Design and synthesis of spiro derivatives of parthenin as novel anti-cancer agents

Article abstract of DOI:10.1016/j.ejmech.2011.04.030

Several novel spiro derivatives of parthenin (1) have been synthesized by the dipolar cycloaddition using various dipoles viz; benzonitrile oxides, nitrones and azides with exocyclic double bond of C ring (α-methylene- γ-butyrolactone). Majority of the compounds exhibited improved anti-cancer activity compared to the parthenin, when screened for their in vitro cytotoxicity against three human cancer cell lines viz., SW-620, DU-145 and PC-3. In vivo screening of select analog revealed improved anti-cancer activity with low mammalian toxicity as compared to parthenin. The results of the cytotoxicity pattern of these derivatives reveals the SAR of these sesquiterpinoid lactones and possible role of α,β-unsaturated ketone of parthenin in inhibiting NF-kB. A mechanistic correlation of anti-cancer activity along with in vivo and western blotting experiments has been described.

Full text of DOI:10.1016/j.ejmech.2011.04.030

European Journal of Medicinal Chemistry 46 (2011) 3210e3217
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design and synthesis of spiro derivatives of parthenin as novel anti-cancer agents
Doma Mahendhar Reddy b, Naveed A. Qazi b, Sanghpal D. Sawant b, Abid H. Bandey b, Jada Srinivas a,
Mannepalli Shankar a, Shashank K. Singh b, Monika Verma b, Gousia Chashoo b, Arpita Saxena b,
,
c
Dilip Mondhe b, Ajit K. Saxena b, V.K. Sethi b, Subhash C. Taneja b, Gulam N. Qazi b
,
,b,
H.M. Sampath Kumar a
*
a Organic Chemistry-I, Indian Institute of Chemical Technology, Hyderabad 500007, India
b Synthetic and Biological Chemistry, Indian Institute of Integrative Medicine, Jammu 180001, India
c 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:
Several novel spiro derivatives of parthenin (1) have been synthesized by the dipolar cycloaddition using
various dipoles viz; benzonitrile oxides, nitrones and azides with exocyclic double bond of C ring
(a-methylene-g-butyrolactone). Majority of the compounds exhibited improved anti-cancer activity
compared to the parthenin, when screened for their in vitro cytotoxicity against three human cancer cell
lines viz., SW-620, DU-145 and PC-3. In vivo screening of select analog revealed improved anti-cancer
activity with low mammalian toxicity as compared to parthenin. The results of the cytotoxicity pattern of
Received 1 February 2011
Received in revised form
7 April 2011
Accepted 9 April 2011
Available online 29 April 2011
these derivatives reveals the SAR of these sesquiterpinoid lactones and possible role of a,b-unsaturated
ketone of parthenin in inhibiting NF-kB. A mechanistic correlation of anti-cancer activity along with
in vivo and western blotting experiments has been described.
Keywords:
Parthenin
Spiro derivatives
Anti-cancer
Ó 2011 Elsevier Masson SAS. All rights reserved.
NF-kB(P65) inhibitors
1. Introduction
presence of different numbers of alkylating structural elements.
Apart from this, other factors such as lipophilicity, molecular
In recent years, the anti-cancer property of various sesquiter-
penes has attracted a great deal of interest and extensive research
work has been carried out to characterize the anti-cancer activity,
the molecular mechanisms and the potential chemo-preventive
and chemo-therapeutic application of sesquiterpenoids [1]. Par-
thenin (1), a sesquiterpene lactone (SL) isolated from Parthenium
hysterophorus L [2], has found interest due to its medicinal prop-
erties viz., anti-cancer, antibacterial, antiamoebic, anti-
inflammatory, lipid peroxidation inhibition, and trypanocidal
activity [3e12]. Cytotoxicity, as many other biological activities of
sesquiterpene lactones, is known to be mediated by the presence of
geometry and the chemical environment or the target sulfhydryl
which may also influence the activity of these compounds.
Using helenalin and parthenolide as models, it has been well
established that DNA binding of NF-kB is prevented by alkylation of
cysteine in the p65/NF-kB subunit, which is considered to be the
general mechanism for SL bearing
a,b-unsaturated carbonyl
structures [19]. Despite the plethora of experimental studies found
in literature on the cytotoxicity of sesquiterpene lactones in general
including few reports on parthenin in particular, little is known
about the individual role of different alkylant structures on the
cytotoxicity in terms of its SAR. This however, would be an
important step in the direction of rational lead optimization.
Parthenin (1) has two active sites; the exocyclic double bond of
potential alkylant structure elements such as an
a-methylene-g-
lactone, an -unsaturated cyclopentenone or a conjugated ester
a,b
capable of reacting covalently with biological nucleophiles, espe-
cially with the cysteine sulfhydryl group of proteins, in a Michael-
type addition (Fig. 1), thereby inhibiting a variety of cellular
functions which directs the cells into apoptosis [13e19].The
differences in activity among individual SLs are attributed to the
a-methylene-g-butyrolactone and double bond of cyclopentenone
ring. Various structural modifications of parthenin skeleton have
been carried out with a view to obtain more potent analogs with
lower toxicity and better activity. In spite of the improved under-
standing of the role of each double bond present on the parthenin
skeleton through such studies, the SAR of parthenin has not been
unequivocally established so far and none of the literature reports
revealed a rational approach to the modification of parthenin.
Furthermore, monofunctional alkylants are known to possess low
* Corresponding author. Tel.: þ91 191 2569000; fax: þ91 191 2569333.
E-mail address: hmskumar@yahoo.com (H.M. Sampath Kumar).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.04.030

D.M. Reddy et al. / European Journal of Medicinal Chemistry 46 (2011) 3210e3217
3211
OH
O
OH
OH
Biological
SH
nucleophile
a
Ar
N
+ O
b
+
O
O
5
O
Ar
O
Biological
c
O
O
HS
N
O
nucleophile
O
2
O
1
Parthenin (1)
Ar
2a
2h
2,6-Cl-C6H3
2-NO2-C6H4
4-NO2-C6H4
2-Me-C6H4
3-Me-C6H4
4-Me-C6H4
4-F-C6H4
2o
2b
2c
2i
2j
4-MeO-C6H4
4-Cl-C6H4
2p
2q
Mono and bis adducts
3,4-OMe-C6H3
Fig. 1. Attack of biological nucleophiles on parthenin.
2k
2l
2d
2e
2f
2-Br-C6H4
-C6H5
3-OMe, 4-OH-C6H3 2r
2-F-C6H4
4-CN-C6H4
2s
degree of side effects such as toxicity or contact dermatitis and
generation of novel monofunctional analogs of parthenin would be
undoubtedly an attractive method for new lead discovery with
minimal toxicity. In this context, efforts from our group to generate
the diversity around endocyclic unsaturation of parthenin through
regioselective Baylis Hillmann adducts resulted in the derivatives
with considerably reduced cytotoxicity [20]. This clearly demon-
strated that tampering with the pharmacophoric cyclopentenone
ring structure either leads to low or complete loss of NF-kB binding
by the ligand and the observed low cytotoxicity in the derivatives
was found to be due to inhibition of telomerase-a case of complete
switch in the target preference possibly due to molecular over-
crowding of the newly created cyclopentane moiety bearing
exocyclic double bond [21].
4-NMe2-C6H4
4-HO-C6H4
2m 3-NO2-C6H4
2n 2-Cl-C6H4
9-Anthracene-
2t
2g
Scheme 2. Synthesis of spiroisoxazoline derivatives of parthenin via 1,3-dipolar
cycloaddition of nitrile oxides to the exocyclic double bond.
derivatives of parthenin thus generated have increased drug-
likeness as revealed by the QSAR parameters and in vivo studies
reveal reduced toxicity.
2. Results and discussion
To establish the role of exo- and endocyclic double bonds
toward the anti-cancer activity, a strategy to selectively react one
of these double bonds has been devised. Out of few chemical
transformational possibilities available to achieve the above goal,
selective addition of dipoles such as nitrile oxide, nitrone and
azide to exocyclic double bond (Scheme 1) has been choosen, with
an initial simple prediction that the newly incorporated nitrogen
framework on the scaffold might also enhance the cytotoxicity.
Although both endocyclic and the exocyclic double bonds are
active toward nucleophilic additions, dipolar cycloaddition takes
place selectively across the exocyclic olefin possibly due to steric
hindrance offered by the substitution at the cyclopentenone ring.
Herein, we report the dipolar cycloaddition of nitrile oxide,
nitrone and azide cycloaddition to the exocyclic double bond of
parthenin to generate a focused library of novel spiro-isoxazolines
(2), spiroisoxazolidine (3) and spiroaziridine (4) presented
(Scheme 2e4). After screening the derivatives for their anti-cancer
activity, we could easily establish the pharmacological importance
2.1. Chemistry
2.1.1. Synthesis of spiroisoxazoline derivatives of parthenin
As illustrated in Scheme 2, spiroisoxazoline derivatives of par-
thenin (2) were prepared through 1,3-dipolar cycloaddition of
OH
OH
O
N+
Ar'
+
Ph
O
O
Ar'
O
N
O
O
Ph
6
O
O
1
3
(mixture of diastereomers)
Ar'
3m
3a 4-Br-C6H4
3g
3h
3-Br, 4-MeO-C6H3
2-Me-C6H4
(94:6)
(95:5)
(92:8)
of cyclopentenone ring over the
a-methylene-g-butyrolactone
C6H5
(97:3)
3n
3o
2-Cl-C6H4
(93:7)
4-Cl-C6H4
3b
ring, which gives clear insight into the SAR of parthenin. The novel
(93:7)
4-F-C6H4
3i 2,6-Cl-C6H3
3c
2-F-C6H4
(92:8)
(94:6)
(65:35)
HO
HO
Aryl-nitrile oxide,
nitrone or azide
3j
3d 4-CN-C6H4
2,6-F-C6H3
3p 2,3,4,5,6-F-C6
(70:30)
(90:10)
(93:7)
O
O
O
R
O
3q
3e
3-Cl-C6H4
3-MeO-C6H4
4-MeO-C6H4
3k
O
O
(95:5)
(90:10)
(91:9)
4-Me-C6H4
3f
3-F-C6H4
4-NO2-C6H4
3l
3r
Ar
Ar'
Ar
O
N
O
N
N
=
(90:10)
R
(90:10)
(92:8)
Ar
Scheme 1. Synthesis of spiro derivatives of parthenin via 1,3-dipolar cycloaddition of
Scheme 3. Synthesis of spiroisoxazolidine derivatives of parthenin via 1,3-dipolar
nitrile oxides, nitrones and azides to the exocyclic double bond.
cycloaddition of nitrone to the exocyclic double bond.

3212
D.M. Reddy et al. / European Journal of Medicinal Chemistry 46 (2011) 3210e3217
Table 1
OH
OH
O
IC50 values of various derivatives of parthenin in
Compound Cell lines Compound Cell lines
SW-620 DU-145 PC-3 SW-620 DU-145 PC-3
mM concentration.
Ar''-N3
O
7
O
O
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
2q
2r
43
41
51
44
45.4
5.1
89
40
9
86
88
11
49
9
38
20
43
18
39.8
4.5
93
34
7
84
71
24
51
19
12
28
14
13
26
4.6
5.3
4.2
7
6
6.4
5
5
7
6.4
7.1
8.1
6.3
4.5
5.1
56
35
49
51
46
3h’
3h’’
3i’
3i’’
3j’
5.3
6
3
5.6
6.5
9
5.7
9
N
Ar''
O
2.4
4.2
2.5
2.3
8.2
12
4.8
9
5.7
8.1
3.5
6.3
2.7
2.5
5.6
5
12
9
8.4
9.3
10.6
86.1
12
52
61.5
10
O
5
6.3
4
5
12
1
6.3 3j’’
20
37
12
25
16
8.2 3m’’
55
12
10
7.2
6.1
10.5
4.8
5.1
6.8
9
5.5
9.2
9
9
7.4
6
3k’
3k’’
3l’
3l’’
3m’
Ar''
4f
5
8.1
6.3
5.4
6
4.1
4.1
6
4a
4b
C6H5
3-Cl-C6H4
2-NO2-C6H4
3-NO2-C6H4
3n’
3n’’
3o’
4g
4h
5
4c 4-MeO-C6H4
3-COOMe-C6H4
15
2.5
3.6
53
4.3
6.7
8.1
7.5
5.1
7.3
6
4.8
5
7.3
4.2
8.7
9
66.6 3o’’
10.2
3
14
27
32
4.9 3q’’
5.8 3r’
2.3 3r’’
7.8 4a
3p’
3p’’
3q’
9.3
6.1
5.5
7.5
6.6
5.7
5.1
87.7
48.4
30
2-MeO-C6H4
4d
4i
4j
4-Me-C6H4
2-Me-C6H4
2s
2t
10
11
5.6
4e
3-Me-C6H4
3a’
3a’’
3b’
3b’’
3c’
3c’’
3d’
3d’’
3e’
3e’’
3f’
3f’’
3g’
3g’’
6
31.4
39.5
78
12
5.7 4c
4b
Scheme 4. Synthesis of spiroaziridine derivatives of parthenin via 1,3-dipolar cyclo-
12
4d
4.8 4e
9.1 4f
5.7 4g
47.3
15
33
27
62.5
28
addition of azide to the exocyclic double bond.
61
30
10.1
10.1
25
12.5
40.3
various nitrile oxides (5) to parthenin [22e29]. The nitrile oxides
(5) were prepared according to literature procedure in which
various aromatic aldehydes converted to corresponding aldoximes
(syn and anti), followed by the reaction with N-chlorosuccinimide
in DMF. Nitrile oxide 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.
Spiroisoxazoline derivatives of parthenin were formed regiospe-
cifically in all cases.
6
4h
30
32
18.5
36
8.5 4i
9.1 4j
15
15
22
30
12
9
1
38.7
31.1
’ major diastereomer, ’’ minor diastereomer.
refluxing condition [32]. Azide 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.
2.1.2. Synthesis of spiroisoxazolidine derivatives of parthenin
As illustrated in Scheme 3, spiroisoxazolidine derivatives of
parthenin (3) were prepared through 1,3-dipolar cycloaddition of
various nitrones (6) to parthenin in dry benzene under reflux
condition [29e32]. Nitrones (6) were prepared according to liter-
ature procedure in which nitrobenzene was reduced in the pres-
ence of Zn/NH4Cl to get phenylhydroxylamine 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 may be due to the
molecular complexicity of parthenin, two diastereomers were
formed. Each diastereomer was isolated after column chroma-
tography or sometimes by preparative TLC and characterized by 1H
NMR, 13C NMR and Mass spectrometry. A clear chemical shift
deviation of benzylic proton adjacent to nitrogen atom in iso-
xazolidine ring between two diastereomers was observed in 1H
NMR. In major isomer this proton appears as a triplet approxi-
All the products synthesized were characterized by IR, 1H, 13C
NMR and mass spectroscopy data.
mately at
d
5, but in the minor isomer this signal shifted toward
4. A series
more shielding region and appears approximately at
d
of nitrones were reacted with parthenin and both diastereomers
of spiroisoxazolidines were isolated in each case.
2.1.3. Synthesis of spiroaziridine derivatives of parthenin
Fig. 2. DNA fragmentation assay of compound 2t in SW-620 cells. Lane-1: Untreated
As illustrated in Scheme 4, spiroaziridine derivatives of par-
thenin (4) were prepared through 1,3-dipolar cycloaddition of
various aromatic azides (7) to parthenin in dry toulene under
Cells, lane-2: treated with 5
compound 2t, lane-4: treated with 50
100 M of compound 2t.
m
M of Camptothecin, lane-3: treated with 10
mM of
m
M of compound 2t, lane-5: treated with
m

D.M. Reddy et al. / European Journal of Medicinal Chemistry 46 (2011) 3210e3217
3213
Fig. 3. Flow cytometric analysis of compound 2t treated with SW-620 cells.
2.2. Evaluation of biological activity
the determination of sub-G1 cell population by propidium iodide
(PI) staining. The DNA cell cycle analysis of compound 2t revealed
a concentration dependent increase in the sub G1 phase of cell cycle
2.2.1. In vitro anti-cancer activity
All derivatives of parthenin were screened for their anti-cancer
activity against three human cancer cell lines (SW-620, DU-145
and PC-3). Most of the spiro derivatives of parthenin exhibited
better anti-cancer activity than the parent parthenin. The results
are summarized in Table 1. In spiroisoxazoline (2) series
compounds 2f, 2o, 2p, 2q, 2r and 2t exhibited better cytotoxicity
than parent molecule. In spiroisoxazolidine (3) series all the
compounds showed better cytotoxicity than parent parthenin
against all the cell lines. In spiroaziridine (4) series compound 4a,
4c, 4f, 4e, 4g, 4h and 4j exhibited better cytotoxicity than parent
parthenin. From the IC50 values it is clear that all the spiro
derivatives of parthenin have anti-cancer activity and most of the
compounds are having better anti-cancer activity than the parent
parthenin.
being 6.58, 33.09, 33.2 and 55% at 1, 5, 10 and 50
mM respectively
and also complete blockage of G1 phase at 50 M concentration
m
after 24 h of incubation in SW-620 cells was observed (Fig. 3).
2.2.4. In vivo study of compound 2t
Solid tumor bearing mice treated with different doses of par-
thenin and its derivative compound 2t exhibited dose dependent
tumor growth inhibition against EAT tumor model. A highly
significant (p < 0.01) tumor growth inhibition up to 35.11% was
observed in EAT bearing mice treated with compound 2t at 100 mg/
kg i.p. dose whereas 200 mg/kg i.p. dose induced mortality of all the
test animals by fourth day of the treatment, on the contrary a high
level of toxicity without significant antitumor activity was recorded
in parthenin treated groups as it caused mortality of all the animals
by second and third day of treatment at 25 and 50 mg/kg i.p. doses
respectively (Table 2).
2.2.2. DNA fragmentation assay
DNA fragmentation is the net result of apoptosis, and is
observed at the late stage. DNA fragmentation assay showed that
compound 2t induced DNA fragmentation in SW-620 cells at 50
In case of Ehrlich ascetic carcinoma (EAC) bearing mice same
pattern of dose dependent tumor growth inhibition and toxicity
was exhibited by both parthenin and its derivative Compound 2t. At
100 mg/kg, i.p. dose of 2t exhibited highly significant (p < 0.01)
tumor growth inhibition up to 60% and its higher dose 200 mg/kg
i.p. induced mortality of all the test animals by fourth day of the
treatment. In this experiment also the parent compound parthenin
induced mortality of all the test animals by 2nd and 3rd day of the
treatment at 25 and 50 mg/kg i.p. doses respectively exhibiting high
level of toxicity to the treated animals without any significant
antitumor activity (Table 3). This clearly indicate the reduced
and 100
taken as a positive control showed significant fragmentation after
6 h at 5 M concentration, however, no fragmentation was
mM concentrations after 24 h of incubation. Camptothecin
m
observed in untreated cells (Fig. 2).
2.2.3. Cell cycle analysis
To address the cell death caused by compound 2t, the extent of
apoptotic death was assessed using FACS flow cytometry through

3214
D.M. Reddy et al. / European Journal of Medicinal Chemistry 46 (2011) 3210e3217
Table 2
Effect of parthenin and its compound 2t on Ehrlich ascitic tumor (EAT) bearing mice.
Test group
Dose (mg/kg)
Tumor weight (mg)
% Tumor growth
Inhibition
Fig. 4. Inhibition of NF-kB-P65 by spiro derivative of parthenin.
Control
1
1
1
2t
2t
2t
2t
2t
5-FU
0.2 mL
10
25
50
10
25
50
100
200
20
1708.57 ꢃ 51.07
e
1542.86 ꢃ 35.24
9.69
All animals died by 4th day
All animals died by 2nd day
1547.14 ꢃ 36.63
3. Conclusion
9.44
15.38
22.03
35.11
1445.71 ꢃ 41.52
The present set of data clearly reveals that spiro derivatives of
parthenin presented here induce concentration dependent
apoptosis in cancer cells. Spiroheterocyclic derivatives of parthe-
nin exhibited improved cytotoxicity as indicated by the in vitro
cell based assay whereas in vivo data of select derivative clearly
indicate reduced mammalian toxicity when tested in mice
models. Isoxazolidine series shown superior cytotoxicity as
compared to other two series viz. spiroisoxazoline and spiro
aziridines. Select spiro derivatives of parthenin completely
inhibited the NFkB-P65 expression confirmed that these deriva-
tives block p65 subunit of NFkB. SAR of parthenin could be
established with clarity as it has been established through the
1332.14 ꢃ 32.59*
1108.57 ꢃ 8.02**
All animals died by 3rd day
832.28 ꢃ 35.21**
51.28
p > 0.05 ¼ Insignificant, * ¼ p < 0.05 ¼ Significant, ** ¼ p < 0.01 ¼ Highly significant.
toxicity and improved anti-cancer activity of the spiro derivative of
parthenin.
2.2.5. Western blot analysis of the inhibition of NF-kB-P65
expression by compound 2t
NFkB-P65 expression is completely inhibited by compound 2t. It
can be presumed that expression is inhibited either on transcrip-
tional level or translational level.
NF-kB is a central mediator of the human immune response. In
the majority of cell types this protein is composed of a p50 and
a p65 subunit. It is retained in an inactive cytoplasmic complex by
binding to I
tory conditions, such as bacterial and viral infections as well as
inflammatory cytokines, rapidly induce NF- B activity.
Active NF-kB is released from the cytoplasmic complex by
phosphorylation, ubiquitination and degradation of the
present study that conservation of
of parthenin is crucial for retaining the anti-cancer activity of the
ligand whereas the modification of the -methylene- -butyr-
a,b-unsaturated ketonic moiety
a
g
olactone would facilitate better protein modulation thus enabling
improved activity and bioavailability through fine tuning of
hydrophilic lipophilic balance. Furthermore, the exocyclic double
bond can be advantageously utilized to incorporate appropriate
structural entities that may enhance the cytotoxicity of the parent
molecule.
kB, its inhibitory subunit. A large variety of inflamma-
k
IkB
4. Experimental protocols
subunit. The activated factor then translocates to the nucleus where
it stimulates the transcription of its target genes. NF-kB regulates
the transcription of various inflammatory cytokines, such as IL-1, IL-
Melting points were recorded on Buchi Melting point apparatus
D-545 and IR spectra (KBr) on Bruker Vector 22 instrument. NMR
spectra were recorded on Bruker DPX500 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 ESI-esquire 3000 Bruker Daltonics instrument. The
progress of all reactions was monitored by TLC on 2 ꢀ 5 cm pre-
coated silica gel 60 F254 plates of thickness 0.25 mm (Merck).
The chromatograms were visualized under UV 254e366 nm and
iodine.
2, IL-6, IL-8 and TNF-a as well as genes encoding cyclo-oxygenase-
II, nitric oxide synthase, immunoreceptors, cell adhesion molecules,
hematopoietic growth factors and growth factor receptors. Phar-
macological inhibition of NF-kB in vivo may thus substantially
attenuate inflammatory processes. The inhibition of NF-kB-P-65 by
compound 2t is shown in Fig. 4.
Western blot analysis of NFkB-p65 in compound 2t treated SW-
620 cells showed decreased expression of protein with the treat-
ment time. It was observed that compound 2t caused early inhi-
bition of translocation of protein in to the nucleus starting from 6h
treatment that was completely inhibited with prolonged exposure.
Interestingly, expression of the protein was also decreased in
cytosolic fraction, thereby indicating that compound 2t doesn’t
only inhibit the translocation of protein from cytosol to nucleus but
may also regulate it at transcriptional levels.
4.1. Synthesis
4.1.1. Synthesis of spiroisoxazolines derivatives of parthenin
In a typical procedure, to a solution of anthracene nitrile oxide
(0.577 g, 1.2 mmol) in THF (10 mL) stirred over a period of 10 min,
maintaining the temperature between 0e5 ꢁC, added parthenin
(0.262 g, 1 mmol) and stirred the reaction mixture at same
temperature for 15 min followed by stirring at ambient tempera-
ture for 2 h. The solvent was evaporated in vaccuo and the crude
was subjected for flash chromatography. The pure product (2t) was
characterized on the basis of 1H NMR, 13C NMR and mass
spectrometry.
Table 3
Effect of Parthenin and its derivative 2t on Ehrlich ascitic carcinoma (EAC) bearing
mice.
Test group Dose (mg/kg) Astic fluid
Tumor cells in
%Tumor growth
volume (mL) ascetic fluid (ꢀ107) inhibition
Control
1
1
1
2t
2t
2t
2t
2t
0.2 mL
10
25
50
10
25
50
100
200
4.57 ꢃ 0.39
85.00 ꢃ 4.75
e
3.92 ꢃ 0.38
74.71 ꢃ 2.77
12.01
4.1.1.1. Compound 2t. Colorless solid, m.p: 162 ꢁC; [
CHCl3); 1H NMR (CDCl3):
a
]
25 ꢂ27 (c 0.6,
D
All animals died by 4th day
All animals died by 2nd day
d
8.54 (s, 1H), 8.15 (d, J ¼ 8 Hz, 2H), 8.02 (d,
J ¼ 8 Hz, 2H), 7.61e7.36 (m, 5H), 6.20 (d, J ¼ 5.7 Hz, 1H), 5.36 (d,
J ¼ 5.5 Hz,1H), 3.80 & 3.77 (s, together integrating to diastereomeric
1H), 3.52 & 3.48 (s, together integrating to diastereomeric 1H),
3.44e3.41 (dd, J ¼ 7.22, 5.51 Hz, 1H), 2.43e2.37 (p, J ¼ 6.74, 1H),
2.35e2.32 (dd, J ¼ 7.04, 6.05, 1H), 1.98e1.9 (q, J ¼ 13.68, 1H),
1.8e1.76 (dd, J ¼ 8.2, 5.51, 1H), 1.54e1.50 (dd, J ¼ 8.71, 5.89, 1H), 1.39
4.07 ꢃ 0.35
3.62 ꢃ 0.32
2.85 ꢃ 0.37
78.42 ꢃ 2.90
68.71 ꢃ 3.16
61.85 ꢃ 4.20*
7.74
19.16
27.23
60.50
2.07 ꢃ 0.33** 33.57 ꢃ 2.60**
All animals died by 3rd day
p > 0.05 ¼ Insignificant, * ¼ p < 0.05 ¼ Significant, ** ¼ p < 0.01 ¼ Highly significant.

D.M. Reddy et al. / European Journal of Medicinal Chemistry 46 (2011) 3210e3217
3215
(s, 3H), 1.15 (d, J ¼ 7.7 Hz, 3H); 13C NMR (CDCl3):
d
212.0, 168.6,
1H), 3.03e2.97 (td, J ¼ 6.54 Hz, 1H), 2.72 (s, 1H), 2.63 (s,
1H), 2.21e2.15 (p, J ¼ 7.11, 1H), 2.12e2.08 (dd, J ¼ 7.86, 6.12, 1H),
164.3, 163.6, 154.2, 132.3, 131.5, 130.1, 130.4, 129.6, 128.4, 127.5,
126.9, 124.0, 90.1, 86.8, 83.4, 78.1, 71.2, 60.2, 49.2, 41.2, 40.4, 31.0,
22.1, 20.4, 18.3; IR (KBr): 3442, 2926, 2870, 1774, 1722, 1626,
1593 cmꢂ1; ESI MS (m/z): 482 (M þ H)þ; Anal. calcd. for C30H27NO5:
C, 74.83; H, 5.65; N, 2.91; Found. C, 74.80; H, 5.60; N, 2.89.
2.03e1.96 (q,
J
¼
13.68, 1H), 1.69e1.65 (dd,
J
¼
8.1, 5.48,
1.33 (s, 3H), 1.12 (d,
d 210.51, 162.23, 144.51, 130.5,
1H), 1.52e1.48 (dd, J ¼ 8.63, 5.24, 1H),
d
J ¼ 7.6 Hz, 3H); 13C NMR (CDCl3):
124.03, 119.1, 116.1, 84.6, 77.82, 60.21, 49.1, 41.12, 35.27, 30.54, 22.48,
20.54, 17.06; IR (KBr): 3439, 2961, 2925, 1779, 1751, 1721, 1590,
1562 cmꢂ1; ESI MS: 376 (M þ Na)þ. Anal. Calcd for C21H23NO4: C,
71.37; H, 6.56; N, 3.96. Found: C, 71.30; H, 6.61; N, 4.0.
4.1.2. Synthesis of spiroisoxazolidine derivative of parthenin
In a typical procedure, to a solution of parthenin (0.5g,1.9 mmol)
in dry benzene (6 mL), was added appropriate nitrone (1.58g,
2.4 mmol) in dry benzene and refluxed the reaction mixture for 8 h.
The solvent was evaporated in vaccuo and the crude was subjected
for flash chromatography. The pure products (3a’ and 3a’’) was
characterized on the basis of 1H NMR, 13C NMR, I.R. and mass
spectrometry.
4.2. Evaluation of in vitro anti-cancer activity
The effect of spiro derivatives of parthenin on the growth of
cancer cell lines was evaluated according to the procedure adopted
by the National Cancer Institute for in vitro anti-cancer drug
screening that uses the protein-binding dye sulforhodamine B to
estimate cell growth [33]. 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 ꢁC and 5% CO2 to allow cell attachment, cultures were treated
with varying concentrations (0.1e100 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 ꢁC 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
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 re-
ported in terms of IC50 values.
4.1.2.1. Compound 3a’. Pale yellow color solid; mp: 185 ꢁC;
25
[
a]
ꢂ12 (c 0.9, CHCl3); 1H NMR (500 MHz, CDCl3):
d 7.52e7.48
D
(m, 3H), 7.39 (d, J ¼ 8.41 Hz, 2H), 7.21-7-17 (t, J ¼ 7.98 Hz, 2H),
6.94e6.92 (t, J ¼ 7.32 Hz, 1H), 6.86 (d, J ¼ 8.20 Hz, 2H), 6,26 (d,
J ¼ 5.86 Hz, 1H), 5.26 (d, J ¼ 5.59 Hz, 1H), 5.16e5.13 (t, J ¼ 8.01 Hz,
1H), 3.21e3.17 (dd, J ¼ 6.51, 5.6 Hz, IH), 2.94e2.90 (dd, J ¼ 6.9,
5.6 Hz, 1H), 2.5e2.45 (dd, J ¼ 9.24, 3.26 Hz, 1H), 2.37e2.31
(p,J ¼ 7.35 Hz, 1H), 2.24e2.17 (td, J ¼ 6.37 Hz, 1H), 1.95e1.86 (q,
J ¼ 13.62 Hz,1H),1.69e1.65 (dd, J ¼ 5.67, 8.49 Hz,1H),1.47e1.43 (dd,
J ¼ 9.44, 5.01 Hz, 1H), 1.34 (s, 3H), 1.09 (d, J ¼ 7.79 Hz, 3H); 13C NMR
(CDCl3): 211.46, 174.42, 164.06, 151.11, 140.03, 132.1, 131.57, 128.84,
128.5, 122.48, 121.65, 114.83, 86.54, 84.19, 79.64, 69.97, 58.99, 49.45,
42.73, 39.9, 31.44, 21.74, 20.12 17.97; IR (KBr): 3451.68, 2962.07,
2925.92, 1774.56, 1754.00, 1722.69, 1596.79, 1488.03, 1011.38,
800.35, 754.89, 713.22 cmꢂ1; ESI MS (m/z): 539 (M þ H)þ; Anal.
Calcd for C28H28BrNO5: C, 62.46; H, 5.24; N, 2.60. Found: C, 62.40;
H, 5.27; N, 2.63.
4.1.2.2. Compound 3a’’. Pale yellow colour solid; mp: 179 ꢁC;
25
[
a]
ꢂ5.5 (c 0.75, CHCl3); 1H NMR (500 MHz, CDCl3): 7.51e7.48
D
(m, 5H), 7.19e7.16 (t, J ¼ 7.55 Hz, 2H), 6.98e6.95 (t, J ¼ 7.38 Hz, 1H),
6.89 (d, J ¼ 7.74 Hz, 2H), 6.21 (d, J ¼ 5.86 Hz, 1H), 5.2 (d, J ¼ 5.48 Hz,
1H), 4.33e4.3 (t, J ¼ 8.21 Hz, 1H), 3.13e3.1 (dd, J ¼ 5.47, 6.2 Hz, 1H),
2.92e2.88 (dd, J ¼ 9.17, 3.95 Hz, 1H), 2.69e2.65 (dd, J ¼ 7.96,
5.06 Hz, 1H), 2.38e2.32 (p, J ¼ 7.35 Hz, 1H), 2.25e2.18 (td,
J ¼ 6.30 Hz, 1H), 1.94e1.86 (q, J ¼ 13.51 Hz, 1H), 1.75e1.71 (dd,
J ¼ 5.67, 8.42 Hz, 1H), 1.58e1.54 (dd, J ¼ 9.55, 4.55 Hz, 1H), 1.37 (s,
3H), 1.1 (d, J ¼ 7.77 Hz, 3H); 13C NMR (CDCl3): 211.21, 174.55,
164.001, 152.01, 140.33, 132.12, 131.42, 128.64, 128.45, 122.46,
121.66, 114.63, 86.51, 84.78, 79.75, 70.09, 59.12, 49.54, 42.72, 40.21,
31.68, 21.91, 20.06 18.05; IR (KBr): 3452.09, 2962.32, 2925.54,
1774.66, 1754.20, 1722.69, 1596.52, 1488.0, 1012.11, 800.91, 754.9,
715.23.cmꢂ1; ESI MS (m/z): 561 (M þ Na)þ; Anal. Calcd for
C28H28BrNO5: C, 62.46; H, 5.24; N, 2.60. Found: C, 62.41; H, 5.27; N,
2.63.
4.3. DNA gel electrophoresis
DNA fragmentation was determined by electrophoresis of
extracted genomic DNA from human colon cancer cell line. Cells
(2 ꢀ 106/6 mL medium/60 mm tissue culture plate) were treated
with Compound 2t at 10, 50 and 100
mM for 24 h. Cells were
harvested, washed with PBS, pellets were dissolved in lysis buffer
(10 mM EDTA, 50 mM tris pH 8.0, 0.5% w/v SDS and proteinase K
(0.5 mg/mL) and incubated at 50 ꢁC for 1 h). Finally the DNA ob-
tained was heated rapidly to 70 ꢁC, supplemented with loading
dye and immediately resolved on to 1.5% agarose gel at 50 V for
2e3 h.
4.4. DNA cell cycle analysis
4.1.3. Synthesis of spiroaziridine derivatives of parthenin
In a typical procedure, to a solution of parthenin (0.05 g,
0.19 mmol) in dry toluene (3 mL), was added phenyl azide (0.045 g,
0.4 mmol) and refluxed the reaction mixture for 15 h. The solvent
was evaporated in vaccuo and the crude product was subjected for
flash chromatography to afford pure product. The pure product (4a)
was characterized on the basis of 1H NMR, 13C NMR, DEPT and mass
spectrometry.
Effect of Compound 2t on DNA content by cell cycle phase
distribution was assessed using SW-620 cells by incubating the
cells 1 ꢀ 106 mL/well with Compound 2t (1, 5, 10 & 50
mM each)
for 24 h. The cells were then washed twice with ice-cold PBS,
harvested, fixed with ice-cold PBS in 70% ethanol and stored
at ꢂ20 ꢁC for 30 min. After fixation, these cells were incubated
with RNase A (0.1 mg/mL) at 37 ꢁC 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 discrimina-
tion 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.
4.1.3.1. Compound 4a. White colour solid; m.p: 245e247 ꢁC;
25
[
a]
ꢂ1 (c 0.75, CHCl3); 1H NMR (500 MHz, CDCl3):
d 7.48 (d,
D
J ¼ 5.36, 1H,), 7.15e7.12 (t, J ¼ 7.81 Hz, 2H,), 7.0e6.95 (t, J ¼ 7.72, 1H),
6.75 (d, J ¼ 7.66 Hz, 2H), 6.18 (d, J ¼ 5.3 Hz, 1H), 5.21 (d, J ¼ 5.66 Hz,

3216
D.M. Reddy et al. / European Journal of Medicinal Chemistry 46 (2011) 3210e3217
4.5. In vivo anti-cancer activity
acacia in their respective group intra-peritonealy for nine consec-
utive days. Another test group was administered 5-FU @ 20 mg/kg
i.p and served as positive control. The tumor control group was
similarly administered normal saline (0.2 mL i.p). The percent
tumor growth inhibition was measured on day 12 with respect to
volume of ascitic fluid and the number of tumor cells in the ascitic
fluid of peritoneal cavity [35].
4.5.1. Animal care and housing
Non-inbred Swiss albino mice from an in-house colony were
used in the present study. The breeding and experimental animals
were housed in standard size polycarbonate cages providing
internationally recommended space for each animal. Animals were
fed balanced mice feed supplied by M/s Ashirwad Industries,
Chandigarh (India) and autoclaved water was available ad libitum.
Animals were cared as per the guide for the care and use of labo-
ratory animals (1996), ILAR, Washington DC. They were housed in
controlled conditions of temperature (23 ꢃ 2 ꢁC), humidity
(50e60%) and 12:12 h of light: dark cycle. The study and the
number of animals used were approved by the institutional animal
ethics committee, IIIM-Jammu, India. The study was conducted as
per the protocols of National Cancer Institute (NCI), USA [34].
The percent tumor growth inhibition was calculated as follow.
%Growth inhibitor ¼ 100ꢀðAverageno:of cellsincontrolgroup
ꢂAverageno:of cellsintestgroupÞ
=Average no:of cellsincontrol group
4.5.4. Preparation of cytosolic and nuclear extracts for the
evaluation of NF-kB by western blotting
SW-620 cells (5 ꢀ 106) were treated with 2t (5
mM) for indicated
time periods. Cells were washed with ice-cold phosphate-buffered
saline and centrifuged. Cytosolic and nuclear lysates were prepared
4.5.2. Anti tumor activity of parthenin and its derivative compound
2t on Ehrlich ascites tumor (EAT)
Parthenin and its derivative 2t were evaluated against solid
Ehrlich Ascites Tumor (EAT) models at different doses. A standard
procedure for experiment was as followed: Animals of the same sex
weighing 20 ꢃ 3 g were injected 1 ꢀ 107 cells collected from the
peritoneal cavity of non-inbred swiss mice, bearing 8e10 days old
tumor cells, into the right thigh, intramuscularly (on Day 1). On next
day animals were randomized and divided into test groups (7
animals in each test group) and one tumor control group (10
animals). Test groups were treated with different doses of Parthenin
(10 mg/kg, 25 mg/kg and 50 mg/kg) and its derivative Compound 2t
(10 mg/kg, 25 mg/kg and 50 mg/kg, 100 mg/kg and 200 mg/kg)
suspension in 1% gum acacia in their respective group intra-peri-
tonealy for nine consecutive days. Another test group was admin-
istered5-FU @20 mg/kg i.p and servedas positivecontrol. The tumor
control group was similarly administered normal saline (0.2 mL i.p).
The percent tumor growth inhibition was measured on day 13 with
respect to their tumor weight. Shortest and longest diameters of the
tumor mass were measured with the help of Vernier caliper and
tumor weight (mg) was calculated by using following formula:
as described earlier [35]. Cell pellets were homogenized in 200 ml of
buffer A (10 mM Hepes, pH 7.9, 1 mM EDTA, 1 mM EGTA, 100 mM
KCl, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride,
5 mM NaF, 1 mM NaVO4 and 10% protease cocktail inhibitor). The
tubes were placed in ice for 10 min. Nonidet P-40 was added (0.5%,
v/v), tubes vortexed briefly and centrifuged at 8000 ꢀ g for 15 min.
The cytosolic supernatants were stored at ꢂ80 ꢁC. The pellets ob-
tained were resuspended in 50 mL of buffer A supplemented with
20% glycerol, 0.4 M KCl, kept on ice for 30 min and centrifuged at
13,000 ꢀ g for 15 min. The supernatants were stored at ꢂ80 ꢁC for
analysis of nuclear NF-kB and AIF. All steps of fractionation were
carried out at 4 ꢁC. Western blot analysis for NF-kB-p65 was per-
formed by as described by Malik et al., 2007.
Acknowledgment
DMR, JS, MS, GC thanks CSIR-New Delhi for the award of
fellowship.
Tumor weightðmgÞ ¼ LengthðmmÞ ꢀ WidthðmmÞ2=2
Appendix. Supplementary data
The average tumor weight for each group was calculated and the
percent tumor growth inhibition in treated groups was calculated
as follows:
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.ejmech.2011.04.030.
%Tumor Growth Inhibitor ¼ 100
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ꢀðAverage tumor weight of control group
ꢂAverage tumor weight of test groupÞ
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