Synthesis and anticancer activity of novel spiro-isoxazoline and spiro-isoxazolidine derivatives of α-santonin

Article abstract of DOI:10.1016/j.ejmech.2013.01.003

In the present study, novel spiro derivatives of α-santonin were prepared and tested for their anticancer activity against a panel of six human cancer cell lines. Spiro-isoxazoline and spiro-isoxazolidine derivatives have been generated on C-ring of α-santonin (α-methylene-γ- butyrolactone) by the 1,3-dipolar cycloaddition of α-santonin derivative 6 with nitrile oxides 7 and nitrones 9 respectively. Among all, compound 10b″ had shown IC₅₀ of 0.01, 0.5 and 0.3 μM against PC-3, THP-1 and MCF-7 cell lines respectively. Further, flow cytometry studies showed that PC-3 cells treated with the spiro-isoxazolidine derivative 10b″ were arrested in the sub G1 phase of the cell cycle in a concentration dependent manner. The spiro-isoxazolidine derivative 10b″ also showed concentration dependent inhibitory activity against NF-κB, p65 with 57% inhibition in 24 h at 10 μM.

Full text of DOI:10.1016/j.ejmech.2013.01.003

European Journal of Medicinal Chemistry 63 (2013) 279e289
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and anticancer activity of novel spiro-isoxazoline and spiro-
isoxazolidine derivatives of -santonin
a
Jabeena Khazir b, Parvinder Pal Singh b, D. Mahendhar Reddya, Irfan Hyder a, Syed Shafi c,
S.D. Sawant b, Gousia Chashoo b, Ajay Mahajan b, M.S. Alam c, A.K. Saxena b, S. Arvinda b,
,
B.D. Gupta b, H.M. Sampath Kumar a
*
a NPC Division, Indian Institute of Chemical Technology, Hyderabad 500007, India
b Medicinal Chemistry Division, Indian Institute of Integrative Medicine, Jammu 180001, India
c Department of Chemistry, Jamia Hamdard University, New Delhi 110062, India
a r t i c l e i n f o
a b s t r a c t
Article history:
In the present study, novel spiro derivatives of a-santonin were prepared and tested for their anticancer
Received 16 November 2012
Received in revised form
26 December 2012
Accepted 3 January 2013
Available online 11 January 2013
activity against a panel of six human cancer cell lines. Spiro-isoxazoline and spiro-isoxazolidine de-
rivatives have been generated on C-ring of -santonin ( -methylene- -butyrolactone) by the 1,3-dipolar
cycloaddition of -santonin derivative 6 with nitrile oxides 7 and nitrones 9 respectively. Among all,
compound 10b00 had shown IC50 of 0.01, 0.5 and 0.3
M against PC-3, THP-1 and MCF-7 cell lines
respectively. Further, flow cytometry studies showed that PC-3 cells treated with the spiro-isoxazolidine
derivative 10b00 were arrested in the sub G1 phase of the cell cycle in a concentration dependent manner.
The spiro-isoxazolidine derivative 10b00 also showed concentration dependent inhibitory activity against
a
a
g
a
m
Keywords:
a
-Santonin
NF-kB, p65 with 57% inhibition in 24 h at 10 mM.
Spiro-derivatives
Anticancer
Ó 2013 Elsevier Masson SAS. All rights reserved.
NF-kB (p65) inhibitors
1. Introduction
it has a highly functionalised A ring with a cross conjugated dien-
one system and a lactone moiety between C(6) and C(12). Chemical
Sesquiterpene lactones (SLs) are the active constituents of a va-
riety of medicinal plants used in traditional medicine for the
treatment of inflammatory diseases [1]. In recent years, sesquiter-
penes has attracted a great deal of interest due to their anticancer
properties and extensive work has been carried to understand the
molecular mechanisms and the potential chemo-preventive and
chemo-therapeutic application of sesquiterpeniods [2e5]. At pre-
sent, many SLs are in different phases of clinical trials such as
Artemisinin 1, Parthenolide 2, Thaipsigargin 3 and many of their
synthetic derivatives [6e8] (Fig. 1). These data indicate that SLs and
related compounds may represent a promising class of anti-
leukaemic agents.
and biochemical transformations of
logical properties have been studied already for over 100 years.
Santonin was widely used in the past as an anthelminthic [9].
Recent studies demonstrate the biological importance of -santo-
nin as antipyretic, anti-inflammatory, and fungicidal [10,11] agent.
In addition 11,13-dehydrosantonin and other -santonin de-
a-santonin and its pharmaco-
a
-
a
a
rivatives show cytotoxicity towards KB cells (cell line derived from
carcinoma of nasopharyx). The cytotoxicity exhibited by these
molecules has been attributed to the presence of
lactone moiety on the scaffold. Further, since
a
-methylene-
g-
a
-santonin bears
several reactive centres, it offers opportunity for further chemical
modification of its scaffold to generate new secondary leads [12e
(ꢀ)-
a
-Santonin (4) is the most important and the most abun-
14]. Several such derivatives of (ꢀ)-
a-santonin have been synthe-
dant of a number of closely related sesquiterpenoid lactones pos-
sessing the eudesmane skeleton that have been isolated from
various Asian plants of the genus Artemisia (especially from Arte-
sized by opening the lactone ring as well and some of the com-
pounds demonstrate potential anticancer activity [15e17].
Our continued interest towards the design and synthesis of
sesquiterpene lactones based anticancer agents [18], we planned
misia maritima). (ꢀ)-
a-Santonin is also commercially available and
the synthesis of novel
direction, we present here, the synthesis and cytotoxic activity of
spiro-isoxazoline and spiro-isoxazolidine derivatives of -santonin
through rational structural modifications of the scaffold. In view of
a-santonin based anticancer agents. In this
a
* Corresponding author. Tel.: þ91 9912901010; fax: þ91 191 2569333.
E-mail addresses: hmskumar@yahoo.com, sampath@iict.res.in (H.M.S. Kumar).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.01.003

280
J. Khazir et al. / European Journal of Medicinal Chemistry 63 (2013) 279e289
O
O
O
O
O
O
H
O
H
O
O
O
OH
OH
O
O
O
O
O
O
O
1. Artemisenin
2. Parthenolide
3. Thiapsigargin
Fig. 1. Few SLs in clinical trials.
Scheme 1. Synthesis of exocyclic double bond on lactone ring of
a-santonin.
the biological importance of spiro compounds together with the
pharmacological significance of nitrogen heterocycles such as iso-
xazolines and isoxazolidines, novel spiro-heterocycles derived from
by the methyl group present on the six membered ring to the
approaching dipole in the transition state for the dipolar cyclo-
addition reaction.
a
-santonin have been planned wherein the spiro-isoxazoline and
As illustrated in Scheme 3, spiro-isoxazolidine derivatives of
santonin were prepared by the 1,3-dipolar cycloaddition between
-santonin derivative 10 and various nitrones 9 in dry benzene
a-
spiro-isoxazolidine derivatives could be generated through 1,3-
dipolar cycloaddition between corresponding nitrile oxides/nitro-
a
nes and a dipolarophile (6) located on the
focussed library of 32 spiro-derivatives has been synthesized and
screened for their anticancer activity against a panel of cancer cell
lines. The novel spiro heterocycle appended derivatives of a-san-
tonin thus designed have shown increased drug likeness as
revealed by the parameters calculated using Schrödinger software.
a
-santonin scaffold. A
under reflux condition [29,30]. Nitrones 9 were prepared according
to literature procedure in which nitrobenzene was reduced in the
presence of Zn/NH4Cl to get phenyl hydroxylamine followed by the
condensation of various aromatic aldehydes. Nitrone cycloaddition
was selectively done to the double bond adjacent to lactone ring,
which is clearly confirmed by the disappearance of the alkene
protons adjacent to lactone ring. Interestingly, in this case, two
diastereomers were formed in 3:1 ratio in all the reactions in which
one was isolated as major (non polar) while the other was minor
(polar). Each diastereomer was isolated after column chromatog-
raphy and characterized by 1H NMR, 13C NMR and mass spec-
trometry. A clear chemical shift deviation of benzylic proton
adjacent to nitrogen atom in isoxazolidine ring between two
2. Results and discussion
2.1. Chemistry
In order to generate the spiro-derivatives,
a-santonin (4) was
converted into 3-oxo-7 H, 6 H,11-(phenylselenyl)-eudesma-1,4-
a
b
dien-6,12-olide 5 on reaction with phenylselenylchloride and LDA
followed by the subsequent oxidation with H2O2 in acetic acid
generated the exocyclic double containing
a-santonin derivative
viz., 3-oxo-7 H, 6 H-eudesma-1,4,11-trien-6,12-olide 6 (Scheme 1)
a
b
[19]. Compound 6 was then taken for 1,3-dipolar cycloaddition
reaction with nitrile oxides and nitrones.
As illustrated in Scheme 2, spiro-isoxozoline derivatives of
santonin (8) were prepared through 1,3-dipolar cycloaddition be-
tween -santonin derivative (6) with various nitrile oxides (7) [20e
a-
a
28]. Nitrile oxides (7) were prepared according to the literature
procedure in which various aromatic aldehydes were converted to
corresponding aldoximes (syn and anti), followed by the reaction
with N-chlorosuccinimide in DMF. Nitrile oxide cycloaddition was
selectively done to the exocyclic double bond adjacent to lactone
ring. The formation of spiro-isoxazoline ring is clearly confirmed by
the disappearance of the alkene protons adjacent to lactone ring
and the appearance of two singlets at
NMR. These spiro-isoxozoline derivatives of
d
3.3e3.2 and
-santonin were
d
4.0e3.9 in 1H
a
formed regiospecifically in all cases. The regiospecific formation of
cycloaddition product with exocyclic double bond present on the
five membered lactone ring is attributed to steric hindrance offered
Scheme 2. Synthesis of spiro-isoxazoline derivatives of a-santonin.

J. Khazir et al. / European Journal of Medicinal Chemistry 63 (2013) 279e289
281
O
N
Ar'
Ar'
Dry Toluene
O
Ph
O
O
N
N
O
120 oC, 6 hrs
O
O
Ar'
O
O
O
O
O
6
9
10
Ar'
10e
10f
3,5-F-C6H4
(81%, R : S = 70 : 30
10a
4-NO2-C6H4
(75%, R : S = 60 : 40)
10b 4-Br-C6H4
(78%, R : S = 70 : 30)
3-Br,4-OMe-C6H4
(75%, R : S = 60 : 40)
10c 2,5-Cl-C6H4
(82%, R : S =70 : 30)
10g 3-NO2-C6H4
(79%, R: S = 70 : 30)
10d 3-Br, 4F-C6H4
(84%, R : S = 65 : 35
10h
1-Napthalene
(86%, R : S = 60 : 40)
Scheme 3. Synthesis of spiro-isoxazolidine derivatives of a-santonin.
diastereomers was observed in 1H NMR. In major isomer this pro-
ton appears as a triplet approximately at 5.2 while in the minor
isomer this signal shifted towards more shielding region and ap-
pears approximately at 4.6. A series of nitrones were reacted with
-santonin and both diastereomers of spiro-isoxazolidine were
d
d
Table 1
IC50 values (
a
m
M) of various spiro-derivatives of
a
-santonin.
MCF-7
isolated in each case. The formation of isoxazolidine ring in major
isomer was confirmed by the appearance of two peaks approx-
Compound
HCT-1
PC-3
THP-1
Hep-1
A-549
imately at
d 3.2 (dd) for one proton and other peak at d 2.5 (dd) for
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
8m
8n
8o
8p
100
55
10
100
29
100
52
78
100
100
100
100
35
100
7.9
21
0.4
0.4
0.6
100
100
0.2
0.8
100
0.1
0.1
0.3
0.5
0.2
3.9
2.3
2.7
25
100
56
27
100
95
0.4
0.02
100
30
0.5
0.9
100
39
0.5
0.2
0.1
0.7
0.9
0.8
0.3
0.1
0.5
0.2
0.7
0.09
3.9
100
50
100
100
15
100
29
100
100
100
100
100
33
100
100
100
61
100
100
100
26
100
100
100
0.6
2.2
2
another proton each with a coupling constant of 9.6 MHz and for-
mation of minor isomer was confirmed by the appearance of two
peaks at
d 3.2e2.8 (dd) for one proton and d 2.5e2.6 (m) for other
13
proton in 1H NMR.
0.7
0.2
100
2.9
8.2
3.1
3.8
100
5.9
45
58
66
66
100
31
77
55
19
100
100
14
8.5
2.7
81
50
2.2. X-ray crystallography
100
100
15
8.2
61
To gain an insight into the structure and stereochemistry of the
spiro-isoxazolidine derivatives of -santonin, crystallisation of
55
40
a
100
0.9
0.3
100
0.5
0.6
0.1
0.01
0.7
0.9
0.9
0.8
0.7
1.4
0.1
0.1
0.3
0.9
0.6
0.7
e
100
7.3
50
100
14
100
100
65
100
0.6
3.7
100
100
100
100
100
100
100
100
100
e
100
45
100
100
100
100
37
10a0
10a00
10b0
10b00
10c0
10c00
10d0
10d00
10e0
10e00
10f0
10f00
10g0
10g00
10h0
10h00
Mytomycine
Adriamycin
5-FU
32
100
100
4.3
0.09
28
100
45
100
48
100
100
0.5
e
0.8
100
100
100
42
14
23
14
82
10
100
59
1.4
3.3
e
e
e
6
2.2
e
0.5
e
e
e
e
1
4.5
4.9
Fig. 2. Molecular conformation of 10e0 in crystal.
00
0R-isomer, S-isomer.

282
J. Khazir et al. / European Journal of Medicinal Chemistry 63 (2013) 279e289
Fig. 3. Flow cytometric analysis of spiro-isoxazolidine derivatives of
a
-santonin 10b0 and 10b00 treated with PC-3 cell.
major diastereomer 10e0 was achieved through slow evaporation of
solution of this compound in methanol containing few drops of
water, and crystal structure of spiro-isoxazolidine derivative of
values (results shown in Table 1). From the IC50 values, it is clear
that majority of the compounds showed significant cytotoxicity
against prostrate, leukaemia, breast and cervix derived cancer cell
lines. However, it may be noted that among all the tested spiro-
a
-santonin 10e0 was determined and its crystal data are presented
in Table 1. X-ray analysis of spiro-isoxazolidine derivative of
a
uration of two newly generated chiral centre at C14 and C16 posi-
tion is S and R respectively.
derivatives of a-santonin 8aep and 10aeh, spiro-isoxazoline de-
-santonin 10e0 (structure given in Fig. 2), shows that the config-
rivative 8g and spiro-isoxazolidine derivatives 10b0, 10b00 and 10e0
showed comparatively more potent IC50 value against HCT-1, PC-3
and MCF-7 cancer cell lines as compared to other derivatives.
Further, spiro-isoxazolidine derivatives of
a-santonin have
2.3. Biological results
shown more promising activity as compared to the spiro-
isoxazoline derivatives. Spiro-isoxazolidine derivatives 10b0 with
4-bromo substitution on the aromatic ring have shown IC50 value of
2.3.1. Evaluation of in vitro anticancer activity
All the compounds were assayed for in vitro cytotoxicity against
a panel of six human cancer cell lines including HCT-1 (colon), PC-3
(prostrate), THP-1 (leukaemia), MCF-7 (breast), Hep-2 (liver) and
A549 (lung). Mitomycin, Adriamycin and 5-FU were taken as ref-
erence compounds and the results are reported in terms of IC50
0.1, 0.3 and 0.8
respectively while its other isomer 10b00 has shown IC50 value of
0.01, 0.5 and 0.3 M against PC-3, THP-1 and MCF-7 cell lines
respectively. Among spiro-isoxazoline derivatives of -santonin 8g
having 4-methoxy substitution also showed promising activity
mM against PC-3, THP-1 and MCF-7 cell lines
m
a

J. Khazir et al. / European Journal of Medicinal Chemistry 63 (2013) 279e289
283
various other cancer cell lines. On account of its promising anti-
cancer activity in PC-3, we choose this cell line to analyse the effect
of spiro-isoxazolidine derivative 10b00 against NF-
kB by using
transcription factor binding assay. Our data showed that PC-3 cells
treated with spiro-isoxazolidine derivative 10b00 displayed con-
centration dependent inhibition of NF-
response element, where, cells treated with 1
20% of reduction in the binding of NF- B with the consensus DNA
sequence, which was further enhanced to 43% at 5 M, and 57% at
10 M (Fig. 4). Thus, our results clearly indicate that spiro-
isoxazolidine derivative 10b00 has a good inhibitory effect on the
activity of NF- B (p65), therefore, spiro-isoxazolidine derivative of
-santonin 10b00 represented new natural product based anticancer
lead.
k
B (p65) binding with DNA
mM showed around
k
m
m
k
a
3. Conclusion
Fig. 4. NF-kB, p65 transcription factor assay. NF-kB, p65 transcription factor assay:
5 ꢁ 106 PC-3 cells were subjected to various concentrations of compound 10b00 for 24 h.
Generation of exocyclic double bond on C ring of
further construction of spiro heterocycles on the
butyrolactone moiety of -santonin would possibly improved the
activity through better protein modulation and enhance the bio-
availability through improved drug likeness. With this aim, the
a
-santonin and
Nuclear lysates were prepared and 20 mg of protein was loaded into each well con-
taining the immobilized DNA response element for NF-
bation, wells were washed and incubated with the primary antibody against NF-
k
B, p65, after overnight incu-
B,
a
-methylene-
g-
k
a
p65, followed by washing and incubation with HP conjugated secondary antibody.
Well were washed again and substrate was added to the wells for 30 min. Optical
density (OD) was taken at 450 nm. The activity of NF-kB, p65 in the control samples
was considered to be 100% and therefore OD value of control was converted to hy-
pothetical value of 100% and other samples were normalized accordingly. Statistical
comparisons were done by using Bonferroni method and p-value <0.05 was consid-
ered to be significant, *p < 0.05, **p < 0.001.
exocyclic double bond was generated on the a-santonin scaffold
through appropriate modifications which was further utilized for
the incorporation of desired heterocyclic structural entities on the
scaffold in order to develop
In this direction, the readily available
a starting material to generate exocyclic double bond on its lactone
ring. The resulting -unsaturated compound 6 with exocyclic
a
-santonin based anticancer agents.
a-santonin (4) was used as
with IC50 value of 0.02 and 0.2
mM against MCF-7 and A549 cell
a,b
lines respectively. From the above results, two most potent spiro-
isoxazolidine derivatives 10b0 and 10b00 were taken for cell cycle
analysis.
double bond on lactone ring was used as a synthon for the prepa-
ration of novel spiro-isoxozoline and spiro-isoxazolidine de-
rivatives. During the synthesis of spiro-isoxazolidine, two
diastereomers were formed whose stereochemistry was deter-
mined through X-ray crystallography. Crystal structure of one of the
major isomer 10e showed the configuration at newly generated
two chiral centres at C14 and C16 position is S and R respectively. All
the synthesised derivatives were evaluated against a panel of hu-
2.3.2. Cell cycle analysis of compounds 10b0 and 10b00
To address the cell death caused by spiro-isoxazolidine de-
rivatives 10b0 and 10b00, the extent of apoptotic death in PC-3 cell
lines was assessed using FACS flow cytometry through the deter-
mination of sub-G1 cell population by propidium iodide (PI)
staining. The DNA cell cycle analysis of spiro-isoxazolidine deriva-
tive 10b0 revealed a concentration dependent increase in the sub G1
man cancer cell lines. Among all the tested spiro-derivatives of a-
santonin 8aep and 10aeh, spiro-isoxazolidine derivative 8g and
spiro-isoxazolidine derivatives 10b0, 10b00 and 10e0 showed com-
paratively more potent IC50 value against HCT-1, PC-3 and MCF-7
cancer cell lines as compared to other derivatives. Further, spiro-
phase of cell cycle being 33.8, 34.7 and 42.8 at 1, 5 and 10
mM
respectively. Spiro-isoxazolidine derivative 10b00 also showed
a concentration dependent increase in the sub G1 phase of cell
isoxazolidine derivatives of a-santonin have shown more promis-
cycle being 25.6, 39.7 and 67.9 at 1, 5 and 10
m
M respectively. Also
ing activity as compared to the spiro-isoxazoline derivatives. Spiro-
complete blockage of G1 phase at 10 M concentration after 24 h of
m
isoxazolidine derivatives 10b0 with 4-bromo substitution on the
incubation in PC-3 cells was observed with spiro-isoxazolidine
aromatic ring has shown IC50 value of 0.1, 0.3 and 0.8
PC-3, THP-1 and MCF-7 cell lines respectively while its other isomer
10b00 shown IC50 value of 0.01, 0.5 and 0.3
M against PC-3, THP-1
mM against
derivative 10b00 (Fig. 3).
m
2.3.3. NF-
In literature, most of SLs having
are NF- B inhibitors [31]. In our study, we visualized that the spiro-
isoxazolidine derivative 10b00 possesses
-unsaturated function-
ality and it might also work through by inhibiting NF- B tran-
k
B (p65), transcription factor assay
and MCF-7 cell lines respectively. Both the compounds showed
dose dependent increase in Sub G1 (apoptotic phase). Spiro-
a,b-unsaturated functionality
k
isoxazolidine derivative 10b00 showed 67.9% apoptosis at 10
mM
a
,b
concentration and also complete blockage of all other phases of cell
cycle was observed with this compound at the same concentration.
Among spiro-isoxozoline series compound 8g was found to be most
k
scription factor. In this direction, the most potent spiro-
isoxazolidine derivative 10b00 was taken for inhibitory NF-
k
B
active and possesses IC50 value of 0.02 and 0.2 mM against MCF-7
(p65) transcription factor activity.
and A549 cell lines respectively. Furthermore, spiro-isoxazolidine
NF-
of transcription factors with five members in mammals viz., Rel (c-
Rel), Rel A (p65), RelB, NF- B1 (p50 and its precursor p105) and NF-
B2 [31a]. NF- B plays a central role in the regulation of cancer cell
proliferation and survival [32]. NF- B has also been shown to play
kB is structurally related and evolutionary conserved group
derivative 10b00 also displayed marked inhibitory activity against
the central regulator of cancer cell growth and survival, NF-kB.
k
Moreover, drug-like parameter calculated using Schödinger soft-
ware also suggested the spiro-isoxazolidine derivative 10b00 pos-
sesses the requisite properties required for clinical candidates.
k
k
k
a vital role in the cancer cell progression by regulating the process
of angiogenesis [33]. Therefore, many new anticancer therapies are
being developed against NF-
showed highest anti-proliferative activity against PC-3 among
These results suggested that spiro-isoxazolidine derivative of a-
santonin 10b00 represents new natural product based promising
candidate which could be taken for further development and
detailed studies.
k
B. Spiro-isoxazolidine derivative 10b00

284
J. Khazir et al. / European Journal of Medicinal Chemistry 63 (2013) 279e289
4. Experimental
J ¼ 4.2, 2.4 Hz), 1.50e1.40 (m, 2H), 1.40 (s, 3H); 13C (100 MHz,
CDCl3): 11.07, 17.43, 19.26, 24.93, 30.32, 37.28, 41.37, 41.76, 54.62,
80.59, 86.04, 125.93, 127.12, 128.33, 129.18, 131.65, 135.07, 150.35,
153.97, 154.86, 171.73, 186.0; Mass QTOF: 454 (M þ Na)þ; Anal.
Calcd for C22H19Cl2NO4: C, 61.12; H, 4.43; N, 3.24. Found: C, 61.09; H,
4.45; N, 3.27.
Melting points were recorded on Buchi Melting point apparatus
D-545. NMR spectra were recorded on Bruker DPX400 instrument
in CDCl3 with TMS as an internal standard. Chemical shift values are
reported in
d (ppm) and coupling constants in Hertz. Mass spectra
were recorded on QTOF Daltonics instrument. The progress of all
reactions was monitored by TLC on 2e5 cm percolated silica gel 60
4.1.4. Compound 8d
25
F254 plates of thickness 0.25 mm (Merck). The chromatograms were
Crystalline white solid; mp: 157e160 ꢂC; [
a
]
D
þ23 (c 0.5,
visualized under UV 254e366 nm and iodine.
CHCl3); yield 75%; IR (KBr, cmꢀ1): 541.34, 667.00, 713.94, 754.66,
786.08, 834.39, 869.02, 946.15, 987.50, 1038.58, 1110.36, 1138.14,
1241.79, 1297.82, 1337.02, 1560.12, 1616.04, 1637.41, 1792.20,
4.1. Synthesis of spiro-isoxazoline derivatives of a-santonin
2932.55, 3400.42; 1H NMR (400 MHz, CDCl3);
d 7.70 (d, 2H,
In a typical procedure, to a solution of benzene nitrile oxide
(0.577 g, 1.2 mmol) in THF (10 mL) stirred over a period of 10 min,
maintaining the temperature between 0 and 5 ꢂC, added santonin
(0.262 g, 1 mmol) and stirred the reaction mixture at same tem-
perature for 15 min followed by stirring at ambient temperature for
2 h. The solvent was evaporated in vacuo and the crude was sub-
jected for flash chromatography ethyl acetate/hexane (3:7). The
pure products (8ae8p) were characterized on the basis of 1H NMR,
13C NMR and mass spectrometry.
J ¼ 6.8 Hz), 7.20 (d, 2H, J ¼ 8.4 Hz), 6.71 (d, 1H, J ¼ 9.6 Hz), 6.30 (d,
1H, J ¼ 9.6 Hz), 5.42 (t, 1H, J ¼ 10 Hz), 3.97 & 3.93 (s, together
integrating to diastereomeric 1H), 3.30 & 3.25 (s, together inte-
grating to diastereomeric 1H), 2.24 (s, 3H), 2.11 (dd, 2H, J ¼ 4.0,
2.3 Hz), 1.93e2.01 (m, 1H), 1.39e1.41 (m, 2H), 1.27 (s, 3H); 13C
(100 MHz, CDCl3): 11.01, 14.05, 17.40, 37.22, 38.72, 41.35, 80.61,
85.72, 116.10, 116.28, 124.32, 125.98, 128.80, 128.973, 129.04, 129.22,
130.89, 139.91, 150.26, 154.77, 155.63, 163.21, 165.21; Mass QTOF:
404 (M þ Na)þ; Anal. Calcd for C22H20FNO4: C, 69.28; H, 5.29; N,
3.67. Found: C, 69.20; H, 5.32; N, 3.70.
4.1.1. Compound 8a
25
Crystalline pale pink solid; mp: 168e170 ꢂC; [
a
]
þ78 (c 1.0,
4.1.5. Compound 8e
D
25
CHCl3); yield 81%; IR (KBr, cmꢀ1): 542.86, 760.18, 834.68, 906.40,
1039.17, 1110.19, 1243.45, 1302.20, 1360.92, 1513.90, 1637.25,
Crystalline white solid; mp: 174e177 ꢂC; [
a
]
þ32 (c 0.5,
D
CHCl3); yield 77%; IR (KBr, cmꢀ1): 758.03, 833.68, 868.62, 905.50,
987.54, 1039.09, 1109.47, 1138.80, 1204.54, 1242.12, 1303.15,
1433.63, 1637.40, 1663.54, 1790.77, 2932.81; 1H NMR (400 MHz,
1663.28, 1791.31, 3368.25; 1H NMR (400 MHz, CDCl3):
d 7.70 (dd,
2H, J ¼ 7.6, 1.6 Hz), 7.40e7.47 (m, 3H), 6.70 (d, 1H, J ¼ 10 Hz), 6.30 (d,
1H, J ¼ 10 Hz), 5.30 (dd, 1H, J ¼ 9.6, 1.2 Hz), 4.00 & 3.96 (s, together
integrating to diastereomeric 1H), 3.32 & 3.28 (s, together inte-
grating to diastereomeric 1H), 2.20 (s, 3H), 1.95 (dd, 1H, J ¼ 4.8,
2.4 Hz), 1.80e1.84 (m, 2H), 1.45e1.70 (m, 2H), 1.37 (s, 3H); 13C
(100 MHz, CDCl3): 11.00, 17.41, 22.97, 23.77, 24.91, 28.93, 30.38,
37.24, 39.35, 41.36, 54.34, 80.63, 85.75, 116.08, 125.97, 128.97,
129.06, 150.29, 154.77, 165.47, 172.18, 185.97; Mass QTOF: 386
(M þ Na)þ; Anal. Calcd for C22H21NO4: C, 72.71; H, 5.82; N, 3.85.
Found: C, 72.69; H, 5.80; N, 3.8.
CDCl3):
d
7.71 (d, 1H, J ¼ 8.4 Hz), 7.54 (s, 1H), 7.32 (d, 1H, J ¼ 2 Hz),
6.70 (d, 1H, J ¼ 9.6 Hz), 6.35 (d, 1H, J ¼ 9.6 Hz), 5.35 (d, 1H,
J ¼ 10.4 Hz), 4.04 & 3.99 (s, together integrating to diastereomeric
1H), 3.54 & 3.50 (s, together integrating to diastereomeric 1H), 2.21
(s, 3H), 2.13 (dd, 2H, J ¼ 5.5, 2.3 Hz), 1.90e1.86 (m, 1H), 1.39e1.32
(m, 2H), 1.27 (s, 3H); 13C (100 MHz, CDCl3): 10.99, 17.50, 24.91,
29.68, 37.25, 41.33, 41.51, 54.42, 80.51, 86.42, 125.99, 126.08, 127.73,
129.26, 130.60, 131.53, 133.61, 137.19, 150.14, 154.71, 155.75, 171.83,
185.93; Mass QTOF: 454 (M þ Na)þ; Anal. Calcd for C22H19Cl2NO4:
C, 61.12; H, 4.43; N, 3.24. Found: C, 61.15; H, 4.39; N, 3.20.
4.1.2. Compound 8b
25
Crystalline yellow solid; mp: 206e210 ꢂC; [
a
]
þ43 (c 1.0,
4.1.6. Compound 8f
D
25
CHCl3); yield 76%; IR (KBr, cmꢀ1): 550.14, 610.38, 765.48, 934.83,
978.34, 1119.87, 1127.16, 1240.45, 1275.63, 1293.41, 1368.42, 1458.11,
1633.48, 1660.67, 1794.66, 2930.21, 3364.61; 1H NMR (400 MHz,
Crystalline white solid; mp: 162e164 ꢂC; [
a
]
þ50 (c 0.5,
D
CHCl3); yield 72%; IR: (KBr, cmꢀ1): 525.76, 570.78, 667.82, 754.44,
832.03, 870.77, 904.40, 988.13, 1039.56, 1077.84, 1077.84, 1138.39,
1244.99, 1345.49, 1382.78, 1464.48, 1587.55, 1615.78, 1637.45,
CDCl3):
d
8.20 (d, 2H, J ¼ 8.8 Hz), 7.80 (d, 2H, J ¼ 8.8 Hz), 6.70 (d, 1H,
J ¼ 9.6 Hz), 6.20 (d, 1H, J ¼ 9.6 Hz), 5.25 (dd, 1H, J ¼ 9.6, 1.2 Hz), 3.94
& 3.84 (s, together integrating to diastereomeric 1H), 3.29 & 3.25 (s,
together integrating to diastereomeric 1H), 2.20 (s, 3H), 1.90 (t, 1H,
J ¼ 13.2 Hz), 1.70 (dd, 2H, J ¼ 4.2, 2.4 Hz), 1.50e1.40 (m, 2H), 1.40 (s,
3H); 13C (100 MHz, CDCl3): 11.02, 17.42, 22.64, 24.94, 37.18, 38.68,
41.36, 54.19, 80.68, 86.67, 100.01, 124.18, 126.02, 127.79, 129.36,
134.07, 149.01, 150.00, 154.71, 155.20, 171.74, 185.93; Mass QTOF:
431 (M þ Na)þ; Anal. Calcd for C22H20N2O6: C, 64.70; H, 4.94; N, 6.8.
Found: C, 64.65; H, 4.90; N, 6.95.
1719.69, 2928.44, 3368.19; 1H NMR (400 MHz, CDCl3):
d 7.45 (d, 3H,
J ¼ 1.2 Hz), 7.20e7.16 (m, 1H), 6.71 (d, 1H, J ¼ 9.6 Hz), 6.34 (d, 1H,
J ¼ 9.6 Hz), 5.32 (t, 1H, J ¼ 8.8 Hz), 3.97 & 3.93 (s, together inte-
grating to diastereomeric 1H), 3.30 & 3.25 (s, together integrating to
diastereomeric 1H), 2.20 (s, 3H), 2.12 (dd, 2H, J ¼ 9.6, 1.5 Hz), 1.94 (t,
1H, J ¼ 13.2 Hz), 1.73 (dd, 2H, J ¼ 2.8, 1.3 Hz), 1.44 (s, 3H); 13C
(100 MHz, CDCl3): 11.01, 14.05, 17.40, 37.21, 38.72, 41.35, 80.61,
85.72, 116.10, 116.28, 124.32, 127.98, 128.80, 128.97, 129.04, 129.22,
130.89, 139.91, 150.26, 154.77, 155.63, 163.21, 165.21; Mass QTOF:
404 (M þ Na)þ; Anal. Calcd for C22H20FNO4: C, 69.28; H, 5.29; N,
3.67. Found: C, 69.26; H, 5.23; N, 3.71.
4.1.3. Compound 8c
25
Crystalline white solid; mp: 163e166 ꢂC; [
a]
þ38 (c 0.5,
D
CHCl3); yield 82%; IR (KBr, cmꢀ1): 541.34, 667.00, 713.94, 754.66,
786.08, 834.39, 869.02, 946.15, 987.50, 1038.58, 1110.36, 1138.14,
1241.79, 1297.82, 1337.02, 1560.12, 1616.04, 1637.41, 1792.20,
4.1.7. Compound 8g
Crystalline pale yellow solid; mp: 196e198 ꢂC; [
a]
25 þ16 (c 0.5,
D
CHCl3); yield 81%; IR (KBr, cmꢀ1): 508.50, 674.95, 771.48, 832.71,
987.14,1039.54,1123.35,1245.99,1374.04,1436.17,1582.22,1622.01,
1637.41, 1664.10, 1721.48, 1792.13, 2928.36, 3368.46; 1H NMR
2932.55, 3400.42; 1H NMR (400 MHz, CDCl3):
d 7.73 (d, 1H,
J ¼ 8.4 Hz), 7.35e7.45 (m, 2H), 6.73 (d, 1H, J ¼ 9.6 Hz), 6.35 (d, 1H,
J ¼ 9.6 Hz), 5.32 (d, 1H, J ¼ 10.4 Hz), 4.02 & 3.97 (s, together inte-
grating to diastereomeric 1H), 3.23 (s, together integrating to dia-
stereomeric 1H), 2.24 (s, 3H), 2.10e2.03 (m, 1H), 1.70 (dd, 2H,
(400 MHz, CDCl3):
d
7.65 (d, 1H, J ¼ 8.8 Hz), 7.60 (dd, 1H, J ¼ 8.4,
2.0 Hz), 6.92 (dd, 2H, J ¼ 10, 8.8 Hz), 6.71 (d, 1H, J ¼ 9.6 Hz), 6.32 (d,
1H, J ¼ 9.6 Hz), 5.30 (d, 1H, J ¼ 8.4 Hz), 3.97 (s, 3H), 3.91 (dd, 1H,

J. Khazir et al. / European Journal of Medicinal Chemistry 63 (2013) 279e289
285
J ¼ 17.5, 6.8 Hz), 3.31 (dd, 1H, J ¼ 17.2, 6.4 Hz), 2.24 (s, 3H), 2.11 (dd,
2H, J ¼ 11.2, 4.8 Hz), 2.00e1.86 (m, 2H), 1.53e1.40 (m, 2H), 1.44 (s,
3H); 13C (100 MHz, CDCl3): 11.07, 17.48, 24.98, 37.31, 37.35, 39.68,
54.46, 55.50, 85.38, 114.44, 120.56, 121.45, 123.34, 126.83, 128.62,
129.24, 150.38, 154.85, 155.35, 157.07, 161.79, 172.45, 186.12; Mass
QTOF: 416 (M þ Na)þ; Anal. Calcd for C23H23NO5: C, 70.21; H, 5.89;
N, 3.56. Found: C, 70.16; H, 5.85; N, 3.60.
3.90 (s, together integrating to diastereomeric 1H), 3.28 & 3.24 (s,
together integrating to diastereomeric 1H), 2.38 (s, 3H), 2.04e1.95
(m, 1H), 1.78e1.70 (m, 2H), 1.60e1.52 (m, 2H), 1.44 (s, 3H); 13C
(100 MHz, CDCl3): 11.02, 12.99, 14.20, 37.20, 41.33, 53.43, 80.60,
86.05, 109.88, 110.10, 117, 117.23, 125.78, 126.04, 129.35, 132.23,
150.02, 154.65, 159.25, 161.77, 171.89, 185.93; Mass QTOF: 482
(M þ Na)þ; Anal. Calcd for C22H19BrFNO4: C, 57.41; H, 4.16; N, 3.04.
Found: C, 57.45; H, 4.20; N, 3.0.
4.1.8. Compound 8h
25
Crystalline yellow solid; mp: 205e208 ꢂC; [
a]
þ56 (c 0.5,
4.1.12. Compound 8l
D
25
CHCl3); yield 78%; IR (KBr, cmꢀ1): 755.71, 832.83, 904.62, 1039.58,
1137.88, 1276.06, 1302.64, 1419.89, 1508.68, 1606.64, 1636.59,
1663.00, 1791.25, 2929.94, 3368.51; 1H NMR (400 MHz, CDCl3):
Crystalline white solid; mp: 167e169 ꢂC; [
a
]
þ24 (c 1.0,
D
CHCl3); yield 82%; IR (KBr, cmꢀ1): 542.73, 610.31, 778.48, 897.65,
985.14, 1107.57, 1125.56, 1247.43, 1275.63, 1276.44, 1345.41, 1434.12,
1634.28, 1665.35, 1778.96, 2935.21, 3456.81; 1H NMR (400 MHz,
d
7.90 (s, 1H), 7.65 (d, 1H, J ¼ 6.8 Hz), 6.95 (d, 1H, J ¼ 7.6 Hz), 6.73 (d,
1H, J ¼ 9.6 Hz), 6.35 (d,1H, J ¼ 9.6 Hz), 5.32 (d,1H, J ¼ 10 Hz), 3.95 (s,
3H), 3.91 & 3.90 (s, together integrating to diastereomeric 1H), 3.27
& 3.23 (s, together integrating to diastereomeric 1H), 2.36 (t, 1H,
J ¼ 4 Hz), 2.12 (s, 3H), 2.03 (d, 1H, J ¼ 8.8 Hz), 1.91 (d, 1H, J ¼ 8.8 Hz),
1.64 (s, 3H), 1.43 (s, 3H); 13C (100 MHz, CDCl3): 11.01, 17.61, 25.08,
37.54, 41.34, 43.98, 53.46, 56.33 69.62, 80.97, 82.02, 112.13, 117.36,
121.63, 126.0, 129.14, 131.61, 132.80, 149.62, 150.70, 154.83, 173.41,
186.09; Mass QTOF: 494 (M þ Na)þ; Anal. Calcd for C23H22BrNO5: C,
58.49; H, 4.69; N, 2.97. Found: C, 58.44; H, 4.73; N, 3.0.
CDCl3):
d
8.10 (d, 1H, J ¼ 3.2 Hz), 7.92 (d, 1H, J ¼ 14 Hz), 7.73 (dd, 2H,
J ¼ 9.6, 6.4 Hz), 6.92 (d, 1H, J ¼ 9.6 Hz), 6.30 (d, 1H, J ¼ 9.6 Hz), 5.32
(d, 1H, J ¼ 11.2 Hz), 3.84 & 3.80 (s, together integrating to diaster-
eomeric 1H), 3.55 & 3.51 (s, together integrating to diastereomeric
1H), 2.34 (t, 1H, J ¼ 11.6 Hz), 2.20 (s, 3H), 2.16 (t, 2H, J ¼ 4.8 Hz),
1.90e1.83 (m, 2H), 1.43 (s, 3H); 13C (100 MHz, CDCl3): 11.14, 18.23,
25.09, 38.32, 39.82, 40.11, 42.97, 48.55, 54.75, 54.83, 82.20, 87.56,
117.48, 120.47, 122.37, 124.3, 126.08, 128.03, 129.15, 130.85, 154.25,
173.94, 188.23; Mass QTOF: 402 (M þ Na)þ; Anal. Calcd for
C22H21NO5: C, 69.64; H, 5.58; N, 3.69. Found: C, 69.62; H, 5.59; N,
4.1.9. Compound 8i
3.71.
25
Crystalline white solid; mp: 182e185 ꢂC; [
a
]
þ64 (c 0.5,
D
CHCl3); yield 76%; IR (KBr, cmꢀ1): 550.45, 612.28, 780.68, 904.83,
981.13, 1113.87, 1128.56, 1239.49, 1277.63, 1293.48, 1379.41, 1453.01,
1631.58, 1658.87, 1787.83, 2930.11, 3369.95; 1H NMR (400 MHz,
4.1.13. Compound 8m
25
Crystalline white solid; mp: 198e201 ꢂC; [
a]
þ53 (c 0.5,
D
CHCl3); yield 77%; IR (KBr, cmꢀ1): 553.73, 615.38, 667.01, 785.48,
904.83, 987.13, 1109.87, 1137.56, 1242.49, 1272.66, 1291.48, 1378.41,
1456.01, 1638.58, 1662.87, 1790.86, 2932.21, 3367.81; 1H NMR
CDCl3):
d
8.02 (dd, 5H, J ¼ 8.4, 2.8 Hz), 7.61e7.54 (m, 2H), 6.77 (d,1H,
J ¼ 9.6 Hz), 6.31 (d,1H, J ¼ 9.6 Hz), 5.32 (t,1H, J ¼ 9.2 Hz), 4.12 & 4.08
(s, 1H together integrating with diastereomeric proton), 3.45 & 3.41
(s, 1H together integrating with diastereomeric proton), 2.21 (s,
3H), 2.12e1.98 (m, 1H), 1.94e1.81 (m, 2H), 1.81e1.70 (m, 2H), 1.63 (s,
2H), 1.56 (dd, 1H, J ¼ 4.4, 1.5 Hz), 1.43 (s, 3H); 13C (100 MHz, CDCl3):
11.03, 17.39, 24.89, 37.21, 39.26, 41.33, 54.42, 80.61, 85.77, 123.32,
125.60, 125.95, 127, 127.59, 127.66, 127.90, 128.49, 128.84, 129.18,
132.86, 134.31, 150.33, 154.78, 156.78, 172.25, 185.98; Mass QTOF:
436 (M þ Na)þ; Anal. Calcd for C26H23NO4: C, 75.53; H, 5.61; N, 3.39.
Found: C, 75.58; H, 5.65; N, 3.43.
(400 MHz, CDCl3):
d
7.61 (d, 1H, J ¼ 8.4 Hz), 7.35 (d, 1H, J ¼ 6.4 Hz),
7.25 (d, 1H, J ¼ 1.2 Hz), 6.72 (d, 1H, J ¼ 9.6 Hz), 6.38 (d, 1H,
J ¼ 9.6 Hz), 5.34 (d, 1H, J ¼ 10.8 Hz), 4.02 & 3.97 (s, together inte-
grating to diastereomeric 1H), 3.23 & 3.18 (s, together integrating to
diastereomeric 1H), 2.24 (s, 3H), 2.10 (t, 1H, J ¼ 12.4 Hz), 2.03e1.94
(m, 2H),1.72e1.69 (m, 2H),1.56 (s, 3H); 13C (100 MHz, CDCl3): 10.92,
12.50, 17.44, 35.27, 287.30, 37.87, 54.67, 80.58, 81.40, 125.92, 125.97,
126.14, 127.13, 128.32, 129.24, 153.96, 154.13, 154.83, 171.72, 186.03;
Mass QTOF: 454 (M þ Na)þ; Anal. Calcd for C22H19Cl2NO4: C, 61.12;
H, 4.43; N, 3.24. Found: C, 61.15; H, 4.39; N, 3.20.
4.1.10. Compound 8j
25
Crystalline white solid; mp: 153e155 ꢂC; [
a
]
þ30 (c 0.5,
4.1.14. Compound 8n
D
CHCl3); yield 72%; IR (KBr, cmꢀ1): 553.58, 588.88, 647.36, 667.70,
797.28, 863.68, 905.73, 1038.62, 1138.90, 1153.90, 1165.94, 1260.38,
1301.85, 1376.34, 1615.73, 1637.59, 1663.42, 1791.14, 2930.71,
Crystalline white solid; mp: 177e178 ꢂC; [
a
]
D
25 þ61 (c 1.0, CHCl3);
yield 80%; IR (KBr, cmꢀ1): 553.73, 615.38, 667.01, 785.48, 904.83,
987.13, 1109.87, 1137.56, 1242.49, 1272.66, 1291.48, 1378.41, 1456.01,
1638.58, 1662.87, 1790.86, 2932.21, 3367.81; 1H NMR (400 MHz,
3367.99; 1H NMR (400 MHz, CDCl3):
d
7.25 (t, 2H, J ¼ 5.6 Hz), 6.90e
6.85 (m, 1H), 6.72 (d, 1H, J ¼ 9.6 Hz), 6.31 (d, 1H, J ¼ 9.6 Hz), 5.34 (t,
1H, J ¼ 9.6 Hz), 3.94 & 3.89 (s, together integrating to diastereo-
meric 1H), 3.27 & 3.23 (s, together integrating to diastereomeric
1H), 2.24 (s, 3H), 2.10e2.03 (m, 1H), 1.93e1.82 (m, 2H), 1.80e1.76
(m, 2H), 1.43 (s, 3H); 13C (100 MHz, CDCl3): 10.96, 14.05, 17.40,
22.49, 23.76, 28.93, 30.37, 37.18, 38.81, 41.33, 54.27, 68.18, 80.61,
86.31, 106.04, 106.29, 109.88, 109.96, 126.05, 128.81, 129.38, 130.90,
131.18, 132.48, 149.97, 155.09, 161.82, 161.94, 164.43, 171.77, 185.93;
Mass QTOF: 422 (M þ Na)þ; Anal. Calcd for C22H19F2NO4: C, 66.16;
H, 4.80; N, 3.51. Found: C, 66.15; H, 4.76; N, 3.54.
CDCl3);
d
7.60e7.54 (m, 1H), 7.51 (d, 1H, J ¼ 1.2 Hz), 7.49 (d, 1H,
J ¼ 3.2 Hz), 7.45 (d, 1H, J ¼ 5.2 Hz), 7.32 (d, 1H, J ¼ 4 Hz), 6.75 (d, 1H,
J ¼ 9.6 Hz), 6.31 (d, 1H, J ¼ 9.6 Hz), 5.36 (d, 1H, J ¼ 10.4 Hz), 4.05 &
4.01 (s, together integrating to diastereomeric 1H), 3.57 & 3.52 (s,
together integrating to diastereomeric 1H), 2.24 (s, 3H), 2.20e2.10
(m, 1H), 2.03e1.94 (m, 2H),1.75 (t, 2H, J ¼ 6.5 Hz), 1.46 (s, 3H); 13C
(100 MHz, CDCl3): 10.93, 12.51, 17.54, 23.13, 24.92, 30.92, 37.31,
41.35, 53.55, 80.41, 86.25, 126.01, 129.27, 130.69, 131.62, 132.93,
150.25, 154.75, 156.62, 171.97, 171.54, 186.05; Mass QTOF: 420
(M þ Na)þ; Anal. Calcd for C22H20ClNO4: C, 66.42; H, 5.07; N, 3.52.
Found: C, 66.45; H, 5.12; N, 3.54.
4.1.11. Compound 8k
25
Crystalline white solid; mp: 234e236 ꢂC; [
a]
þ18 (c 0.5,
4.1.15. Compound 8o
D
CHCl3); yield 75%; IR (KBr, cmꢀ1): 556.45, 615.28, 781.58, 914.43,
978.13,1120.87,1132.56,1245.40,1276.63,1293.38,1376.41,1468.01,
1645.58, 1660.87, 1792.86, 2934.21, 3363.81; 1H NMR (400 MHz,
Crystalline transparent solid; mp: 220e224 ꢂC; [
a]
25 þ38 (c 1.0,
D
CHCl3); yield 75%; IR (KBr, cmꢀ1): 553.73, 615.38, 785.48, 904.83,
987.13, 1109.87, 1137.56, 1242.49, 1272.66, 1291.48, 1378.41, 1456.01,
1638.58, 1662.87, 1790.86, 2932.21, 3367.81; 1H NMR (400 MHz,
CDCl3):
d
7.90 (s, 1H), 7.61 (s, 1H), 7.34 (d, 1H, J ¼ 8 Hz), 6.72 (d, 1H,
J ¼ 9.6 Hz), 6.32 (d, 1H, J ¼ 9.6 Hz), 5.30 (d, 1H, J ¼ 9.6 Hz), 3.94 &
CDCl3):
d
8.11 (d,1H, J ¼ 8.0 Hz), 8.05 (d,1H, J ¼ 8.8 Hz), 7.62 (dd, 2H,

286
J. Khazir et al. / European Journal of Medicinal Chemistry 63 (2013) 279e289
J ¼ 5.6, 1.2 Hz), 7.51 (dd, 1H, J ¼ 10.4, 6.8 Hz), 7.35 (s, 1H), 6.88 (d, 1H,
J ¼ 9.6 Hz), 6.31 (d, 1H, J ¼ 9.6 Hz), 5.45 (d, 1H, J ¼ 9.6 Hz), 4.12 &
4.08 (s, together integrating to diastereomeric 1H), 3.44 & 3.39 (s,
together integrating to diastereomeric 1H), 2.42 (t, 1H, J ¼ 7.6 Hz),
2.20e2.11 (m, 3H), 1.70e1.66 (m, 4H), 1.5 (s, 3H); 13C (100 MHz,
CDCl3): 11.2, 18.6, 27.3, 37.9, 39.0, 41.3, 43.2, 75.6, 83.4, 126.0, 126.7,
127.0,127.3,127.6,128.3,128.9,129.0,129.2,130.3,130.6,131.7,141.0,
155.6, 156.2, 162.9, 172.3, 187.9; Mass QTOF: 486 (M þ Na)þ; Anal.
Calcd for C30H25NO4: C, 77.74; H, 5.44; N, 3.02. Found: C, 77.77; H,
5.48; N, 3.0.
13C (100 MHz, CDCl3): 10.91, 19.50, 24.98, 37.21, 41.12, 42.34, 51.68,
68.59, 78.32, 83.22, 117.93, 124.35, 126.12, 127.95, 128.79, 129.58,
146.23, 147.93, 149.32, 149.63, 154.41, 174.16, 185.87; Mass QTOF:
507 (M þ Na)þ; Anal. Calcd for C28H26N2O4: C, 69.12; H, 5.39; N,
5.76. Found: C, 69.10; H, 5.32; N, 5.81.
4.2.3. Compound 10b0
25
Crystalline yellow solid; mp: 232e236 ꢂC; [
a
]
þ56 (c 0.5,
D
CHCl3); IR (KBr, cmꢀ1): 693.43, 758.43, 831.61, 1041.08, 1106.00,
1157.69, 1265.10, 1454.05, 1488.25, 1596.40, 1635.75, 1662.62,
1791.03, 2924.14, 3368.73; 1H NMR (400 MHz, CDCl3):
d 7.62 (d, 2H,
4.1.16. Compound 8p
J ¼ 8 Hz), 7.42 (d, 2H, J ¼ 8 Hz), 7.35e7.26 (m, 2H), 7.25 (d, 1H,
J ¼ 6.8 Hz), 7.05 (d, 1H, J ¼ 8.8 Hz), 6.75 (d, 1H, J ¼ 12.5 Hz), 6.32 (d,
1H, J ¼ 12 Hz), 5.35e5.30 (dd, 1H, J ¼ 12, 1.2 Hz), 4.85 (t, 1H,
J ¼ 10.5 Hz), 3.15 (dd, 1H, J ¼ 12.8, 7.2 Hz), 2.55 (dd, 1H, J ¼ 12.8,
8.8 Hz), 2.25e2.18 (m, 1H), 2.10 (s, 3H), 2.00 (t, 2H, J ¼ 11 Hz), 1.9e
1.6 (m, 2H), 1.4 (s, 3H); 13C (100 MHz, CDCl3): 11.01, 17.61, 25.08,
37.54, 40.24, 41.34, 43.98, 53.46, 56.33, 69.62, 80.97, 82.02, 111.53,
112.13, 117.36, 121.63, 124.07, 126.0, 127.27, 128.78, 129.14, 131.61,
132.80, 149.62, 150.70, 154.83, 155.73, 173.41, 186.09; Mass QTOF:
543 (M þ Na)þ; Anal. Calcd for C28H26BrNO4: C, 64.62; H, 5.04; N,
2.69. Found: C, 64.66; H, 5.09; N, 2.62.
25
Crystalline white solid; mp: 214e217 ꢂC; [
a]
þ59 (c 0.5,
D
CHCl3); yield 73%; IR (KBr, cmꢀ1); 694.33, 756.59, 831.44, 1038.58,
1103.78, 1158.48, 1245.73, 1488.24, 1597.24, 1636.84, 1663.84,
1788.47, 2926.88, 3368.52; 1H NMR (400 MHz, CDCl3):
d 7.61 (q, 4H,
J ¼ 6.8 Hz), 6.74 (d, 1H, J ¼ 9.6 Hz), 6.35 (d, 1H, J ¼ 9.6 Hz), 5.34 (d,
1H, J ¼ 9.6 Hz), 3.92 & 3.89 (s, together integrating to diastereo-
meric 1H), 3.46 & 3.42 (s, together integrating to diastereomeric
1H), 2.22 (s, 3H), 2.12e2.05 (m, 1H), 1.71e1.64 (m, 2H), 1.53e1.41
(m, 2H), 1.40 (s, 3H); 13C (100 MHz, CDCl3): 11.2, 19.6, 27.2, 38.5,
39.1, 41.0, 42.5, 83.5, 125.4, 128.7, 131.8, 132.0, 132.5, 156.7, 157.3,
162.6, 172.4, 186.2; Mass QTOF: 464 (M þ Na)þ; Anal. Calcd for
C
22H20BrNO4: C, 59.74; H, 4.56; N, 3.17. Found: C, 59.81; H, 4.52; N,
4.2.4. Compound 10b00
25
3.15.
Crystalline yellow solid; mp: 186e188 ꢂC; [
a]
ꢀ48 (c 0.5,
D
CHCl3); IR (KBr, cmꢀ1): 758.47, 831.04, 1040.94, 1105.58, 1488.31,
4.2. Synthesis of spiro-isoxazolidine derivative of santonin
1636.17, 1662.97, 1790.92, 2853.20, 2923.67, 3438.45; 1H NMR
(400 MHz, CDCl3):
d
7.52 (d, 1H, J ¼ 8.4 Hz), 7.35 (d, 1H, J ¼ 8.4 Hz),
In a typical procedure, to a solution of
a
-santonin (0.5 g,
7.20e7.17 (m, 5H), 7.05 (q, 2H, J ¼ 12 Hz), 6.71 (d, 1H, J ¼ 9.6 Hz),
6.30 (d, 1H, J ¼ 9.6 Hz), 4.80e4.74 (m, 2H), 2.80e2.85 (dd, 1H,
J ¼ 12.4, 6.4 Hz), 2.75 (dd, 1H, J ¼ 12.4, 10 Hz), 2.60e2.65 (m, 1H),
2.32 (d, 3H, J ¼ 4.8 Hz), 2.00 (t, 2H, J ¼ 12 Hz),1.51e1.06 (m, 2H),1.45
(s, 3H); 13C (100 MHz, CDCl3): 10.91, 19.49, 24.97, 37.25, 41.12, 42.44,
51.98, 68.99, 78.22, 82.92, 118.18, 122.21, 124.18, 126.10, 128.63,
128.76,129.53,132.25,137.62,149.53,149.80,154.46,174.36,185.95;
Mass QTOF: 543 (M þ Na)þ; Anal. Calcd for C28H26BrNO4: C, 64.62;
H, 5.04; N, 2.69. Found: C, 64.66; H, 5.09; N, 2.62.
1.9 mmol) in dry benzene (6 mL) was added appropriate nitrone
(1.58 g, 2.4 mmol) in dry benzene and refluxed the reaction mixture
for 8 h. The solvent was evaporated in vacuo to afford the crude
product which was further purified by flash chromatography using
ethyl acetate and hexane (2:8) as solvent system. The pure products
(10a0 and 10a00) were isolated in good yields (75%) and charac-
terized on the basis of 1H NMR, 13C NMR and mass spectrometry.
4.2.1. Compound 10a0
25
Crystalline yellow solid; mp: 210e213 ꢂC; [
a
]
þ43 (c 0.5,
4.2.5. Compound 10c0
D
CHCl3); IR (KBr, cmꢀ1): 695.70, 752.29, 837.21, 856.23, 1040.85,
1105.95, 1158.20, 1267.59, 1346.69, 1453.72, 1489.70, 1521.06,
1598.02, 1636.28, 1663.06, 1790.55, 2926.44; 1H NMR (400 MHz,
Crystalline pink solid; mp: 195e197 ꢂC; [
a
]
25 þ54 (c 0.5, CHCl3);
D
IR (KBr, cmꢀ1): 692.93, 755.33, 863.54, 906.64, 1039.28, 1103.18,
1136.17, 1158.65, 1200.93, 1244.60, 1489.21, 1597.18, 1616.19,
CDCl3):
d
8.31 (d, 2H, J ¼ 8.8 Hz), 7.70 (d, 2H, J ¼ 8 Hz), 7.36e7.25 (m,
1663.43, 1789.40, 2933.93; 1H NMR (400 MHz, CDCl3):
d 7.75 (d, 1H,
3H), 7.12 (d, 1H, J ¼ 7.2 Hz), 6.92 (t, 1H, J ¼ 8.8 Hz), 6.74 (d, 1H,
J ¼ 9.6 Hz), 6.34 (d, 1H, J ¼ 9.6 Hz), 5.30e5.35 (dd, 1H, J ¼ 10.8,
1.2 Hz), 5.10e5.15 (t,1H, J ¼ 8 Hz), 3.24 (dd,1H, J ¼ 12.5, 7.2 Hz), 2.53
(dd, 1H, J ¼ 12.5, 8.4 Hz), 2.35 (s, 3H), 1.84 (dd, 1H, J ¼ 12, 6.4 Hz),
1.6e1.55 (m, 4H), 1.4 (s, 3H); 13C (100 MHz, CDCl3): 11.02, 17.52,
25.09, 37.42, 41.31, 43.66, 53.06, 69.46, 80.96, 82.34, 116.78, 124.21,
124.30, 126.04, 127.97, 128.80, 128.99, 129.27, 147.24, 147.79, 149.31,
150.37, 154.71, 173.11, 186; Mass QTOF: 507 (M þ Na)þ; Anal. Calcd
for C28H26N2O4: C, 69.12; H, 5.39; N, 5.76. Found: C, 69.10; H, 5.32;
N, 5.81.
J ¼ 8 Hz), 7.45 (d, 2H, J ¼ 8 Hz), 7.30e7.24 (m, 4H), 7.14 (q, 1H,
J ¼ 6.8 Hz), 6.95 (t, 1H, J ¼ 6.8 Hz), 6.72 (d, 1H, J ¼ 9.6 Hz), 6.34 (d,
1H, J ¼ 9.6 Hz), 5.32e5.37 (t, 1H, J ¼ 8.4 Hz), 4.76e4.83 (dd, 1H,
J ¼ 12, 1.2 Hz), 3.24 (dd, 1H, J ¼ 12.8, 7.2 Hz), 2.51 (dd, 1H, J ¼ 12.8,
8.4 Hz), 2.20 (d, 3H, J ¼ 6.8 Hz), 2.13e2.00 (m, 1H, J ¼ 3.2 Hz), 2.00e
1.93 (m, 2H), 1.60e1.56 (m, 2H), 1.40 (s, 3H); 13C (100 MHz, CDCl3):
10.99, 17.51, 25.03, 37.42, 41.29, 41.62, 53.08, 66.04, 80.73, 82.57,
115.52, 116.72, 118.01, 127.75, 128.72, 128.71, 128.77, 128.93, 128.97,
129.13, 129.20, 129.70, 136.81, 149.44, 150.502, 154.45, 172.95,
186.02; Mass QTOF: 532 (M þ Na)þ; Anal. Calcd for C28H25Cl2NO4:
C, 69.12; H, 5.39; N, 5.76. Found: C, 69.10; H, 5.32; N, 5.81.
4.2.2. Compound 10a00
25
Crystalline yellow solid; mp: 202e205 ꢂC; [
a]
ꢀ28 (c 0.5,
4.2.6. Compound 10c00
D
CHCl3); IR (KBr, cmꢀ1): 695.13, 753.56, 834.07, 855.33, 989.99,
1039.12, 1106.25, 1158.58, 1268.44, 1346.11, 1382.11, 1453.94,
1489.36, 1522.56, 1598.59, 1637.24, 1663.35, 1724.24, 1789.38,
Crystalline pink solid; mp: 187e191 ꢂC; [
a]
25 ꢀ30 (c 0.5, CHCl3);
D
1H NMR (400 MHz, CDCl3):
d
7.75 (d, 1H, J ¼ 8.4 Hz), 7.40 (d, 1H,
J ¼ 8.4 Hz), 7.33e7.27 (m, 4H), 7.05 (dd, 3H, J ¼ 5.6 Hz), 6.73 (d, 1H,
J ¼ 9.6 Hz), 6.34 (d, 1H, J ¼ 9.6 Hz), 5.32 (t, 1H, J ¼ 8.4 Hz), 4.82 (dd,
1H, J ¼ 12, 1.2 Hz), 2.81e2.76 (dd, 1H, J ¼ 12, 7.2 Hz), 2.53e2.48 (m,
1H), 2.20 (d, 3H, J ¼ 7.2 Hz), 1.93 (t, 2H, J ¼ 12.4 Hz), 1.50e1.45 (m,
2H), 1.34 (s, 3H); 13C (100 MHz, CDCl3): 10.90, 17.54, 19.54, 22.99,
24.98, 30.91, 37.44, 39.89, 41.07, 41.30, 41.65, 51.78, 53.11, 64.82,
66.07, 68.17, 80.74, 83.51, 116.75, 123.42, 126.10, 127.75, 127.99,
2924.92, 3400.49; 1H NMR (400 MHz, CDCl3):
d 8.31 (dd, 2H,
J ¼ 8 Hz), 7.72 (d, 2H, J ¼ 8.4 Hz), 7.34 (dd, 3H, J ¼ 9.6, 5.6 Hz), 7.12
(m, 2H), 6.75 (d, 1H, J ¼ 9.6 Hz), 6.34 (d, 1H, J ¼ 9.6 Hz), 5.00e5.05
(dd, 1H, J ¼ 9.6, 6.4 Hz), 4.80e4.85 (dd, 1H, J ¼ 12, 1.2 Hz), 2.87e2.95
(dd, 1H, J ¼ 12.4, 6.4 Hz), 2.67e2.75 (dd, 1H, J ¼ 12.4, 10.4 Hz), 2.59e
2.66 (m,1H), 2.1 (s, 3H), 2.05e1.93 (m, 2H),1.62 (m, 2H),1.45 (s, 3H);

J. Khazir et al. / European Journal of Medicinal Chemistry 63 (2013) 279e289
287
128.73,128.79,129.60,129.78, 133.16,134.39, 135.69, 149.35,149.72,
154.52, 154.73, 173.83, 185.94, 206.89; Mass QTOF: 532 (M þ Na)þ;
Anal. Calcd for C28H25Cl2NO4: C, 69.12; H, 5.39; N, 5.76. Found: C,
69.10; H, 5.32; N, 5.81.
8.4 Hz), 2.60 (t, 1H, J ¼ 3.2 Hz), 2.23 (s, 3H), 1.85 (dd, 2H, J ¼ 8.4,
4.4 Hz), 1.63e1.57 (m, 2H), 1.30 (d, 3H, J ¼ 12.8 Hz); 13C (100 MHz,
CDCl3): 10.83, 17.81, 19.25, 25.19, 30.89, 37.44, 37.68, 41.29, 43.24,
50.31, 69.34, 80.28, 80.65, 103.59, 103.79, 110.62, 110.67, 118.55,
124.66, 126.04, 129.24, 143.02, 147.83, 150.64, 154.34, 164.21, 173.00,
186.10; Mass QTOF: 422 (M þ 23)þ; Anal. Calcd for C28H25F2NO4: C,
70.43; H, 5.28; N, 2.93. Found: C, 70.39; H, 5.32; N, 2.96.
4.2.7. Compound 10d0
25
Crystalline white solid; mp: 153e156 ꢂC; [
a
]
þ51 (c 0.5,
D
CHCl3); IR (KBr, cmꢀ1): 510.46, 550.37, 667.37, 694.02, 833.28,
859.69, 983.34, 1039.37, 1119.11, 1216.06, 1247.56, 1378.93, 1454.60,
1490.93, 1598.66, 1626.05, 1665.26, 1723.71, 1789.80, 2856.96,
4.2.11. Compound 10f0
25
Crystalline yellow solid; mp: 152e156 ꢂC; [
a
]
þ35 (c 0.5,
D
2928.69; 1H NMR (400 MHz, CDCl3):
d
7.31 (m, 2H), 7.12 (dd, 3H,
CHCl3); IR (KBr, cmꢀ1): 694.13, 756.70, 832.48, 1038.69, 1103.91,
1103.69, 1257.60, 1454.05, 1496.66, 1609.33, 1787.86, 2930.11,
3368.60; 1H NMR (400 MHz, CDCl3): 7.80 (dd, 1H, J ¼ 12, 6.8 Hz),
7.42 (m, 1H), 7.25 (dd, 2H, J ¼ 9.6, 2.4 Hz), 7.15 (t,1H, J ¼ 8.4 Hz), 7.00
(t,1H, J ¼ 7.2 Hz), 6.92 (d, 2H, J ¼ 7.2 Hz), 6.72 (d,1H, J ¼ 9.6 Hz), 6.34
(d,1H, J ¼ 9.6 Hz), 5.35 (dd,1H, J ¼ 9.6 Hz), 4.94 (t,1H, J ¼ 8 Hz), 3.90
(s, 3H), 3.14 (dd, 1H, J ¼ 12.5, 7.2 Hz), 2.55 (dd, 1H, J ¼ 12.5, 8.4 Hz),
2.36e2.15 (m, 1H), 2.14 (s, 3H), 2.00e1.85 (m, 2H), 1.65e1.54 (m,
2H), 1.40 (s, 3H); 13C (100 MHz, CDCl3): 11.01, 17.61, 25.08, 37.54,
41.34, 43.98, 53.46, 56.33, 69.62, 80.97, 82.02, 111.53, 112.13, 117.36,
121.63, 124.07, 126.0, 127.27, 128.78, 129.14, 131.61, 132.80, 149.62,
150.70, 154.83, 155.73, 173.41, 186.09; Mass QTOF: 573 (M þ Na)þ;
Anal. Calcd for C29H28BrNO5: C, 63.28; H, 5.13; N, 2.54. Found: C,
63.26; H, 5.09; N, 2.57.
J ¼ 14.8, 8 Hz), 7.32e7.25 (m, 4H), 6.95 (dd, 2H, J ¼ 8.4, 0.8 Hz), 6.76
(d, 1H, J ¼ 9.6 Hz), 6.36 (d, 1H, J ¼ 9.6 Hz), 5.30e5.35 (d, 1H,
J ¼ 10.8 Hz), 4.95e5.00 (t, 1H, J ¼ 8 Hz), 3.15e3.12 (dd, 1H, J ¼ 12.8,
4.4 Hz), 2.40 (dd, 1H, J ¼ 12.8, 8.4 Hz), 2.23 (s, 3H), 2.05e1.90 (m,
1H), 1.60e1.54 (m, 4H), 1.35 (s, 3H); 13C (100 MHz, CDCl3): 10.96,
14.05, 17.44, 22.97, 37.39, 38.73, 41.32, 53.05, 69.24, 80.93, 82.28,
103.30, 103.71, 109.59, 109.70, 116.56, 123.91, 125.93, 128.80, 128.90,
144.15, 149.46, 150.62, 154.88, 162.33, 164.41, 173.16, 186.05; Mass
QTOF: 560 (M þ Na)þ; Anal. Calcd for C28H25BrFNO4: C, 62.40; H,
4.68; N, 2.60. Found: C, 62.43; H, 4.70; N, 2.65.
4.2.8. Compound 10d00
25
Crystalline white solid; mp: 178e181 ꢂC; [
a
]
ꢀ24 (c 0.5,
D
CHCl3); IR (KBr, cmꢀ1): 693.88, 757.02, 832.83, 984.06, 1040.99,
1118.82, 1248.88, 1462.85, 1491.30, 1598.24, 1624.90, 1664.02,
4.2.12. Compound 10f00
1789.72, 2924.31, 3394.40; 1H NMR (400 MHz, CDCl3):
d
7.71e7.68
Crystalline brown solid; mp: 164e167 ꢂC; [
a
]
ꢀ37 (c 0.5,
25
D
(m, 3H), 7.12e7.00 (m, 4H), 6.85 (dd, 1H, J ¼ 7.2, 3.4 Hz), 6.72 (d, 1H,
J ¼ 9.6 Hz), 6.34 (d, 1H, J ¼ 9.6 Hz), 5.32 (t, 1H, J ¼ 7.6 Hz), 4.76 (dd,
1H, J ¼ 9.6, 2.4 Hz), 3.14 (dd, 1H, J ¼ 12.5, 7.2 Hz), 2.54e2.48 (m, 1H),
CHCl3); IR (KBr, cmꢀ1): 514.78, 686.56, 756.02, 831.32, 906.81,
1039.37, 1138.30, 1244.51, 1303.04, 1403.39, 1500.30, 1615.59,
1636.72, 1663.22, 1791.42, 2852.88, 2923.26, 3435.92; 1H NMR
2.33 (s, 3H), 2.00 (t, 2H, J ¼ 6 Hz), 1.61e1.57 (m, 2H), 1.30 (s, 3H); 13
C
(400 MHz, CDCl3):
d
7.30e7.25 (m, 2H), 7.12 (dd, 2H, J ¼ 9.6, 6.8 Hz),
(100 MHz, CDCl3): 11.12, 14.67, 15.53, 21.65, 36.73, 37.90, 38.45,
40.41, 51.30, 67.65, 80.76, 81.23, 101.23, 106.56, 107.83, 109.21,
114.31, 121.67, 127.71, 128.00, 128.30, 143.25, 149.29, 151.12, 156.73,
161.83, 167.41, 175.16, 186.15; Mass QTOF: 560 (M þ Na)þ; Anal.
Calcd for C28H25BrFNO4: C, 62.40; H, 4.68; N, 2.60. Found: C, 62.43;
H, 4.70; N, 2.65.
7.00 (d, 2H, J ¼ 6.24 Hz), 6.84 (t, 1H, J ¼ 6.8 Hz), 6.71 (d, 1H,
J ¼ 9.6 Hz), 6.32 (d, 1H, J ¼ 9.6 Hz), 5.30 (d, 1H, J ¼ 9.6 Hz), 5.00 (t,
1H, J ¼ 8.4 Hz), 3.93 (s, 3H), 2.95e3.00 (dd, 1H, J ¼ 12.5, 7.2 Hz),
2.80e2.83 (dd, 1H, J ¼ 12.4, 7.8 Hz), 2.75e2.72 (dd, 1H, J ¼ 12.5,
6.4 Hz), 2.57e2.65 (m, 1H), 2.20 (d, 3H, J ¼ 4 Hz), 2.10 (t, 2H,
J ¼ 4 Hz), 2.00e1.84 (m, 2H), 1.40 (s, 3H); 13C (100 MHz, CDCl3):
10.90, 19.51, 25.06, 37.31, 41.16, 42.46, 43.56, 52.03, 56.34, 68.73,
78.24, 82.89, 100.01, 112.18, 112.35, 118.69, 124.24, 126.05, 127.49,
128.09, 129.52, 131.61, 132.80, 149.62, 150.70, 154.83, 155.73, 173.41,
186.09; Mass QTOF: 573 (M þ Na)þ; Anal. Calcd for C29H28BrNO5: C,
63.28; H, 5.13; N, 2.54. Found: C, 63.26; H, 5.09; N, 2.57.
4.2.9. Compound 10e0
25
Crystalline yellow solid; mp: 145e148 ꢂC; [
a
]
þ47 (c 0.5,
D
CHCl3); IR (KBr, cmꢀ1): 694.02, 756.60, 831.26, 1038.96, 1104.96,
1159.33, 1245.20, 1491.37, 1597.43, 1636.36, 1663.37, 1788.27,
2927.74, 3368.49; 1H NMR (400 MHz, CDCl3):
d
7.75 (dd, 1H, J ¼ 6.8,
2.4 Hz), 7.35 (dd, 1H, J ¼ 9.6, 7.6 Hz), 7.25 (dd, 2H, J ¼ 16.8, 8.4 Hz),
7.15 (d, 1H, J ¼ 7.6 Hz), 7.00 (d, 1H, J ¼ 10 Hz), 6.94 (d, 2H, J ¼ 10 Hz),
6.72 (d, 1H, J ¼ 9.6 Hz), 6.34 (d, 1H, J ¼ 9.6 Hz), 5.30 (dt, 1H, J ¼ 11.2,
1.2 Hz), 4.97e4.95 (t, 1H, J ¼ 8 Hz), 3.15e3.13 (dd, 1H, J ¼ 12.8,
7.2 Hz), 2.47e2.40 (dd, 1H, J ¼ 12.8, 8.8 Hz), 2.20 (s, 3H), 2.00 (d, 1H,
J ¼ 12.5 Hz), 1.80 (dd, 2H, J ¼ 8.4, 1.2 Hz), 1.60e1.57 (m, 2H), 1.30 (d,
3H, J ¼ 12.5 Hz); 13C (100 MHz, CDCl3): 11.01, 17.53, 25.07, 29.69,
37.46, 41.32, 43.86, 53.18, 69.22, 80.94, 82.17, 109.58, 109.79, 116.83,
117.04, 124.13, 126.0, 127.45, 127.52, 128.89, 129.17, 131.81, 137.09,
137.13, 149.47, 150.54, 154.77, 157.49, 159.96, 173.24, 186.02; Mass
QTOF: 422 (M þ Na)þ; Anal. Calcd for C28H25F2NO4: C, 70.43; H,
5.28; N, 2.93. Found: C, 70.39; H, 5.32; N, 2.96.
4.2.13. Compound 10g0
25
Crystalline brown solid; mp: 203e205 ꢂC; [
a
]
þ47 (c 0.5,
D
CHCl3); IR (KBr, cmꢀ1): 693.80, 756.61, 778.50, 832.65, 1040.62,
1104.01, 1156.40, 1246.13, 1379.07, 1453.63, 1490.38, 1597.29,
1636.79, 1790.12, 2925.71; 1H NMR (400 MHz, CDCl3): 8.40 (s, 1H),
8.21 (dd, 1H, J ¼ 12, 6.8 Hz), 7.80 (dd, 1H, J ¼ 12, 6.8 Hz), 7.63 (t, 1H,
J ¼ 6.8 Hz), 7.25e7.20 (m, 4H), 7.10 (d, 1H, J ¼ 7.6 Hz) 7.15 (t, 1H,
J ¼ 8.4 Hz), 6.92 (d, 2H, J ¼ 7.2 Hz), 6.74 (d, 1H, J ¼ 9.6 Hz), 6.30 (d,
1H, J ¼ 9.6 Hz), 5.44 (dd, 1H, J ¼ 10.8, 1.2 Hz), 5.13 (t, 1H, J ¼ 8.4 Hz),
3.20 (dd, 1H, J ¼ 12.8, 7.2 Hz), 2.55 (dd, 1H, J ¼ 12.8, 7.8 Hz), 2.25 (d,
3H, J ¼ 4 Hz), 2.10e2.00 (m, 1H), 2.00 (t, 2H, J ¼ 8.4 Hz), 1.62e1.57
(m, 2H), 1.30 (s, 3H); 13C (100 MHz, CDCl3): 9.41, 11.02, 14.85, 15.74,
17.55, 22.62, 25.09, 30.32, 31.67, 37.43, 39.39, 41.32, 43.72, 69.43,
80.97, 82.35, 117.07, 121.82, 123.18, 124.32, 126.10, 128.98, 129.22,
130.05; Mass QTOF; 509 (M þ Na)þ; Anal. Calcd for C28H26N2O4: C,
69.12; H, 5.39; N, 5.76. Found: C, 69.10; H, 5.32; N, 5.81.
4.2.10. Compound 10e00
25
Crystalline yellow solid; mp: 183e186 ꢂC; [
a]
ꢀ83 (c 0.5,
D
CHCl3); IR (KBr, cmꢀ1): 691.02, 753.64, 834.12, 1035.56, 1112.76,
1154.30, 1249.24, 1497.57, 1592.40, 1632.26, 1660.27, 1785.37,
2923.54, 3365.39; 1H NMR (400 MHz, CDCl3):
d
7.75e7.72 (m, 3H),
4.2.14. Compound 10g00
25
7.20 (d, 2H, J ¼ 6.4 Hz), 7.10 (t, 1H, J ¼ 7.2 Hz), 6.90 (d, 1H, J ¼ 8 Hz),
6.80 (t, 1H, J ¼ 9.6 Hz), 6.73 (d, 1H, J ¼ 9.6 Hz), 6.30 (d, 1H,
J ¼ 9.6 Hz), 5.31 (dd, 1H, J ¼ 10.8, 6.4 Hz), 4.30 (t, 1H, J ¼ 8.4 Hz),
2.92e2.97 (dd, 1H, J ¼ 13.2, 7.2 Hz), 2.75e2.82 (dd, 1H, J ¼ 13.2,
Crystalline brown solid; mp: 213e216 ꢂC; [
a]
ꢀ43 (c 0.5,
D
CHCl3); IR (KBr, cmꢀ1): 693.80, 756.61, 778.50, 832.65, 1040.62,
1104.01, 1156.40, 1246.13, 1379.07, 1453.63, 1490.38, 1597.29,
1636.79, 1790.12, 2925.71; 1H NMR (400 MHz, CDCl3):
d 8.32 (t, 2H,

288
J. Khazir et al. / European Journal of Medicinal Chemistry 63 (2013) 279e289
J ¼ 8 Hz), 7.62 (t, 1H, J ¼ 9.6), 7.32e7.28 (m, 5H), 7.10 (d, 2H,
J ¼ 7.8 Hz), 6.82 (d, 1H, J ¼ 6.8 Hz), 6.71 (d, 1H, J ¼ 9.6 Hz), 6.74 (d,
1H, J ¼ 9.6 Hz), 6.32 (d, 1H, J ¼ 9.6 Hz), 5.10 (dd, 1H, J ¼ 9.6, 6Hz),
4.51 (t, 1H, J ¼ 8.4 Hz), 2.90e2.95 (dd, 1H, J ¼ 12.0, 5.2 Hz), 2.70 (dd,
1H, J ¼ 12.4, 2.4 Hz), 2.67e2.62 (m,1H), 2.20 (d, 3H, J ¼ 4 Hz), 2.15 (t,
2H, J ¼ 4 Hz), 1.60e1.47 (m, 2H), 1.30 (s, 3H); 13C (100 MHz, CDCl3):
11.04, 14.11, 17.81, 19.50, 29.69, 31.53, 32.52, 37.21, 37.45, 41.14, 41.31,
42.38, 43.23, 49.71, 49.97, 68.38, 68.60, 80.35, 83.19, 116.52, 116.99,
118.31, 122.02, 122.96, 123.33, 123.41, 124.52, 124.90, 126.03, 173.10,
185.94; Mass QTOF: 509 (M þ Na)þ; Anal. Calcd for C28H26N2O4: C,
69.12; H, 5.39; N, 5.76. Found: C, 69.10; H, 5.32; N, 5.81.
recorded on ELISA reader at 540 nm. The cell growth was calculated
by subtracting mean OD value of the respective blank from the
mean OD value of experimental set. Percentage of growth in the
presence of test material was calculated considering the growth in
the absence of any test material as 100% and the results are
reported in terms of IC50 values.
4.3.2. DNA cell cycle analysis
Effect of compounds 10b0 and 10b00 on DNA content by cell
cycle phase distribution was assessed using PC-3 cells by incu-
bating the cells 1 ꢁ 106 mL/well with compounds 10b0 and 10bØ
(1, 5 &10 mM each) for 24 h. The cells were then washed twice with
4.2.15. Compound 10h0
ice-cold PBS, harvested, fixed with ice-cold PBS in 70% ethanol and
stored at 20 ꢂC for 30 min [35]. After fixation, these cells were
incubated with RNase A (0.1 mg/mL) at 37 ꢂC for 30 min, stained
with propidium iodide (50 mg/mL) for 30 min on ice in dark, and
then measured for DNA content using BD-LSR flow cytometer
(Becton Dickinson, USA) equipped with electronic doublet dis-
crimination capability using blue (488 nm) excitation from Argon
laser. Data were collected in list mode on 10,000 events for FL2-A
vs FL2-W.
25
Crystalline yellow solid; mp: 145e147 ꢂC; [
a
]
þ28 (c 0.5,
D
CHCl3); IR (KBr, cmꢀ1): 694.02, 756.60, 831.26, 1038.96, 1104.96,
1159.33, 1245.20, 1491.37, 1597.43, 1636.36, 1663.37, 1788.27,
2927.74, 3368.49; 1H NMR (400 MHz, CDCl3):
d 7.90 (t, 2H,
J ¼ 7.2 Hz), 7.83 (d, 2H, J ¼ 8.4 Hz), 7.30 (q, 3H, J ¼ 7.2 Hz), 7.10 (d,
1H, J ¼ 6.8 Hz), 7.00e6.96 (m, 5H), 6.75 (d, 1H, J ¼ 9.6 Hz), 6.34 (d,
1H, J ¼ 9.6 Hz), 5.47 (dd, 1H, J ¼ 9.6, 1.2 Hz), 5.23 (t, 1H, J ¼ 8.4 Hz),
3.28 (dd, 1H, J ¼ 12.5, 7.2 Hz), 2.90 (dd, 1H, J ¼ 12.5, 7.8 Hz), 2.15 (s,
3H), 2.10e2.00 (m, 1H), 1.65e1.45 (m, 4H), 1.43 (s, 3H); 13C
(100 MHz, CDCl3): 11.20, 16.65, 19.30, 24.50, 30.89, 36.35, 37.60,
41.32, 42.15, 47.45, 70.96, 78.36, 80.32, 102.21, 109.12, 110.62,
111.23, 114.51, 121.21, 125.31, 128.54, 147.08, 147.56, 152.43, 160.98,
171.76, 185.21; Mass QTOF: 514 (M þ Na)þ; Anal. Calcd for
4.3.3. Preparation of nuclear lysates
PC-3 cells (5 ꢁ 106) were incubated with the test compounds for
24 h at different concentrations. Cells were lysed with hypotonic
buffer (10 mM HEPES/KOH pH 7.9, 2 mM MgCl2, 1 mM EDTA, 1 mM
NaF, 1 mM Na3OV4, 10 mM KCl, 1 mM DTT, 0.5 mM PMSF, 1% (v/v)
eukaryotic protease inhibitor cocktail) for 10 min on ice. Cell sus-
pension was centrifuged at 15,000 g for 30 s at 4 ꢂC and the nuclear
C
32H29NO4: C, 78.19; H, 5.95; N, 2.85. Found: C, 78.21; H, 5.93; N,
2.90.
4.2.16. Compound 10h00
pellet was resuspended into 100 mL of ice cold saline buffer (50 mM
25
Crystalline yellow solid; mp: 153e155 ꢂC; [
a]
ꢀ23 (c 0.5,
D
HEPES/KOH pH 7.9, 50 mM KCL, 300 mM NaCl, 1 mM EDTA, 1 mM
NaF, 1 mM Na3OV4, 10% glycerol, 1 mM DTT, 0.5 mM PMSF, 1% (v/v)
eukaryotic protease inhibitor cocktail) on ice for 30 min. The sus-
pension was centrifuged at 15,000 g for 5 min at 4 ꢂC and the su-
pernatant was collected as nuclear fraction.
CHCl3); IR (KBr, cmꢀ1): 694.02, 756.60, 831.26, 1038.96, 1104.96,
1159.33, 1245.20, 1491.37, 1597.43, 1636.36, 1663.37, 1788.27,
2927.74, 3368.49; 1H NMR (400 MHz, CDCl3):
d 7.90 (t, 2H,
J ¼ 7.2 Hz), 7.85 (d, 2H, J ¼ 8.4 Hz), 7.30 (q, 3H, J ¼ 7.2 Hz), 7.15 (d,1H,
J ¼ 6.8 Hz), 7.00e6.96 (m, 5H), 6.75 (d, 1H, J ¼ 9.6 Hz), 6.30 (d, 1H,
J ¼ 9.6 Hz), 5.40 (d, 1H, J ¼ 9.6 Hz), 5.15 (t, 1H, J ¼ 8.4 Hz), 3.05e3.15
(m, 1H), 2.90e2.97 (dd, 1H, J ¼ 12.5, 4.4 Hz), 2.10 (s, 3H), 2.00e1.93
4.3.4. NF-
Effect of the test compound on the binding of NF-
consensus DNA sequence was checked by using NF-
kB (p65), transcription factor assay
k
B to its
(m, 1H), 1.90 (t, 2H, J ¼ 8.4, 5 Hz), 1.65e1.53 (m, 3H), 1.45 (s, 3H); 13
C
k
B (p65),
(100 MHz, CDCl3): 11.07, 15.45, 19.89, 22.31, 32.34, 35.46, 37.38,
40.98, 41.23, 46.67, 67.45, 75.90, 80.76, 104.64, 103.21, 107.54,
110.98, 113.32, 121.54, 127.65, 129.43, 149.11, 152.40, 152.87, 160.78,
173.76, 186.53; Mass QTOF: 514 (M þ Na)þ; Anal. Calcd for
transcription factor assay Kit from Cayman Chemical. Briefly, equal
quantity of nuclear protein was loaded into each well containing
immobilized DNA consensus sequence for NF-
overnight at 4 ꢂC. Wells were washed and incubated with primary
antibody against NF- B (p65) for 1 h. HP-conjugated secondary
kB and incubated for
C
32H29NO4: C, 78.19; H, 5.95; N, 2.85. Found: C, 78.21; H, 5.93; N,
k
2.90.
antibody was added for 1 h to the wells after washing. Wells were
incubated with the substrate, reaction was stopped after 30 min
and reading was taken at 450 nm.
4.3. Biological assays
4.3.1. Evaluation of in vitro anticancer activity
The effect of spiro-derivatives of
a
-santonin on the growth of
4.4. X-ray crystallography of spiro-isoxazolidine derivative of a-
cancer cell lines was evaluated according to the procedure adopted
by the National Cancer Institute for in vitro anticancer drug
screening that uses the protein-binding dye sulforhodamine B to
estimate cell growth [34]. Briefly, cells in their log phase of growth
were harvested, counted and seeded (104 cells/well in 100 mL
medium) in 96-well microtitre plates. After 24 h of incubation at
37 ꢂC and 5% CO2 to allow cell attachment, cultures were treated
santonin 10e0
A single crystal of spiro-isoxazolidine derivative 10e0 was
obtained by slow evaporation at room temperature, from a mixture
of methanol/water. The X-ray data was collected from a dry crystal
mounted on an ‘Xcalibur, Sapphire3’, Oxford diffractometer. The
crystal structure was solved by direct method using SHELXS-97
followed by full matrix anisotropic least square refinement using
SHELXL-97 [36]. All the proton atoms were located from difference
Fourier map except the methyl groups. For methyl group the proton
atom was fixed geometrically and refined in the final cycle as riding
over the heavy atom it is bonded. All the relevant crystallographic
data collection parameters and structure refinement details, bond
lengths and bond angles for 10e0 are given in Supplementary
information.
with varying concentrations (0.1e10 mM) of test samples made with
1:10 serial dilutions. Four replicate wells were set up for each
experimental condition. Test samples were left in contact with the
cells for 48 h under same conditions. Thereafter cells were fixed
with 50% chilled TCA and kept at 4 ꢂC for 1 h, washed and air-dried.
Cells were stained with sulforhodamine B dye. The adsorbed dye
was dissolved in tris buffer and the plates were gently shaken for
10 min on a mechanical shaker. The optical density (OD) was

J. Khazir et al. / European Journal of Medicinal Chemistry 63 (2013) 279e289
289
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Acknowledgements
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Authors DMR and GC are grateful to CSIR (India) for Senior
Research Fellowships.
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