Substituent and solvent effects in the 1,3-dipolar cycloadditions for synthesis of anti-influenza agent peramivir and its analog

Article abstract of DOI:10.1016/j.tet.2019.06.023

Influenza remains a health problem to humans. Peramivir is a FDA approved anti-influenza drug targeting the virus neuraminidase. The (3 + 2) cycloaddition reaction of 2-ethylbutanenitrile oxide with the cyclopentene dipolarophile derived from Vince lactam is a key step in the conventional synthesis of peramivir. Our study showed that conducting the (3 + 2) cycloaddition reactions with either aliphatic or aromatic nitrile oxide in hexane solution provided high percentage of the desired regioisomer, and the N-substituent having electron-withdrawing property is also beneficial to the regioselectivity. This study also demonstrated an alternative synthetic pathway of (?)-peramivir and the analog having a phenyl group in place of the 3-pentyl moiety.

Full text of DOI:10.1016/j.tet.2019.06.023

Accepted Manuscript
Substituent and solvent effects in the 1,3-dipolar cycloadditions for synthesis of anti-
influenza agent peramivir and its analog
Chien-Liang Chen, Tzu-Wei Chiu, Yung-Wen Chen, Jim-Min Fang
PII:
S0040-4020(19)30683-0
https://doi.org/10.1016/j.tet.2019.06.023
TET 30415
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 6 April 2019
Revised Date: 15 June 2019
Accepted Date: 17 June 2019
Please cite this article as: Chen C-L, Chiu T-W, Chen Y-W, Fang J-M, Substituent and solvent effects in
the 1,3-dipolar cycloadditions for synthesis of anti-influenza agent peramivir and its analog, Tetrahedron
(2019), doi: https://doi.org/10.1016/j.tet.2019.06.023.
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ACCEPTED MANUSCRIPT
Graphic Abstract
Substituent and solvent effects in the 1,3-dipolar cycloadditions for synthesis of anti-influenza
agent peramivir and its analog

ACCEPTED MANUSCRIPT
Substituent and solvent effects in the 1,3-dipolar cycloadditions for synthesis of anti-
influenza agent peramivir and its analog
a, †
a, †
a
a, b,
Chien-Liang Chen, Tzu-Wei Chiu, Yung-Wen Chen, Jim-Min Fang
*
a
Department of Chemistry, National Taiwan University, Taipei 106, Taiwan
The Genomics Research Center, Academia Sinica, Taipei 115, Taiwan
b
ABSTRACT
Influenza remains a health problem to humans. Peramivir is a FDA approved anti-influenza
drug targeting the virus neuraminidase. The (3+2) cycloaddition reaction of 2-
ethylbutanenitrile oxide with the cyclopentene dipolarophile derived from Vince lactam is a
key step in the conventional synthesis of peramivir. Our study showed that conducting the
(3+2) cycloaddition reactions with either aliphatic or aromatic nitrile oxide in hexane solution
provided high percentage of the desired regioisomer, and the N-substituent having electron-
withdrawing property is also beneficial to the regioselectivity. This study also demonstrated
an alternative synthetic pathway of (−)-peramivir and the analog having a phenyl group in
place of the 3-pentyl moiety.
Keywords:
Peramivir; Nitrile oxide; Cycloaddition; Influenza; Organic synthesis.
_
*
___________
Corresponding author.
E-mail address: jmfang@ntu.edu.tw (J.-M. Fang)
C.-L.C and T.-W.C contributed equally to this work.
†
1

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1
. Introduction
Seasonal influenza is a major cause of mortality and morbidity among patients with acute
upper respiratory diseases [1,2]. Influenza virus belongs to the family Orthomyxoviridae and
has a single-stranded segmented RNA genome, which comprises eight negative-sense single-
stranded RNA segments to produce at least 11 proteins [3,4]. Hemagglutinin (HA) and
neuraminidase (NA) are glycoproteins anchored on the cell surface of influenza virus.
Genetic diversity of influenza viruses is due to high mutation rates and frequent genetic
reassortments which lead to high variable HA and NA antigens.
NA is responsible for cleaving the linkage between influenza virus and the sialyl receptor
of host cell. Inhibition of the viral NA will suppress the spread of viruses to other host cells
[
5]. There are 11 subtypes of influenza NAs bearing conserved active sites. Therefore, an
effective NA inhibitor may act as a universal anti-influenza drug to all subtypes [6].In



addition to zanamivir (Relenza ) [7] and oseltamivir (Tamiflu ) [8], peramivir (Repiacta ,
Fig. 1) [9−12] is a potent NA inhibitor and approved for treating influenza infection by
intravenous administration [13].
Fig. 1. Chemical structures of peramivir (1), analogue (1a), and the synthetic precursors.
Peramivir was first synthesized by Babu and coworkers via a key step of (3+2)
cycloaddition reaction of 2-ethylbutanenitrile oxide (4a) and cyclopentene 3a, which was
2

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readily prepared from (−)-2-azabicyclo[2.2.1]hept-5-en-3-one (Vince lactam) [9]. As shown
in Eq. 1, the nitrile oxide 4a was generated in situ from 2-ethyl-1-nitrobutane (4) using
phenyl isocyanate as the dehydrating reagent, and the (3+2) cycloaddition with cyclopentene
3
a in benzene (reflux, 24 h) was promoted by Et N to give the major product 6a (61%
3
isolated yield), along with the regioisomer 6b (11%) and their stereoisomers (5%) [14].
Alternatively, the research teams of Miller and Chen treated 2-ethylbutanal oxime with
NaOCl (bleach) to give the corresponding chlorination compound 5a, which produced nitrile
oxide 4a in the presence of Et N and underwent the 1,3-dipolar cycloaddition with alkene 3a
3
in CH Cl solution (reflux or room temperature, 20 h) [15,16].
2
2
The (3+2) cycloaddition between nitrile oxide and alkene is an efficient way to prepare
isoxazoline, which can be reduced to provide the functionality of 3-amino alcohol [17−19].
The synthesis of peramivir benefits from the strategy that employs the (3+2) cycloaddition of
2
-ethylbutanenitrile oxide for simultaneous formation of two covalent bonds and
implantation of latent amino and hydroxyl groups. In this study, we further investigated
various factors, such as the dipolarophile substrates of mono- and bicyclic alkenes, nitrile
oxide 1,3-dipoles, solvents and reaction conditions, which may influence the regio- and
stereochemistry of the (3+2) cycloadditions for the synthesis of peramivir and its analogs [20].
2
. Results and discussion
2
.1. Method for generation of nitrile oxide
3

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In the synthesis of peramivir, the required 2-ethylbutanenitrile oxide was either prepared
by dehydration of 2-ethyl-1-nitrobutane [14] or by dehydrochlorination of 2-ethyl-N-
hydroxybutanimidoyl chloride [15, 16]. In general, nitrile oxide is an active species and
easily undergoes self-dimerization to give 1,2,5-oxadiazole 2-oxide [17−20]. In addition to
NaOCl, some other halogenation reagents, such as chloramine-T, N-bromosuccinimide and
(diacetoxyiodo)benzene have been reported for conversion of oximes to hydroximoyl
chlorides [21−23]. In our study, 2-ethylbutanal oxime and benzaldehyde oxime was treated
with N-chlorosuccinimide (NCS) to give relatively stable chlorination compounds of
hydroximoyl chlorides 5a and 5b, which were isolated and prepared as dilute solution in
appropriate solvent for slow injection to a solution containing Et N to generate the
3
corresponding nitrile oxides 4a or 4b for the in situ (3+2) cycloaddition reactions with the
alkene substrates.
2
.2. Cycloaddition reactions of Vince lactam
We speculated that Vince lactam having a [2.2.1] bicyclic skeleton might render better
stereochemical control and facilitate the (3+2) cycloaddition by releasing the ring strain. We
first tested the (3+2) cycloaddition by adding hydroximoyl chloride 5a to a toluene solution
o
containing lactam (±)-2 and Et N. However, the reaction at 60 C for 8 h only resulted in a
3
complicated mixture containing less than 20% of cycloaddition products.
4

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We then investigated the (3+2) cycloaddition reactions of N-protected Vince lactam
(2a−2c) with benzonitrile oxide (4b). Thus, N-hydroxybenzimidoyl chloride (5b) was
dissolved in toluene solution and slowly injected via syringe pump into a toluene solution
containing N-Boc lactam (±)-2a and Et N (Eq. 2). The desired (3+2) cycloaddition proceeded
3
smoothly at room temperature to give compounds (±)-7a and (±)-7b. The possible
dimerization of reactive nitrile oxide was significantly suppressed by slow addition of
hydroximoyl chloride to Et N for controlled generation of nitrile oxide, which was readily
3
consumed by excess amount of alkene. Though the cycloaddition was much facilitated and
no stereoisomers were formed, the regioselectivity of products was poor (7a/7b = 57:43). The
isomers 7a and 7b were separated by silica gel chromatography, and the desired isomer 7a
(
as racemic mixture) was further recrystallized from CH Cl –hexane for X-ray diffraction
2 2
analysis (supplementary Fig. S1), thus confirming the regio- and stereochemistry.
On using N-Troc lactam (±)-2b as the dipolarophile, the regioselectivity of cycloaddition
products (±)-8a and (±)-8b was slightly increased to 63:37. In contrast, the (3+2)
cycloaddition of lactam (±)-2c bearing an electron-donating benzyl substituent favored the
product of undesired regioisomer (±)-9b. Nonetheless, the minor isomer 9a (as racemic
mixture) was isolated and recrystallized from CH Cl –hexane to verify the regio- and
2
2
stereochemistry by X-ray crystallography (supplementary Fig. S2).
On the basis of photoelectron spectroscopic study [24], Novak and Kovač concluded that
no transannular interaction exists between the π-moiety and amide group of Vince lactam,
though the electronic effects are prominent in norbornadiene system. However, certain
inductive effect might involve in the (3+2) cycloaddition reactions of N-substituted Vince
lactam because the regioselectivity varies with different electronic properties of the
substituents (e.g. Boc, Troc and benzyl groups in Eq. 2).
5

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2
.3. Effects of N-substituents in (3+2) cycloaddition reactions of cyclopentene dipolarophiles
Without success on using bicyclic alkenes 2a−2c to establish the desired regiochemistry
in the (3+2) cycloadditions with nitrile oxides, we re-examined the possible effects of N-
substituents in the cyclopentene dipolarophiles 3a−3g. The substituents include electron-
withdrawing groups, such as tert-butoxycarbonyl (Boc), 2,2,2-trichloroethoxycarbonyl (Troc),
(2-nitrophenyl)sulfonyl (Nosyl), (trifluoromethyl)sulfonyl (Tf), trifluoroacetyl, acetyl and
phthalyl (Phth). The (3+2) cycloaddition reactions were similarly carried out with either
benzonitrile oxide or 2-ethylbutanenitrile oxide, which were in situ generated from the
corresponding hydroximoyl chlorides (Table 1). In comparison with the bicyclic
dipolarophile (e.g. 2a), the monocyclic dipolarophile (e.g. 3a) was less reactive. The
dimerization of nitrile oxides, once generated from hydroximoyl chlorides, could compete
with the desired (3+2) cycloaddition reactions. Thus, more amount of hydroximoyl chloride
was applied to ensure good conversion of cyclopentene dipolarophiles (Table 1).
Table 1
a
Substituent effect in the regioselectivity of (3+2) cycloaddition reactions
3
1
2
R C(=NOH)Cl conv.
c
entry
alkene (R , R )
b
ratio of isomers
(equiv)
(%)
d
d
1
2
3
4
5
6
7
8
9
3a (Boc, H)
3b (Troc, H)
3c (Nosyl, H)
3d (CF SO , H)
3e (CF CO, H)
3f (CH CO, H)
3g (Phth)
3a (Boc, H)
3b (Troc, H)
5a (2)
5a (6)
5a (8)
5a (8)
5a (7)
5a (8)
5a (16)
5b (5)
5b (4)
95
75
95
95
95
95
45
75
90
6a/6b = 85:15
10a/10b = 76:24
11a/11b = 68:32
12a/12b = 68:32
13a/13b = 80:20
14a/14b = 80:20
15a/15b = 64:36
16a/16b = 67:33
17a/17b = 74:26
3
2
3
3
6

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1
1
1
1
1
0
1
2
3
4
3c (Nosyl, H)
5b (6)
5b (4)
5b (4)
5b (4)
5b (8)
75
75
90
95
60
18a/18b = 74:26
19a/19b = 66:34
20a/20b = 87:13
21a/21b = 76:24
22a/22b = 56:44
3d (CF SO , H)
3
2
3e (CF CO, H)
3
3f (CH CO, H)
3
3g (Phth)
a
b
c
d
The reactions of alkenes 3a−3g with the nitrile oxides generated in situ from hydroximoyl
chlorides 5a and 5b at room temperature.
The conversion was estimated by H NMR spectral analysis based on the remaining alkene
3a−3g) in the crude product mixture.
The ratio of regioisomers was calculated based on the characteristic proton signals, such as
H-3a, H-6a and CO CH , in the crude product mixture (Table 2).
1
(
2
3
o
The reaction was conducted at 60 C for 24 h.
For the 1,3-dipolar cycloaddition of 3a bearing an N-Boc group in this study (Table 1,
entry 1), the nitrile oxide 4a was produced by adding hydroximoyl chloride 5a to a toluene
solution containing Et N, different from the previously reported method using 2-ethyl-1-
3
nitrobutane as the precursor of 4a [9,14]. Nonetheless, both methods gave the cycloaddition
o
products 6a and 6b at 60 C in a ratio of 85:15. We further found that the related (3+2)
cycloaddition reaction of alkene (3a−3g) occurred readily at room temperature by slow
injection of hydroximoyl chloride (5a or 5b) to the toluene solution containing Et N (Table
3
1
). The conversion of alkenes to cycloadducts was generally good (75−95%) as estimated by
1
H NMR spectral analysis, except for the reactions of alkene 3g carrying phthalimido
substituent (≤ 60% conversion).
1
The crude product mixture was subject to H NMR spectral analysis to determine the
ratio of regioisomers based on their characteristic protons, such as H-3a, H-6a and CO CH3
2
(Table 2). In general speaking, most a-series isomers exhibited the H-6a at a lower field than
that of b-series isomers, presumably because the H-6a in a-series isomers was deshielded by
the adjacent carboxylate group. The similar deshielding effect was observed for the H-3a of
b-series isomers. Some isomers could be separated by repeated silica gel chromatography,
and 2D NMR spectra were applied to establish the chemical structures and assignment of
protons.
7

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Table 2
Comparison of chemical shifts of characteristic protons in a- and b-series regioisomers.
a
isomer a
H-6a
5.20
5.20
5.19
5.15
5.14
5.15b
NA
isomer b
H-6a
compound
a/6b
H-3a
3.58
3.64
3.72b
NA
3.60
3.60b
NA
CO CH
H-3a
3.89
3.88
3.90
CO CH
2
3
2
3
6
3.76
3.75
3.77
3.78
3.75
3.75
3.74
3.78
3.80
3.81
3.82
3.82
3.80
3.76
4.83
3.79
3.70
3.79
3.79
3.76
3.76
3.73
3.81
3.84
3.87
3.88
3.86
3.67
3.71
b
1
1
1
1
1
1
1
1
1
1
2
2
2
0a/10b
1a/11b
2a/12b
3a/13b
4a/14b
5a/15b
6a/16b
7a/17b
8a/18b
9a/19b
0a/20b
1a/21b
2a/22b
NA
4.80
4.98
4.85
4.83
b
NA
3.69
3.69
b
b
NA
NA
4.20
4.28
4.36
4.37
4.26
4.27b
5.44
5.43
5.44
5.38
5.39
5.39b
NA
4.48
4.46
4.51
4.31
4.37
5.04
5.12
5.08
5.21
5.08
4.87
b
NA
b
b
NA
NA
NA
a
1
Adapted from the H NMR spectra (400 MHz) measured in CDCl solution.
3
b
NA is not available, due to complication by other signals.
2
.4. Effects of solvent, reaction temperature and catalyst
The regiochemistry in 1,3-dipolar cycloaddition reactions of nitrile oxides can be
influenced by several factors such as solvent, reaction temperature, metal catalyst and Lewis
acid [17−19,25,26]. In our present study, the solvent effect is most prominent (Table 3). The
cycloaddition reaction appeared to proceed well in nonpolar solvent, such as hexane and
toluene, to attain high regioselectivity. For example, the 1,3-dipolar cycloaddition of alkene
1
3
b (R = Troc) with benzonitrile oxide in toluene or a mixed toluene−hexane system (Table 3,
entries 7 and 8) showed higher regioselectivity for the desired product 17a than that
conducted in more polar solvent systems (Table 3, entries 9−12). In agreement with this trend,
1
1
the cycloaddition reactions of alkenes 3a (R = Boc) and 3e (R = CF CO) in hexane solution
3
also provided higher percentage of the desired a-series regioisomers than that were
8

ACCEPTED MANUSCRIPT
performed in toluene solution. Though a previous report has demonstrated that the 1,3-
dipolar cycloadditions of nitrile oxides with certain alkenes can proceed in aqueous solution
[
25], we did not test the possibility of using aqueous solvent in our case.
Table 3
a
Solvent effect in the regioselectivity of (3+2) cycloaddition reactions
3
1
R C(=NOH)Cl
conv.
c
entry
alkene (R )
solvent
b
ratio of isomers
(equiv)
(%)
d
d
1
2
3
4
5
6
7
8
9
3a (Boc)
3a (Boc)
3e (CF CO)
5a (2)
5a (4)
5a (7)
5a (7)
5b (5)
5b (4)
5b (4)
5b (4)
5b (4)
5b (4)
5b (4)
5b (4)
5b (4)
5b (4)
5b (4)
PhMe
hexane
PhMe
hexane
PhMe
95
6a/6b = 85:15
6a/6b = 91:9
97
95
95
75
95
90
90
20
60
90
90
90
95
95
13a/13b = 80:20
13a/13b = 90:10
16a/16b = 67:33
16a/16b = 86:14
17a/17b = 74:26
17a/17b = 84:16
17a/17b = 62:38
17a/17b = 63:37
17a/17b = 68:32
17a/17b = 68:32
20a/20b = 87:13
20a/20b = 91:9
20a/20b = 94:6
3
3e (CF CO)
3
3a (Boc)
3a (Boc)
3b (Troc)
3b (Troc)
3b (Troc)
3b (Troc)
3b (Troc)
3b (Troc)
hexane
PhMe
PhMe/hex (1:1)
DMF
1
1
1
1
1
1
0
1
2
3
4
5
CH CN
3
THF
CH Cl
2
2
3e (CF CO)
3e (CF CO)
3e (CF CO)
PhMe
PhMe/hex (1:2)
hexane
3
3
3
a
d
−
Referring to the footnotes of Table 1.
The reaction temperature was not crucial to regioselectivity of the 1,3-dipolar
cycloadditions in the present study. For example, the cycloaddition between 3b and
benzonitrile oxide in toluene solution (Table 3, entry 7) gave the products 17a/17b in a ratio
of 74:26, which was not appreciably affected by performing the reaction at −78, 0, 25 or 95
o
C (supplementary Table S3).
It is well known that 1,3-dipolar cycloadditions of nitrile oxides with allylic alcohols or
electron-deficient alkenes (e.g. α,β-unsaturated ester) can be facilitated by incorporation of
9

ACCEPTED MANUSCRIPT
additives, such as metal-catalyst and Lewis acid, to gain good regio- and stereoselectivities
[
18,21,27]. Hydroximoyl chlorides 5a and 5b were treated with base Et N to generate the
3
corresponding nitrile oxides for in situ cycloaddition with non-activated alkenes 3a−3g,
therefore, Lewis acid catalysis would not be compatible to the basic system in the present
i
study. Indeed, none of additives MgBr , Ti(O Pr) , Zn(OAc) or Zn(acac) could enhance the
2
4
2
2
yield of the desired regioisomer 17a (supplementary Table S3).
1
As an approach to peramivir and its analogs, the key (3+2) cycloadditions of 3a (R =
Boc) were realized in hexane solution to give the desired products 6a and 16a in high
regioselectivity (Table 3, entries 2 and 6). In comparison with the previously reported (3+2)
cycloadditions of 3a with nitrile oxide 4a in toluene (or benzene) solution [14−16], our
current method provided the desired isomers 6a with somewhat better regioselectivity by
conducting the cycloadditions in hexane solution. To our satisfaction, the (3+2)
1
cycloadditions of alkene 3e (R = CF CO) with nitrile oxides 4a and 4b in hexane solution
3
afforded the desired products 13a and 20a in optimized regioselectivity (Table 3, entries 4
and 15). This synthetic procedure could be performed in gram scale to provide 13a and 20a
in ~80% isolated yield after purification by silica gel chromatography.
2
.5 Rationale for regioselectivity
The 1,3-dipolar cycloadditions of nitrile oxides to disubstituted alkenes generally lead to
mixtures of regioisomers, unlike the more regioselective reactions with monosubstituted
alkenes and conjugated dipolarophiles, which have more distinct steric and electronic effects
[
28]. Although the regioselectivity has been interpreted by the HOMO, LUMO or combined
HOMO–LUMO controlled modes [28], it is still difficult to predict the regioselectivity in the
3+2) cycloaddition of disubstituted alkene by frontier orbital theory.
(
1
0

ACCEPTED MANUSCRIPT
In comparison, the (3+2) cycloaddition reactions of alkene 3e bearing trifluoroacetamido
substituent tended to attain high percentage of a-series isomers (13a and 20a), whereas the
(
3+2) cycloaddition reactions of alkene 3g carrying phthalimido substituent resulted in low
regioselectivity (Table 1, entries 7 and 14). We speculate that the high regioselectivity in
3+2) cycloaddition of 3e might be attributable to the intramolecular hydrogen bonded form,
(
which is devoid in 3g. Our preliminary density functional theory (DFT) calculation supports
the preferable structure of 3e (Fig. 2) having the trifluoroacetamide and ester moieties in
proximity to form intramolecular hydrogen bond (N-H•••OCO). The intramolecular hydrogen
bonded form also explains why the regioselectivity increased when (3+2) cycloaddition
reaction was conducted in nonpolar solvent. As the conformation of cyclopentene (e.g. 3e) is
rigidified by intramolecular bonding, the influence of electron-withdrawing substituent (e.g.
trifluoroacetyl), either through bonds or through space, on the C=C double bond would
become substantial [24].
Fig. 2. DFT calculation for the optimized geometry of compound 3e. The functional CAM-
B3LYP and the basis set 6-31G(d) were used in the calculation. The number of imaginary
frequencies = 0. Gibbs free energy = –928.430500 Hartree.
2
.6. Alternative approach to peramivir (−)-1 and analog (−)-1a
In the conventional synthesis of (−)-peramivir [14], the key intermediate (+)-6a is
prepared in 61% yield by the (3+2) cycloaddition of alkene (−)-3a with nitrile oxide 4a in
refluxing toluene (or benzene) solution for 24 h (Eq. 1). In this study (Scheme 1), we found
1
1

ACCEPTED MANUSCRIPT
that the cycloaddition could be performed in hexane solution at room temperature for 4 h to
provide a better yield (66%) of (+)-6a because of the improved regioselectivity (Table 3,
entry 2).
Scheme 1. Synthesis of peramivir (−)-1 and analog (−)-1a bearing a phenyl group at C-1′
position. Reagents and conditions: (a) Et N, hexane, rt, 4 h; isolated yield: (+)-6a, 66%; (+)-
3
1
3a, 80%; (+)-16a, 52%; (+)-20a, 83%; (b) HCl in MeOH, reflux, 23 h; (c) Boc O, Et N,
2 3
o
CH Cl , 0 C → rt, 1.5 h; isolated yield: (+)-6a, 74%; (+)-16a, 73%; (d) Raney nickel, H ,
2
2
2
o
o
THF, rt, 30 min; (e) Ac O, Et N, MeOH, 0 C → rt, 12 h; (f) CF CO H, CH Cl , 0 C → rt,
2
3
3
2
2
2
2
0 min; (g) 1,3-bis(tert-butoxycarbonyl)-2-methyl-2-thiopseudourea, HgCl , Et N, CH Cl ,
2 3 2 2
1
4 h; 32% yield from (+)-16a; (h) NaOH, 30 min; (i) H O, reflux, 1.5 h; 99% yield.
2
1
As shown in Table 3, the (3+2) cycloadditions of alkene 3e (R = CF CO) with nitrile
3
oxides were conducted in hexane solution to afford the desired products 13a and 20a in
optimized regioselectivity. We thus explored the possibility in utilizing the isoxazolines 13a
and 20a for syntheses of peramivir and its analog 1a bearing a phenyl group at C-1′ position.
Accordingly [14], optically active Vince lactam was subjected to solvolysis in
methanolic HCl, and the resulting amino ester was treated with trifluoroacetic anhydride in
1
2

ACCEPTED MANUSCRIPT
the presence of Et N to give (−)-3e bearing a trifluoroacetamido substituent. The (3+2)
3
cycloaddition reaction was carried out by slow injection of a hexane solution of hydroximoyl
chloride 5a via syringe pump into a mixture of alkene (−)-3e and Et N in hexane to afford
3
isoxazoline isomers 13a and 13b (90:10). The desired product (+)-13a was isolated in 80%
yield by silica gel column chromatography. Compound (+)-13a was heated in methanolic
HCl to release the amino group, followed by treatment with di-tert-butyl dicarbonate to give
7
4% yield of the N-Boc derivative (+)-6a. The physical and spectral properties of as-
synthesized (+)-6a were in agreement with that prepared by the conventional procedure [14].
Thus, the formal synthesis of peramivir (−)-1 was realized, though no better yield of
isoxazoline (+)-6a was obtained by the two-step preparation via intermediacy of (+)-13a.
For the synthesis of peramivir analog 1a, benzonitrile oxide was generated from
hydroximoyl chloride 5b in the presence of Et N and reacted in situ with (−)-3a to form
3
isoxazoline (+)-16a in 52% yield. Alternatively, isoxazoline (+)-20a was prepared in high
yield by the (3+2) cycloaddition of alkene (−)-3e with benzonitrile oxide. The trifluoroacetyl
group of (+)-20a was removed by acid-catalyzed solvolysis, and subsequently converted to
N-Boc group to afford (+)-16a. The two-step sequence gave a 61% overall yield of (+)-16a
1
from (−)-3e (R = CF CO), better than that derived from a direct (3+2) cycloaddition of (−)-
3
3
a (R = Boc).
The isoxazoline ring in (+)-16a was subjected to hydrogenolysis over Raney nickel to
furnish N–O cleavage and C=N reduction [14−16,20]. On treatment with acetic anhydride,
the amino group of intermediate product was converted to acetamide, giving a mixture of
1
diastereomers 23a and 23b (85:15) differing at the C-1′ center. In H NMR spectra, the major
isomer 23a with (1′R)-configuration exhibited its H-1′ signal at δ 5.47, whereas isomer 23b
displayed the H-1′ at a higher field (δ 5.24).
1
3

ACCEPTED MANUSCRIPT
The Boc group in 23a/23b was subsequently removed on treatment with trifluoroacetic
acid. The resulting aminium salt was treated with a thiopseudourea reagent, N,N′-bis-(tert-
butoxycarbonyl)-S-methylisothiourea, to afford the bis-Boc-guanidine compound 24a/24b.
The desired product (−)-24a was obtained by column chromatography in 32% overall yield
starting from (+)-16a. After saponification of (−)-24a, the Boc protecting groups were
successfully removed by heating in water [20,29] to yield the peramivir analog (−)-1a
bearing a phenyl group at the C-1′ position.
In an attempt to obtain (−)-24a via a shorter pathway (Scheme 2), (+)-20a was reduced
by Raney nickel under an atmosphere of hydrogen. The resulting amino alcohol was treated
with pentafluorophenyl acetate to afford compound 25a and its (1′S)-diastereomer (85:15).
However, the selective hydrolysis of trifluoroaceamide in the presence of acetamide and
methyl ester posed a problem [30]. So far, we have tried several reaction conditions for the
hydrolysis of 25a in either acidic or basic conditions, including refluxing in 1 M HCl aqueous
solution, refluxing in 35% NH methanolic solution, refluxing or stirring with K CO in
3
2
3
anhydrous or moist MeOH, without success to obtain the desired product 26a.
Scheme 2. An attempted conversion of (+)-20a to (−)-24a via a short pathway.
3
. Conclusion
1
4

ACCEPTED MANUSCRIPT
We demonstrate in this study that chlorination of oximes with NCS gave the relatively
stable hydroximoyl chlorides 5a and 5b as convenient precursors of nitrile oxides 4a and 4b
for in situ (3+2) cycloadditions with alkenes. Because our initial plan of using Vince lactam
as a reactive 1,3-dipolarphile could not afford good regioselectivity, we turned to investigate
the (3+2) cycloaddition reactions of the cyclopentene dipolarophiles 3a−3g equipped with
various N-substituents. A suitable electron withdrawing substituent (e.g. Boc, Troc and
trifluoroacetyl) on the dipolarophiles was an important factor to induce the regioselectivity.
Both aliphatic nitrile oxide (4a) and aromatic nitrile oxide (4b) showed a similar trend of
regioselectivity in the (3+2) cycloaddition reactions. The cycloaddition reactions proceeded
smoothly in nonpolar solvent (e.g. hexane and toluene) to give high percentage of the desired
1
a-series regioisomers. Up to 94:6 regioselectivity of the (3+2) cycloaddition between 3e (R
=
CF CO) and benzonitrile oxide was obtained by stirring in hexane solution at room
3
temperature for 4 h (Table 3, entry 15).
The key intermediate (+)-6a for the synthesis of (−)-peramivir was obtained from the
1
cycloaddition of (−)-3a (R = Boc) in hexane solution, showing better regioselectivity (91:9)
than that performed in toluene solution (compared entries 1 and 2 in Table 3). We also
carried out the 1,3-dipolar cycloadditions of (−)-3e in high regioselectivity to give
isoxazolines (+)-13a and (+)-20a, and demonstrated how to convert them into the N-Boc
analogs (+)-6a and (+)-16a as an alternative approach to the syntheses of (−)-peramivir and
the analog (−)-1a having phenyl group in place of the 3-pentyl moiety.
4
. Experimental section
4
.1. General
1
5

ACCEPTED MANUSCRIPT
All the reagents and solvents were reagent grade and used without further purification
unless otherwise specified. Dichloromethane (CH Cl ) was distilled from CaH . All air or
2
2
2
moisture sensitive experiments were performed under an atmosphere of argon. Analytic thin-
layer chromatography (TLC) was performed on 0.25 mm E. Merck silica gel 60 F254 glass
plates. TLC plate visualization was performed with UV light, potassium permanganate,
ninhydrin or phosphomolybdic acid (PMA). E. Merck silica gel 60 (0.040–0.063 mm particle
sizes) was used for column chromatography. High-performance liquid chromatography
(HPLC) was conducted on a Schambeck SFD GmbH SDS2100 instrument equipped with
Schambeck SFD GmbH S-3210 UV/Vis detector set at absorption wavelength of 365 nm.
TM
Dikma column (250 × 10.0 mm) packed with Inspire silica (10 ꢀm) was used for HPLC,
and an eluent of hexane/EtOAc (86:14) at a flow rate of 1.5 mL/min was applied.
Melting points were recorded on a Yanaco micro apparatus and are uncorrected. Optical
rotations were measured on digital polarimeter of Japan JASCO Co. P-2000. [α] values are
D
–
1
2
–1
given in the unit of 10 deg cm g . Infrared (IR) spectra was recorded on Nicolet Manga
50-II. Nuclear magnetic resonance (NMR) spectra were obtained on Varian Unity Plus-400
400 MHz) or Bruker A VIII (500 MHz) NMR. Chemical shifts (δ) are given in parts per
million (ppm) relative to residual CDCl solvent: δ 7.24 and δ 77.0 (the central line of
5
(
3
H
C
triplet). Coupling constants (J) are given in hertz (Hz) and the splitting patterns are reported
as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), and br (broad). The
electrospray ionization high resolution mass spectra (ESI‒HRMS) experiments were
conducted on a Bruker Daltonics BioTOF III high-resolution mass spectrometer.
4
.2. Representative procedure A: 1,3-dipolar cycloaddition of N-protected Vince lactam with
benzonitrile oxide generated in situ from hydroximoyl chloride
1
6

ACCEPTED MANUSCRIPT
A solution of N-Boc lactam (±)-2a (418 mg, 2.0 mmol) and triethylamine (0.83 mL, 6.0
mmol) in toluene (2.0 mL) was stirred at room temperature under an atmosphere of argon. A
solution of hydroximoyl chloride 5b (467 mg, 3.0 mmol) in toluene (12 mL) was injected via
syringe pump at a rate of 3.0 mL/h. After completion of injection, the mixture was extracted
with EtOAc and brine. The organic phase was dried over MgSO , filtered, and concentrated
4
1
under reduced pressure. H NMR spectral analysis indicated that the crude product mixture
contained cycloaddition products (±)-7a and (±)-7b in a ratio of 57:43. Compounds 7a and
7
b were separated by silica gel chromatography (CH Cl ), and 7a was further recrystallized
2 2
from CH Cl –hexane for X-ray diffraction analysis.
2
2
4
2
.3. Representative procedure B: 1,3-dipolar cycloaddition of N-protected 4-aminocyclopent-
-ene-1-carboxylate with nitrile oxide generated in situ from hydroximoyl chloride
A solution of alkene (±)-3e (24 mg, 0.1 mmol) and triethylamine (110 ꢀL, 0.8 mmol) in
hexane (1.0 mL) was stirred at room temperature under an atmosphere of argon. A solution of
-ethyl-N-hydroxybutanimidoyl chloride (5a, 104 mg, 0.7 mmol) in hexane (8.0 mL) was
2
injected via syringe pump at a rate of 2.0 mL/h. After completion of injection, the mixture
was extracted with EtOAc and brine. After completion of injection, the mixture was extracted
with EtOAc and brine. The organic phase was dried over MgSO , filtered, and concentrated
4
1
under reduced pressure. H NMR spectral analysis indicated the crude product mixture
contained cycloaddition products (±)-13a and (±)-13b in a ratio of 90:10.
4
.4. Representative procedure C: conversion of N-trifluoroacetyl protected cycloaddition
product to N-Boc protected analog
1
7

ACCEPTED MANUSCRIPT
Trifluoroacetamide (+)-20a (1.02 g, 3.1 mmol) was dissolved in 4 M HCl methanolic
solution and heated at boiling for 23 h. The solution was concentrated under reduced pressure.
The residue was dissolved in CH Cl , added Et N (1.3 mL, 9.3 mmol) and Boc O (0.93 mL,
2
2
3
2
o
4
.0 mmol) at 0 C. The mixture was stirred at room temperature for 1.5 h, and then
concentrated under reduced pressure. The residue was re-dissolved in CH Cl , washed with
2
2
H O, dried over MgSO , and filtered. The filtrate was concentrated by rotatory evaporation
2
4
under reduced pressure. The residue was purified by silica gel column chromatography
hexane/EtOAc = 10:1) to afford carbamate (+)-16a (819 mg) in 73% overall yield from (+)-
(
2
4
4
0a.
.5. Synthetic procedure and compound characterization
.5.1. Tert-Butyl 3-oxo-2-azabicyclo[2.2.1]hept-5-ene-2-carboxylate (2a)
To a solution of lactam (±)-2 (307 mg, 3.0 mmol) and DMAP (183 mg, 1.5 mmol) in
acetonitrile (3 mL) was added Boc O (687 mg, 3.15 mmol). The mixture was stirred at room
2
temperature for 3 h, concentrated under reduced pressure, and purified by silica gel
chromatography (hexane/EtOAc = 5:1) to afford the Boc derivative (±)-2a (531 mg, 85%
o
yield). C H NO ; white solid; mp 53–54 C; TLC (hexane/EtOAc = 5:1) R = 0.46; IR ν
1
1
15
3
f
max
–1
1
(
neat) 3384, 2980, 2939, 2880, 1790, 1710, 1369, 1335, 1152, 991, 935 cm ; H NMR (400
MHz, CDCl ) δ 6.76 (1 H, ddd, J = 5.6, 2.4, 0.4 Hz), 6.53 (1 H, ddd, J = 5.2, 3.2, 1.6 Hz),
3
4
1
5
.85–4.79 (1 H, m), 3.27–3.22 (1 H, m), 2.21 (1 H, dt, J = 8.4, 1.6 Hz), 2.02 (1 H, dt, J = 8.8,
1
3
.6 Hz), 1.36 (9 H, s); C NMR (100 MHz, CDCl ) δ 176.1, 150.2, 139.8, 138.0, 82.4, 62.3,
3
4.8, 54.2, 27.9 (3 ×); ESI−HRMS calcd for C H NO Na: 232.0948, found m/z 232.0944
1
1
15
3
+
[
M + Na] .
1
8

ACCEPTED MANUSCRIPT
4
.5.2. 2,2,2-Trichloroethyl 3-oxo-2-azabicyclo[2.2.1]hept-5-ene-2-carboxylate (2b)
To a solution of lactam (±)-2 (109 mg, 1.0 mmol) and triethylamine (0.50 mL, 3.6 mmol)
o
in CH Cl (5 mL) was added 2,2,2-trichloroethyl chloroformate (0.46 mL, 3.3 mmol) at 0 C
2
2
under an atmosphere of argon. The mixture was stirred at room temperature for 12 h, and
extracted with EtOAc and brine. The organic phase was dried over MgSO , filtered, and
4
concentrated under reduced pressure. The residue was purified by silica gel chromatography
(EtOAc/hexane = 1:10) to afford the Troc derivative (±)-2b (68.0 mg, 24% yield).
o
C H Cl NO ; white solid; mp 99–100 C; TLC (hexane/EtOAc = 5:1) R = 0.38; IR ν (neat)
9
8
3
3
f
max
–
1 1
3
007, 2956, 2876, 1803, 1380, 1338, 1276, 1182, 1121, 999, 882, 756, 714 cm ; H NMR
(
400 MHz, CDCl ) δ 6.88 (1 H, dd, J = 5.2, 2.4 Hz), 6.69–6.61 (1 H, m), 5.10–5.04 (1 H, m),
3
4
8
1
.79 (1 H, d, J = 12.0 Hz), 4.73 (1 H, d, J = 12.0 Hz), 3.44–3.39 (1 H, m), 2.38 (1 H, dt, J =
13
.4, 1.6 Hz), 2.21 (1 H, dt, J = 8.8, 1.6 Hz); C NMR (100 MHz, CDCl ) δ 175.5, 148.4,
3
39.9, 138.2, 94.5, 74.7, 62.8, 54.5, 53.7; ESI−HRMS calcd for C H Cl NO : 283.9639,
9
8
3
3
+
found m/z 283.9643 [M + H] .
4
.5.3. 2-Benzyl-2-azabicyclo[2.2.1]hept-5-en-3-one (2c)
Under an atmosphere of argon, sodium hydride (80 mg of a 60% suspension in mineral
oil, 2.0 mmol) was placed in a flask, washed with hexane, and added anhydrous THF (4 mL).
A solution of lactam (±)-2 (109 mg, 1.0 mmol) in anhydrous THF (2 mL) was added
dropwise. The mixture was stirred at room temperature for 30 min, and benzyl bromide (0.24
mL, 2.0 mmol) was added dropwise. After stirring for 8 h, the mixture was extracted with
EtOAc and H O. The organic phase was dried over MgSO , filtered, and concentrated under
2
4
reduced pressure. The residue was purified by silica gel chromatography (hexane/EtOAc =
:1) to afford the benzyl derivative (±)-2c (144 mg, 72% yield). C H NO; brown oil; TLC
5
1
3
13
(
hexane/EtOAc = 5:1) R = 0.22; IR νmax (neat) 3065, 3027, 3005, 2948, 2868, 1719, 1558,
f
1
9

ACCEPTED MANUSCRIPT
–
1 1
1
7
3
2
1
2
496, 1455, 1391, 1356, 1232, 1075, 930, 836, 757, 699 cm ; H NMR (400 MHz, CDCl ) δ
3
.30–7.19 (3 H, m), 7.17–7.13 (2 H, m), 6.62–6.44 (2 H, m), 4.40 (1 H, d, J = 14.8 Hz), 4.01–
.97 (1 H, m), 3.93 (1 H, d, J = 14.8 Hz), 3.37–3.32 (1 H, m), 2.25 (1 H, dt, J = 7.6, 1.6 Hz),
1
3
.03 (1 H, dt, J = 7.6, 1.6 Hz); C NMR (100 MHz, CDCl ) δ 179.9, 139.4, 137.1, 136.3,
3
28.4 (2 ×), 128.2 (2 ×), 127.4, 62.5, 58.2, 53.6, 47.8; ESI−HRMS calcd for C H NO:
1
3
14
+
00.1072, found m/z 200.1075 [M + H] .
4
.5.4. Methyl 4-((tert-butoxycarbonyl)amino)cyclopent-2-ene-1-carboxylate (3a)
Lactam (±)-2 (109 mg, 1.0 mmol) was placed in a flask under an atmosphere of argon,
and hydrochloride in methanol (1.25 M, 3.0 mL) was added. The mixture was heated under
reflux for 12 h, and then concentrated under reduced pressure. The residue was dissolved in
CH Cl (5 mL), and treated successively with triethylamine (170 ꢀL, 1.2 mmol) and Boc O
2
2
2
(0.28 mL, 1.2 mmol). The mixture was stirred at room temperature for 1 h, and extracted with
CH Cl , 1 M HCl and water. The organic phase was dried over MgSO , filtered, and
2
2
(aq.)
4
concentrated under reduced pressure. The residue was purified by silica gel chromatography
EtOAc/hexane = 1:20) to afford the Boc derivative (±)-3a (190.0 mg, 79% yield).
C H NO ; colorless oil; TLC (hexane/EtOAc = 5:1) R = 0.32; IR ν (neat) 3374, 3067,
(
12
19
4
f
max
–
1 1
2
5
978, 2928, 1737, 1521, 1436, 1330, 1157, 1061, 860 cm ; H NMR (400 MHz, CDCl ) δ
3
.95–5.74 (2 H, m), 4.87 (1 H, br), 4.76 (1 H, br), 3.69 (3 H, s), 3.49–3.41 (1 H, m), 2.47 (1
1
3
H, dt, J = 14.0, 8.8 Hz), 1.84 (1 H, dt, J = 14.0, 4.0 Hz), 1.42 (9 H, s); C NMR (100 MHz,
CDCl ) δ 174.6, 154.9, 134.6, 130.8, 79.0, 55.6, 51.9, 48.9, 34.3, 28.2 (3 ×); ESI−HRMS
3
+
calcd for C H NO Na: 264.1212, found m/z 264.1216 [M + Na] .
1
2
19
4
4
.5.5. Methyl 4-(((2,2,2-trichloroethoxy)carbonyl)amino)cyclopent-2-ene-1-carboxylate (3b)
2
0

ACCEPTED MANUSCRIPT
By a procedure similar to that for 3a, lactam (±)-2 (109 mg, 1.0 mmol) was heated with
hydrochloride in methanol (1.25 M) under reflux for 12 h, and the intermediate product was
treated with 2,2,2-trichloroethyl chloroformate (0.17 mL, 1.2 mmol) in CH Cl solution in the
2
2
presence of Et N to give the Troc derivative (±)-3b (200 mg, 63% yield). C H Cl NO ;
3
10 12
3
4
°
white solid; mp 66–67 C; TLC (hexane/EtOAc = 8:1) R = 0.18; IR ν (neat) 3345, 3074,
f
max
–
1 1
2
953, 2844, 1732, 1520, 1436, 1328, 1093, 1052, 942, 819, 727, 569 cm ; H NMR (400
MHz, CDCl ) δ 5.80–5.91 (2 H, m), 5.51 (1 H, d, J = 9.2 Hz), 4.79 (1 H, td, J = 8.8, 3.2 Hz),
3
4
.68 (1 H, d, J = 12.0 Hz), 4.64 (1 H, d, J = 12.0 Hz), 3.66 (3 H, s), 3.46 (1 H, ddd, J = 8.4,
13
4
.0, 0.8 Hz), 2.47 (1 H, dt, J = 14.0, 8.4 Hz), 1.90 (1 H, dt, J = 14.0, 4.0 Hz); C NMR (100
MHz, CDCl ) δ 174.7, 153.7, 133.9, 131.9, 95.5, 74.3, 56.4, 52.2, 49.1, 34.3; ESI−HRMS
3
+
calcd for C H Cl NO Na: 337.9706, found m/z 337.9724 [M + Na] .
1
0
12
3
4
4
.5.6. Methyl 4-((2-nitrophenyl)sulfonamido)cyclopent-2-ene-1-carboxylate (3c)
By a procedure similar to that for 3a, lactam (±)-2 (218 mg, 2.0 mmol) was heated with
hydrochloride in methanol (1.25 M) under reflux for 12 h, and the intermediate product was
treated with 4-nitrobenzenesulfonyl chloride (485 mg, 2.2 mmol) in CH Cl solution in the
2
2
presence of Et N (0.34 mL, 2.4 mmol) and DMPA (24 mg, 0.2 mmol) to give the Nosyl
3
°
derivative (±)-3c (239 mg, 47% yield). C H N O S; white solid; mp 117–118 C; TLC
1
3
14
2
6
(
EtOAc/hexane = 1:3) R = 0.15; IR νmax (neat) 3648, 3308, 3098, 2955, 2849, 1732, 1693,
f
–
1 1
1
538, 1442, 1360, 1166, 1061, 1005, 914, 786, 731, 656, 598, 562 cm ; H NMR (400 MHz,
CDCl ) δ 8.15–8.08 (1 H, m), 7.84–7.77 (1 H, m), 7.75–7.68 (2 H, m), 5.89–5.80 (2 H, m),
3
5
.66 (1 H, dt, J = 5.6, 2.4 Hz,), 4.65–4.55 (1 H, m), 3.65 (3 H, s), 3.42–3.34 (1 H, m), 2.28 (1
13
H, dt, J = 14.4, 8.4 Hz), 1.83 (1 H, dt, J = 14.0, 3.6 Hz); C NMR (100 MHz, CDCl ) δ
3
1
74.0, 147.7, 134.7, 133.6, 133.1, 132.9, 132.9, 130.4, 125.2, 59.4, 52.3, 49.0, 34.0; ESI−
+
HRMS calcd for C H N O SNa: 349.0460, found m/z 349.0465 [M + Na] .
1
3
14
2
6
2
1

ACCEPTED MANUSCRIPT
4
.5.7. Methyl 4-((trifluoromethyl)sulfonamido)cyclopent-2-ene-1-carboxylate (3d)
By a procedure similar to that for 3a, lactam (±)-2 (218 mg, 2.0 mmol) was heated with
hydrochloride in methanol (1.25 M) under reflux for 12 h, and the intermediate product was
treated with trifluoromethanesulfonic anhydride (0.19 mL, 1.1 mmol) in CH Cl solution in
2
2
the presence of Et N to give the triflate derivative (±)-3d (164 mg, 60% yield). C H F NO S;
3
8
10
3
4
colorless liquid; TLC (EtOAc /hexane = 1:8) R = 0.20; IR ν (neat) 3650, 3296, 3233, 3959,
f
max
–
1 1
2
900, 2851, 1721, 1452, 1384, 1235, 1065, 1009, 928, 788, 741, 613, 572 cm ; H NMR
(
400 MHz, CDCl ) δ 6.02–5.95 (2 H, m), 5.91 (1 H, d, J = 8.4 Hz), 4.64 (1 H, t, J = 8.8 Hz),
3
3
.72 (3 H, s), 3.55–3.48 (1H, m), 2.41 (1 H, dt, J = 14.4, 8.0 Hz), 2.09 (1 H, dt, J = 14.4, 1.6
13
1
Hz); C NMR (100 MHz, CDCl ) δ 175.5, 133.5, 133.2, 119.6 ( J = 319 Hz), 60.1, 52.7,
3
CF
19
4
9.0, 34.9; F NMR (376 MHz, CDCl ) δ −78.3 (3 ×); ESI−HRMS calcd for C H F NO S:
3 8 9 3 4
–
2
72.0204, found m/z 272.0200 [M – H] .
4
.5.8. Methyl 4-(2,2,2-trifluoroacetamido)cyclopent-2-ene-1-carboxylate (3e)
Under an atmosphere of argon, a mixture of lactam (±)-2 (1.09 g, 10.0 mmol) and
hydrochloride in methanol (1.25 M, 20 mL) was heated under reflux for 12 h. The mixture
was then concentrated under reduced pressure. To the residue were added CH Cl (10 mL)
2
2
and triethylamine (4.66 mL, 33.5 mmol). Trifluoroacetic anhydride (3.34 mL, 24.0 mmol)
o
was then added dropwise at 0 C. The mixture was stirred at room temperature for 18 h to
complete the acylation reaction. The mixture was extracted with CH Cl and brine. The
2
2
organic phase was dried over MgSO , filtered, and concentrated under reduced pressure. The
4
residue was purified by silica gel chromatography (hexane/EtOAc = 10:1) to afford (±)-3e,
1
.76 g, 75% yield). By a similar procedure, optically active lactam (−)-2 underwent
solvolysis in 4 M HCl methanolic solution for 2.5 h, followed by acylation with
2
2

ACCEPTED MANUSCRIPT
2
0
D
trifluoroacetic anhydride, to give (−)-3e. C H F NO ; colorless oil; [α] = −47.4 (c = 2.0,
9
10
3
3
CHCl ); TLC (EtOAc/hexane = 1:8) R = 0.15; IR ν (neat) 3315, 3081, 2958, 2918, 2849,
3
f
max
–
1 1
1
720, 1551, 1439, 1349, 1215, 1161 cm ; H NMR (400 MHz, CDCl ) δ 7.26 (1 H, br), 5.99
3
(1 H, dd, J = 5.6, 2.8 Hz), 5.94 (1 H, dt, J = 5.4, 2.0 Hz), 5.06 (1 H, t, J = 4.4 Hz), 3.72 (3 H,
s), 3.55 (1 H, dq, J = 8.0, 2.0 Hz), 2.42 (1 H, dt, J = 14.4, 8.4 Hz), 1.99 (1 H, dt, J = 14.0, 2.0
13
2
1
Hz); C NMR (100 MHz, CDCl ) δ 175.2, 156.0 ( J = 37 Hz) , 133.4, 132.9, 115.8 ( J =
3
CF
CF
1
9
2
86 Hz), 54.5, 52.5, 49.2, 33.9; F NMR (376 MHz, CDCl ) δ −76.6 (3 ×); ESI−HRMS
3
+
calcd for C H F NO : 238.0691, found m/z 238.0696 [M + H] .
9
11
3
3
4
.5.9. Methyl 4-acetamidocyclopent-2-ene-1-carboxylate (3f)
By a procedure similar to that for 3a, lactam (±)-2 (218 mg, 2.0 mmol) was heated with
hydrochloride in methanol (1.25 M) under reflux for 12 h, and the intermediate product was
treated with acetic anhydride (0.28 mL, 3.0 mmol) in CH Cl solution in the presence of Et N
2
2
3
to give the acetamide derivative (±)-3f (234 mg, 64% yield). C H NO , white solid; mp 59–
9
13
3
°
6
0 C; TLC (EtOAc/hexane = 2:1) R = 0.29; IR ν (neat) 3587, 3378, 3273, 3065, 2953,
f max
–1 1
2
847, 1738, 1652, 1541, 1437, 1375, 1174, 1065, 1009, 927, 741, 603 cm ; H NMR (400
MHz, CDCl ) δ 6.01 (1 H, br), 5.91–5.80 (2 H, m), 5.09–5.01 (1 H, m), 3.69 (3 H, s), 3.53–
3
3
.44 (1 H, m), 2.43 (1 H, dt, J = 14.0, 8.4 Hz), 1.93 (3 H, s), 1.85 (1 H, dt, J = 14.0, 3.2 Hz);
1
3
C NMR (100 MHz, CDCl ) δ 175.4, 169.1, 134.6, 131.6, 54.2, 52.3, 49.2, 34.3, 23.4; ESI−
3
+
HRMS calcd for C H NO Na: 206.0793, found m/z 206.0796 [M + Na] .
9
13
3
4
.5.10. Methyl 4-(1,3-dioxoisoindolin-2-yl)cyclopent-2-ene-1-carboxylate (3g)
Under an atmosphere of argon, lactam (±)-2 (218 mg, 2.0 mmol) was placed in a flask,
and hydrochloride in methanol (1.25 M, 2.5 mL) was added. The mixture was heated under
reflux for 12 h, and then concentrated under reduced pressure. The residue was dissolved in
2
3

ACCEPTED MANUSCRIPT
1
1
,2-dichloroethane (5 mL), and phthalic anhydride (163 mg, 1.1 mmol) and DMAP (146 mg,
.2 mmol) were successively added. The mixture was stirred at room temperature for 17 h,
and extracted with CH Cl and 1 M HCl and water. The organic phase was dried over
(aq.)
2
2
MgSO , filtered, and concentrated under reduced pressure. The residue was purified by silica
4
gel chromatography (hexane/EtOAc = 10:1) to afford the phthalimide derivative (±)-3g (117
°
mg, 43% yield). C H NO ; white solid; mp 72–73 C; TLC (EtOAc/hexane = 1:3) R = 0.37;
1
5
13
4
f
–
IR νmax (neat) 2953, 1771, 1710, 1467, 1392, 1370, 1203, 1172, 1113, 933, 891, 718, 700 cm
1
1
;
H NMR (400 MHz, CDCl ) δ 7.82–7.76 (2 H, m), 7.71–7.65 (2 H, m), 6.14 (1 H, dt, J =
3
5
.6, 2.4 Hz), 5.78 (1 H, dt, J = 5.6, 2.4 Hz), 5.38–5.29 (1 H, m), 3.77 (3 H, s), 3.68–3.59 (1 H,
1
3
m), 2.66 (1 H, dt, J = 14.0, 9.2 Hz), 2.50 (1 H, dt, J = 13.6, 6.4 Hz); C NMR (100 MHz,
CDCl ) δ 173.3, 167.8 (2 ×), 133.8 (2 ×), 132.0, 131.8, 130.6 (2 ×), 123.1 (2 ×), 54.7, 52.0,
3
+
4
9.5, 31.0; ESI−HRMS calcd for C H NO : 272.0923, found m/z 272.0907 [M + H] .
1
5
14
4
4
.5.11. 2-Ethyl-N-hydroxybutanimidoyl chloride (5a)
To a solution of 2-ethylbutanal oxime (575 mg, 5.0 mmol) in DMF (5.0 mL) was added
N-chlorosuccinimide (800.0 mg, 6.0 mmol) at room temperature. The mixture was stirred for
h, and concentrated under reduced pressure. The residue was extracted with EtOAc and
4
brine. The organic phase was dried over MgSO , filtered, and concentrated under reduced
4
pressure to afford hydroximoyl chloride 5a (566 mg, 76% yield). C H ClNO; colorless
6
12
liquid; TLC (EtOAc/hexane = 1:2) R = 0.17; IR ν (neat) 3440, 3062, 3028, 2981, 2891,
f
max
2
7
1
2
753, 2602, 1955, 1882, 1811, 1633, 1578, 1493, 1445, 1304, 1289, 1210, 1074, 967, 869,
–
1 1
56, 692 cm ; H NMR (400 MHz, CDCl ) δ 8.66 (1 H, br), 2.37 (1 H, tt, J = 9.2, 5.2 Hz),
3
1
3
.66–1.44 (4 H, m), 0.85 (6 H, t, J = 7.2 Hz); C NMR (100 MHz, CDCl ) δ 145.98, 49.51,
3
5.26 (2 ×), 11.47 (2 ×).
2
4

ACCEPTED MANUSCRIPT
4
.5.12. N-Hydroxybenzimidoyl chloride (5b)
To a solution of benzaldehyde oxime (605 mg, 5.0 mmol) in DMF (5 mL) was added N-
chlorosuccinimide (730 mg, 5.5 mmol) at room temperature. The mixture was stirred for 4 h,
and concentrated under reduced pressure. The residue was extracted with EtOAc and brine.
The organic phase was dried over MgSO , filtered, and concentrated under reduced pressure
4
to afford hydroximoyl chloride 5b as a mixture of E- and Z-isomers (713 mg, 92% yield).
C H ClNO; oily solid; TLC (EtOAc/hexane = 1:5) R = 0.57 and 0.61; IR ν (neat) 3405,
7
6
f
max
3
9
7
063, 2972, 2934, 2877, 1766, 1635, 1606, 1461, 1383, 1279, 1172, 1146, 1089, 1059, 971,
–
1 1
23, 816 cm ; H NMR (400 MHz, CDCl , containing E-and Z-isomers) δ 8.14 (1 H, s),
3
1
3
.60–7.53/7.96–7.93 (2 H, m), 7.41–7.35/7.45–7.39 (3 H, m); C NMR (100 MHz, CDCl ,
3
containing E-and Z-isomers) δ 140.6, 132.2, 130.7, 128.4 (2 ×), 127.1(2 ×).
4
.5.13.
Tert-Butyl
6-oxo-3-phenyl-3a,6,7,7a-tetrahydro-4,7-methanoisoxazolo[4,5-
c]pyridine-5(4H)-carboxylate (7a) and tert-butyl 5-oxo-3-phenyl-4,5,7,7a-tetrahydro-4,7-
methanoisoxazolo[5,4-c]pyridine-6(3aH)-carboxylate (7b)
According to the representative procedure A, the cycloaddition of (±)-2a (418 mg, 2.0
mmol) with benzonitrile oxide, which was generated in situ from hydroximoyl chloride 5b
(467 mg, 3.0 mmol), was carried out to give a crude product containing (±)-7a and (±)-7b in
1
a ratio of 57:43 as shown by the H NMR analysis. Some 7a (121 mg, 18% yield) and 7b (54
mg, 8% yield) were isolated by repeated silica gel chromatography (CH Cl ). Compound (±)-
2
2
7
a was further recrystallized from CH Cl –hexane for X-ray diffraction analysis.
2 2
o
Compound 7a: C H N O ; white solid; mp 174–176 C; TLC (CH Cl ) R = 0.35; IR
18
20
2
4
2
2
f
–1 1
νmax (neat) 2975, 2924, 2852, 1793, 1702, 1358, 1298, 1146, 887, 773 cm ; H NMR (400
MHz, CDCl ) δ 7.83–7.75 (2 H, m), 7.45–7.40 (3 H, m), 5.23 (1 H, dt, J = 8.4, 1.6 Hz), 4.79–
3
4
.74 (1 H, m), 4.24 (1 H, d, J = 8.0 Hz), 3.32–3.27 (1 H, m), 2.00 (1 H, dt, J = 11.2, 1.6 Hz),
2
5

ACCEPTED MANUSCRIPT
1
3
1
1
.87 (1 H, dt, J = 11.2, 1.6 Hz), 1.58 (9 H, s); C NMR (100 MHz, CDCl ) δ 170.8, 154.7,
3
49.0, 130.7, 129.0 (2 ×), 127.8, 126.7 (2 ×), 84.1, 83.9, 61.1, 58.8, 54.8, 31.8, 28.1 (3 ×);
+
ESI−HRMS calcd for C H N O Na: 351.1321, found m/z 351.1337 [M + Na]
1
8
20
2
4
o
Compound 7b: C H N O ; white solid; mp 200–204 C; TLC (CH Cl ) R = 0.57; IR
18
20
2
4
2
2
f
–1 1
νmax (neat) 3063, 2979, 2931, 1793, 1715, 1349, 1294, 1149, 962, 765 cm ; H NMR (400
MHz, CDCl ) δ 7.72–7.67 (2 H, m), 7.44–7.38 (3 H, m), 5.13 (1 H, dt, J = 8.0, 1.6 Hz), 4.74–
3
4
1
1
.69 (1 H, m), 4.23 (1 H, d, J = 8.0 Hz), 3.06–3.01 (1 H, m), 2.01 (1 H, dt, J = 11.6, 1.6 Hz),
1
3
.77 (1 H, dt, J = 11.6, 1.6 Hz), 1.52 (s, 9 H); C NMR (100 MHz, CDCl ) δ 172.7, 155.0,
3
48.5, 130.5, 128.9 (2 ×), 127.3, 126.7 (2 ×), 85.4, 83.7, 62.6, 53.1, 49.1, 31.7, 27.9 (3 ×);
+
ESI−HRMS calcd for C H N O Na: 351.1321, found m/z 351.1332 [M + Na] .
1
8
20
2
4
4
.5.14. 2,2,2-Trichloroethyl 6-oxo-3-phenyl-3a,6,7,7a-tetrahydro-4,7-methanoisoxazolo[4,5-
c]pyridine-5(4H)-carboxylate (8a) and 2,2,2-trichloroethyl 5-oxo-3-phenyl-4,5,7,7a-
tetrahydro-4,7-methanoisoxazolo[5,4-c]pyridine-6(3aH)-carboxylate (8b)
According to the representative procedure A, the cycloaddition of (±)-2b (87 mg, 0.31
mmol) with benzonitrile oxide, which was generated in situ from hydroximoyl chloride 5b
(115 mg, 0.74 mmol), was carried out to give a crude product containing (±)-8a and (±)-8b in
1
a ratio of 63:37 as shown by the H NMR analysis (partial, 400 MHz, CDCl ) δ 5.25 (0.63 H,
3
d, J = 8.0 Hz, H-7)/ 5.21 (0.37 H, d, J = 8.0 Hz, H-7), 4.35 (0.63 H, d, J = 8.0 Hz, H-4)/ 4.27
(0.37 H, d, J = 8.0 Hz, H-4).
4
.5.15. 5-Benzyl-3-phenyl-4,5,7,7a-tetrahydro-4,7-methanoisoxazolo[4,5-c]pyridin-6(3aH)-
one (9a) and 6-benzyl-3-phenyl-3a,6,7,7a-tetrahydro-4,7-methanoisoxazolo[5,4-c]pyridin-
(4H)-one (9b).
5
2
6

ACCEPTED MANUSCRIPT
According to the representative procedure A, the cycloaddition of (±)-2c (62 mg, 0.31
mmol) with benzonitrile oxide, which was generated in situ from hydroximoyl chloride 5b
(102 mg, 0.66 mmol), was carried out to give a crude product containing (±)-9a and (±)-9b in
1
a ratio of 37:63 as shown by the H NMR analysis. Some 9a (9 mg, 7% yield) and 9b (43 mg,
3
1% yield) were isolated by repeated silica gel chromatography (CH Cl /MeOH = 100:1).
2 2
Compound (±)-9a was further recrystallized from CH Cl –hexane for X-ray diffraction
2
2
analysis.
Compound 9a: C H N O ; white solid; mp 136–138 C; TLC (hexane/EtOAc = 2:1) R
f
o
2
0
18
2
2
=
0.25; IR νmax (neat) 2947, 2925, 2853, 1707, 1496, 1456, 1410, 1355, 1219, 980, 884, 766,
–
1 1
6
4
3
93 cm ; H NMR (400 MHz, CDCl ) δ 7.42–7.32 (10 H, m), 5.12 (1 H, dt, J = 8.0, 1.6 Hz),
3
.52 (1 H, d, J = 14.8 Hz), 4.35 (1 H, d, J = 14.8 Hz), 3.88–3.95 (1 H, m), 3.73–3.68 (1 H, m),
1
3
.32–3.27 (1 H, m), 1.95 (1 H, dt, J = 10.4, 1.6 Hz), 1.79 (1 H, dt, J = 10.4, 1.6 Hz); C
NMR (100 MHz, CDCl ) δ 173.4, 155.0, 135.8, 130.3, 129.2 (2 ×), 128.9 (2 ×), 128.7 (2 ×),
3
1
28.4, 128.0, 126.4 (2 ×), 85.2, 61.4, 58.4, 54.1, 45.8, 34.7; ESI−HRMS calcd for
+
C H N O : 319.1447, found m/z 319.1441 [M + H] .
20
19
2
2
o
Compound 9b: C H N O ; white solid; mp 160–161 C; TLC (hexane/EtOAc = 2:1) R
f
20
18
2
2
=
0.31; IR νmax (neat) 2971, 2924, 2853, 1705, 1496, 1456, 1409, 1356, 1216, 967, 884, 767,
–
1 1
6
94 cm ; H NMR (400 MHz, CDCl ) δ 7.76–7.67 (2 H, m), 7.43–7.21 (8 H, m), 4.78 (1 H,
3
dt, J = 8.4, 1.6 Hz), 4.51 (1 H, d, J = 14.8 Hz), 4.20 (1 H, d, J = 14.8 Hz), 4.10 (1 H, d, J =
.4 Hz), 3.95–3.89 (1 H, m), 3.32–3.27 (1 H, m), 1.91 (1 H, dt, J = 10.8, 1.6 Hz), 1.68 (1 H,
8
1
3
dt, J = 10.8, 1.6 Hz); C NMR (100 MHz, CDCl ) δ 175.2, 155.5, 136.2, 130.4, 128.9 (2 ×),
3
1
28.9 (2 ×), 128.1 (2 ×), 128.0, 127.6, 126.8 (2 ×), 85.8, 63.0, 54.4, 48.3, 45.3, 34.3; ESI−
+
HRMS calcd for C H N O : 319.1447, found m/z 319.1450 [M + H] . The structural
2
0
19
2
2
1
1
assignment of 9b was confirmed by H− H COSY analysis.
2
7

ACCEPTED MANUSCRIPT
4
.5.16. Methyl 4-tert-butoxycarbonylamino-3-(1-ethylpropyl)-4,5,6,6a-tetrahydro-3aH-
cyclopenta[d]isoxazole-6-carboxylate (6a) and methyl 6-((tert-butoxycarbonyl)amino)-3-
(pentan-3-yl)-3a,5,6,6a-tetrahydro-4H-cyclopenta[d]isoxazole-4-carboxylate (6b) [14].
According to the representative procedure B, the cycloaddition of (±)-3a (750 mg, 3.1
mmol) with 2-ethylbutanenitrile oxide, which was generated in situ from hydroximoyl
chloride 5a (2.77 g, 18.6 mmol), was performed in hexane to give a crude product containing
1
(
±)-6a and (±)-6b in a ratio of 91:9 as shown by the H NMR analysis (partial, 400 MHz,
CDCl ) δ 5.20 (0.83 H, d, J = 8.4 Hz, H-6a)/ 4.83 (0.17 H, d, J = 8.6 Hz, H-6a), 3.58 (0.83 H,
3
d, J = 9.2 Hz, H-3a)/ 3.89 (0.17 H, m, H-3a), 3.76 (2.49 H, s, CO Me)/ 3.79 (0.51 H, s,
2
CO Me). Compound (±)-6a (727 mg, 66% yield) was isolated by silica gel chromatography
2
(EtOAc/hexane 1:9 to 1:6 gradients).
Alternatively, optically active (+)-6a was prepared from trifluoroacetamide (+)-13a
according to the representative procedure C. Therefore, trifluoroacetamide (+)-13a (94 mg,
.27 mmol) was dissolved in 4.6 M HCl methanolic solution and heated at boiling for 24 h.
0
The solution was concentrated under reduced pressure. The residue was dissolved in CH Cl2
2
o
(
1 mL), added Et N (0.12 mL, 0.84 mmol) and Boc O (0.08 mL, 0.36 mmol) at 0 C. The
3
2
mixture was stirred at room temperature for 4 h, and then concentrated under reduced
pressure. The residue was re-dissolved in CH Cl , washed with H O, dried over MgSO , and
2
2
2
4
filtered. The filtrate was concentrated by rotatory evaporation under reduced pressure. The
residue was purified by silica gel column chromatography (hexane/EtOAc = 10:1) to afford
carbamate (+)-6a (70 mg, 74% yield).
20
D
1
Compound 6a: C H N O ; pale yellow oil; [α] = +58.3 (c = 2.0, CHCl ); H NMR
18
30
2
5
3
(
(
(
400 MHz, CDCl
3
) δ 5.57 (1 H, d, J = 7.2 Hz), 5.16 (1 H, d, J = 8.4 Hz ), 4.17 (1 H, br), 3.71
3 H, s), 3.54 ( 1 H, d, J = 9.2 Hz), 3.14 (1 H, d, J = 7.2 Hz), 2.46−2.45 (1 H, m), 2.10−2.03
1
3
1 H, m), 2.00−1.96 (1 H, m), 1.71−1.53 (4 H, m), 1.40 (9 H, s), 0.90−0.83 (6 H, m); C
2
8