Discovery of coumarin-monastrol hybrid as potential antibreast tumor-specific agent

Article abstract of DOI:10.1016/j.ejmech.2012.11.044

Development of new, targeted antibreast cancer drug which can treat both the hormone receptor (positive and negative) breast cancers is a very challenging task. The concept of molecular hybridization led us to discover a novel class of coumarin-monastrol

Full text of DOI:10.1016/j.ejmech.2012.11.044

Accepted Manuscript
Discovery of coumarin-monastrol hybrid as potential antibreast tumor-specific agent
Koneni V. Sashidhara, Srinivasa Rao Avula, Komal Sharma, Gopala Reddy Palnati,
Surendar Reddy Bathula
PII:
S0223-5234(12)00721-0
10.1016/j.ejmech.2012.11.044
EJMECH 5870
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 5 September 2012
Revised Date: 10 November 2012
Accepted Date: 30 November 2012
Please cite this article as: 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, European Journal
of Medicinal Chemistry (2013), doi: 10.1016/j.ejmech.2012.11.044.
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Research Highlights
•
•
Discovery of coumarin-monastrol hybrids with anti-breast cancer activity.
Compound 10 inhibits the proliferation of both primary and metastatic breast cancer
cells.
•
Compound 10 induces apoptosis in breast cancer cells and arrests cell cycle at G1 phase.

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Graphical abstract
Discovery of coumarin-monastrol hybrid as potential antibreast
tumor-specific agent
Koneni V. Sashidhara *, Srinivasa Rao Avula, Komal Sharma, Gopala Reddy Palnati, and Surendar Reddy
Bathula *
A novel series of coumarin-monastrol hybrids were synthesized and evaluated for their in
vitro anticancer activity, the compound 10 exhibited selective and potent antibreast cancer
activity.

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Discovery of coumarin-monastrol hybrid as potential antibreast
tumor-specific agent*
Koneni V. Sashidhara*a, Srinivasa Rao Avula,a Komal Sharma,b Gopala Reddy Palnati,a and Surendar
Reddy Bathula*b
a Department of Medicinal and Process Chemistry, b Department of Pharmaceutics,
CSIR-Central Drug Research Institute, Lucknow-226001 (INDIA)
Abstract
Development of new, targeted antibreast cancer drug which can treat both the hormone
receptor (positive and negative) breast cancers is a very challenging task. The concept of
molecular hybridization led us to discover a novel class of coumarin-monastrol hybrid, as a
novel breast cancer agent which selectively induce apoptosis in both primary and metastatic
breast cancer cell lines.
Keywords: Coumarin, Monastrol, Hybrids, Antibreast tumor.
* Part XXIV in the series, “Advances in drug design and discovery”.
*Corresponding author. Tel.: +91 9919317940; Fax: +91-522-2628493.
Email: sashidhar123@gmail.com, kv_sashidhara@cdri.res.in, bsreddy@cdri.res.in
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1. Introduction
Breast cancer is the most common malignancy and the second leading cause of cancer deaths
in women today [1]. Since estrogens are well known to play crucial roles in breast cancer
development, much effort has been devoted to block estrogen formation [2]. The most widely
used therapy for antagonizing estrogen receptor (ER) function is the antiestrogen tamoxifen
(TAM), which binds to the ER and blocks downstream signaling [3]. However, current breast
cancer therapies with TAM achieve meaningful clinical results in only 40% of patients
because drug resistance usually develops within couple of years of treatment. This resistance
occurs via several mechanisms, such as induction of estrogen-independent pathways for
breast cancer cell growth, over expression of human epidermal growth factor receptor 2
(HER2), the functional crosstalk between ER and HER2, etc [4]. In recent years, combination
chemotherapy has been a common clinical strategy adopted to treat cancer and/ or to
overcome drug resistance (e.g. the combination of an antiestrogen TAM with another
cytotoxic drug, such as anastrozole, an aromatase inhibitor) [5]. But then, patients still relapse
and cancer reoccurs, therefore the development of more potent antibreast cancer agents that
combine the desired, tissue selective effects with novel structures or new mechanism of
action are still needed. Currently, molecules used to treat either primary or metastatic breast
cancers are limited and the development of new antibreast cancer molecules is still very
challenging.
In the design of new drug prototypes, the concept of molecular hybridization is a useful
tool and is based on the combination of pharmacophoric moieties of different bioactive
substances to produce a new hybrid compound with improved affinity and efficacy [6].
This strategy has resulted in compounds with modified selectivity profile, different and/or
dual mode of action and reduced undesired side effects. Recently, Musa et al. have
reviewed the application of potential coumarin derivatives in the pharmacotherapy of
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breast cancer using this concept [7]. Also , Lee et al. have reported the isolation of neo-
tanshinlactone, coumarin containing compound, showing significant inhibition activity
against two ER+ human breast cancer cell lines in comparison to TAM (i.e 10-fold more
potent and 20-fold more selective than TAM) [8]. On the other hand monastrol, a
structurally simple dihydropyrimidinone (DHPM), has been identified as a novel cell
permeable molecule that caused mitotic arrest by blocking bipolar mitotic spindle in
mammalian cells [9]. It is the first mitotic kinesin Eg5 (also called kinesin-5 or kinesin
spindle protein [KSP]) inhibitor (IC50=14µM) causing a specific and reversible cell cycle
block. Moreover, it is considered as a new lead in anticancer drug development as it
specifically inhibits mitotic kinesin Eg5 motor protein. Although, the antimitotic activity
of monastrol is not very high, the development of more potent, specific and cell
permeable monastrol analogues with enhanced kinesin inhibitory and anti-proliferative
properties has been carried out in recent years with encouraging results [10,11].
In continuation of our anticancer drug discovery programme [12], in order to further
explore the structure-activity relationship (SAR) of the coumarin ring system [12b], we
envisioned construction of coumarin-monastrol hybrids, the prototype structure of which
is shown in Fig 1.
2. Chemistry
The detailed reaction conditions for the synthesis of coumarin-monastrol hybrids are
illustrated in Scheme 1. The synthesis of intermediate compounds (2a-b) was achieved by the
Duff formylation reaction of o-substituted phenols in the presence of hexamethylenetetramine
(HMTA) and trifluoroacetic acid (TFA) at 120 ºC. Reaction of compounds (2a-b) with
different substituted phenyl acetic acids in the presence of cyanuric chloride and N-methyl
morpholine (NMM) in DMF for 1 h afforded the respective 3-aryl coumarin aldehydes [13]
(3a-d) in excellent yield. Finally, the target compounds were synthesized via the Biginelli
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reaction involving 3-aryl coumarin aldehydes, ethyl aceto acetate and thiourea/urea in ethanol
in the presence of catalytic amount of pTSA to afford coumarin-monastrol hybrid compounds
(4-10). All the new synthesized compounds were characterized using NMR, IR and mass
spectrometry (see supporting information). Spectral data of all synthesized hybrid compounds
were in good agreement with the proposed structures. IR spectrum revealed the presence of
NH bond (3400-3580 cm-1), C=O and C=S (1690–1715 cm-1) functional groups in the
1
synthesized hybrid compounds. This was further confirmed by H NMR spectrum, which
displayed two additional singlets around δ 5-6 and δ 9-11 for chiral CH and NH in
dihydropyrimidone ring respectively. The molecular formula of all the synthesized
compounds was confirmed by HRMS data analysis. The purity of the compounds was
ascertained by HPLC and were found to be >95% pure
3. Results and discussion
3.1. Antiproliferative activity
The new hybrids were evaluated for their in vitro anticancer activity against cancerous
and non-cancerous cells at four different molar concentrations (2.5µM, 5µM, 10µM,
20µM). The cell lines used in this investigation include breast cancer cells (MCF-7, T47D
and MDA-MB-231) lung cancer cell line (A549), Human prostate lines (PC-3 and DU-
145), Human hepatocellular liver carcinoma cell line (HepG2) as well as non cancer cells
(NIH3T3, HEK-293 and isolated primary mouse skin fibroblasts). In this experiment, the
cells were treated with compounds dissolved in DMSO+PEG 400 cocktail at different
concentrations and kept in serum media for 48 h to observe antiproliferative activity using
3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. IC50 values
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of the new hybrids in µM against various cell lines are given in Table 1.
The results revealed that some of the coumarin-monastrol hybrids demonstrated efficient
antiproliferative activities by inhibiting cell growth with IC50 values in the micromolar
range. An increase in anticancer activity of thiourea containing coumarin-monastrol
hybrids (C5, C7, C9, C10) was observed with increasing number of methoxy group on the
benzene ring attached to the coumarin segment and the similar trend was observed in case
of urea containing derivatives except derivative 4 in hormone responsive breast cancer
cells. Also, coumarin-thiodihydropyrimidone with tertiary butyl group on the benzene
ring of coumarin segment, compound 10 (C10), significantly inhibited the proliferation of
MCF-7 (IC50~2.4µM), T47D (IC50~3.1µM) and MDA-MB-231 (IC50~3.9µM) breast
cancer cell lines at all concentrations in a time dependant manner (Fig. 2). Interestingly, it
did not show significant anticancer activity in other cancer cells such as prostate (PC-3
and DU-145), lung (A549) and hepatocellular (HepG2) at all tested concentrations (Table
1). Furthermore it displays no activity (inactive) in the non-cancer cells (NIH/3T3, HEK-
293 and isolated primary mouse skin fibroblasts). The comparative viability studies of
C10 and other known antibreast cancer drugs such as TAM and epirubicin in various cell
lines at 5µM concentration are illustrated in Fig. 3.
Toxicity result shows that the anti proliferative effects of C10, TAM and Epirubicin are
cell specific. In MCF-7 cells growth inhibitory effect of C10, TAM and epirubicin are
same but in drug resistant MDA-MB-231, TAM did not induce significant
antiproliferative effect, where as C10 and epirubicin are showing equal amount of
anticancer activity. In support of the above data, we also found the similar results in C10
treated estrogen receptor positive T47D cells. C10 and epirubicin equally inhibited the
growth of T47D cells, where as TAM growth inhibitory effect is not high at 5µM (Fig. 3).
From the above data it is obvious that C10 exhibits efficient, yet selective, anticancer
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activity in all form of breast cancer cells compared to tamoxifen while epirubicin showed
only marginally higher activity compared to C10.
3.2. Apoptosis studies
Towards finding the possible reason of how C10 could kill cancer cells, we have analyzed
the apoptosis induction in C10 treated breast cancer cells MCF-7 & MDA-MB-231.
Apoptosis is one of the major cell death pathways induced by anticancer agents and we
have evaluated apoptosis inducing effects of derivative C10 by Annexin V/Propidium
iodide (PI) binding. As can be seen in Fig. 4, the compound treatment (5µM) increased
the percentage of apoptotic cells in MCF-7 cells from 5.90% in control untreated cells to
54.58% in treated cells. In MDA-MB-231 (Fig. 4) cells the treatment of 5µM C10 also
increased the percentage of apoptotic cells in comparison to untreated control from 0.05%
to 13.41%. In case of normal cells like NIH3T3 there were no difference in the percentage
of apoptotic cells in C10 treated and untreated cells. From the above data it is evident that
C10 treatment truly induced apoptosis specifically in breast cancer cells irrespective of
ER status and did not induce apoptosis in normal cells.
3.3. Caspase-3 Activation assay
We have measured caspase-3 activation to further confirm the fact that C10 treatment
induces apoptosis in both primary as well as metastatic breast cancer cells MCF-7 and
MDA-MB-231. Caspase-3 can acts as an indicator of apoptosis induction since active
caspase-3 is present in apoptotic cells both by extrinsic and intrinsic pathways [14]. Fig. 5
shows the effect of C10, at the level of caspase-3 induction. There is a concentration
dependent increase in caspase-3 activity with the C10 treatment as compared with the
untreated control in cancer cells. These results suggested that the hybrid C10 treatment
induce caspase-3 activation and apoptosis in a concentration dependent manner in both
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the breast cancer cells MCF-7 & MDA-MB-231 but not in noncancer cells NIH3T3.
3.4. Cell Cycle analysis
The induction of apoptosis in C10 treated MCF-7 cells was further verified by flow
cytometric analysis of DNA content. Loss of DNA is a typical feature of apoptotic cells
[15]. Fig. 6 shows the DNA content histograms obtained after PI staining of
permeabilized cells that were treated with 5µM, 10µM and 20µM of hybrid C10 for 24 h.
In agreement with FITC-Annexin V binding results, a typical sub-diploid apoptotic peaks
were observed in MCF-7 cells treated with C10 at all treated concentrations (Fig. 6). The
FACS analysis of untreated MCF-7 cells, on the other hand, showed prominent G1,
followed by S and G2/M phases (Fig. 6). Only 4.90% of these cells showed hypodiploid
DNA (sub Go/G1 peaks). This value of 4.90% hypodiploid DNA in control cells
increased to 8.70% at 5µM, 26.21% at 10µM and 39.92% at 20µM of C10 treatment.
Along with increase in hypodiploid DNA in response to the C10 treatment the decrease of
the cells at S and G2 phase of cell cycle was also observed. The result hence indicates
that derivative 10 treatment arrests MCF-7 cell cycle at G1phase.
3.5. Effect of compound 10 on Cell Viability of Tamoxifen pretreated in MCF-7 and T47D
cells.
In order to check whether the growth-inhibitory activity of C10 is estrogen receptor (ER)
mediated or not, we have compared antiproliferative effect of derivative 10 in two
different ER positive breast cancer cells (MCF-7 and T47D) independantly, at tamoxifen
pretreated and untreated conditions. If the compound-10 growth inhibitory effect is ER
mediated, tamoxifen pretreatment can promote the proliferation of C10 treated MCF-7
cells. Fig. 7 shows the comparative antiproliferative efficiencies of C10 in the MCF-7
cells with or without pre- treatment of 5 µM tamoxifen. In MCF-7cells not treated with
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tamoxifen, the extent of cell viability was in general high compared to tamoxifen-
pretreated cells at all used concentrations and it was found to be statistically significant
(p< 0.05) at all concentrations. In support of this, we have performed the tamoxifen pre-
treatment experiment in another ER positive breast cancer cell T47D (Fig. 7B). In this
experiment we also found that the extent of C10 treated T47D cell viability was in
general high, compared to tamoxifen pre-treated cells. These results indicate that the C10
selective toxicity is ER independent in both the breast cancer cells.
4. Conclusion
In conclusion, we report the latent ability of coumarin-monastrol hybrid as a new class of
selective anti breast cancer agent. The mechanistic studies that underlie their selective
anti breast cancer activity are under investigation, this unique hybrid provides an
impending possibility to develop single agent therapy for hormone positive and hormone
negative primary and advanced breast cancer cells.
5. Experimental Methods
5.1. General information
All reagents were commercial and used without further purification. Chromatography was
carried on silica gel (60-120 and 100-200 mesh). All reactions were monitored by TLC, silica
gel plates with fluorescence F254 were used. Melting points were taken in open capillaries on
Complab melting point apparatus and are presented uncorrected. Infrared spectra were
1
13
recorded on a Perkin-Elmer FT-IR RXI spectrophotometer. H NMR and C NMR spectra
were recorded using Bruker Supercon Magnet DRX-300 spectrometers (operating at 300
MHz for 1H; 75 MHz respectively for 13C) using CDCl3, and DMSO-d6 as solvents and TMS
as internal standard. Chemical shifts are reported in parts per million. Splitting patterns are
described as singlet (s), broad singlet (brs), doublet (d), triplet (t), quartet (q), and multiplet
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(m). Electrospray ionization mass spectra (ESIMS) were recorded on Thermo Lcq Advantage
Max-IT. High resolution mass spectra (HRMS) were recorded on 6520 Agilent Q T of LC
MS/MS (Accurate mass). All the synthesized compounds were analyzed for their purity (>
95% area) by high performance liquid chromatography on Waters HPLC instrument
equipped with 1525 binary HPLC pump, 717 plus auto sampler and Thermo Synchronis C18
column (4.6 mm *250 mm, 5 µM). Peaks were analyzed and integrated using waters 2487
PDA detector from 200 nm to 400 nm with isocratic flow of water and acetonitrile (40:60)
with flow rate of 1 mL/min for 20 minutes.
Representative procedure for the synthesis of compound (2a/2b):
o-Cresol / 2-tert-Butyl phenol (1 equiv) and hexamethylenetetramine (1.2 equiv) were
dissolved in TFA (25 mL) and the solution was heated at 120 ºC for 5 h. After cooling to
room temperature, 10 % aq.H2SO4 (40 mL) was added and again the temperature maintained
(at 90-100 ºC) for two h. The solution was neutralised with NaHCO3 and extracted with
EtOAc. The combined organic layers were dried on Na2SO4, filtered, and concentrated to
dryness under reduced pressure. The crude products were purified over column
chromatography (60-120 mesh) to afford required compounds (2a/2b).
4-hydroxy-5-methylisophthalaldehyde (2a);
White solid; yield: 54%; 1H NMR (CDCl3, 300 MHz): δ 2.33 (s, 3H, CH3), 7.31 (s, 1H, Ar-
H), 7.98 (s, 1H, Ar-H), 9.91 (s, 1H, CHO), 9.99 (s, 1H, CHO), 11.83 (s, 1H, OH);
5-Tert-butyl-4-hydroxyisophthalaldehyde (2b);
Oily, yield 60%; 1H NMR (CDCl3, 300 MHz) δ: 1.46 (s, 9H, C(CH3)3), 7.99 (brs, 1H, Ar-H),
8.07 (brs, 1H, Ar-H), 9.93 (s, 1H, CHO), 9.99 (s, 1H, CHO), 12.39 (s, 1H, OH);
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Representative procedure for the synthesis of compounds (3a-3d):
To a mixture of cyanuric chloride (1.0 equiv) and N-methyl morpholine (1.5 equiv) in DMF
appropriate substituted phenyl acetic acid (1 equiv) was added and stirred at room
temperature for 10 min. After that, aldehyde (2a/2b, 1 equiv) was added, subsequently, the
resulting reaction mixture was refluxed for 45 min (monitored by TLC). The reaction mixture
was diluted with water and extracted with EtOAc. The combined organic layers were dried
on Na2SO4, filtered, and concentrated to dryness under reduced pressure. The residue was
purified over column chromatography (Al2O3, 70-230 mesh, neutral, hexane/DCM) provided
the pure compounds (3a-3d)
3-(4-Methoxyphenyl)-8-methyl-2-oxo-2H-chromene-6-carbaldehyde (3a):
Light yellow solid, yield: 96%; mp: 148-150 ºC; IR (KBr): 3033 (=C—H stretching), 1725
1
(C=O stretching), 1624 (ester), 1594 (C=C stretching), 1021(C—O—C stretching) cm-1; H
NMR (CDCl3, 300 MHz) δ: 2.53 (s, 3H, CH3), 3.84 (s, 3H, O-CH3), 6.96 (d, J = 8.5 Hz, 2H,
Ar-H), 7.67 (d, J = 8.9 Hz, 2H, 2 x Ar-H), 7.79 (s, 1H, Ar-H), 7.85-7.87 (m, 2H, Ar-H, CH
(vinylic)), 9.98 (s, 1H, CHO); 13C NMR (CDCl3, 75 MHz) δ: 15.6, 55.4, 114.1, 119.8, 126.5,
127.3, 128.1, 128.6, 129.9, 132.3, 132.4, 137.9, 155.4, 159.9, 160.5, 190.6;
3-(3,4-Dimethoxyphenyl)-8-methyl-2-oxo-2H-chromene-6-carbaldehyde (3b):
Light yellow solid, yield: 98%; mp: 178-180 ºC; IR (KBr): 3053 (=C—H stretching), 1730
1
(C=O stretching), 1630 (ester), 1590 (C=C stretching), 1026 (C—O—C stretching) cm-1; H
NMR (CDCl3, 300 MHz) δ: 2.56 (s, 3H, CH3), 3.94 (s, 3H, O-CH3), 3.95 (s, 3H, O-CH3),
6.96 (d, J = 9.0 Hz, 1H, Ar-H), 7.32–7.29 (m, 2H, Ar-H), 7.84 (s, 1H, Ar-H), 7.88-7.91 (m,
2H, Ar-H, CH (vinylic)), 10.01 (s, 1H, CHO); 13C NMR (CDCl3, 75 MHz) δ: 15.5, 56.0, 56.1,
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111.2, 119.8, 121.4, 126.8, 127.3, 128.0, 128.7, 132.4, 132.5, 138.2, 148.9, 150.2, 155.4,
159.9, 190.5;
8-Methyl-2-oxo-3-(3,4,5-trimethoxyphenyl)-2H-chromene-6-carbaldehyde (3c):
White solid, yield: 99%; mp: 196-198 ºC; IR (KBr): 3074 (=C—H stretching), 1728 (C=O
1
stretching), 1632 (ester), 1594 (C=C stretching), 1010 (C—O—C stretching) cm-1; H NMR
(CDCl3, 300 MHz) δ: 2.57 (s, 3H, CH3), 3.91 (s, 3H, O-CH3), 3.93 (s, 6H, 2 x O-CH3), 6.96
(s, 2H, 2 x Ar-H), 7.86 (s, 1H, Ar-H), 7.91-7.93 (m, 2H, Ar-H, CH (vinylic)), 10.02 (s, 1H,
13
CHO); C NMR (CDCl3, 75 MHz) δ: 15.6, 56.4, 61.0, 106.2, 119.7, 127.4, 128.2, 129.0,
129.6, 132.6, 132.8, 139.1, 139.4, 153.4, 155.5, 159.8, 190.5;
8-tert-butyl-2-oxo-3-(3,4,5-trimethoxyphenyl)-2H-chromene-6-carbaldehyde (3d):
Pale yellow colour crystals, yield: 90%; mp: 169-171 ºC; IR (KBr): 3091 (=C—H stretching),
1710 (C=O stretching), 1628 (ester), 1575 (C=C stretching), 1023 (C—O—C stretching) cm-
1; 1H NMR (CDCl3, 300 MHz) δ: 1.57 (s, 9H, C(CH3)3), 3.90 (s, 3H, O-CH3), 3.92 (s, 6H, 2 ×
O-CH3), 6.97(s, 1H, Ar-H), 7.87 (s, 1H, Ar-H), 7.95 (s, 1H, Ar-H), 8.03 (s, 1H, CH, (vinylic)
), 10.03 (s, 1H, CHO); 13C NMR (CDCl3, 75 MHz) δ: 29.7, 35.2, 56.3, 60.9, 106.0, 120.3,
128.2, 129.1, 129.4, 132.3, 139.1, 139.2, 139.5, 153.2, 155.8, 159.2, 190.6;
General procedure for the synthesis of coumarin-monastrol derivatives (4-10): To a solution
of 3-arylcoumarin-aldehyde (3a-3d) (1.0 equiv) in acetonitrile, ethylacetoacetate (1.0 equiv)
and urea/thiourea (1.5 equiv) were added. The reaction mixture was refluxed for 6-8 h. After
completion of reaction (monitored by TLC), the reaction mixture was cooled to room
temperature. Solid thus obtained was filtered and recrystallized in methanol, to afford pure
coumarin-dihydropyrimidinone derivatives (4-10) in good yields.
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(±)Ethyl4-(3-(4-methoxyphenyl)-8-methyl-2-oxo-2H-chromen-6-yl)-6-methyl-2-oxo-1,2,3,4-
tetrahydropyrimidine-5-carboxylate (4): White solid; yield: 69%; mp: 210-212 °C; IR (KBr,
cm-1): 3334 (N—H stretching), 3128 (=C—H stretching), 2973 (C—H stretching), 1691 (C=O
stretching), 1638 (ester), 1605 (C=C (Ar) stretching), 1450 (C—H bending), 1105 (C—O—C
stretching); 1H NMR (DMSO-d6, 300 MHz):
δ 1.10 (t, 3H, J = 7.0 Hz, O-CH2-CH3), 2.29 (s,
3H, Ar-CH3), 2.39 (s, 3H, =C-CH3, dihydropyrimidone ring), 3.81 (s, 3H, O-CH3), 3.99 (q,
2H, J = 6.1Hz, O-CH2-CH3), 5.20 (s, 1H, CH), 7.01 (d, 2H, J = 8.8 Hz, 2 × Ar-H), 7.35 (s,
1H, Ar-H), 7.42 (s, 1H, Ar-H), 7.71-7.77 (m, 3H, 2 × Ar-H, NH), 8.23 (s, 1H, CH (vinylic)),
13
9.23 (s, 1H, NH); C NMR (DMSO-d6, 75 MHz): 14.5, 15.5, 18.3, 54.1, 55.6, 59.6, 99.2,
114.0, 119.5, 124.0, 125.1, 126.5, 127.2, 130.2, 131.2, 139.8, 141.2, 149.1, 150.7, 152.2,
160.0, 160.3, 165.7; ESI-MS (m/z): 449 (M+H)+ (100%), 433 (50%); HRMS(m/z): calcd for
C25H24N2O6 (M+H) +: 449.1713, Found: 449.1709.
(±)Ethyl4-(3-(4-methoxyphenyl)-8-methyl-2-oxo-2H-chromen-6-yl)-6-methyl-2-thioxo-
1,2,3,4-tetrahydropyrimidine-5-carboxylate (5): White solid; yield: 72%; mp: 205 - 207 ºC;
IR (KBr, cm-1): 3434 (N—H stretching), 3178 (=C—H stretching), 2921 (C—H stretching),
1704 (C=S stretching), 1630 (ester), 1599(C=C (Ar) stretching), 1465 (C—H bending), 1114
1
(C—O—C stretching); H NMR (DMSO-d6, 300 MHz):
δ
:1.10 (t, 3H, J = 7.0 Hz, O-CH2-
CH3), 2.34 (s, 3H, Ar-CH3), 2.39 (s, 3H, =C-CH3, dihydropyrimidone ring), 3.81 (s, 3H, O-
CH3), 4.01 (q, 2H, J = 7.0 Hz, O-CH2-CH3), 5.23 (s, 1H, CH), 7.01 (d, 1H, J = 8.9 Hz, Ar-H),
7.33 (s, 1H, Ar-H), 7.41 (d, 1H, J = 1.7 Hz, Ar-H), 7.71 (d, 1H, J = 2.0 Hz, Ar-H), 7.74 (d,
13
1H, J = 1.9 Hz, Ar-H), 8.22 (s, 1H, CH (vinylic)), 9.67 (s, 1H, NH), 10.35 (s, 1H, NH); C
NMR (DMSO-d6, 75 MHz): 14.5, 15.6, 17.4, 54.1, 55.6, 60.0, 100.5, 114.0, 119.7, 124.2,
125.3, 126.7, 127.2, 130.2, 131.3, 139.7, 145.9, 150.9, 160.0, 160.2, 165.4, 174.4; ESI-MS
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(m/z): 465 (M+H)+.(100%), 433 (6%), 389 (8%); HRMS(m/z): calcd for C25H24N2O5S (M+H)
+ 465.1484, Found: 465.1484.
(±)Ethyl4-(3-(3,4-dimethoxyphenyl)-8-methyl-2-oxo-2H-chromen-6-yl)-6-methyl-2-oxo-
1,2,3,4-tetrahydropyrimidine-5-carboxylate (6): Pale green solid; yield: 63%; mp: 223-225
°C; IR (KBr, cm-1): 3427 (N—H stretching ), 3018 (=C—H stretching), 2963 (C—H
stretching), 1700 (C=O stretching), 1645 (ester), 1602 (C=C (Ar) stretching), 1463 (C—H
bending), 1095 (C—O—C stretching); 1H NMR (DMSO-d6, 300 MHz):
δ 1.10 (t, 3H, J = 7.0
Hz, O-CH2-CH3), 2.29 (s, 3H, Ar-CH3), 2.39 (s, 3H, =C-CH3, dihydropyrimidone ring), 3.80-
3.82 (m, 6H, 2 × O-CH3), 3.9 (q, 2H, J = 7.0 Hz, O-CH2-CH3), 5.21 (s, 1H, CH), 7.03 (d, 1H,
J = 8.1 Hz, Ar-H), 7.35-7.42 (m, 4H, 4 × Ar-H), 7.77 (s, 1H, NH), 8.26 (s, 1H, CH (vinylic)),
9.22 (s, 1H, NH); 13C NMR (CDCl3, 75 MHz): 14.2, 15.5, 18.7, 55.1, 55.9, 56.0, 60.2, 101.0,
111.0, 111.7, 119.3, 121.2, 123.4, 126.2, 127.2, 127.6, 130.7, 138.8, 139.7, 146.6, 148.7,
149.7, 151.1, 153.5, 160.7, 165.5; ESI-MS (m/z): 479 (M+H)+ (100%), 447 (10%) ;
HRMS(m/z): calcd for C26H26N2O7 (M+H) +: 479.1818, Found: 479.1806.
(±)Ethyl4-(3-(3,4-dimethoxyphenyl)-8-methyl-2-oxo-2H-chromen-6-yl)-6-methyl-2-thioxo-
1,2,3,4-tetrahydropyrimidine-5-carboxylate (7): Pale green solid; yield: 68%; mp: 197-199
ºC; IR (KBr, cm-1): 3524 (N—H stretching), 3093 (=C—H stretching), 2965 (C—H
stretching), 1713 (C=S stretching), 1612 (ester), 1605 (C=C (Ar) stretching), 1459 (C—H
bending), 1186 (C—O—C stretching); 1H NMR (DMSO-d6, 300 MHz):
δ 1.15 (t, 3H, J = 7.1
Hz, O-CH2-CH3 ), 2.40 (s, 3H, Ar-CH3), 2.45 (s, 3H, =C-CH3, dihydropyrimidone ring), 3.86
(s, 3H, O-CH3), 3.87 (s, 3H, O-CH3), 4.06 (q, 2H, J = 6.9 Hz, O-CH2-CH3), 5.29 (s, 1H, CH),
7.08 (d, 1H, J = 8.4 Hz, Ar-H), 7.39-7.46 (m, 4H, 4 × Ar-H), 8.31 (s, 1H, CH (vinylic)), 9.73
(s, 1H, NH), 10.41 (s, 1H, NH) 13C NMR (CDCl3, 75 MHz): 14.1, 15.6, 18.6, 55.6, 55.9,
56.0, 60.6, 102.7, 111.0, 111.7, 119.6, 121.2, 123.5, 126.6, 127.2, 127.9, 130.8, 138.3, 138.7,
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142.7, 148.7, 149.8, 151.4, 160.5, 165.1, 174.6; ESI-MS (m/z): 495 (M+H)+ (100%), 463
(12%); HRMS(m/z): calcd for C26H26N2O6S (M+H) +: 495.1590, Found: 495.1576.
(±)Ethyl
6-methyl-4-(8-methyl-2-oxo-3-(3,4,5-trimethoxyphenyl)-2H-chromen-6-yl)-2-oxo-
1,2,3,4-tetrahydropyrimidine-5-carboxylate (8): Pale green solid; yield: 61%; mp: 192- 194
ºC; IR (KBr, cm-1): 3400 (N—H stretching), 3139 (=C—H stretching), 2927 (C—H
stretching), 1700 (C=O stretching), 1644 (ester), 1504 (C=C (Ar) stretching), 1458 (C—H
bending), 1123 (C—O—C stretching); 1H NMR (DMSO-d6, 300 MHz):
δ 1.05 (t, 3H, J = 7.0
Hz, O-CH2-CH3 ), 2.29 (s, 3H, Ar-CH3), 2.40 (s, 3H, =C-CH3, dihydropyrimidone ring), 3.71
(s, 3H, O-CH3), 3.83 (s, 6H, 2 × O-CH3), 4.00 (q, 2H, J = 6.8 Hz, O-CH2-CH3), 5.21 (s, 1H,
CH), 7.09 (s, 2H, 2 × Ar-H), 7.37-7.43 (m, 2H, 2 × Ar-H), 7.78 (s, 1H, NH), 8.30 (s, 1H, CH
(vinylic)), 9.22 (s, 1H, NH); 13C NMR (DMSO-d6, 75 MHz): 14.5, 15.5, 18.4, 54.2, 56.5,
59.7, 60.5, 99.1, 106.7, 119.4, 124.0, 125.1, 126.7, 130.4, 131.7, 138.4, 141.0, 141.3, 149.1,
150.7, 152.2, 152.9, 160.1, 165.7; ESI-MS (m/z): 509 (M+H)+ (100%), 493 (12%);
HRMS(m/z): calcd for C27H28N2O8 (M+H) +: 509.1924, Found: 509.1906.
(±)Ethyl 6-methyl-4-(8-methyl-2-oxo-3-(3,4,5-trimethoxyphenyl)-2H-chromen-6-yl)-2-thioxo-
1,2,3,4-tetrahydropyrimidine-5-carboxylate (9): Pale green solid, yield: 65%; mp: 183-185
ºC; IR (KBr, cm-1): 3580 (N—H stretching), 3187 (=C—H stretching), 2965 (C—H
stretching), 1714 (C=S stretching), 1623 (ester), 1579 (C=C (Ar) stretching), 1458 (C—H
bending), 1160 (C—O—C stretching); 1H NMR (DMSO-d6, 300 MHz):
δ 1.09 (t, 3H, J = 7.0
Hz, O-CH2-CH3), 2.34 (s, 3H, Ar-CH3), 2.40 (s, 3H, =C-CH3, dihydropyrimidone ring), 3.71
(s, 3H, O-CH3), 3.83 (s, 6H, 2 × O-CH3), 4.00 (q, 2H, J = 6.9 Hz, O-CH2-CH3), 5.24 (s, 1H,
CH), 7.08 (s, 2H, 2 × Ar-H), 7.36 (s, 1H, Ar-H), 7.41 (s, 1H, Ar-H), 8.30 (s, 1H, CH
(vinylic)), 9.68 (s, 1H, NH), 10.36 (s, 1H, NH); 13C NMR (CDCl3, 75 MHz): 14.1, 15.5, 18.4,
55.5, 56.3, 60.6, 60.9, 102.5, 106.0, 119.3, 123.7, 126.5, 127.9, 129.9, 131.1, 138.5, 138.8,
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139.5, 143.0, 151.4, 153.1, 160.4, 165.1, 174.3; ESI-MS (m/z): 525 (M+H)+ (100%), 493
(16%); HRMS(m/z): calcd for C27H28N2O7S (M+H) +: 525.1695, Found: 525.1702.
(±)Ethyl-4-(8-tert-butyl-2-oxo-3-(3,4,5-trimethoxyphenyl)-2H-chromen-6-yl)-6-methyl-2-
thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate: (10)
Pale green coloured solid, yield: 72%; mp: 202 - 204 ºC; IR (KBr, cm-1): 3510 (N—H
stretching), 3099 (=C—H stretching), 2967 (C—H stretching), 1715 (C=S stretching), 1623
1
(ester), 1583 (C=C (Ar) stretching), 1445 (C—H bending), 1118 (C—O—C stretching); H
NMR (CDCl3, 300 MHz): δ 1.17 (t, 3H, J = 7.0 Hz, O-CH2-CH3), 1.51 (s, 9H, C(CH3)3), 2.39
(s, 3H, =C-CH3, dihydropyrimidone ring), 3.89 (s, 3H, O-CH3), 3.92 (s, 6H, 2 × O-CH3), 4.10
(q, 2H, J = 6.9 Hz, O-CH2-CH3), 5.46 (s, 1H, CH), 6.98 (s, 2H, 2 × Ar-H), 7.32 (d, 1H, J =
1.8 Hz, Ar-H), 7.43 (d, 1H, J = 1.8 Hz, Ar-H), 7.79 (s, 1H, CH (vinylic)), 7.88 (s, 1H, NH),
8.39 (s, 1H, NH); 13C NMR (CDCl3, 75 MHz): 14.1, 18.3, 29.7, 35.0, 55.6, 56.2, 60.6, 60.9,
102.7, 105.9, 120.3, 124.1, 127.3, 127.6, 129.9, 138.2, 138.3, 138.7, 140.0, 143.1, 151.7,
153.0, 159.8, 165.1, 174.4; ESI-MS (m/z): 567 (M+H)+ (100%), 535 (12%); HRMS(m/z):
calcd for C30H34N2O7S (M+H)+: 567.2165, Found: 567.2159.
5.2. Biological experiments
5.2.1. Materials and Methods
MCF-7, MDA-MB-231, T47D, A549, DU-145, PC-3, HepG2, and NIH/3T3 cell lines used in
the present study was obtained from ATCC (American Type Culture Collection, USA).
Annexin V FITC Apoptosis Detection Kit was purchased from Calbiochem. MTT dye and
Caspase-3 Activation Kit was procured from Sigma-Aldrich. Absorbance was recorded using
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Eliza Plate Reader. Flow cytometry was performed using a FACScan (Becton Dickinson,
Mountain View, CA) flowcytometer.
5.2.2. Cell Culture
MCF-7, MDA-MB-231, T47D, A549, skin fibroblasts cells were routinely maintained in
Dulbecco's Modified Eagle Medium DMEM (Sigma Aldrich) while PC-3, DU-145, HepG2,
NIH/3T3 cells were maintained in RPMI 1640 (Merck) supplemented with 10% FBS (Merck)
and 1% Antibiotic-Antimycotic Solution (Merck) at 37 °C in a humidified incubator with 5%
CO2. All stock solution of compounds were prepared in cell culture grade DMSO and stored
in -20 °C. Compounds were diluted in culture media prior to use in experiments.
5.2.3. Antiproliferative activity
For general anticancer screening and also for other bio-chemical assays coumarin- monastrol
hybrids were dissolved in DMSO: PEG400 cocktails (4:6 ratios). We have screened all the 10
compounds against cancer cells such as MCF-7, MDA-MB-231, T47D, A549, DU-145, PC-3
and HepG2, non-cancer cells like NIH/3T3, HEK-293 and mouse skin fibroblasts. Cell
viability of the compound treated cells was measured by using a MTT assay. Briefly, cells (4
× 103 cells /well) were cultured in 96 well tissue culture plates and treated with different
concentrations of compounds for 48 h. At the end of incubations, 10µL of MTT (10mg/mL)
was added to the wells and incubated for 3 h. Absorbance was recorded at 540 nm using Eliza
Plate Reader. All the experiments were repeated at least thrice independently. IC50 values
were calculated using Graph pad prism 5.0 software.
5.2.4. Apoptosis studies
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Quantitation of apoptotic cells by Annexin V staining was carried out according to the
manufacturer’s instructions (Calbiochem). Briefly, MCF-7, MDA-MB-231, NIH/3T3 cells (5
× 105 cells / well) were seeded in 6 well plates and treated with 5ꢀM of compound 10 for 24
h. After the incubations, cells prepared as a suspension in 500ꢀL of cold PBS, centrifuged for
5 min. at 1000 × g then resuspended in 500ꢀL cold 1x binding buffer and added 1.25µL of
Annexin V FITC and incubated for 15 min at RT in dark, centrifuge at 1000×g for 5 min. at
RT, remove supernatant, gently resuspend in 500ꢀL cold 1x binding buffer and added 10ꢀL
PI. Flow cytometry was performed using a FACScan (Becton Dickinson, Mountain View,
CA) flowcytometer, equipped with a single 488nm argon laser. Annexin V-FITC were
analyzed using excitation and emission settings of 488nm and 535nm (FL-1 channel); PI, 488
nm and 610 nm (FL-2 channel). Debris and clumps were gated out using forward and
orthogonal light scatter. The experiment was repeated three times independently. Values are
expressed as mean ± SEM.
5.2.5. Caspase-3 Activation Assay
The activity of caspase-3 was determined using caspase-3 colorimetric assay kit (Sigma
Aldrich) according to the manufacturer’s protocol. Briefly, MCF-7 and MDA-MB-231 and
NIH/3T3 cells (1 × 106cells/well) were treated with 5ꢀM and 10ꢀM and 20ꢀM compound 10
for 24 h. The cells were harvested and lysed by addition of lysis buffer. The protein content
in the cell lysates was determined by Bio-Rad protein assay. Samples of the cell lysates were
mixed with colorimetric substrate (Ac-DEVD-pNA) and incubated at 37 °C in the dark for 4
h. The absorbance was measured at 405 nm in an ELISA reader. Caspase-3 activity was
expressed as the change of the activity compared to the control.
5.2.6. Cell cycle analysis
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For cell cycle distribution studies post-treatment, cells were fixed in 70% ethanol, rehydrated
in PBS, and stained with PBS containing PI (50ꢀg/mL) and ribonuclease A (100ꢀg/mL) for
30 min at room temperature. Cells were collected and analyzed with FACScan flow
cytometer (Becton Dickinson, Mountain View, CA) equipped with an argon laser to give 488
nm light. Values are given as the mean of three independent experiments, each done in
duplicate.
5.2.7. Effect of compound 10 on Cell Viability of Tamoxifen pretreated in MCF-7 and T47D
cells
Cell viability of the treated cells was measured by using a MTT assay. Briefly, both MCF-7
& T47D cells (4 × 103 cells /well) were cultured in 96 well tissue culture plates and treated
with different concentrations of compounds for 48 h. In one row before addition of
compound 10, cells were treated with tamoxifen (5µM) for 2 h, the media was removed from
these wells and then compound 10 was added and kept for 48 h. At the end of incubations,
10µL of MTT (10mg/mL) was added to the wells and incubated for 3 h. Absorbance was
recorded at 540 nm using Eliza Plate Reader. All the experiments were repeated at least thrice
independently. Values are expressed as mean ± SEM. *p<0.05 considered statistically
significant.
5.3. Statistical Analysis
Each experiment was observed in triplicate. The data are presented as mean ± SD and
compared using student’s t-test. p<0.05 or less was considered to be statistically significant
Acknowledgement
The authors are grateful to the Director, CDRI, Lucknow, India for constant encouragement
in drug development program, S.P. Singh for technical support, SAIF for NMR, IR, and Mass
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spectral data. S.R.A, and G.R.P. are thankful to CSIR and K.S is thankful to DST-INSPIRE,
New Delhi, India for financial support. This is C.D.R.I. Communication No. 140/2012/KVS
Supplementary data
Spectral data of all the compounds associated with this article is available as supplementary
data
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Figure Captions
Figure 1. Chemical structures of some potent anticancer compounds and our hybrid
molecules.
Figure 2. Representation of time dependant viabilities of compound 10 (C10) treated MCF-7
cells. Values are expressed as mean ± SD.
Figure 3. Representation of viabilities of compound 10 (C10) 5µM-treated breast cancer
cells (MCF-7, T47D and MDA-MB-231), prostate cancer (DU-145 and PC-3), hepatocellular
liver carcinoma cell line (HepG2), human lung adenocarcinoma epithelial cell line (A549)
and non cancer cells NIH3T3, isolated Primary mouse skin fibroblasts and human embryonic
kidney cells (HEK-293) compared with 5µM tamoxifen and 5µM epirubicin.
Figure 4. Flow cytometry was done for detection of apoptosis in MCF-7, MDA-MB-231
cells and NIH3T3 cells after treatment with 5µM of coumarin- monastrol hybrid derivative
10.
Figure 5. Coumarin-monastrol hybrid (compound 10) activates homogeneous caspase in
MCF7 and MDA-MB-231, but not in NIH/3T3 cells: 1 × 105cells/well with 70–80%
confluence were grown and treated with 0, 5, 10 and 20µM of compound 10 for 24h.
Colorimetric determination of general caspase was done as described in materials and
methods each data point is the representation of triplicate treatments. ** Represents p<0.005
and * represents p=0.05.
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Figure 6. Cell cycle profiles of coumarin- monastrol hybrid derivative 10 (0µM, 5µM,10µM,
20µM) treated MCF-7 cells. Cellular DNA was stained with 50 mg/mL PI in PBS. Stained
cells were analyzed by FACScan flow cytometer (Becton Dickinson, USA). All the
experiments were performed at least three times. Untreated cells (blank) were used as control.
Data are expressed as % of cell count in each phase of cell cycle induced by compound 10 in
each concentration.
Figure 7. Antiproliferative effect of C10 in both TAM pretreated and untreated cells. MCF
(Fig. 7A) and T47D (Fig. 7B) cells (4 × 103 cells /well) were cultured in 96 well tissue
culture plates and treated with different concentrations of compounds for 48 h. In one row
before addition of compound 10, cells were treated with tamoxifen (5µM) for 2 h, the media
was removed from these wells and then compound 10 was added and kept for 48 h. At the
end of incubations Cell viability of the treated cells was measured by using MTT assay.
Absorbance was recorded at 540 nm using Eliza Plate Reader.
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Table 1. IC50 values of coumarin- monastrol hybrids in µM against various cell lines.
Mouse
Skin
Fibro
blast
Compounds
MDA-
IC50
MCF-7
T47D
DU-145
PC-3
A549
HepG2 NIH/3T3
MB-231
(µM)
2.093
>20
>20
8.57
3.03
11.87
2.42
5.5
9.241
>20
>20
>20
19.85
>20
>20
>20
18.97
>20
>20
>20
>20
>20
>20
12.0
9.6
>20
>20
>20
>20
>20
>20
>20
6.3
>20
>20
>20
>20
17.14
>20
>20
11.4
4.2
>20
>20
>20
>20
>20
>20
>20
>20
>20
4
5
>20
17.28
>20
19.82
14.59
>20
>20
6
5.68
13.23
7.15
3.13
13.0
4.3
9.8282
19.31
>20
7
10.78
>20
8
>20
9
10
3.978
11.4
5.6
>20
>20
5.5
10
TAM
3.7
9.9
5.7
4.6
Epirubicin
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Figure 1: Chemical structures of some potent anticancer compounds and our hybrid molecules.
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Figure 2.
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Figure 3.
120
100
80
60
40
20
0
C10(5µM)
Tamoxifen
Epirubicin
Breast Cancer Cells
Other Cancer Cells
Non Cancer Cells
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Figure 4.
27