Synthesis and biological evaluation of novel Δ2-isoxazoline fused cyclopentane derivatives as potential antimicrobial and anticancer agents

Article abstract of DOI:10.1039/c4md00525b

As a part of our endeavour toward the synthesis of new heterocyclic bioactive agents, three series of Δ²-isoxazoline fused cyclopentane derivatives (27 compounds) were synthesized and characterized by IR, ¹H NMR, ¹³C NMR and MS analysis. The newly synthesized target compounds were evaluated for their preliminary in vitro antimicrobial and anticancer activities. The results indicated that compounds 4b, 4h, 4i, 5d and 5g displayed remarkable anti-microbial activity with respect to their standard drugs Ampicillin, Gentamycin and Amphotericin B. In preliminary MTT cytotoxicity studies, compound 4i was found to be equipotent to the standard drug Etoposide against MCF-7. The influence of the most active cytotoxic compound 4i on the cell cycle distribution was evaluated in the MCF-7 cell line, which displayed a cell cycle arrest at the S phase. Moreover, acridine orange/ethidium bromide staining, annexin V binding assay and mitochondrial membrane potential revealed that compound 4i can induce cell apoptosis in MCF-7 cells. Compounds 4b and 4h are potential leads as antimicrobials owing to no significant cell toxicity observed in the present study. Docking studies revealed that compound 4i binds to Phe1145, Glh698, Met696, Cys1191, Met1169 and Ile1167 on DNA methyltransferase (DNMT1) protein and inhibition of DNMT1 could be the possible mechanism of action for these compounds.

Full text of DOI:10.1039/c4md00525b

View Article Online
View Journal
MedChemComm
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: N. B. Bathini, S.
K. Prajapti, S. Shrivastava, U. Bihade, A. K. Gupta, V.G. M. Naidu and U. C. Banerjee, Med. Chem.
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
Accepted Manuscripts are published online shortly after
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the Ethical guidelines still
apply. In no event shall the Royal Society of Chemistry be held
responsible for any errors or omissions in this Accepted Manuscript
or any consequences arising from the use of any information it
contains.
www.rsc.org/medchemcomm

Page 1 of 7
Medicinal Chemistry Communications
View Article Online
DOI: 10.1039/C4MD00525B
MedChemComm
RSCPublishing
ARTICLE
Synthesis and biological evaluation of novel ꢀ2ꢁisoxazoline
fused cyclopentane derivatives as potential antimicrobial
and anticancer agents
Cite this: DOI: 10.1039/x0xx00000x
Santosh Kumar Prajaptia, Shweta Shrivastavab, Umesh Bihadec, Ajay Kumar
Guptaa, V.G.M. Naidub, Uttam Chand Banerjeec, Bathini Nagendra Babua,*
Received 00th January 2012,
Accepted 00th January 2012
DOI: 10.1039/x0xx00000x
As a part of our endeavour toward the synthesis of new heterocyclic bioactive agents, three series of
ꢀ2ꢁisoxazoline fused cyclopentane derivatives (27 compounds) were synthesized and characterized
by IR, 1H NMR, 13C NMR and MS analysis. The newly synthesized target compounds were
evaluated for their preliminary in vitro antimicrobial and anticancer activities. The results indicated
www.rsc.org/
that the compounds 4b, 4h, 4i, 5d and 5g displayed remarkable antiꢁmicrobial activity with respect
to their standard drugs Ampicillin, Gentamycin and Amphotericin B. In preliminary MTT
cytotoxicity studies, compound 4i was found to be equipotent to the standard drug Etoposide against
MCFꢁ7. The influence of the most active cytotoxic compound 4i, on the cell cycle distribution was
evaluated in the MCFꢁ7 cell line, which displayed a cell cycle arrest at S phase. Moreover, Acridine
orange/ethidium bromide staining, annexin V binding assay and mitochondrial membrane potential
revealed that the compound 4i can induce cell apoptosis in MCFꢁ7 cells. Compounds 4b and 4h are
potential leads as antimicrobials owing to no significant cell toxicity observed in present study.
Docking studies revealed that compound 4i binds to Phe1145, Glh698, Met696, Cys1191, Met1169
and Ile1167 on DNA methyltransferase (DNMT1) protein and inhibition of DNMT1 could be the
possible mechanism of action for these compounds.
as antibiotics.14,15 In addition, isoxazolines are versatile masked
structural entities and allow easy access to amino alcohols,16,17
Introduction
The microbial resistance to the currently available antimicrobial
drugs is the major concern area in the treatment of microbial
infections. These resistant strains curtail the life span of the
drugs.1 On the other hand, cancer is one of the leading causes of
death worldwide. It accounted for 8.2 million deaths (around
17% of all deaths) in 2012 and with an estimated 14 million
deaths in 2030.2 The current scenario highlights the need for the
discovery and development of new chemical entity with novel
mode of actions.3 The identification of novel molecule that can
be more effective and reliable is still a major challenge for
medicinal chemistry researchers.
hydroxy ketones18,19 and isoxazolidines,20,21 which have been
played a crucial role in natural product synthesis and several
pharmacologically active heterocyclic compounds.22 On the
other hand, synthesis of cyclopentane derivatives has attracted
tremendous interest for decades among the researchers due to
their potential applications in medicinal chemistry as
therapeutic agents. The functionalized cyclopentane derivatives
are known to posses various biological activities such as
anticancer (by inhibiting Akt phosphorylation23 and steroid
metabolizing
enzymes
AKR1C1
and
AKR1C324),
antimicrobial,25 antimalarial,25 antiviral,26 human neurokininꢁ1
(hNK1) inhibiotors27 and so on. Also, it is revealed from the
literature survey that the isoxazoline fused heterocyclic hybrids
as well as different fused nitrogen containing heterocycles,
showed a broad range of therapeutic activity (Fig. 1).28ꢁ32
Despite several reports on fused heterocycles, there is
continuing demand for development of new methods for the
synthesis of novel fused heterocycles due to their plethora of
medicinal applications. Thus, based on aforementioned results
and taking into account of these structural aspects of
In recent years, there has been increased attention towards the
synthesis of isoxazole/isoxazoline derivatives, since they
displayed a significant role in organic and medicinal chemistry.
Organic compounds containing isoxazole/isoxazoline motif as a
core unit are known to exhibit a wide range of biological
activities such as antibacterial,4,5 antifungal,6 anticancer,7
anticonvulsant,8
antiꢁinflammatory,9
antiviral,10,11
antidepressant,12 and antithrombic activity.13 Isoxazole moiety
has been found in various drugs such as cloxacillin,
dicloxacillin, flucloxacillin which are currently in clinical use
This journal is © The Royal Society of Chemistry 2013
J. Name., 2013, 00, 1-3 | 1

Medicinal Chemistry Communications
Page 2 of 7
View Article Online
ARTICLE
Journal Name
DOI: 10.1039/C4MD00525B
ecyclopentane and isoxazolines, we anticipate that the hybrid analogs, exo adducts were subjected to oxidative opening of
emanating from the incorporation of isoxazoline moiety to C=C bond. For that purpose, we have utilized two different path
cyclopentane scaffold might exhibit clinically useful for the oxidative cleavage of C=C bond of cycloadducts 3a
antimicrobial and anticancer activity. In this communication, obtain target compounds 4a 5a , and 6a . PathꢁA involves
we describe the synthesis, antimicrobial and anticancer activity cis-dihyroxylation of cycloadducts exoꢁ3a in presence of
of novel ꢀ2ꢁisoxazoline fused cyclopentane derivatives 4a
catalytic amount of osmium tetraoxide and
5a and 6a . Among these, the most effective cytotoxic methylmorpholine
ꢁoxide36 followed by oxidative opening
compound 4i was also evaluated for acridine orange/ethidium using sodium periodate37 and then reduction38 of respective diꢁ
bromide staining, annexin V binding assay, mitochondrial aldehydes to afford series of diol products of fused
. IR bands ranging from 3250 to 3400 cmꢁ1
–i to
–
i,
–
i
–i
–
i
–
i,
Nꢁ
–
i
–
i
N
membrane potential, and cell cycle analysis.
isoxazolines 4a–i
indicated the presence of hydroxyl group in the synthesized
compounds. The appearance of multiplet signal around δ 3.5–
4.0 ppm depicted the presence of CH2 protons attached to fused
bicyclic system of 4a
C=C bond in cycloadducts exoꢁ3a
–
i, confirmed the oxidative opening of
. In 13C NMR, peak from δ
–
i
155–162 ppm indicated the presence C=N of dihydroisoxazole.
PathꢁB involves potassium permanganate for direct opening of
C=C bond39 of exoꢁ3a
–
i
to get series of biꢁcarboxylic acid
substituted isoxazoline fused cyclopentane analogs 5a , which
–
i
on further treatment with catalytic amount of sulfuric acid in
methanol40 yielded methyl ester analog of isoxazoline fused
cyclopentane analogs 6a
–
i
in high yields. Disappearance of
olefin peak in H NMR confirmed the oxidative opening of
cycloadducts exoꢁ3a . Furthermore, the appearance of peak in
the range of δ 171−174 in 13C NMR confirmed the presence of
carbonyl carbon in 5a . IR bands in range of 2900–3120 and
1690–1720 cmꢁ1 indicated O–H and C=O stretching
respectively. Compounds 6a were confirmed by the
Fig. 1 Structure of some representative cyclopentane, isoxazole and fused
isoxazoline derivatives.
1
–
i
Result and discussion
–
i
Chemistry
All isoxazoline fused cyclopentane derivatives 4a
6a were synthesized as racemates by utilizing the [3+2]
cycloaddition of nitrile oxide to norbornadiene as depicted in
Scheme 1. The aldehydes (1a ) were first converted to
corresponding aldoximes 2a via reaction with
hydroxylamine in presence of sodium hydroxide.33 The
resulted aldoximes 2a were treated with sodium
–i, 5a–i, and
–
i
1
–
i
characteristic peak of –OCH3 at range of δ 3.6–3.8 in H NMR.
The IR spectra showed strong C=O absorption at 1720–1740
cmꢁ1. Furthermore, δ 172–173 in 13C NMR indicated the
–
i
(
–i)
presence of carbonyl carbon of ester in compounds 6a–i.
HO
O
(
–i)
Path-A (iii) (iv) (v)
N
O
hypochlorite to generate respective hydroimoyl chloride,
followed by dehydrohalogenation with the removal of HCl
using 4ꢁdimethylaminopyridine (DMAP) to produce nitrile
oxide, which underwent [3+2] cycloaddition with
norbornadiene to provide a racemate of exo and endo isomer of
cycloadduct in a ratio of about ~80:20 [7,22]. The mixture of
adducts were separated by silica gel column chromatography
using 1–5% of EtOAc:Hexane. The structure of cycloadducts
were confirmed by 1H NMR and as per the literature
precedents.34,35 In its spectrum, two double doublet (dd) peaks
in range of δ ~6.0–6.5 ppm correspond to olefin protons and the
chemical shift (δ) at ~5 ppm and ~4 ppm depicted the presence
N
OH
H
R
HO
O
N
O
(i)
(ii)
4(a-i)
R
R
H
R
OH
exo-3(a-i)
O
O
O
O
O
O
1(a-i)
2(a-i)
endo-3(a-i)
(vii)
O
(vi)
N
N
Path-B
(~80:20)
R
R
OH
5(a-i)
6(a-i)
R =
(a) 4ꢀ methoxyphenyl
(b) 3,4ꢀdimethoxy phenyl
(c) 4ꢀmethylphenyl
(d) 4ꢀfluorophenyl
(e) 4ꢀchlorophenyl
(f) 2ꢀchlorophenyl
(g) 4ꢀbromophenyl
(h) 4ꢀcyanophenyl
(i) 2ꢀnaphthalenyl
Scheme 1 Synthesis of ∆2ꢀisoxazoline fused cyclopentane derivatives;
Reagents and conditions: (i) NH2OH•HCl, NaOH, EtOHꢀH2O (1:1), rt, 4 hr;
(ii) Norbornadiene, NaOCl, DMAP, DCM, 0°C to rt, 12 h; (iii) OsO4, NMO,
acetoneꢀwater (7:3), rt, 24 h; (iv) NaIO4, DCMꢀ aq. NaHCO3 (15:1), 0 °C,
30 min; (v) NaBH4, MeOH, 0 °C, 2 h; (vi) KMnO4, acetoneꢀH2O (8:2), 0 °C,
3 h (vii) Cat. H2SO4, MeOH, 6 hr.
of protons at fused position of cycloadducts 3a
–
i
, which comes
= ~8
as doublet (d) having the same coupling constant value (
J
Hz) confirmed the stereochemistry of exo cycloadducts, while
double doublet (dd) peak at δ ~5 and δ ~4 with couping
constant (J = ~4 Hz, ~9 Hz) was observed in case of endo
Biological evaluation
cycloadduct. IR bands in the region of 1585–1620 cmꢁ1,
confirmed the presence of C=N bond in cycloadducts. In all
cases, exo adduct was found to be the major product and these
cycloadducts were utilized as key intermediate to carry out
further reactions. To obtain isoxazoline fused cyclopentane
In vitro antimicrobial activity. In vitro antimicrobial screening
of the newly synthesized compounds 4aꢁi, 5aꢁi and 6aꢁi were
examined against two Gramꢁpositive bacteria viz; Bacillus
subtilis and Bacillus megaterium, two Gramꢁnegative bacteria
viz; Escherichia coli and Pseudomonas Spp. and one fungal
2 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 2012

Page 3 of 7
Journal Name
Medicinal Chemistry Communications
View ArticAle ROnTliInCeLE
DOI: 10.1039/C4MD00525B
strain viz; Candida albicans. Cup plate diffusion method41,42 µg/mL) and E.coli (MIC 78 µg/mL), while 4ꢁchloro phenyl
was used for the determination of the preliminary antibacterial substituted compound 5e showed inhibitory activity only
and antifungal activities. Ampicillin, Gentamycin and against Gram positive bacterial strains, Bacillus subtilis (MIC
AmphotericinꢁB were used as reference drugs. The results 60 µg/mL), Bacillus megaterium (MIC 30 µg/mL). Diols with
illustrated in Table 1 revealed that most of the tested 2ꢁchloro phenyl 4f and 4ꢁbromo phenyl 4g substitution showed
compounds displayed variable inhibitory effects on the growth moderate activity against Gram positive bacterial strains.
of the tested Gram positive and Gram negative bacterial strains, However, the bicarboxylic ꢀ2ꢁisoxazolines bearing 4ꢁbromo
and also against antifungal strains. Particularly compounds 4h
phenyl 5g substitution displayed excellent activity against
Bacillus subtilis (MIC 10.54 µg/mL), Bacillus megaterium
(MIC 10.54 µg/mL), E. coli (MIC 2.2 µg/mL) along with good
inhibitory action against Pseudomonas Spp. (MIC 22 µg/mL)
Table 1: In vitro antimicrobial activity of compounds 4(a-i), 5(a-
i) and 6(a-i)
.
Minimum inhibitory concentration (µg/mL)a
and Candida albicans (MIC 8.78 µg/mL). Compound with 4ꢁ
Compound Gram positive
Gram negative
Fungi
cyano phenyl substitution 4h exhibited significant activity
against Gram positive and Gram negative bacterial strain as
compared to their reference drugs. The ꢀ2ꢁisoxazoline diol 4h
also showed good activity against Escherichia coli and Candida
albicans (MIC 11 µg/mL), whereas its carboxylic acid analog
5h lost their activity against all tested microbial strains. The
compound with naphthalenyl substitution 4i in ꢀ2ꢁisoxazoline
diol displayed equipotent activity to their reference drug
Ampicillin, specifically against Gram positive bacteria, while
activity was lost in case of its bicarboxylic acid analog 5i. On
the other hand, the antimicrobial activity was lost against all
tested microbial strains in methyl ester analogs of ꢀ2ꢁ
isoxazoline 6aꢁi. It is worth mentioning that the compound ꢀ2ꢁ
isoxazoline diol substituted with planar and bulky group
(naphthalenyl) 4i displayed selectivity towards Gram positive
bacterial strain. Similarly, the selectivity for Gram positive
bacterial strain was also observed in bicarboxylic analog of ꢀ2ꢁ
isoxazoline tethered electron withdrawing with bulky group (4ꢁ
chloro phenyl) 5e. Introduction of two methoxy groups
(electron donating group) on phenyl ring 4b in ꢀ2ꢁisoxazoline
diol enhanced its selectivity to inhibit Gram negative bacterial
and fungal growth, while introducing electron withdrawing
group (cyano) at para position of phenyl ring 4h showed broad
spectrum antimicrobial activity. Moreover, bicarboxylic analog
of ꢀ2ꢁisoxazoline substituted with bulky group 5g (4ꢁbromo
phenyl) showed activity against all tested microbial strains.
Bs
ꢁ
46.8
ꢁ
ꢁ
ꢁ
74.4
48.6
21
26
ꢁ
ꢁ
ꢁ
ꢁ
Bm
ꢁ
46.8
ꢁ
ꢁ
ꢁ
ꢁ
48.6
21
26
ꢁ
ꢁ
ꢁ
ꢁ
30
Ec
ꢁ
0.92
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
2.1
ꢁ
ꢁ
88.4
ꢁ
78
ꢁ
ꢁ
2.2
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
NT
Ps
ꢁ
1.2
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
2.1
ꢁ
ꢁ
44.2
ꢁ
7.8
ꢁ
ꢁ
22
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
NT
Ca
ꢁ
4.6
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
11
ꢁ
ꢁ
81.6
ꢁ
ꢁ
ꢁ
ꢁ
8.7
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
4a
4b
4c
4d
4e
4f
4g
4h
4i
5a
5b
5c
5d
5e
5f
5g
5h
5i
6a
6b
6c
6d
6e
6f
6g
6h
6i
60
ꢁ
10.5
ꢁ
10.5
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
NT
Ampicillin 25
25
Gentamyci
n
Amphoteri
cin B
NT
NT
NT
2
8
NT
NT
NT
NT
5
Bs: Bacillus subtilis, Bm: Bacillus megaterium, Ec: Escherichia coli
,
Ps: Pseudomonas Spp., Ca: Candida albicans
a(ꢁ): Inactive ( MIC > 100 µg/mL).
NT: Not tested.
.
In vitro anticancer activity. All newly synthesized compounds
were evaluated for their in vitro cytotoxic activity against five
cancer cell lines HTꢁ29 (colon), HeLa (cervix), MCFꢁ7 (breast),
A549 (lung) and PCꢁ3 (prostate) cancer cell lines by employing
MTT assay.43 Etoposide was taken as the reference in this
study. Concentration response course analysis was performed to
determine drug concentrations required to inhibit the growth of
cancer cells by 50% (IC50) after incubation for 48 h. The results
of in vitro anticancer activity revealed that, some of the
synthesized compounds exhibited different levels of anticancer
properties (Table 2). From the close analysis of the IC50 values,
and 5g have shown broad spectrum activity. It was observed
that, the diol series of ꢀ2ꢁisoxazoline substituted with 3,4
dimethoxy phenyl 4b showed superior activity against E. coli
(MIC 0.92 µg/mL), Pseudomonas Spp. (MIC 1.2 µg/mL) and
Candida albicans (MIC 4.6 µg/mL) as compared to their
reference drugs, while moderate inhibition was observed
against Gram positive bacterial strains. Replacing the diol
substitution with bicarboxylic acid 5b displayed decreased
activity against all tested organism. Substitution of ꢀ2ꢁ
.
it was observed that, ꢀ2ꢁisoxazolines, bearing
pꢁmethoxy
phenyl substituent of diol series 4a showed inhibitory activity
against MCFꢁ7 and Aꢁ549; however its bicarboxylic compound
5a lost cytotoxicity against all cancer cell line and its methyl
ester analog 6a displayed activity specifically against HTꢁ29.
ꢀ2ꢁIsoxazoline, substituted with 3,4 dimethoxy phenyl, only
isoxazoline diols with pꢁmethoxy phenyl 4a, tolyl 4c, 4ꢁfluoro
phenyl 4d and 4ꢁchloro phenyl 4e led to loss in antimicrobial
activity. However, the bicarboxylic analog of ꢀ2ꢁisoxazoline
substituted with 4ꢁfluoro phenyl 5d was active specifically
against Gram negative bacteria, Pseudomonas Spp. (MIC 7.8
This journal is © The Royal Society of Chemistry 2012
J. Name., 2012, 00, 1-3 | 3

Medicinal Chemistry Communications
Page 4 of 7
View Article Online
ARTICLE
DOI: 10.1039J/Co4uMrnDa00l 5N2a5Bme
bicarboxylic analog 5b showed moderate cytotoxicity against observations may promote a further development of ꢀ2ꢁIsoxazoline
HTꢁ29. In case of tolyl substitution, only 4c was shown to be and may lead to compounds with better pharmacological profile than
active against HeLa and PCꢁ3. Enhancement in anticancer the standard anticancer drugs.
activity was observed in ꢀ2ꢁisoxazoline diol against HTꢁ29 and Notably, the compounds 4b and 4h, which showed potent
Aꢁ549 when, fluoro group was introduced at para position of antimicrobial activity, were accompanied with no significant
phenyl ring 4d. Complete loss of activity was observed in case cytotoxicity against cancer as well as normal mammalian cells,
of its biꢁcarboxylic acid derivative 5d; however its ester analog which might be useful as potential lead for the development of novel
6d exhibited moderate activity against HeLa and MCFꢁ7.
antimicrobial agents. However, the compounds 4i and 5g showing
remarkable antimicrobial activity cannot be considered as good
antimicrobials because of their potential cytotoxicity.
Acridine Orange and Ethidium Bromide (AO/EB) staining. The
most active cytotoxic compound 4i was also investigated for the
inhibition of cell proliferation and cytotoxicity to understand
whether it is due to apoptotic induction or nonspecific necrosis,
morphological features were analyzed through Phaseꢁcontrast
microscopy and AO/EB staining.44 The morphological abnormalities
were studied under a phaseꢁcontrast microscope. Cells treated with 5
Table 2. In vitro anticancer activity of compounds 4a-i, 5a-i and 6a-i.
IC50 (µg/mL)a
Comp
HTꢁ29
HeLa
MCFꢁ7
A549
PCꢁ3
ꢁ
ꢁ
4a
4bc
4c
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
23.7±3.25
36.6±4.98
ꢁ
ꢁ
ꢁ
ꢁ
26.5±3.51
28.0±2.54
4d
4e
17.8±4.21
22.9±6.74
ꢁ
ꢁ
ꢁ
31.1±5.62
ꢁ
ꢁ
ꢁ
ꢁ
14.7±2.15
ꢁ
4f
33.8±7.84
ꢁ
ꢁ
ꢁ
4g
37.3±
ꢁ
14.3±5.32
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
4hc
4i
ꢁ
and 10 µg/ml of compound 4i for 48
h showed obvious
24.5±8.43
11.5±1.45
48.4±5.32
morphological changes, with chromatin condensation, fragmentation
and formation of apoptotic bodies. However, the control group
(without test compound) showed normal healthy shape with intact
nuclei and without any abnormalities (Fig. 2a). Most of the treated
cells in all the test concentrations exhibited similar symptoms of
apoptosis but the damage was severe in the cells exposed to highest
concentration of 10µg/ml (Fig. 2c). The results of light microscopy
were consistent with that of fluorescence microscopy using AO/EB
(Fig. 3a–c). The bright condensed chromatin identified by AO
staining was a clear indication of early apoptosis leading to
margination of chromatin into a horseshoe shaped structure. Cells
treated with high concentration 10 µg/ml of compound 4i exhibited
intense nuclear fragmentation followed by the formation of apoptotic
bodies.
5a
5b
5c
5d
5e
5f
5g
5h
5i
6a
6b
6c
6d
6e
6f
6g
6h
6i
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
40.6±7.12
ꢁ
ꢁ
ꢁ
ꢁ
19.0±4.35
28.5±1.87
30.0±6.74
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
45.6±8.32
33.3±5.63
30.3±2.54
29.7±4.87
28.9±3.65
ꢁ
ꢁ
ꢁ
ꢁ
31.8±2.21
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
20.1±2.56
39.4±4.38
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
27.4±3.48
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
Etopo
4.21±0.53
2.35±0.25
11.95±2.63
8.9±1.87
7.2±1.37
ꢁsideb
a(ꢁ): Nonꢁcytotoxic (IC50 > 50 µg/mL).
bEtoposide was used as reference drug.
cCompound tested against normal mammalian breast epithelium cell line
(MCFꢁ10A) [IC50 > 100 µg/mL]
Furthermore, compound substituted with chloro at para position 4e
showed good inhibitory activity against MCFꢁ7 along with HTꢁ29,
while bicarboxylic derivative 5e was active against HeLa, MCFꢁ7
and Aꢁ549 and loss of cytotoxicity was observed in case of its ester
analog 6e. Substitution of phenyl with chloro at ortho 4f and bromo
Fig. 2 Morphological changes in MCFꢀ7 cells treated with and without compound
4i for 48 h. Light microscopy: (a) Control, cells with intact nuclei; (b) Early signs
of apoptosis characterized at 5 ꢁg/ml by extremely condensed chromatin;(c) cell
membrane blebbing, nuclear fragmentation and chromatin condensation at 10
at para position 4g of ꢀ2ꢁisoxazoline diol displayed cytotoxicity ꢁg/ml.
specifically against HTꢁ29. Cytotoxicity was lost in ortho chloro
phenyl substituted bicarboxylic 5f and its methyl esters analog 6f
whereas, pꢁbromo substituted carboxylic and ester analog 5g, 6g
were shown to be active specifically against HeLa. In case of 4ꢁCN
phenyl substituted ꢀ2ꢁisoxazoline, only acid derivative 5h was found
to be active against HTꢁ29 and HeLa. Replacement of phenyl group
with the naphthalenyl ring, the fused isoxazoline diol 4i displayed
cytotoxic activitiy towards HTꢁ29, HeLa, MCFꢁ7 and PCꢁ3.
Fig. 3 Morphological changes in MCFꢀ7 cells treated with and without compound
However, its acid derivative 5i was active only against HTꢁ29 and 4i for 48 h. Fluorescence microscopy: (a) Green live cells show normal
morphology of control; (b) Early signs of apoptosis characterized at 5 ꢁg/ml by
extremely condensed chromatin, which marginated into a horseshoeꢀshaped
MCFꢁ7 and complete loss of activity was observed in its ester analog
6i. Significantly, compound 4i was found to be most potent having
comparable IC50 value (11.5 ± 1.45 µg/mL) with that of the reference
drug, Etoposide (IC50 11.95±2.63 µg/mL) in MCFꢁ7. These
structure and cell membrane blebbing, (c) Destructive fragmentation of the
nuclei at 10 ꢁg/ml and irregular distribution of chromatin, late apoptotic cells
exhibited condensed chromatin and their nuclei, stained red with EB.
4 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 2012

Page 5 of 7
Journal Name
Medicinal Chemistry Communications
View Article Online
ARTICLE
DOI: 10.1039/C4MD00525B
Annexin V binding assay. In the next series of experiment, the fluorescence by JCꢁ1 dye where healthy mitochondria gives out o
apoptotic activity of compound 4i on human breast cancer (MCFꢁ7) fluorescence because of aggregates of JCꢁ1 dyes and
J
cells was proved using the apoptosis assay based on the annexin dead/apoptotic cells show green fluorescence because of lack of
V/propidium iodide staining.45 MCFꢁ7 cells were treated with 5 and mitochondrial membrane potential (Fig. 6). Changes in the ratio
10 µg/ml of compound 4i for 48 h. As depicted in Fig. 4, the between the measurement at wavelengths of 590 nm (red) and 540
compound 4i increased the percentage of early apoptosis stage nm (green) fluorescence intensities were in agreement with
(6.00% to 24.94% and 48.32%, respectively) with the concentration cytotoxicity potency by inducing significant depolarization of
which indicated that compound 4i induced apoptosis of MCFꢁ7 in a mitochondrial membrane potential in a doseꢁdependent manner in
dose dependent manner (Fig. 4).
MCFꢁ7 cells.
Fig. 6 Determination of mitochondrial membrane potential through JCꢀ1 staining
and detection using fluorescent microscopy. Cells were exposed to 0, 5 and 10
ꢂg/ml of compound 4i for 12 h followed by JC‑1 dye incubation for the final 20
min. After treatment, the mitochondrial membrane potential was found to be
interrupted, as evidenced by the migration of JC‑1 dye from the mitochondria
into the cytoplasm of treated cells, and the subsequent reduction in the
mitochondrial red fluorescence signals.
Fig. 4 Annexin V binding assay by MuseTM Cell Analyzer after 48 h of treatment
with compound 4i in MCFꢀ7. The dual parameter dot plots combining annexin Vꢀ
fluorescein isothiocyanate (FITC) and PI fluorescence show the viable cell
population in the lower left quadrant (annexin VꢀPIꢀ), apoptotic cells in the lower
right quadrant (annexin V+ PIꢀ) and the upper right quadrant (annexin V+ PI+ ),
and necrotic cells in the upper left quadrant (annexin VꢀPI+).
Cell cycle analysis. To gain insight into the mechanisms through
which tested compounds reduced the number of adherent cells, flow
cytometric analysis46 was performed after incubation of MCFꢁ7 cells
with compound 4i at selected concentrations for 48 h. Separation of
cells in G0/G1, S and G2/M phase was based on fluorescence
intensity after staining with propidium iodide. Representative
profiles are shown in Fig. 5. In untreated cells, a predominant
number of MCFꢁ7 cells were accumulated in G0/G1 phase.
Compound 4i induced a marked reduction in the number of cells in
the G0/G1 phase and an accumulation of cells in the S phase of the
cell cycle. MCFꢁ7 cells were treated with 5 & 10 µg/ml of
compound 4i for 48 h, percentage of cell arrest in S phase increased
progressively from 12.7, 14.9, and 27.5% respectively (Fig. 5). At 48
h of exposure, the number of cells increased in the Sꢁphase by 2.16
fold, when compared to controls.
Molecular docking studies. Biological studies revealed that the
molecule induce cell death by arresting cell cycle at Sꢁphase and
mitochondrial apoptotic pathway. In order to investigate the possible
mechanism, molecular docking was performed for compound 4i into
the binding pocket of the cofactor of human DNA methyltransferase
(DNMT1). Coordinates of protein structure were obtained from the
Protein Data Bank (PDB ID: 3PTA).48 Geometry of the molecules
was optimized by using the optimized potentials for liquid
simulationsꢁ2005 (OPLSꢁ2005) force field. Compound 4i was
flexibly docked into the crystallographic structure of the
methyltransferase domain of human DNMT1 using Docking Glide
Extra Precision (XP), version 6.049 (Schrödinger, 2013). For the
purpose of docking protocol validation, Sꢁadenosylhomocysteine
(SAH) bound to the crystal structure was removed from the binding
pocket and docked back into the cofactor binding site. Docking
results showed a similar docking pose to the pose of coꢁcrystal
ligand with RMSD of 1.11 Å which is in the acceptable range to
reproduce the binding mode of SAH. As depicted in Figure 7, the
compound interacted with the SAH binding site formed from the
Glu1168, Glh1189, Val144, Ile1167, Asn1192, Asp1190, Cys1191,
Leu1247, Phe1145, Met1169, Trp1170, Glh698, Met696 and
Ala696. The πꢁπ stacking exhibited by adenine moiety with Phe1145
in SAH is compensated by naphthalene ring of compound 4i. The –
OH group of ꢀ2ꢁisoxazoline ring displayed hydrogen bonding
interaction with Glh698 and Met696. Beside this, docking pose of
compound 4i showed hydrophobic interaction with Met1169,
Ile1167, Val1144 and Cys1191. Docking studies of these
compounds on DNMT1 protein suggest that DNMT1 inhibition
could be the possible mechanism of action for these compounds. The
binding pose for molecule 4i in DNMT1 protein is shown in Fig. 7.
Fig. 5 Effect of compound 4i on cell cycle progression of MCFꢀ7 cells, (a)
Control cells; (b, c) Cells treated with 5 and 10 ꢁg/ml for 48 h followed by
analysis of cell cycle distribution using propidium iodide cell staining method.
Cell population in each cell cycle phase was numerically depicted. Data
represent one of three independent experiments.
Analysis of mitochondrial membrane potential (MMP). In
addition, the effect of compound 4i on mitochondrial membrane
potential loss in MCFꢁ7 by was investigated by JCꢁ1 staining
followed by fluorescent microscopy.47 Change in the mitochondrial
membrane potential was determined by red verses green
This journal is © The Royal Society of Chemistry 2012
J. Name., 2012, 00, 1-3 | 5

Medicinal Chemistry Communications
Page 6 of 7
View Article Online
ARTICLE
DOI: 10.1039J/Co4uMrnDa00l 5N2a5Bme
The following supplementary data are available: synthetic procedure and
characterization data of synthesized compounds; procedure for in vitro
biological activity of antimicrobial and anticancer.
1. A. L. Demain and S. Sanchez, Microbial drug discovery: 80 years of
progress, J. Antibiot., 2009, 62, 5.
2. C. A. Co and I. L. In, Facts & Figures 2014.
3. P. Heffeter, M. A. Jakupec, W. Orner, S. Wild, N. G. Keyserlingk, L.
Elbling, H. Zorbas, A. Korynevska, S. Knasmuller, H. Sutterluty, M.
Micksche, B. K. Keppler and W. Berger, Biochem. Pharmacol., 2006,
71, 426.
4. G. T. Zitouni, A. Ozdemir and K. Guven, Arch. Pharm. (Weinheim),
2005, 338, 96.
5. H. Agirbas and S. Gunner, Bioorg. Med. Chem., 2007, 15, 2322.
6. M. Babu, K. Pitchumani and P. Ramesh, Eur. J. Med. Chem., 2012, 47,
608.
7. A. Kamal, E. Vijaya Bharathi, J. Surendranadha Reddy, M. Janaki
Ramaiah, D. Dastagiri, M. Kashi Reddy, A. Viswanath, T.
Lakshminarayan Reddy, T. Basha Shaik, S. N. C. V. L. Pushpavalli and
M.P. Bhadra, Eur. J. Med. Chem., 2011, 46, 691.
8. Z. Ozdemir, H. B. Kandilici, B. Gumusel, U. Calis and A. A. Bilgin, Eur.
J. Med. Chem., 2007, 42, 373.
Fig.7 Top view of 3ꢀD image showing interactions of compound 4i with human
DNMT1 protein (PDB ID: 3PTA).
9. K. M. Amin, M. M. Kamel, M. M. Anwar, M. Khedr and Y. M. Syamb,
Eur. J. Med. Chem., 2010, 45, 2117.
Conclusion
In the present study, a series of ꢀ2ꢁisoxazoline fused cyclopentane
analogs were synthesized and evaluated for their in vitro
antimicrobial and anticancer potentials. From the results of the tested
compounds, compounds 4b, 4h, 4i, 5d and 5g displayed promising
antimicrobial activity with reference to standard drugs Ampicillin,
Gentamycin and Amphotericin B respectively. Interstingly, the
compounds 4b and 4h were shown to be nonꢁcytotoxic towards
cancer and normal cell lines, demonstrated the potential candidates
for antimicrobials. In preliminary MTT cytotoxicity studies,
compound 4i was found to be equipotent to the standard drug
Etoposide against MCFꢁ7 cancer cell line. The cell cycle analysis
revealed that the compound 4i arrested the MCFꢁ7 cell cycle at S
10. B. L. Deng, T. L. Hartman, R.W. Buckheit, C. Pannecouque, E. De
Clercq and M. Cushman, J. Med. Chem., 2006, 49, 5316.
11. Y. S. Lee and B. H. Kim, Bioorg. Med. Chem. Lett., 2002, 12, 1395.
12. J. Liu, L. ꢁF. Yu, J. B. Eaton, B. Caldarone, K. Cavino, C. Ruiz, M.
Terry, A. Fedolak, D. Wang and A. Ghavami, J. Med. Chem., 2011, 54,
7280.
13. S. Batra, A. K. Roy, A. Patra, A. P. Bhaduri, W. R. Surin, S. A. V.
Raghavan, P. Sharma, K. Kapoor and M. Dikshit, Bioorg. Med. Chem.,
2004, 12, 2059.
14. S. Cicchi, F. M. Cordero and D. Giomi, Prog. Heterocycl. Chem., 2003,
15, 261.
15. R. Sutherland, E. A. P. Croydon and G. N. Rolinson, Br. Med. J., 1970,
4, 455.
16. A. A. Fuller, B. Chen, A. R. Minter and A. K. Mapp, J. Am. Chem. Soc.,
2005, 127, 5376.
17. A. KellaꢁBennani, M. Soufjaoui, A. Kerbal, S. FkihꢁTetouani and N.
Bitit, Tetrahedron, 1995, 51, 10923.
phase in
a doseꢁdependent manner. Furthermore, Acridine
18. G. G. Tsantali, J. Dimtsas, C. A. Tsoleridis and I. M. Takakis, Eur. J.
Org. Chem., 2007, 258.
19. A. Ferna´ndezꢁMateos, G. P. Coca and R. R. Gonza´lez, Tetrahedron,
2005, 61, 8699.
20. T. Itoh, M. Watanabe and T. Fukuyama, Synlett, 2002, 1323.
21. A. Cerri, D. Micheli and R. Gandolfi, Synthesis, 1974, 710.
22. K. Kaur, V. Kumar, A. K. Sharma and G. K. Gupta, Eur. J. Med. Chem.,
2014, 77, 121.
23. M. M. Alam, E. ꢁH. Joh, Y. Kim, Y. I. Oh, J. Hong, B. Kim, D.ꢁH. Kim
and Y. S. Lee, Eur. J. of Med. Chem., 2012, 47, 485.
24. B. Stefane, P. Brozic, M. Vehovc, T. L. Rizner and S. Gobec, Eur. J.
Med. Chem., 2009, 44, 2563.
orange/ethidium bromide staining, annexin V binding assay and
mitochondrial membrane potential studies evidenced that compound
4i inhibit the cancer cell growth in MCFꢁ7 through apoptosis.
Docking studies of 4i on DNMT1 protein suggested that DNMT1
inhibition could be the possible mechanism for the cytotoxic activity
of these compounds. These preliminary results encourage further
investigation on synthesized compounds aiming to the development
of new potential bioactive agents.
25. S. Hanessian, R. R. Vakiti, A. K. Chattopadhyay, S. Dorich and C.
Lavallée, Bioorg. Med. Chem., 2013, 21, 1775.
Acknowledgments
We thank the Department of Pharmaceuticals (Ministry of 26. P. Chand, P. L. Kotian, A. Dehghani, Y. E. Kattan, T. H. Lin, T. L.
Hutchison, Y. S. Babu, S. Bantia, A. J. Elliott and J. A. Montgomery, J.
Med. Chem., 2001, 44, 4379.
27. L. C. Meurer, P. E. Finke, K. A. Owens, N. N. Tsou, R. G. Ball, S. G.
Chemicals and Fertilizers) for providing funds and also CSIRꢁIndian
Institute of Chemical Technology, Hyderabad for providing the
facilities.
Mills, M. MacCoss, S. Sadowski, M. A. Cascieri, K. L. Tsao, G. G.
Chicchi, L. A. Egger, S. Luell, J. M. Metzger, D. E. MacIntyre, N. M. J.
Rupniak, A. R. Williamse and R. J. Hargreaves, Bioorg. Med. Chem.
Lett., 2006, 16, 4504.
Notes and references
aDepartment of Medicinal Chemistry, National Institute of Pharmaceutical 28. A. Kamal, J. S. Reddy, M. J. Ramaiah, D. Dastagiri, E. V. Bharathi, M.
Education and Research (NIPER), Balanagar, Hyderabad 500037, India.
A. Azhar, F. Sultana, S. N. C. V. L. Pushpavalli, M. P. Bhadra, A.
Juvekar, S. Sen and S. Zingde, Eur. J. Med. Chem., 2010, 45, 3924.
bDepartment of Pharmacology and Toxicology, National Institute of
Pharmaceutical Education and Research (NIPER), Balanagar, Hyderabad 29. N. K. Bejjanki, A. Venkatesham, J. Madda, N. Kommu, S. Pombala, C.
500037, India.
G. Kumar, K. R. Prasad and J. B. Nanubolu, Bioorg. Med. Chem. Lett.,
2013, 23, 4061.
30. R. Baruchello, D. Simoni, P. Marchetti, R. Rondanin, S. Mangiola, C.
Costantini, M. Meli, G. Giannini, L. Vesci and V. Carollo, Eur. J. Med.
Chem., 2014, 76, 53.
cDepartment of Pharmaceutical Technology, National Institute of
Pharmaceutical Education and Research (NIPER), Sector 67, S. A. S. Nagar,
Punjab 160062, India.
6 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 2012

Page 7 of 7
Journal Name
Medicinal Chemistry Communications
View ArticAle ROnTliInCeLE
DOI: 10.1039/C4MD00525B
31. H. Agirbas, S. Guner, F. Budak, S. Keceli, F. Kandemirli, N. Shvets, V. 41. A. L. Barry, The Antimicrobic Susceptibility Test: Principles and
Kovalishyn and A. Dimoglo, Bioor. Med. Chem., 2007, 15, 2322. Practices; Ilus Lea & Febiger: Philadelphia, 1976. p 180.
32. S. Castellano, D. Kuck, M. Viviano, J. Yoo, F. LopezꢁVallejo, P. Conti, 42. P. M. Reddy, Y. P. Ho, K. Shanker, R. Rohini and V. Ravinder, Eur. J.
L. Tamborini, A. Pinto, J. L. MedinaꢁFranco and G. Sbardella, J. Med.
Chem., 2011, 54, 7663.
33. P. Aschwanden, D. E. Frantz and E. M. Carreira, Org. Lett., 2000, 2,
2331.
Med. Chem., 2009, 44, 2621.
43. S. Shrivastava, P. Kulkarni, D. Thummuri, M. K. Jeengar, V. G. M.
Naidu, M. Alvala, G.B. Redddy and S. Ramakrishna, Apoptosis, 2014,
19, 1148.
34. P. Mayo, T. Hecnar and W. Tam, Tetrahedron, 2001, 57, 5931.
35. G. K. Tranmer and W. Tam, Org. Lett., 2002, 4, 4101.
36. G. A. Molander and R. Figueroa, Org. Lett., 2006, 8, 75.
37. W. Van Brabandt, M. Vanwalleghem, M. D'Hoogh and N. De Kimpe, J.
Org. Chem., 2006, 71, 7083.
38. R. P. Dickinson, K. N. Dack, C. J. Long and J. Steele, J. Med. Chem.,
1997, 40, 3442.
39. F. Caputo, F. Clerici, M. L. Gelmi, S. Pellegrino and D. Pocar,
Tetrahedron: Asymmetry, 2006, 17, 1430.
44. D. Ribble, N. B. Goldstein, D. A. Norris and Y. G. Shellman, BMC
Biotechnol., 2005, 5, 5.
45. B. Kloesch, T. Becker, E. Dietersdorfer, H. Kiener and G. Steiner, Int.
Immunopharmacol., 2013, 15, 400.
46. E. ꢁJ. Lee, S. ꢁY. Oh and M. ꢁK. Sung, Food Chem. Toxicol., 2012, 50,
4136.
47. M. T. Binet, C. J. Doyle, J. E. Williamson and P. Schlegel, J. Exp. Mar.
Biol. Ecol., 2014, 452, 91.
48. J. Song, O. Rechkoblit, T. H. Bestor and D. J. Patel, Science, 2011, 331,
1036.
49. Glide, version 6.0, Schrödinger, LLC, New York, NY, 2013.
40. A. L. Whiting, N. M. Neufeld and F. Hof, Tetrahedron Lett., 2009, 50,
7035.
This journal is © The Royal Society of Chemistry 2012
J. Name., 2012, 00, 1-3 | 7