Exploring the anti-breast cancer potential of flavonoid analogs

Article abstract of DOI:10.1039/c6ra14428d

In the course of our search for new antitumor agents for breast cancer, novel flavone derivatives were synthesized, characterized and examined for their antitumor activities against breast cancer cell lines. In initial screening, analogs 7a [3-(5-amino-1,3,4-thiadiazol-2-yl)methoxy-2-phenyl-4H-chromen-4-one] and 7b [3-(5-amino-1,3,4-thiadiazol-2-yl)methoxy-2-(4-methoxyphenyl)-4H-chromen-4-one] were found to be effective against the estrogen receptor negative cell line (MDA-MB 453), which was followed by their evaluation in five dose assays. In addition, mechanistic studies of 7a and 7b were performed by cytometric analysis and electrophoretic studies and it was observed that apoptosis is a mechanism of cell death, confirmed morphologically by acridine orange/ethidium bromide double staining and TUNEL analysis. Further in vivo evaluation of the anti-tumor activity of compound 7a and 7b by Ehrlich Ascites Carcinoma (EAC) model and related studies confirms the anti-breast cancer potential of flavonoid analogs.

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ARTICLE
Exploring the Anti-Breast Cancer Potential of
Flavonoid Analogs
a
a
b
b
Vanrajsinh Thakor , Mayur Poddar , Sumit Dey , S. N. Manjula , SubbaRao V.
*
**
Madhunapantula c, Rahul Pawara d, Harun M. Patel d , Malleshappa N. Noolvi a
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
Abstract
www.rsc.org/advance
In the course of our search for new antitumor agents in breast cancer, novel flavone derivatives
were synthesized, characterized and examined for the antitumor activities against breast cancer
cell lines. In initial screening, analogs 7a [3ꢀ(5ꢀaminoꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)methoxy)ꢀ2ꢀphenylꢀ
4Hꢀchromenꢀ4ꢀone] and 7b [3ꢀ(5ꢀaminoꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)methoxy)ꢀ2ꢀ(4ꢀmethoxyphenyl)ꢀ
4Hꢀchromenꢀ4ꢀone] was found to be effective against estrogen receptor negative cell line (MDAꢀ
MB 453) followed by their evaluation in five dose assay. In addition, mechanistic study of 7a and
7b was done with cytometric analysis and electrophoretic study and it was observed that
apoptosis is a mechanism of cell death, confirmed morphologically by acridine orange/ethidium
bromide double staining and TUNEL analysis. Further In-vivo evaluation of antiꢀtumor activity
of compound 7a and 7b by Ehrlich Ascites Carcinoma (EAC) model and related studies confirm
the antiꢀbreast cancer potential of flavonoid analogs.
Keywords: Synthesis Flavones, Breast Cancer, MDAꢀMB 453, Anticancer agent, Flavonoid
a Department of Pharmaceutical Chemistry, Shree Dhanvantary Pharmacy College, Kim (Surat)-394110, Gujarat, India
b Department of Pharmacology, JSS College of Pharmacy, Mysore-570015, Karnataka, India
c Department of Biochemistry, JSS Medical College, Mysore-570015, Karnataka, India
d Department of Pharmaceutical Chemistry, R. C. Patel Institute of Pharmaceutical Education and Research, Shirpur,
District Dhule-425 405, Maharashtra, India
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1. Introduction
Breast cancer is the most commonly diagnosed malignancy among women with more than one
million new cases diagnosed per year throughout the world. [1,2] Despite advances in the early detection
of breast cancer and the advent of novel targeted therapies, breast cancer still remains a significant
public health problem due to the involvement of multiple aberrant and redundant signaling
pathways in the tumorigenesis and the development of resistance to the existing therapeutic
agents. The currently available breast cancer therapies achieve meaningful clinical results in only
30–40% of the patients [3]. The efficacy of current chemotherapeutics is low and undesirable side
effects are still unacceptably high [4]. Hence, the development of novel, efficient, and less toxic
antiꢀbreast cancer agents remains an important and challenging goal of medicinal chemists
worldwide.
The female hormone estrogen stimulates breast cell division leading to the increase in risk of permanent
damage to DNA [5]. Compounds that can regulate the apoptosis of cancer cells are of a high medical
significance [6]. Natural products (NPs) have played a valuable role in the drug discovery and
development [7ꢀ9]. Newman and Cragg [10] reported that in the case of cancer around 79% of FDAꢀ
approved drugs during a period of 1981ꢀ2010 are either natural products or their based/mimickedꢀ
compounds. NPs are chosen through evolutionary process via lead optimization to interact with
various enzymes/proteins and thus represent biologically relevant regions of the vast chemical space
[11ꢀ13]. Flavopiridol, a semisynthetic flavones analog, acts as CDK9 inhibitor, is FDAꢀapproved orphan
drug for acute myeloid leukaemia. It has been reported that Myricetin, (flavonoid compound) could
decrease pancreatic cancer growth via induction of cell apoptosis [14]. LY294002 (Flavonoid analogue)
entered clinical trials as a potential antineoplastic agent [15]. Effects of phytoestrogens in cancer
prevention have been reported for decades [16ꢀ18]. Since then many molecular mechanisms underlying
these effects have been identified. Targets of phytoestrogens comprise steroid receptors, steroid
metabolising enzymes, elements of signal transduction and apoptosis pathways, and even the DNA
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processing machinery [18]. Phytoestrogens include chalcones (A), flavones (B) and isoflavones (C) which
are nonꢀsteroidal compounds possessing antiꢀestrogenic activity (Fig.1) [19].
In light of these findings and in continuation of our research for novel antiꢀcancer agents [20ꢀ23] in the
present study, new series of flavone derivatives has been synthesized and screened in vitro for
cytotoxicity by sulphorhodamine B assay. Five dose assay in Estrogen Receptor negative cell line (MDAꢀ
MB 453) and determination of IC50 by SRB assay was also performed. In addition, mechanistic study was
done with cytometric analysis and electrophoretic determination of apoptosis. Further in-vivo activity
evaluation of antiꢀtumor activity of selected synthetic compounds by Ehrlich Ascites Carcinoma (EAC)
model and related studies was performed.
2. Rationale and Design
Estrogens have long been recognized to play a key role in the development, growth, and function of
female sex organs, and mammary gland [24ꢀ25]. Estrogens have also an important role in the skeletal,
cardiovascular, and central nervous systems [26ꢀ28]. Since estrogens are known to play a predominant
role in breast cancer development and growth [29ꢀ30], a logical approach for the treatment of estrogenꢀ
sensitive breast cancer is the use of antiꢀestrogens, which block the interaction of estrogens with their
specific receptors.
Tamoxifen, the compound in general use for treatment of breast cancer, possesses mixed agonistꢀ
antagonist activities, thus limiting its efficacy as a blocker of estrogen action since it exerts estrogenic
activity at various organs in different species [31ꢀ34]. The most serious adverse events attributable to
tamoxifen are the increased risk of uterine cancer and thromboembolic phenomena. Furthermore, up to
one half of ERꢀpositive breast cancers and the majority of ERꢀnegative cancers are not prevented with
tamoxifen [35]. The use of a pure selective estrogen receptor modulator (SERM) as preventive and
therapeutic agent should also have positive effects on the skeletal and cardiovascular systems while
decreasing the risk as well as treating breast and uterine cancer [36].
Acolbifene (EMꢀ652.HCl) is a fourth generation SERM of the benzopyran class which has been found to
have no estrogen agonist effects in either the breast or endometrium [37ꢀ40]. Acolbifene and its prodrug
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(EMꢀ800) have been associated with reduction of growth of tumor xenografts [41] as well as the
incidence of DMBAꢀinduced rat mammary cancer [42]. The lack of estrogen agonist activity in the uterus
of EMꢀ800 as well as reported activity in tamoxifenꢀresistant metastatic disease [40] made it an attractive
agent for assessment for treatment and prevention. Preclinical and clinical data indicate that Acolbifene
(EMꢀ652) possesses characteristics superior to tamoxifen and raloxifene for breast and uterine cancer
prevention and treatment as well as for hormone replacement therapy at menopause [43ꢀ45]. Encouraged
with the above findings we have concentrated our efforts to synthesis the further derivatives of
Acolbifene (EMꢀ652), where we tried to modified 4ꢀhydroxyphenyl (Cꢀ3) of acolbifene with different
1,3,4ꢀthiadiazoles and imidazo[2,1ꢀb][1,3,4]thiadiazoles with hope to get better analogue of this category
(Fig.2).
3. Chemistry
General scheme to synthesize proposed flavone derivatives is described in Scheme 1. The most common
synthetic routes to the chromone structure occur via a chalcone intermediate or via the Bakerꢀ
Venkataraman rearrangement. The chalcone pathway implicates the baseꢀcatalyzed aldol condensation of
2ꢀ hydroxy acetophenones with aromatic or conjugated aldehydes. The condensation of substituted oꢀ
hydroxy acetophenone 1 and 2 with substituted benzaldehyde 3 was carried out by vigorous stirring in
sodium hydroxide solution (1gm/5 ml) and 95% ethanol to give chalcone 4(a-g). The resulting chalcone
4(a-g) is cyclized to the corresponding 3ꢀhydroxyflavone 5(a-g), using alkaline hydrogen peroxide
solution, via the AlgarꢀFlynnꢀ Oyamada reaction. Further treatment of 5(a-g) with chloroacetic acid in
sodium hydroxide solution gives chromone carboxylic acid 6(a-g). Later chromone 6(a-g) is converted in
to the corresponding thiadiazoles 7(a-g) using POCl3 and thiosemicarbazide. Substituted phenacyl
bromides eventually react with thiadiazoles 8(a-g) to yield imidazoꢀthiadiazoles 7(a-n).
1
13
The structures of final derivatives 7(a-g) and 8(a-n) were confirmed spectral by IR, H NMR, C NMR
and Mass study. IR absorption peak at ~3000 cmꢀ1 for aromatic CꢀH, disappearance of acid peak at 1700
cmꢀ1 and primary amine peak above 3200 cmꢀ1 confirm the synthesis of 7(a-g). C=N of thiadiazole and
imidazole are identified individually at around 1600 cmꢀ1 and 1500 cmꢀ1. 1HNMR spectra revealed all the
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corresponding peaks at δ = 6ꢀ8 ppm for aromatic protons while methoxy protons shows peak at δ= 3 ꢀ 4.5
ppm. 13C NMR and Mass gave valuable information about the cyclization of substituted chalcone to
flavone and thiadiazole to imidazo thiadiazole ring system.
4. Results and Discussion
4.1 In vitro Cytotoxicity study
4.1.1 In vitro Cytotoxicity study of compounds 7(a-g) and 8(a-n) by Sulphorhodamine B assay using
MDA-MB 453 cells (Breast Cancer Cell Line)
Preliminary screening of compounds 7 (a-g) and 8 (a-n) was determined in MDAꢀMB 453 cells (Breast
Cancer Cell Line) by sulphorhodamine B assay. The percentage cytotoxicity for all compounds was
assessed at different time point 24, 48 and 72h as shown in Table 1. Compound 7a and 7b showed
maximum % Cytotoxicity compared to other compounds.
4.1.2 Five dose assay of selected compounds in MDA-MB453 cells (Breast Cancer Cell Line) by SRB
assay
Amongst all the tested compounds 7 (a-g) and 8 (a-n),7a and 7b was the most effective, the cytotoxic
potency of the compounds 7a and 7b at different time points on MDAꢀMB453 cells exhibited marked
growth inhibition at 72 h when compared to 24 and 48 h, in a doseꢀdependent fashion. In case of standard
DiꢀAllyl Di Sulphide (DADS) a dose dependent and time dependent increase in the cytotoxicity activity
was observed as evident by the IC50 values. Therefore, it was selected for detailed mechanistic study
(Table 2).
4.1.3 Electrophoretic determination of apoptosis
For confirmation of nuclear fragmentation, DNA laddering assay was performed. As illustrated in Fig. 3,
agarose gel electrophoresis of DNA extracted from MDA-MB453 cells treated with different
concentration of 7a and 7b (50, 150, 300ꢁm) for 72h, revealed a progressive increase in the nonꢀrandom
fragmentation of DNA. 7b at 150 and 300ꢁM showed a characteristic fragmentation in DNA, whereas 7a
showed an effective degradation in DNA at 300ꢁM when compared to 50 and 150 ꢁM treated
concentration. The extracted DNA was electrophoresed on 1% agarose gel at 60 V, 400 mA for 90
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min.the gel were captured under UV illumination in Gel Doc system (Syngene gel documentation
system).
4.1.4 Influence of 7a and 7b on the cell cycle progression through cell cycle after 48 h treatment on
MDA-MB453 cells
The assay was performed to assess the cell cycle specificity of compound (7a and 7b) on MDA-MB453
cells. The data in Figure 4 demonstrates that the compound 7a arrested cells in G2/M stage of the cell
cycle as evidenced by a significant increase in G2/M population when cells were exposed to 50 ꢁM. But,
a significant increase in G0/G1 and Sꢀphase population was also observed when the 7a concentration is
elevated to 150 ꢁM, which could be due to increased cell death (~45% cell death, please compare the IC50
Table. 2) at this concentration. Similar to compound 7a, compound 7b also arrested the cells in G2/M
phase of the cell cycle at 50 ꢁM concentration.
DADS, which is used as a positive control, arrested cells in G2/M phase of the cell cycle. Though there is
a slight increase in Sꢀphase population, the difference between control and DADS treated cells is not
significant. Furthermore, no significant difference was seen in G0/G1 phase of the cell cycle.
4.1.5 Maximum tolerable dose of selected compounds (7a and 7b) by acute toxicity study
Maximum tolerable dose was determined by acute toxicity study according to OECD guidelineꢀ425. After
14 days administration of both compounds there were no sign of mortality but slight decline in body
weight (<9.0%) was found in 2000mg/kg treated mice whereas 500 mg/kg dosed animals were found to
be safe without any sign of mortality hence the MTD was calculated.
4.2 In Vivo evaluation of anti-cancer activity by Ehrlich’s ascites (EAC) Model
4.2.1 Effect of 7a and 7b on Viability of tumor cells
On day 15th ascitic fluid was collected and checked for percentage viability of cells, there were increased
number of viable cells in negative control compared to day 0. Standard decreased the increase in number
of % viable cells compared to negative control. The treatment with 7a and 7b at 25 mg/kg and 10 mg/kg
significantly reduced the % increase in viable cells compared to negative control. The efficacy of 7b at
10mg/kg was comparable with standard (Fig.5).
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4.2.2 Effect of 7a and 7b on change in body weight of (EAC) inoculated Mice
Substantial gain in body weight of EAC inoculated negative control mice was found to be maximum gain
of (35.00±1.06) on day 15 when compared to day 0. Standard DiꢀAllyl Di Sulphide (DADS) at 50mg/kg
reduced the body weight (24.80±0.16) compared to negative control. The treatment with 7a and 7b at
10mg/kg and 25mg/kg significantly reduced the % increase in the body weight when compared to
negative control and the efficacy was comparable to standard (Fig.6.)
4.2.3 The effect of 7a and 7b on Hematological parameters in EAC inoculated mice
In EAC inoculated negative control, the reduction in total RBC, hemoglobin (Hb) content and increase in
total WBC count were significant when compared with normal mice. DADS (50 mg/kg i.p) significantly
reversed and normalized the EAC induced hematological changes. 7a and 7b at both doses (10 and 25
mg/kg) significantly reversed the elevated WBC count and increase in RBC and Hb content was
significant when compared to negative control (Fig.7.).
4.8.4 Effect of 7a and 7b on Mean Survival time and % increase in life span of EAC inoculated mice
Effective decrease in mean survival time of EAC inoculated negative control mice was found
(15.0±0.516) days, standard DADS at 50mg/kg showed an increase in MST (22.8±0.872) when compared
to negative control, treatment with 7a at 25mg/kg effectively enhanced the MST (28.8±0.654) when
compared to negative control mice. While treatment with 7b at 10mg/kg prolonged the MST (22.3±0.333)
when compared to negative control. Treatments with 7a at 25mg/kg showed significant increase in %
increase in life span (93.10 %) (Fig. 8,9).
4.8.5 Effect of 7a and 7b on Serum Enzyme levels
In EAC inoculated negative control mice, enhancement in ALP, SGOT and SGPT were significantly high
when compared with normal. DADS (50 mg/kg i.p) significantly reversed the elevation in EAC induced
serum level changes. Treatment with 7a and 7b at both doses (10 and 25 mg/kg) significantly reversed the
elevated serum enzymes level. Higher dose of test compounds was highly effective when compared to
lower dose (Fig.10).
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4.8.6 Effect of 7a and 7b on Liver antioxidant markers
In EAC treated negative control mice, the decrease in SOD, GSH, CAT and increase in LPO were
significantly high when compared with normal. DADS (50mg/kg i.p) significantly increased the decline
in EAC induced liver endogenous enzymes while significantly reduced the elevated level of LPO.
Treatment with 7a and 7b at both doses (10 and 25 mg/kg) significantly reversed the declined liver
enzymes level (Fig.11).
4.8.7 In-vivo mechanistic studies of EAC tumor cells (DNA fragmentation assay and fluorescent
imaging of EAC cells following treatment with 7a and 7b
Analysis of DNA isolated from EAC cell of animals treated with 7a , 7b and DADS showed fragmented
DNA predominantly in 7a treated animals compared to 7b treated animals indicating the induction of
apoptosis further For accurate determination of live/dead nucleated cell concentration in heterogeneous
samples dualꢀfluorescence staining of EAC cells was performed using the dyes acridine orange/ethidium
bromide. 7a and 7b (10 &25 mg/kg) treated EAC cells displayed shrinkage as observed under varying
magnifications of fluorescent imaging (Fig.12,13). The standard DADS also showed similar morphology
when examined.
4.8.8 Structure Activity Relationship (SAR)
Structure activity relationship (SAR) study indicated that the different substitution on flavone (4Hꢀ
chromenꢀ4ꢀone) ring, exerted varied effect on antiꢀbreast cancer activities. The antiꢀbreast cancer activity
of the newly synthesized compounds revealed that 1,3,4ꢀthiadiazole substitution at Cꢀ3 position of 4Hꢀ
chromenꢀ4ꢀone is more potent compared to the imidazo[2,1ꢀb][1,3,4]thiadiazole (Compound 7a, 7b and
8a, 8b). From the data shown in Table 1 and 2, it is clear that the presence of 4ꢀmethoxy phenyl at 2nd
position of 4Hꢀchromenꢀ4ꢀone ring of 1,3,4ꢀthiadiazole series enhances the antiꢀbreast cancer activity
(Compound 7b) as compare to the plane phenyl substitution (7a). The possible enhancement in the antiꢀ
breast cancer activity can be further accomplished by slender variation in the ring substituents and/or
extensive additional fictionalization warrants further investigations.
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5. CONCLUSION
Conclusively, a new series of flavone analogs 7(a-g) and 8(a-n) have been successfully synthesized from
2ꢀhydroxy acetophenone and substituted benzaldehydes. The yields of flavone derivatives were found to
1
be ranging between 35ꢀ70%. The structure of compounds was characterized and confirmed by IR, Hꢀ
13
NMR, CꢀNMR, and Mass spectral studies. In initial screening, analogs 7a [3ꢀ(5ꢀaminoꢀ1,3,4ꢀthiadiazolꢀ2ꢀ
yl)methoxy)ꢀ2ꢀphenylꢀ4Hꢀchromenꢀ4ꢀone]
and
7b
[3ꢀ(5ꢀaminoꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)methoxy)ꢀ2ꢀ(4ꢀ
methoxyphenyl)ꢀ4Hꢀchromenꢀ4ꢀone] was found to be effective against estrogen receptor negative cell line
(MDAꢀMB 453) followed by their evaluation in five dose assay. For confirmation of nuclear fragmentation,
DNA laddering assay was performed. Compound 7b at 150 and 300ꢁM showed a characteristic
fragmentation in DNA, whereas 7a showed an effective degradation in DNA at 300ꢁM when compared to
50 and 150 ꢁM treated concentration. Cell cycle specificity of compound (7a and 7b) on MDA-MB453
cells has also been performed and results demostrated that percentage of G2/M phase significantly
increased with 150 ꢁM and 50 ꢁM of 7b and at 50 ꢁM of 7a compared to control. In-vivo EAC model
study demonstrated that the treatment with 7a and 7b at 10 mg/kg and 25 mg/kg significantly reduced the
% increase in the body weight, significantly reversed the elevated WBC count and increase in RBC and
Hb content; considerably inverted the elevated serum enzymes and liver enzyme level. It was concluded
that the synthesized flavone derivatives have potential to act as an anticancer agents and the activity of
various compounds varied according to the substituent attached. These preliminary encouraging results of
biological screening of the tested compounds could offer an excellent framework in this field that may
lead to discovery of potent antitumor agent.
6. Experimental
6.1. Materials and Methods
The chemicals employed in the synthetic work i.e. 5ꢀmethoxy 2’ꢀhydroxy acetophenone was purchased
from SigmaꢀAldrich while all other chemicals i.e. thiosemicarbazide, 2’ꢀhydroxy acetophenone, other
benzaldehydes, 2ꢀ chloro acetic acid, Bromine, H2O2 and POCl3 etc. were purchased from Spectrochem.
All the solvents were used after distillation. Most of the solvents and chemicals used were of LR or
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Analytical grade. The purity of the compounds was confirmed by thin layer chromatography using
precoated TLC plates and solvent systems like, Chloroform: Methanol: Formic acid (7:2.5:0.5),
Chloroform: Methanol (8:2) and their different ratios. The spots were visualized under ultraviolet lamp
and by using of Iodine chamber. 1H NMR and 13C NMR spectra of synthesized compounds were recorded
in DMSO solution Bruker Model (400 MHz) instrument at CSMCRI, Bhavnagar and SAIF, Punjab
University, Chemical shifts are reported as parts per million (ppm) using tetramethylsilane (TMS) as an
internal standard. Column chromatography separations were progressed on silica gel (200ꢀ300 mesh). A
series of molecules were synthesized, molecules (flavones) were dissolved in dimethyl Sulphoxide
(DMSO) to a stock concentration of 100 mM. Working standard for in-vitro study was made up in serum
containing media. The final concentration of DMSO in cell culture did not exceed 0.1 %.
6.1.1 General procedure for the synthesis of 2-(3,4-disubstitutedphenyl)-3-hydroxy-6-substituted-4H-
chromen-4-one 5(a-g) and 2-((6-substituted-4-oxo-2-phenyl-4H-chromen-3-yl)oxy)acetic acid 6(a-g)
It was synthesized as per the method given by Gupta et al [46].
6.1.2 General procedure for the synthesis of 3-((5-amino-1,3,4-thiadiazol-2-yl)methoxy)-6-substituted-
2-(3,4-substitutedphenyl)-4H-chromen-4-one 7(a-g)
A mixture of flavone acid 6 (a-g) (0.01 mol), thiosemicarbazide (0.01 mol) and POCl3 (10 mol) are
refluxed at 75 ℃ for 30 min. After cooling down to room temperature, sufficient water was added. The
reaction mixture was then reflux at 75℃ for 3 hrs. After cooling reaction mixture was basified to pH 8 by
drop wise addition of 50% NaOH solution under stirring. The precipitate was filtered and recrystallized
with ethanol.
6.1.2.1 3-((5-amino-1,3,4-thiadiazol-2-yl)methoxy)-2-phenyl-4H-chromen-4-one (7a)
Yield 69%, mp 237ꢀ239℃, IR (KBr) Vmax 3400 (NH strech), 2922 (CH aro. strech), 2851 (CH ali. strech),
1
1642 (C=O) cmꢀ1, H NMR(DMSOꢀd6) δ ppm: 6.52ꢀ8.21 (m, 9H, ArꢀH), 6.12 (s, 2H, NH2), 5.54 (s, 2H,
CH2); 13C NMR (DMSOꢀd6) δ ppm: 172.12, 169.34, 163.12, 159.29, 156.22, 148.12, 138.92, 136.22,
135.22, 134.12, 132.22, 130.82, 128.27, 127.45, 125.48, 123.44, 122.46, 121.33, 120.37, 118.13, 116.23,
66.39, 55.38; HRMS (ESI) m/z calcd for C18H13N3O3S: 351.0678; found: 351.0683.
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6.1.2.2 3-((5-amino-1,3,4-thiadiazol-2-yl)methoxy)-2-(4-methoxyphenyl)-4H-chromen-4-one(7b)
Yield 39%, mp 245ꢀ247℃, IR (KBr) Vmax 3338 (NH strech), 3141 (CH aro. strech), 2918 (CH ali. strech),
1642.69 (C=O) cmꢀ1, 1H NMR(DMSOꢀd6) δ ppm: 6.38ꢀ8.23 (m, 8H, ArꢀH), 6.11 (s, 2H, NH2), 5.13(s, 2H,
CH2), 3.92(s, 3H, CH3); 13C NMR (DMSOꢀd6) δ ppm: 175.02, 169.20, 163.26, 161.28, 156.49, 154.72,
145.89, 142.42, 130.48, 128.48, 125.38, 123.24, 122.46, 121.77, 118.61, 116.42, 114.62, 65.35, 56.81
; HRMS (ESI) m/z calcd for C19H15N3O4S: 381.0783; found: 381.0789.
6.1.2.3 3-((5-amino-1,3,4-thiadiazol-2-yl)methoxy)-2-(3,4-dimethoxyphenyl)-4H-chromen-4-one(7c)
Yield 47%, mp 248ꢀ251℃, IR (KBr) Vmax 3339 (NH strech), 3142 (CH aro. strech), 2917 (CH ali. strech),
1
1668 (C=O) cmꢀ1, H NMR(DMSOꢀd6) δ ppm: 6.58ꢀ8.34 (m, 8H, ArꢀH), 6.09 (s, 2H, NH2), 5.15(s, 2H,
CH2), 3.92(s, 3H, OCH3), 3.82(s, 3H, OCH3) ; 13C NMR (DMSOꢀd6) δ ppm: 176.12, 167.78, 166.82,
161.28, 158.97, 156.82, 152.27, 139.37, 135.92, 125.27, 123.12, 122.13, 121.12, 120.21, 118.78, 114.16,
112.17, 65.28, 56.28, 56.01; HRMS (ESI) m/z calcd for C20H17N3O5S: 411.0889; found: 411.0895.
6.1.2.4 3-((5-amino-1,3,4-thiadiazol-2-yl)methoxy)-6-methoxy-2-phenyl-4H-chromen-4-one(7d)
Yield 48%, mp 244ꢀ246℃, IR (KBr) Vmax 3321 (NH strech), 3111 (CH aro. strech), 2932 (CH ali. strech),
1
1662 (C=O) cmꢀ1, H NMR(DMSOꢀd6) δ ppm: 6.62ꢀ8.10 (m, 8H, ArꢀH), 6.14 (s, 2H, NH2), 5.25(s, 2H,
CH2), 3.76 (s, 3H, OCH3) ; 13C NMR (DMSOꢀd6) δ ppm: 174.18, 169.18, 164.17, 159.91, 156.23, 149.51,
140.12, 132.13, 130.16, 128.12, 127.12, 124.32, 122.12, 120.55, 111.62, 64.23, 55.81; HRMS (ESI) m/z
calcd for C19H15N3O4S: 381.0783; found: 381.0788.
6.1.2.5 3-((5-amino-1,3,4-thiadiazol-2-yl)methoxy)-6-methoxy-2-(4-methoxyphenyl)-4H-chromen-4-one (7e)
Yield 63%, mp 235ꢀ237℃, IR (KBr) Vmax 3348 (NH strech), 3028 (CH aro. strech), 2934 (CH ali. strech),
1
1683 (C=O) cmꢀ1, H NMR(DMSOꢀd6) δ ppm: 6.82ꢀ8.12 (m, 7H, ArꢀH), 6.15 (s, 2H, NH2), 5.01(s, 2H,
CH2), 3.71(s, 3H, OCH3), 3.92(s, 3H, OCH3); 13C NMR (DMSOꢀd6) δ ppm: 178.12, 168.23, 167.12,
161.23, 159.112, 156.43, 150.51, 139.12, 128.28, 128.01, 124.12, 122.34, 122.01, 120.12,
114.12, 112.12, 110.12 ,64.26, 56.81, 55.82; HRMS (ESI) m/z calcd for C20H17N3O5S: 411.0889;
found: 411.0881.
6.1.2.6 3-((5-amino-1,3,4-thiadiazol-2-yl)methoxy)-2-(3-methoxyphenyl)-4H-chromen-4-one (7f)
11
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Yield56%, mp 239ꢀ242℃, IR (KBr) Vmax 3328 (NH strech), 3011 (CH aro. strech), 2934 (CH ali. strech),
1
1676 (C=O) cmꢀ1, H NMR(DMSOꢀd6) δ ppm: 6.82ꢀ8.38 (m, 8H, ArꢀH), 6.10 (s, 2H, NH2), 5.16(s, 2H,
13
CH2), 3.91(s, 3H, OCH3); C NMR (DMSOꢀd6) δ ppm: 172.12, 169.12, 165.12, 162.12, 159.12, 158.12,
140.12, 138.12, 130.12, 129.16, 125.58, 123.84, 121.47, 120.22, 116.51, 113.55, 112.57, 64.25, 55.18 ;
HRMS (ESI) m/z calcd for C19H15N3O4S: 381.0783; found: 381.0792.
6.1.2.7 3-((5-amino-1,3,4-thiadiazol-2-yl)methoxy)-6-methoxy-2-(3-methoxyphenyl)-4H-chromen-4-one (7g)
Yield 59%, mp 244ꢀ246℃, IR (KBr) Vmax 3312 (NH strech), 3021 (CH aro. strech), 2973 (CH ali. strech),
1
1681 (C=O) cmꢀ1, H NMR(DMSOꢀd6) δ ppm: 6.72ꢀ8.29 (m, 7H, ArꢀH), 6.11 (s, 2H, NH2), 5.06(s, 2H,
CH2), 3.81(s, 3H, OCH3), 3.91(s, 3H, OCH3); 13C NMR (DMSOꢀd6)
δ
ppm:
173.12,168.12,165.12,162.12,159.38,158.93,150.12,140.38,130.43,129.12,126.34,124.12,124.72,
122.43,116.23,114.23,110.61, 63.52,55.92,55.18;
411.0889; found: 411.0895.
HRMS (ESI) m/z calcd for C20H17N3O5S:
6.1.3 General procedure for the synthesis of 2-(3,4-disubstitutedphenyl)-6-substituted-3-((6-(2,3,4-
trisubstitutedphenyl)imidazo[2,1-b][1,3,4]thiadiazol-2-yl)methoxy)-4H-chromen-4-one 8(a-n)
A mixture of equimolar quantities of flavone thiadiazole 7(a-g) (0.01 mol) and 2ꢀbromoꢀ1ꢀ(substitutedꢀ
aryl) ethanone (0.01 mol) was refluxed in dry ethanol for 20hr. The excess of solvent was distilled off and
the solid hydrobromide that separated was collected by filtration, suspended in water and neutralized by
10% sodium carbonate solution to get free base. It was filtered, washed with water, dried and crystallized
from ethanol.
6.1.3.1 3-((6-phenylimidazo[2,1-b][1,3,4]thiadiazol-2-yl)methoxy)-2-phenyl-4H-chromen-4-one(8a)
Yield 57%, mp 252ꢀ254℃, IR (KBr) Vmax 3048 (CH aro. strech), 2913 (CH ali. strech), 1691 (C=O) cmꢀ1,
1H NMR(DMSOꢀd6) δ ppm: 6.28ꢀ8.29 (m,14H, ArꢀH), 8.41 (s,1H, CHꢀImidazole), 5.18 (m, 2H, CH2); 13C
NMR (DMSOꢀd6) δ ppm: 178.12, 169.12, 158.12, 148.12, 138.12, 158.12, 148.21, 142.21, 138.12, 134.12,
132.12, 130.12, 129.12, 128.12, 126.12,, 125.12, 124.12, 122.12, 121.12, 120.12, 118.11, 66.91; HRMS
(ESI) m/z calcd for C26H17N3O3S: 451.0991; found: 451.0997.
6.1.3.2 3-((6-phenylimidazo[2,1-b][1,3,4]thiadiazol-2-yl)methoxy)-2-(4-methoxyphenyl)-4H-chromen-4-one (8b)
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Yield 63%, mp 259ꢀ261℃, IR (KBr) Vmax 3056 (CH aro. strech), 2910 (CH ali. strech), 1684 (C=O) cmꢀ1,
1H NMR(DMSOꢀd6) δ ppm: 6.46ꢀ8.36 (m,13H, ArꢀH), 8.44 (s,1H, CHꢀImidazole), 5.12 (m, 2H, CH2),
3.91 (s,3H, OCH3); 13C NMR (DMSOꢀd6) δ ppm: 172.12, 169.12, 162.12, 158.38, 156.12, 148.12, 142.12,
138.12, 136.12, 134.10, 130.12, 129.12, 128.71, 126.51, 124.18, 122.41, 122.16, 120.31, 120.17, 118.11,
116.12, 64.19, 55.18; HRMS (ESI) m/z calcd for C27H19N3O4S: 481.1096; found: 481.1091.
6.1.3.3 3-((6-(3-methoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazol-2-yl)methoxy)-2-phenyl-4H-chromen-4-one (8c)
Yield 31%, mp 252℃, IR (KBr) Vmax 3034 (CH aro. strech), 2921 (CH ali. strech), 1689 (C=O) cmꢀ1, 1H
NMR (DMSOꢀd6) δ ppm: 6.36ꢀ8.23 (m,13H, ArꢀH), 8.32 (s,1H, CHꢀImidazole), 5.10 (m, 2H, CH2), 3.89
(s,3H, OCH3); 13C NMR (DMSOꢀd6) δ ppm: 175.01, 169.10, 166.11, 159.91, 156.21, 148.11, 136.91,
135.12, 134.12, 134.01, 130.31, 129.21, 128.16, 127.19, 126.19, 125.81, 123.14, 122.23, 121.67, 119.78,
116.31, 114.33, 113.66, 64.92, 55.28; HRMS (ESI) m/z calcd for C27H19N3O4S: 481.1096; found:
481.1090.
6.1.3.4 3-((6-(3-methoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazol-2-yl)methoxy)-2-(4-methoxyphenyl)-4H-chromen-
4-one (8d)
Yield 34%, mp 248ꢀ250℃, IR (KBr) Vmax 3012 (CH aro. strech), 2934 (CH ali. strech), 1678 (C=O) cmꢀ1,
1H NMR(DMSOꢀd6) δ ppm: 6.36ꢀ8.23 (m,12H, ArꢀH), 8.32 (s,1H, CHꢀImidazole), 5.14 (m, 2H, CH2),
3.91 (s,3H, OCH3), 3.81 (s,3H, OCH3); 13C NMR (DMSOꢀd6) δ ppm: 174.23, 169.23, 164.48, 162.91,
159.91, 158.12, 148.13, 136.92, 134.22, 133.22, 132.03, 130.22, 128.49, 126.82, 123.45,, 122.26, 121.33,
120.72, 119.82, 116.12, 114.23, 114.12, 112.16, 64.92, 56.22, 55.08; HRMS (ESI) m/z calcd for
C28H21N3O5S: 511.1202; found: 511.1206.
6.1.3.5 3-((6-(2-methoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazol-2-yl)methoxy)-2-phenyl-4H-chromen-4-one (8e)
Yield 54%, mp 251ꢀ253℃, IR (KBr) Vmax 3048 (CH aro. strech), 2923 (CH ali. strech), 1681 (C=O) cmꢀ1,
1H NMR(DMSOꢀd6) δ ppm: 6.48ꢀ8.10 (m,13H, ArꢀH), 8.28 (s,1H, CHꢀImidazole), 5.04 (m, 2H, CH2),
3.92 (s,3H, OCH3) ; 13C NMR (DMSOꢀd6) δ ppm: 176.01, 169.02, 159.92, 156.31, 155.22, 148.23, 142.91,
138.22, 136.22, 132.11, 130.33, 129.37, 128.36, 127.39, 126.29, 125.38, 123.44, 122.43, 121.67, 120.35,
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119.39, 118.14, 114.13, 65.92, 56.23; HRMS (ESI) m/z calcd for C27H19N3O4S: 481.1096; found:
481.1090.
6.1.3.6 3-((6-(2-methoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazol-2-yl)methoxy)-2-(4-methoxyphenyl)-4H-chromen-
4-one (8f)
Yield 42%, mp 247ꢀ250℃, IR (KBr) Vmax 3012 (CH aro. strech), 2921 (CH ali. strech), 1686 (C=O) cmꢀ1,
1H NMR(DMSOꢀd6) δ ppm: 6.28ꢀ8.16 (m,12H, ArꢀH), 8.35 (s,1H, CHꢀImidazole), 5.09 (m, 2H, CH2),
3.96 (s,3H, OCH3), 3.86 (s,3H, OCH3) ; 13C NMR (DMSOꢀd6) δ ppm: 175.23, 169.02, 160.12, 159.28,
158.93, 157.33, 156.32, 148.13, 138.92, 136.32, 135.42, 133.15, 120.27, 128.48, 127.48, 126.48, 123.44,
120.33, 120.39, 118.13, 116.22, 114.23, 112.13, 65.92, 56.21, 55.08; HRMS (ESI) m/z calcd for
C28H21N3O5S: 511.1202; found: 511.1208.
6.1.3.7 3-((6-(2,4-dimethoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazol-2-yl)methoxy)-2-phenyl-4H-chromen-4-one
(8g)
Yield 44%, mp 245ꢀ246℃, IR (KBr) Vmax 3053 (CH aro. strech), 2921 (CH ali. strech), 1682 (C=O) cmꢀ1,
1H NMR(DMSOꢀd6) δ ppm: 6.38ꢀ8.36 (m,12H, ArꢀH), 8.41 (s,1H, CHꢀImidazole), 5.16 (m, 2H, CH2),
3.91 (s,3H, OCH3), 3.85 (s,3H, OCH3) δ ppm: 176.23, 171.02, 165.62, 162.23, 159.92, 158.22, 148.12,
138.29, 136.22, 135.23, 132.25, 130.43, 128.56, 128.36, 126.92, 125.83, 123.34, 122.43, 121.47, 118.13,
116.23, 114.23, 112.13, 110.12, 66.92, 56. 38, 55.12; HRMS (ESI) m/z calcd for C28H21N3O5S:
511.1202; found: 511.1208.
6.1.3.8 3-((6-(2,4-dimethoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazol-2-yl)methoxy)-2-(4methoxyphenyl)-
4H-chromen-4-one (8h)
Yield 42%, mp 244ꢀ248℃, IR (KBr) Vmax 3063 (CH aro. strech), 2942 (CH ali. strech), 1672 (C=O) cmꢀ1,
1H NMR(DMSOꢀd6) δ ppm: 6.18ꢀ8.06 (m,11H, ArꢀH), 8.21 (s,1H, CHꢀImidazole), 4.98 (m, 2H, CH2),
3.92 (s,3H, OCH3), 3.84 (s,3H, OCH3), 3.81 (s,3H, OCH3); 13C NMR (DMSOꢀd6) δ ppm: 178.10, 169.23,
167.82, 164.81, 162.23, 160.13, 159.33, 156.42, 149.14, 142.92, 138.22, 136.22, 134.35, 130.28, 128.28,
126.42, 124.36, 122.33, 120.27, 119.18, 118.24, 114.22, 112.23, 111.12, 110.82, 65.39, 56.12, 55.88,
55.10; HRMS (ESI) m/z calcd for C29H23N3O6S: 541.1308; found: 541.1302.
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6.1.3.9 3-((6-(4-chlorophenyl)imidazo[2,1-b][1,3,4]thiadiazol-2-yl)methoxy)-2-phenyl-4H-chromen-4-one (8i)
Yield 75%, mp 267ꢀ269℃, IR (KBr) Vmax 3024 (CH aro. strech), 2912 (CH ali. strech), 1684 (C=O), 712
(CꢀCl) cmꢀ1, 1H NMR(DMSOꢀd6) δ ppm: 6.12ꢀ8.21 (m,13H, ArꢀH), 8.41 (s,1H, CHꢀImidazole), 4.99 (m,
2H, CH2); 13C NMR (DMSOꢀd6) δ ppm: 178.23, 169.23, 158.92, 156.22, 148.12, 142.91, 138.12, 136.21,
134.13, 132.11, 130.23, 129.33, 128.49, 128.16, 127.92, 127.29, 125.28, 123.24, 122.13, 121.17, 118.12,
66.19; HRMS (ESI) m/z calcd for C26H16ClN3O3S: 485.0601; found: 485.0607.
6.1.3.10 3-((6-(4-chlorophenyl)imidazo[2,1-b][1,3,4]thiadiazol-2-yl)methoxy)-2-(4-methoxyphenyl)-4H-chromen-
4-one (8j)
Yield 68%, mp 266ꢀ268℃, IR (KBr) Vmax 3034 (CH aro. strech), 2910 (CH ali. strech), 1681 (C=O), 701
1
(CꢀCl) cmꢀ1, H NMR(DMSOꢀd6) δ ppm: 6.19ꢀ8.26 (m,12H, ArꢀH), 8.38 (s,1H, CHꢀImidazole), 5.09 (m,
2H, CH2), 3.84 (s,3H, OCH3); 13C NMR (DMSOꢀd6) δ ppm: 172.60, 162.23, 160.38, 154.41, 152.31,
145.52, 138.12, 133.33, 130.68, 129.33, 124.69,124.31, 123.54, 121.30, 118.18, 114.43, 113.91, 63.55,
55.27; HRMS (ESI) m/z calcd for C27H18ClN3O4S: 515.0707; found: 515.0701.
6.1.3.11 3-((6-(4-fluorophenyl)imidazo[2,1-b][1,3,4]thiadiazol-2-yl)methoxy)-2-phenyl-4H-chromen-4-one (8k)
Yield 39%, mp 268ꢀ272℃, IR (KBr) Vmax 3012 (CH aro. strech), 2923 (CH ali. strech), 1684 (C=O), 1201
1
(CꢀF) cmꢀ1, H NMR(DMSOꢀd6) δ ppm: 6.12ꢀ8.21 (m,13H, ArꢀH), 8.41 (s,1H, CHꢀImidazole), 4.99 (m,
2H, CH2); 13C NMR (DMSOꢀd6) δ ppm: 176.12, 169.12, 161.27, 159.91, 156.22, 149.12, 141.92, 138.22,
136.21, 132.31 130.61, 129.16, 128.92, 126.19, 125.26, 124.28, 122.43, 120.23, 119.27, 118.21, 116.20,
64.92; HRMS (ESI) m/z calcd for C26H16FN3O3S: 469.0896; found: 469.0891.
6.1.3.12 3-((6-(4-fluorophenyl)imidazo[2,1-b][1,3,4]thiadiazol-2-yl)methoxy)-2-(4-methoxyphenyl)-4H-
chromen-4-one (8l)
Yield 45%, mp 218ꢀ220℃, IR (KBr) Vmax 3064 (CH aro. strech), 2963 (CH ali. strech), 1689 (C=O), 1223
1
(CꢀF) cmꢀ1, H NMR(DMSOꢀd6) δ ppm: 6.23ꢀ8.32 (m,12H, ArꢀH), 8.38 (s,1H, CHꢀImidazole), 5.12 (m,
2H, CH2), 3.91 (s,3H, OCH3) ; 13C NMR (DMSOꢀd6) δ ppm: 178.12, 167.23, 164.92, 162.83, 160.92,
158.22, 148.21, 142.19, 138.22, 136.32, 130.12, 129.26, 128.16, 126.18, 124.14, 122.33, 121.26, 120.71,
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118.21, 116.03, 114.32, 64.92, 55.18; HRMS (ESI) m/z calcd for C27H18FN3O4S: 499.1002; found:
499.1009.
6.1.3.13 3-((6-(4-methoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazol-2-yl)methoxy)-2-phenyl-4H-chromen-4-one (8m)
Yield 34%, mp 249ꢀ253℃, IR (KBr) Vmax 3084 (CH aro. strech), 2985 (CH ali. strech), 1684 (C=O) cmꢀ1,
1H NMR(DMSOꢀd6) δ ppm: 6.13ꢀ8.32 (m,13H, ArꢀH), 8.34 (s,1H, CHꢀImidazole), 5.15 (m, 2H, CH2),
3.81 (s,3H, OCH3); 13C NMR (DMSOꢀd6) δ ppm: 178.12, 168.01, 165.62, 158.92, 156.22, 148.12,
138.92, 136.22, 135.22, 130.23, 129.36, 128.25, 127.29, 126.93, 125.28, 124.32, 122.43, 120.23, 119.73,
118.13, 116.83, 65.39, 55.38; HRMS (ESI) m/z calcd for C27H19N3O4S: 481.1096; found: 481.1090.
6.1.3.14 3-((6-(4-methoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazol-2-yl)methoxy)-2-(4-methoxyphenyl)-
4H-chromen-4-one (8n)
Yield 48%, mp 259ꢀ262 ºC, IR (KBr) Vmax 3042 (CH aro. strech), 2978 (CH ali. strech), 1678 (C=O)cmꢀ
1, 1H NMR(DMSOꢀd6) δ ppm: 6.23ꢀ8.23 (m,12H, ArꢀH), 8.30 (s,1H, CHꢀImidazole), 5.05 (m, 2H, CH2),
3.86 (s,3H, OCH3), 3.92 (s,3H, OCH3) ; 13C NMR (DMSOꢀd6) δ ppm: 178.12, 169.12, 164.61, 160.18,
159.91, 158.12, 152.11, 142.91, 138.12, 136.21, 130.18, 128.51, 126.81, 125.31, 123.14, 122.36, 120.31,
119.71, 118.12, 116.81, 114.21, 65.91, 55.84, 55.02; HRMS (ESI) m/z calcd for C28H21N3O5S: 511.1202;
found: 511.1209.
6.2 In vitro anticancer activity
6.2.1 Cell lines
The human breast cancer cell line MDAꢀMB 453 was obtained from the National center of cell sciences
(NCCS), Pune, India. MDAꢀMB231 cells were cultured in DMEM medium (Gibco) supplemented with
10% foetal bovine serum (FBS) and 100 IU/mL penicillin. All cells were cultured at 37ºC with 5% CO2.
6.2.2 Sulphorhodamine-B Assay
The cytotoxic potential of test compounds on growth of breast cancer cells (MDAꢀMB 453) was assessed
by SRB assay. A fixed number of cells (5000), taken in a volume of 100ꢁL were added to each well of a
flatꢀbottomed 96ꢀwell plate, and kept in CO2 incubator for adherence of the cells for 48 h. The test
compounds were dissolved in DMSO (0.1%) and further diluted with the medium. After 48 h, cells were
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treated with 100 µL of two different concentrations (250 and 500 µM) of test compounds at different time
points (24, 48 and 72h). Cells in the control wells received the same volume of medium containing 0.1%
DMSO. Diꢀallyl di Sulphide (DADS) was used as a positive control [47]. After incubation cells were
fixed with 10% TCA and stained with 0.4% of SRB for 30 min, bound SRB was solubilized in 10 mM
Tris base solution and the absorbance was measured at 490 nm in a BioꢀRad plate reader. Percentage
inhibition was calculated using formula:
Percentage Inhibition = C – T ×100
C
C= Absorbance of DMSO vehicle control treated cells
T= Absorbance of compound treated cells
SRB assay for optimization of dose with the selected compounds by five dose assay
The promising compounds (7a and 7b) identified from the preliminary screening were further subjected
to five dose assay by SRB assay at an extended time points like 24,48 and 72hr on MDAꢀMB 453 cells to
determine the time dependent activity of compounds and to optimize the dose for further assay, IC50 was
determined.
6.2.3 DNA Fragmentation Assay
Optimum number of MDAꢀMB 453 cells (1×106) was incubated for 48h in DMEM media supplemented
with 10% FBS. After 48h incubation, media was aspirated and treated with different concentrations (50,
150 and 300 ꢁM) of the compounds (7a and 7b) for 72h. After treatment media containing cells were
subjected to centrifugation. The adherent cells in the flasks as well as cell pellets in the centrifuge tube
were washed with PBS and 25ꢁl of RNAse solution was added to the centrifuge tube kept at 57ºC for 1h.
Cell lysate containing DNA sample was loaded into each well of the agarose gel and electrophoresis was
carried at 60 V, 400mA for 90 min. Images were captured using gel documentation system.
6.2.4 Flow Cytometry Analysis
A fixed number of MDAꢀMB 453 cells (1×106) were treated with different concentrations of 7a and 7b
(50 and 150 µM). After treatment, cells were harvested and washed twice with PBS and reꢀsuspended in
70% EtOH while vortexing and stored until analysis. At the time of analysis, cells were washed with PBS
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to remove alcohol and incubated with FACS reagent containing RNAse (100 µg/ml), Triton X (0.1%) and
trisodium citrate (0.1%) for 75 min at 37 ºC. After incubation cells were stained with propidium iodide (2
mg/ml) for 15 min in iceꢀcold dark condition, cells were then mixed thoroughly and subjected to flow
cytometer (BD FACS Calibur, Mountain View, USA) for acquisition. The relative DNA content per cell
was obtained by measuring the fluorescence of PI that bound stoichiometrically to DNA. The DNA
histograms were analyzed using cell Proquest software, the experiment was conducted in triplicates and
DiꢀAllyl Di Sulphide (DADS) was used as standard.
6.2.5 Determination of Maximum tolerable dose of the selected compounds (7a and 7b)
Acute toxicity of selected compounds was done according to OECD guidelines 425, Swiss albino mice of
either sex was selected. Mice were deprived of the food 4h prior to administration of test compounds and
till 2h after administration. On the day Zero, test compounds were administered to animals at a dose of
2000mg/kg and 550mg/kg and animals were observed for death for 4, 6 and 8h upto 14 days following
administration of compounds, MTD was calculated.
6.3 In Vivo Study
In-vivo study Ehrlich’s ascites carcinoma (EAC) cells were originally obtained from Amala cancer
Research Centre, Thrissur, India, were maintained and propagated as ascites tumor in swiss albino mice
by serial intraꢀperitoneal transplantation at animal quarantine house, JSS College of pharmacy, Mysore,
India.
6.3.1 Animals
Eight to ten week old swiss albino mice weighing between 25 and 30 g were procured form central animal
facility, JSS medical college, animals were maintained under the controlled conditions of temperature (23
± 30ºC), humidity (50 ± 5%), light and dark respectively. The animals were provided with sterile food
and water ad libitium. Six animals were housed in each polypropylene cage containing paddy husk as
bedding. Animal care and handling was done according to guidelines issued by CPCSEA, Government of
India. Experiments were performed after obtaining ethical clearance from IAEC [IAEC approval no:
154/2014].
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6.3.2 Anticancer activity of selected compounds (7a and 7b) against EAC inoculated mice
EAC cells obtained from the peritoneal cavity of an EAC bearing mouse, after 15 days of tumor
transplantation. Aspirated EAC cells were diluted in PBS and fixed number of viable EAC cells (2.5×106
cells/mouse) was injected intraperitoneally into each mouse. After 24h of tumor inoculation the animals
were randomly divided into following groups of six mice each and treated with test compounds (7a and
7b) at different concentration based on MTD.
Group I
:
:
:
:
:
:
:
Normal
Group II
Group III
Group IV
Group V
Group VI
Group VII
EAC Cells + DMSO i.p for 7 days
EAC Cells + DADS (50mg/kg) i.p for 7 days
EAC Cells + 7aꢀ14 (25mg/kg) i.p for 7 days
EAC Cells + 7aꢀ14 (10mg/kg) i.p for 7 days
EAC Cells + 7bꢀ4 (25mg/kg) i.p for 7 days
EAC Cells + 7bꢀ4 (10mg/kg) i.p for 7 days
The test compounds (7a and 7b) were administered for seven days, i.p, alternatively starting from day 1 of
tumor inoculation. DiꢀAllyl Di Sulphide (DADS) at dose of 50 mg/kg was administered i.p. which served
as standard drug. Body weight was assessed every third day after tumor inoculation to assess the increase
in tumor growth. The animals were monitored for 15 days and various parameters were evaluated. i.e.
haematological parameters (RBC, WBC, Hb content), viable tumor cell count was done to check the
number of viable cell among the treated group, mortality was assessed to calculate Mean survival time
[MST] and percentage increase in life span using formula.
Cell count = (No. of cells x Dilution) / (Area x thickness of liquid film)
Percentage increase in body wt = (body weight on respective day - body weight on day
0)*100 / Body weight on day 0
% ILS = [(MST Test – MST Con)] * 100/ MST (Con.)
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6.3.3 Biochemical Tests of EAC inoculated mice
On completion of treatment Serum alanine transaminase (ALT), aspartate transaminase (AST) and ALP
activities were determined using a commercial kit (Merck), liver homogenate glutathione (GSH) was
determined by the method of Alapati et al [48]. Malondialdehyde (MDA) was determined in liver tissue
homogenate superoxide dismutase (SOD) and catalase (CAT) activity was assayed
spectrophotometrically [48].
6.3.4 DNA Fragmentation assay on isolated EAC cells
DNA fragmentation assay on isolated EAC cells was done by nonꢀenzymatic method, adequate volume of
EAC cells was aspirated and diluted with PBS and centrifuged for 3 min at 3000 rpm, supernatent was
discarded and obtained pellets was washed with PBS, 100ꢁl of 10% SDS & TE mixture was added to
pellets and incubated for 30 min, after incubation 1.6 ml of 8 M potassium acetate was added and
centrifuged at 7000 rpm for 1hr.The obtained supernatent was separated out and to that organic
propionate mixture of (phenol: chloroform: isoamyl alcohol) was added, to above mixture 2 vol of ice
cold ethanol was added and centrifuged at 10000 rpm for 30 min . Cell lysate containing DNA was
dissolved into 50ꢁL of Tris EDTA (TE) buffer and sample was loaded into each well of the agarose gel
and electrophoresis was carried at 60 V, 400 mA for 90 min. Images were captured using gel
documentation system.
6.3.5 Fluorescent imaging of EAC cells using dual staining
Dual staining with ethidium bromide and acridine orange was performed. Aspirated EAC cells suspended
in PBS (25ꢁL) from each group was mixed with 1ꢁL AO/EB for 15 min prior to microscopic evaluation.
Thin smear of stained cells was prepared on microscopic slides, Images were captured using fluorescent
microscope.
Acknowledgements
The authors would like to thank Advisor and Member Secretary, Gujarat Council on Science and
Technology, Gandhinagar (Grant No. GUJCOST/MRP/12ꢀ13/65/1332) for funding this project, H. G.
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Shiva Kumar, Principal JSS College of Pharmacy, Mysore for rendering us necessary laboratory facilities
and Natco Pharma, Hyderabad for providing necessary drugs.
Authors Harun M. Patel and Rahul Pawara would like to thanks “Science and Engineering Research
Board (SERB) of Department of Science and Technology (DST) Govt. of India” (Grant
No.YSS/2015/002017) for funding the project.
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Table 1. Preliminary screening of compounds 7(a-g) and 8(a-n) on MDA-MB 453 cells (Breast
Cancer Cell Line) at different time points.
Compounds
Percentage (%) cytotoxicity of MDA-MB453 cells at different time points
24h
48h
72h
500µM
250µM
27.0±1.3
500µM
39.7±0.67
57.5±0.12
ꢀ12.7±0.09
ꢀ28.9±0.98
ꢀ24.6±0.31
ꢀ10.29±0.78
ꢀ2.3±1.2
250µM
45.4±0.76
37.3±0.23
ꢀ12.9±0.12
ꢀ9.09±0.1
ꢀ12.9±1.23
ꢀ9.0±2.1
500µM
56.5±1.1
250µM
7a
7b
7c
7d
7e
7f
45.44±0.23
48.8±1.34
ꢀ16.7±0.9
ꢀ8.9±2.321
ꢀ14.0±0.34
ꢀ4.23±1.22
ꢀ6.6±1.034
44.05±1.23
29.90±0.12
ꢀ10.90±0.78
ꢀ10.2±0.98
ꢀ19.09±0.34
ꢀ10.09±1.98
33.3±0.72
ꢀ14.2±0.87
ꢀ4.91±1.32
ꢀ10.6±0.87
ꢀ7.9±1.23
ꢀ1.3±0.99
63.1±0.56
ꢀ11.3±0.98
ꢀ9.6±3.2
ꢀ12.7±0.45
ꢀ6.7±0.23
ꢀ7.6±0.34
ꢀ9.4±1.29
ꢀ6.2±0.29
3.7±1.23
ꢀ9.0±2.1
ꢀ10.098±1.09
ꢀ3.098±1.07
ꢀ6.098±2.09
12.90±1.06
ꢀ6.78±1.05
ꢀ9.12±2.12
ꢀ23±1.08
7g
8a
8b
8c
8d
8e
8f
ꢀ8.9±1.3
ꢀ3.3±3.21
ꢀ8.8±3.89
10.0±0.9
ꢀ8.0±1.23
ꢀ7.0±1.8
ꢀ5.4±1.23
ꢀ0.3±0.01
ꢀ8.7 ± 1.89
ꢀ1.3±0.0.87
ꢀ1.9±0.08
13.1±1.42
2.3±1.23
29.3±0.98
0±0.01
11.0±3.2
ꢀ7.1±1.23
ꢀ4.5±1.89
ꢀ25±0.98
ꢀ16±0.87
ꢀ34.8±1.2
ꢀ6.91±1.68
ꢀ9.27±0.76
ꢀ12.2±0.89
ꢀ11±0.27
ꢀ6.27±0.98
ꢀ5.2±1.34
ꢀ8.1±1.08
ꢀ4.6±0.48
ꢀ29.4±1.09
ꢀ30.2±1.02
ꢀ31.4±1.2
ꢀ6.2±4.8
ꢀ3.23±1.48
ꢀ23.2±0.33
ꢀ17.8±0.98
ꢀ67.2±1.45
ꢀ6.9±2.56
ꢀ11.7±3.1
ꢀ12.8±0.5
ꢀ12.8±0.92
ꢀ12.7±2.1
ꢀ19.6±0.98
ꢀ9.38±0.99
ꢀ43.7±2.1
ꢀ19.0±0.98
ꢀ46.8±1.2
ꢀ22.8±0.91
ꢀ10.1±0.12
ꢀ11.7±0.19
ꢀ11.0±0.12
ꢀ13.1±0.09
ꢀ34.8±1.2
ꢀ43±3.21
ꢀ23±2.43
ꢀ13.0±1.34
ꢀ8.01±0.8
ꢀ4.5±2.3
ꢀ32±1.09
8g
8h
8i
ꢀ56.96±1.12
ꢀ9.23±0.62
ꢀ9.90±0.91
ꢀ11.90±0.98
ꢀ21±2.09
ꢀ12.3±1.8
ꢀ12.3±0.94
ꢀ12.2±12.02
ꢀ10.3±1.2
ꢀ33±1.23
8j
ꢀ11.9±0.16
ꢀ21±1.43
ꢀ6.5±1.3
8k
8l
ꢀ8.90±0.89
ꢀ9.08±0.67
8m
8n
ꢀ6.7±1.32
All the values are expressed as mean ± SEM of triplicates compared to control
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Table 2. Five Dose assays of selected compounds in MDA-MB453 cells by SRB assay
MDA MB 453 IC50(µM) (Breast Cancer Cell Line)
Compounds
24h
48h
72h
478±24.1a
578±7.77a
929.2±1.05
185±2.20a
167±83.4a
724.2±11.05
240±25.3a
315±66.9
324.2±32.40
7a
7b
DADS
All values are expressed as mean ± SEM, n = 3. Inhibitory concentration 50 (IC50) was calculated by
plotting % cytotoxicity vs. concentration using linear regression. Three independent experiments were
carried out. Data was analyzed by one way ANOVA followed by post Dunnet’s test; a = p<0.05, as
compared to standard Di-Allyl Di Sulphide (DADS)
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Fig. 1. A) Anticancer Flavonoid leads B) Structural similarities between Chalcone (A), Flavone
(B) and Estradiol (C)
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Fig. 2. Structural comparison between acolbifene and proposed molecule.
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Fig. 3. Agarose gel electrophoresis nucleosomal DNA fragmentation of MDAꢀMB453 cells
induced by selected compounds 7a and 7b at 72h.
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