Investigations of regio- and stereoselectivities in the synthesis of cytotoxic isoxazolidines through 1,3-dipolar cycloadditions of nitrones to dipolarophiles bearing an allylic oxygen

Article abstract of DOI:10.1016/j.tet.2006.12.076

Regio- and stereoselectivities in cycloadditions of nitrones to dipolarophiles bearing an allylic oxygen, which furnishes substituted-isoxazolidine analogs of the furanose ring of nucleosides, have been investigated. Although the obtained regioselectiviti

Full text of DOI:10.1016/j.tet.2006.12.076

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Tetrahedron 63 (2007) 2283–2291
Investigations of regio- and stereoselectivities in the synthesis
of cytotoxic isoxazolidines through 1,3-dipolar cycloadditions
of nitrones to dipolarophiles bearing an allylic oxygen
Rajinder Singh,a Surinderjit Singh Bhella,a A. K. Sexana,b M. Shanmugavel,b
Abdul Faruka and M. P. S. Ishara,
*
aBio-organic and Photochemistry Laboratory, Department of Pharmaceutical Sciences, Guru Nanak Dev University,
Amritsar 143 005, Punjab, India
bPharmacology Division, Regional Research Laboratory, Jammu 184 001, J & K, India
Received 1 June 2006; revised 21 December 2006; accepted 21 December 2006
Available online 27 December 2006
Abstract—Regio- and stereoselectivities in cycloadditions of nitrones to dipolarophiles bearing an allylic oxygen, which furnishes
substituted-isoxazolidine analogs of the furanose ring of nucleosides, have been investigated. Although the obtained regioselectivities are
anticipated, a rationalization of the preferred formation of endo-cycloadducts necessitates the involvement of an allylic oxygen in secondary
interaction. The obtained isoxazolidines display cytotoxic activities against a number of human cancer cell lines.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
Consequently, synthetic studies on isoxazolidines have
drawn considerable attention and 1,3-dipolar cycloadditions
of nitrones afford the most straightforward route to isox-
azolidines. For instance, in one of the approaches, nitro-
phosphonate 1 has been reacted with vinyl acetate to
obtain a mixture of epimeric isoxazolidines 2 and 3
(Scheme 1), which have been utilized as precursors to the
synthesis of reverse-transcriptase inhibitors.1e
There has been an ever-increasing quest for modified
nucleosides due to their potential applications in antiviral
and anticancer therapies.1 In a recent approach to modified
nucleosides, the furanose ring has been replaced by other
heterocyclic analogs.2 Among these N and O containing
five-membered heterocycles, isoxazolidines, and isoxazo-
line derivatives have emerged as important candidates, and
have been shown to display useful anticancer and antiviral
properties.3 It is pertinent to mention here that substituted
isoxazolidines have been known to display cytotoxicity as
in the case of alkaloids such as pyrinodemin-A and isoxazo-
lidinium salts employed in the therapy of malignant tumors
(Fig. 1).4
(EtO)2OP
N
H
PO(OEt)2
OAc
O
OAc
2
+
O
N
-
Me
(EtO)2OP
1
N
O
3
OAc
Scheme 1.
R'
H
H
O
X-
+
N
N
R''
N
The formation of epimeric products is the outcome of two
different approaches of the dipole to the dipolarophile.
In the above example (Scheme 1), with the nitrone reacting
in its most stable Z-form, the cis-product 2 arises from an
exo-transition state and the trans-product 3 from an endo-
transition state; the exo-adduct 2 was reported to be the ma-
jor product.1e Similar predominance of an exo-adduct has
also been reported in the addition of a-(2-pyridyl)-N-benzyl-
nitrone to allyl alcohol.2c Avariety of secondary factors have
O
(C8H16
)
(C9H18
)
H
Isoxazolidinium salt
N
Pyrinodemin A
Figure 1.
Keywords: Cycloadditions; Nitrone; Stereoselectivities; Secondary interac-
tions; Isoxazolidines; Anticancer activity.
* Corresponding author. Tel.: +91 183 2258802x3321; fax: +91 183
2258820; e-mail: mpsishar@yahoo.com
0040–4020/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2006.12.076

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R. Singh et al. / Tetrahedron 63 (2007) 2283–2291
been invoked to explain stereoselectivities in 1,3-dipolar
cycloadditions.5 The predominant occurrence of endo selec-
tivity in the cycloaddition reactions has generally been at-
tributed to the role of secondary orbital interactions (SOI)
or some secondary interactions (SI), however, contribution
of such interactions is disputed and recently it has been con-
tended that the existence of such SOI/SI is negated by
closed-shell repulsions.5a,c However, more recent calcula-
tions favor the existence of such interactions in controlling
stereoselectivity.6 Consequently, the importance of second-
ary interactions in controlling stereoselectivities is far from
settled. Recently, the involvement of oxygen atoms at allylic
and homo-allylic positions in secondary interactions with
LUMO-dipole has been reported to control the stereochemi-
cal outcome of nitrone addition to such dipolarophiles lead-
ing to the predominance of endo-adducts,5d,e which is in
contradiction with the stereoselectivities reported in the
nitrone cycloadditions discussed above.1e,2c We have inves-
tigated stereoselectivities in 1,3-dipolar cycloadditions of a
varietyofa-aryl-N-phenylnitroneswithanumberofdipolaro-
philes possessing oxygen at the allylic position so as to
investigate the proposed involvement of allylic oxygen in
secondary interactions with nitrogen of the nitrone and the
obtained substituted isoxazolidines have also been evaluated
for cytotoxic activity against a number of human cancer
cells lines.
o-allyloxy-/crotyloxy-/cinnamyloxy-acetophenones 4a–c
were carried out by refluxing (30–35 h) their equimolar
solutions in dry benzene. After the completion of reactions
(TLC), column chromatography afforded the isoxazolidines
6a–d, 7a–d, and 8a,c (Scheme 2 and Table 1). The obtained
isoxazolidines were characterized spectroscopically. The
observed regio- and stereochemical outcomes of these
cycloadditions were delineated by detailed NMR spectro-
1
scopic analyses involving extensive H-decoupling experi-
1
1
ments and establishing H–1H and H–13C connectivities
by 2D NMR techniques.
The formation of the cycloadducts was established by the 1H
NMR and Mass spectra. The assigned regiochemistry of
addition in the case of (6a–d) is based, besides H NMR
1
spectra, on the presence of two methine (CH–) resonances,
one in the range of d 69–70 (C3) and the other in the range
Table 1. Reaction times and yields of the products from cycloadditions of
nitrones 5a–d with dipolarophiles 4a–c
Entry Dipolarophile Nitrone Reaction
time (h)
% Yields of adducts
6
7
8
1
2
3
4
5
6
7
8
4a
4a
4a
4a
4b
4b
4b
4b
4c
4c
5a
5b
5c
5d
5a
5b
5c
5d
5a
5c
30
32
30
35
32
33
31
35
33
31
65
63
70
60
—
—
—
—
—
—
—
—
—
—
64
62
68
60
—
—
—
—
—
—
—
—
—
—
65
67
2. Results and discussion
Initially, cycloadditions of nitrones 5a–d (prepared by the
reaction of phenylhydroxylamine with the corresponding
aldehydes and characterized spectroscopically), with
9
10
R'
1
O
O
Ph
2
N
5
Reflux
Ph
R'
Ar
N
+
H
O
dry benzene
3
O
4
H
Ha
Hb
Ar
4a
5a-d
6a-d
1
O
H
2
N
5
Ph
Ph
Me
Reflux
R'
Ar
3
4
N
+
O
CH3
H
dry benzene
O
H
Ar
O
R'
4b
5a-d
7a-d
1
O
H
2
N
5
Ph
Ph
Reflux
R'
Ph
+
Ar
N
O
Ph
dry benzene
4
3
O
H
O
H
Ar
R'
4c
5a,c
8a,c
(a) Ar = C6H5
O
(b) Ar = p-OMe-C6H4
(c) Ar = p-NO2-C6H4
(d) Ar = p-Cl-C6H4
Me
R' =
Scheme 2.

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R. Singh et al. / Tetrahedron 63 (2007) 2283–2291
2285
R'
C3–H), and a 1H doublet of quartet at d 4.34 (J¼8.6,
6.2 Hz, C5–H); the chemical shift value of C5–H was critical
for the assigned regiochemistry of addition. A 1H multiplet
at d 2.70 was due to C4–H and the C5–Me was located as
a 3H doublet at d 1.51 (J¼6.2 Hz). The characteristic fea-
tures of its 13C NMR were the presence of methine carbons
of the isoxazolidine ring d 77.0 (C5), d 72.7 (C3), d 61.1
(C4), besides, –OCH2 at d 66.8 and the carbonyl carbon reso-
nance at d 198.7. Stereochemically, the substituent at C4 and
the methyl at C5 have to be trans because of the concerted
nature of nitrone cycloaddition. Again, the obtained cou-
pling constant value J3,4¼6.5 Hz is lower than the value of
coupling constants J5,4¼8.6 Hz for a trans relationship
between C5–H and C4–H, thereby, signifying that C3–H is
also trans to C4–H.
H
O
Ph
+
Ar
SI
N
O
Ph
H
N
O
H
R'
O
H
H
Ha
H
A
Hb
Ar
6
Ph
SI
O
Ph/Me
H
N
Ph
Ar
R'
N
O
H
H
O
Ar
Me/Ph
R'
O
H
H
H
B
7 & 8
Ph
O
H
Ph
Ar
N
N
O
Ph/Me
R'
H
H
Ar
H
Me/Ph
The investigations have clearly established that the regio-
chemistry of addition of nitrones to o-allyloxyacetophenone,
leading to isoxazolidines 6a–d, is different from addition to
o-crotyloxy/cinnamyloxy-acetophenones, the latter afford-
ing isoxazolidines 7a–d and 8a,c and these regiochemical
variations are anticipated.8 The stereochemical outcomes
of the cycloadditions in all the cases are derived from an
endo-mode of addition as far as the –CH2–O–R0 moiety is
concerned, involving the Z-isomers of nitrones (Fig. 2).
Thus, addition of nitrone in its Z-form8,9 to o-allyloxy-/crotyl-
oxy-/cinnamyloxy-acetophenonesinvolvesanendo-transition
state, possibly stabilized by recently proposed5d,e secondary
interaction involving allylic oxygen (A and B, Fig. 2) and
N of dipole and/or avoidance of steric encumbrance, particu-
larly, in the case of addition to o-crotyloxy-/cinnamyloxy-
acetophenones, with altered regiochemistry (C).
O
H
H
O
exo-adduct
R'
C
O
Me
R' =
Figure 2.
of d 76–77 (C5). Another resonance of an oxygen-linked car-
bon (CH2) appeared in the range of d 68–70 (R0–O–CH2–).
The 1H NMR spectrum of 6c revealed the –CH2–O– protons
as double doublet at d 4.28 (J¼10.1, 6.4 Hz) and double dou-
blet d 4.11 (J¼10.1, 4.5 Hz) and C3–H resonance as a dd at
1
d 4.97 (J¼8.1, 6.5 Hz), and H multiplet at d 4.76 (C5–H).
The C4–Hs of the isoxazolidine ring appeared as two sepa-
rate ddds at d 2.28 (Jgem¼12.3 and J¼8.2, 6.5 Hz, C4–Hb)
and d 3.05 (Jgem¼12.3 and J¼8.2, 7.3 Hz, C4–Ha). The as-
signed stereochemical dispositions are based on J values
In order to further investigate the observation of complete
endo selectivity in the above cycloadditions and to substan-
tiate the possible role of the secondary orbital interactions
and steric factors, the investigations were extended to the
cycloadditions of nitrones to dipolarophiles such as allyl
alcohol and allyl-acetate 9a,b. Here, two stereoisomers
i.e., endo- (10a, major product) and exo-adduct 11a were
isolated from a,N-diphenylnitrone addition to allyl alcohol
and only endo-adduct 10b was obtained in the case of
nitrone addition to allyl-acetate (Scheme 3).
1
and H-connectivities worked out from decoupling experi-
ments, and derived from the basic premise that cis vicinal
1H couplings are always higher than the trans in the case
of isoxazolidines.7 For instance, in 6c the values of coupling
constants J5,4a¼7.3 Hz, J5,4b¼8.2 Hz indicate that C5–H is
cis to the C4–Hb and J3,4a¼8.1 Hz, J3,4b¼6.5 Hz allude to
trans relationship between C3–H and C4–Hb, thereby, estab-
lishing that both C5–H and C3–H are trans to each other.
The assigned stereochemistry in the case of 10a,b and 11a
are based on the determination of vicinal coupling constants
(Table 2). Thus, in the case of 10a the values of coupling
constants J5,4a¼7.2 Hz, J5,4b¼7.8 Hz indicate that C5–H is
cis to the C4–Hb and J3,4a¼7.8 Hz, J3,4b¼5.4 Hz allude to
a trans relationship between C3–H, and C4–Hb, thereby, es-
tablishing that both C5–H and C3–H are trans to each other.
The regio- and stereochemical assignments in the case of
7a–d and 8a,c have been similarly achieved. Thus, the H
NMR spectrum of 7c revealed, besides the resonances in
the aromatic region, the presence of a doublet at d 4.14
(2H, J¼5.2 Hz, –OCH2), a resonance d 4.86 (d, J¼6.5 Hz,
1
R'
1
O
O
1
O
H
2
N
5
2
N
O
R'
Ph
5
Ph
Ph
Reflux
+
H
R'
Ph
N
+
O
4
3
4
dry benzene
3
O
H
Ha
H
Ha
Hb
Hb
Ph
9
5a
Ph
(a) R' = H
(b) R' = -COMe
endo-adduct
(10a, 85%)
(10b, 87%)
exo-adduct
(11a, 10%)
Scheme 3.

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R. Singh et al. / Tetrahedron 63 (2007) 2283–2291
Table 2. Some critical coupling constant values in isoxazolidines 10a,b
and 11a
allyloxyacetophenone derived isoxazolidines 6a–d. It is per-
tinent to mention here that in the reported formation of exo-
adducts in the addition of a-(2-pyridyl)-N-butyl nitrone to
allyl alcohol, no discussion of the NMR spectroscopic data
was recorded and also the reported2c chemical shifts pattern
for C3–H and C5–H is different i.e., C5–H is located down-
field (d 4.49 for exo-adduct and d 4.28 for endo-adduct) than
C3–H (d 4.19 forendo- and d 4.11 for exo-adduct),2c which is
in reverse order as far as data recorded for various adducts in
the present investigations. Even the theoretical calculations
in the above report2c did not validate the experimental re-
sults. The present findings, on the other hand, are in conso-
nance with the endo selectivity observed in the additions of
nitrones, including some rigid ones, to dipolarophiles bear-
ing allylic and homo-allylic oxygen.5d,e
Compound
Coupling constant values (Hz)
J3H–4Ha
J3H–4Hb
J5H–4Ha
J5H–4Hb
10a
10b
11a
7.8
7.8
8.7
5.4
5.1
6.1
7.2
7.2
6.9
7.8
8.1
6.5
But in case of 11a the values of coupling constants
J5,4a¼6.9 Hz, J5,4b¼6.5 Hz indicate that C5–H is cis to the
C4–Ha and J3,4a¼8.7 Hz, J3,4b¼6.1 Hz establish that C3–H
is also cis to the C4–Ha, thereby, both C5–H and C3–H are
1
cis to each other. Critical H couplings in the case of 10b
(Table 2) establish its structure as an endo-cycloadduct.
It appears that both steric factors and secondary interactions
may be important in determining the sterochemical out-
come. Thus, in the case of addition to allyl alcohol the in-
volvement of proposed5d,e secondary interaction involving
allylic oxygen leads to preferred formation of an endo-
adduct 10a with an exo isomer 11a as the minor product.
Secondary interaction coupled with steric encumbrance in
the cycloadditions to allyl-acetate may be responsible for
the preferred formation of endo-adduct 10b. In these cyclo-
additions, the regiochemistry of addition of nitrone to di-
polarophiles 9a,b is similar to that obtained in the case of
3. Biological activity
The in vitro inhibitory potential of the obtained isoxazol-
idines 6–8 was evaluated using eight human cancer cell
lines,10 representing different organs/tissues, according to
the method of Skehan et al.10b as reported previously.11
The cytotoxicity was determined at 1ꢀ10ꢁ4, 1ꢀ10ꢁ5, and
1ꢀ10ꢁ6 M. Mitomycin C, Paclitaxel, and 5-Fluorouracil
were used as the positive controls (1ꢀ10ꢁ5 M, Table 3).
The isoxazolidines 6–8 exhibited a dose dependent cell
Table 3. In vitro cytotoxicity of isoxazolidines (6a–d, 7a,c,d, and 8a,c) against human cancer cell lines10a
Compounds/standard
drugs
Concn (M)
% Growth inhibition against human cancer cell lines
HT-29
(colon)
SW-620
(colon)
DU-145
(prostate)
KB
(oral)
Hep-2
(liver)
MCF-7
(breast)
A-549
(lung)
HOP-62
(lung)
6a
6b
6c
6d
7a
7c
7d
8a
8c
1ꢀ10ꢁ6
1ꢀ10ꢁ5
1ꢀ10ꢁ4
0
0
33
0
2
21
22
39
43
15
21
54
12
19
44
9
22
45
4
18
43
0
0
0
1ꢀ10ꢁ6
1ꢀ10ꢁ5
1ꢀ10ꢁ4
32
30
50
—
—
—
19
30
42
1
12
0
—
—
—
—
—
—
0
0
0
—
—
—
1ꢀ10ꢁ6
1ꢀ10ꢁ5
1ꢀ10ꢁ4
1
19
22
0
0
0
9
14
19
5
7
2
9
22
32
13
28
38
19
27
18
0
0
1
1ꢀ10ꢁ6
1ꢀ10ꢁ5
1ꢀ10ꢁ4
0
14
34
0
0
4
22
22
36
22
35
52
8
21
41
10
28
42
0
1
50
0
0
5
1ꢀ10ꢁ6
1ꢀ10ꢁ5
1ꢀ10ꢁ4
44
57
64
0
0
10
22
25
32
0
0
0
11
29
59
11
26
51
28
30
33
1
0
7
1ꢀ10ꢁ6
1ꢀ10ꢁ5
1ꢀ10ꢁ4
5
18
39
0
0
22
0
5
0
0
5
4
17
30
46
9
26
38
25
32
34
2
2
6
1ꢀ10ꢁ6
1ꢀ10ꢁ5
1ꢀ10ꢁ4
10
42
50
—
—
—
19
21
59
10
8
4
—
—
—
—
—
—
26
30
51
—
—
—
1ꢀ10ꢁ6
1ꢀ10ꢁ5
1ꢀ10ꢁ4
0
20
36
0
8
17
58
59
61
11
0
0
14
28
40
9
29
37
8
7
40
0
0
0
1ꢀ10ꢁ6
1ꢀ10ꢁ5
1ꢀ10ꢁ4
3
8
43
0
0
32
13
18
19
0
36
0
14
23
40
9
20
42
28
35
38
0
0
0
Mitomycin C
Paclitaxel
5-Flourouracil
1ꢀ10ꢁ5
1ꢀ10ꢁ5
1ꢀ10ꢁ5
31
—
34
90
—
8
89
85
—
90
86
—
88
—
12
93
—
31
80
63
—
70
—
3