).
(300 MHz, CDCl3):
d
8.00 (d, 1H, J ¼ 2.2 Hz, Ar–H), 7.91 (dd, 1H,
J ¼ 2.2 and 8.9 Hz, Ar–H), 7.34 (d, 2H, J ¼ 8.0 Hz, Ar–H), 7.21 (d, 2H,

2670
S. Wang et al. / European Journal of Medicinal Chemistry 45 (2010) 2663–2670
4.1.31. 5-[4-(4-Tert-butylbenzyloxy)-3-cyanophenyl]isoxazole-
3-carboxylic acid (11e)
Acknowledgments
Similar to the procedure used to prepare 5, hydrolysis of 10e
provided 11e in a yield of 73.5% as a yellow solid, m.p. 165.0–
The authors are grateful to Wen Li, Aihua Song, and Dr. Weiming
Cheng (Analytical Department of Shenyang Pharmaceutical
University, Shenyang, China) for the 1H NMR, IR, and TOF-HRMS
spectroscopy data. We also would like to thank Molegro ApS for
providing us free evaluation of their software package.
166.1 ꢁC (from ethanol). 1H NMR (DMSO-d6):
d 8.39 (d, 1H,
J ¼ 2.2 Hz, Ar–H), 8.22 (dd, 1H, J ¼ 2.3 and 8.9 Hz, Ar–H), 7.54 (d, 1H,
J ¼ 9.1 Hz, Ar–H), 7.48–7.40 (m, 5H, Ar–H, H-4), 5.34 (s, 2H, CH2),
1.29 (s, 9H, 3CH3); IR (KBr, cmꢀ1): 3453, 2229, 1732, 1692, 1618,
1501, 1451, 1384, 1302, 1277, 1123; TOF-HRMS m/z ¼ 399.1313
[M þ Na]þ(C22H20N2NaO4 requires 399.1321).
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