Improved synthesis of 3-aryl isoxazoles containing fused aromatic rings

Article abstract of DOI:10.1016/j.tet.2012.09.084

A critical comparison of methods to prepare sterically hindered 3-aryl isoxazoles containing fused aromatic rings using the nitrile oxide cycloaddition (NOC) reveal that modification of the method of Bode, Hachisu, Matsuura, and Suzuki (BHMS), utilizing either triethylamine as base or sodium enolates of the diketone, ketoester, and ketoamide dipolarophiles, respectively, was the method of choice for this transformation.

Full text of DOI:10.1016/j.tet.2012.09.084

Tetrahedron 68 (2012) 10360e10364
Contents lists available at SciVerse ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Improved synthesis of 3-aryl isoxazoles containing fused aromatic rings
Yousef R. Mirzaei, Matthew J. Weaver, Scott A. Steiger, Alison K. Kearns, Mariusz P. Gajewski,
*
Kevin C. Rider, Howard D. Beall, N.R. Natale
Medicinal Chemistry Graduate Program, Department of Biomedical and Pharmaceutical Sciences, The University of Montana, Missoula, MT 59812, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 14 August 2012
Received in revised form 14 September 2012
Accepted 17 September 2012
Available online 25 September 2012
A critical comparison of methods to prepare sterically hindered 3-aryl isoxazoles containing fused
aromatic rings using the nitrile oxide cycloaddition (NOC) reveal that modification of the method of
Bode, Hachisu, Matsuura, and Suzuki (BHMS), utilizing either triethylamine as base or sodium enolates of
the diketone, ketoester, and ketoamide dipolarophiles, respectively, was the method of choice for this
transformation.
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
line growth inhibition (GI50) against the full panel in the NCI60 cell
line antitumor screening protocol,8 and as a benchmark for com-
Isoxazoles continue to be of interest for both their biological
activities and synthetic utility.1e4 The synthesis of aryl-isoxazoles is
also the subject of on-going improvements.1,5e7 In connection with
our studies on aryl isoxazole amides (AIMs)8e9 we have reported
lead compounds, which possess (1) useful antitumor activity and
(2) photophysical properties, which are of potential diagnostic use
as ‘tumor paint’.10
An illustration of the application of AIMs is shown in Fig. 1. AIM
NSC 728558 has exhibited a mid-graph mean point of ꢀ5.71e5.75
(log scale, which translate to single digit micromolar) for tumor cell
parison clinically used agents 5-fluorodeoxyuridine, bleomycin and
rubidazone gave values of ꢀ4.7, ꢀ5.2, and ꢀ5.8, respectively, in the
same standard assay. The calculated physical property data for NSC
728558 fall within or close to values with useful bioavailability
(radar graph, Symyx Draw v3.1; top right panel). Laser Scanning
Cytometry shows that NSC 728558 does in fact permeate human
glioma SNB-19 cells (the AIM is blue) and is localized within the
nucleus. Surgical resection of many cancers, especially brain tumors
or gliomas, have a poor success rate because of the difficulty of
judging the border between cancerous and healthy tissue. Fluo-
rescent agents with antitumor activity, such as the AIMs hold
promise for improvements in visualizing tumors during surgery.10b,c
We required both improvements in the efficiency of the prep-
aration of the isoxazole and concomitant economy of scale to pre-
pare amounts of material sufficient to expand the scope of our
investigations toward in vivo studies.
Isoxazoles prepared via nitrile oxide cycloaddition (NOC)11 are
most often prepared by dehydration of
a-methylene nitro pre-
cursors,5 or chlorination/dehalogenation of oximes1,6,7 to form the
nitrile oxide 1.
Fig. 1. Structure, Symyx radar graph, and Laser Scanning Cytometry of NSC 728558.
Among the unsymmetrical dipolarophiles 2, which usually give
rise to regioselective cycloaddition are enamines. First pioneered by
Stork and McMurry,12 the use of enamines in the NOC works well for
aliphatic and aromatic nitrile oxides on mole scales.1 We have
* Corresponding author. Tel.: þ1 406 243 4132; fax: þ1 406 243 5228; e-mail
address: nicholas.natale@umontana.edu (N.R. Natale).
0040-4020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2012.09.084

Y.R. Mirzaei et al. / Tetrahedron 68 (2012) 10360e10364
10361
employed modifications of this synthesis in the preparation
of isoxazoleeoxazolines,13 antihypertensive 4-isoxazolyl-1,4-
dihydropyridines,14 and we have championed the reaction as
a learning tool for aspiring chemists.15 Our previous reports in the
AIM series have used enamines as dipolarophiles,16 while for most
cases modest yields in the range of 30e40% overall of 3 were
obtained, sufficient quantities of material were isolated for phar-
macology studies. Upon scale-up however, we have observed
a consistent diseconomy of scale using enamines and nitrile oxides
derived from fused ring aromatics, to the extent that even a ten-fold
increase in scale (from ca. 10e100 mM) produced only marginally
more desired product, and purified yields plummeted to 10e20%.
We have found this is especially a limitation when using nitrile oxides
derived from fused aromatic systems. Careful examination of the re-
action by-products after chromatographic isolation and character-
ization by LCeMS revealed that as the stability of aryl nitrile oxides 1
increases17,16b their consequent reactivity to dipolarophiles slows,
with concomitant increases in amine trapping of the nitrile oxide/
oximidoyl chloride to give 7, as well as self-condensation of the
nitrile oxide to yield 8 as shown in Scheme 1.
yield compared to 30e40% for the enamine method for which the
by-products complicated purification and lowered the yield of de-
sired isoxazole (Scheme 1). Similar results were observed for 3g the
yields were 80% (Table 1, entry 8) for the BHMS verses 35% for en-
amine (Scheme 1). In cases where the BHMS yields are modest, the
enamine methodology gave only traces or no desired product at all
that is, the enamine entries (not shown) corresponding to Table 1,
entries 3 9,and 10, would correspond to traces at best).
The comparison was especially dramatic for the anthryl isoxazole
cases, shown in Table 2. Excellent yields were obtained for anthryl
isoxazoles 3j, 10-chloro analog 3k, and 10-bromo analog 3m. The
yields with the enamine procedures were 35e4516 and 10e20%, for
3j and 3k, respectively. Table 2, entry 3 illustrates the direct synthesis
of a fused aromatic containing a C-4 tertiary amide, which is an un-
common example of a ketamide used as dipolarophile in the NOC.22
The ketamide was prepared from diketene and pyrrolidine,18 and
cycloaddition provided the C-4 amide in synthetically reasonable
yield. The sluggish reactivity we previously reported for nucleophilic
addition to the C-4 ester of the 3-anthryl-isoxazole is exemplified by
(1) slow hydrolysis rates (i.e., 60 h using refluxing aqueous LiOH)9
and (2) the observation that n-BuLi deprotonates the C-5 methyl at
ꢀ78 ꢁC without noticeable addition to the C-4 ester.19 The C-4
functionalization prior to the NOC stage is a definite advantage for
the preparation of AIMs, and will be the subject of further study.
In summary, we have found that the BHMS protocol produced
superior results, especially in sterically encumbered examples
containing fused ring aromatic nitrile oxides and that these con-
ditions represent the methodology of choice when standard enamine
methodology fails. We will report on the chemical and pharma-
cological application of these novel isoxazoles in due course.
O
N
R
Ar
4. R = -CHO
NH2OH
NCS
CO2Et
5. R = -CHNOH
H3C
6
N+ O-
1. R =
C
Ar
O
N
2. Experimental
2.1. General
N
O
N
+
O
+
CH3
Ar
Ar
N+
O-
OH
All reactions were performed under inert atmosphere. Purifi-
cation was carried out by column chromatography. Chemicals were
purchased from TCI or Aldrich Chemical Company, all commercial
reagents are routinely examined for purity by NMR and TLC, and
recrystallized or distilled as appropriate. Solvents were reagent
grade. Melting points were determined in open capillary tubes on
a Melt-Temp apparatus and are uncorrected. NMR spectra were
obtained using either a Varian 400 MHz Unity Plus or a Varian NMR
systems 500 MHz spectrometer, in deuteriochloroform unless
otherwise noted. Infrared spectra were obtained on a thermo-
Nicolet 633 FT-IR spectrometer.
CO2Et
Ar
N
8b.14%
8g. 6%
8k.
3b. Ar = 2-Naphthyl 30-40%
3g. Ar = 2-MeO-Naphthyl 35%
3k. Ar= 10-Cl--anthr-9-yl 10-20%
7b. 46%
7g.
7k. 29%
Scheme 1. Synthesis of the isoxazoles used for the present study.
We therefore examined the methodology of Bode, Hachisu,
Matsuura, and Suzuki (BHMS), which use either the sodium eno-
lates of ketoesters,6 or tertiary amines as base.7 The use of sodium
enolates in the NOC was pioneered by Renzi and Dal Piaz,20 and
recently applied in 2,6-disubstituted aryl cases for the preparation
of growth hormone secretagogue receptor antagonists.21 The BHMS
procedure has been used to prepare particularly hindered un-
symmetrical 2,6-disubstituted aryl examples, including examples,
which appear to exist as atropisomers,7 which recommended its
application to our examples.
Using the BHMS procedure, the yields in all cases where critical
comparison was made were dramatically improved (Table 1). Sym-
metrical diketones (Table 1, entries 1, 4, 5 and 11e13), un-
symmetrical arylalkyl ketones (Table 1, entries 3, 9 and 10) and
ketoesters (Table 1, entries 2, and 6e8) all produced the desired
isoxazole products. In particularly hindered situations the triethyl
amide protocol7 often gave higher yields than the sodium ethoxide
method.6 (Table 1, entries 1, 3, 4 and 7, and compare entries 10 to 9),
although in a select few cases we have observed that sodium iso-
propoxide to produce slightly higher yield (exemplified by Table 1,
entries 11 and 14). All of the BHMS methods gave superior results to
our experience using enamines as dipolarophiles for fused 3-aryl
isoxazoles: for 3b the BHMS conditions gave 70% (Table 1, entry 2)
Chemical shifts (
d
) are reported using CHCl3 (7.26 ppm for 1H),
CDCl3 (77 ppm for 13C) as references. High resolution mass spectra
(HRMS) were obtained using a Micromass electrospray ionization
(ESI)/time-of-flight mass spectrometry (LCTOF). Mass spectrometer
samples were introduced using a Waters model 2690 separations
module HPLC fitted with a C-18 reversed phase column (2.1 mm
i.d., 5 cm). Elemental analyses for C, H, and N were performed by
Midwest Microlab, Indianapolis, IN. All reactions were monitored
by Thin Layer Chromatography (TLC). Purification was performed
by flash column chromatography, and analytical samples were
prepared by PTLC. Analytical LCMS (UV at 254 nm) and NMR were
used to establish the purity of targeted compounds. All compounds
that were evaluated in biochemical and biophysical assays had
>95% purity as determined by 1H NMR and LCMS NSC 728558 was
prepared from 3k as previously described.8,9
2.1.1. 1-(Pyrrolidin-1-yl)butane-1,3-dione.18 Pyrolidine
(0.539 g,
7.5 mmol) is dissolved in 10 mL of anhydrous DCM and diketene
(0.925 g, 11.00 mmol) is added. The mixture is stirred at ambient
temperature for 2.5 h, followed by removal of the solvent under

10362
Y.R. Mirzaei et al. / Tetrahedron 68 (2012) 10360e10364
Table 1
Nitrile oxide cycloaddition synthesis of hindered 3-naphthyl-isoxazoles
Entry
Nitrile oxide
Dipolarophile
Conditionsa
Et3N/EtOH
Product
Yield %
40
1
2
3
1a, R2¼CH3
1a, R2¼CH3
2b, R1¼OCH2CH3
2c, R1¼C6H5
Na/EtOH
Et3N
3b, R1¼OCH2CH3; R2¼CH3
3c, R1¼C6H5; R2¼CH3
70
40
4
5
1a, R2¼CH3
Et3N
71
70
2a, R1¼CH3
Na/EtOH
6
7
8
9
1b, R2¼OCH3
1b, R2¼OCH3
1b, R2¼OCH3
1b, R2¼OCH3
1b, R2¼OCH3
2e, R1¼OCH3
2e, R1¼OCH3
2b, R1¼OCH2CH3
2c, R1¼C6H5
2c, R1¼C6H5
Na/MeOH 2.5 h
Et3N
Na/EtOH
Na/EtOH
Et3N/
3f, R1¼OCH3; R2¼OCH3
3f, R1¼OCH3; R2¼OCH3
3g, R1¼OCH2CH3; R2¼OCH3
3h, R1¼C6H5; R2¼OCH3
3h, R1¼C6H5; R2¼OCH3
60
62
80
15
22
10
EtOH
11
12
1b, R2¼OCH3
2c, R1¼C6H5
NaOi-Pr
3h, R1¼C6H5; R2¼OCH3
43
1b, R2¼OCH3
Na/EtOH
42
13
14
1b, R2¼OCH3
1b, R2¼OCH3
2d
2d
Et3N
NaOi-Pr
3i
3i
53
57
a
The reaction was conducted in anoxic conditions.
vacuum to afford (1.163 g, 99%) as red-brown liquid. 1H NMR
3.46 (t, J¼6.62 Hz, 2H), 3.45 (s, 2H), 3; 13C NMR
Table 2
Nitrile oxide cycloaddition to formed hindered 3-anthracenyl-isoxazoles
(500 MHz, CDCl3):
(125 MHz, CDCl3):
d
d
202.6, 165.0, 88.8, 51.3, 47.2, 45.9, 30.4, 26.0,
24.4; IR cmꢀ1; mass calculated for C8H13NO2 155.0946, found m/z
156.1902 (Mþ1, 100%).
Entry Nitrile oxide Dipolarophile Conditionsa
Product
Yield (%)
2.2. NOC: sodium enolate method
1
2
Na/EtOH
Na/EtOH
88e94
2.2.1. (3-(Anthracen-9-yl)-5-methylisoxazol-4-yl)(pyrrolidin-1-yl)
methanone, 3l. To a solution of sodium ethoxide, prepared from
0.199 g of Na in 44.2 mL of absolute EtOH, was added 1-(pyrrolidin-
1-yl)butane-1,3-butane-1,3-dione (1.015 g, 6.54 mmol), and 9-
anthrahydroximinoylchloride, (1.06, 4.105 mmol) successively and
the resulting red solution was stirred under Ar atmosphere for 4 h
at ambient temperature. The red-brown solution was extracted
with ethyl acetate (4ꢂ20 mL), washed with DI water (2ꢂ50 mL)
and brine (50 mL), and dried over anhydrous Na2SO4. Filtration and
concentration afforded the desired product 3l as red-brown oil
(1.40 g, 95%), which was further purified by silica column flash
chromatography (hexanes/EtOAc; 10:1), and PTLC to afford 3l as off
white solid (0.7375 g, 50%).
90e92
3
4
Na/EtOH
50
2.3. NOC: triethylamine method
Na/MeOH
Et3N
82
72
2.3.1. Synthesis of 1-(5-methyl-3-(2-methylnaphthalene-1-yl)iso-
xazol-4-yl)ethanone, 3a. To 2,4-pentanedione (0.42 g, 4.11 mmol) in
absolute ethanol (21 mL) at ambient temperature was added
5
1e
2e
3m
a
The reaction was conducted in anoxic conditions.

Y.R. Mirzaei et al. / Tetrahedron 68 (2012) 10360e10364
10363
triethylamine (0.47 g, 4.584 mmol) followed by addition of nitrile
NMR (500 MHz, CDCl3):
d
7.96 (d, J¼8.99 Hz, 1H), 7.81 (d, J¼8.07 Hz,
oxide (0.6 g, 3.28 mmol). The temperature was raised to 53 ꢁC and the
mixture stirred under Ar atmosphere for 72 h. The pale yellow solu-
tion was extracted with chloroform (4ꢂ20 mL), washed with DI water
(2ꢂ50 mL) and brine (50 mL), dried over anhydrous Na2SO4. Filtra-
tion, concentration and silica column flashchromatography (hexanes/
EtOAc/DCM; 5:1:1) afforded the desired product 3a (0.289 g, 40%).
1H), 7.50 (d, J¼8.55 Hz, 1H), 7.39 (dd, J¼11.00, 6.84 Hz 1H), 7.34 (d,
J¼9.05 Hz, 2H), 3.96 (q, J¼7.06 Hz, 2H), 3.88 (s, 3H), 2.82 (s, 3H),
0.79 (t, J¼7.06 Hz, 3H); 13C NMR (125 MHz, CDCl3):
d 175.3, 161.9,
158.6, 155.3, 133.3, 131.3, 128.6, 127.9, 127.1, 124.1, 123.7, 112.7, 111.8,
110.6, 60.1, 56.5, 13.5, 13.3; ESI-MS calcd for C18H17NO4þH m/z
312.1428 (Mþ1, 100%). Anal. Calcd for C18H17NO4: C, 69.44; H, 5.50;
N, 4.50. Found: C, 69.58; H, 5.47; N, 4.54.
2.4. Analytical data for 3-fused aryl isoxazole products
2.4.8. Phenyl (3-(2-methoxynaphthalen-1-yl)-5-methylisoxazol-4-
2.4.1. 1-(5-Methyl-3-(2-methylnaphthalene-1-yl)isoxazol-4-yl)etha-
yl) methanone, 3h. Mp 141e142 ꢁC; 1H NMR (500 MHz, CDCl3):
none, 3a. Oil, TLC SiO2 hexane/DCM/EtOAc 4:1:1 Rf 0.77. 1H NMR
d
8.06 (d, J¼8.50, 1H), 7.73 (d, J¼8.80 Hz, 2H), 7.51 (t, J¼8.40 Hz, 1H),
(500 MHz, CDCl3)
d
7.80e7.75 (m, 2H), 7.38e7.32 (m, 4H), 2.76 (s, 3H),
192.3, 175.7,
7.44 (d, J¼8.30 Hz, 2H), 7.36 (t, J¼7.55 Hz, 1H), 6.96 (t,
J¼7.80 Hz, 2H), 6.90 (d, J¼9.30 Hz, 1H), 3.61 (s, 3H), 2.69 (s, 3H);
2.26 (s, 3H), 1.56 (s, 3H); 13C NMR (125 MHz, CDCl3)
d
159.6, 135.3, 132.3, 131.4, 129.5, 127.8, 126.8, 125.2, 124.0, 117.5, 28.6,
19.8, 13.6; HRMS calcd for C17H16NO2 266.1181; found: 266.1193.
13C NMR (125 MHz, CDCl3):
d 189.8, 173.2, 157.8, 154.5, 137.1,
132.7, 132.4, 131.7, 128.7, 128.5, 128.0, 127.7, 127.5, 124.2, 117.6, 111.6,
78.1, 55.5, 29.7; ESI-MS for C22H17NO3þH m/z 344.1008
(Mþ1, 100%).
2.4.2. Ethyl 3-(2-methylnaphthalen-1-yl)-5-methylisoxazole-4-
carboxylate, 3b. Oil, TLC SiO2 hexane/DCM/EtOAc 4:1:1 Rf 0.6. 1H
NMR (CDCl3)
(s, 3H); 2.303 (s, 3H); 0.651 (t, 3H). 13C NMR (125 MHz, CDCl3)
175.7, 161.4, 160.7, 135.0, 132.5, 131.4, 128.9, 127.9, 127.7, 126.2,
124.8, 124.6, 124.5, 110.1, 59.9, 20.1, 13.2, 13.0. HRMS calcd for
C18H18NO3: 296.1287; found: 296.1297.
d
7.826 (d, 2H); 7.25e7.398 (m, 4H); 3.87 (q, 2H); 2.841
2.4.9. 6,6-Dimethyl-3-(2-methoxynaphthalen-1-yl)-6,7-
dihydrobenzo[d]isoxazol-4(5H)-one, 3i. Oil, TLC SiO2 hexane/DCM/
d
EtOAc 4:1:1 Rf 0.46. 1H NMR (500 MHz, CDCl3)
d
7.96 (d, J¼9.1, 1H),
d
7.81 (d, J¼7.8 Hz,1H), 7.69 (d, J¼8.6 Hz,1H), 7.43 (dt, J¼9.8 Hz,1.5 Hz,
1H), 7.36 (dd, J¼6.8 Hz, 1.3 Hz, 1H), 7.34 (d, J¼9.3 Hz, 2H), 3.85 (s,
3H), 2.90 (s, 2H), 2.36 (q, J¼14.7 Hz, 2H), 1.17 (s, 3H), 1.14 (s, 3H); 13C
2.4.3. Phenyl (5-methyl-3-(2-methylnaphthalen-1-yl)isoxazol-4-yl)
NMR (125 MHz, CDCl3) d 190.7, 180.2, 155.7, 155.4, 132.6, 131.7,
methanone, 3c. Oil, TLC SiO2 hexane/DCM/EtOAc 4:1:1 Rf 0.79. 1H
131.6, 128.6, 128.1, 128.0, 127.9, 127.8, 127.6, 127.2, 126.9, 123.9,
123.7, 123.4, 115.2, 112.9, 112.8, 112.6, 109.9, 56.4, 56.2, 52.3, 36.7,
35.1, 28.3, 28.0. HRMS calcd for C20H20NO3 322.1443; found:
322.1458.
NMR (500 MHz, CDCl3):
d
7.65 (d, J¼7.90, 1H), 7.60 (d, J¼8.50 Hz, 1H),
7.55 (d, J¼8.00 Hz,1H), 7.37 (t, J¼6.60 Hz,1H), 7.33 (m, 3H), 7.16 (m, 2H),
6.94 (t, J¼8.00 Hz, 2H), 3.38 (s, 3H), 2.64 (s, 3H); 13C NMR (125 MHz,
CDCl3): d 189.8,173.7,171.0,160.3,137.1,135.5, 132.4,132.3,131.4,129.3,
129.2, 128.1, 128.0, 127.9, 127.8, 127.5, 125.0, 124.5, 124.4, 123.6, 117.8,
20.4, 14.0; HRMS calcd for C22H18NO2: 328.1338; found: 328.1370.
2.4.10. Ethyl
3j. Mp 121e122 ꢁC; TLC SiO2 hexane/EtOAc 10:1, Rf 0.30. 1H NMR
(500 MHz, CDCl3):
(d, J¼8.55 Hz, 2H), 7.40e7.49 (m, 4H), 3.71 (q, J¼7.21 Hz, 2H), 2.93
(s, 3H), 0.32 (t, J¼7.10 Hz, 3H); 13C NMR (500 MHz, CDCl3): 176.2,
161.5, 160.5, 131.0, 130.8, 128.7, 128.5, 126.3, 125.4, 125.2, 122.7,
111.4, 60.1, 13.5, 12.8, spectral data are in accord with those
reported previously.16a ESI-MS for C21H17NO3þH m/z 332.1441
(Mþ1, 100%).
3-(anthracen-9-yl)-5-methylisoxazole-4-carboxylate,
d
8.58 (s, 1H), 8.05 (d, J¼8.31 Hz, 2H), 7.65
2.4.4. 6,6-Dimethyl-3-(2-methylnaphthalen-1-yl)-6,7-dihydrobenzo
[d]isoxazol-4(5H)-one 3d. Oil, TLC SiO2 hexane/DCM/EtOAc 4:1:1 Rf
0.71. 1H NMR (500 MHz, CDCl3)
d
7.87 (d, J¼8.3, 1H), 7.84 (d, J¼8.3,
1H), 7.36e7.43 (m, 4H), 3.01 (s, 2H), 2.40 (d, J¼8.3, 2H), 2.31 (s, 3H),
1.22 (s, 3H), 1.19 (s, 3H); 13C NMR (125 MHz, CDCl3)
190.8, 180.9,
d
171.1, 157.9, 135.6, 132.3, 131.7, 129.7, 129.6, 128.4, 128.2, 128.1, 128.0,
125.2,125.0,124.5,124.3,122.9, 52.4, 37.0, 35.5, 28.6, 28.5, 28.1, 28.0,
21.0, 14.2. HRMS calcd for C20H20NO2: 306.1494; found: 306.1516.
2.4.11. Ethyl 3-(10-chloroanthracen-9-yl)-5-methylisoxazole-4-
carboxylate, 3k. Mp 123e124 ꢁC, lit. Mp 119e120.16b TLC SiO2
2.4.5. 1-(3-(2-Methoxynaphthalene-1-yl)-5-methylisoxazol-4-yl)
hexane/EtOAc 10:1, Rf 0.28. 1H NMR (500 MHz, CDCl3):
d 8.59 (d,
ethanone, 3e. Oil, TLC SiO2 hexane/DCM/EtOAc 4:1:1 Rf 0.53. 1H
J¼8.80 Hz, 2H), 7.53e7.60 (m, 4H), 7.43 (d, J¼8.80 Hz, 2H), 3.72 (q,
J¼7.1 Hz, 2H), 2.93 (s, 3H), 0.39 (t, J¼7.1 Hz, 3H); 13C NMR (500 MHz,
CDCl3): 177.3, 161.3, 161.2, 134.1, 131.1, 128.3, 126.7, 125.9, 125.1,
122.6, 111.5, 60.2, 13.2; ESI-MS for C21H16ClNO3þH, m/z 366.0886
(Mþ1, 95%).
NMR (500 MHz, CDCl3)
d
8.01 (d, J¼9.30 Hz, 1H), 7.84 (d, J¼7.80 Hz,
1H), 7.48 (d, J¼8.30 Hz, 1H), 7.44 (t, J¼8.30 Hz, 1H), 7.39 (d,
J¼7.90 Hz, 1H), 7.36 (d, J¼8.80 Hz, 1H), 3.90 (s, 3H), 2.81 (s, 3H), 1.76
(s, 3H); 13C NMR (125 MHz, CDCl3)
d 193.2, 175.4, 157.7, 155.3, 133.3,
132.1, 128.7, 128.1, 127.8, 124.2, 123.9, 118.3, 112.6, 111.4, 56.4, 28.8,
14.0. HRMS calcd for MþH, C17H16NO3 282.1130; found: 282.1150
(Mþ1, 100%). Anal. Calcd for C17H15NO3: C, 72.58; H, 5.37; N, 4.98;
found: C, 71.81; H, 5.52; N, 4.56.
2.4.12. [3-(Anthracen-9-yl)-5-methylisoxazol-4-yl](pyrrolidin-1-yl)
methanone, 3l. Mp 194e195 ꢁC. 1H NMR (500 MHz, CDCl3):
d 8.54
(s, 1H), 7.99 (d, J¼9.29 Hz, 2H), 7.95 (d, J¼10.0 Hz, 2H), 7.48e7.45
(m, 4H), 3.10 (t, J¼6.87 Hz, 2H), 2.73 (s, 3H), 2.60 (t, J¼6.61 Hz, 2H),
1.42 (pentet, J¼6.85 Hz, 2H), 1.29 (pentet, J¼6.60 Hz, 2H); 13C NMR
2.4.6. Methyl 3-(2-methoxynaphthalene-1-yl)-5-methylisoxazole-4-
carboxylate, 3f. Oil, TLC SiO2 hexane/DCM/EtOAc 4:1:1 Rf 0.56. 1H
(125 MHz, CDCl3): d 170.1, 161.3, 158.3, 131.0, 130.5, 129.4, 128.5,
NMR (500 MHz, CDCl3):
d
7.96 (d, J¼9.05 Hz, 1H), 7.82 (d, J¼9.99 Hz,
126.9, 126.7, 125.8, 125.3, 125.0, 121.9, 116.5, 47.7, 45.3, 25.6, 23.7,
12.4; ESI-MS for C23H20N2O2þH, m/z 357.0757 [Mþ1, (100)]. Anal.
Calcd for C23H20N2O2: C, 77.51; H, 5.66; N, 7.86. Found: C, 77.37; H,
5.45; N, 7.99.
1H), 7.55 (d, J¼9.99 Hz,1H), 7.40 (t, J¼9.99 Hz,1H), 7.35 (t, J¼9.99 Hz,
2H), 3.86 (s, 3H), 3.54 (s, 3H), 2.81 (s, 3H); 13C NMR (500 MHz,
CDCl3):
d 175.1, 162.1, 158.5, 155.3, 133.0, 131.3, 128.6, 127.9, 127.0,
124.0, 123.6, 112.6, 111.4, 110.4, 56.4, 51.3, 13.3; IR cmꢀ1; HRMS calcd
for C17H16NO4: 298.1079; found 298.1111. Anal. Calcd for C17H15NO4:
C, 68.68; H, 5.09; N, 4.71; found: C, 66.81; H, 5.00; N, 4.53.
2.4.13. Methyl 3-(10-bromoanthracen-9-yl)-5-methylisoxazole-4-
carboxylate, 3m. Rf 0.45 hexane/EtOAc/DCM 4:1:1. 1H NMR
(500 MHz, CDCl3): 8.63e8.60 (d, 2H), 7.62e7.59 (m, 4H), 7.47e7.43
(m, 2H), 3.32 (s, 3H), 2.93 (s, 3H). 13C NMR (125 MHz, CDCl3):
176.08, 163.32, 161.53, 131.12, 129.90, 127.96, 126.88, 126.39, 125.60,
2.4.7. Ethyl 3-(2-methoxynaphthalen-1-yl)-5-methylisoxazole-4-
carboxylate, 3g. Yellow solid (1.8 g, 60%): mp 103e104 ꢁC; 1H

10364
Y.R. Mirzaei et al. / Tetrahedron 68 (2012) 10360e10364
123.12, 51.35, 13.42. HRMS calcd for MþH C20H14NO379Br þ H, m/z
2. The Kurth group has provided a useful catalog of the bioactive isoxazole
literature in their NOC synthesis papers over the years, the following are
illustrative examples: (a) Yu, G. J.; Yang, B.; Verkman, A. S.; Kurth, M. J. Synlett
2010, 1063e1066; (b) Meng, L.; Lorsbach, B. A.; Sparks, T. C.; Fettinger, J. C.;
Kurth, M. J. J. Comb. Chem. 2010, 12, 129e136; (c) Butler, J. D.; Donald, M. B.;
Ding, Z.; Fettinger, J. C.; Kurth, M. J. Tetrahedron Lett. 2009, 50, 5110e5112; (d)
Choung, W.; Lorsbach, B. A.; Sparks, T. C.; Ruiz, J. M.; Kurth, M. J. Synlett 2008,
3036e3040; (e) Quan, C.; Kurth, M. J. Org. Chem. 2004, 69, 1470e1474; (f)
Hwang, S. H.; Kurth, M. J. J. Org. Chem. 2002, 67, 6564e6567.
3. In the course of their studies on glutamate receptor and transporter ligand
pharmacology, the Krogsgaard-Larsen group has greatly expanded the tool box
for isoxazole synthesis:Glutamate and GABA Receptors and Transporters,
Structure Function and Pharmacology; Egebjerg, J., Schousboe, A., Krogsgaard-
Larsen, P., Eds.; Taylor and Francis: New York, NY, 2002.
4. Pinho e Melo, T. M. V. D. Curr. Org. Chem. 2005, 9, 925e958.
5. Trogu, E.; Cecchi, L.; De Sarlo, F.; Guideri, L.; Ponticelli, F.; Machetti, F. Eur. J. Org.
Chem. 2009, 5971e5978 and cited references.
6. Bode, J. W.; Hachisu, Y.; Matsuura, T.; Suzuki, K. Org. Lett. 2003, 5, 391e394.
7. Bode, J. W.; Hachisu, Y.; Matsuura, T.; Suzuki, K. Tetrahedron Lett. 2003, 44,
3555e3558.
8. Han, X.; Li, C.; Mosher, M. D.; Rider, K. C.; Zhou, P.; Crawford, R. L.; Fusco, W.;
Paszczynski, A.; Natale, N. R. Bioorg. Med. Chem. 2009, 17, 1671e1680.
9. Gajewski, M. P.; Beall, H.; Schnieder, M.; Stranahan, S. M.; Mosher, M. D.; Rider,
K. C.; Natale, N. R. Bioorg. Med. Chem. Lett. 2009, 19, 4067e4069.
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396.0227; found: 396.0235.
2.5. Laser Scanning Cytometry methods
SNB-19 human glioblastoma cells (American Type Cell Culture
Cat No. CRL-2266) were plated at a density of 2000 cells/mL on
cover slips in RPMI medium (with L-glutamine and penicillin/
streptomycin and supplemented with 10% fetal bovine serum). Cell
culture medium and supplements were obtained from VWR (West
Chester, PA). The cells were incubated at 37 ꢁC under a humidified
atmosphere containing 5% CO2 and allowed to adhere. Medium was
aspirated and replaced with 1 mM NSC 728558 for 24-h exposure.
Drug was removed and cells were washed twice with phosphate
buffered saline (PBS). Cells were fixed in 4% paraformaldehye
(15 min, 21 ꢁC) and washed once with PBS. Following two addi-
tional washes, slides were inverted to microscope slides and sealed
using FluorSave Reagent (Calbiochem). Images were generated
from scans from a CompuCyte iCys Laser Scanning Cytometer.
(CompuCyte, Westwood, MA). Cells were scanned with a 405 nm
30 mW Diode laser. Fluorescent signals were measured in photo-
multiplier detectors following a 440/30 Band-pass (BP) filter to
detect NSC 728558 presence. Light absorption was also measured to
produce a differential interference contrast (DIC)-like image for cell
morphology. Each signal was given a pseudo-color and overlaid to
produce images.
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Acknowledgements
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The authors thank the NIH for grants R21NS067466,
P20RR017670 and P30NS055022 for financial support of this work.
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Supplementary data
Representative HPLC trace for 3m. Characterization data for by-
products 7 and 8, representative spectra for products 3. Supple-
mentary data related to this article can be found at http://
dx.doi.org/10.1016/j.tet.2012.09.084.
21. (a) Liu, B.; Liu, G.; Xin, Z.; Serby, M. D.; Zhao, H.; Schaefer, V. G.; Falls, D.;
Kaszubska, W.; Collins, C. A.; Sham, H. L. Bioorg. Med. Chem. Lett. 2004, 14,
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References and notes
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