Synthesis and cytotoxicity evaluation of regioisomeric substituted N-phenyl-3′-(chrom-4-one-3-yl)-isoxazolidines: Induction of apoptosis through a mitochondrial-dependent pathway

Article abstract of DOI:10.1039/c3md00055a

Regio- and stereoselective 1,3-dipolar cycloadditions of C-(chrom-4-one-3-yl)-N-phenylnitrones (5) with different mono-substituted, disubstituted and cyclic dipolarophiles were carried out to obtain substituted N-phenyl-3′-(chrom-4-one-3-yl)-isoxazolidines (7a-p, 10a-c and 12a-c). Some of the obtained isoxazolidines display significant cytotoxicity against several human cancer cell lines. The preliminary bioassay results revealed that compound 10c showed higher cytotoxicity than paclitaxel against the A549 cell line with an IC₅₀ value of 0.7 μM. Compound 10c induces apoptosis through a mitochondrial-dependent pathway in human promyelocytic leukemia (HL-60) cells as revealed by induced apoptotic bodies' formation, DNA ladder, increased Sub-G₀ DNA fraction and loss of mitochondrial membrane potential (ΔΨ_m) in HL-60 cells.

Full text of DOI:10.1039/c3md00055a

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CONCISE ARTICLE
Synthesis and cytotoxicity evaluation of regioisomeric
substituted N-phenyl-30-(chrom-4-one-3-yl)-
isoxazolidines: induction of apoptosis through a
mitochondrial-dependent pathway†
Cite this: Med. Chem. Commun., 2013,
4, 972
Gagandeep Singh,a Amritpal Kaur,a Vishal Sharma,a Nitasha Suri,b
b
b
a
*
Parduman R. Sharma, Ajit. K. Saxena and Mohan Paul S. Ishar
Regio- and stereoselective 1,3-dipolar cycloadditions of C-(chrom-4-one-3-yl)-N-phenylnitrones (5) with
different mono-substituted, disubstituted and cyclic dipolarophiles were carried out to obtain
substituted N-phenyl-30-(chrom-4-one-3-yl)-isoxazolidines (7a–p, 10a–c and 12a–c). Some of the obtained
isoxazolidines display significant cytotoxicity against several human cancer cell lines. The preliminary
bioassay results revealed that compound 10c showed higher cytotoxicity than paclitaxel against the A549
cell line with an IC50 value of 0.7 mM. Compound 10c induces apoptosis through a mitochondrial-
dependent pathway in human promyelocytic leukemia (HL-60) cells as revealed by induced apoptotic
bodies' formation, DNA ladder, increased Sub-G0 DNA fraction and loss of mitochondrial membrane
potential (DJm) in HL-60 cells.
Received 18th February 2013
Accepted 18th April 2013
DOI: 10.1039/c3md00055a
www.rsc.org/medchemcomm
group of naturally occurring oxygen containing heterocyclic
compounds called avones.7 Because of their structural features
Introduction
Cancer cells are genetically unstable, which results in tumor
masses of heterogeneous cells and makes the cancer a “moving
target” for drug therapy.1 Despite the availability of improved
drugs, including targeted cancer therapies, cancer is still one of
the leading causes of mortality worldwide. A number of nucle-
oside analogues have been used clinically as anticancer agents
while new nucleoside analogues continue to show promise.2
These nucleoside analogues exert cytotoxic activity either by
altering the DNA and RNA biosynthesis or by interfering, as
inhibitors/modulators, with key cellular enzymes involved in
the synthesis of nucleic acids.3 Isoxazolidines, which are ve
membered heterocyclic analogs of the furanose ring, have
attracted attention as these possess signicant cytotoxic
activity.4 Some anticancer isoxazolidines include isoxazolidinyl
polycyclic aromatic hydrocarbons (1), having remarkable
apoptosis enhancing activity caused by anti-fas antibody addi-
tion.5 Earlier we have reported some isoxazolidines (2a–c) which
display signicant cytotoxic activity.6
and diverse biological activities attributed to avones, they
occupy an important place in the realm of natural products,
medicinal and synthetic organic chemistry.8 Benzopyrone based
anticancer agents (Fig. 1) include cytotoxic chromanyl-dithia-
zoles (3)9 and chloro/uoro substituted chromones (4)10 as
potential topoisomerase II inhibitor, cytotoxic agents.
Keeping in view, the reported signicant anticancer activity
of the isoxazolidines and chromone derivatives, it was decided
to re-synthesize earlier reported chromanyl-isoxazolidines 7a–i
by regio- and stereoselective 1,3-dipolar cycloadditions,11 along
with some new derivatives (7j–p, 10a–c and 12a–c) and evaluate
their cytotoxic activity against various human cancer cell lines
and probe the plausible mode of action.
On the other hand, the chromone moiety, a privileged
structure, represents the basic skeleton of the most abundant
aBio-Organic and Photochemistry Laboratory, Department of Pharmaceutical Sciences,
Guru Nanak Dev University, Amritsar-143 005, Punjab, India. E-mail: mpsishar@
yahoo.com; Fax: +91-183-2258820; Tel: +91-183-2258802 ext. 3321
bPharmacology Division, Indian Institute of Integrative Medicine, Jammu 180 001,
J & K, India
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c3md00055a
Fig. 1 Some cytotoxic isoxazolidine and chromone derivatives.
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Results and discussion
Chemistry
C-(Chrom-4-one-3-yl)-N-phenylnitrones (5) were reacted with
monosubstituted (6), disubstituted (9) and cyclic (11) dipolar-
ophiles by stirring in dry dichloromethane (DCM) at room
temperatureleadingto the synthesisof isoxazolidines (7a–p, 10a–
c, 12a–c, Scheme 1). All the products were separated and puried
by column chromatography, and characterized by spectroscopic
analysis i.e. 1H and 13C NMR, IR and mass spectroscopy.
The assigned regio- and stereochemistry in cycloadducts
(7a–p, 10a–c and 12a–c) is based on rigorous analysis of the
spectroscopic data. The 13C NMR resonance of methylene
carbon (C40) in 7n appeared at d 41.5, clearly indicating that it is
not attached to any hetero-atom. The cis relationship between
the chromone moiety at C30 and the substituent at C50 in 7a–p is
based on 1H NMR couplings involving C30-, C40- and C50-Hs and
it follows from the consistent observation that the cis vicinal 1H
coupling is always higher (5–9 Hz) than the trans (0–6 Hz) in the
case of isoxazolidines.12 For instance, analysis of vicinal
coupling constants extracted from the spectra of compound 7n
Fig. 2 The preferred conformation of cis isoxazolidine (7n).
In this conformation, the chromone and ethoxy groups
occupy the equatorial and pseudo-equatorial positions respec-
tively, thus the cis relationship of the substituents at C30-H and
C50-H in 7n was unambiguously proved. In view of the preferred
Z-form of phenylnitrone (5) under the reaction conditions, the
cis-cycloadducts can be described as derived from an exo-mode
of addition, whereas the trans-adducts can be described as
derived from an endo-approach (Fig. 3).
In compounds 10a–c, the hydrogen, which is cis to C50-
0
`
methyl (C4 -Ha), appears upeld vis-a-vis the proton cis to
methoxycarbonyl function and its trans relationship with C30-H
0
0
is indicated by its lower vicinal coupling constant value J3 ,4 b
¼
0
0
6.6 Hz as compared to J3 ,4 a ¼ 7.5 Hz as observed in the case of
10c. Mechanistically, the compounds 12a–c can be described as
the endo-cycloadducts and 13a–c as the corresponding exo-
cycloadducts derived from the addition of phenylnitrone in the
0
0
0
0
0
0
[ J3 ,4 a ¼ 8.5 Hz, J3 ,4 b ¼ 1.5 Hz, J5 ,4 a ¼ 5.7 and the lack of any
observable vicinal coupling between C50-H and C40-Hb] shows
that the isoxazolidine ring exists in the preferred E3 confor-
mation (Fig. 2).
Scheme 1 Synthesis of chromanyl-isoxazolidines.
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Biological activity evaluation
Initial screening for in vitro cytotoxicity. All the synthesized
compounds (7a–p, 10a–c and 12a–c) were evaluated according
to the protocol of Skehan et al.13 at different concentrations of 1,
10, 30, 50 and 100 mM for determining IC50 values (mM); against
lung (A-549), leukemia (THP 1), prostate (PC-3) and colon
(Caco2) cancer cell lines using paclitaxel, mitomycin-C and 5-
uorouracil as positive controls and cytotoxic results are
summarized in Table 1. All the synthesized chromanyl-iso-
xazolidines exhibit cytotoxic activities with observable varia-
tions due to different substitutions at C50 and C6 and were
found to be highly active against lung (A-549) and leukemia
Fig. 3 Regioselective addition of dipolarophiles to N-phenylnitrone (5).
Z-form.11 The production of endo-adduct (12a–c) as the major (THP 1) cancer cells and moderately active against prostate (PC-
product from the addition of phenylnitrone (5) to N-phenyl- 3) and colon (Caco2) cancer cells.
maleimide (11) represents a very interesting and clear case of
The obtained results revealed that some of the compounds
very high endo-selectivity because of steric factors (Fig. 3). The possess excellent antiproliferative activity against selected
major compound 12a displayed a 1H singlet at d 5.85 which was human cancer cell lines. Among them, two representative
attributed to C30-H and the lack of any observable vicinal disubstituted and bicyclic isoxazolidines, compounds 10c and
coupling signied its trans-relationship with C40-H; such a lack 12b, displayed much higher antitumor activity against lung
of any observable coupling between trans-vicinal hydrogens in (A549) and colon (Caco2) cancer cell lines than positive
the case of isoxazolidines, particularly, in the case of rigid control paclitaxel and 5-uorouracil, respectively. In partic-
systems derived from the addition of nitrones to cyclic dipo- ular, compound 10c exhibited
a broad spectrum anti-
larophiles has precedents. Both C40-H and C50-H were present proliferative activity against A549, THP1 and Caco2 with
1
as H doublets at d 4.08 and d 4.80, respectively, displaying a IC50 values of 0.7, 7.3 and 19.7 mM, respectively. In the
mutual splitting of 7.5 Hz.
case of monosubstituted isoxazolidines, compounds bearing
Table 1 In vitro cytotoxicity of synthesized compounds 7a–p, 10a–c and 12a–c against various human cancer cell lines
Nitrone (5)
Cytotoxicitya (IC50 mM)
Lung
A549
Leukemia
Prostate
PC3
Colon
Compound no.
X1
X2
Dipolarophile
THP1
Caco-2
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
H
CH3
H
CH3
H
Cl
H
Cl
CH3
F
Br
Cl
F
Br
Cl
H
F
Br
CH3
F
Br
CH3
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
Cl
H
H
H
H
H
H
H
6; R ¼ –Ph
R ¼ –Ph
R ¼ –OEt
R ¼ –OEt
R ¼ –CO2Me
R ¼ –CO2Me
R ¼ –CONH2
R ¼ –CN
R ¼ –OiBu
R ¼ –Ph
R ¼ –Ph
R ¼ –Ph
R ¼ –OEt
R ¼ –OEt
R ¼ –OEt
R ¼ –Pyridyl
9
>60
49
>60
>60
>60
41
>60
>60
>60
35
>60
>60
>60
39
>60
>60
>60
41
>60
31.7
>60
31
>60
>60
>60
39
>60
20.7
>60
>60
>60
40
17.1
51.5
51
21.5
54.7
60
37.7
49
60
>60
60
>60
23.5
>60
>60
>60
37
29.5
>60
19.7
>60
20.3
>60
—
43
38
32
26.4
21.5
19.8
19.3
20.3
9.9
0.7
13.5
17
>60
>60
16.5
16.4
47
14.7
7.3
43.5
22.3
>60
1.5
31.7
>60
>60
46
31.3
>60
>60
>60
13.3
11.1
1.5
—
10a
10b
10c
12a
12b
12c
Mitomycin-C
Paclitaxel
5-Fluorouracil
9
9
11
11
11
>60
0.4
2.7
—
—
—
21
—
—
a
IC50 mM: concentration required to inhibit cancer cell proliferation by 50% aer exposure of cells to test compounds.
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non-polar hydrophobic groups at C50 show good cytotoxic
activity. For instance compounds such as 7d and 7i bearing
ethoxy and isobutoxy groups at C50 display a signicant cyto-
toxic activity with IC50 values of 20.7 and 17.1 mM, respectively,
against the A549 cancer cell line. Similarly, compounds 7l and
7o bearing phenyl and ethoxy groups at C50 and the chloro
group at C6 showed good antiproliferative activity with IC50
values of 43 and 19.8 mM, respectively, against the lung (A549)
cell line. The cytotoxicity difference in compounds 7l and 7o is
attributed to the difference in the hydrophobic character of
the phenyl/ethoxy group at C50. Compound 7o also displays
signicant cytotoxicity against leukemia (THP1) cancer cell
lines with an IC50 value of 16.5 mM and interestingly,
compound 7l shows a broad spectrum antiproliferative activity
against THP1, PC3 and Caco2 cancer cell lines with IC50 values
of 38, 32 and 23.5 mM, respectively. Compound 7p bearing the
pyridyl group at C50 displays signicant cytotoxicity against
lung (A549) and leukemia (THP1) cancer cell lines with IC50
values of 19.3 and 16.4 mM, respectively. Furthermore, in the
case of disubstituted and bicyclic isoxazolidines, compound
10b showed good antiproliferative activity against A549 and
THP1 cancer cell lines with IC50 ¼ 9.9 and 14.7 mM. The
difference in the cytotoxicity of compounds 10c and 10b is
attributed to the presence of methyl and bromo groups at C6.
Fig. 4 Effect of compound 10c on the nuclear morphology of HL-60 cells as
revealed by DAPI fluorescence staining.
However, among bicyclic isoxazolidines, compound 12a positive control and the cells were treated for 6 h. The
showed good cytotoxic inhibition against the lung (A549) untreated cells (A) have large sized intact nuclei. Compound
cancer cell line (IC50 ¼ 13.5 mM) and compound 12c displays 10c induced the condensation and fragmentation of nuclei
cytotoxicity with an IC50 value of 11.1 mM against the prostate (arrow) due to apoptosis (C–E). Higher numbers of apoptotic
(PC-3) cancer cell line.
cells were seen in treatment with compound 10c at 50 and
Although in most of the cases, the exact mechanism of 100 mM (Fig. 4D and E). The morphological analysis of HL-60
cytotoxic activity is not known for chromone derivatives and cells treated with compound 10c displayed the morphological
isoxazolidines, various modes of action of chromone derivatives events characteristic of apoptosis as revealed by uorescence
have been reported, which include the inhibition of DNA repair microscopic studies.
enzyme DNA-dependent protein kinase (DNA-PK) causing
DNA fragmentation test. One of the most commonly used
apoptosis of concerned cells;14 the chromone derivatives exhibit techniques for conrmation of apoptosis is DNA fragmenta-
binding activity for the surface pocket of the Bcl-2 protein and tion ascertained through identication of DNA ladders. To
induce apoptosis of tumor cells.15 Furthermore, the iso- elucidate whether compound 10c decreases cell survival by the
xazolidine derivatives exhibit cytotoxic and apoptotic properties induction of DNA fragmentation, genomic DNA was isolated
and have been shown to bind to calf-thymus DNA and two from cells exposed to different concentrations of compound
synthetic double-stranded polynucleotides, poly-d(AT)2 and 10c from HL-60 cells and electrophoresed. The compound 10c
poly-d(GC)2.16 Compound 10c showed signicant anti- treatment of HL-60 cancer cells indicated internucleosomal
proliferative activity against all the four human cancer cell lines DNA breakdown, leading to DNA fragmentation as expected for
of different tissue origins and it was therefore selected for apoptotic cells (Fig. 5). The minimal concentration inducing
mechanistic studies to probe the plausible mechanism of its DNA fragmentation was evident at 50 mM and the investigation
action on cancerous cells. To ascertain the mechanism of cell conrmed that compound 10c induces DNA laddering, a
death by compound 10c, the human promyelocytic leukemia hallmark of apoptosis.
cell line (HL-60) was used.
Compound 10c increases the Sub-G0 DNA fraction of cell
Effect of compound 10c on the nuclear morphology of HL-60 cycle phase distribution. The development of cytouorimetric
cells. Apoptosis is a major form of cell death, characterized by approaches to monitor these multiple cell alterations permits
a series of stereotypic morphological changes such as forma- a precise and reliable quantication of apoptosis. Apoptotic
tion of apoptotic bodies, chromatin condensation, shrinkage cells exhibit some morphological modications that are
of cells and bleb formation, internucleosomal DNA fragmen- readily detected by ow cytometry according to their light
tation and Sub-G0 DNA accumulation.17 Morphological anal- scattering properties (FSC/SCC).18 To further determine and
ysis of 10c treated promyelocytic leukemia (HL-60) cells was conrm whether or not the growth inhibitory activity of
carried out with an inverted uorescence microscope, to gain compound 10c was related to induction of apoptosis, the
insight into morphological alterations caused by the test subdiploid (character of apoptosis) fraction was detected by
compound (Fig. 4). Camptothecin (1 mM) was used as a ow cytometry. The presence of the Sub-G0 peak in the
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histogram of DNA distribution indicated that a population of
cells was dying by apoptosis (Fig. 6).
Compound 10c induces the loss of mitochondrial
membrane potential. The loss of mitochondrial membrane
potential (DJm) is largely because of the opening of mito-
chondrial permeability transition pores (PTP), which conduit
the leakage of cytochrome c and proapoptotic proteins from
mitochondria to the cytosol.19 Mitochondria are known to
modulate and synchronize Ca2+ signaling. Massive accumula-
tion of Ca2+ in the mitochondria leads to apoptosis. The Ca2+
dynamics of mitochondria appears to be modulated by the Bcl-
2 family proteins, a key factor involved in apoptosis. The
number and morphology of mitochondria are precisely
controlled through mitochondrial fusion and ssion processes
by numerous mitochondria-shaping proteins. Mitochondrial
ssion accompanies apoptotic cell death and appears to be
important for progression of the apoptotic pathway.20 Mito-
chondrial function was indirectly assessed by variation in
mitochondrial trans-membrane potential using Rh-123, a cell
permeable cationic dye, which preferentially partitions into
mitochondria because of the highly negative mitochondrial
membrane potential (DJm).21,22 Depolarization of DJm results
in the loss of Rh-123 from the mitochondria and a decrease in
intracellular uorescence. Compound 10c caused concentra-
tion-dependent mitochondrial damage and loss of mitochon-
drial membrane potential in HL-60 cells that showed the role
of PTP in the loss of DJm. There was a signicant decrease of
DJm from 7% in control; 53.3% in positive control (Campto-
thecin 1 mM) and 7.9%, 36.6% to 53.9%, in cells treated
with 10, 30, to 50 mM of compound 10c respectively, aer
24 hours (Fig. 7). These observations proved that compound
10c primarily induces mitochondrial dependent cell death
Fig. 5 Compound 10c induced DNA fragmentation in the HL-60 cells. Frag-
mentation of genomic DNA was studied in HL-60 cells exposed to the indicated
concentrations of compound 10c for 24 hours. Genomic DNA was isolated and
electrophoresed. Lane 1, control; lane 2, Camptothecin 1 mM (positive control);
lane 3, comp. 10c 10 mM; lane 4, comp. 10c 30 mM; lane 5, comp. 10c 50 mM; lane
6, comp. 10c 100 mM. A prominent DNA ladder was observed at 50 mM treatment.
Data are representative of 1 of 3 similar experiments.
Fig. 6 Cell cycle analysis of compound 10c-treated HL-60 cells; briefly, 1 ꢀ 106 cells were exposed to different doses (10, 30, 50 and 100 mM) of comp. 10c for 24 hours.
Followingincubation,thecellswereharvested, treatedwithRNase,stainedwithpropidiumiodide(25mgmLꢁ1),andanalyzedusingaBD-FACSCaliberflow-cytometer. Sub-G0
population indicative of DNA damage was analyzed from the hypo diploid fraction (<2n DNA) of DNA cell cycle analysis. Data are representative of 1 of 3 similar experiments.
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Fig. 7 Analysis of compound 10c-induced alterations in mitochondrial membrane potential (DJm); exponentially growing cells were treated with the indicated
concentration of compound 10c for 24 hours. For the assessment of loss in DJm, trypsinized cells were incubated with fluorophore rhodamine-123 for 30 minutes at 37
ꢂC in the dark. The unbound dye was removed by washing the cells with chilled phosphate-buffered saline and analyzed by flow cytometry. Graphs show the
percentages of rhodamine-123 negative cells.
which is mediated by mitochondrial outer membrane poten-
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Conclusions
In conclusion, we have synthesized a library of chromanyl-iso-
xazolidines by 1,3-dipolar cycloaddition reaction and biological
evaluation of obtained compounds indicated that these mole-
cules possess signicant cytotoxicity against a number of
human cancer cell lines. Compound 10c showed higher cyto-
toxicity than paclitaxel against the A549 cell line with an IC50
value of 0.7 mM and compound 12b also exhibited a broad
spectrum antitumor activity against A549, THP1, PC3 and Caco2
with IC50 values of 17, 22.3, 13.3 and 20.3 mM, respectively.
Furthermore, in vitro biological evaluation revealed that
compound 10c induces apoptotic bodies' formation, DNA
fragmentation, increased Sub-G0 DNA fraction and loss of
mitochondrial membrane potential (DJm) in HL-60 cells. These
observations have established that compound 10c induced
apoptosis through the mitochondrial-dependent pathway in
HL-60 cells.
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