An effective synthesis of a (pyridin-3-yl)isoxazole via 1,3-dipolar cycloaddition using ZnCl2: Synthesis of a (2-aminopyridin-3-yl) isoxazole derivative and its antifungal activity

Article abstract of DOI:10.1246/cl.2010.1033

We described a rare example of an effective isoxazole synthesis via a 1,3-dipolar cycloaddition using ZnCl₂, which allowed us to synthesize novel (2-aminopyridin-3-yl)isoxazole derivatives effectively. In addition, these compounds demonstrated potent antifungal activity in vitro against both Candida albicans and Aspergillus fumigatus.

Full text of DOI:10.1246/cl.2010.1033

doi:10.1246/cl.2010.1033
Published on the web August 21, 2010
1033
An Effective Synthesis of a (Pyridin-3-yl)isoxazole via 1,3-Dipolar Cycloaddition Using ZnCl2:
Synthesis of a (2-Aminopyridin-3-yl)isoxazole Derivative and Its Antifungal Activity
Keigo Tanaka,* Satoshi Inoue, Norio Murai, Syuji Shirotori, Kazutaka Nakamoto, Shinya Abe, Takaaki Horii,
Mamiko Miyazaki, Katsura Hata, Naoaki Watanabe, Makoto Asada, and Masayuki Matsukura
Discovery Research Laboratories, Eisai Co., Ltd., 5-1-3 Tokodai, Tsukuba, Ibaraki 300-2635
(Received July 5, 2010; CL-100605; E-mail: k6-tanaka@hhc.eisai.co.jp)
We described a rare example of an effective isoxazole
synthesis via a 1,3-dipolar cycloaddition using ZnCl2, which
allowed us to synthesize novel (2-aminopyridin-3-yl)isoxazole
derivatives effectively. In addition, these compounds demon-
strated potent antifungal activity in vitro against both Candida
albicans and Aspergillus fumigatus.
there are only a few reports that indicate an improvement in
chemical yield,3 and the addition of a Lewis acid generally has
little effect. The decrease in the reactivity of the nitrile oxide due
to complexation with the Lewis acid is thought to be the major
reason.13 In the course of our research toward the development of
novel antifungal agents, we found a rare example of an effective
isoxazole synthesis via a 1,3-dipolar cycloaddition using ZnCl2.
In this letter, we describe the details of this reaction and its
application to the development of novel antifungal agents.
(2-Aminopyridin-3-yl)isoxazoles 1 were selected as target
compounds because they were expected to possess high
antifungal potency and to be patentable because of their unique
structure (Figure 1).14
Because both conventional methods for a 1,3-dipolar-
cycloaddition reaction and the improved methods described
above3 did not work well for the synthesis of 1, we endeavored
to develop a more effective method to synthesize 1.15 Sub-
sequently, we found that the addition of ZnCl2 resulted in both a
decreased amount of by-products and an increased amount of the
target compound 1aa (Table 1). These effects were reflected in a
dramatic improvement in the chemical yield of 1aa based on
Isoxazoles are important compounds in medicinal chemistry
because of their pharmacological activity, and are widely used in
clinical practice.1 Numerous approaches to the synthesis of
isoxazoles have been reported,2 including 1,3-dipolar cyclo-
addition,3 the cyclization of an ethynyloxime,4 the reaction of
hydroxylamine with an ¡,¢-unsaturated carbonyl compound or
a 1,3-dicarbonyl compound,5 the reaction of O-methylhydroxyl-
amine with an ¡,¢-unsaturated carbonyl compound followed by
electrophilic cyclization,6 the reaction of cyclopropyl oximes in
the presence of phosphorous oxychloride,7 the reaction between
a halogenated cyclopropane and a nitrosyl cation,8 and the
reaction of an oxime-derived dianion with an ester9 or an
amide.10 Among these approaches, the 1,3-dipolar-cycloaddition
reaction promises to be the most useful for exploratory synthesis
in medicinal chemistry because the reaction is compatible with a
wide range of functional groups,11 and because divergent
compound libraries can be synthesized in one step by combining
a variety of alkynes and nitrile oxides.
R
N
O
N
O
O
R =
In the category of 1,3-dipolar-cycloaddition reactions, there
are many reports regarding the improvement in chemical yield by
the addition of Lewis acids.12 On the other hand, in the case of
1,3-dipolar cycloaddition yielding a 3,5-disubstituted isoxazole,
N
NH2
1
1aa
1ab
Figure 1. (2-Aminopyridin-3-yl)isoxazole derivatives 1.
Table 1. Effect of ZnCl2 on isoxazole synthesis using ethynylpyridine 2a and hydroximoyl chloride 316
R
ZnCl2
Structure A:
N
O
HO
+
N
Cl
Et3N, THF
R
N
NH2
O
O
N
30 °C, 2 h
Structure B:
3a: R = Structure A
3b: R = Structure B
1aa: R = Structure A
1ab: R = Structure B
N
NH2
2a
Yielda/%
Hydroximoyl
chloride (equiv)
ZnCl2
(equiv)
Et3N
(equiv)
Yield of 1 based
on recovered 2a/%
Entry
Product
Product 1
Recovery 2
1
2
3
4
5
6
7
8
3a (1.4)
3a (3.0)
3a (1.4)
3a (1.4)
3a (1.4)
3a (3.0)
3b (3.0)
3b (3.0)
None
None
0.5
1.4
2.0
2.0
2.0
3.0
2.5
3.5
2.5
2.5
2.5
3.5
3.5
3.5
b
1aa
1aa
1aa
1aa
1aa
1aa
1ab
1ab
c
14
45
25
34
41
78
93
88
88
88
24 (23b)
22
29 (25b)
46
36
42
54
55
33
25
59 (57b)
66 (69b,c
63
)
28
aDetermined by HPLC versus internal standard. Isolated yield. rt, 18 h.
Chem. Lett. 2010, 39, 1033-1035
© 2010 The Chemical Society of Japan
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1034
HO
R
R
N
Et3N (2.5 equiv)
N
HO
+
+
+
N
THF, 30 °C, 4 h
Cl
O
R
N
NH2
O
R =
N
NH2
N
NH2
2a
3a (1.4 equiv)
1aa: 23%
4: 13%
R
HO
R
N
N
O
+ O-
O
N
N
+
+
+
R
R
N
O
N
O
N
O
N
NH2
NC
5: 11%
NC
6: 9.5%
NC
7: 4.7%
2a (recovered): 25%
8: 16% based on using 3a
R
R
R
Scheme 1. Isolated products in the isoxazole synthesis without ZnCl2.
Table 2. Isoxazole synthesis using alkyne 2 and hydroximoyl chloride 3a with or without ZnCl2
O
N
ZnCl2
HO
+
Cl
Et3N (2.5 equiv)
THF
30 °C, 2 h
N
O
X
Y
O
X
Y
X
Y
1aa
1b
1c
NH2
H
NH2
H
2a
2b
2c
2d
NH2
H
NH2
H
X
Y
N
N
CH
CH
N
N
CH
CH
3a (1.4 equiv)
1d
Yielda/%
ZnCl2
(equiv)
Yield of 1 based
on recovered 2a/%
Entry
Alkyne
Product
Product 1
Recovery 2
1
2
3
4
5
6
7
8
2a
2a
2b
2b
2c
2c
2d
2d
None
2.0
None
2.0
None
2.0
None
2.0
1aa
1aa
1b
1b
1c
1c
1d
1d
14
42
45
62
30b
24b
30
28
45
55
33
29
14b
36b
29
33
25
93
67
87
35b
38b
42
42
aDetermined by HPLC versus internal standard (Entries 1 and 2). Determined by NMR versus internal standard (Entries 3-8). bIsolated
yield.
recovered starting material from 25% to 93% (Entries 1 and 5).
The optimal amount of ZnCl2 utilized was 2.0 equivalents
(Entries 3-5, 7, and 8). Utilizing 3.0 equivalents of hydroximoyl
chloride 3a gave a better yield of 1aa than 1.4 equivalents
(Entry 6). The optimized reaction method also worked well for
hydroximoyl chloride 3b (Entry 7).
These reactions were analyzed in detail as shown below. In
the case of the reaction without ZnCl2, several products were
obtained (Scheme 1).17 Along with isoxazole 1aa (23%) and
recoverd 2a (25%), ethynyloxime 4 (13%), cyanohydrin deriv-
atives 5 (11%), 6 (9.5%), and 7 (4.7%) were isolated.18 In
addition, furazan 8 was obtained in 16% yield based on using 3a.
Then 1H NMR monitoring of these reactions in THF-d8 with
or without ZnCl2 was conducted. Several bits of information
were obtained as follows: 1) in the case of reaction with ZnCl2 (2
equiv), only isoxazole 1aa and furazan 8 could be detected as
products and other products such as compounds 4-7 were not
detected throughout the reaction; 2) immediately after Et3N was
added to the reaction mixture containing 2a, 3a, and ZnCl2 (both
1 and 2 equiv) at 0 °C, 3a was converted to the corresponding
nitrile oxide which gradually disappeared at 30 °C concurrent
with the production of 1aa and 8; 3) the proton on the terminal
alkyne in 2a was detected throughout the reaction with a
reasonable mass balance, which indicated that Zn-acetylide was
not formed in this reaction; 4) protons on the pyridine ring, the
terminal alkyne, and the isoxazole ring in 1aa and 2a led to a
significant low-field shift in the 1H NMR by the addition of ZnCl2
(both 1 and 2 equiv) compared with the ZnCl2-free conditions;
and 5) protons on the pyridine ring and the isoxazole ring in 1aa
caused a shift to a lower field by using 2 equivalents of ZnCl2
compared with 1 equivalent of ZnCl2, whereas the chemical shift
of all protons on 2a was almost the same in both cases.
Judging from these results, in the case of reaction without
ZnCl2, not only did 1,3-dipolar cycloaddition, but also nucle-
ophilic attack of the terminal alkyne and primary amine on the
nitrile oxide occurred. On the other hand, in the case of reaction
Chem. Lett. 2010, 39, 1033-1035
© 2010 The Chemical Society of Japan
www.csj.jp/journals/chem-lett/

1035
J. Org. Chem. 2005, 70, 7761.
Table 3. Antifungal activities of isoxazoles 1
4
5
a) L. Birkofer, K. Richtzenhain, Chem. Ber. 1979, 112, 2829. b)
R. J. Linderman, K. S. Kirollos, Tetrahedron Lett. 1989, 30,
2049.
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Bamoharram, Synth. Commun. 2008, 38, 135. b) R. F. Kurangi,
R. Kawthankar, S. Sawal, V. G. Desai, S. G. Tilve, Synth.
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R. Valero, Tetrahedron 2002, 58, 4975.
¹1 a
MIC/¯g mL
C. albicansb
Compound
A. fumigatusc
1aa
1ab
0.20
0.050
0.39
1.6
0.20
0.20
Fluconazole
Amphotericin B
>25
0.78
6
7
J. P. Waldo, R. C. Larock, Org. Lett. 2005, 7, 5203.
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Y. He, N.-H. Lin, Synthesis 1994, 989.
aMinimum inhibitory concentrations (MIC) were defined as the
lowest concentrations at which a prominent decrease in
turbidity could be observed visually compared to that in the
8
9
b
c
control well. CAF2-1 strain. Tsukuba strain.
10 T. J. Nitz, D. L. Volkots, D. J. Aldous, R. C. Oglesby, J. Org.
Chem. 1994, 59, 5828.
with ZnCl2, the 1,3-dipolar cycloaddition was found to be
preferable. One might conclude that the electron density of the
2-aminopyridine moiety and the terminal alkyne in 2a became
lowered by the chelation of the 2-aminopyridine with ZnCl2, and
which prevented the undesired nucleophilic addition reaction.
Then we studied the effects of substrates (Table 2). The
addition of ZnCl2 also worked well in the reaction using
ethynylpyridine 2b (Entries 3 and 4). On the other hand, in the
case of ethynylaniline 2c and ethynylbenzene 2d, the addition of
ZnCl2 showed less impact on the reaction (Entries 5-8). These
results indicate that the nitrogen atom on the pyridine ring in 2-
aminopyridine 2a might play an important role in the effective
1,3-dipolar cycloaddition using ZnCl2.
We next evaluated the in vitro antifungal activity of (2-
aminopyridin-3-yl)isoxazole derivatives 1 (Table 3). Isoxazoles
1aa and 1ab demonstrated more potent antifungal activity
against both Candida albicans and Aspergillus fumigatus
compared with fluconazole and amphotericin B which are
commonly used antifungal agents in clinical practice. Studies on
the development of (2-aminopyridin-3-yl)isoxazole derivatives 1
as antifungal agents are underway.
11 R. V. Stevens, Tetrahedron 1976, 32, 1599.
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Shirotori, S. Abe, PCT Int. Appl. WO2007/052615, 2007.
15 Variables changed in optimization included: Lewis acids [Pref-
erable; ZnCl2 and ZnBr2. Less effective; CeCl3, ZnI2, MgBr2,
CuBr2, BF3¢OEt2, Ti(Oi-Pr)4, Yb(OTf)3, Et2AlCl, Cu(OTf)2,
Sc(OTf)3, AlCl3, and ZnMe2]; solvents [Preferable; THF and
DME. Less effective; CH3CN, t-BuOMe, i-PrOH, toluene, and
CH2Cl2]; bases [Most preferable; Et3N. Preferable; i-Pr2NEt and
N-methylmorpholine. Undesirable; n-BuLi. No addition of the
base results in lower yield of product 1].
16 Typical procedure of the ZnCl2-mediated isoxazole synthesis
using 2-amino-3-ethynylpyridine and a hydroximoyl chloride
(Table 1): To a stirred solution of 2-amino-3-ethynylpyridine
(0.17 mmol) in tetrahydrofuran (2 mL) were successively added
hydroximoyl chloride 3a (0.51 mmol), ZnCl2 (0.34 mmol), and
triethylamine (0.59 mmol) at 0 °C, then the reaction mixture was
stirred for 2 h at 30 °C. To the reaction mixture was added
aqueous ammonium chloride at room temperature, which was
then extracted with ethyl acetate. The organic layer was washed
with saturated aqueous sodium hydrogen carbonate, followed by
brine, and the solvent was evaporated under a reduced pressure.
The residue was purified by NH silica gel column chromatog-
raphy (ethyl acetate:heptane = 1:1) to obtain (2-aminopyridin-3-
yl)isoxazole (1a).
17 The reaction mixture was purified by silica gel column
chromatography directly without aqueous workup.
18 For a plausible reaction mechanism for the formation of
cyanohydrin derivatives, see Supporting Information. Supporting
Information is available electronically on the CSJ-Journal Web
site, http://www.csj.jp/journals/chem-lett/index.html.
In summary, we synthesized novel (2-aminopyridin-3-
yl)isoxazole derivatives 1 effectively via 1,3-dipolar cyclo-
addition using ZnCl2. In addition, we demonstrated potent in
vitro antifungal activity of 1 against both Candida albicans and
Aspergillus fumigatus.
We thank Mr. Masaki Kato and Ms. Yumi Asai in the
Analytical Research, Eisai Co., Ltd., for the confirmation of the
chemical structure of by-products.
References and Notes
1
2
3
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64, 1231.
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Isoxazoles, ed. by E. C. Taylor, A. Weissberger, Wiley, New
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Chem. Lett. 2010, 39, 1033-1035
© 2010 The Chemical Society of Japan
www.csj.jp/journals/chem-lett/