Structural Investigation of 3,5-Disubstituted Isoxazoles by 1H-Nuclear Magnetic Resonance

Article abstract of DOI:10.1002/jhet.5570400621

HIV-1 integrase (IN) is a very promising and validated target for the development of therapeutic agents against AIDS. In an effort to design and synthesize biological isosteric analogs of β-diketoacid-containing inhibitors of IN, we prepared a series of s

Full text of DOI:10.1002/jhet.5570400621

Nov-Dec 2003
1097
Structural Investigation of 3,5-Disubstituted Isoxazoles by
1
H-Nuclear Magnetic Resonance
1
1
1
1
Mario Sechi, * Luciano Sannia, Maria Orecchioni, Fabrizio Carta,
1
2
Giuseppe Paglietti and Nouri Neamati
1Dipartimento Farmaco Chimico Tossicologico, Via Muroni 23/A, 07100 Sassari, Italy and
2Department of Pharmaceutical Sciences, University of Southern California, School of Pharmacy,
1985 Zonal Avenue, PSC 304BA, Los Angeles, CA 90089, USA
Received August 8, 2003
This work is dedicated to the memory of Prof. Paolo Sanna
HIV-1 integrase (IN) is a very promising and validated target for the development of therapeutic agents
against AIDS. In an effort to design and synthesize biological isosteric analogs of β-diketoacid-containing
inhibitors of IN, we prepared a series of substituted isoxazole carboxylic acids. Several of these compounds
inhibited catalytic activities of purified IN at micromolar concentration range. With an aim to prepare a large
number of analogues based on the isoxazole pharmacophore we focused our study on a series of 3,5-disub-
1
stituted isoxazole isomers. For a rapid structural analysis we discovered a convenient H-nmr method for
distinguishing between isomeric structures based on their H-4 assignments. This "finger print" approach to
isomer identification will be useful in combinatorial chemistry settings where a mixture can be further
derivatized.
J. Heterocyclic Chem., 40, 1097 (2003).
Introduction.
general structure 2 and observed that some of these com-
pounds inhibited purified IN at micromolar concentration
range. Details of these results will be published elsewhere
[11].
HIV-1 integrase (IN) is an essential enzyme for effective
viral replication. IN catalyses the integration of the viral
DNA into the host genome through coordinated reactions
of processing and joining. IN does not have a human
homologue and is considered a promising target for the
development of new antiretroviral drugs [1-3].
In recent years a plethora of IN inhibitors were identi-
fied by systematic screening of natural and synthetic prod-
ucts against purified enzyme [4-6]. In particular, a class of
compounds bearing a diketo acid moiety (1) was indepen-
dently discovered by the scientists from Shionogi & Co.
and Merck as selective IN inhibitors [7-10].
Results and Discussion.
The preparation of these compounds can be envisaged
using the so-called [3+2] atom fragments synthetic route,
subtype CCC+NO [12-14], by condensation of an appro-
priate β-dicarbonyl intermediate (3) with hydroxylamine
hydrochloride followed by alkaline hydrolysis (Figure 2).
At this stage it was important to study the isoxazole-car-
boxylic acid moiety in order to establish the correct struc-
ture before further synthesis.
The diketoacid functionality is not only responsible for
antiviral activity, but it also, unfortunately, contributes to
cytotoxicity. Therefore, replacement of the diketo group
with a pertinent bioisostere, endowed with reduced cyto-
toxicity, is of paramount importance in drug discovery tar-
geting IN. Our goal in this study was to take advantage of
bioisosteric replacement strategy to develop compounds
with desirable pharmaceutical properties. In this context,
our current research project was aimed at developing novel
inhibitors by incorporating the 1,3-diketo moiety into a
constrained isoxazole ring (2) (Figure 1).
In conjunction with this project we extended our study
to investigate some chemical aspects arising from the
above synthetic approach. In general, preparation of isoxa-
zoles from unsymmetrical disubstituted β-diketones (3)
and hydroxylamine is less straightforward and can a priori
lead to two isomeric 3,5-disubstituted isoxazoles 4 and 5
(Figure 2), [12-17].
A review of the literature on the structural assignments
reveals that data are scanty and controversial [15,18-21].
For example, the oximes originated from 1-aryl-3-phenyl-
propane-1,3-diones have subsequently been demonstrated
to give rise either to one of the two possible isoxazoles or
to a mixture of both isomers [22]. From a survey of the
cases reported in the literature, it became obvious that
many factors can influence the yield and regioselectivity
of the isomers. Also, it has been observed that the direction
of enolization may be a governing factor in the ratio of the
products obtained as well as the site-selectivity [15,19,23].
In fact this reaction proceeds through an attack of hydrox-
With this rational in mind we designed and synthesized a
series of aryl and heteroaryl isoxazole-carboxylic acids of
Figure 1

1098
M. Sechi, L. Sannia, M. Orecchioni, F. Carta, G. Paglietti and N. Neamati
Vol. 40
Figure 2. Formation of 3,5-disubstituted isoxazoles by reaction of β-diketones with hydroxylamine.
ylamine on the electrophilic center, and this is important
for the subsequent ring closure to isoxazole. Since this
reaction depends on the nature of R and R', all the vari-
ables which affect enolization (such as acidity or alkalinity
of the medium) may influence the ratio in which the two
isomers are formed [12]. According to Barnes et al. [19],
in the highly enolized diketones, which possess alternative
H-bonded (tautomeric structures), the structure of the prin-
cipal reaction product can be predicted on the basis of the
more or less electrophilic character of the two carbon
atoms bearing R and R'. Nevertheless, although a rigorous
study on the direction of enolization of disubstituted β-
diketones was reported [24], in many cases it is not easy to
predict if such a reaction can be regiospecific. Also, when
the substituents are not electronically very different, the
separation of the 3,5-disubstituted isomers is difficult.
In the course of our combinatorial drug design efforts
targeted against HIV-1 IN we investigated the structure of
several isoxazoles. Interestingly, when we generated com-
pounds 4a, 4e and 5a (Scheme 1) by reaction of β-dike-
tones and hydroxylamine using different conditions, we
obtained different results in accordance with the previ-
ously reported observations. For example, the reaction of
the regioselective product 4a was obtained on treating the
starting 3a with hydroxylamine hydrochloride (1.1 mole
eq) in pyridine at 50 °C (Method B [26]) (Scheme 1). This
was in contrast with previous reports where regioselectiv-
ity was observed for Method A but not for Method B
[15,26]. The ester 4e was instead obtained regioselectively
using both methods.
Although the spectral data for compounds 4a [27] and
4e [25] were previously reported, we unambigously
assigned their structures on the basis of NOE-difference
and NOESY data of corresponding N-Methyl isoxa-
zolium salts 6a and 6e. In fact, we thought that the posi-
tion of the N-methyl group could be detectable by NOE
experiments. Both experiments showed NOE correla-
+
tions between N -CH and the methyl in position-3 of
3
isoxazole ring (6a) and the methyl ester group in posi-
tion-3 of 6e (Figure 3). According to our prediction no
NOE interactions between the N-methyl isoxazolium
group and the 2',6'-aromatic protons could be observed
for compounds 6a and 6e.
The isoxazolium salts 6a and 6e, necessary for NOE
experiments, were prepared by heating in toluene the cor-
responding isoxazole derivatives 4a and 4e with dimethyl-
Scheme 1
Reagents and Conditions: A) NH OH HCl (3 eq), MeOH, reflux, 8h for 4a, 5a or 1h for 4e; B) NH OH HCl (1.1 eq), Pyridine, 50 °C, 1.5h.
2
2
benzoylacetone (3a) with hydroxylamine hydrochloride (3
mole eq) in methanol under reflux (Method A [25])
afforded both isoxazole isomers 4a and 5a. However only
sulfate. The isoxazolium methansulfate intermediates 7a
and 7e were then converted to the respective tetraflurobo-
rates [28] according to the reaction of Scheme 2.

Nov-Dec 2003
Structural Investigation of 3,5-Disubstituted Isoxazoles
1099
An explanation of these results may be due to the nature
of the “aromaticity” of isoxazole nucleus, which is consid-
erably less aromatic than other five-membered heterocycles
[39-41], and its low resonance energy [42]. This aspect
favours a stabilization of the double bond at position-4,5
thus causing a direct interaction between H-4 with the sub-
stituents at position-5. This turns out to influence the chem-
ical shift of H-4 as also documented by Battaglia et al. [31].
Figure 3. NOE correlations for compounds 6a and 6e.
Scheme
2
Reagents and Conditions: 1. Dimethylsulfate, anhydrous toluene, reflux, 46h for 6a or 70h for 6e; 2. NaBF , 0-5 °C.
4
Table 1
Assignments of H-NMR Chemical Shifts for H-4
13
Previously C-nmr studies were used to distinguish
1
1
between isoxazole isomers [29,30]. Moreover, H-nmr
was used to compare the electronic effects of the sub-
stituents based on evaluation of their Hammett σ-constant
parameters [31]. Unfortunately, the 60-MHz instrument
used was not sufficient to unequivocally discern the split-
ting patterns for each isomer. In this context we have now
1
discovered a robust and convenient H-nmr method to
identify 3,5-disubstituted isoxazole isomers.
Compound
R
H (δ)
Lit. Ref.
4
We examined a series of unsymmetrical 3(5)-substi-
tuted-3(5)-phenyl-isoxazole derivatives (4 and 5 series),
where R is an electron-donating or electron-withdrawing
substituent in order to study the influence of these groups
on H-4 chemical shift of isoxazole ring (Table 1).
Although the interatomic distances between substituents
and H-4 are the same in both series, signals were depen-
dent on the nature of the substituents.
4a
5a
4b
5b
4c
5c
4d
5d
4e
5e
4f
CH
CH
NH
NH
OCH
OCH
6.37
6.29
6.18
5.38
6.12
5.51
6.80
6.77
6.94
7.25
6.67
6.98
6.83
7.10
6.92
7.03
[a, 27]
[a, 27]
[32]
[32]
[33]
[34]
[22]
[22]
[a, 25]
[35]
[36]
[37]
[a]
3
3
2
2
3
3
4-CH C H
3
6
4
4
4-CH C H
3
6
CO CH
2
3
3
CO CH
2
In the case of isoxazole isomers bearing two different
substituents, one of which being more electron-donating
CF
CF
3
5f
3
4g
5g
4h
5h
CN
CN
than the other one (e.g. CH , NH , OCH , 4-CH -phenyl
3
2
3
3
[38]
[22]
[22]
vs Phenyl), the H-4 resonance is shifted upfield for the iso-
mers where these electron-donating groups are located on
position-5. In fact, the H-4 signal for compounds 5a-d was
shifted by 0.03 – 0.80 ppm to higher fields compared with
the corresponding isomers 4a-d. Opposite results were
obtained for those compounds containing stronger elec-
4-NO C H
2
6
4
4
4-NO C H
2
6
[a] This study.
Spectral data of model compounds of Table 1 were
either previously reported (4b-d,f,h and 5b-h) or
obtained after chemical synthesis in this study (4a,e,g
and 5a). The only unknown compound 4g was synthe-
sized using the sequence of reactions depicted in the
Scheme 3 that involves a classical transformation of an
ester into a nitrile [43,44].
tron-withdrawing groups (such CO CH , CF , CN,
2
3
3
4-NO -Phenyl vs Phenyl). In these cases the H-4 signal
2
shifted downfield by 0.11 – 0.31 ppm for compounds bear-
ing an electron-withdrawing substituent on position-5 of
isoxazolic ring (5e-h) compared with those of the corre-
sponding isomers 4e-h. These results correlated with the
electronic properties of the substituents.

1100
M. Sechi, L. Sannia, M. Orecchioni, F. Carta, G. Paglietti and N. Neamati
Vol. 40
Scheme 3
Reagents and Conditions: i) 30% NH , r.t, 12h; ii) POCl , reflux, 24h.
3
3
A mixture of benzoylacetone (3a) (1 g, 6.17 mmol) and
hydroxylamine hydrochloride 1.3 g, 3 mole eq) in methanol (25
mL) was refluxed for 8 h. After evaporation of the solvent, the
solid obtained was purified by silica gel flash chromatography
(petroleum ether:ethyl acetate = 9:1) to give a white solid that
was a mixture of the isomers 4a and 5a. These were separated by
silica gel flash chromatography (petroleum ether:ethyl acetate =
9.5:0.5) to give 4a and 5a in 17 and 13% yield, respectively.
Conclusion.
1
The H-nmr analysis of two series of 3,5-disubstituted
isoxazole isomers showed that the resonance values of H-4
on the isoxazole ring could be used as an important para-
meter to distinguish the isoxazole isomers. Analysis of
spectral data showed that changes in H-4 chemical shifts
on the isoxazole ring is due to electronic effects of the sub-
stituents through the heterocyclic system. Particularly, the
electronic effect of a substituent is significant when it is
directly connected to H-4 through a π bond on the 4- and
5-positions of the isoxazole ring.
3-Methyl-5-phenylisoxazole (4a).
This compound was obtained as white solid, mp 65-67°; Rf
0.43 (petroleum ether:ethyl acetate = 9.5:0.5); ir (Nujol): 1610
-1 1
(isoxazole), 1378 (CH bending symm) cm ; H-nmr (CDCl ): δ
3
3
7.73–7.78 (m, 2H, Ar-H aromatic), 7.43–7.46 (m, 3H, Ar-H aro-
matic), 6.37 (s, 1H, H-4 isoxazole), 2.36 (s, 3H, CH ); gc/ms: m/z
3
+
159 (M ).
EXPERIMENTAL
Anal. Calcd. for C H NO (159.18). C, 75.45; H, 5.70; N,
10
9
8.80. Found: C, 75.21; H, 5.83; N, 8.87.
General.
5-Methyl-3-phenylisoxazole (5a).
Anhydrous solvents and all reagents were purchased from
Aldrich, Merck or Carlo Erba. All reactions involving air- or
moisture-sensitive compounds were performed under nitrogen
atmosphere using oven-dried glassware and syringes to transfer
solutions. Melting points (mp) were determined using an
Electrothermal melting point or a Köfler apparatus and are uncor-
rected. Infrared (ir) spectra were recorded as thin films or nujol
mulls on NaCl plates with a Perkin-Elmer 781 IR spectropho-
This compound was obtained as a white solid, mp 38-40°; Rf
0.52 (petroleum ether:ethyl acetate = 9.5:0.5); ir (Nujol): 1605
-1 1
(isoxazole), 1373 (CH bending symm) cm ; H-nmr (CDCl ): δ
3
3
7.75– 7.81 (m, 2H, Ar-H aromatic), 7.42–7.47 (m, 3H, Ar-H aro-
matic), 6.29 (s, 1H, H-4 isoxazole), 2.48 (s, 3H, CH ); gc/ms: m/z
3
+
159 (M ).
Anal. Calcd. for C H NO (159.18). C, 75.45; H, 5.70; N,
10
9
-1
tometer and are expressed in ν (cm ). Nuclear magnetic reso-
8.80. Found: C, 75.37; H, 5.74; N, 8.69.
1
nance ( H-nmr, NOE-difference and NOESY) spectra were deter-
Method A.
mined in CDCl , DMSO or CDCl /DMSO (in the ratio 1:3) and
3
3
Synthesis of Methyl 5-phenylisoxazole-3-carboxylate (4e).
were recorded on a Varian XL-200 (200 MHz). Chemical shifts (δ
scale) are reported in parts per million (ppm) downfield from
tetramethylsilane (TMS) as internal standard. Splitting patterns
are designated as follows: s, singlet; d, doublet; t, triplet; q,
quadruplet; m, multiplet; br s, broad singlet; dd, double doublet.
The assignment of exchangeable protons (OH and NH) was con-
A solution of the methyl 2,4-dioxo-4-phenylbutanoate [25]
(3e) (1 g, 4.85 mmol) and hydroxylamine hydrochloride (1.01 g,
3 mole eq) in methanol (20 mL) was refluxed for 1 h. After evap-
oration in vacuo a yellow solid was obtained that was purified by
silica gel flash chromathography (hexane:ethyl acetate = 8:2).
The crude product was recrystallized from water-ethanol to give
4e as yellow crystals (39% yield), mp 81-82°; Rf 0.57 (petroleum
ether:ethyl acetate = 8.5:1.5); ir (Nujol): 1728 (C=O, ester), 1610
firmed by the addition of D O. Electron ionization mass spectra
2
(70 eV) were recorded on a Hewlett-Packard 5989 Mass Engine
Spectrometer. Analytical thin-layer chromatography (TLC) was
performed on Merck silica gel F-254 plates. Pure compounds
showed a single spot on TLC. For flash chromatography Merck
Silica gel 60 was used with a particle size 0.040-0.063 mm (230-
400 mesh ASTM). Elemental analyses were performed on a
Perkin-Elmer 2400 instrument at Laboratorio di Microanalisi,
Dipartimento di Chimica, Università di Sassari (Italy), and the
results were within ±0.4% of the theoretical values.
-1
1
(isoxazole) cm ; H-nmr (CDCl ): δ 7.77–7.85 (m, 2H, Ar-H
3
aromatic), 7.47–7.55 (t, 3H, Ar-H aromatic), 6.94 (s, 1H, H-4
+
isoxazole), 4.01 (s, 3H, CO CH ); gc/ms: m/z 203 (M ).
2
3
Anal. Calcd. for C H NO (203.19). C, 65.02; H, 4.46; N,
11
9
3
6.89. Found: C, 65.26; H, 4.28; N, 6.72.
Method B.
Synthesis of 3-Methyl-5-phenylisoxazole (4a) and Methyl 5-
Phenylisoxazole-3-carboxylate (4e).
General Procedure for Preparation of Isoxazoles.
Method A.
To a solution of benzoylacetone (3a) (for 4a) or methyl 2,4-
dioxo-4-phenylbutanoate [25] (3e) (for 4e) (10 mmol) in pyri-
dine (10 mmol) was added a saturated aqueous solution of
Synthesis of 3-Methyl-5-phenylisoxazole (4a) and 5-Methyl-3-
phenylisoxazole (5a).

Nov-Dec 2003
Structural Investigation of 3,5-Disubstituted Isoxazoles
1101
hydroxylamine hydrochloride (11 mmol in 5 mL of deionized
water). The mixture was stirred for 1.5 h at 50 °C. A solid was
obtained that after purification by silica gel flash chromatogra-
phy (petroleum ether:ethyl acetate = 9:1) gave compounds 4a
(78% yield) or 4e (69% yield) identical (mixed mp, ir, H-nmr)
to the above samples.
obtained was purified by silica gel flash chromatography
(hexane:ethyl acetate = 8:2) to give 4g as a pale orange solid (37%
yield), mp 87-89°; Rf 0.57 (Petroleum ether:Ethyl acetate = 9:1);
-1
1
ir (Nujol): 2260 (CN), 1610 (isoxazole) cm ; H-nmr (CDCl ): δ
3
1
7.76–7.83 (m, 2H, Ar-H aromatic), 7.50–7.56 (m, 3H, Ar-H aro-
+
matic), 6.83 (s, 1H, H-4 isoxazole); gc/ms: m/z 170 (M ).
Anal. Calcd. for C H N O (170.17). C, 70.58; H, 3.55; N,
16.46. Found: C, 70.77; H, 3.22; N, 16.50.
10
6 2
General Procedure for the Synthesis of N-Methylisoxazolium
Tetrafluoroborates Salts (6a and 6e).
Acknowledgments.
A mixture of the appropriate isoxazole (4a or 4e) (2 mmol) and
dimethylsulfate (1.1 mole eq) in anhydrous toluene (5 mL) was
refluxed, under nitrogen atmosphere, for 46 h (for 6a) or 70 h (for
6e). Subsequently the toluene layer was decanted, and the oily
residue was dissolved in water, and it was washed three times
with ethyl ether (for 6a) or ethyl acetate (for 6e). To this aqueous
solution was added a solution of sodium tetrafluoroborate
(4 mole eq) in water and, after cooling with ice, a yellow solid
was separated. Compounds 6a or 6e were collected in 31 and
11% yield, respectively.
We express our gratitude to Ms Paola Manconi for mass spec-
trometric analysis and to Mr Domenico Serra for hplc analyses.
This work was financially supported by the Ministero
dell'Istruzione, dell'Università e della Ricerca (MIUR), Rome,
Italy.
REFERENCES AND NOTES
*
To whom correspondence should be addressed: Dipartimento
Farmaco Chimico Tossicologico, Università di Sassari, Via Muroni 23/A,
07100 Sassari, Italy; e-mail: mario.sechi@uniss.it; Tel: +39 079
228753; Fax: +39 079 228720.
[1] N. Neamati, C. Marchand and Y. Pommier, in Advances in
Pharmacology, Academic Press, 2000, pp. 147-165.
2,3-Dimethyl-5-phenylisoxazolium Tetrafluoroborate (6a) [28].
This compound was obtained as a white solid, mp 165-166°; ir
(Nujol): 1610 (isoxazole), 1375 (CH bending symm.), 1060
3
-
-1 1
(BF ) cm ; H-nmr (DMSO): δ 8.00 (d, 2H, Ar-H aromatic),
4
[2] Y. Pommier, C. Marchand and N. Neamati, Antiv. Res., 47,
139 (2000).
7.78 (s, 1H, H-4 isoxazole), 7.60–7.74 (m, 3H, Ar-H aromatic),
+
4.32 (s, 3H, N-CH ), 2.68 (s, 3H, CH ); gc/ms: m/z 174 (M )
3
3
[3] N. Neamati, C. Marchand, H. E. Winslow and Y. Pommier, in
Antiretroviral Theraphy, ASM Press, Washington D.C., 2001, pp. 83-104.
[4] N. Neamati, Exp. Opin. Invest. Drugs, 10, 281 (2001).
[5] N. Neamati, Exp. Opin. Ther. Pat., 12, 709 (2002).
[6] J. d'Angelo, J. F. Mouscadet, D. Desmaele, F. Zouhiri and H.
Leh, Pathol. Biol., 49, 237 (2001).
[7] D. J. Hazuda, P. Felock, M. Witmer, A. Wolfe, K. Stillmock, J.
A. Grobler, A. Espeseth, L. Gabryelski, W. Schleif, C. Blau and M. D.
Miller, Science, 287, 646 (2000).
[8] J. S. E. Wai, L. S. Payne, T. E. Fisher, M. W. Embrey, L. O.
Tran, J. Y. Melamed, H. M. Langford, J. P. Guare, Jr., L. Zhuang, V. E.
Grey, J. P. Vacc, M. K. Holloway, A. M. Naylor-Olsen, D. J. Hazuda, P. J.
Felock, A. L. Wolfe, K. A. Stillmock, W. A. Schleif and L. J. Gabryelski,
J. Med. Chem., 23, 4923 (2000).
[9] G. C. G. Pais, X. Zhang, C. Marchand, N. Neamati, K.
Cowansage, E. S. Svarovskaia, V. K. Pathak, Y. Tang, M. Nicklaus, Y.
Pommier and T. R. Burke, Jr., J. Med. Chem., 45, 3184 (2002).
[10] G. C. G. Pais and T. R. Burke, Jr., Drugs of the Future, 27,
1101 (2002).
[11] M. Sechi, L. Sannia, M. Derudas, R. Dallocchio, A. Dessì, F.
Carta, T. Sanchez and N. Neamati, manuscript in preparation.
[12] S. A. Lang, Jr. and Y.-I Lin, Isoxazoles and their Benzo
Derivatives, in Comprehensive Heterocyclic Chemistry, Vol 6, A. R.
Katritzky and C. W. Rees, Pergamon Press, Oxford, 1984, p 61-62.
[13] B. J. Wakefield and D. J. Wright, Isoxazole Chemistry since
1963, in Advances in Heterocyclic Chemistry, Vol 25, A. R. Katritzky
and A. J. Boulton, Academic Press, New York, 1979, p 149.
[14] R. R. Gupta, M. Kumar and V. Gupta, Heterocyclic Chemistry
Volume II: Five-membered Heterocycles, Springer-Verlag, Berlin, 1999,
p 458.
(cation).
Anal. Calcd. for C H NO•BF (261.03). C, 50.62; H, 4.63;
N, 5.37. Found: C, 50.77; H, 4.42; N, 5.41.
11 12
4
3-(Methoxycarbonyl)-2-methyl-5-phenylisoxazol-2-ium
Tetrafluoroborate (6e).
This compound was obtained as a white solid, mp 175-176°; ir
(Nujol): 1742 (C=O ester), 1605 (isoxazole), 1370 (CH bending
symm), 1060 (BF ) cm ; H-nmr (DMSO): δ 8.45 (s, 1H, H-4
isoxazole), 8.15 (d, 2H, Ar-H aromatic), 7.63–7.84 (m, 3H, Ar-H
aromatic), 4.60 (s, 3H, N-CH ), 4.08 (s, 3H, COOCH ); gc/ms:
m/z 218 (M ) (cation).
Anal. Calcd. for C H NO •BF (305.04). C, 47.25; H, 3.97;
N, 4.59. Found: C, 47.44; H, 3.68; N, 4.33.
3
-
-1 1
4
3
3
+
12 12
3
4
Synthesis of 5-Phenylisoxazole-3-carboxamide (8).
A suspension of methyl 5-phenylisoxazole-3-carboxylate (4e)
(0.30 g, 1.48 mmol) and 30% ammonia aqueous solution (15 mL)
was stirred at room temperature overnight. The solid obtained
was triturated with a mixture of petroleum ether and ethyl ether to
give 8 as a white solid (65% yield), mp 200-201°; Rf 0.38 (petro-
leum ether:ethyl acetate = 6:4); ir (Nujol): 3400 (NH ), 3230
2
-1
1
(NH ), 1660 (C=O, amide), 1610 (isoxazole) cm ; H-nmr
2
(CDCl ): δ 7.78–7.85 (d, 2H, Ar-H aromatic), 7.46–7.55 (m, 3H,
3
Ar-H aromatic), 6.97 (s, 1H, H-4 isoxazole), 6.79 (br s, 1H, NH ,
exchangeable with D O), 5.76 (br s, 1H, NH , exchangeable with
2
2
2
+
D O); gc/ms: m/z 188 (M ).
2
[15] A. Quilico, in Five- and six-membered compound with nitro-
gen and oxygen (excluding oxazole), A. Quilico, G. Speroni, L. C. Behr
and R. L. Mc Kee, John Wiley & Sons, N.Y.-London, 1962, pp 6-9.
[16] A. R. Katritzky and A. F. Pozharskii, Handbook of
Heterocyclic Chemistry 2nd edition, Pergamon Press, Amsterdam, 2000,
p 556.
Anal. Calcd. for C H N O (188.18). C, 63.82; H, 4.28; N,
14.89. Found: C, 63.63; H, 4.39; N, 14.98.
10
8 2 2
Synthesis of 5-Phenylisoxazole-3-carbonitrile (4g).
A mixture of 5-phenylisoxazole-3-carboxamide (8) (0.15 g,
0.80 mmol) in 2.55 mL of POCl (27.0 mmol) was heated at
[17] J. A. Joule and K. Mills, Heterocyclic Chemistry 4th edition,
Blackwell Science, London, 2000, p 440.
3
reflux for 24 h. After cooling the solution was poured on ice and
made alkaline with aqueous sodium carbonate. The orange solid
[18] W. S. Johnson and W. E. Shelberg, J. Am. Chem. Soc., 67,

1102
M. Sechi, L. Sannia, M. Orecchioni, F. Carta, G. Paglietti and N. Neamati
Vol. 40
1745 (1945).
Chem., 7, 721 (1970).
[19] R. P. Barnes and A. S. Spriggs, J. Am. Chem. Soc., 67, 134
(1945).
[20] B. Eistert and E. Merkel, Chem. Ber., 86, 825 (1953).
[21] R. P. Barnes and J. T. Snead, J. Am. Chem. Soc., 67, 138
(1945).
[22] T. Bandiera, P. Grünager and F. Marinone Albini, J.
Heterocyclic Chem., 29, 1423 (1992).
[23] V. Bertolasi, P. Gilli, V. Ferretti and G. Gilli, J. Am. Chem.
Soc., 113, 4917 (1991).
[32] Z. Tanee Fomum, P. Forsche Asobo, S. R. Landor and P. D.
Landor, J. Chem. Soc., Perkin Trans. 1, 1079 (1984).
[33] R. G. Micetich and C. G. Chin, Can. J. Chem., 48, 1371
(1970).
[34] M. L. Purkayastha, L. Bhat, H. Ila and H. Junjappa, Synthesis,
641 (1995).
[35] V. Yedidia and C. C. Leznoff, Can. J. Chem., 58, 1144 (1980).
[36] K. Tanaka, H. Masuda and K. Mitsuhashi, Bull. Chem. Soc.
Jpn., 57, 2184 (1984).
[24] R. M. Cravero, M. Gonzalez-Sierra and A. C. Olivieri, J.
Chem. Soc., Perkin Trans. 2, 1067 (1993).
[37] H.-P. Guang, X.-Q. Tang, B.-H. Luo and C.-M. Hu, Synthesis,
1489 (1997).
[25] A. Tanaka, T. Terasawa, H. Hagihara, Y. Sakuma, N. Ishibe,
M. Sawada, H. Takasugi and H. Tanaka, J. Med..Chem., 41, 2390 (1998).
[26] M. A. P. Martins, A. F. C. Flores, R. Freitag and N. Zanatta, J.
Heterocyclic Chem., 32, 731 (1995).
[27] C. Kashima, S. Shirai, N. Yoshiwara and Y. Omote, J. Chem.
Soc. Chem. Comm., 826 (1980).
[28] A. Alberola, J. M. Bañez, L. Calvo, M. T. Rodriguez
Rodriguez and M. C. Sañudo, J. Heterocyclic Chem., 30, 461 (1993).
[29] A. L. Baumstark, D. R. Chrisope, R. A. Keel and D. W.
Boykin, J. Heterocyclic Chem., 17, 1719 (1980).
[38] A. Baranski, Pol. J. Chem. 56, 257 (1982).
[39] S.A. Lang, Jr. and Y.-I Lin, Isoxazoles and their Benzo
Derivatives, in Comprehensive Heterocyclic Chemistry, A. R. Katritzky
and C. W. Rees, Vol 6, Pergamon Press, Oxford, 1984, p 3.
[40] G. Del Re, J. Chem. Soc., 3324 (1962).
[41] G. Berthier and G. Del Re, J. Chem. Soc., 3109 (1965).
[42] D. T. Clark and D. M. J. Lilly, Chem. Phys. Lett., 9, 234
(1975).
[43] M. Israel, E. C. Zoll, N. Muhammad and E. J. Modest, J. Med.
Chem., 16, 1 (1973).
[30] D. R. Chrisope, R. A. Keel, A. L. Baumstark and D. W.
Boykin, J. Heterocyclic Chem., 18, 795 (1981).
[44] O. Vitse, F. Laurent, T. M. Pocock, V. Bénézech, L. Zanik, K.
R. F. Elliot, G. Subra, K. Portet, J. Bompart, J. P. Chapat, R. C. Small, A.
Michel and P. A. Bonnet, Bioorg. Med. Chem., 7, 1059 (1999).
[31] A. Battaglia, A. Dondoni and F. Taddei, J. Heterocyclic