2-(3′,4′-Dimethoxybenzylidene)tetralone induces anti-breast cancer activity through microtubule stabilization and activation of reactive oxygen species

Article abstract of DOI:10.1039/c6ra02663j

Breast cancer is a leading cause of women mortality worldwide. Diverse analogues of 2-benzylidene tetralone have been synthesized and evaluated for anti-cancer activity against a panel of cancer cell lines. Among these, compounds 13, 15 and 19 exhibited p

Full text of DOI:10.1039/c6ra02663j

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2-(3´, 4´-dimethoxybenzylidene) tetralone induces anti-breast
cancer activity through microtubule stabilization and activation of
reactive oxygen species
Yashveer Gautam,a† Sonam Dwivedi,b† Ankita Srivastava,a Hamidullah,b Arjun Singh,a D. Chanda,a
Jyotsna Singh,b Smita Rai,b Rituraj Konwar,b* and Arvind S. Negia*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
Breast cancer is a leading cause of women mortality worldwide. Diverse analogues of 2-benzylidene tetralone have been
synthesized and evaluated for anti-cancer activity against a panel of cancer cell lines. Among these, compounds 13, 15 and
19 exhibited potent anti-cancer activity (IC50=1-3μM) against human breast cancer cells, MDA-MB-231 and non-toxic
towards non-malignant cell line, HEK-293. Compounds 13, 15 and 19 significantly stabilized tubulin polymerization and
arrested cell cycle progression at G0/G1 phase instead of typical mitotic arrest at G2/M phase as observed in case of
tubulin polymerization modulators suggesting induction of mitotic slippage followed by cell death. Further, mechanistic
studies revealed that compound 13 induced reactive oxygen species generation and apoptosis in breast cancer cells.
Inhibition of ROS by N-acetyl-L-cysteine prevented compound 13 induced cytotoxicity. Compound 13 showed potent anti-
cancer activity (74-79% tumour reduction) in syngeneic rat mammary tumour model without any adverse side effect. It
was well tolerated up to 1000 mg/kg dose in acute oral toxicity. The identified lead molecule 13 may further be optimized
for better activity.
Introduction
considered as one of the most suitable targets for cancer drug
Breast cancer is the most common invasive cancer in female development.3, 4
worldwide. It accounts 26% of female cancer cases and causes The present study describes development of anti-cancer agents
about 18.2% female cancer deaths. Breast cancer affects one in through microtubule modulation. Microtubules are tube like rigid
eight women during their lives. It caused 1.7 million patients and hollow cylindrical filamentous structures built by polymerisation of
about 0.52 million deaths in 2012.1 The disease has high complexity tubulin dimers.5 Microtubules serve as cytoskeletons of the cells
of more than 18 sub-types of breast cancer. Among various risk and also form framework for structures like spindle apparatus that
factors for developing breast cancer life style, environmental, helps in chromosome segregation during cell division. Owing to
genetic and biological are predominant. There are a number of their crucial role in mitotic process, microtubules have become an
treatments available to tackle this cancer including surgery, attractive target especially in the treatment of cancer.6
radiation therapy, chemotherapy, hormonal therapy and targeted We designed 2-substituted 1-tetralone derivatives as possible
therapy. Based on responsiveness, breast cancer is subdivided into antitubulins. The basis of designing was inspired from structure-
hormone responsive (ER/PR/Her-2) and hormone non-responsive. activity relationship of combretastatin A4, a natural antitubulin
In chemotherapy, cytotoxic drugs like paclitaxel, taxotere, isolated from African Willow tree i.e. Combretum caffrum.7
doxorubicin etc., and hormone antagonists like tamoxifen, According to its SAR, a diaryl system should be separated through a
fulvestrant and aromatase inhibitors like anastrozole, letrozole etc. double bond (restricted rotation) and a 3,4,5-trimethoxyphenyl unit
are in use.2 However, multimodality approach is used to eradicate should be present at one of the phenyl ring.8 The 3,4,5-
residual breast cancer cells to prevent recurrence of the disease. trimethoxyphenyl fragment is an important motif to interact with
Several anti-breast cancer drugs have been developed, but the microtubule and induces antitubulin effect.6 Based on these facts
morbidity and mortality of the disease is so high that it is still a we planned to synthesize 2-benzylidene tetralones as possible anti-
challenge to scientific fraternity. Microtubules have been cancer agent through microtubule modulation.
Results and Discussion
Chemistry
a.CSIR-Central Institute of Medicinal and Aromatic Plants (CSIR-CIMAP), Kukrail
Picnic Spot Road, P.O. CIMAP, Lucknow-226015, India.
2-Benzylidene tetralones were prepared as per Scheme 1. 6-
Methoxytetralone (1) was treated with various aromatic
benzaldehydes (2-11) using 3% alkaline KOH to afford
corresponding 2-benzylidenetetralones (12-21) in good yields (68-
86%) through Claisen-Schmidt reaction. Compounds were purified
b.CSIR-Central Drug Research Institute (CSIR-CDRI), B.S. 10/1, Sector 10, Jankipuram
Extension, Sitapur Road, Lucknow-226031, India.
† Equal authorship.
Electronic Supplementary Information (ESI) available: All the 1H, 13C and Mass
spectra of potential compounds i.e. 13, 15 and 19 will be available online. See
DOI: 10.1039/x0xx00000x
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through column chromatography and confirmed through cancer) and non-cancer cell line, HEK-293 (human embryonic
spectroscopy.
kidney) using MTT assay to assess cell inhibition.9 Results are shown
in Table 1. Out of fifteen synthesized analogues ten analogues
showed significant cytotoxicity (IC50=1-47μM) against MDA-MB-231,
LA-7, HCT116 and H1299 cells rest were inactive at 50µM
concentration. However, compounds 13, 15 and 19 showed potent
cytotoxicity (IC50=1-3.2μM) against highly aggressive breast cancer
cell line (MDA-MB-231 cells). Compounds 15 effectively inhibited
growth of MCF-7, MDA-MB-231, HCT116 and HCT1299 cells.
Compound 25 was selective to both breast cancer cell lines.
However, compounds 13 and 19 more effectively inhibited MDA-
MB-231 cells as compared to other cell lines. Among the entire
compounds screened, compound 13 was the most active analogue
in this series with lowest IC50 (1.0μM) against MDA-MB-231 cells
and IC50 (7µM) against LA-7 cells. Furthermore, compound 13 did
not show cytotoxicity against non-malignant cells (HEK-293).
Collectively, these cytotoxicity data suggest that compound 13 was
the most potent against MDA-MB-231 and LA-7 cells.
O
O
O
a
A.
R
H
+
R
H3CO
H3CO
1
2: R=H;
3: R=3,4-dimethoxy;
12: R=H;
13: R=3,4-dimethoxy;
14: R=3,4,5-trimethoxy;
15: R=4-trifluoromethyl;
16: R= 4-N,N-dimethylamino;
17: R=4-Fluoro;
18: R=3-Nitro;
19: R=4-Nitro;
20: R=3,4-methylenedioxy;
21: R=2,3-methylenedioxy
4: R=3,4,5-trimethoxy;
5: R=4-trifluoromethyl;
6: R= 4-N,N-dimethylamino;
7: R=4-Fluoro;
8: R=3-Nitro;
9: R=4-Nitro;
10: R=3,4-methylenedioxy;
11: R=2,3-methylenedioxy
Scheme 1: Reagents and conditions: a) 3% KOH in MeOH, RT, 2-5h, 68-86%;
The best analogue of the series i.e. compound 13 was further
modified as per scheme 2, to get some structural clues to 2-
benzyltetralone (25) using catalytic hydrogenation (Pd/C). On
reduction with NaBH4-TFA, compound 13 afforded 2-benzyltetralin
(26). 6,7-Dimethoxy-2-benzylidene tetralone (22) was also prepared
as for compounds 12-21. Compound 22 was selectively
demethylated with anhydrous aluminium chloride to get phenolic
products 23 and 24.
[Table 1. Cell inhibiting activity of the compounds (IC50 SEM in µM)]
placed at last
Structure activity relationship
Scheme 2
A.
O
These analogues had structural diversity mainly at benzylidene ring.
However, some definite trends were observed. These compounds
mostly exhibited potent cytotoxicity against MDA-MB-231 i.e.
hormone independent breast cancer cell line. Hence, for SAR we
focused on trend against this cell line. Analogue 12 possessing no
substitution in benzylidene ring showed marginal activity
(IC50=40μM) against MDA-MB-231 cells. While, compound 13 with
3,4-dimethoxy substitution at benzylidene ring was the best
analogue (IC50=1μM). But, on introducing another methoxy group at
C5´ position of benzylidene ring (analogue 14) activity (IC50=42μM)
was reduced drastically as compared to 12. Introducing a Nitro
(withdrawing) group at meta position (Analogue 18), activity was
slightly enhanced (IC50=36μM) but changing the position of nitro
group from meta to para (analogue 19) activity (IC50=1.5μM) was
enhanced remarkably. Similarly, another withdrawing group
trifluoromethyl at benzylidene ring (analogue 15) also exhibited
potent cytotoxicity (IC50=3.2μM). But, in case of p-fluoro
substitution (analogue 17) activity was abolished.
OCH3
H3CO
OCH3
13
b
a
O
OCH3
OCH3
OCH3
OCH3
H3CO
H3CO
26
25
H
O
O
O
H3CO
H3CO
H3CO
H3CO
OCH3
OCH3
c
B.
+
OCH3
OCH3
22
1
3
d
O
O
H3CO
HO
OCH3
OCH3
H3CO
HO
OCH3
+
OH
23
24
Scheme 2: Reagents and conditions: a) 10% Pd-C, THF, RT, quantitative; b)
NaBH4-TFA, 0oC to RT, 53%; c) 3% KOH in MeOH, RT, 3h, 79%; d) AlCl3-DCM
RT, 23:24=1:1, 87%.
Analogues 13 exhibited best activity (IC50=1.0μM). On introducing
another methoxy group at C7 (analogue 22) activity was reduced.
Partial reduction of analogue 13 to 2-benzyl derivative (25),
retained potential cytotoxicity (IC50=8.9μM). While reducing the C1-
ketone, to C1-methylene (tetralin 26) compound was inactive.
Overall, from these modifications, it may be concluded that C1
ketone is essentially required in the tetralone skeleton and
presence of 3,4-dimethoxy in benzylidene ring enhances the
cytotoxicity.
Biological evaluation
Cytotoxicity evaluation
All the synthesized compounds were evaluated for anti-cancer
activity against breast cancer cell lines MCF-7 (estrogen responsive
proliferative breast cancer model) and MDA-MB-231 (estrogen
independent aggressive breast cancer model), LA7 (rat mammary
tumour), HCT116 (human colon cancer), H1299 (human lung
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Figure 1. Tubulin polymerisation kinetics of compounds 13, 15 and 19.
Figure 2. Effect of compounds 13, 15 and 19 on cell cycle progression in MDA-MB-231 cells. Cells were treated with compounds 13, 15 and 19 at 1µM for
24h and 48h stained with PI and cell cycle was analyzed by flow cytometry.
Antimitotic activity
disturbance created in this dynamic equilibrium by the microtubule
Microtubule dynamics is a crucial aspect of mitosis phase in cell modulators (stabilizers or inhibitors) induces cell death.12, 13
cycle. Microtubule interfering agents induce mitotic arrest which
leads to increase in 4N cells at G2/M phase.6 Therefore, we Cell cycle analysis
examined cell cycle phase distribution of MDA-MB-231 cells Flow cytogram results revealed that compounds 13, 15 and 19
followed by compounds 13, 15 and 19 treatments in MDA-MB-231 induced G0/G1 cells cycle arrest as compared to untreated vehicle
cells by PI staining using flow cytometry.10 In the kinetics graph control cells (Figure 2), while, there was subsequent decrease of
(Figure 1), compounds 13, 15 and 19 showed interference in tubulin synthetic phase (S-phase) of cells cycle. Usually, microtubule
15
polymerization. Paclitaxel was taken as a positive control. Results stabilizers like paclitaxel are known to induce G2/M arrest.14,
showed that compounds 13, 15 and 19 stabilized tubulin assembly Following mitotic arrest, cells may have two fates, culminating into
at indicated concentrations in comparison to control groups. apoptosis or go for mitotic exit without proper cell division (mitotic
Microtubule assembly is vital for many fundamental cellular slippage) depending on the inhibitor concentration and check point
processes.11 The conversion of tubulin to microtubule is in dynamic status. In case of mitotic slippage, aberrant mitosis occurs and cells
equilibrium which is very much crucial for mitotic process. Any appear arrested in G1 phase due to pseudo G1 accumulation.16
Since our microtubule polymerization results clearly showed
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induction of microtubule stabilization, in view of this we believe
that the G1 arrest in cell cycle analysis may suggest mitotic slippage
into pseudo G1 followed by cell death.17,18 Low concentration of Activation of Reactive Oxygen Species
paclitaxel has also been showed to induce G1 arrest.19,20 In addition, Effect of compound 13 was evaluated on ROS generation by ROS
microtubule stabilization and microtubule destabilization leading to sensitive probe DCFH-DA staining using flow cytometry in MDA-MB-
mitotic arrest may not be the only processes for inducing anti- 231 cells.24 There was increased ROS generation detected in
tubulin effects. There can be a mixed mechanism which is not fully compound 13 treated cells as compare to untreated vehicle control
understood so far, this opens new avenues to explore this area. cells (Figure 4). Thus, ROS play as a key mediator in compound 13
However, induction of cell cycle arrest in cancer cell lines mediated apoptosis induction and cancer cell inhibition. Activation
constitutes one of the most prevalent strategies to stop or limit of ROS by compound 13 and subsequent apoptosis induction
cancer spreading.21,22
demonstrates a possible pathway leading to cell death by these
compounds. Role of ROS in regulation of apoptosis is well accepted
and widely documented.25,26 Activated of ROS in response to
Apoptosis induction
In order to evaluate nuclear morphology in response to compound paclitaxel and other microtubule stabilizer is also documented to be
13, Hoechst 33258 staining was carried out using microscopy in associated with apoptosis in cancer cells.27 Therefore, interference
MDA-MB-231 cells. Shrunken nucleus and peripherally clumped and in tubulin polymerization induced by compound 13 and subsequent
fragmented chromatin were seen following compound 13 apoptosis is probably mediated by ROS.
treatments, a typical characteristic of cells undergone apoptosis
(Figure 3A). To further confirm whether cell growth inhibition was
associated with physiological apoptosis or non-specific necrosis
AnnexinV-FITC and PI dual staining was carried out using flow
cytometer.23 Compound 13, the most potent compound of this
series induced time dependent increase of apoptotic cells
(AnnexinV-FITC stained) and no necrotic (PI stained) population was
observed in compound 13 treatment as compare to untreated
vehicle control cells (Figure 3B). Mitotic arrest commonly observed
as G2/M arrest directly lead to apoptosis called as mitotic death. Figure 4. Effect of compound 13 at 1 µm for 24h, and 48h on ROS generation
in MDA-MB-231 cells. ROS level was evaluated using DCFH-DA dye by flow
cytometry in response to compound 13.
Here, in our case, treatments of compounds probably induce
mitotic slippage which is followed by apoptosis rather than
senescence or resistance.
To decipher role of increased ROS generation in compound 13
induced cytotoxicity, MDA-MB-231 cells were treated with NAC, a
ROS scavenger followed by compound 13 treatment for 48h and
cytotoxicity was evaluated using MTT assay. Result showed that in
presence of ROS scavenger compound 13-mediated cytotoxicity
was abolished (Figure 5). This clearly suggests that ROS generation
have a major role during compound 13 -induced cytotoxicity.
Figure 3. Effect of compound 13 on nuclear morphology and apoptosis in
MDA-MB-231 cells. (A) Cells were treated with compound 13 at 1µm and
nuclear morphology was checked by hoechst staining by microscopy at 40x.
(B) Cells were treated with compound 13 at 1µM for 24h and 48h and
apoptosis was evaluated using AnnexinV-FITC/PI dual staining kit by flow
cytometry.
Figure 5. ROS is required in compound 13-mediated cytotoxicity in MDA-
MB-231 for 48h
In vivo anti-tumour activity against rat mammary tumour
LA7 induced syngenic Sprague Dawley rat mammary tumour
model was used to determine in vivo anti-cancer efficacy of
compound 13.24 Daily oral administration of 5 mg/kg and 10
mg/kg of compound 13 resulted in significant regression of
tumour as compared to untreated vehicle control group (Figure
6). In addition, significant reduction of final tumor weight was
also observed in the compound 13 treated group. Haematoxylin
and Eosin staining of tumor tissue section showed densely
growing tumor cells with pleomorphic, hyperchromatic nuclei
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and low cytoplasmic content in untreated vehicle control tumor
section (Figure 7). Tumor tissue section of the compound 13
treated group showed decreased cellular density with
vacuolization and elastosis as compared to untreated vehicle
control. The histopathological recovery of tumor cellularity was
dose dependent. No significant change in terms of body weight
was observed in the treated group. Collectively, these in vivo
results suggest that compound 13 shows potent anti-tumour
effect on mammary tumour cells without any apparent
cytotoxicity.
Figure 6 Effect of compound 13 on tumor regression in syngenic rat mammary tumor model. (A) Compound 13 was daily administered orally and tumor was measured twice a
week. At the end, animals were sacrificed, tumors were excised from animals and the tumor was photographed. (B) Tumor volume was calculated and plotted in response to days
of treatment. (C) After end of experiment tumor weight was taken and graph was plotted. (D) Body weight was measured twice a week and graph was plotted.
Figure 7. Histopathology (H & E staining) images of tumor sections of compound 13 tumor tissues shows morphological differences between untreated vehicle control and
treated tumor tissues. Magnifications at 20X
In vivo acute oral toxicity
2 & Fig. 8). Animals on gross pathological study showed no changes
Acute oral toxicity is used to assess the ability of a substance to in any of the organs
cause adverse effects within a short period of time following dosing studied including their absolute and relative weight (Figure 8).
or exposure.28 In acute oral toxicity experiment of compound 13, Therefore, the experiment showed that compound 13 was well
there were no observational changes, morbidity and mortality tolerated by the Swiss albino mice up to the dose level of 1000
throughout the experimental period in all the groups of mg/kg body weight as a single acute oral dose. However, sub-acute
experimental animals up to the tested dose levels of 1000 mg/kg. and or chronic experiment with the test drug needs to be carried
Blood and serum samples upon analysis showed non-significant out to look for any adverse effect on repeated exposure to
changes in all the parameters studied like haemoglobin level, RBC compound 13 in view of significant increase in serum albumin and
count, WBC count, differential leucocytes count, SGPT, ALP, SGOT level in animals treated with the test drug at 1000 mg/kg.
creatinine, triglycerides, cholesterol, albumin, serum protein (Table
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Table 2. Effect of compound 13 as a single acute oral dose at 5, 50, 300 and 1000 mg/kg on body weight, haematological and serum biochemical parameters
in Swiss albino mice (Mean±SE, n=6)
Parameters
Dose of compound 13 at mg/kg body weight as a single oral dose
Control
22.43±0.34
13.72±0.79
6.53±0.24
5 mg/kg
23.26±0.69
15.59±1.27
6.76±0.19
50 mg/kg
23.44±0.59
13.41±1.37
6.77±0.17
300 mg/kg
23.03±1.03
14.01±0.33
6.78±0.21
1000 mg/kg
23.36±0.76
14.44±.017
7.13±0.25
Body wt. 7th day (g)
Haemoglobin (g/dL)
RBC (106/mm3)
WBC (1000/mm3)
10.69±0.80
297.01±12.10
30.34±2.22
31.17±1.94
2.15±0.05
10.09±0.33
301.33±18.29
30.21±1.82
27.71±1.33
1.97±0.11
9.75±0.22
9.24±0.32
9.42±0.33
ALP (U/L)
317.73±15.63
29.30±2.43
26.87±1.04
2.08±0.05
298.05±17.44
26.68±1.56
31.93±2.50
1.97±0.12
307.50±20.63
29.60±2.15
27.27±1.32
2.13±0.10
SGPT (U/L)
SGOT (U/L)
Albumin (g/dL)
Creatinine (mg/dL)
Triglycerides (mg/dL)
Serum Protein (mg/ml)
0.31±0.02
0.43±0.09
0.60±0.06
0.47±0.04
0.63±0.06
268.94±39.6
3.09±0.23
245.49±40.9
2.95±0.29
335.96±23.6
3.01±0.15
211.03±27.7
2.96±0.33
303.85.1±29.1
3.98±0.30
Cholesterol (mg/dL)
219.02±26.6
191.15±28.92
193.68±25.45
205.02±29.93
203.62±25.86
* P<0.05 compared to control; a P<0.05 compared to control, 5, 50, 300, 1000 mg/kg) .
Figure 8. Effect of compound 13 as a single acute oral dose at 5, 50, 300 and 1000 mg/kg on (A) absolute and (B) relative organ weights (C) differential
leucocytes counts in Swiss albino mice (n=6, Non significant changes were found compared to control).
ceric sulphate in 10% sulphuric acid (aqueous). Compounds were
Experimental section
General experimental Procedures
purified through Flash chromatography system (CombiFlash Rf200i,
Teledyne-ISCO, USA) using glass columns (13cm lengthx2cm i.d.)
The starting substrate 6-methoxytetralone was procured from and silica gel (230-400mesh) using UV detector (254nm & 280nm)
Sigma Chemicals India. Reagents and other chemicals were and characterised by 1H and 13C NMR, ESI-MS, and ESI-HRMS. The
purchased from Avra Synthesis India and used without purification. purity of the final compound benzylidene indanone
1 was
Melting points were determined on E-Z Melt automated melting ascertained by Reversed Phase-HPLC. NMR spectra were obtained
point apparatus in open glass capillaries and were uncorrected. on Bruker Avance-DRX300 MHz instrument with tetramethylsilane
Reactions were monitored on pre-coated silica gel TLC-GF254 (TMS) as an internal standard. Chemical shifts are given in δ ppm
1
aluminium sheets (E. Merck, Germany) and visualization was done values. H–1H coupling constant (J) values are given in Hz. ESI mass
under UV light (254nm and 365nm) and further charring with 2% spectra were recorded on APC3000 LC-MS-MS (Applied Biosystem)
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and High Resolution Mass (HRMS) on Agilent 6520Q-TOF after 153.48, 164.00, 188.44; ESI-MS (MeOH) for C21H22O5: 355 [M+H]+;
dissolving the compounds in methanol. FT-IR spectra were recorded Negative mode: 353 [M-H]-.
on Perkin–Elmer SpectrumBX.
2-(4´-Trifluoromethylbenzylidene)-6-methoxy-3,4-
DMEM and FBS were purchased from Gibco, India. RNaseA, Crystal dihydronaphthalen-1(2H)-one (15)
1
Violet Dye, HEPES, Trypsin-EDTA, Antibiotic-Antimycotic (Ab-Am) yield=70%; m.p.=136oC; H NMR (CDCl3, 300MHz): δ2.38 (bs, 2H, 3-
Solution, Phosphate Buffer Saline (PBS), Citric Acid and Propidium CH2), 2.51 (bs, 2H, 4-CH2), 3.33 (s,3H, OCH3), 6.16 (s, 1H, 5-CH), 6.36
Iodide (PI) were acquired from Sigma-Aldrich, USA. Sodium (d, 1H, 7-CH, J=8.7Hz), 6.97 (d, 2H, 2´ & 6´-CH of benzylidene ring,
bicarbonate (NaHCO3), Agar, Sodium Citrate and Di-sodium J=8.6Hz), 7.11 (d, 2H, 3´ & 5´-CH of benzylidene ring); 7.26 (s, 1H, 2-
Hydrogen Phosphate were obtained from Himedia Laboratories, benzylidene CH=), 7.58 (d, 1H, 8-CH, J=8.7Hz), 13C NMR (CDCl3,
India. Solvents including ethanol and isopropanol were procured 75MHz): δ27.60, 29.61, 55.87, 112.76, 113.94, 125.71, 125.76,
from Merck, India Ltd. In-vivo experiments were conducted on 127.20, 130.24, 131.29, 134.42, 137.96, 146.10, 164.23, 186.71; ESI-
Swiss-albino mice after the approval of Institute’s Animal Ethical MS (MeOH) for C19H15F3O2: 333.3 [M+H]+, 355.2 [M+Na]+, 371.1
Committee.
[M+K]+.
Chemical syntheses
2-(4´-N,N-Dimethylaminobenzylidene)-6-methoxy-3,4-
General procedure for the synthesis of benzylidene tetralones (12- dihydronaphthalen-1(2H)-one (16)
22)
yield=68%; m.p.=151oC; 1H NMR (CDCl3, 300MHz): δ2.90 (t, 2H, 3-
6-Methoxytetralone (352mg, 2mmol) was taken in 10mL of 3% CH2, J=6.2Hz), 3.01 (s, 6H, N-(CH3)2), 3.13 (bs, 2H, 4-CH2), 3.86 (s,3H,
potassium hydroxide in methanol (w/v) in an ice-bath (5-10oC). To OCH3), 6.69 (bs, 3H, 5-CH and 2´ & 6´-CH of 2-benzylidene ring),
this stirred reaction mixture aromatic benzaldehyde (2-11, 2mmol) 6.86 (d, 1H, 7-CH, J=8.7Hz), 7.41 (d, 2H, 3´ & 5´-CH of benzylidene
was added and stirred for 2-5h. On completion, solvent was ring, J=8.4Hz), 7.81 (s, 1H, 2-benzylidene CH=), 8.10 (d, 1H, 8-CH,
evaporated and water (10mL) was added to it and acidified with 5% J=8.7Hz); 13C NMR (CDCl3, 75MHz): δ 27.79, 29.62, 30.11, 40.61,
dil. HCl (10mL). The reaction mixture was extracted with ethyl 55.81, 112.10, 112.56, 113.44, 124.26, 127.93, 130.93, 131.73,
acetate, washed with water and organic layer was dried over 132.33, 137.57, 145.81, 150.86, 163.62, 187.21; ESI-MS (MeOH) )
anhydrous sodium sulphate. On evaporated, a residue was obtained for C20H21NO2: 308.2 [M+H]+, 330.2 [M+Na]+, 346.0 [M+K]+.
which was purified through column chromatography over silica gel. 2-(4´-Fluorobenzylidene)-6-methoxy-3,4-dihydronaphthalen-
On elution with ethyl acetate-hexane the pure product (12-22) was 1(2H)-one (17)
1
obtained in good yield.
yield=82%; m.p.=105oC; H NMR (CDCl3, 300MHz): δ2.84 (bs, 2H, 3-
2-Benzylidene-6-methoxy-3,4-dihydronaphthalen-1(2H)-one (12)
CH2), 2.98 (bs, 2H, 4-CH2), 3.79 (s,3H, OCH3), 6.62 (s, 1H, 5-CH), 6.78
yield=83%; m.p.= 86oC; H NMR (CDCl3, 300MHz): δ2.89 (bs, 2H, 3- (d, 1H, 7-CH, J=8.4Hz), 7.14-7.17 (d, 2H, 2´ & 6´-CH of benzylidene
CH2, J=6.3Hz), 3.11 (bs, 2H, 4-CH2, J=5.7Hz), 3.86 (s,3H, OCH3), 6.71 ring, J=8.7Hz), 7.79 (s, 1H, 2-benzylidene CH=), 8.09 (d, 1H, 8-CH,
(s, 1H, 5-CH), 6.87 (d, 1H, 7-CH, J=8.7Hz), 7.26-7.38 (m, 5H, 2- J=8.9Hz), 8.2 (d, 2H, 3´ & 5´-CH of benzylidene ring); 13C NMR
benzylidene phenyl ring), 7.84 (s, 1H, 2-benzylidene CH=), 8.18 (d, (CDCl3, 75MHz): δ27.55, 29.61, 55.85, 112.70, 113.79, 115.75,
1H, 8-CH, J=8.7Hz); 13C NMR (CDCl3, 75MHz): δ27.63, 29.71, 55.86, 116.04, 127.37, 131.18, 132.00, 132.11, 132.52, 135.22, 135.82,
112.70, 113.77, 127.44, 128.30, 129.83, 130.23, 130.55, 131.19, 146.02, 161.28, 164.06, 164.58, 186.97; ESI-MS (MeOH) for
136.03, 136.44, 146.19, 164.02, 187.26; ESI-MS (MeOH) for C18H15FO2: 283.3 [M+H]+, 305.3 [M+Na]+, 565.6 [2M+H]+.
C18H16O2: 265.0 [M+H]+, 287.1 [M+Na]+, 303.0 [M+K]+, 551.2 2-(3´-Nitrobenzylidene)-6-methoxy-3,4-dihydronaphthalen-1(2H)-
1
[2M+Na]+.
one (18)
2-(3´,4´-Dimethoxybenzylidene)-6-methoxy-3,4-
yield=73%; m.p.=163oC; 1H NMR (CDCl3, 300MHz): δ2.92 (t, 2H, 3-
CH2, J=6.2Hz), 3.06 (t, 2H, 4-CH2, J=5.9Hz), 3.85 (s,3H, OCH3), 6.69 (s,
dihydronaphthalen-1(2H)-one (13)
1
yield=86%; m.p.=112oC; H NMR (CDCl3, 300MHz): δ2.91 (bs, 2H, 3- 1H, 5-CH), 6.85 (d, 1H, 7-CH, J=8.7Hz), 7.57 (t, 1H, CH of 3´-CH of
CH2), 3.21 (bs, 2H, 4-CH2), 3.85 (s,3H, OCH3), 3.90 (s,6H, 2xOCH3), benzylidene ring, J=8.0Hz), 7.68 (d, 1H, 2´-CH benzylidene ring,
6.69-8.10 (m, 7H, all aromatic protons and 2- benzylidene CH); 13C J=7.5Hz); 7.78 (s, 1H, 2-benzylidene CH=), 8.08 (d, 1H, 8-CH,
NMR (CDCl3, 75MHz): δ27.70, 29.60, 55.82, 56.33, 112.64, 112.81, J=8.7Hz),, 8.16 (d, 1H, 4´-CH of benzylidene ring), 8.26 (s, 1H, 6´-CH
113.65, 123.54, 127.55, 129.25, 131.06, 134.39, 136.53, 145.93, of benzylidene ring); 13C NMR (CDCl3, 75MHz): δ27.56, 29.48, 55.90,
149.14, 149.86, 163.89, 187.05; ESI-MS (MeOH) for C20H20O4: 325 112.76, 114.05, 123.29, 124.46, 127.05, 131.32, 133.21, 136.03,
[M+H]+, 347 [M+Na]+. HRMS (ESI-TOF) m/z [M+H]+ calcd for 138.12, 138.50, 146.03, 148.72, 164.32, 186.37; ESI-MS (MeOH) for
C20H21O4 325.1440, found 325.1430.
C18H15NO4: 310 [M+H]+, 332 [M+Na]+.
2-(3´,4´,5´-Trimethoxybenzylidene)-6-methoxy-3,4-
dihydronaphthalen-1(2H)-one (14)
2-(4´-Nitrobenzylidene)-6-methoxy-3,4-dihydronaphthalen-1(2H)-
one (19)
yield=78%; m.p.=108oC; 1H NMR (CDCl3, 300MHz): δ2.90-2.94 (t, 2H, yield=71%; m.p.=187oC; H NMR (CDCl3, 300MHz): δ2.73 (dt, 2H, 3-
3-CH2, J=6.6Hz), 3.09-3.14 (t, 2H, 4-CH2, J=6.2Hz), 3.85 (s,3H, OCH3), CH2, J=6.3), 2.93, t, 2H, 4-CH2, J=5.9Hz), 3.87 (s,3H, OCH3), 6.60 (s,
3.88 (s,3H, OCH3), 3.89 (s,3H, OCH3), 6.66 (bs, 1H, 5-CH), 6.71 (bs, 1H, 5-CH), 6.88 (d, 1H, 7-CH, J=8.4Hz), 7.14-7.17 (d, 2H, 2´ & 6´-CH
1H, 7-CH), 6.94 (s, 2H, 2´ & 6´-CH of benzylidene ring, J=8.6Hz), 7.76 of benzylidene ring, J=8.7Hz), 7.79 (s, 1H, 2-benzylidene CH=), 8.09
(s, 1H, 2-benzylidene CH=), 8.09 (m, 1H, 8-CH); 13C NMR (CDCl3, (d, 1H, 8-CH, J=8.9Hz), 8.2 (d, 2H, 3´ & 5´-CH of benzylidene ring);
75MHz): δ27.75, 29.74, 55.83, 56.49, 56.61, 61.22, 107.69, 112.68, 13C NMR (CDCl3, 75MHz): δ27.71, 29.49, 55.91, 112.79, 114.07,
127.42, 128.58, 131.10, 131.90, 136.30, 136.59, 138.96, 146.38, 124.06, 124.06, 127.01, 130.74, 130.74, 131.34, 133.36, 139.18,
143.14, 146.06, 147.59, 164.36, 186.32; ESI-MS (MeOH) for
1
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C18H15NO4: 310.3 [M+H]+, 332.3 [M+Na]+, 348.1 [M+K]+, 619.5 146.78, 150.96, 188.00; ESI-MS (MeOH) for C19H16O5: 327.3 [M+H]+,
[2M+H]+, 641 [2M+Na]+, 657.4 [2M+K]+.
2-(3´,4´-Methylenedioxybenzylidene)-6-methoxy-3,4-
dihydronaphthalen-1(2H)-one (20)
349.2 [M+Na]+, 365.2 [M+K]+.
2-(3´,4´-dimethoxybenzyl)-6-methoxy-3,4-dihydronaphthalen-
1(2H)-one (25)
yield=82%; m.p.=173oC; 1H NMR (CDCl3, 300MHz): δ2.92 (t, 2H, 3- Benzylidene tetralone 13 (200mg, 0.62mmol) was taken in dry THF
CH2, J=6.3Hz), 3.10 (t, 2H, 4-CH2, J=5.9Hz), 3.87 (s,3H, OCH3), 6.01 (s, (10mL). To this stirred solution Pd-C (10%, 100mg) was added.
2H, O-CH2-O), 6.72 (s, 1H, 5-CH), 6.86-6.97 (m, 4H, 4xCH, aromatic), Hydrogen gas was supplied through balloon at room temperature.
7.73 (s, 1H, 2-CH of benzylidene); 8.07 (d, 1H, 8-CH, J=8.7Hz); ESI- The reaction was stirred for an hour. On completion the reaction
MS (MeOH) for C19H16O4: 309.3 [M+H]+, 331.3 [M+Na]+, 347.1 mixture was filtered through celite bed, washed with ethyl acetate
[M+K]+.
and evaporated. The residue thus obtained was recrystallised with
chloroform-hexane (1:3) to get 25 as amorphous solid.
2-(2´,3´-Methylenedioxybenzylidene)-6-methoxy-3,4-
dihydronaphthalen-1(2H)-one (21)
yield=179mg (89%); m.p.=99oC; 1H NMR (CDCl3, 300MHz): ): δ1.43
yield=86%; m.p.=101oC; H NMR (CDCl3, 300MHz): δ2.47 (bs, 2H, 3- (bs, 2H, CH2, benzylic), 2.02 (bs, 1H, 2-CH), 2.90 (bs, 2H, 3-CH2), 3.14
CH2), 2.54 (bs, 2H, 4-CH2), 3.41 (s,3H, OCH3), 5.54 (s, 2H, O-CH2-O), (bs, 2H, 4-CH2), 3.94 (bs, 9H, 3xOCH3), 6.68 (s, 2H, CH2 of benzylic),
6.23 (s, 1H, 5-CH), 6.23-6.41 (bs, 4H, CH, aromatic), 7.26 (s, 1H, CH 6.77-7.11 (m, 3H, aromatic), 7.77 (s, 1H, 5-CH), 8.09 (s, 1H, 8-CH);
of benzylidene ring, J=8.6Hz), 7.67 (bs, 1H, CH aromatic); 13C NMR 13C NMR (CDCl3, 75MHz): δ27.70, 29.75, 30.09, 55.80, 56.31x2,
(CDCl3, 75MHz): δ28.24, 29.71, 55.84, 101.35, 108.96, 112.72, 111.33, 112.63, 113.64, 123.55, 127.52, 129.23, 131.06, 134.35,
113.72, 118.84, 121.77, 122.97, 127.40, 129.48, 131.17, 137.64, 136.57, 145.93, 149.12, 149.86, 163.89, 187.06; ESI-MS (MeOH) for
146.44, 148.03, 163.99, 186.69; ESI-MS (MeOH) for C19H16O4: 309 C20H22O4: 327 [M+H]+.
1
[M+H]+.
2-(3´,4´-dimethoxybenzyl)-6-methoxy-1,2,3,4-
tetrahydronaphthalen (26):
2-(3´,4´-Dimethoxybenzylidene)-6,7-dimethoxy-3,4-
dihydronaphthalen-1(2H)-one (22)
To a stirred solution of benzylidene tetralone 13 (200mg,
yield=79%; m.p.=345oC (Blackens); H NMR (CDCl3, 300MHz): δ2.89 0.62mmol) in TFA (1mL) at 5-10oC, sodium borohydride
(t, 2H, 3-CH2, J=6.3Hz), 3.14 (t, 2H, 4-CH2, J=5.9Hz), 3.91 (s, 12H, (150mg,4mmol) was added in portions over a period of 20 min. and
4xOCH3), 6.66 (s, 1H, =CH of benzylidene), 6.88 (d, 1H, 3´-CH of further stirred for an hour. On completion the reaction mixture was
benzylidene phenyl, J=8.4Hz), 6.97 (s, 1H, CH, aromatic), 7.04 (d, poured to crushed ice and extracted with ethyl acetate (15mLx2),
1H, 2´-CH of benzylidene phenyl, J=8.4Hz), 7.62 (s, 1H, 5-CH), 7.77 washed with water and dried over anhydrous sodium sulphate. The
(s, 1H, 8-CH); 13C NMR (CDCl3, 75MHz): δ27.93, 28.97, 56.35, 56.47, dried organic layer was evaporated in vacuo and crude mass was
110.12, 110.28, 111.39, 123.49, 127.14, 129.30, 134.25, 136.50, purified through column chromatography over silica gel (100-200
138.39, 148.67, 149.18, 149.87, 153.87, 187.03; ESI-MS (MeOH) for mesh) to get pure 26.
1
C21H22O5: 354 [M+H]+.
yield=102mg (53%); m.p.=74oC; 1H NMR (CDCl3, 300MHz): δ1.49
(bs, 2H, CH2, benzylic), 2.00 (m, 1H, 2-CH), 2.62 (bs, 2H, 3-CH2), 2.79
Synthesis of 23 and 24
Compound 22 (200mg, 0.56mmol) was taken in dry DCM (10mL). To (bs, 2H, 4-CH2), 3.76 (s, 3H, OCH3), 3.88 (s, 6H, 2xOCH3), 6.66-6.96
this stirred solution anhydrous aluminium chloride (200g, 1.5mmol) (m, 6H, CH, aromatic); 13C NMR (CDCl3, 75MHz): δ, 29.50, 29.78,
was added and further stirred for 20 minutes to get products 23 35.54, 37.00, 42.85, 55.64, 56.25, 56.32, 111.50, 112.27, 112.75,
and 24 in 3:1.
113.83, 121.43, 129.07, 130.36, 133.85, 138.17, 147.64, 149.16,
157.92; ESI-MS (MeOH) for C20H24O3: 313 [M+H]+, 335 [M+Na]+, 351
[M+K]+.
2-(3´,4´-Dimethoxybenzylidene)-6-hydroxy-7-methoxy-3,4-
dihydronaphthalen-1(2H)-one (23)
Yield=108mg (56%); m.p.= 149oC; 1H NMR (CDCl3, 300MHz): δ2.89
(t, 2H, 3-CH2), 3.15 (t, 2H, 4-CH2), 3.91 (s,3H, OCH3), 3.94 (s, 6H, Biological assays
2xOCH3), 3.96 (s, 3H, OCH3),6.67 (s, 1H,CH of benzylidene ring), Cell cultures
6.97-6.98 (m, 3H, CH aromatic of benzylidene phenyl ring), 7.63 (s, Human breast cancer cell lines MCF-7( ER+ve) and MDA-MB-231 (
1H, 5-CH), 7.78 (s, 1H, 8-CH); 13C NMR (CDCl3, 75MHz): δ28.24, ER-ve), LA-7 (rat mammary tumour), PC-3 (human prostate cancer),
29.71, 55.84, 101.35, 108.96, 112.72, 113.72, 118.84, 121.77, HeLa (human cervical cancer), Ishikawa (human endometrial
122.97, 127.40, 129.48, 131.17, 137.64, 146.44, 148.03, 163.99, cancer), HCT116 (human colorectal cancer), H1299 (human small
186.69; ESI-MS (MeOH) for C19H18O4: 341 [M+H]+.
non-small lung cancer) and U935 (human histocytic lymphoma),
were originally obtained from American type of cell culture
2-(3´-methoxy-4´-hydroxybenzylidene)-6-hydroxy-7-methoxy-3,4-
dihydronaphthalen-1(2H)-one (24): Compound 22 (200mg, collection (ATCC) and human embryonic kidney cell (HEK-293) were
0.56mmol) was taken in dry DCM (10mL). To get compound 24 in obtained from Institute’s cell repository of animal tissue culture
higher quantity, aluminium chloride (600mg, 4.5mmol) is added and facility (CSIR-CDRI, Lucknow). Cells were cultured in DMEM
reaction time is for an hour to get 23:24 in 1:5.
(Dulbecco modified eagle medium, Sigma) supplemented with 10%
yield=134mg (73%); m.p.=167oC; 1H NMR (CDCl3, 300MHz): δ2.88 FBS (GIBCO BRL Laboratories, New York, USA) and 1% antibiotic-
(bs, 2H, 3-CH2), 3.14 (bs, 2H, 4-CH2), 3.91 (s, 3H, OCH3), 3.95 (s, 3H, antimycotic solution (Sigma Chemical CO., St Louis, MO, USA) at
OCH3), 6.78 (s, 1H, =CH of benzylidene), 6.89-6.97 (m, 3H, 370C with 5% CO2 in humidified chamber. LA7 cells were cultured in
aromatic), 7.65 (s, 1H, 5-CH), 7.76 (s, 1H, 8-CH); 13C NMR (CDCl3, DMEM supplemented with 5%FBS, 5 µg/ml insulin and 50 nM
75MHz): δ27.90, 28.71, 56.39, 56.62, 109.99, 113.11, 114.83, hydrocortisone.
116.17, 124.09, 126.99, 134.08, 136.73, 139.15, 146.30, 146.64,
8 | J. Name., 2012, 00, 1-3
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In vitro cell proliferation assay
were stained with hoechst33258 and images were captured using
The anti-cancer activity of tetralone derivatives was determined florescence microscope (Leica).
using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide
(MTT) assay as described earlier.9 Briefly, cells (1×104 cells) were Tubulin polymerisation assay
seeded in 96-well microculture plates in 200µL DMEM for 24h. Tubulin polymerization experiment was done as per reported
Compounds were diluted to desired concentrations in culture method using ‘assay kit’ from Cytoskeleton, USA.5,11 In brief, tubulin
medium DMEM without phenol red free DMEM culture medium protein (3 mg/mL) in tubulin polymerization buffer (80 mM PIPES,
and incubated for 24h and 48h. In case of inhibitor study, cells were pH 6.9, 2mM MgCl2, 0.5 mM EGTA, 1 mM GTP and 15% glycerol)
pre-treated with 10mM NAC for 2h followed by compound 13 for was placed in pre-warmed 96-well microtiter plates at 370C in the
48h. At the end of incubation, 20µL of 5mg/mL was added to each presence of test compounds with variable concentrations. All
well and the plates were further incubated for 3h. Thereafter, samples were mixed well and polymerization was monitored
supernatant from each well was carefully removed and formazan kinetically at 340nm every min for 1h using Spectramax plate
crystals were dissolved in 200µL of dimethylsulphoxide (DMSO) reader. paclitaxel (taxol) was used as standard stabilizer of tubulin
using plate shaker (Biosan) and absorbance was recorded in a polymerisation.
microplate reader at 570nm wavelength (Microquant; BioTek).
In vivo tumor regression
Cell cycle analysis
Animal studies were coordinated by prior approval from the
Cell cycle analysis was carried out to check distribution of the cells Institutional Animal Ethics committee (IAEC), CSIR-CDRI, Lucknow.
in different phases induced by compound 13, 15 and 19 using Adult sprague dawley (SD) RAT were used to develop syngenic
propidium iodide (PI) staining method. MDA-MB-231 cells (1×106 mammary tumor model.24 Briefly, LA-7 (6x106) cells were inoculated
cells) were seeded in T-25 culture flask and allowed to grow for 24h. into mammary fat pad of female SD rats. After one week, when
After 24h, cells were treated with compound 13 for 24h and 48h. At tumor size were measurable, animals were randomly grouped.
end of incuabtion, cells were harvested, fixed in ice cold 70% 5mg/kg and 10mg/kg body weight of compound 13 was orally
ethanol and incubated for 1 h at 40C. Thereafter, cells were administred daily. Control group receive vehicle only. During this
centrifuged and resuspended in 300µL PBS and incubated with 30μg period, tumors size were measured twice a week and tumor volume
of RNAseA and 15μg of PI for 30 minutes at room temperature in was calculated using formula; V=[(length)X(width)2]/2.
dark. Samples were analyzed by flow cytometry using FACSCalibur
instrument (BD biosciences).
Hematoxylin and eosin (H & E) staining
Formalin-fixed tumor tissue was used for histopathological studies.
After deparaffinization and dehydration, tissue samples were
Apoptosis analysis
To determine effect of compound 13 on apoptosis in MDA-MB-231 stained with Harris-hematoxylin and eosin stains. The images were
cells, AnnexinV-FITC/PI dual staining was carried out by flow taken under 20X magnification using light microscopy
cytometry.24 Cells (1x106 cells) were seeded in six well plates and (OlympusBX51, Tokyo, Japan). For each group, sections from at
allowed to grow for 24h. Cells were treated with compound 13 for least three rats (n=3) were examined for histopathological analysis.
24h and 48h. At the end of incubation cells were harvested, washed
with PBS and stained with AnnexinV-FITC and PI using apoptosis Acute oral toxicity
detection kit (sigma). Samples were acquired by flow cytometer In view of potent anti-cancer activity of compound 13, acute oral
using FACSCalibur instrument (BD biosciences).
toxicity of the same was carried out in Swiss albino mice for its
further development as drug candidate. Experiment was
conducted in accordance with the Organization for Economic Co-
a
ROS generation assay
ROS generation was measured using 2,7-Dichlorodihydrofluorescein operation and Development (OECD) test guideline No 423 (1987).
diacetate dye by flow cytometer.24 MDA-MB-231 cells (1x106) were Briefly, 30 mice (15 male and 15 female) were taken and divided
seeded in 6 well plates for 24h. Cells were treated with compound into four groups comprising 3 male and 3 female mice in each group
13 for 24h and 48h. At the end of incubation, cells were harvested weighing between 20-25 g. The animals were maintained at 22±50C
by trypisinization, fixed in cold methanol and incubated with with humidity control and also on an automatic dark and light cycle
30µg/mL of DCFH-DA dye for 30 minutes in dark at room of 12 hours. The animals were fed with the standard mice feed and
temperature. After the end of incubation, cells were centrifuged, provided ad libitum drinking water. Mice of group 1 (Group I) were
resuspended in 300µL of PBS. Samples were analyzed by flow kept as control and animals of groups 2, 3, 4 and 5 (Groups II, III, IV
cytometry using FACSCalibur instrument (BD biosciences).
& V) were kept as experimental. The animals were acclimatized for
7 days in the experimental environment prior to the actual
experimentation. Compound 13 was dissolved in minimum volume
Hoechst staining
Nuclear fragmentation was detected by staining nuclei with of DMSO, diluted with 0.7% CMC (carboxymethylcellulose) and was
hoechst33258. MDA-MB-231 cells (1x106) were seeded in six well given at 5, 50, 300 and 1000 mg/kg body weight to animals of
plates for 24h. Thereafter, cells were treated with compound 13 for groups 2, 3, 4 and 5 (Groups II, III, IV & V) respectively once orally.
24h and 48h. Cells were washed with PBS and fixed with 4% Control animals received only vehicle. The animals were checked
paraformaldehyde and permeabilized with 0.5% tritonX-100. Cells for mortality and any signs of ill health at hourly interval on the day
of administration of drug and there after a daily general case side
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clinical examination was carried out for changes in skin, mucous
membrane, eyes, occurrence of secretion and excretion and also References
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Conclusions
In conclusion, 2-benzylidene tetralones have been explored as 13. M. K. Gardener, B. D. Charlebois, I. M. Janosi, J. Howard, A. J.
potent anti-breast cancer agents. The representative compounds 13 Hunt, D. J. Odde. Cell 2011, 146 , 582.
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compound of this series, compound 13 induced stabilization of 15. B. A. Weaver. Perspective on Cell Biology and Human Health
tubulin polymerization similar to paclitaxel along with cell cycle
arrest and apoptosis. However, compound 13 showed G1 arrest
unlike other microtubule inhibitors possibly due to mitotic slippage
associated secondary apoptosis. Tubulin stabilizers are known to
activate ROS and we observed a significant induction of ROS by
compound 13. ROS dependent cell death was further confirmed
with inhibition of ROS using its scavenger that abrogated compound
induced apoptosis. This suggested that interference in tubulin
polymerisation by compound 13 is possibly linked with ROS
generation and cancer cell death. Most interestingly, compound 13
reduced rat mammary tumour growth in vivo by 74-79% at both
5mg/kg and 10mg/kg. The additional data on acute oral toxicity
suggested that compound 13 can be well tolerated by the mice up
to 1000mg/kg dose without any adverse effects. Compound 13
could serve as promising lead molecule for targeting breast cancer
inhibition through tubulin stabilization and microtubule exit without
significance in-vitro and in-vivo toxicity.
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We are grateful to SERB-DST for funding support. Senior Research
Fellowship to the authors (YG) and (H) from Council of Scientific and
Industrial Research (CSIR) and Department of Biotechnology
respectively is duly acknowledged. We also thank Mr. AL
Vishwakarma, SAIF, CSIR-CDRI for flow cytometric analysis.
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Supplementary Information
1
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……………………
and 19 will be available online......
Notes
The authors declare no competing financial interest.
10 | J. Name., 2012, 00, 1-3
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DOI: 10.1039/C6RA02663J
ARTICLE
Table 1. Cell inhibiting activity of compounds (IC50 ±SEM in µM)
Comp
12
MCF-7
24 h
MDA-MB-231
LA-7
24h
PC-3
24h
HeLa
24h
Ishikawa
24h
HCT116
24h
H1299
24h
U937
24h
HEK-293
24 h
48 h
24 h
40
48 h
48h
48h
48h
48h
48h
48h
48h
48 h
>50
>50
>50
ND*
ND
>50
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
>50
>50
±0.01
>50
13
>50
>50
1
>50
7
>50
25
>50
18
>50
34
>50
6.12
>50
7.5
>50
19
>50
>50
0.03
0.05
0.03
0.01
0.04
0.02
0.01
±0.01
>50
3.25
±0.01
>50
14
15
>50
>50
>50
>50
>50
ND
>50
ND
15
0.02
>50
>50
ND
29
0.02
ND
>50
ND
20
0.05
ND
>50
ND
40
0.02
ND
>50
ND
ND
>50
ND
ND
>50
ND
22
0.03
>50
>50
>50
>50
12.5
±0.01
>50
9.76
0.02
10.2
0.01
18
19
20
>50
>50
36.2
±0.02
>50
ND
>50
ND
ND
>50
>50
>50
ND
ND
>50
ND
ND
ND
>50
ND
ND
ND
>50
ND
ND
ND
>50
ND
ND
ND
>50
ND
ND
>50
>50
>50
>50
>50
>50
>50
>50
1.53
±0.01
>50
18
0.01
30
0.03
25
0.02
42
0.02
22.4
0.07
15.3
0.03
25
0.05
21.6
±0.02
>50
>50
ND
ND
ND
ND
ND
ND
ND
21
22
>50
>50
>50
>50
>50
ND
ND
ND
ND
>50
>50
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
>50
>50
>50
>50
>50
43.51
±0.02
>50
25
25.9
0.02
1
0.01
2.60
0.01
12.6
0.03
0.32
0.02
1.5
8.91
0.01
0.41
0.01
6
ND
ND
1.2
0.02
11
0.05
>50
ND
1.1
0.03
>50
ND
ND
ND
ND
2.9
0.02
28.7
0.01
ND
ND
0.1
0.02
38
0.05
ND
ND
4
0.01
25
0.02
ND
ND
1.5
0.01
8.9
0.03
>50
35.15
0.02
18
0.02
30
0.03
Paclit
axel
3.6
2.4
1.7
0.04
>50
1
0.65
5.2
0.4
7.5
1
25
0.01
10
0.02
0.01
20
0.07
0.01
40
0.04
0.03
31
0.02
0.04
32.2
0.03
0.01
45
0.03
0.03
30
0.01
0.02
5.7
0.04
0.01
26
0.01
Tamo
xifen
0.02
0.01
* ND= Not Determined
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Graphical Abstract
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2-(3´, 4´-dimethoxybenzylidene) tetralone induces anti-breast
Leave this area blank for abstract info.
cancer activity through microtubule stabilization and activation
of reactive oxygen species
Yashveer Gautam, Sonam Dwivedi, Ankita Srivastava,
Hamidullah, Arjun Singh, D. Chanda, Jyotsna Singh,
Smita Rai, Rituraj Konwar, and Arvind S. Negi
Microtubule stabilization
In-vitro cytotoxicity
MDA-MB-231, IC50=1µM
HEK-293, IC50=>50µM
Cell cycle-G0/G1 phase arrest
O
OCH3
OCH3
Acute oral toxicity
Non-toxic up to 1000mg/kg dose
H3CO
13
Induces apoptosis
Tumour reduction by 74% at 5mg/kg & 79% at 10mg/kg dose
In-vivo LA-7 induced rat mammary carcinoma
Induces ROS
1