Antiproliferative efficacy of curcumin mimics through microtubule destabilization

Article abstract of DOI:10.1016/j.ejmech.2018.03.063

Curcumin possesses an attractive chemical structure with highly conjugated diferuloylmethane core. Curcumin mimics have been designed and prepared with an additional bridged phenyl ring in conjugation. Fourteen diverse analogues were evaluated against a p

Full text of DOI:10.1016/j.ejmech.2018.03.063

Accepted Manuscript
Antiproliferative efficacy of curcumin mimics through microtubule destabilization
Sadiya Khwaja, Kaneez Fatima, Hassanain, Chittaranjan Behera, Avneet Kour, Arjun
Singh, Suaib Luqman, Jayanta Sarkar, Debabrata Chanda, Karuna Shanker, A.K.
Gupta, D.M. Mondhe, Arvind S. Negi
PII:
S0223-5234(18)30306-4
10.1016/j.ejmech.2018.03.063
EJMECH 10328
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 17 November 2017
Revised Date: 20 March 2018
Accepted Date: 21 March 2018
Please cite this article as: S. Khwaja, K. Fatima, Hassanain, C. Behera, A. Kour, A. Singh, S. Luqman,
J. Sarkar, D. Chanda, K. Shanker, A.K. Gupta, D.M. Mondhe, A.S. Negi, Antiproliferative efficacy of
curcumin mimics through microtubule destabilization, European Journal of Medicinal Chemistry (2018),
doi: 10.1016/j.ejmech.2018.03.063.
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Graphical Abstract
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Antiproliferative efficacy of curcumin mimics through
microtubule destabilization
Sadiya Khwaja, Kaneez Fatima, Hassanain, Chittaranjan Behera,
Avneet Kour, Arjun Singh, Suaib Luqman, Jayanta Sarkar, Debabrata
Chanda, Karuna Shanker, A. K. Gupta, D. M. Mondhe, Arvind S. Negi
Destabilizes microtubule
filaments
Cell cycle
* Cytotoxicity
Epidermoid carcinoma
G2/M phase arrest
IC50=1.5µM
IC50=4.68µM
OCH3
O
O
OCH3
Curcumin mimics, 6a
In-vivo efficacy
Non-toxic/safe
upto 300mg/kg dose
78.6% tumour reduction
at 80mg/kg dose.

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Antiproliferative efficacy of curcumin mimics through microtubule
destabilization
Sadiya Khwajaa, Kaneez Fatimaa, Hassanainb, Chittaranjan Beherac, Avneet Kourc, Arjun Singha, Suaib
Luqmana, Jayanta Sarkarb, Debabrata Chandaa, Karuna Shankera, A. K. Guptaa, D. M. Mondhec*, Arvind S.
Negi*a
aCSIR-Central Institute of Medicinal and Aromatic Plants (CSIR-CIMAP), Kukrail Picnic Spot Road, P.O. CIMAP,
Lucknow-226015, India.
bCSIR-Central Drug Research Institute (CSIR-CDRI), B.S. 10/1, Sector-10, Janakipuram Extension, Sitapur Road,
Lucknow-226031, India.
cCSIR-Indian Institute of Integrative Medicine (CSIR-IIIM), Canal Road, Jammu-180001, India.
Abstract
Curcumin possesses an attractive chemical structure with highly conjugated diferuloylmethane core.
Curcumin mimics have been designed and prepared with an additional bridged phenyl ring in
conjugation. Fourteen diverse analogues were evaluated against a panel of human cancer cell lines.
The best analogue of the series i.e. compound 6a exhibited potent cytotoxicity against A431,
epidermoid carcinoma cell line (IC50=1.5 µM) and DLD1, colorectal adenocarcinoma cell line
(IC50=6.9 µM). In tubulin kinetics experiment, compound 6a destabilized polymerisation process
(IC50=4.68 µM). In cell cycle analysis, compound 6a exerted G2/M phase arrest in A431cells and
induced apoptosis. In Ehrlich Ascites Carcinoma in Swiss-albino mice, compound 6a showed
78.6% tumour reduction at 80 mg/kg dose and 57% solid tumour reduction at 150 mg/kg dose.
Further, in acute-oral toxicity experiment in rodent model, compound 6a was given in three
different oral doses to Swiss albino mice. There were non-significant changes in various
biochemical parameters and major body organs studied, including their absolute and relative
weights. It was tolerable up to 300 mg/kg dose in Swiss-albino mice. The present study shows that
the novel curcumin mimic 6a is a safe and efficacious anticancer compound. However, it needs to
be optimized for better efficacy.
Keywords: curcumin mimics, anticancer; preclinical studies, cell cycle; microtubule destabilization; acute
oral toxicity
1. Introduction
Skin cancer is the most common form of cancer accounting approximately 40% of cases. It is an
abnormal growth of skin cells occurring due to the development of abnormal cells that have the
ability to spread to other parts of the body. There are three main types of skin cancer i.e. basal cell
carcinoma, squamous cell carcinoma and melanoma. Among these, melanoma is the most
aggressive. According to WHO, there are 2-3 million non-melanoma skin cancer and 0.23 million
melanoma skin cancer cases globally at present [1,2]. More than 90% of skin cancer cases are
caused by exposure to UV radiation victimizing mostly light skin people. Common treatments are
surgical removal, radiation therapy and topical medication of US-FDA approved drugs like
................................................................................
*Corresponding authors: Tel: +91-522- 2342676 x 327; Fax: +91-522-2342666;
E mail: arvindcimap@rediffmail.com (a.s. negi), dmmondhe@iiim.ac.in (dm mondhe); CIMAP Communication No.:
CIMAP/PUB/2017/22
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fluorouracil, imiquimod, cobimetinib etc [3]. Globally, skin cancer causes around one lakh deaths
annually. There is still a need for an efficient anticancer drug to tackle this disease.
Curcumin is a well known plant phenolic compound abundantly available in the rhizomes of
Curcuma longa (Zinziberaceae). Curcumin, demethoxycurcumin and bisdemethoxycurcumin are
three main components known as curcuminoids. Curcumin has been a hot molecule due to its broad
range of pharmacological properties [4] like anticancer [5], anti-inflammatory [6],
immunomodulatory, antibacterial [7], antimalarial activities. However, its most of the
pharmacological properties are attributed to its antioxidant activity. Despite of potential anticancer,
antiinflammatory and immunomodulatory activities, curcumin could not be developed as a drug
because of its poor bioavailability and sometimes referred as pseudo-drug [8].
Curcumin possesses an attractive chemical structure with highly conjugated diferuloylmethane core.
Two phenolic aromatic rings are linked through a conjugated aliphatic linker chain. It is in keto-
enol form which is stabilized through hydrogen bonding. Researchers have utilized the structural
features of curcuminoids to prepare various curcumin mimics [4]. In the present communication, we
have done detailed pharmacology of potential curcumin mimics possessing an additional aromatic
ring in between the aliphatic linker chain to enhance the conjugation of the system for better
efficacy. Analogue 6a exhibited potential cytotoxicity against A431 skin cancer cell line, which was
further evaluated for mechanistic study, and preclinical efficacy and toxicity in rodent models.
[Figure 1]
2. Results
2.1 Chemistry
2.1.1 Synthesis of curcumin mimics
The synthetic strategy was as depicted in scheme-1. Three different approaches were adopted to
prepare these curcumin mimics. For prototype-I, 1,4-terephthaldehyde was the starting substrate,
which was condensed with various acetophenones in 7% methanolic alkali to afford desired
compounds 3a-3g in 53-96% yields. 1,3-Diacetylbenzene was the starting substrate for prototype-II
compounds. The same methodology was used i.e. 7% methanolic KOH to get compounds 6a-6c in
moderate yields (60-78%). Some of the representative compounds of prototype-II were prepared by
using 3% conc. HCl in dioxane to yield the desired curcumin mimics 6d-6g in 68-96% yield. All
the products were purified through flash chromatography using silica gel (230-400 mesh) as
adsorbent and ethyl acetate-hexane as eluants. Compounds were confirmed by spectroscopy.
[Scheme 1]
2.1.2 Isolation of curcuminoids
Curcumin (CUR), demethoxycurcumin (DC) and bisdemethoxycurcumin (BDC) were isolated from
the rhizomes of Curcuma longa obtained from Central Research Farm, CIMAP, Lucknow. The
dried and ground rhizomes (100 g) were extracted with chloroform (1000 mL) in a soxhlet
apparatus for 5h to get curcumin rich extract (3.84g). The crude mass was purified through Flash
chromatography using glass column (230 mmΧ25 mm, lengthΧdia) and silica gel (230-400 mesh,
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118 g) and eluted successively with 20%, 40%, 60%, 80% chloroform-hexane, chloroform, 1%, 2%,
3%, 4%, 5%, 6% acetone-chloroform and 1%, 2%, 3% 4% methanol-chloroform. The similar
fractions were pooled to get CUR (3% acetone-chloroform), DC (5% acetone-chloroform) and BDC
(3% methanol-chloroform). The solvent was distilled off to get pure compounds in powder form.
These were confirmed by spectroscopy.
2.1.3 Purity profile of compound 6a by UPLC
The integration on the peaks was performed at λmax i.e. 300 nm with data acquisition in PDA (190-
400 nm). The normalized peak area of total impurities, the retention time of 6a, and the resolutions
between 6a and its adjacent peaks were selected as optimization criteria. Under optimised
chromatographic condition, 6a was eluted at 2.150 min without any interference of neighbouring
peaks. As a reference method, the functions for peak purity analysis in the chromatographic data
processing by Empower were applied. The purity of the synthesized 6a was found to be 97.30%
2.2 Biological evaluation
2.2.1 Antiproliferative activity against human cancer cell lines
All the curcumin mimics (3a-3g and 6a-6g) were evaluated for cytotoxicity against a panel of
human cancer cell lines by MTT assay [9]. Human cancer cell lines, A549 (Lung), MCF-7 (Breast),
WRL-68 (Liver), DLD1 (Colorectal), and A431 (Epidermoid) were originally obtained from
American type of cell culture collection (ATCC), USA and stock were maintained in laboratory.
Tamoxifen and podophyllotoxin were used as standard anticancer drugs (positive control) for
cytotoxicity while natural CUR, DC, and BDC were used for comparison purpose. IC50>100 µM
was considered as inactive. For structure activity relationship, we mainly took A431 cell line data
into consideration. We had two structural differentiated series i.e. Series I of 4-bis (E/E) phenyl)-3-
oxo-benzene (compounds 3a-3g) and Series II of 1,3-bis (E/E) phenyl-1-oxo-2-enyl benzene
(Compounds 6a to 6g). Series I compounds were not much effective as compared to Series II
compounds. In Series II compounds 6a, 6b, 6f and 6g exhibited potential cytotoxicity. All the four
analogues possessed methoxy or methylenedioxy substitutions in both the terminal phenyl rings.
But, more the number of alkoxy substitutions less was the cytotoxicity. Compound 6a with one
alkoxy group was the best analogue (IC50=1.5 µM) of the series. Presence of two methoxy groups in
both the terminal phenyl rings decreased cytotoxicity of compounds 6f (IC50=6.9 µM) while
introducing third methoxy group as in compound 6g cytotoxicity was reduced drastically (IC50=10
µM). Presence of other substitutions like hydroxyl or N,N-dimethylamino in the both terminal
phenyl rings either reduced the activity or were inactive at 100 µM concentration.
Compound 6a and 6f were further evaluated for interaction with tubulin polymerisation process
(cytoskeleton USA) [10, 11]. Podophyllotoxin (PDT), a standard microtubule destabilizer was used
as control positive for antitubulin effect and curcumin was used for comparison purpose. The
cytotoxicity and antitubulin data are represented in Table 1.
[Table 1]
2.2.2 Soft agar colony assay
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Soft agar assay allows for semi-quantitative evaluation by utilizing the ability of cells to grow in
soft agar and form colony. It is the gold standard assay for cellular transformation in-vitro [12].
Hence, the cytotoxicity of compound 6a was further confirmed by Soft agar colonisation assay [13].
It suppressed growth of A431 cells by 13-99% at 1.5-50 µg/mL concentrations (IC50=6.2 µg/mL)
(Table 2). The inhibition of A431 colony formation by 6a clearly indicated potential cytotoxicity of
it, which is quite significant and concentration dependent (Figure 2). Soft agar colonisation assay is
used to confirm cellular anchorage-independent growth in-vitro and it is an astringent method to
detect the tumorigenic potential of transformed cells and tumour suppressive effects on transformed
cells [14].
[Table 2], [ Figure 2]
2.2.3 Cell cycle analysis
Cell division cycle is an ordered set of events culminating in cell growth and cell division into two
daughter cells after DNA duplication. In cell cycle analysis compound 6a (Figure 3) induced G2/M
phase arrest at concentration dependent manner. Flow cytogram clearly showed induction of
apoptosis at all experimental concentrations. There was S phase arrest also which was relatively less
than G2/M phase arrest.
[ Figure 3]
2.2.4 Apoptosis induction by Annexin V FITC
Compound 6a induced early apoptosis by 1.1% and late apoptosis by 2.5% while necrosis was
21.6% at its IC50 (1.5 µM) while at 2ΧIC50 (3 µM) early and late apoptosis were much pronounced
(6% & 9.8% ) as compared to necrosis (20%) (Figure 4)
[Figure 4]
2.2.5 Tubulin polymerisation assay
Microtubules are polymers of α/β-tubulins existing in dynamic equilibrium. This dynamic
equilibrium of microtubules is very crucial for cellular functions especially cell transport and cell
division which achieved with help of several microtubule associated proteins. Any impede in
tubulin-microtubule equilibrium disrupts tubulin dynamics which leads to cell cycle arrest and
apoptosis. The microtubule interfering agents induce mitotic arrest which leads to increase in 4N
cells in G2/M phase in cell cycle. Compound 6a showed inhibition (IC50=4.68 µM) of
polymerisation of tubulins to microtubules (Figure 5). Its destabilization effect was better than the
curcumin (IC50=5.12 µM) but much lower than the standard depolymerisation compound
podophyllotoxin (PDT, IC50=0.78 µM). Another compound 6f showed very poor destabilization
effect (IC50=14.39 µM).
[Figure 5]
2.2.6 Effect of 6a on actin–tubulin cytoskeleton network
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Further, the phenotypic effect of compound 6a was observed on actin-tubulin cytoskeleton network.
A431 cancer cells were immune-stained and analysed under Confocal microscope (Figure 6).
Compound 6a exhibited destabilization effect on microtubule filaments on concentration dependent
manner which was clearly observed at 20 µM concentration. This effect was quite similar to
curcumin (20 µM). But this destabilization effect was much lower than colchicine (1 µM).
[Figure 6]
2.2.7 In-vivo anticancer activity against Ehrlich Ascites Carcinoma (EAC)
Ehrlich Ascites Carcinoma is a spontaneous murine mammary adenocarcinoma adapted to ascites
form and performed by serial intraperitoneal passages in outbred mice [15]. In our first experiments
of EAC with potent curcumin mimic 6a, there was no loss of weight and mortality of animals
(Tables 3 and 4). Compound 6a reduced EAC by 47.95%, 68.53% and 77.67% at 20 mg/kg, 40
mg/kg and 60 mg/kg doses respectively (Tables 3a & 3b). Further enhancement of doses of 6a to 80
mg/kg and 100 mg/kg showed only slight improvement in efficacy but one mortality (01/07) at 100
mg/kg dose group and loss in bodyweight were also observed (Table 4a and 4b).
The results of second experiment of compound 6a against EAC with solid tumour were much
encouraging. Compound 6a reduced the tumour by 43% and 56.86% at 100 mg/kg and 150 mg/kg
doses respectively with no mortality (Tables 5a & 5b). In case of 5-fluorouracil 51% tumour was
reduced at 22 mg/kg dose with two mortalities (02/07). The efficacy of 6a was quite comparable to
clinical drug 5-flurouracil though at relatively higher doses but lower toxicity.
[Table 3a], [Table 3b], [Table 4a], [Table 4b], [Table 5]
2.2.8 Safety studies
In the present experiment, acute oral toxicity of the lead compound 6a was carried out in Swiss
albino mice at 5, 50 and 300 mg/kg once orally. No mortality and morbidity was observed
throughout the experimental period in the animals studied. There were no changes observed in gait,
posture and response of animals receiving up to 300 mg/kg dose. Blood and serum biochemistry
samples upon analysis showed non-significant changes in all the blood and serum parameters
studied like haemoglobin (Hb), RBC, WBC, differential leukocyte count (DLC), SGPT, ALP,
creatinine, triglycerides, cholesterol, albumin, total bilirubin and serum protein (Table 6 & Fig. 8).
Animals on gross pathological study showed no significant changes in any of the major body organs
studied, including their absolute and relative weights (Figure 7). Therefore, the experiment showed
that 6a is well tolerated by the Swiss albino mice up to the dose level of 300 mg/kg body weight as
a single acute oral dose.
[Table 6], [Figure 7], [Figure 8]
3. Discussion
Plants are a rich source of diverse pharmacologically active secondary metabolites. Drug
development from natural resources has a major contribution and it is much more in case of
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anticancer drug development from plants. Turmeric, widely used in traditional Asian kitchen also
exhibits
diverse
pharmacological
activities.
Curcumin,
demethoxycurcumin
and
bisdemethoxycurcumin are the three main components of turmeric. The purpose of this study was to
prepare compounds as curcumin mimics possessing an extra aromatic ring at centre with better
anticancer efficacy.
The pharmacological activity of curcuminoids is mainly due to their antioxidant property
[16]. Presence of phenolic groups was considered important in some studies while others pointed
out the β-diketone. Recently, Cai et al. (2017) has reported some of these compounds as
antidiabetic agents through α-glycosidase inhibition [17].
Our synthesized curcumin mimics exhibited poor antioxidant property as compared to curcuminoids
[data not shown]. However, these compounds exhibited significant cytotoxicity as compared to
curcuminoids. Compound 6a showed potential anticancer activity against skin cancer and colorectal
cancer cells. The cytotoxicity of 6a was further confirmed by soft agar colony assay where it killed
10-99% of skin cancer cells in concentration dependent manner.
As evident from tubulin polymerisation kinetic experiment, compound 6a exhibited potential
cytotoxicity through microtubule destabilization and induction of apoptosis in A431 cells.
Previously, curcumin has been reported to depolymerise interphase microtubules at 25 µM and 40
µM concentrations [18]. Later, it was found that curcumin bioactivity involves non-specific
microtubule disruption, similar to that of colchicines and other antitubulin drugs used to combat
uncontrolled cancerous cell growth [19]. Confocal microscopy showed degradation of microtubules
clearly at 20 µM concentration of 6a.
Cell cycle is an important phenomenon for cell growth and proliferation. Induction of cell cycle
arrest in cancer cells through various mechanisms is a strategy to control cancer cell proliferation.
Cell cycle check points play an important role in regulation of cell cycle [20]. These checkpoints
control system by sensing defects that occur during essential processes such as DNA replication,
chromosome segregation etc. and inducing cell cycle arrest in response until the defects are repaired
[21]. Some compounds interfere with CDK/cyclin complexes leaving cells stuck at the G2/M phase
border, while others affect CaMKII phosphorylation, inducing G1 phase arrest. Among several
other mechanisms interference with RNA function and inhibition of protein synthesis are important.
As a result of cell cycle interruption, many compounds ultimately induce apoptosis [22]. Compound
6a induced mainly G2/M phase arrest in A431epidermoid carcinoma cells in concentration
dependent manner. In general, interference in microtubule dynamics disturbs mitosis and such
compounds cause G2/M phase arrest [23]. Induction of cell cycle arrest in cancer cells constitutes
one of the most prevalent strategies to stop or limit cancer spreading [24, 25].
EAC is also known as undifferentiated carcinoma which is more malignant than a differentiated
carcinoma and difficult to treat. In in-vivo efficacy studies compound 6a exhibited potential
cytotoxicity. Its efficacy was comparable to clinical drug 5-fluorouracil though relatively at higher
doses. But the toxicity of compound 6a was relatively much less than 5-fluorouracil. Ehrlich ascites
tumor cells are much more difficult to break than most nonmalignant somatic cells [26].
Drug safety is an important parameter while assessing efficacy of an anticancer agent. Acute oral
toxicity is used to assess the ability of a substance to cause adverse effects within a short period of
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time following dosing or exposure. When in-vivo testing is done, estimation of rodent acute toxicity
is an important task in safety assessment of drug candidates. Compound 6a was well tolerable up to
300 mg/kg dose in Swiss-albino mice. However, sub-acute, chronic or sub-chronic experiments
with compound 6a needed to be carried out for any adverse effect on prolonged exposure [27].
4. Conclusion
The present study showed that introduction of bridged phenyl ring enhances anticancer efficacy of
curcumin mimics. Compound 6a, the best representative of the series exhibits potential cytotoxicity
against human epidermoid carcinoma cells. Its cytotoxic efficacy was through microtubule
destabilization. Its in-vivo efficacy was quite comparable to clinical drug 5-fluorouracil at relatively
higher doses. In-vivo toxicity studies showed it quite safe up to 300 mg/kg dose in mice model. All
these results clearly suggest that compound 6a has significant potential for further translation into
clinical application. Also it may further be optimized for better efficacy and lower toxicity in future.
5. Materials and methods
5.1 General
Curcumin, demethoxycurcumin and bisdemethoxycurcumin were isolated from the rhizomes of
Curcuma longa. For synthesis, substrates, reagents and other chemicals were procured from Avra
Chemicals India and used without purification. Melting points were determined in open glass
capillaries on E-Z Melt automated melting point apparatus, Stanford Research System USA and
were uncorrected. Reactions were monitored on pre-coated silica gel TLC-GF254 aluminium sheets
(Merck) and visualized done under UV light (254 nm and 365 nm) and subsequent charring with
2% ceric sulphate in 10% sulphuric acid (aqueous). Compounds were purified through column
chromatography and Flash chromatography system (CombiFlash Rf200i, Teledyne-ISCO, USA)
using glass columns (13 cm lengthx2 cm i.d.) and silica gel (230-400 mesh, Avra Chemicals) using
UV detector (230 nm and 254 nm). The NMR spectra were obtained on Bruker Avance-300 MHz &
500MHz instruments with tetramethylsilane (TMS) as an internal standard. Chemical shifts are
1
given in δ ppm values. H–1H coupling constant (J) values are given in Hz. ESI mass spectra were
recorded on APC3000 LC-MS-MS (Applied Biosystem) and High Resolution Mass (HRMS) on
Agilent 6520Q-TOF after dissolving the compounds in methanol. The purity profile was determined
on Ultra Performance Liquid Chromatography (UPLC) ACQUITY UPLC H-Class Bio System,
Waters USA. DMEM (Dulbecco’s Modified Essential Eagle Medium) and FBS (Fetal Bovine
Serum) were purchased from Gibco, India. RNaseA, Crystal Violet Dye, Trypsin-EDTA, HEPES,
Antibiotic-Antimycotic (Ab-Am) Solution, Phosphate Buffer Saline (PBS), Citric Acid and
Propidium Iodide (PI) were acquired from Sigma-Aldrich, USA. Sodium bicarbonate (NaHCO3),
Agar, Sodium Citrate and Di-sodium Hydrogen Phosphate were obtained from Himedia
Laboratories, India. Solvents including DMSO, ethanol and isopropanol were procured from Merck,
India Ltd.
5.2 Chemical synthesis
5.2.1 General procedure for the synthesis of curcumin mimics 3a-3g by alkaline method
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1,4-Terephthaldehyde (300 mg, 1.12 mmol) in 7% methanolic KOH (10 mL) was stirred at room
temperature. To this acetophenone derivative (2.4mmol) was added and reaction mixture was
further stirred for 7-8 h. On completion methanol was evaporated and water was added to it,
acidified with 5% HCl (10 mL) and extracted with ethyl acetate (3X20 mL). Organic layer was
washed with water, dried over anhydrous sodium sulphate and dried in-vacuo. The residue was
purified through column chromatography using ethyl acetate-hexane as eluents to get pure
compound.
1,4-Bis-(E/E)-[1-(4-methylphenyl)-prop-3-oxo-1-enyl]benzene (3a): Yield=64%; m.p.=234-235oC;
1H NMR (300MHz, CDCl3): δ2.43 (s, 6H, 2XCH3), 7.24-7.31 (m, 6H, CH, aromatic), 7.53-7.58 (d,
2H, =CH, J=15.9Hz), 7.66 (s, 2H, CH, aromatic), 7.75-7.781 (d, 2H, CH=, J=15.9Hz), 7.94 (m, 4H,
CH, aromatic); ESI-MS (MeOH): For C26H22O2 , 367 [M+H]+, 405[M+K]+; HRMS (ESI-TOF) m/z
[M+H] calcd for C26H22O2 367.1698 , found 367.1696.
1,4-Bis-(E/E)-[1-(4-methoxylphenyl)-prop-3-oxo-1-enyl]benzene (3b): Yield=92%; m.p.=239-
242oC; 1H NMR (300MHz, CDCl3): δ3.90 (s, 6H, 2XOCH3), 7.01 (d, 4H, CH, aromatic), 7.51-7.56
(d, 2H, =CH, J=14.7Hz), 7.61 (s, 4H, CH, aromatic), 7.77-7.83 (d, 2H, CH=, J=15.9Hz), 8.10 (d,
4H, CH, aromatic); ESI-MS (MeOH): For C26H22O4 , 399 [M+H]+.
1,4-Bis-(E/E)-[1-(3,4-dimethoxylphenyl)-prop-3-oxo-1-enyl]benzene (3c): Yield=61%; m.p.=218-
1
220oC; H NMR (300MHz, CDCl3): δ4.02 (s, 12H, 4XOCH3), 6.93-6.96 (d, 2H, CH, aromatic,
J=8.7Hz), 7.47-7.52 (d, 2H, =CH, J=15Hz), 7.73-7.78 (d, 2H, CH=, J=15Hz), 7.52-8.11 (m, 8H,
CH, aromatic), ; ESI-MS (MeOH): For C28H26O6 , 481[M+Na]+; HRMS (ESI-TOF) m/z [M+H]
calcd for C28H26O6 459.1807, found 459.1801.
1,4-Bis-(E/E)-[1-(3,4,5-trimethoxylphenyl)-prop-3-oxo-1-enyl]benzene
(3d):
Yield=96%;
m.p.=216-218oC; H NMR (300MHz, CDCl3): δ3.93 (s, 18H, 6ΧCH3), 7.27 (s, 4H, CH, aromatic),
7.48-7.53 (d, 2H, =CH, J=15.6Hz), 7.76 (s, 4H, CH, aromatic), 7.76-7.81 (d, 2H, CH=, J=15.6Hz);
13C NMR (75MHz, CDCl3): δ56.8, 61.3, 106.6, 123.1, 129.3, 133.7, 137.3, 143.2, 143.8, 153.6,
189.2; ESI-MS (MeOH): For C30H30O8 , 519 [M+H]+, 541[M+Na]+.
1
1,4-Bis-(E/E)-[1-(3,4-methylenedioxyphenyl)-prop-3-oxo-1-enyl]benzene
(3e):
Yield=53%;
m.p.=242-245oC; H NMR (300MHz, CDCl3): δ6.10 (s, 4H, 2xO-CH2-O), 6.92-6.95 (d, 2H, CH,
aromatic, J=8.1Hz), 7.53-7.58 (d, 2H, =CH, J=15.6Hz), 7.57 (d, 4H, CH, aromatic) 7.68-7.70 (d,
4H, CH, aromatic, J=6.9Hz), 7.79-7.84 (d, 2H, CH=, J=15.6Hz); ESI-MS (MeOH): For C26H18O6 ,
465[M+K]+.
1
1,4-Bis-(E/E)-[1-(4-fluorophenyl)-prop-3-oxo-1-enyl]benzene (3f): Yield=94%; m.p.=242-245oC;
1H NMR (300MHz, CDCl3): δ7.27-7.33 (m, 6H, CH, aromatic), 7.60-7.65 (d, 2H, =CH, J=15.6Hz),
7.77 (bs, 4H, CH, aromatic), 7.86-7.92 (d, 2H, CH=, J=15.6Hz), 8.12-8.15 (m, 2H, CH, aromatic);
ESI-MS (MeOH): For C24H16F2O2, 397 [M+Na]+; Negative mode: 373 [M-H]-; HRMS (ESI-TOF)
m/z [M+H] calcd for C24H16O2 375.1196 , found 399.1194.
1,4-Bis-(E/E)-[1-(4-bromophenyl)-prop-3-oxo-1-enyl]benzene (3g): Yield=63%; m.p.=262-264oC;
1H NMR (300MHz, CDCl3): δ7.48--7.53 (d, 2H, =CH, J=15.6Hz), 7.63-7.68 (m, 6H, CH,
aromatic), 7.71-7.82 (m, 4H, CH, 2XCH= and 2xaromatic), 7.94 (m, 4H, CH, aromatic); ESI-MS
(MeOH): For C24H16Br2O2, 535[M+K]+; negative mode: 495 [M-H]-.
8

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5.2.2 General procedure for the synthesis of curcumin mimics 6a-6c by alkaline method
1,3-Dicetylbenzene (400 mg, 2.47 mmol) was taken in 7% methanolic KOH (10 mL). To this
stirred solution benzaldehyde derivative (5mmol) was added and reaction mixture was further
stirred at room temperature for 7-8 h. The work up and purification procedure was followed as in
section 2.2.1 to get the pure product.
1,3-Bis-(E/E)-[3-(2-methoxyphenyl)-1-oxo-2-enyl]-benzene (6a): Yield=78%; oil; 1H NMR
(300MHz,
CDCl3):
δ3.90
(s,
6H,
2XOCH3),
6.93
-7.01 (m, 4H, CH, aromatic), 7.37-7.40 (bs, 2H, 2X =CH, J=14Hz), 7.62-7.68 (m, 4H, CH,
aromatic), 8.14-8.17 (d, 2H, 2XCH=, J=16Hz), 8.20-8.22 (m, 3H, CH aromatic), 8.63 (s, 1H, CH,
aromatic); 13C NMR (75MHz, CDCl3): δ55.6, 111.3, 120.8, 122.5, 123.8, 128.5, 129.0, 129.5,
132.0, 132.3, 138.8, 141.2, 158.9, 190.5; ESI-MS (MeOH): For C26H22O4 421 [M+Na]+,
,
437[M+K]+. HRMS (ESI-TOF) m/z [M+H] calcd for C26H22O4 399.1596, found 399.1597.
1,3-Bis-(E/E)-[3-(3,4-methylenedioxyphenyl)-1-oxo-2-enyl]-benzene (6b): Yield=60%; m.p.=205-
1
208oC; H NMR (300MHz, CDCl3): δ6.16 (s, 4H, 2XO-CH2-O), 6.83 (m, 2H, CH, aromatic), 7.12
(s, 2H, CH, aromatic, J=8.1Hz), 7.43 (d, 2H, CH, aromatic), 7.76-7.81 (d, 2H, =CH, J=16.5Hz),
13
7.90-7.95 (d, 2H, CH=, J=15.5Hz), 8.26 (d, 2H, CH, aromatic), 8.46 (d, 2H, CH, aromatic); C
NMR (75MHz, CDCl3): δ101.5, 102.5, 107.9, 108.1, 108.6, 209.4, 120.4, 127.1, 128.7, 129.9,
130.1, 133.4, 137.4, 139.2, 145.6, 148.9, 150.6, 189.6; ESI-MS (MeOH): For C26H18O6 , 449
[M+Na]+, 465[M+K]+.
1,3-Bis-(E/E)-[3-(4-N,N-dimethylphenyl)-1-oxo-2-enyl]-benzene (6c): Yield=71%; m.p.=149-
1
152oC; H NMR (300MHz, CDCl3): δ3.04 (s, 12H, 4XN-CH3), 6.68 (d, 4H, CH, aromatic,
J=8.7Hz), 7.35-7.40 (d, 2H, =CH, J=15.6Hz), 7.56-7.61 (d, 5H, CH, aromatic), 7.80--7.85 (d, 2H,
13
CH=, J=15.6Hz), 8.15-8.18 (d, 2H, CH, aromatic), 8.60 (s, 1H, CH, aromatic); C NMR (75MHz,
CDCl3): δ40.4, 111.1, 112.2, 113.3, 116.8, 122.88, 128.3, 129.1, 131.1, 132.1, 139.7, 146.9, 152.6,
190.3; ESI-MS (MeOH): For C28H28N2O2 , 425 [M+H]+, 447[M+Na]+, 463[M+K]+.
5.2.3 General procedure for the synthesis of curcumin mimics 6d-6g by acidic method
1,3-Dicetylbenzene (500 mg, 3.08 mmol) and particular benzaldehyde derivative (6.2 mmol) was
taken in dioxane (10 mL). To this conc. hydrochloric acid (0.5 mL) was added and reaction mixture
was refluxed for 4-5 h. On completion, water was added to reaction mixture, extracted with ethyl
acetate, washed with water and dried over anhydrous sodium sulphate.
1
1,3-Bis-(E/E)-[3-(4-hydroxyphenyl)-1-oxo-2-enyl]-benzene (6d): Yield=69%; m.p.=235-238oC; H
NMR (300MHz, CDCl3): δ6.92-7.08 (d, 4H, CH, aromatic), 7.69-7.76 (bd, 8H, CH, aromatic &
13
2X=CH), 8.20 (bd, 2H, CH, 2XCH=), 8.62 (bs, 1H, CH, aromatic); C NMR (75MHz, CDCl3):
δ116.3, 119.1, 127.0, 128.2, 129.4, 131.2, 132.4, 139.4, 145.3, 189.1; ESI-MS (MeOH): For
C26H22O2 , 371 [M+H]+, 393 [M+Na]+, 409[M+K]+.
1,3-Bis-(E/E)-[3-(3-hydroxy-4-methoxyphenyl)-1-oxo-2-enyl]-benzene (6e): Yield=79%; m.p.=147-
1
150oC; H NMR (300MHz, CDCl3): δ3.91 (s, 6H, 2XOCH3), 7.84-7.86 (bd, 2H, CH, aromatic,
J=8.1Hz), 7.13-7.16 (bd, 2H, CH, aromatic), 7.33-7.39 (d, 2H, =CH, J=16.8Hz), 7.58 (bs, 1H, CH,
9

ACCEPTED MANUSCRIPT
aromatic), 7.73-7.78 (d, 2H, CH=, J=15.6Hz), 8.16-8.19 (bs, 2H, CH, aromatic), 8.60 (s, 1H, CH,
aromatic); 13C NMR (75MHz, CDCl3): δ56.4, 111.1, 113.8, 120.3, 123.3, 128.7, 129.4, 132.6,
139.2, 146.1, 146.4, 149.5, 190.2; ESI-MS (MeOH): For C26H22O6, 431 [M+H]+, 453 [M+Na]+;
HRMS (ESI-TOF) m/z [M+H] calcd for C26H22O6 431.1494, found 431.1485.
1
1,3-Bis-(E/E)-[3-(3,4-dimethoxyphenyl)-1-oxo-2-enyl]-benzene (6f): Yield=72%; oil; H NMR
(300MHz, CDCl3): δ3.93 (s, 6H, 2XOCH3), 3.96 (s, 6H, 2XOCH3), 6.91 (bd, 2H, CH, aromatic),
7.08-7.34 (d, 4H, CH, aromatic), 7.41-7.46 (d, 2H, =CH, J=15.6Hz), 7.61 (t, 1H, CH, aromatic),
13
7.78-7.84 (d, 2H, CH=, J=15.6Hz), 8.19 (bd, 2H, CH, aromatic), 8.62 (s, 1H, CH, aromatic); C
NMR (75MHz, CDCl3): δ56.4, 110.6, 111.6, 119.9, 123.9, 128.1, 129.3, 132.6, 139.3, 146.3, 149.7,
152.1, 190.3; ESI-MS (MeOH): For C28H26O6 , 459 [M+H]+, 481 [M+Na]+, 497[M+K]+; HRMS
(ESI-TOF) m/z [M+H] calcd for C28H26O6 459.1807, found 459.1802.
1
1,3-Bis-(E/E)-[3-(3,4,5-trimethoxyphenyl)-1-oxo-2-enyl]-benzene (6g): Yield=96%; oil; H NMR
(300MHz, CDCl3): δ4.02 (s, 18H, 6XOCH3), 6.98 (s, 4H, CH, aromatic), 7.36-7.41 (d, 2H,
CH=,J=15Hz), 7.68 (bs, 1H, CH, aromatic), 7.76-7.81 (d, 2H, =CH, J=15.6Hz), 8.22 (bs, 2H, CH,
aromatic), 8.64 (s, 1H, CH, aromatic); 13C NMR (75MHz, CDCl3): δ56.3, 61.0, 105.9, 120.9, 128.3,
129.1, 130.1, 132.4, 138.8, 146.0, 153.5, 189.9; ESI-MS (MeOH): For C30H30O8 , 519 [M+H]+, 541
[M+Na]+, 557[M+K]+.
5.2.4 Purity profile of 6a by UPLC
The purity profile of most potent compound 6a was checked by reverse phase Ultra Performance
Liquid Chromatography (UPLC, Waters USA) using C-18 column (BEH130Å, 1.7X 50 mm, 1.7
µm, Waters, USA, thermostated at 35±0.1°C). Column analysis was performed with a using
isocratic elution of acetonitrile and water (70:30, v/v) at flow rate of 0.20 mL/min. The injection
volume was 2 µL. The purity of the compounds was calculated by peak integration.
5.3 Biological evaluation
4.3.1 Antiproliferative activity by MTT assay
The assay was performed as per reported method [9]. Human cancer cell lines, A549 (Lung), MCF-
7 (Breast), DLD1 (Colorectal), A431 (epidermoid) and WRL-68 (Liver) were originally obtained
from American type of cell culture collection (ATCC), USA and grown at 37˚C in DMEM
supplemented with 10% FBS and Ab-Am (antibiotic-antimitotic) solution in a CO2 incubator (New
Brunswick/Eppendorff, Germany) under 5%CO2 and 95% relative humidity. Briefly, 2X103
cells/well were incubated in the 5% CO2 incubator for 24 h to enable them to adhere properly to the
96 well polystyrene microplate (Grenier, Germany). Test compounds 3a-3g and 6a-6g were
dissolved in dimethylsulphoxide (DMSO, Merck, Germany), in at least five concentrations, were
added into the wells and left for 4 h. After the incubation, the compound and media were replaced
with fresh media and the cells were incubated for another 24h in the CO2 incubator at 37oC. The
concentration of DMSO was always kept below 1.25%, which was found to be non-toxic to cells.
Then, from 5 mg/ml 10 µL MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]
was added to each well and plates were incubated at 37oC for 4 h. 100 µL DMSO was added to all
10

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wells and mixed thoroughly to dissolve the formazan crystals. The plates were read on SpectraMax
190 Microplate reader (Molecular Devices Inc. USA) at 570 nm within 1h of DMSO addition.
The cytotoxic effect of compound was calculated as % inhibition in cell growth as per formula: [1-
(Absorbance of drug treated cells/Absorbance of untreated cells) X100]. Dose dependent curves
were used to determine 50% inhibitory concentration (IC50).
5.3.2 Soft agar colony assay
Briefly, human epidermoid cancer A431 cells (5 X 104 per mL) were seeded separately in 24 well
plates with or without compound 6a treatment for 24h [13]. A bottom jelly layer was made with
0.72% agar and 4mL media in a 90 mm Petri dish followed by the formation of top layer with cells
and 3mL media in 0.36% agar. Petri dishes were incubated in a CO2 incubator for 15-20 days till
the appearance of the colonies followed by staining with Crystal Violet (0.04% in 2% ethanol) and
incubation for 1h at room temperature. After the incubation period, colony was counted using
inverted microscope and pictures were captured accordingly.
5.3.3 Cell cycle analysis
The effect of compound 6a on cell division cycle of A431 cells was assessed by flow cytometry
with PI-stained cellular DNA [28]. In brief, cells (2 X 106 per mL) were grown in a 6 well plate at
37oC in 5%CO2 with or without compound treatment for 24 h. After incubation, cells were collected
and washed with PBS followed by fixing in 70% ethanol (add drop wise through vortexing) for
overnight at 4˚C. The fixed cells were centrifuged at high rpm to remove ethanol followed by
washing with PBS and addition of DNA extraction buffer. The samples were incubated for 5 min at
room temperature and centrifuged again. The obtained pellet was dissolved in PI staining solution
followed by RNaseA (200 µg/mL) treatment and incubation for 30 min at room temperature.
Finally, cell cycle analysis was done using Flow Cytometric approach on a BD Biosciences LSR II
(San Jose, CA, USA) and the analysis was performed through Facs Diva Software, version 6.1.3.
Both the soft agar colony formation and cell cycle experiment was performed in duplicates and
results are expressed as Mean ± SD.
5.3.4 Apoptosis Vs Necrosis induction by 6a by Annexin V-FITC assay
For Annexin V-FITC apoptosis assay by Flow cytometry [29], 1 X 106 cells/mL cells of A431 were
seeded in 6-well plate and left overnight before treatment with compound 6a at various
concentrations (IC50 value and 2XIC50) for 24h. All cells apoptotic and adherent were collected and
centrifuged at 5000 rpm for 5min at 4ºC and washed twice with cold PBS. Pellets were dissolved in
100µL 1X binding buffer and incubated with 5µL FITC-annexin V and 5 µL propidium iodide (PI)
(protocol followed as described in BD Bioscience Kit) for 15 min. Samples were maintain with 500
µL 1x binding buffer and stained cells were analysed by FACS Diva software of flow cytometer
within 1h. The annexin V-FITC binds to phosphatidiylserine present on membrane surface in case
of apoptotic cells whereas PI labelled the cellular DNA in necrotic cell. This combination allows the
differentiation among viable cells (annexin V negative, PI negative), early apoptotic cells (annexin
11

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V positive, PI negative), late apoptotic cells (annexin V positive, PI positive), and necrotic cells
(annexin V positive, PI positive).
5.3.5 Tubulin Polymerisation assay
Tubulin kinetics (polymerization) experiment was performed using ‘assay kit’ from Cytoskeleton,
USA, as per reported protocol [10, 11]. In brief, tubulin protein (3 mg/mL) in tubulin
polymerization buffer (80 mM PIPES, pH 6.9, 2 mM MgCl2, 0.5 mM EGTA, 1 mM GTP and 15%
glycerol) was placed in pre-warmed 96-well microtiter plates at 37°C in the presence of test
compounds with variable concentrations. All samples were mixed well and polymerization was
monitored kinetically at 340 nm every min for 1h using Spectramax plate reader. Podophyllotoxin
(PDT) was used as standard destabilizer and paclitaxel as standard stabilizer of tubulin polymerase
and DMSO as negative control. The IC50 value was determined from dose-dependent analysis and is
defined as the concentration that inhibits the rate of polymerization by 50%.
5.3.6 In vivo anticancer activity of 6a against Ehrlich Ascites Carcinoma (EAC)
The in-vivo anticancer assay was performed through Ehrlich Ascites Carcinoma (EAC) model [30].
EAC cells were collected from the peritoneal cavity of the female Swiss mice (each weighing 20-28
g) harbouring 8-10 days old ascitic tumour. 1X107 EAC cells were injected intraperitoneally in each
of the Swiss mice selected for the experiment on day zero. The next day, animals were randomized
and divided into different groups. The treatment groups contained 7 animals each and control group
contained 10 animals. Three treatment groups were treated with 20 mg/kg, 40 mg/kg and 60 mg/kg
of potent compound 6a intra-peritoneally from day 1-9. One more of the treatment group received
anticancer drug 5-fluorouracil (20 mg/kg, i.p) and it served as positive control. The tumour bearing
control group was similarly administered normal saline (0.2 mL, i.p.). On day 12, animals were
sacrificed and ascitic fluid was collected from peritoneal cavity of each mouse for the evaluation of
tumour growth. Percent tumour growth inhibition was calculated based on the total number of
tumour cells present in the peritoneal cavity as on day 12 of the experiment using the following
formula.
Avg. no. of cells in control gp – Avg. no. of cells in treated gp
% tumour growth inhibition =
--------------------------------------------------------------------- X 100
Avg. no. of cells in control group
The experiment was further done at higher doses of 80 mg/kg and 100 mg/kg of 6a with some
marginal improvement in the activity.
5.3.7. In vivo anticancer Activity of curcumin mimic 6a against Ehrlich tumour (Solid)
Ehrlich ascites carcinoma (EAC) cells were collected from the peritoneal cavity of the Swiss mice
harbouring 8-10 days old ascitic tumour. 1X107 EAC cells were injected intramuscularly in right
thigh of 24 Swiss male mice (20-28 g each) selected for the experiment on day 0. The next day,
animals were randomized and divided into three groups. Two treatment groups contained 7 animals
12

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each and one control group contained 10 animals. One of the treatment groups was treated with
potent compound 6a (100 mg/kg, and 150 mg/kg, i/p) from day 1-9. Another treatment group was
treated with 5-fluorouracil (22 mg/kg, i.p) from day 1-9 and it served as positive control. The
control group was similarly administered normal saline (0.2 mL, i.p.) from day 1-9. On day 9 and
13, tumour bearing thigh of each animal was shaved and longest and shortest diameters of the
tumour were measured with the help of Vernier callipers. Tumour weight of each animal was
calculated using the following formula.
Length (mm) X [width(mm)]2
Tumour weight (mg) = ---------------------------------------
2
The percent tumour growth inhibition was calculated on day 13 by comparing the average values of
treated groups with that of control group. Tumour growth in saline treated control animals was
taken to be 100%.
5.3.8 Acute oral toxicity
Compound 6a, the most potent anticancer compound of this series was further evaluated for safety
studies through acute oral toxicity in Swiss albino mice. The Experiment was conducted in
accordance with the Organization for Economic Co-operation and Development (OECD) test
guideline No 423 (1987). For the acute oral toxicity study, 24 mice (12 male and 12 female) were
taken and divided into four groups comprising 3 male and 3 female mice in each group weighing
between 20-25 g. The animals were maintained at 22±5°C with humidity control and also on an
automatic dark and light cycle of 12 h. The animals were fed with the standard mice feed and
provided ad libitum drinking water. Mice of group 1 (Group I) were kept as controls and animals in
groups 2, 3 and 4 (Groups II, III & IV) were kept as experimental. The animals were acclimatized
for 7 days prior for the experimental environment adaptation. Compound 6a was dissolved in few
drop of DMSO then solubilised in 0.7% CMC and was given at 5, 50 and 300 mg/kg body weight
of animals of groups 2, 3 and 4 (Groups II, III & IV) respectively once orally. Control animals
received only vehicle.
The animals were checked for mortality and any signs of ill-health at hourly interval on the day of
administration of the drug and thereafter a daily general case side clinical examination was carried
out including changes in the skin, mucous membrane, eyes, occurrence of secretion and excretion
and also responses like lachrymation, piloerection, respiratory patterns etc. Also, changes in gait,
posture and response to handling were also recorded [31]. In addition to the observational study,
body weights were recorded, blood and serum samples were collected from all the animals on the
7th day of the experiment. The samples were analyzed for total RBC, WBC, differential leukocyte
count, haemoglobin percentage and biochemical parameters like ALP, SGPT, SGOT, total
cholesterol, triglycerides, creatinine, bilirubin and serum protein. The animals were then sacrificed
and were necropsied for any gross pathological changes. Weights of vital organs like liver, heart,
kidney etc. were recorded [32].
5.3.9 Statistical analysis
13

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Data expressed as mean or representative of one of three similar experiments unless otherwise
indicated. The MTT, Soft agar colony formation, Annexin V FITC, and cell cycle experiments were
performed in duplicates and results are expressed as Mean ±SD. The MTT absorbance was taken
from the SoftMax Pro Microplate data acquisition and analysis software version 5.3 (Moleular
Devices Corporation., Sunnyvale CA, USA). The IC50 value was calculated from Table curve 2D
windows version 4.07 (SPSS Inc., Chicago, IL, USA). Cell cycle was performed on a BD
Bioscience LSR II (San Jose, CA, USA) and analysis was done through Facs Diva Software,
version 6.1.3. For multiple comparisons each value was compared by one way ANOVA following
Dunnett test, Tukey test and Student t-test in GraphPad Instat version 3.06.
Acknowledgements
The financial support for a part of work from National Medicinal Plant Board Project, GAP-249 is
duly acknowledged.
Supplementary information
The spectra (1H NMR, 13C NMR, DEPT135 and ESI-HRMS) of compound 6a are being submitted
as Supplementary information.
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Table, Figure and Scheme Legends
Table 1: In-vitro cytotoxicity of curcumin mimics against various human cancer cell lines by MTT assay (IC50
in µM).
Table 2: Effect of compound 6a on Percent colony formation in A431 cells in soft agar after 24h incubation (no.
of seeded cells=5x104 cells/mL, area of 60 mm plate=2826 mm2)
Table 3a: Effect of compound 6a on body weight of mice bearing Ehrlich Ascites Carcinoma
Table 3b: In-vivo efficacy of compound 6a on against Ehrlich Ascites Carcinoma
Table 4a: Effect of compound 6a on body weight of mice bearing Ehrlich Ascites Carcinoma at higher doses
Table 4b: In-vivo efficacy of compound 6a on against Ehrlich Ascites Carcinoma at higher doses
Table 5: Effect on body weight of mice and In-vivo efficacy of compound 6a on against Ehrlich Ascites
Carcinoma (solid tumour)
Table 6. Effect of 6a as a single acute oral dose at 5, 50 and 300 mg/kg body weight on hematological and
serum biochemical parameters in swiss albino mice (Mean±SE; n=6)
Figure 1: Structure of three clinical drugs of skin cancer, curcuminoids and designed prototypes 1 & 2 as
curcumin mimics
Figure 2: Antiproliferative activity of 6a by soft agar colony assay against A431 cells;
Control 6a: 1.5µg/mL, 6a: 5µg/mL, 6a: 10 µg/mL, 6a: 20 µg/mL, 6a: 50 µg/mL
Figure 3: Cell cycle analysis of compound 6a in A431 cells.
Figure 4: Induction of apoptosis and necrosis by compound 6a in A431 cells by Annexin V-FITC assay
Figure 5: Tubulin polymerisation kinetics graph of 6a at various concentrations, curcumin was taken for
comparison, Paclitaxel (PAC) as standard microtubule stabilizer and podophyllotoxin (PDT as standard
microtubule inhibitor.
Figure 6: Effect of compound 6a on actinetubulin cytoskeleton structure with confocal microscopy. (Curcumin
at 20 µM, colchicine at 1 µM, and 6a at 5 µM, 10 µM and 20 µM.
Figure 7: Effect of 6a as a single acute oral dose at 5, 50 and 300 mg/kg on absolute and relative organ weight
in Swiss albino mice (Mean±SE; n=6).
Figure 8: Effect of 6a as a single acute oral dose at 5, 50 and 300 mg/kg body weight on differential leukocyte
counts in Swiss albino mice (Mean±SE; n=6).
Scheme 1: i) 7% KOH in MeOH, RT, 7-8h, 53-96%; ii) 7% KOH in MeOH, RT, 7-12h, 60-78%; iii) 5% Con
HCl in Dioxane, reflux, 7-8h, 69-96%.
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Table 1.
S.
A549
A431
WRL-68
MCF-7
DLD1
Tubulin
no.
Polymerisat
ion
inhibition
IC50 in µM
---
---
---
---
---
---
---
4.68
---
---
14.39
---
5.12
---
---
1.
2.
3.
4.
5.
6.
7.
8.
Inactive
Inactive
85.6
68.7
Inactive
96.7
Inactive
Inactive
41.7
70.9
80.1
81.8
72.2
Inactive
Inactive
Inactive
56.7
Inactive
64.6
Inactive
Inactive
30.6
Inactive
9.2
16.1
Inactive
21.2
10.8
26.6
22.1
92.1
48.9
4.16
8.61
>40
>40
>40
16.1
>40
>40
>40
6.9
16.5
16.2
9.7
>40
>40
>40
>40
0.11
16.3
3a
3b
3c
3d
3e
3f
3g
6a
6b
6e
6f
6g
CUR
32
18.6
Inactive
Inactive
85.2
30.7
18.7
20.3
>40
Inactive
43.9
28.2
Inactive
Inactive
Inactive
1.5
10.0
16.9
9.
10.
11.
12.
13.
14.
15.
16.
17.
6.9
10.0
38.7
27.8
26.3
45.4
Inactive
Inactive
Inactive
Inactive
Inactive
DC
BDC
ND
5.61
17.3
Podophyllotoxin
Tamoxifen
3.02
0.78
---
IC50>100µM considered as inactive.
Table 2
Condition
Concentration Avg. % live
A431 live
A431
IC50 in
µg/mL
In µg/mL
cells
cells
(% dead
cells)
Control
---
100
0
Compd. 6a
1.5
86.86
13.14±4.9**
6.21
5
61.46
34.73
5.59
38.54±0.31**
65.25±2.60**
94.41±1.16**
99.25±0.03**
10
20
50
0.75
**p<0.01 (Dunnett test);
2

ACCEPTED MANUSCRIPT
Table 3a:
Treatment
Dose
Body weight (g)
Day 1
22.0
Day 5
23.7
Day 9
28.66
30.14
30.42
30.0
Day 12
32.5
Control
0.2 ml N.S. i/p
20 mg/kg i/p
40 mg/kg i/p
60 mg/kg i/p
20 mg/Kg i/p
Compd. 6a
Compd. 6a
Compd. 6a
5-FU
23.28
23.14
22.85
22.85
24.42
25.14
25.42
22.57
32.42
32.71
35.33
21.42
21.71
Table 3b:
Treatment
Dose
Day 12
Av.
Av.
Av. No. of
% Tumor % Tumor Mortality
Volume
of ascitic
fluid (ml)
Weight of
ascitic
fluid (g)
13.24
tumor cells
cell
growth
growth
inhibition
Control
0.2 ml N.S. i/p 13.44
415.04 x 107
216.0 x 107***
130.61 x 107***
92.64 x 107***
13.17 x 107***
100
--
0/10
0/7
0/7
0/7
0/7
Compd. 6a
Compd. 6a
Compd. 6a
5-FU
20 mg/kg i/p
40 mg/kg i/p
60 mg/kg i/p
12.85
12.05
10.08
12.21
52.05
47.95
68.53
77.67
96.82
12.08
31.47
22.33
3.18
9.75
20 mg/Kg i/p 0.47
0.42
***p<0.001 (Dunnett test);
Table 4a
Treatment
Dose
Body weight (g)
Day 1
24.77
25.85
25.85
25.85
Day 5
26.33
27.57
27.14
25.85
Day 9
Day 12
35.5
Control
0.2 ml N.S. i/p
80 mg/kg i/p
100 mg/kg i/p
20 mg/Kg i/p
30.55
28.42
27.57
24.57
Compd. 6a
Compd. 6a
5-FU
30.33
28.57
24.14
3

ACCEPTED MANUSCRIPT
Table 4b
Dose
Day 12
Treatment
Av.
Av.
Av. No. of
% Tumor % Tumor Mortalit
Volume of Weight of
tumor cells
cell
growth
y
ascitic
ascitic
growth
inhibition
fluid (ml)
fluid (g)
Control
0.2 ml N.S. i/p 12.62
12.03
340.31 x 107
100
--
0/10
0/7
1/7
0/7
Compd. 6a
Compd. 6a
5-FU
80 mg/kg i/p
100 mg/kg i/p
20 mg/Kg i/p
6.91
6.28
0.58
6.91
6.22
0.69
72.85 x 107*** 21.41
62.44 x 107*** 18.35
12.36 x 107*** 3.64
78.59
81.65
96.36
***p<0.001 (Dunnett test);
Table 5
Treatment Groups
Av. Body weights (g) of
animals on days
Day 13
%Tumor
Growth
Mortality
Inhibition
1
5
9
Av. Body
Av. Tumor
weights (g) weights (mg)
Compd. 6a
27.0
27.14 26.71
26.00 26.14
25.14 24.71
27.4
592.35**
448.5***
509.58***
1039.8
43.03
56.86
50.99
--
0/7
0/7
(100 mg/kg i/p)
Compd. 6a
26.57
26.42
25.0
27.0
(150 mg/kg i/p)
5-FU(22 mg/kg i/p)
24.85
26.9
2/7
Normal Control
NS ( 0.2 ml i/p)
26.7
26.7
0/10
**p<0.01 (Dunnett test); ***p<0.001 (Dunnett test);
4

ACCEPTED MANUSCRIPT
Table 6
Effect of 6a in various parameters as a single oral dose in Swiss
albino mice
5 mg/kg
Parameters
Control
24.87±0.49
11.50±0.41
7.01±0.48
11.80±0.31
336.0±23.94
25.79±2.39
24.08±3.16
2.23±0.18
1.69±0.28
0.19±0.01
0.42±0.03
50 mg/kg
24.08±0.63
11.67±0.42
7.40±0.35
12.38±0.53
300 mg/kg
20.80±1.37
11.91±0.46
6.78±0.27
11.42±0.94
24.51±0.72
11.85±0.46
8.08±0.45
Body weight (gm)
Haematological
Haemoglobin (gm/dL)
RBC (million/mm3)
WBC (1000*/mm3)
ALP (U/L)
parameters
12.59±0.77
348.23±25.62
29.62±1.26
20.91±1.04
2.21±0.08
268.57±17.12 321.95±23.11
Liver Function test
29.68±2.42
27.37±1.82
2.20±0.10
1.39±0.12
0.21±0.02
0.32±0.02
31.87±2.06
32.83±1.89
1.97±0.10
1.57±0.15
0.27±0.02
0.39±0.03
SGOT (U/L)
SGPT (U/L)
Albumin (g/dL)
1.68±0.23
Serum Protein (mg/ml)
Total Bilirubin (mg/dL)
Creatinine (mg/dL)
0.20±0.04
0.45±0.04
Kidney Function test
Cholesterol profile
Triglycerides (mg/dL)
Cholesterol (mg/dL)
156.79±15.13
80.12±6.06
145.70±11.82
82.26±6.54
152.84±10.57 146.29±14.76
87.25±5.10 79.67±7.27
Figure 1
HN
H
NH2
F
F
N
N
N
OH
O
F
N
NH
O
HN
NH
O
I
F
5-Fluorouracil
Imiquimod
Cobimetinib
O
O
O
OH
R
R1
R2
R
R
R
HO
OH
O
O
R1=OCH3, R2=OCH3, Curcumin;
R1=H, R2=OCH3, Demethoxycurcumin;
R1=R2=H, Bisdemethoxycurcumin
Prototype-2
Prototype-1
5

ACCEPTED MANUSCRIPT
Figure 2
Control
6a:1.5 µg/mL,
6a: 5 µg/mL,
6a: 10 µg/mL,
6a: 20 µg/mL,
6a: 50 µg/mL
Figure 3
Figure 4
6

ACCEPTED MANUSCRIPT
Figure 5:
Figure 6:
Figure 7:
7

ACCEPTED MANUSCRIPT
Figure 8:
Scheme 1.
Scheme-1
O
O
O
R
O
O
i
CH3
(A)
+
R
H
H
R
2a: R=CH3; 2b: R=4-OCH3;
2c: R=3,4-dimethoxy;
1
3a: R=CH3; 3b: R=4-OCH3;
3c: R=3,4-dimethoxy;
3d: 3,4,5-trimethoxy;
3e: 3,4-methylenedioxy;
3f: 4-Fluoro; 3g: 4-Bromo
2d: 3,4,5-trimethoxy;
2e: 3,4-methylenedioxy;
2f: 4-Fluoro; 2g: 4-Bromo
O
O
O
O
O
ii
(B)
H
R
+
R
R
H3C
CH3
5a: R=2-OCH3;
5b: R=3,4-methylenedioxy;
5c: R=4-N,N-dimethyl;
6a: R=2-OCH3;
6b: R=3,4-methylenedioxy;
6c: R=4-N,N-dimethylamino;
O
O
O
(C)
O
O
iii
H
R
+
R
R
H3C
CH3
5d: R=4-hydroxy;
6d: R=4-hydroxy;
5e: R=3-hydroxy,4-methoxy;
5f: R=3,4-dimethoxy;
5g: R=3,4,5-trimethoxy
6e: R=3-hydroxy,4-methoxy;
6f: R=3,4-dimethoxy;
6g: R=3,4,5-trimethoxy
8

ACCEPTED MANUSCRIPT
Highlights
* Fourteen curcumin mimics with a bridged phenyl ring.
* Five compounds exhibited potential antiproliferative activity.
* Compound 6a, novel, cytotoxicity skin cancer (IC50=1.5µM), microtubule destabilization.
* In-vivo anticancer efficacy 78.6% tumour reduction at 80mg/kg dose.
* In-vivo toxicity studies-safe up to 300mg/kg dose.