Synthesis, structure-activity relationship of iodinated-4-aryloxymethyl- coumarins as potential anti-cancer and anti-mycobacterial agents

Article abstract of DOI:10.1016/j.ejmech.2013.12.061

A series of new iodinated-4-aryloxymethylcoumarins 6, 8 and 10 have been obtained from the reaction of various 4-bromomethylcoumarins 4 with 2-iodophenol 5, 3-iodophenol 7 and 4-iodophenol 9 respectively. All the title compounds were screened for anticancer activity against two cancer cell lines (MDA-MB human adenocarcinoma mammary gland and A-549 human lung carcinoma) and two mycobacterial strains (Mycobacterium tuberculosis H₃₇ RV and Mycobacterium phlei). The SAR results indicate that nine compounds are potent, among these 10h and 10i having chlorine are most effective. This is the first report assigning in vitro anti-mycobacterial, anticancer and structure-activity relationship for this new class of iodinated-4-aryloxymethyl-coumarins.

Full text of DOI:10.1016/j.ejmech.2013.12.061

Accepted Manuscript
Synthesis, structure–activity relationship of iodinated-4-aryloxymethyl-coumarins as
potential anti-cancer and anti-mycobacterial agents
Mahantesha Basanagouda, Vishwanath B. Jambagi, Nivedita N. Barigidad, Sandeep
S. Laxmeshwar, Venkatesh Devaru, Narayanachar
PII:
S0223-5234(14)00035-X
10.1016/j.ejmech.2013.12.061
EJMECH 6657
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 5 August 2013
Revised Date: 25 December 2013
Accepted Date: 31 December 2013
Please cite this article as: M. Basanagouda, V.B. Jambagi, N.N. Barigidad, S.S. Laxmeshwar, V.
Devaru, Narayanachar, Synthesis, structure–activity relationship of iodinated-4-aryloxymethyl-coumarins
as potential anti-cancer and anti-mycobacterial agents, European Journal of Medicinal Chemistry (2014),
doi: 10.1016/j.ejmech.2013.12.061.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Graphical Abstract:
I
O
R
O
O
Iodo-4-aryloxymethylcoumarins
The thirty title compounds were synthesized and screened for their anticancer and
anti-mycobacterial activity. Among these 10h and 10i having chlorine most effective.

ACCEPTED MANUSCRIPT
Synthesis, structure–activity relationship of iodinated-4-aryloxymethyl-coumarins
as potential anti-cancer and anti-mycobacterial agents
a,٭
a
a
Mahantesha Basanagouda, Vishwanath B. Jambagi, Nivedita N. Barigidad,
b
c
d
Sandeep S. Laxmeshwar, Venkatesh Devaru, Narayanachar
a
P.G. Department of Chemistry, P.C. Jabin Science College, Hubli-580031, Karnataka,
India.
b
P.G. Department of Studies and Research in Polymer Science, Sir, M. V. PG Centre,
Mandya-571402, University of Mysore, , Karnataka, India.
c
Department of Physics, L.V.D. College, Raichur-584103, Karnataka, India.
d
Department of Chemistry, L.V.D. College, Raichur-584103, Karnataka, India.
Abstract:
A series of new iodinated-4-aryloxymethylcoumarins 6, 8 and 10 have been
obtained from the reaction of various 4-bromomethylcoumarins 4 with 2-iodophenol 5, 3-
iodophenol 7 and 4-iodophenol 9 respectively. All the title compounds were screened for
anticancer activity against two cancer cell lines (MDA-MB human adenocarcinoma
mammary gland and A-549 human lung carcinoma) and two mycobacterial strains
(
Mycobacterium tuberculosis H RV and Mycobacterium Phlei). The SAR results
37
indicate that nine compounds are potent, among these 10h and 10i having chlorine most
effective. This is the first report assigning in vitro anti-mycobacterial, anticancer and
structure-activity relationship for this new class of iodinated-4-aryloxymethyl-coumarins.
Keywords: Coumarin; heterocycles; iodine; anticancer; anti-mycobacterial; SAR.
٭
Corresponding author: Tel.: +91 9448760184.
E-mail address: mahanteshachem@gmail.com (Mahantesha Basanagouda)
1

ACCEPTED MANUSCRIPT
1
. Introduction
The cancer and tuberculosis are called the ‘big killer and intractable diseases’.
Cancer, one of the most life-threatening diseases, has more than 200 distinct types
associated with it, affecting over 60 human organs. More than 90% of all cancer-related
deaths occur from metastasis of the primary cancer tumor. The early stages of cancer
development carry the maximum potential for therapeutic intervention. Therefore,
detecting premalignant or premetastatic malignant tumors when they are still confined
within organ(s) is critical to enable effective treatment and improving survival rate.
Among all cancers, lung cancer continues to be the most prevalent and life threatening
globally. The disease has a severe impact on the quality of life, due to reduced
oxygenation levels and a higher incidence of metastasis due to high blood flow in the
lungs. It affects more than a million people worldwide and accounts for about 25% of all
cancer deaths [1]. Great advances have been made mapping out the cellular pathways
altered in tumors and the pathways that respond to cancer therapeutics. The obvious
importance of the components of DNA damage response pathways as potential cancer
therapeutic targets has stimulated researchers and pharmaceutical companies to develop
numerous chemical inhibitors for many of the proteins involved in these pathways [2].
Tuberculosis (TB) remains the leading cause of mortality due to a bacterial
pathogen, Mycobacterium tuberculosis. Approximately one-third of the world’s
population has been infected with the causative organism M. tuberculosis (MTB), eight
million become sick with TB every year, and globally it accounts for almost three million
deaths annually. One-fifth of all deaths of adults in developing countries are due to TB,
2

ACCEPTED MANUSCRIPT
which is a reemerging problem particularly in many industrialized countries. It is
estimated that between 2005 and 2020, one billion people will be newly infected, over
1
25 million people will get sick and 30 million will die of tuberculosis if control is not
further strengthened. In addition, the evolution of its new virulent forms like multidrug
resistant (MDR-TB) and extremely drug resistant (XDR-TB) has become a major threat
to human kind. Among HIV infected people, the resurgence of TB is alarming due to the
development of pathogenic synergy. The worsening situation has prompted the World
Health Organization (WHO) to declare tuberculosis a global public health crisis [3].
Among the oxygen heterocycles, coumarin derivatives are important motifs,
widely found in many natural products, many of them displaying diverse biological
activities such as antimicrobial, anti-inflammatory, analgesic, antioxidant, antimalarial,
anticancer, antituberclosis and anti-HIV properties, which have been reviewed [4-12].
This type of special coumarin structure enables its derivatives readily interact with a
diversity of enzymes and receptors in organisms through weak bond interactions, thereby
exhibit wide potentiality as medicinal drugs [12]. The coumarin derivatives in recent
studies have been reported to possess the potent anticancer effect through different
mechanisms. The tricyclic coumarin sulfamate (STX64), (IC = 8 nM) a nonsteroid-
5
0
based irreversible aromatase-steroid sulfatase (STS) inhibitor provides remarkable
activity for the cure of prostate cancer, and most encouragingly, its clinical trials have
been accomplished in 2011 [13-15]. For instance, 3,8-dibromo-7-hydroxy-4-methyl
coumarin (DBC) (IC = 100 nM) is treated as a CK2 inhibitor to suppress neoplastic
5
0
growth [16,17]. Novobiocin, a known DNA gyrase inhibitor, binds to a nucleotide-
3

ACCEPTED MANUSCRIPT
binding site located on the Hsp90 C-terminus and induces degradation of Hsp90-
dependent client proteins at ~700 µM in breast cancer cells [18-21]. 7-O-Alkoxy-4-
methylumbelliferone derivatives with longer chains, especially nonyl and decyl have
good inhibitory activity against mycobacterium tuberculosis [22,23]. Some biologically
active anticancer [24-27] and anti-tuberculosis [28-31] agents having coumarin moiety
are presented in figure 1 and 2, respectively.
Halogenation is an important approach in lead optimization for drug development
and about half of the molecules used in high-throughput screening are halogenated. The
biomedical implications of the findings are discussed with respect to vast potential
applications in biomolecular design and drug discovery [32,33]. The biological
importance of iodine in the compound have been well reported in the literature such as
binding ability [34], cannabinoid receptor antagonists [35], as anti-cancer agent [36].
Amiodarone is an antiarrhythmic drug used for the treatment of tachyarrhythmias [37].
The amiodarone analogue (KB130015) was reported as a antiarrhythmic drug with an
improved toxicity profile compared with amiodarone [38]. Three new iodinated
tryptophan derivatives, plakohypaphorines have been isolated from the Caribbean sponge
Plakortis simplex and evaluated for their antihistamine activity [39].
In the light of the above facts and in continuation of our interest in designing
oxygen heterocycle based biologically active molecules [40-43], we planned to
synthesize a new series of iodinated-4-aryloxymethyl coumarins in order to study their
structure activity relationship and hoping that the new compounds might show significant
anticancer and anti-tuberculosis activity.
4

ACCEPTED MANUSCRIPT
2
. Chemistry
The required substituted-4-bromomethylcoumarins 4 were prepared by the
Pechmann cyclisation of substituted phenols 3 with 4-bromoethylacetoacetate 2 using
sulphuric acid as the condensing agent. The 4-bromoethylacetoacetate 2 in turn was
o
obtained by the bromination of ethylacetoacetate 1 in dry ether at 0-5 C.
The nucleophilic displacement of 4-bromomethylcoumarins with three phenols
viz., 2-iodophenol 5, 3-iodophenol 7 and 4-iodophenol 9, resulted the 4-
aryloxymethylcoumarins 6, 8 and 10, respectively under standard acetone-potassium
carbonate conditions at room temperature (Scheme 1). Formation of ethers was indicated
1
by the difference in H-NMR chemical shifts. The methylene protons in 4-
bromomethylcoumarins 4 resonated around 4.6 δ ppm where as the corresponding 4-
aryloxymethylcoumarins 6, 8 and 10 exhibited this peak around 5.4 ppm. Further these
1
3
were confirmed by their IR, C NMR and mass spectral data.
3
3
. Pharmacology
.1 Anticancer screening
All the title compounds 6(a-j), 8(a-j) and 10(a-j) were screened for their in vitro
anticancer activity against two cancer cell lines MDA-MB human adenocarcinoma
mammary gland and A-549 human lung carcinoma by using MTT assay for the
determination of MIC values and the results are presented in Table 1. All the thirty
compounds were screened in the present study, MIC ranging from 1.56 to 100 µg/mL.
5

ACCEPTED MANUSCRIPT
3
.1.1 MDA-MB human adenocarcinoma mammary gland
In the series 6(a-j), the compounds bearing iodine on the second position of the
phenoxy moiety with the halogens (6h, 6i, 6j) and mono-methyl group (6a, 6b) on
th
th
coumarin at 6 and 7 positions exhibited potent activity (MIC 12.5 µg/mL). Whereas
decrease in activity were observed on varying the substituent by methoxy (6d, 6e), benzo
(
6f, 6g) and dimethyl (6c) with MIC 25 µg/mL.
The change in the position of iodine from second to third position, i.e., the series
of compounds 8(a-j), the activity increased from MIC 12.5 to 6.25 µg/mL for compounds
bearing chlorine and bromine (8h, 8i, 8j). The decrease in activity were observed for
mono methyl (8a, 8b) compounds from 12.5 to 25 µg/mL, methoxy (8d, 8e) and benzo
(
8f, 8g) compounds from MIC 25 to 50 µg/mL. Whereas the activity of compound 8c was
unaffected.
Further, the change in the position of iodine from third to fourth position, i.e., the
series of compounds 10(a-j), the activity increased from MIC 6.25 to 1.56 µg/mL for
compounds bearing chlorine (10h, 10i) and to 3.125 µg/mL bromine (10j) on coumarin.
The increase in activity were observed for compounds 10a, 10b, 10c from 25 to 12.5
µg/mL and for compounds 10d, 10e, 10f, 10g from MIC 50 to 25 µg/mL.
In the library of thirty compounds, 6(a-j), 8(a-j) and 10(a-j), the chloro (10h, 10i)
compounds and bromo (10j) compound displayed potent activity with MIC 1.56 and
3
.125 µg/mL respectively. The chloro (8h, 8i) and bromo (8j) compounds showed
moderate activity with MIC 6.25 µg/mL.
6

ACCEPTED MANUSCRIPT
3
.1.2 A-549 human lung carcinoma
In the series of compounds 6(a-j), bearing iodine on the second position of the
th
th
phenoxy moiety with the halogens (6h, 6i, 6j) on coumarin at 6 and 7 positions
exhibited potent activity with MIC 12.5 µg/mL. The moderate activity was exhibited by
the methoxy (6d, 6e) and benzo (6f, 6g) compounds with MIC 25 µg/mL. The mono-
methyl (6a, 6b) and dimethyl (6c) compounds exhibited very weak activity with MIC 50
µg/mL.
The change in the position of iodine from second to third in the phenoxy moiety,
i.e., the series of compounds 8(a-j), the activity increased from MIC 12.5 to 6.25 µg/mL
for compounds bearing chlorine and bromine (8h, 8i, 8j). The increase in activity was
also observed for compounds 8(d-g) and 8(a-c) from MIC 25 to 12.5 µg/mL and MIC 50
to 25 µg/mL, respectively.
The change in the position of iodine from third to fourth in the phenoxy moiety,
i.e., the series of compounds 10(a-j), the activity increased in all the compounds, from
MIC 6.25 to 3.125 µg/mL for compounds bearing chlorine and bromine (10h, 10i, 10j).
The increase in activity was also observed for compounds 10(d-g) and 10(a-c) from MIC
1
2.5 to 6.25 µg/mL and MIC 25 to 12.5 µg/mL, respectively.
In general the halogenated (10h, 10i, 10j) compounds were exhibited potent
activity with MIC 3.125 µg/mL. The halogenated (8h, 8i, 8j), methoxy (10d, 10e) and
benzo (10f, 10g) compounds were moderate activity with MIC 6.25 µg/mL.
th
th
In concise, the compounds bearing the chlorine at 6 and 7 position and bromine
th
at 6 position on coumarin have the potential impact in improving the anti-cancer activity
7

ACCEPTED MANUSCRIPT
th
compared to methyl, methoxy, benzo-substitutions. The iodine atom at 4 position on
rd
phenoxy moiety play an important role in enhancing the activity compared to its 3 and
nd
2
position.
3
.2 Anti-mycobacterial screening
The in vitro results of anticancer activity encouraged us to evaluate their anti-
mycobacterial effect against Mycobacterium tuberculosis H RV and Mycobacterium
3
7
Phlei by Microplate Alamar Blue Assay (MABA) for the determination of MIC values of
all the synthesized compounds along with standard drugs streptomycin and pyrizanamide
for the comparison are presented in Table 2. All the thirty compounds were screened in
the present study, MIC ranging from 1.56 to 100 µg/mL.
3
.2.1 Mycobacterium tuberculosis H RV
37
In the series 6(a-j), the compounds bearing iodine on the second position of the
th
th
phenoxy moiety with the halogens (6h, 6i, 6j) on coumarin at 6 and 7 positions
exhibited potent activity with MIC 6.25 µg/mL. The mono-methyl (6a, 6b) and methoxy
(
6d, 6e) compounds showed the moderate activity with MIC 12.5 µg/mL. The MIC 25
µg/mL exhibited by dimethyl compound 6c. The very weak activity were observed by
benzo (6f, 6g) compounds with MIC 50 µg/mL.
The change in the position of iodine from second to third in the phenoxy moiety,
i.e., the series of compounds 8(a-j), the activity decreased from MIC 6.25 to 12.5 µg/mL
for compounds bearing chlorine and bromine (8h, 8i, 8j). The decrease in activity was
8

ACCEPTED MANUSCRIPT
also showed by mono methyl (8a, 8b) and methoxy (8d, 8e) compounds from 12.5 to 25
µg/mL. The increase in activity was observed for compounds 8f, 8g from MIC 50 to 25
µg/mL. The compound 8c was unaffected.
The change in the position of iodine from third to fourth in the phenoxy moiety,
i.e., the series of compounds 10(a-j), the activity increased for chloro (10h, 10i) and
bromo (10j) compounds from MIC 12.5 to 1.56 and 6.25 µg/mL respectively. The
increase in activity were also observed for compounds 10a, 10c with MIC 25 to 12.5
µg/mL. Whereas the methyl (10 b), methoxy (10d, 10e) and benzo (10f, 10g) compounds
were unaffected in their MIC with 25 µg/mL.
The chloro (10h, 10i) compounds showed excellent activity with MIC 1.56 µg/mL
and were more potent than standard drugs streptomycin (MIC of 6.25 µg/mL),
pyrizanamide (MIC of 3.125 µg/mL). The chloro (6h, 6i) and bromo (6j, 10j) compounds
showed moderate activity with MIC 6.25 µg/mL against Mycobacterium tuberculosis.
3
.2.2 Mycobacterium Phlei
In the series of compounds 6(a-j), bearing iodine on the second position of the
th
th
phenoxy moiety with the chlorine (6h, 6i) on coumarin at 6 and 7 positions exhibited
potent activity with MIC 25 µg/mL. The moderate activity was exhibited by the dimethyl
(
6c), methoxy (6d, 6e), benzo (6f, 6g), and bromo (6j) compounds 50 µg/mL. Whereas
the mono methyl (6a, 6b) compounds showed least active with MIC greater than 100
µg/mL.
9

ACCEPTED MANUSCRIPT
The change in the position of iodine from second to third position, i.e., the series
of compounds 8(a-j), the chloro (8h, 8i) and bromo (8j) compounds exhibited potent
activity in the series with increase in activity from 25 to 6.25 and 50 to 6.25 µg/mL
respectively. The dimethyl (8c) and benzo (8f, 8g) compounds exhibited moderate
activity in this series with increase in activity MIC from 50 to 25 µg/mL. Similarly the
mono methyl (8a, 8b) compounds were moderate active in series with increase in activity
with MIC from greater than 100 to 12.5 µg/mL. The change in the position of iodine was
unaffected on activity of methoxy (8d, 8e) compounds MIC 50 µg/mL.
Further, the change in the position of iodine from third to fourth position, i.e., the
series of compounds 10(a-j), the activity increased from MIC 6.25 to 3.125 µg/mL for
compounds bearing chlorine (10h, 10i). The activity remains same for bromo (10i)
compound with 6.25 µg/mL. The methyl (10a, 10b) compounds exhibited moderate
activity with MIC 12.5 µg/mL which were unaffected. Whereas dimethyl (10c) and
methoxy (10d, 10e) compounds showed moderate activities and increased the activity
from 25 to 12.5 and 50 to 12.5 µg/mL respectively. The lease activity was exhibited by
benzo (10f, 10g) compounds with MIC 25 µg/mL which were unaffected.
The chloro (10h,10i) compounds showed excellent activity with MIC 3.125
µg/mL and were more potent than standard drugs streptomycin (MIC of 6.25 µg/mL),
and equal potent to pyrizanamide (MIC of 3.125 µg/mL). The chloro (8h, 8i) and bromo
(
8j, 10j) compounds with MIC 6.25 µg/mL showed moderate activity against
Mycobacterium Phlei.
1
0

ACCEPTED MANUSCRIPT
To summarize, it is interesting to note that, the compounds having the chlorine at
th
th
th
6
and 7 position and bromine at 6 position on coumarin have the exhibited potent
activity compared to methyl, methoxy, benzo-substitutions. The similar trend was
th
observed as in anti-cancer screening, the iodine atom at 4 position on phenoxy moiety
rd
nd
play an important role in enhancing the activity compared to its 3 and 2 position.
4
. Results and Discussion
The IR spectrum of 4-(2-iodo-phenoxymethyl)-6-methyl-chromen-2-one (6a) (R =
-
1
1
6
-CH ) showed lactone carbonyl at 1710 cm . The H-NMR spectrum exhibited three
3
singlets at 2.41, 5.50 and 6.82 δ ppm due to C6-CH , C4-CH and C3-H, respectively.
3
2
The aromatic protons were resonated as multiplet in the range of 6.84-7.85 δ ppm. This
1
3
was further confirmed by C NMR spectrum which agrees with the number of carbons
and by its mass spectrum that shows the molecular ion peak m/z 393 (M+1), agrees with
the molecular weight of the compound (Supplementary material, spectrum 1-4).
The IR spectrum of 4-(3-iodo-phenoxymethyl)-6-methyl-chromen-2-one (8a) (R =
-
1
1
6
-CH ) exhibited lactone carbonyl at 1715 cm . The H-NMR spectrum showed singlets
3
at 2.40, 5.40 and 6.57 δ ppm due to C6-CH , C4-CH and C3-H respectively. The
3
2
remaining protons were resonated as a multiplet in the range of 7.11-7.77 δ ppm.
1
3
Formation of the product was confirmed by its C NMR and mass spectral data
(
Supplementary material, spectrum 9-12).
The IR spectrum of 4-(4-iodo-phenoxymethyl)-6-methyl-chromen-2-one (10a) (R
-
1
1
=
6-CH ) exhibited lactone carbonyl at 1704 cm . The H-NMR spectrum showed
3
1
1

ACCEPTED MANUSCRIPT
singlets at 2.40, 5.40 and 6.53 δ ppm due to C6-CH , C4-CH and C3-H respectively. The
3
2
remaining aromatic protons resonated as a multiplet in the range of 7.01-7.68 δ ppm. The
LCMS of compound showed a peak at m/z 393 (M+1) confirming its molecular weight
(
Supplementary material, spectrum 17-20).
Conclusions
The series of synthesized title compounds were characterized by spectral data and
evaluated for their anticancer and anti-mycobacterial activity. By results, it is interesting
th
th
to note that in general the compounds having the chlorine at 6 and 7 position on
th
coumarin and bromine 6 position on coumarin have the exhibited potent activity
compared to other substitutions. The careful observation on SAR, the compounds 10h
th
th
th
and 10i having the chlorine at 6 and 7 position on coumarin and iodine at 4 position
on phenoxy moiety exhibited potent anticancer and anti-mycobacterial activity. These
two compounds are even more anti-mycobacterial than standard drugs under
investigation. The higher activities of these compounds may lead to new anticancer and
anti-mycobacterial drugs in future.
5
5
. Experimental protocols
.1. Chemistry
The melting points were determined by open capillary method and are
uncorrected. The IR spectra (KBr disc) were recorded on a Nicolet-5700 FT-IR
1
13
spectrophotometer. H NMR and C NMR spectra were recorded on Bruker 400 and 300
1
2

ACCEPTED MANUSCRIPT
MHz spectrometer using CDCl DMSO-d as solvents and TMS as an internal standard.
3
,
6
The chemical shifts are expressed in δ ppm. The mass spectra were recorded using
Agilent-single Quartz LC-MS. The elemental analysis was carried out using Heraus CHN
rapid analyzer. The purity of the compound was checked by T.L.C. All the chemicals
purchased were of analytical grade, and were used without further purification unless
otherwise stated.
5
.1.1. Synthesis of substituted-4-bromomethyl coumarins 4(a-j)
The required substituted-4-bromomethyl-coumarins [40, 44] have been
synthesized by the Pechmann cyclization of substituted phenols with 4-
bromoethylacetoacetate [45].
5
.1.2 General procedure for the synthesis of iodinated-4-aryloxymethyl-coumarins 6
(
a-j), 8 (a-j) and 10 (a-j):
A mixture of 2-iodophenol 5 (1.08g, 10 mmol) and anhydrous potassium
carbonate (1.38g, 10 mmol) was stirred for 30 minutes in dry acetone (30 mL). To this, 6-
methyl-4-bromomethylcoumarin 4a (2.53g, 10 mmol) was added and the stirring was
continued for 24 h. Then, the resulting reaction mixture was poured to crushed ice. The
separated solid was filtered, washed with 1:1 HCl (30 mL) and with water. Then product
6
a was recrystallised from suitable solvent.
1
3

ACCEPTED MANUSCRIPT
The similar procedure was followed for the synthesis of 4-
aryloxymethylcoumarins 8 (a-j) and 10 (a-j) by the reaction of the substituted-4-
bromomethylcoumarins 4 (a-j) with 3-iodophenol 7 and 4-iodophenol 9 respectively.
5
.1.2.1 4-(2-Iodo-phenoxymethyl)-6-methyl-chromen-2-one (6a)
o
-1
Colourless (Ethanol), m.p. 241 C, yield 95 %; IR (KBr, ν in cm ): 1710 (lactone C=O);
1
H NMR (400 MHz, DMSO-d ): δ 2.41 (s, 3H, C6-CH ), 5.50 (s, 2H, C4-CH ), 6.82 (s,
6
3
2
1
3
1
1
1
3
5
H, C3-H), 6.84-7.85 (m, 7H, Ar-H); C NMR (CDCl , 75 MHz): 20.97, 66.77, 112.40,
3
13.80, 113.85, 116.87, 117.15, 123.25, 128.94, 132.96, 133.18, 134.08, 134.23, 149.30,
51.83, 152.05, 160.73. LCMS m/z: 393 [M+1]; Anal.calcd. for C H IO ; C, 52.06; H,
1
7
13
3
.34. Found: C, 52.16; H, 3.42.
.1.2.2 4-(2-Iodo-phenoxymethyl)-7-methyl-chromen-2-one (6b)
o
-1
Colourless (Ethanol), m.p. 190 C, yield 90 %; IR (KBr, ν in cm ): 1706 (lactone C=O);
1
H NMR (400 MHz, DMSO-d ): δ 2.42 (s, 3H, C7-CH ), 5.56 (s, 2H, C4-CH ), 6.61 (s,
6
3
2
1
3
1
1
1
3
5
H, C3-H), 6.88-8.06 (m, 7H, Ar-H); C NMR (100 MHz, DMSO-d ): 21.21, 66.79,
6
10.92, 113.45, 117.05, 117.15, 121.38, 126.41, 129.93, 131.51, 133.11, 133.88, 144.27,
46.09, 152.29, 160.79. LCMS m/z: 393 [M+1]; Anal.calcd. for C H IO ; C, 52.06; H,
1
7
13
3
.34. Found: C, 52.22; H, 3.48.
.1.2.3 4-(2-Iodo-phenoxymethyl)-7,8-dimethyl-chromen-2-one (6c)
o
-1
Colourless (Chloroform), m.p. 238 C, yield 83 %; IR (KBr, ν in cm ): 1712 (lactone
1
C=O); H NMR (400 MHz, DMSO-d ): δ 2.40 (s, 6H, C7 & C8-CH ), 5.52 (s, 2H, C4-
6
3
1
4

ACCEPTED MANUSCRIPT
CH ), 6.95 (s, 1H, C3-H), 7.16-7.94 (m, 6H, Ar-H); LCMS m/z: 407 [M+1]; Anal.calcd.
2
for C H IO ; C, 53.22; H, 3.72. Found: C, 53.36; H, 3.86.
1
8
15
3
5
.1.2.4 4-(2-Iodo-phenoxymethyl)-6-methoxy-chromen-2-one (6d)
o
-1
Colourless (Chloroform), m.p. 214 C, yield 88 %; IR (KBr, ν in cm ): 1701 (lactone
1
C=O); H NMR (400 MHz, DMSO-d ): δ 3.84 (s, 3H, C6-OCH ), 5.58 (s, 2H, C4-CH ),
6
3
2
1
3
6
6
1
5
5
.98 (s, 1H, C3-H), 7.16-8.01 (m, 7H, Ar-H); C NMR (100 MHz, DMSO-d ): 56.74,
6
6.89, 109.57, 112.83, 113.48, 117.64, 118.87, 119.98, 121.81, 129.14, 132.14, 144.18,
45.26, 148.94, 155.63, 160.16. LCMS m/z: 409 [M+1]; Anal.calcd. for C H IO ; C,
17
13
4
0.02; H, 3.21. Found: C, 50.24; H, 3.29.
.1.2.5 4-(2-Iodo-phenoxymethyl)-7-methoxy-chromen-2-one (6e)
o
-1
1
Yellow (Ethanol), m.p. 166 C, yield 86 %; IR (KBr, ν in cm ): 1704 (lactone C=O); H
NMR (400 MHz, DMSO-d ): δ 3.89 (s, 3H, C6-OCH ), 5.61 (s, 2H, C4-CH ), 7.06 (s,
6
3
2
1
3
1
1
1
3
5
H, C3-H), 7.19-8.24 (m, 7H, Ar-H); C NMR (100 MHz, DMSO-d ): 56.92, 66.93,
6
09.74, 112.92, 113.59, 117.77, 118.94, 120.08, 121.96, 129.46, 132.62, 144.56, 145.69,
48.98, 155.82, 160.28. LCMS m/z: 409 [M+1]; Anal.calcd. for C H IO ; C, 50.02; H,
1
7
13
4
.21. Found: C, 50.32; H, 3.33.
.1.2.6 1-(2-Iodo-phenoxymethyl)-benzo[f]chromen-3-one (6f)
o
-1
Pale yellow (Chloroform), m.p. 226 C, yield 91 %; IR (KBr, ν in cm ): 1711 (lactone
1
C=O); H NMR (400 MHz, DMSO-d ): δ 5.91 (s, 2H, C4-CH ), 7.36 (s, 1H, C3-H), 7.14-
6
2
8
3
5
.56 (m, 10H, Ar-H); LCMS m/z: 429 [M+1]; Anal.calcd. for C H IO ; C, 56.10; H,
20 13 3
.06. Found: C, 50.22; H, 3.11.
.1.2.7 4-(2-Iodo-phenoxymethyl)-benzo[h]chromen-2-one (6g)
1
5

ACCEPTED MANUSCRIPT
o
-1
Yellow (Chloroform), m.p. 218 C, yield 83 %; IR (KBr, ν in cm ): 1708 (lactone C=O);
1
H NMR (400 MHz, DMSO-d ): 5.66 (s, 2H, C4-CH ), 7.21 (s, 1H, C3-H), 7.18-8.45 (m,
6
2
1
0H, Ar-H); LCMS m/z: 429 [M+1]; Anal.calcd. for C H IO ; C, 56.10; H, 3.06.
20 13 3
Found: C, 50.36; H, 3.19.
5
.1.2.8 6-Chloro-4-(2-iodo-phenoxymethyl)-chromen-2-one (6h)
o
-1
Colourless (Chloroform), m.p. 244 C, yield 61 %; IR (KBr, ν in cm ): 1712 (lactone
1
C=O); H NMR (400 MHz, DMSO-d ): δ 5.55 (s, 2H, C4-CH ), 7.01 (s, 1H, C3-H), 7.11-
6
2
1
3
7
1
.96 (m, 7H, Ar-H); C NMR (100 MHz, DMSO-d ): 66.59, 112.32, 118.08, 118.63,
6
21.01, 126.06, 128.10, 128.17, 129.36, 131.21, 131.49, 143.52, 152.17, 159.64. LCMS
m/z: 413 [M+1]; Anal.calcd. for C H ClIO ; C, 46.57; H, 2.44. Found: C, 46.70; H,
1
6
10
3
2
.49.
5
.1.2.9 7-Chloro-4-(2-iodo-phenoxymethyl)-chromen-2-one (6i)
o
-1
Colourless (Chloroform), m.p. 225 C, yield 59 %; IR (KBr, ν in cm ): 1708 (lactone
1
C=O); H NMR (400 MHz, DMSO-d ): δ 5.57 (s, 2H, C4-CH ), 7.04 (s, 1H, C3-H), 7.12-
6
2
7
2
5
.99 (m, 7H, Ar-H); LCMS m/z: 413 [M+1]; Anal.calcd. for C H ClIO ; C, 46.57; H,
16 10 3
.44. Found: C, 46.62; H, 2.56.
.1.2.10 6-Bromo-4-(2-iodo-phenoxymethyl)-chromen-2-one (6j)
o
-1
Colourless (Ethanol), m.p. 208 C, yield 56 %; IR (KBr, ν in cm ): 1728 (lactone C=O);
1
H NMR (400 MHz, DMSO-d ): δ 5.56 (s, 2H, C4-CH ), 7.03 (s, 1H, C3-H), 7.14-8.01
6
2
(
m, 7H, Ar-H); LCMS m/z: 459 [M+2]; Anal.calcd. for C H BrIO ; C, 42.05; H, 2.21.
16 10 3
Found: C, 42.21; H, 2.29.
1
6

ACCEPTED MANUSCRIPT
5
.1.2.11 4-(3-Iodo-phenoxymethyl)-6-methyl-chromen-2-one (8a)
o
-1
Colourless (Ethanol), m.p. 164 C, yield 82 %; IR (KBr, υ in cm ): 1715 (lactone C=O);
1
H NMR (400 MHz, DMSO-d ): δ 2.40 (s, 3H, C6-CH ), 5.40 (s, 2H, C4-CH ), 6.57 (s,
6
3
2
1
3
1
1
1
3
5
H, C3-H), 7.11-7.77 (m, 7H, Ar-H); C NMR (CDCl , 75 MHz): 21.67, 66.62, 112.27,
3
12.68, 113.61, 114.67, 117.57, 123.05, 125.54, 128.93, 133.06, 134.18, 143.27, 149.46,
51.98, 153.76, 160.83. LCMS m/z: 393 [M+1]; Anal.calc. for C H IO ; C, 52.06; H,
1
7
13
3
.34. Found: C, 52.24; H, 3.49
.1.2.12 4-(3-Iodo-phenoxymethyl)-7-methyl-chromen-2-one (8b)
o
-1
Colourless (Ethanol), m.p. 170 C, yield 92 %; IR (KBr, υ in cm ): 1711 (lactone C=O);
1
H NMR (400 MHz, DMSO-d ): δ 2.43 (s, 3H, C7-CH ), 5.39 (s, 2H, C4-CH ), 6.46 (s,
6
3
2
1
3
1
1
1
3
5
H, C3-H), 7.05-7.71 (m, 7H, Ar-H); C NMR (100 MHz, DMSO-d ): 21.12, 66.08,
6
10.84, 113.53, 117.24, 117.08, 121.21, 126.36, 129.82, 131.40, 133.02, 133.81, 144.22,
46.01, 152.17, 160.14. LCMS m/z: 393 [M+1]; Anal.calc. for C H IO ; C, 52.06; H,
1
7
13
3
.34. Found: C, 52.28; H, 3.40
.1.2.13 4-(3-Iodo-phenoxymethyl)-7,8-dimethyl-chromen-2-one (8c)
o
-1
Colourless (Ethanol), m.p. 186 C, yield 83 %; IR (KBr, υ in cm ): 1714 (lactone C=O);
1
H NMR (400 MHz, DMSO-d ): δ 2.41 (s, 6H, C7 & C8-CH ), 5.40 (s, 2H, C4-CH ),
6
3
2
6
.47 (s, 1H, C3-H), 7.04-7.92 (m, 6H, Ar-H); LCMS m/z: 407 [M+1]; Anal.calc. for
C H IO ; C, 53.22; H, 3.72. Found: C, 53.38; H, 3.80.
1
7
13
3
5
.1.2.14 4-(3-Iodo-phenoxymethyl)-6-methoxy-chromen-2-one (8d)
o
-1
Colourless (Ethanol), m.p. 167 C, yield 83 %; IR (KBr, υ in cm ): 1707 (lactone C=O);
1
H NMR (400 MHz, DMSO-d ): δ 3.87 (s, 3H, C6-OCH ), 5.38 (s, 2H, C4-CH ), 6.62 (s,
6
3
2
1
7

ACCEPTED MANUSCRIPT
1
3
1
1
1
3
5
H, C3-H), 6.88-7.39 (m, 7H, Ar-H); C NMR (100 MHz, DMSO-d ): 56.09, 66.42,
6
09.46, 112.38, 113.42, 117.57, 118.37, 119.85, 121.38, 129.89, 132.60, 144.25, 145.78,
48.55, 155.89, 160.83. LCMS m/z: 409 [M+1]; Anal.calc. for C H IO ; C, 50.06; H,
1
7
13
4
.21. Found: C, 50.21; H, 3.25.
.1.2.15 4-(3-Iodo-phenoxymethyl)-7-methoxy-chromen-2-one (8e)
o
-1
Colourless (Ethanol), m.p. 158 C, yield 69 %; IR (KBr, υ in cm ): 1709 (lactone C=O);
1
H NMR (400 MHz, DMSO-d ): δ 3.88 (s, 3H, C7-OCH ), 5.39 (s, 2H, C4-CH ), 6.60
6
3
2
1
3
(
s, 1H, C3-H), 6.97-7.67 (m, 7H, Ar-H); C NMR (100 MHz, DMSO-d ): 56.97, 66.82,
6
1
1
3
5
09.62, 112.81, 113.52, 117.70, 118.83, 120.03, 121.83, 129.39, 132.51, 144.48, 145.60,
48.84, 155.80, 160.16. LCMS m/z: 409 [M+1]; Anal.calc. for C H IO ; C, 50.06; H,
1
7
13
4
.21. Found: C, 50.29; H, 3.31.
.1.2.16 1-(3-Iodo-phenoxymethyl)-benzo[f]chromen-3-one (8f)
o
-1
1
Grey (Ethanol), m.p. 168 C, yield 91 %; IR (KBr, υ in cm ): 1717 (lactone C=O); H
NMR (400 MHz, DMSO-d ): δ 5.46 (s, 2H, C4-CH ), 6.63 (s, 1H, C3-H), 7.19-8.69 (m,
6
2
1
0H, Ar-H); LCMS m/z: 429 [M+1]; Anal.calc. for C H IO ; C, 56.10; H, 3.06. Found:
20 13 3
C, 56.29; H, 3.22
5
.1.2.17 4-(3-Iodo-phenoxymethyl)-benzo[h]chromen-2-one (8g)
o
-1
1
Brown(Acetone), m.p. 206 C, yield 90 %; IR (KBr, υ in cm ): 1713 (lactone C=O); H
NMR (400 MHz, DMSO-d ): δ 5.47 (s, 2H, C4-CH ), 6.64 (s, 1H, C3-H), 7.15-8.65 (m,
6
2
1
0H, Ar-H); LCMS m/z: 429 [M+1]; Anal.calc. for C H IO ; C, 56.10; H, 3.06. Found:
20 13 3
C, 56.32; H, 3.26.
5
.1.2.18 6-Chloro-4-(3-iodo-phenoxymethyl)-chromen-2-one (8h)
1
8

ACCEPTED MANUSCRIPT
o
-1
1
Grey (Ethanol), m.p. 203 C, yield 65 %; IR (KBr, υ in cm ): 1712 (lactone C=O); H
NMR (400 MHz, DMSO-d ): δ 5.38 (s, 2H, C4-CH ), 6.51 (s, 1H, C3-H), 7.10-7.76 (m,
6
2
1
3
7
1
H, Ar-H); C NMR (100 MHz, DMSO-d ): 66.02, 112.26, 118.04, 118.57, 121.16,
6
26.18, 128.24, 128.11, 129.18, 131.16, 131.40, 143.48, 152.08, 159.57. LCMS m/z: 413
[
M+1]; Anal.calc. for C H ClIO ; C, 46.57; H, 2.44. Found: C, 46.36; H, 2.36
16 10 3
5
.1.2. 19 7-Chloro-4-(3-iodo-phenoxymethyl)-chromen-2-one (8i)
o
-1
Colourless (Ethanol), m.p. 195 C, yield 62 %; IR (KBr, υ in cm ): 1710 (lactone C=O);
1
H NMR (400 MHz, DMSO-d ): δ 5.38 (s, 2H, C4-CH ), 6.53 (s, 1H, C3-H), 7.06-7.68
6
2
(
m, 7H, Ar-H); LCMS m/z: 413 [M+1]; Anal.calc. for C H ClIO ; C, 46.57; H, 2.44.
16 10 3
Found: C, 46.29; H, 2.31.
5
.1.2.20 6-Bromo-4-(3-iodo-phenoxymethyl)-chromen-2-one (8j)
o
-1
Colourless (Ethanol), m.p. 214 C, yield 58 %; IR (KBr, υ in cm ): 1708 (lactone C=O);
1
H NMR (400 MHz, DMSO-d ): δ 5.24 (s, 2H, C4-CH ), 6.49 (s, 1H, C3-H), 7.13-7.89
6
2
(
m, 7H, Ar-H); LCMS m/z: 459 [M+2]; Anal.calc. for C H BrIO ; C, 42.05; H, 2.21.
16 10 3
Found: C, 42.31; H, 2.34.
5
.1.2.21 4-(4-Iodo-phenoxymethyl)-6-methyl-chromen-2-one (10a)
o
-1
Colourless (Ethyl acetate), m.p. 192 C, yield 94 %; IR (KBr, υ in cm ): 1704 (lactone
1
C=O); H NMR (400 MHz, DMSO-d ): δ 2.40 (s, 3H, C6-CH ), 5.40 (s, 2H, C4-CH ),
6
3
2
1
3
6
6
.53 (s, 1H, C3-H), 7.01-7.68 (m, 7H, Ar-H); C NMR (100 MHz, DMSO-d ): 20.69,
6
6.90, 112.93, 116.53, 116.63, 120.86, 125.88, 129.41, 130.99, 132.59, 133.36, 143.75,
1
9

ACCEPTED MANUSCRIPT
1
3
5
45.57, 151.77, 160.26.LCMS m/z: 393 [M+1]; Anal.calc. for C H IO ; C, 52.06; H,
1
7
13
3
.34. Found: C, 52.24; H, 3.38.
.1.2.22 4-(4-Iodo-phenoxymethyl)-7-methyl-chromen-2-one (10b)
o
-1
Green (Ethyl acetate), m.p. 148 C, yield 89 %; IR (KBr, υ in cm ): 1708 (lactone C=O);
1
H NMR (400 MHz, DMSO-d ): δ 2.40 (s, 3H, C7-CH ), 5.48 (s, 2H, C4-CH ), 6.72 (s,
6
3
2
1
3
1
1
1
3
5
H, C3-H), 7.07-7.69 (m, 7H, Ar-H); C NMR (100 MHz, DMSO-d ): 21.10, 66.12,
6
10.83, 113.21, 117.01, 117.12, 121.23, 126.36, 129.62, 131.36, 133.02, 133.69, 144.16,
46.01, 152.18, 160.68. LCMS m/z: 393 [M+1]; Anal.calc. for C H IO ; C, 52.06; H,
1
7
13
3
.34. Found: C, 52.49; H, 3.44.
.1.2.23 4-(4-Iodo-phenoxymethyl)-7,8-dimethyl-chromen-2-one (10c)
o
-1
Colourless (Ethyl acetate), m.p. 232 C, yield 83 %; IR (KBr, υ in cm ): 1708 (lactone
1
C=O); H NMR (400 MHz, DMSO-d ): δ 2.43 (s, 3H, C7 & C8-CH ), 5.41 (s, 2H, C4-
6
3
CH ), 6.61 (s, 1H, C3-H), 6.80-7.85 (m, 7H, Ar-H); LCMS m/z: 407 [M+2]; Anal.calc.
2
for C H IO ; C, 53.22; H, 3.72. Found: C, 53.06; H, 3.60.
1
8
15
3
5
.1.2.24 4-(4-Iodo-phenoxymethyl)-6-methoxy-chromen-2-one (10d)
o
-1
Pale yellow (Ethyl acetate), m.p. 196 C, yield 94 %; IR (KBr, υ in cm ): 1707 (lactone
1
C=O); H NMR (400 MHz, DMSO-d ): δ 3.87 (s, 3H, C6-OCH ), 5.25 (s, 2H, C4-CH ),
6
3
2
1
3
6
6
1
5
.68 (s, 1H, C3-H), 6.91-7.86 (m, 7H, Ar-H); C NMR (100 MHz, DMSO-d ): 56.02,
6
6.30, 109.18, 112.29, 113.40, 117.51, 118.33, 119.76, 121.24, 129.68, 132.46, 144.21,
45.62, 148.61, 155.77, 160.12. LCMS m/z: 409 [M+1]; Anal.calc. for C H IO ; C,
1
7
13
3
0.02; H, 3.21. Found: C, 50.14; H, 3.32.
2
0

ACCEPTED MANUSCRIPT
5
.1.2.25 4-(4-Iodo-phenoxymethyl)-7-methoxy-chromen-2-one (10e)
o
-1
Pale yellow (Ethyl acetate), m.p. 188 C, yield 88 %; IR (KBr, υ in cm ): 1711 (lactone
1
C=O); H NMR (400 MHz, DMSO-d ): δ 3.89 (s, 3H, C7-OCH ), 5.29 (s, 2H, C4-CH ),
6
3
2
1
3
6
6
1
5
5
.70 (s, 1H, C3-H), 6.73-7.91 (m, 7H, Ar-H); C NMR (100 MHz, DMSO-d ): 57.08,
6
6.10, 109.18, 112.24, 113.46, 117.58, 118.80, 120.01, 121.78, 129.21, 132.58, 144.41,
45.73, 148.69, 155.76, 160.10. LCMS m/z: 409 [M+1]; Anal.calc. for C H IO ; C,
1
7
13
3
0.02; H, 3.21. Found: C, 50.26; H, 3.36.
.1.2.26 1-(4-Iodo-phenoxymethyl)-benzo[f]chromen-3-one (10f)
o
-1
Green (Ethyl acetate), m.p. 188 C, yield 86 %; IR (KBr, υ in cm ): 1706 (lactone C=O);
1
H NMR (400 MHz, DMSO-d ): δ 5.88 (s, 2H, C4-CH ), 7.34 (s, 1H, C3-H), 7.11-8.12
6
2
(
m, 10H, Ar-H); LCMS m/z: 429 [M+1]; Anal.calc. for C H IO ; C, 56.10; H, 3.06.
20 13 3
Found: C, 56.01; H, 3.02.
5
.1.2.27 4-(4-Iodo-phenoxymethyl)-benzo[h]chromen-2-one (10g)
o
-1
Yellow (Ethyl acetate), m.p. 240 C, yield 84 %; IR (KBr, υ in cm ): 1701 (lactone
1
C=O); H NMR (400 MHz, DMSO-d ): δ 5.65 (s, 2H, C4-CH ), 7.28 (s, 1H, C3-H), 6.93-
6
2
8
3
5
.05 (m, 10H, Ar-H); LCMS m/z: 429 [M+1]; Anal.calc. for C H IO ; C, 56.10; H,
20 13 3
.06. Found: C, 56.03; H, 3.04.
.1.2.28 6-Chloro-4-(4-iodo-phenoxymethyl)-chromen-2-one (10h)
o
-1
Yellow (Ethyl acetate), m.p. 196 C, yield 76 %; IR (KBr, υ in cm ): 1716 (lactone
1
C=O); H NMR (400 MHz, DMSO-d ): δ 5.17 (s, 2H, C4-CH ), 6.74 (s, 1H, C3-H), 6.90-
6
2
1
3
7
.71 (m, 7H, Ar-H); C NMR (100 MHz, DMSO-d ): 66.70, 112.94, 118.02, 118.79,
6
2
1

ACCEPTED MANUSCRIPT
1
21.06, 126.13, 128.14, 128.26, 129.44, 131.34, 131.72, 143.63, 152.30, 159.73. LCMS
m/z: 413 [M+1]; Anal.calc. for C H ClIO ; C, 46.57; H, 2.44. Found: C, 46.36; H, 2.38.
1
6
10
3
5
.1.2.29 7-Chloro-4-(4-iodo-phenoxymethyl)-chromen-2-one (10i)
o
-1
Pale yellow (Ethyl acetate), m.p. 210 C, yield 71 %; IR (KBr, υ in cm ): 1713 (lactone
1
C=O); H NMR (400 MHz, DMSO-d ): δ 5.21 (s, 2H, C4-CH ), 6.81 (s, 1H, C3-H), 6.97-
6
2
7
2
5
.85 (m, 7H, Ar-H); LCMS m/z: 413 [M+1]; Anal.calc. for C H ClIO ; C, 46.57; H,
16 10 3
.44. Found: C, 46.41; H, 2.40.
.1.2.30 6-Bromo-4-(4-iodo-phenoxymethyl)-chromen-2-one (10j)
o
-1
Colourless (Ethyl acetate), m.p. 179 C, yield 66 %; IR (KBr, υ in cm ): 1713 (lactone
1
C=O); H NMR (400 MHz, DMSO-d ): δ 5.42 (s, 2H, C4-CH ), 6.72 (s, 1H, C3-H), 6.90-
6
2
7
2
.74 (m, 7H, Ar-H); LCMS m/z: 459 [M+2]; Anal.calc. for C H BrIO ; C, 42.05; H,
16 10 3
.21 Found: C, 42.16; H, 2.34.
5
.2 Anti-Cancer activity
4
In all the experiments, different cell lines were seeded at a final density of 2 x 10
cells/well, in 96 well microtiter plates. Cytotoxicty was measured using the MTT [3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay, according to the method
4
of Mosmann (1983) [46]. Briefly, the cells (2 x 10 ) were seeded in each well containing
0
.1 mL of medium in 96 well plates. After overnight incubation, the cells were treated
with different test concentrations of test compounds (5-200 mg/mL) at identical
conditions.. The cell viability was assessed after 24 h, by adding 10 mL of MTT (5
o
mg/mL) per well. The plates were incubated at 37 C for additional 3 h. The medium was
2
2

ACCEPTED MANUSCRIPT
discarded and the formazan blue, which formed in the cells, was dissolved with 100 mL
of DMSO. The rate of colour production was measured at 570 nm in a spectrophotometer
(
Spectra MAX Plus; Molecular Devices; supported by SOFTmax PRO-5.4). The percent
inhibition of cell viability was determined with reference to the control values (without
test compound). The data were subjected to linear regression analysis and the regression
lines were plotted for the best straight-line fit. The IC (inhibition of cell viability)
5
0
concentrations were calculated using the respective regression equation.
5
.3 Anti-mycobacterial activity
The anti-mycobacterial activity of compounds was assessed against M.
tuberculosis H RV and M. phlei using Microplate Alamar Blue Assay (MABA) [47].
3
7
This methodology is non-toxic, uses a thermally stable reagent and shows good
correlation with proportional and BACTEC radiometric method. Briefly, 200 µL of
sterile deionized water was added to all outer perimeter wells of sterile 96-well plate to
minimize evaporation of medium in the test wells during incubation. The 96-well plate
received 100 µL of the Middlebrook 7H9 broth, and serial dilution of compounds was
made directly on plate. The final drug concentrations tested were 100–0.2 µg ⁄ mL. Plates
o
were covered and sealed with parafilm and incubated at 37 C for 5 days. After this time,
2
5 µL of freshly prepared 1:1 mixture of Almar Blue reagent and 10% tween 80 were
added to the plate and incubated for 24 h. A blue colour in the well was interpreted as no
bacterial growth, and pink colour was scored as growth. The MIC was defined as lowest
drug concentration that prevented the colour change from blue to pink.
2
3

ACCEPTED MANUSCRIPT
Acknowledgements
The author Mahantesha B, thankful to the Principal, P.C. Jabin Science College, Hubli
and K.L.E. Society, Belgaum.
References:
[
1] S.K. Arya, S. Bhansali, Lung cancer and its early detection using biomarker-based
biosensors, Chem. Rev. 111 (2011) 6783–6809.
[
(
[
2] M. Ljungman, Targeting the DNA damage response in cancer, Chem. Rev. 109
2009) 2929–2950.
3] M. Jeyachandran, P. Ramesh, D. Sriram, P. Senthilkumar, P. Yogeeswari, Synthesis
and in vitro antitubercular activity of 4-aryl/alkylsulfonylmethylcoumarins as inhibitors
of mycobacterium tuberculosis. Bioorg. Med. Chem. Lett. 22 (2012) 4807–4809.
[
4] A. Lacy, Studies on coumarins and coumarin-related compounds to determine their
therapeutic role in the treatment of cancer, Curr. Pharma. Des. 10 (2004) 3797-3811.
5] F. Borges, F. Roleira, N. Milhazes, L. Santana and E. Uriarte, Simple coumarins and
[
analogues in medicinal chemistry: occurrence, synthesis and biological activity, Curr.
Med. Chem. 12 (2005) 887-916.
[
6] I. Kostova, Synthetic and natural coumarins as cytotoxic agents, Anti-Cancer Agents
in Medicinal Chemistry, 5 (2005) 29-46.
2
4

ACCEPTED MANUSCRIPT
[
7] M.V. Kulkarni, G.M. Kulkarni, C.H. Lin, C.M. Sun, Recent advances in coumarins
and 1-azacoumarins as versatile biodynamic agents, Curr. Med. Chem. 13 (2006) 2795-
818.
8] M.A. Musa, J.S. Cooperwood, M.O. F. Khan, A review of coumarin derivatives in
pharmacotherapy of breast cancer, Curr. Med. Chem. 15 (2008) 2664-2679.
9] L. Wu, X. Wang, W. Xu, F. Farzaneh, R. Xu, The structure and pharmacological
functions of coumarins and their derivatives, Curr. Med. Chem. 16 (2009) 4236-4260.
10] M.E. Riveiro, N.D. Kimpe, A. Moglioni, R. Vazquez, F. Monczor, C. Shayo, C.
2
[
[
[
Davio, Coumarins: old compounds with novel promising therapeutic perspectives, Curr.
Med. Chem. 17 (2010) 1325-1338.
[
(
[
11] C. Kontogiorgis, A. Detsi, D.H. Litina, Coumarin-based drugs: a patent review
2008 - present), Expert Opinion on Therapeutic Patents, 22 (2012) 437-454.
12] X.M. Peng, G.L.V. Damu, C.H. Zhou, Current developments of coumarin
compounds in medicinal chemistry, Curr. Pharma. Des. 19 (2013) 3884-3930.
13] L.W.L. Woo, A. Purohit, B. Malini, M.J. Reed, B.V.L. Potter, Potent active site-
[
directed inhibition of steroid sulphatase by tricyclic coumarin-based sulphamates, Chem.
Biol. 7 (2000), 773-791.
[
14] B. Malini, A. Purohit, D. Ganeshapillai, L.W.L. Woo, B.V.L. Potter, M.J. Reed,
Inhibition of steroid sulphatase activity by tricyclic coumarin sulphamates, J. Steroid
Biochem. Mol. Biol. 75 (2000), 253-258.
2
5

ACCEPTED MANUSCRIPT
[
15] A. Purohit, L.W.L. Woo, B.V.L. Potter, M.J. Reed, In vivo inhibition of estrone
sulfatase activity and growth of nitrosomethylureainduced mammary tumors by 667
COUMATE, Cancer Res. 60 (2000) 3394-3396.
[
16] F. Meggio, M.A. Pagano, S. Moro, G. Zagotto, M. Ruzzene, S. Sarno, G. Cozza, J.
Bain, M. Elliott, A.D. Deana, A.M. Brunati, L.A. Pinna, Inhibition of protein kinase CK2
by condensed polyphenolic derivatives. An in vitro and in vivo study, Biochemistry. 43
(
[
2004) 12931-12936.
17] A. Chilin, R. Battistutta, A. Bortolato, G. Cozza, S. Zanatta, G. Poletto, M.
Mazzorana, G. Zagotto, E. Uriarte, A. Guiotto, L.A. Pinna, F. Meggio, S. Moro,
Coumarin as attractive casein kinase 2 (CK2) inhibitor scaffold: an integrate approach to
elucidate the putative binding motif and explain structure-activity relationships, J. Med.
Chem. 51 (2008) 752-759.
[
18] G.L. Bras, C. Radanyi, J.F. Peyrat, J.D. Brion, M. Alami, V. Marsaud, B. Stella,
J.M. Renoir, New novobiocin analogues as antiproliferative agents in breast cancer cells
and potential inhibitors of heat shock protein 90, J. Med. Chem. 50 (2007) 6189-6200.
[
19] A.C. Donnelly, J.R. Mays, J.A. Burlison, J.T. Nelson, G. Vielhauer, J. Holzbeierlein,
B.S.J. Blagg, The design, synthesis, and evaluation of coumarin ring derivatives of the
novobiocin scaffold that exhibit antiproliferative activity, J. Org. Chem. 73 (2008) 8901–
8
920.
20] H.P. Zhao, A.C. Donnelly, B.R. Kusuma, G.E.L. Brandt, D. Brown, R.A. Rajewski,
G. Vielhauer, J. Holzbeierlein, M.S. Cohen, B.S.J. Blagg, Engineering an antibiotic to
[
2
6

ACCEPTED MANUSCRIPT
fight cancer: optimization of the novobiocin scaffold to produce anti-proliferative agents,
J. Med. Chem. 54 (2011), 3839-3853.
[
21] H.P. Zhao, B. Yan, L.B. Peterson, B.S.J. Blagg, 3-Arylcoumarin derivatives
manifest anti-proliferative activity through Hsp90 inhibition, ACS Med. Chem. Lett. 3
(
[
2012) 327−331.
22] S. Ananthan, E.R. Faaleolea, R.C. Goldman, J.V. Hobrath, C.D. Kwong, B.E.
Laughon, J.A. Maddry, A. Mehta, L. Rasmussen, R.C. Reynolds, J.A. Secrist III, N.
Shindo, D.N. Showe, M.I. Sosa, W.J. Suling, E.L. White. High-throughput screening for
inhibitors of mycobacterium tuberculosis H37Rv. Tuberculosis, 89 (2009) 334-353.
[
23] R.C. Reynolds, S. Ananthan, E. Faaleolea, J.V. Hobrath, C.D. Kwong, C. Maddox,
L. Rasmussen, M.I. Sosa, E. Thammasuvimol, E.L. White, W. Zhang, J.A. Secrist III.
High throughput screening of a library based on kinase inhibitor scaffolds against
Mycobacterium tuberculosis H37Rv. Tuberculosis, 92 (2012) 72-83.
[
24] F. Borges, F. Roleira, N. Milhazes, L. Santana, E. Uriarte, Simple coumarins and
analogues in medicinal chemistry: occurrence, synthesis and biological activity Curr.
Med. Chem. 12 (2005) 887-916.
[
25] H. Kohno, R. Suzuki, M. Curini, F. Epifano, F. Maltese, S.P. Gonzales, T. Tanaka,
Dietary administration with prenyloxycoumarins, auraptene and collinin, inhibits colitis-
related colon carcinogenesis in mice. Int. J. Cancer, 118 (2006) 2936–2942.
[
26] W. Liu, J. Hua, J. Zhou, H. Zhang, H. Zhu, Y. Cheng, R. Gust, Synthesis and in vitro
antitumor activity of novel scopoletin derivatives, Bioorg. Med. Chem. Lett. 22 (2012)
008–5012.
5
2
7

ACCEPTED MANUSCRIPT
[
27] K.V. Sashidhara, S.R. Avula, K. Sharma, G.R. Palnati, S. R. Bathula, Discovery of
coumarin-monastrol hybrid as potential antibreast tumor-specific agent. Euro. J. Med.
Chem. 60 (2013) 120-127.
[
28] Z.Q. Xu, W.W. Barrow, W.J. Suling, L. Westbrook, E. Barrow, Y.M. Lin, M.T.
Flavin, Anti-HIV natural product (+)-calanolide A is active against both drug-susceptible
and drug-resistant strains of Mycobacterium tuberculosis. Bioorg. Med. Chem. 12 (2004)
1
199–1207.
[
29] A. Manvar, A. Malde, J. Verma, V. Virsodia, A. Mishra, K. Upadhyay, H. Acharya,
E. Coutinho, A. Shah, Synthesis, anti-tubercular activity and 3D-QSAR study of
coumarin-4-acetic acid benzylidene hydrazides. Euro. J. Med. Chem. 43 (2008) 2395-
2
403.
[
30] S.H. Cardoso, M.B. Barreto, M.C.S. Lourenco, M.G.M.O. Henriques, A.L.P.
Candea, C.R. Kaiser, M.V.N. Souza, Antitubercular activity of new coumarins. Chem.
Biol. Drug Des. 77 (2011) 489-493.
[
31] R.J. Naik, M.V. Kulkarni, K.S.R. Pai, P.G. Nayak, Click chemistry approach for bis-
chromenyl triazole hybrids and their antituberculosis activity. Chem. Bio. Drug Des. 80
(
[
2012) 516-523.
32] Y. Lu, Y. Wang, W. Zhu, Nonbonding interactions of organic halogens in biological
systems: implications for drug discovery and biomolecular design, Phys. Chem. Chem.
Phys. 12 (2010) 4543–4551.
2
8