Design, synthesis and cytotoxic activity of certain novel chalcone analogous compounds

Article abstract of DOI:10.1016/j.ejmech.2016.09.099

A series of chalcone analogous compounds were designed and synthesized. Replacing/substituting the enone or ethylenic bridge of the parent chalcone with rigid heterocyclic moieties or substituted aromatic amines gave nineteen target compounds. Their cytot

Full text of DOI:10.1016/j.ejmech.2016.09.099

Accepted Manuscript
Design, synthesis and cytotoxic activity of certain novel chalcone analogous
compounds
S. El-Meligie, Azza T. Taher, Aliaa M. Kamal, A. Youssef
PII:
S0223-5234(16)30862-5
10.1016/j.ejmech.2016.09.099
EJMECH 8965
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 17 June 2016
Revised Date: 29 September 2016
Accepted Date: 30 September 2016
Please cite this article as: S. El-Meligie, A.T. Taher, A.M. Kamal, A. Youssef, Design, synthesis and
cytotoxic activity of certain novel chalcone analogous compounds, European Journal of Medicinal
Chemistry (2016), doi: 10.1016/j.ejmech.2016.09.099.
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ACCEPTED MANUSCRIPT
Design, Synthesis and Cytotoxic Activity of Certain Novel Colchicine Analogous
Compounds
S. El-Meligie a, Azza T. Taher a, b, Aliaa M. Kamala*, A. Youssef c
a Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Cairo University,
11561, Cairo, Egypt
b Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, October University for
Modern Sciences and Arts (MSA), Giza, Egypt
c
Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy and Drug Manufacturing,
Misr University for Science and Technology, Giza, Egypt
HN
NH
S
. Replacing the enone bridge (Type 1) or the ethylenic bridge (Type 2&3) with either 3,4-
dihydropyrimidine-2-thione, oxirane, pyrazoles, thiirane or diphenyl ethanolamine afforded
compounds with excellent antitumor activity and showed remarkable inhibition of TUBb
polemerization.

ACCEPTED MANUSCRIPT
Design, Synthesis and Cytotoxic Activity of Certain Novel Chalcone Analogous Compounds
S. El-Meligie a, Azza T. Taher a, b, Aliaa M. Kamala*, A. Youssef c
a Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Cairo University,
11561, Cairo, Egypt
b Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, October University for Modern
Sciences and Arts (MSA), Giza, Egypt
c
Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy and Drug Manufacturing, Misr
University for Science and Technology, Giza, Egypt
*Corresponding author:
Dr. Aliaa M. Kamal (Ph.D. Pharmaceutical Organic Chemistry)
Pharmaceutical Organic Chemistry Department,
Faculty of Pharmacy, Cairo University, 11561, Cairo, Egypt.
Fax: +202-23628426
Telephone (Mobile): +201280007280
E-mail: Aliaa.kamal@pharma.cu.edu.eg; K_aliaa2511@hotmail.com
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Abstract
A series of chalcone analogous compounds were designed and synthesized. Replacing/substituting the
enone or ethylenic bridge of the parent chalcone with rigid heterocyclic moieties or substituted aromatic
amines gave nineteen target compounds. Their cytotoxic activities were screened against both breast and
liver cancer cells as well as breast and liver normal cells. Target compounds were also evaluated for
their inhibition activity of tubulin beta polymerization. Target compound 2e, 3a, 3b, 3c, 4a-4d, 5a, 5b
and 6 showed broad spectrum excellent anticancer activity against both MCF-7 and HepG2. Compound
4a showed the most TUBb inhibition activity.
Key words: Chalcone derivatives; replacing enone bridge; cytotoxic activity; cancer cell lines; normal
cell lines; tubulin beta polymerization inhibitors.
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1.Introduction
Cancer, the uncontrolled, rapid and pathological proliferation of abnormal cells, is one of the most
formidable afflictions in the world. Targeted therapies that interfere with a single biological molecule or
pathway have been successfully utilized in treating cancer [1, 2]. Tubulin heterodimers, the major
component of microtubules, are a molecular target of anticancer therapy based on microtubule-targeting
agents. Inhibition of tubulin polymerization or interfering with microtubule assembly/disassembly
disrupts several cellular functions. Tubulin targeting agents constitute an important class of anticancer
drugs and it includes several agents, such as colchicine (I), combretastatin A-4 (II), MDL-27048 (III)
(Figure 1), vinca alkaloids and a number of structurally unrelated small molecules. Two different
binding sites on tubulin are known, namely the colchicine and the vinca alkaloid binding sites [3-19].
Figure 1
Chalcones (a series of biaryl propenones) have important effects on cancer cell growth and proliferation.
Many mechanisms of action have been identified, including the inhibition of tubulin assembly,
inhibition of angiogenesis, induction of apoptosis, anti-estrogenic activity and reversal of multidrug
resistance – or a combination of these mechanisms. Chalcones were powerful tubulin assembly
inhibitors, with almost similar potency to combretastatin A4 (II, CA-4). Chalcone and related
compounds as MDL-27048, CA-4, chalcone epoxides, related dienones and piperazine nucleus
containing novel chalcones have been reported to possess great cytotoxic activity [7-10, 12, 14, 15, 18].
Many structure activity relationship studies (SAR) and a number of ligand- and structure based docking
studies have been reported concerning the tubulin targeting agents [6, 20].
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During the last decade, several chalcone analogs and derivatives have been designed with the enone
moiety replaced by a heterocyclic ring in order to get rigid analogs [1].
Pyrimidines, pyrazoles and epoxides nuclei exhibited remarkable pharmacological activities. Literature
indicates that compounds having pyrimidine nucleus have wide range of therapeutic uses that include
anticancer activity [21-24].
Structure-activity relationship (SAR) studies indicated ring A and ring C are crucial for colchicine-
tubulin binding, whereas ring B, although important, did not emerge as essential for the activity and was
shown to be tolerant to structural modifications where the hydroxyl group was not essential while the 4-
methoxyphenyl group seemed crucial for cytotoxicity [1, 25].
Docking studies of enormous colchicine site inhibitors (CSI) showed that, 7-point pharmacophore
connected the different structural classes of CSI, based on consistent structural features and recurring
tubulin–ligand interactions [1, 5-6]. Three hydrogen bond acceptors, one hydrogen bond donor, two
hydrophobic centers and one planar group were the 7-point pharmacophores. Hydrophobic centers and
planar groups’ points represented the rigid portion of the molecular scaffold, while the other features
(the different heterocyclic of substituted amino alcohol bridge) represented critical interactions with the
protein, thus being responsible for binding specificity. It should be noted that, none of the CSI drugs
possessed all of the 7-points pharmacophore, suggesting that the discovery of more potent tubulin
destabilizing agents could be a feasible task. [1, 5-6]
The target of this work was the development of novel compounds bearing chalcone moieties hybridized
with epoxides, pyrimidines or pyrazoles via modification of the chalcones` structure involved either the
enone (type 1)- or the alkene (type 2&3)-functionalized parts of parent chalcones. (Figure 2).
Figure 2
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All the synthesized compounds (Schemes 1) were characterized by two aromatic rings (A&C) linked by
various fragments e.g. heterocyclic three, five or six membered rings and chalcone derivatives.
2. Results and discussion:
2.1. Chemistry
In the present study, compounds (E)-3-(4-substituted phenyl)-1- n-(methoxyphenyl) prop-2-en-1-ones
(1a- i) were obtained by the Claisen-Schmidt condensation of substituted acetophenones and
benzaldehydes, affording exclusively the E-isomer in high yields [14]. Replacing the enone moiety with
pyrimidine and hydroxypyrazole heterocycles took place by reacting the parent chalcones 1a-i with
thiourea to afford 2a-g or reacting the epoxide derivatives of the parent chalcone 3a-d with either phenyl
hydrazine, hydrazine hydrate or thiourea to yield compounds 4a-f and compound A respectively
(Scheme 1). Upon using the conventional method for preparing compounds 2a-g, it took very long time
to complete the reaction with moderate yield. A novel highly efficient rapid method with very mild
reaction conditions was used herein to synthesize compounds 2a-g in high yield, high atom economy
(less chemical wastes) and following environmental friendly protocol. Under microwave irradiation, the
reaction time reduced to 3-4 hours and the product was afforded in almost high yield. The progress of
the reaction was monitored by TLC after every 1 hour (Scheme 1).
The structure of the compounds 2a-g was confirmed by element analysis, IR, 1H-NMR, 13CNMR and
mass spectroscopy. The spectral data revealed the formation of the 3,4-dihydropyrimidinethione rather
1
than the fully unsaturated pyrimidine. H-NMR of compounds 2a-g showed the appearance of two
doublets corresponded to C4 proton due to geometrical isomerism and a doublet at δ 6.81-6.92 ppm due
to C5 proton of the pyrimidine ring. 13CNMR showed the characteristic C=S signal at δ 173.7-175.7
ppm and the characteristic signal of C4 at δ 60.4-60.6 ppm.
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Scheme 1
Replacement of the ethylenic bridge with oxirane ring via epoxidation of α,β−unsaturated ketones gave
3-aryl-2-substituted-acetophenoyl-oxiranes 3a-d. Reacting compounds 1a-d with hydrogen peroxide
28% in a mixture of polar solvents (methanol and acetone) at 0 ºC afforded compounds 3a-d in excellent
yield. The structure of the products 3a-c was confirmed by element analysis, IR, 1HNMR, 13CNMR and
mass spectroscopy. IR spectra of 3a-c showed an absorption band at 3446-3462 cm-1 indicating the
presence of C-H of oxirane ring functionality. 1HNMR spectra of 3a-c showed two doublet signals at δ
4.13-4.17ppm and δ 4.76-4.82 ppm corresponding to CH-O-CH of oxirane ring. Epoxides are versatile
intermediate in organic synthesis as their ring can be easily opened by a variety of nucleophiles e.g.
amines and hydrazines. To replace the enone bridge with hydroxypyrazole moiety; hydrazinolysis of the
oxirane derivatives 3a-d using either phenyl hydrazine or hydrazine hydrates was carried out to give
compounds 5-aryl-N1-substituted-3-(substituted-methoxy-phenyl)-4-hydroxypyrazolines 4a- f in good
yield. The reaction of the hydrazine derivatives and epoxy chalcone analogues was believed to proceed
via the formation of the intermediate hydrazone. IR, 1HNMR, 13CNMR and mass spectroscopy and
element analysis were used to identify compounds 4a-f. The 1HNMR spectra of 4a-f showed signals
corresponding to C4 proton, C5 proton of pyrazole ring and the hydroxyl group. More over and as a
result of the geometrical isomerism, two adjacent singlet signals of the methoxy methyl group were
appeared.
Replacing the Enone Bridge with fully saturated pyrimidinone thione compound A was believed to be
via reacting the oxirane derivatives of chalcone with thiourea, but upon carrying out this reaction a
different product was formed. The spectral data of the obtained products proved the formation of
thiirane derivatives of the chalcone 5a-b rather than compound A. 1HNMR spectra the 5a-b should two
doublet signals due to C2 and C3 protons of the thiirane ring at δ 4.15-4.17, 4.81-4.82 ppm. 13CNMR
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spectrum of compound 5a showed the characteristic signals of C2 and C3 protons of the thiirane ring at
δ 38.9 and 59.7 ppm. Aminolysis of the epoxide chalcones by amines resulted in β-amino alcohols,
which are important intermediates in the synthesis of a large number of biologically active natural and
synthetic products. To replace the ethylenic bridge with substituted amine, compound 3b was reacted
with substituted aniline to give 3-(4-bromophenyl)-3-(3-chloro-4-fluorophenylamino)-2-hydroxy-1-(3-
methoxyphenyl) propan-1-one (6) in very good yield. 1HNMR of compound 6 showed four doublet
signals at δ 4.78, 5.04, 5.84 and 6.36 ppm corresponds to CH-N, CH-O, OH and NH respectively.
2.2. Discussion of the cytotoxic activity of the target compounds
All the test compounds were evaluated for their cytotoxic activity against MCF-7 and HepG2 cell lines.
They showed excellent cytotoxic activity, much more potent than or even as good as the reference drug
colchicine.
2.2.1. Activity against the breast cancer cell line (MCF-7):
In the present study, we synthesized nineteen novel compounds. They were tested for their cytotoxic
activity against MCF-7 cell line using colchicine as a reference drug.
Replacement of Enone Bridge of compounds 1a-i with 3,4-dihydropyrimidine-2-thione moiety (Type 1)
represented compounds 2a-g which showed cytotoxic activity as potent as or more potent than the
reference drug. Compounds 2d-g (with trimethoxyphenyl (TMP) moiety (ring A)) showed higher
cytotoxic activity against MCF-7 cell line than 2a-c. Compound 2e (IC50 0.0770 µM) with TMP (ring A)
and bromophenyl (ring C) was the most active amongst compounds 2a-g nearly double the potency of
the reference drug (IC50 0.1504 µM).
Replacing the ethylenic bridge with oxirane resulted in compounds 3a-d (type 2). Compounds 3a-c
showed excellent cytotoxic activity against MCF-7 much more potent than colchicine. The most active
amongst this group were 3b and 3c with IC50 0.0130 µM and IC50 0.0140 µM respectively, about ten
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folds more potent than colchicine (IC50 0.1504 µM). It was noticed that changing of the halogen group
(ring C) from Br/F (compounds 3b and 3c) to Cl (compound 3a) reduced the potency nearly to the half.
Replacing the Enone Bridge by hydroxypyrazole afforded compounds 4a-f (type 1) which were far more
potent than the reference drug. Compounds 4a-d with N1-phenyl moiety revealed excellent
antiproliferative activity against MCF-7 cell line (ten folds more potent), while 4e and 4f showed
moderate activity in comparison to compound 4a, the parent of this group. In fact, compounds 4a and 4b
were amongst the most active ones against MCF-7 of all the test compounds.
Upon replacing the ethylenic bridge with thiirane heterocycle, compounds 5a and 5b resulted (type 2).
The activity of compound 5a against MCF-7 was one and half the cytotoxic activity of compound 5b.
Substituting the ethylenic bridge with diphenyl ethanolamine moiety (type 3) resulted in one of the most
potent test compounds 6 (Table 1).
Table 1
2.2.2. Activity against human liver cancer cell line (HepG2):
Generally, all the novel synthesized compounds showed more potent cytotoxic activity against human
liver carcinoma cell line (HepG2) than MCF-7 cell line. Replacing the enone bridge (Type 1) or the
ethylenic bridge (Type 2&3) with either 3,4-dihydropyrimidine-2-thione, oxirane, pyrazoles, thiirane or
diphenyl ethanolamine afforded compounds 2a-g, 3a-d, 4a-f, 5a-b and 6 respectively.
In spite of being moderately active against MCF-7; compounds 2c, 2d, and 2e were the most active of
this group (2a-g) with IC50 ( 0.0300, 0.0110 and 0.0095 µM) respectively against human hepatic
carcinoma cell line (HepG2). Moreover, Compounds 3a and 3c showed remarkable antiproliferative
activity with IC50 (0.0150 and 0.0170 µM), while compound 3b with IC50 (0.0300 µM) showed only one
third of their activity. Concerning compounds 4a-f; Compounds 4a-d revealed a higher potency than
compounds 4e and 4f. Compounds 4a, 4b and 4d have nearly the same cytotoxic activity against HepG2
8

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cell line with IC50 (0.0110, 0.0099, 0.0130, and 0.0102 µM). Also compounds 5a (0.0130 µM) and 5b
(0.0110 µM) showed excellent activity as potent as compounds 4a-d. Compound 6 disclosed very good
antiproliferative activity (Table 1).
To summarize the cytotoxic activity of all the test compounds against both cell lines, compounds 2e, 3a,
3b, 3c, 4a-4d, 5a, 5b and 6 showed broad spectrum excellent anticancer activity against both MCF-7
and HepG2 (Table 1).
2.3. Enzyme-linked Immunosorbent Assay for tubulin Beta (TUBb):
This assay was employed to predict the possible mode of action of representative target compounds. The
percentage inhibition of tubulin polymerization of representative of the most active target compounds
against cancer cell lines was measured. Compounds 3b, 3c, 4a, 4b, 5a and 6 (Figure 3) with the highest
cytotoxic activity against human breast cancer cell line (MCF-7) were tested for inhibiting tubulin beta
(TUBb) polymerization using colchicine as a reference drug. All the selected tested compounds showed
excellent TUBb polymerization inhibition activity. They were more potent than colchicine as TUBb
polymerization inhibitors and this was in accordance with the in vitro cytotoxic activity of these
compounds against MCF-7 cell line. Compounds 3b, 4a and 6 (Figure 3) were excellent TUBb
polymerization inhibitors with percentage inhibition 92.62997%, 92.29742% and 92.16634%
respectively, while the percentage of TUBb inhibition of colchicine was 86.2803% (Table 2).
Table 2
All the selected test compounds with outstanding TUBb polymerization inhibition activity have almost
the same common features; 3/4-methoxyphenyl (ring A), halo phenyl (ring C) and a bridge with a
carbonyl group and/or a hydroxyl group (Figure 3). Compounds 5a, 3c and 4b showed remarkable
inhibition activity as well as great potencies against MCF-7.
Figure 3
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In case of compounds that revealed excellent cytotoxic activity against human liver carcinoma (HepG2),
TUBb polymerization inhibition assay was carried out for representative compounds 2a, 2c, 2d, 2e, 4a,
4b, 5a, 5b and 6. In spite of not being the most active compound amongst the representative target
compounds, compound 4a showed outstanding inhibition activity for TUBb polymerization with
percentage inhibition 93.02209%. As they showed excellent cytotoxic activity against HepG2,
compounds 2e, 2d, 2c and 2a (Figure 4) showed very high percentage of TUBb polymerization
inhibition (92.77821%, 92.36795%, 91.43021% and 90.58723%) much more potent than colchicine
(82.52266%). Compounds 5a, 6 and 4b were more potent as TUBb polymerization inhibitors than
colchicine (Table 2).
Figure 4
2.4. Effect of representative target compounds on normal human breast cells and hepatic cells:
One of the main problems of cancer chemotherapy is the unwanted damage to normal cells caused by
the high toxicities of anticancer drugs. Representative target compounds that were the most active
against MCF-7 and HepG2 and cause the highest percentage inhibition of TUBb polymerization were
selected to be tested against normal breast cell line (Hs 371.T) and normal liver (AML12) cell line and
their IC50 were determined using colchicine as reference drug. Since all the target compounds were
more potent against HepG2, six representative compounds were evaluated for their toxicity towards
AML12 using colchicine as reference drug. Furthermore, three representative target compounds were
evaluated for their cytotoxic effect on Hs 371.T (Breast N cells) via MTT assay. It was worthy to
mention that, the IC50 doses of all the representative target compounds against normal breast and liver
cells were very high in comparison to their IC50s doses of the cancer cell lines, 2.5-9 folds more than the
anticancer does (Table 3).
Table 3
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3. Conclusion
In this study, a group of novel compounds were synthesized according to structure based-drug design to
be colchicine site inhibitors (CSI). Compounds were tested for their cytotoxic activity against MCF-7
and HepG2 cell lines. Target compounds exhibited potent inhibitory activity at nanomolar level.
Generally, target compounds showed more potent activity against HepG2 cell line than MCF-7 cell line.
Target compounds 2e, 3a, 3b, 3c, 4a-4d, 5a, 5b and 6 showed broad spectrum excellent anticancer
activity against both MCF-7 and HepG2. Inhibition of TUBb polymerization test was used to confirm
the mechanism of action of the representative broad spectrum antiproliferative active compounds.
Compound 4a showed the most TUBb inhibition activity. It is worthy to mention that, the replacement
of either the enone or ethylenic bridge of the chalcone with rigid heterocyclic moieties resulted in
superior antitumor potential ligands that may eventually allow the design and synthesis of chalcone
derivatives with superior anticancer activity. The effect of the most active target compounds against
normal breast cells and normal hepatic cells was tested and it was clear that the IC50 doses of the target
compounds against cancer cell lines were safe doses for normal cells.
Experimental Section
4. Experimental
4.1. Chemistry
Melting points were determined on Stuart apparatus and the values given are uncorrected. Column
Chromatography was used for purification by using solvent system benzene-acetone 90% (9:1). IR
spectra were determined on Shimadzu IR 4000s spectrophotometer (KBr, cm-1) at Faculty of Pharmacy,
Misr University for Science and Technology, Egypt. 1HNMR spectra were carried out using a Mercury,
a Gemini 300-BB 300 MHz and Joel (eca), 300 or 400 MHz spectrophotometers using TMS as internal
standard. Chemical shift values were recorded in ppm on δ scale, Microanalytical center, Cairo
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University, Egypt, Main laboratory of the War Chemical, Chemical War Department, Ministry of
Defense and National Research Centre, and Faculty of Pharmacy, Cairo University. 13CNMR spectra
were carried out using Mercury and Gemini 300-BB 75-100 MHz spectrophotometers using TMS as
internal standard. Chemical shift values were recorded in ppm on δ scale, Micro analytical center, Cairo
University, Egypt, and Faculty of Pharmacy, Cairo University. Mass spectra were recorded on Hewlett
Packard 5988 spectrometer, Microanalytical Center, Cairo University, Egypt. Elemental analyses were
carried out at the Microanalytical Center, Faculty of Pharmacy, Al Azhar University, Egypt. Scientific
microwave MSA2 was used irradiated under 300-400 watt microwave irradiation, operating at 2450Hz
intermittently at 60 °C. Progress of the reactions was monitored using TLC sheets precoated with UV
fluorescent silica gel Merck 60F 254. The solvent system was benzene, benzene: acetone and benzene:
acetone: chloroform: TEA with different ratios and spots were visualized using UV lamp. The docking
was performed using Auto Dock empirically derived program. Evaluation of the cytotoxic activity was
performed at the Egyptian National Cancer Institute using MCF-7 and HPEG2 cell lines. Enzyme essay
was carried out at VACSERA, Giza, Egypt using SEB870Hu 96 Tests Enzyme-linked Immunosorbent
Assay Kit for Tubulin Beta (TUBb). Compounds 1a-i and 3d were prepared according to reported
methods [14, 26]
4.1.1. General procedure for preparation of compounds 2a-g
A mixture of compounds 1a, 1b, 1e, 1f, 1g, 1h or 1i (0.02 mol) and thiourea (1.52 g, 0.02 mol) in
ethanolic sodium hydroxide (8%, 10 mL) was placed in a conical flask. The conical flask was covered
with a funnel and then the flask was placed in a scientific microwave MSA2. The reaction mixture was
irradiated under 300-400 watt, operating at 2450 Hz intermittently at 60 °C for 3-4 h.
4.1.1.1. 4-(4-Chlorophenyl)-6-(3-methoxyphenyl)-3,4-dihydropyrimidine-2(1H)-thione (2a)
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Yield, 88 %; mp 188-200 °C; IR (KBr) ν= 3394 (NH), 3183, 3073 (aromatic CH) cm-1. 1H NMR
(DMSO-d6) ppm δ: 3.82 (s, 3H, OCH3), 5.14, 5.41 (2d, 1H, J= 2.56, 4.76 Hz), 6.89 (d, 1H, J= 7.92 Hz),
6.97- 7.47 (m, 8H, Ar-H), 9.14(s, 1H, NH, D2O exchangeable), 9.28, 9.92 (2s, 2H, 2NH, D2O
exchangeable). 13C NMR (DMSO-d6) ppm δ: 175.7, 173.8, 159.6, 140.0, 135.1, 134.9, 129.9, 129.1,
+
128.8, 118.7, 115.5, 114.5, 111.4, 60.5, 55.6. GCMS m/z (% rel. abundance): 330.05 (M . , 100%).
Anal. Calcd for C17ClH15N2OS (330.83): C, 61.72; H, 4.57; N, 8.47. Found: 61.52; H, H 4.53; N, 8.51.
4.1.1.2. 4-(4-Bromophenyl)-6-(3-methoxyphenyl)-3,4-dihydropyrimidine-2(1H)-thione (2b)
Yield, 85 %; mp 212-214 °C; IR (KBr) ν= 3392 (NH), 3197 (aromatic CH) cm-1. 1H NMR (DMSO-d6)
ppm δ: 3.85 (s, 3H, OCH3), 5.13, 5.42 (2d, 1H, J= 4.36, 4.44 Hz) 6.92 (d, 1H, J= 7.96 Hz) 7.00-7.99 (m,
8H, Ar-H), 9.13, 9.93 (2s, 2H, 2NH, D2O exchangeable). 13C NMR (DMSO-d6) ppm δ: 173.7, 159.6,
143.2, 137.7, 135.0, 131.7, 130.3, 129.7, 122.1, 120.0, 115.4, 112.4, 56.5, 60.6, 54.1. GCMS m/z (% rel.
+
abundance): 374.90 ((M . , 100%). Anal. Calcd for C17BrH15N2OS (375.27): C, 54.41; H, 4.03; N,
7.46. Found: 54.57; H, 4.01; N, 7.52.
4.1.1.3. 4-(4-Bromophenyl)-6-(4-methoxyphenyl)-3,4-dihydropyrimidine-2(1H)-thione (2c)
Yield, 50 %; mp 210-212 °C; IR (KBr) ν= 3399 (NH), 3189 (aromatic C-H) cm-1. 1H NMR (DMSO-d6)
ppm δ: 3.81 (s, 3H, OCH3), 5.09, 5.27 (2d, 1H, J= 4.68, 4.92 Hz) 6.89 (d, 1H, J= 4.92 Hz), 7.01-7.67(m,
+
.
8H, Ar-H), 9.05, 9.88 (2s, 2H, 2NH, D2O exchangeable). GCMS m/z (% rel. abundance): 375.95 (M
, 43.07%), 219.00 (100%). Anal. Calcd for C17BrH15N2OS (375.28): C, 54.41; H, 4.03; N, 7.46. Found:
54.60; H, 4.06; N, 7.41.
4.1.1.4. 4-(4-Chlorophenyl)-6-(3,4,5-trimethoxyphenyl)-3,4-dihydropyrimidine-2(1H)-thione (2d)
Yield, 50 %; mp 185-187 °C; IR (KBr) ν= 3398 (NH), 3196 (aromatic CH) cm-1. 1H NMR (DMSO-d6)
ppm δ: 3.78, 3.82 (2s, 9H, 3OCH3), 5.14, 5.45 (2d, 1H, J= 3.48, 4.52 Hz) 6.82 (d, 1H, J= 4.52 Hz),
6.94-8.39 (d, 6H, Ar-H), 9.00, 9.94 (2s, 2H, 2NH, D2O exchangeable). 13C NMR (DMSO-d6) ppm δ:
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175.5, 173.7, 153.1, 152.8, 142.8, 138.4, 134.8, 132.6, 129.1, 128.8, 123.0, 112.5, 103.8, 100.8, 60.5,
56.4. GCMS m/z (% rel. abundance): 332.00 (100%). Anal. Calcd for C19ClH19N2O3S (390.88): C,
58.38; H, 4.90; N, 7.17. Found: 58.66; H, 4.61; N, 7.15.
4.1.1.5. 4-(4-Bromophenyl)-6-(3,4,5-trimethoxyphenyl)-3,4-dihydropyrimidine-2(1H)-thione (2e)
Yield, 55 %; mp 208-210 °C; IR (KBr) ν= 3389 (NH), 3176 (aromatic CH) cm-1. 1H NMR (DMSO-d6)
ppm δ: 3.73, 3.82 (2s, 9H, 3OCH3), 5.21, 5.44 (2d, 1H, J= 2.32, 4.36 Hz), 6.81(d, 1H, J=4.68 Hz), 7.23-
7.62 (m, 6H, Ar-H), 9.11, 9.93 (2s, 2H, 2NH, D2O exchangeable). 13C NMR (DMSO-d6) ppm δ: 175.5,
153.2, 143.9, 138.4, 134.7, 132.1, 129.2, 129.0, 121.1, 103.7, 100.7, 60.4, 56.4, 54.6. GCMS m/z (% rel.
+
abundance): 435.95 (M . , 51.78%), 279.05 (100%). Anal. Calcd for C19ClH19N2O3S (435.33): C,
52.42; H, 4.40; N, 6.43. Found: 52.65; H, 4.10; N, 6.63.
4.1.1.6. 4-(4-Flurophenyl)-6-(3,4,5-trimethoxyphenyl)-3,4-dihydropyrimidine-2(1H)-thione (2f)
Yield, 60 %; mp 179-181°C; IR (KBr) ν= 3399 (NH), 3191 (aromatic C-H) cm-1. 1H NMR (DMSO-d6)
ppm δ: 3.77, 3.82, 3.91 (3s, 9H, 3OCH3), 5.14, 5.45 (2d, 1H, J= 2.48, 4.44 Hz), 6.81 (d, 1H, J= 3.76 Hz,
pyrimidine), 7.08-7.44 (m, 6H, Ar-H), 8.98, 9.85(2s, 2H, 2NH, D2O exchangeable). 13C NMR (DMSO-
d6) ppm δ: 175.4, 163.3, 160.9, 153.1, 140.8, 138.4, 134.7, 128.9, 115.8, 103.7, 101.0, 60.5, 56.4, 54.4.
+
GCMS m/z (% rel. abundance): 374.05 ((M . 100%). Anal. Calcd for C19FH19N2O3S (374.43): C,
60.95; H, 5.11; N, 7.48. Found: 60.77; H, 5.10; N, 7.73.
4.1.1.7. 4-(4-Methoxyphenyl)-6-(3,4,5-trimethoxyphenyl)-3,4-dihydropyrimidine-2(1H)-thione (2g)
Yield, 95 %; mp 216-218 °C; IR (KBr) ν= 3395 (NH), 3171 (aromatic C-H) cm-1. 1H NMR (DMSO-d6)
ppm δ: 3.82, 3.84, 3.90 (3s, 12 H, 4OCH3), 5.05, 5.41 (2d, 1H, J= 4.00, 4.68 Hz) 6.81 (d, 1H, J= 4.68
Hz), 6.94-7.23 (m, 6 H, Ar-H), 9.02, 9.83 (2s, 2H, NH, D2O exchangeable). 13C NMR (DMSO-d6) ppm
δ: 175.1, 159.2, 153.0, 138.3, 136.7, 134.4, 129.2, 128.3, 114.4, 106.5, 103.7, 101.4, 60.4, 56.3, 55.6,
14

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+
54.7. GCMS m/z (% rel. abundance): 386.05 (M . , 100%). Anal. Calcd for C20H22N2O4S (386.46): C,
62.16; H, 5.74; N 7.25. Found: 62.55; H, 5.69; N, 7.27.
4.1.2. General procedure for preparation of compounds 3a-c
To a well stirred solution of chalcone 1a, 1b, 1c or 1d (0.04 mol) in a mixture of acetone (25 mL) and
methanol (10 mL) at 0°C, a solution of hydrogen peroxide (28 %, 10 mL) in sodium hydroxide solution
(4N, 5 mL) was added over a period of time. After completion of addition, the reaction mixture was
stirred for an additional one hour, and then poured onto ice-cold water (100 mL). The separated product
was collected by filtration, washed with water and crystallized from ethanol to give compounds 3a-d.
4.1.2.1. [3-(4-Chlorophenyl)oxiran-2-yl](3-methoxyphenyl)methanone (3a)
Yield, 95 %; reaction time 1h; mp 90-92 °C; IR (KBr) ν= 3462- 3446 (C-H of oxirane ring), 1689 (C=O)
cm-1. 1H NMR (DMSO-d6) ppm δ: 3.80 (s, 3H, OCH3), 4.16 (d, 1H, J= 2.1 Hz), 4.82 (d, 1H, J= 2.1 Hz),
7.24-7.63 (m, 8H, Ar-H). 13C NMR (DMSO-d6) ppm δ: 192.6, 159.5, 136.5, 134.6, 133.5, 130.1, 128.5,
+
128.2, 120.8, 120.0, 112.6, 59.7, 57.9, 55.3. GCMS m/z (% rel. abundance): 287.90 (M-1 . , 12.57%),
135.00 (100%). Anal. Calcd for C16 Cl H13O3 (288.73): C, 66.56; H, 4.54. Found: C, 66.71; H, 4.59.
4.1.2.2. [3-(4-Bromophenyl)oxiran-2-yl](3-methoxyphenyl)methanone (3b)
Yield, 95 %; reaction time 1h; mp 93-95 °C; IR (KBr) ν= 3446-3421 (C-H of oxirane ring), 1689 (C=O)
cm-1. 1H NMR (DMSO-d6) ppm δ: 3.81 (s, 3H, OCH3), 4.15 (d, 1H, J= 2.1 Hz), 4.82 (d, 1H, J= 1.8 Hz),
+
7.29 -7.63 (m, 8H, Ar-H). GCMS m/z (% rel. abundance): 333.90 (M . , 8.61%), 135.05 (100%). Anal.
Calcd for C16 Br H13O3 (333.18): C, 57.68; H, 3.93. Found: 57.80; H, 3.91.
4.1.2.3. [3-(4-Flurophenyl)oxiran-2-yl](4-methoxyphenyl)methanone (3c)
Yield, 71 %; reaction time 4 h; mp 86-88 °C; mp 75-77; IR (KBr) ν= 3462-3446 (C-H of oxirane ring),
1680 (C=O) cm-1. 1H NMR (DMSO-d6) ppm δ: 3.85 (s, 3H, OCH3), 4.13 (d, 1H, J= 2.4 Hz), 4.76 (d, 1H,
J= 2.4 Hz), 7.06-7.27 (m, 4H, Ar-H), 7.47-7.52 (m, 2H, Ar-H), 8.01-8.04 (m, 2H, Ar-H). GCMS m/z (%
15

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+
rel. abundance): 272.00 (M . , 5.65%), 135.05 (100%). Anal. Calcd for C16 F H13O3 (272.27): C,
70.58; H, 4.81. Found: 70.69; H, 4.89.
4.1.3. General procedure for preparation of compounds 4a-f
A solution of Oxirane 3a, 3b, 3c or 3d (0.02 mol) in absolute ethanol (30 mL) was refluxed with either
phenyl hydrazine (0.02 mol, 1.97 mL) or hydrazine hydrate (99 %, 0.02 mol, 0.64 mL) for 1-2 h. The
solvent was evaporated under reduced pressure and the residue was purified either by crystallization
from ethanol/ether mixture (2:1) for compounds 4a, 4b and 4e or by column chromatography using
solvent system benzene: acetone (90%) for compounds 4c, 4d and 4f.
4.1.3.1. 5-(4-Chlorophenyl)-3-(3-methoxyphenyl)-1-phenyl-4,5-dihydro-1H-pyrazol-4-ol (4a)
Yield, 86 %; mp 80-82 °C; IR (KBr) ν= 3317 (OH), 2951-2931 (aliphatic CH), 1597 (C=N) cm-1. 1H
NMR (DMSO-d6) ppm δ: 3.86 (s, 3H, OCH3), 5.07 (dd, 1H, J= 2.70, 3.00 Hz), 5.24 (d, 1H, J= 2.4 Hz),
6.52 (d, 1H, J= 7.5 Hz, OH, D2O exchangeable), 6.80-7.50 (m, 13H, Ar-H). 13C NMR (DMSO-d6) ppm
δ: 159.4, 147.9, 143.3, 137.9, 132.9, 132.2, 129.6, 129.0, 127.7, 119.2, 118.5, 114.3, 112.9, 111.0, 81.8,
+
+
71.7, 55.1. GCMS m/z (% rel. abundance): 379.30 (M+1 . , 0.77%), 378.30 (M . , 1.61%),
81.00(100%). Anal. Calcd for C22 Cl H19N2O2 (378.85): C, 69.75; H, 5.05; N, 7.39. Found: 69.92; H,
5.12; N, 7.53.
4.1.3.2. 5-(4-Bromophenyl)-3-(3-methoxyphenyl)-1-phenyl-4,5-dihydro-1H-pyrazol-4-ol (4b)
Yield, 83 %; mp 82-84 °C; IR (KBr) ν= 3311 (OH), 3041 (aliphatic CH), 1593 (C=N) cm-1. 1H NMR
(DMSO-d6) ppm δ: 3.86 (s, 3H, OCH3), 5.09 (d, 1H, J= 7.20 Hz), 5.26 (s, 1H), 6.53 (d, 1H, J= 7.50 Hz,
+
.
OH, D2O exchangeable), 6.81-7.61 (m, 13H, Ar-H). GCMS m/z (% rel. abundance): 423.90 (M
,
5.37%), 404. 00 (100%). Anal. Calcd for C22 Br H19N2O2 (423.30): C, 62.42; H, 4.52; N, 6.62. Found:
62.58; H, 4.55; N, 6.71.
4.1.3.3. 5-(4-Flurophenyl)-3-(4-methoxyphenyl)-1-phenyl-4,5-dihydro-1H-pyrazol-4-ol (4c)
16

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Yield, 63 %; mp 60-62 °C; IR (KBr) ν= 3446 (OH), 2933 (aliphatic CH) cm-1. 1H NMR (DMSO-d6)
ppm δ: 3.86, 3.92 (2s, 3H, OCH3), 5.07 (s, 1H), 5.19 (s, 1H), 6.44 (d, 1H, OH, J= 7.2 Hz, D2O
+
exchangeable), 6.80- 7.86 (m, 13H, Ar-H). GCMS m/z (% rel. abundance): 363.05 (M+1 . , 19.80%),
+
362.05 (M . , 74.70%), 77.00 (100%). Anal. Calcd for C22 F H19N2O2 (362.40): C, 72.91; H, 5.28; N,
7.73. Found: 73.12; H, 5.32; N, 7.91.
4.1.3.4. 5-(4-Methoxyphenyl)-3-(4-methoxyphenyl)-1-phenyl-4,5-dihydro-1H-pyrazol-4-ol (4d)
Yield, 54 %; mp 66-68 °C; IR (KBr) ν= 3394 (OH), 2931 (aliphatic CH), 1597 (C=N) cm-1. 1H NMR
(DMSO-d6) ppm δ: 3.77 (s, 3H, OCH3), 3.86, 3.91 (2s, 3H, OCH3), 5.02 (s, 1H), 5.12 (s, 1H), 6.40 (d,
1H, OH, J= 6.3 Hz, D2O exchangeable), 6.79-7.86 (m, 13H, Ar-H). GCMS m/z (% rel. abundance):
+
374.10 (M . , 3.48%), 77.00 (100%). Anal. Calcd for C18H22N2O3 (374.43): C, 73.78; H, 5.92; N, 7.48.
Found: 74.04; H, 5.99; N, 7.56.
4.1.3.5. 5-(4-Chlorophenyl)-3-(3-methoxyphenyl)-4,5-dihydro-1H-pyrazol-4-ol (4e)
Yield, 71 %; mp 136-138 °C; IR (KBr) ν= 3444-3282 (NH and OH), 1598 (C=N) cm-1. 1H NMR
(DMSO-d6) ppm δ: 3.81, 3.82 (s, 3H, OCH3), 4.53 (d, 1H, J= 6.30 Hz), 4.92 (d, 1H, J= 6.3 Hz), 6.01 (d,
1H, J= 7.80 Hz, OH, D2O exchangeable), 6.84-7.48(m, 8H, Ar-H), 7.77 (d, 1H, J= 3.0 Hz, NH, D2O
+
+
exchangeable). GCMS m/z (% rel. abundance): 304.20 (M+2 . , 0.57%), 302.20 (M+ . , 1.06%),
75.05 (100%). Anal. Calcd for C16 Cl H15N2O2 (302.76): C, 63.47; H, 4.99; N, 9.25. Found: 63.65; H,
5.07; N, 9.33.
4.1.3.6. 5-(4-Bromophenyl)-3-(3-methoxyphenyl)-4,5-dihydro-1H-pyrazol-4-ol (4f)
Yield, 55 %; mp 130-132 °C; IR (KBr) ν= 3435-2926 (NH and OH), 1577 (C=N) cm-1. 1H NMR
(DMSO-d6) ppm δ: 3.84 (s, 3H, OCH3), 4.13 (d, 1H, J= 8.1 Hz), 4.81(s, 1H), 7.26 (d, 1H, J= 8.1 Hz,
OH, D2O exchangeable), 7.12-7.84 (m, 8H, Ar-H), 8.69 (s, 1H, NH, D2O exchangeable). 13C NMR
(DMSO-d6) ppm δ: 160.7, 158.4, 142.3, 136.9, 131.9, 127.7, 129.8, 120.8, 122.3, 116.3, 113.9, 83.9,
17

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+
56.9, 55.9. GCMS m/z (% rel. abundance): 347.10(M+ . , 3.91%), 135.10(100%). Anal. Calcd for C16
Br H15N2O2 (347.21): C, 55.38; H, 4.35; N, 8.07. Found: 55.46; H, 4.39; N, 8.14.
4.1.4. General procedure for preparation of compounds 5a-b
Compounds 5a and 5b were prepared adopting the same procedure used for compounds 4a-f using 3a or
3b as starting materials, and thiourea (1.52 g, 0.02 mol) as a cycling agent instead of phenyl hydrazine
or hydrazine hydrate. Compounds 5a-b were crystallization from a mixture of ethanol/ether (2:1)
4.1.4. 1. (3-(4-Chlorophenyl)thiiran-2-yl)(3-methoxyphenyl)methanone (5a)
Yield, 58 %; mp 101-103 °C; IR (KBr) ν= 3059 (aromatic CH), 1685 (C=O), 1263 cm-1. 1H NMR
(DMSO-d6) ppm δ: 3.80 (s, 3H, OCH3), 4.16 (d, 1H, J= 2.8 Hz), 4.82 (d, 1H, J= 2.8 Hz), 7.26-7.64 (m,
8H, Ar-H). 13C NMR (DMSO-d6) ppm δ: 192.5, 159.4, 136.5, 134.6, 133.4, 130.5, 129.8, 128.4, 120.7,
119.9, 112.6, 59.7, 57.8, 55.3. Anal. Calcd for C16Cl H13O2S (304.79): C, 63.05; H, 4.30; Found: 63.04;
H, 4.34.
4.1.4.2. (3-(4-Bromophenyl)thiiran-2-yl)(3-methoxyphenyl)methanone (5b)
Yield, 50 %; mp 106-108 °C; IR (KBr) ν= 3060 (aromatic CH), 1664 (C=O) cm-1. 1H NMR (DMSO-d6)
ppm δ: 3.81 (s, 3H, OCH3), 4.15 (d, 1H, J= 2.4 Hz), 4.81 (d, 1H, J= 2.4 Hz), 7.26-7.63 (m, 8H, Ar-H).
Anal. Calcd for C16BrH13O2S (349.24): C, 55.03; H, 3.75. Found: 55.26; H, 3.89.
4.1.5. 3-(4-Bromophenyl)-3-[(3-chloro-4-fluorophenyl)amino]-2-hydroxy-1-(3-methoxyphenyl)
propan-1-one (6)
A solution of oxirane 3b (6.66 g, 0.02mol) in absolute ethanol (20mL) was heated with 3-chloro-4-fluro-
aniline (5.82 g, 0.04mol) for 2 h. The reaction mixture was filtered while hot, concentrated under
vacuum and then cooled. The separated product was filtered and crystallized from ethanol to give
compound 6.
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Yield, 77 %; mp 136-138 °C; IR (KBr) ν= 3408-3358 (OH, NH), 1677 (C=O) cm-1. 1H NMR (DMSO-
d6) ppm δ: 3.80 (s, 3H, OCH3), 4.78 (d, 1H, J= 8.40 Hz), 5.04 (t, 1H), 5.05 (d, 1H, J= 8.40 Hz), 5.84 (d,
1H, J= 8.40 Hz, NH, D2O exchangeable), 6.36 (d, 1H, J= 12.4 Hz, OH, D2O exchangeable), 6.45-6.62
(m, 1H, Ar-H), 6.62 (dd, 1H, J= 8.40 Hz, J= 8.40 Hz, Ar-H), 6.96-7.02 ( m, 1H, Ar-H), 7.20-7.61 (m,
8H, Ar-H). 13C NMR (DMSO-d6) ppm δ: 197.2, 160.3, 147.3, 144.4, 142.7, 134.6, 131.6, 129.3, 127.8,
122.5, 121.9, 121.5, 118.7, 117.0, 115.5, 114.5, 90.5, 63.9, 55.7. GCMS m/z (% rel. abundance): 481.30
+
+
+
+
(M+3 . , 15.99%), 480.30 (M+2 . , 19.19%), 479.30 (M+1 . , 15.70%), 478.30 (M+ . , 27.33%),
337.30 (100%). Anal. Calcd for C22BrClFH18NO3 (478.74): C, 55.19; H, 3.79; N, 2.93. Found: 55.34; H,
3.81; N, 2.97.
4.2. Cytotoxic activity
The newly synthesized compounds were evaluated on the in-vitro growth inhibition of two solid human
tumor cell lines representing different tumor types, namely, breast adenocarcinoma (MCF-7) and liver
hepatocellular carcinoma (HepG2) using colchicine as reference drug using SRB assay. Moreover, the
toxic effect of representative target compounds was evaluated on normal breast (Hs. 371.T) and liver
(AML 12) cell lines using MTT assay.
4.2.1. Cell Cultures:
The antitumor activity was determined for the newly synthesized compounds in Egyptian National
Cancer Institute (NCI) for in-vitro detection of IC50 of their antitumor activity. The breast tumor cell line
0
(MCF-7) and hepatic tumor cell line HepG2 were obtained frozen in liquid nitrogen (-180 C) from the
American Type Culture Collection (ATCC) and was maintained in the National Cancer Institute, Cairo,
Egypt, by serial sub-culturing. All chemicals used in this study are of high analytical grade. They were
obtained from (either Sigma-Aldrich or Bio-Rad).
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The cytotoxicity of the test compounds was determined using SRB assay applying the method of Skehan
et al [27]. These testing procedures were carried out at pharmacology lab at Cancer Biology Unit in
Egyptian National Cancer Institute. The Cells were plated in 96-multiwell plate (104 cells/ well) for 24h
before treatment with the compounds to allow the attachment of cells to the wall of the plate. Different
concentrations of each compound (0, 1, 2.5, 5 and 10 µg/mL) were added to the cell monolayer triplicate
wells were prepared for each individual dose. Monolayer cells were incubated with the compounds for
48h at 37oC and in atmosphere of 5% CO2. After 48h, cells were fixed, washed, and stained with
sulforhodamine B stain. Excess stain was washed with acetic acid and attached stain was recovered with
tris EDTA buffer. Color intensity was measured in an ELISA reader. The relation between surviving
fraction and drug concentration was plotted to get the survival curve of each tumor cell line, The IC50
value was calculated using sigmoidal dose response curve-fitting models (Graph Pad, Prizm software
incorporated), each concentration was repeated 3 times. (Table 1)
4.3. Tubulin enzyme assay
MCF-7 and HepG2 cell lines were obtained from American Type Culture Collection, they were cultured
using DMEM (Invitrogen/Life Technologies) supplemented with 10% FBS (Hyclone), 10 µg/mL of
insulin (Sigma), and 1% penicillin-streptomycin. Plate cells (cells density 1.2 – 1.8 × 10,000 cells/well)
in a volume of 100µl complete growth medium and 100 µl of the tested compound per well in a 96-well
plate for 18–24 hours before the enzyme assay for Tubulin . The microtiter plate provided in this kit has
been pre-coated with an antibody specific to TUBb. Standards or samples are then added to the
appropriate microtiter plate wells with a biotin-conjugated antibody specific to TUBb. Next, Avidin
conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After
TMB substrate solution is added, only those wells that contain TUBb, biotin-conjugated antibody and
enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated
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by the addition of sulphuric acid solution and the color change is measured spectrophotometrically at a
wavelength of 450nm ± 10nm. The concentration of TUBb in the samples is then determined by
comparing the O.D. of the samples to the standard curve. [28]
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24

ACCEPTED MANUSCRIPT
Figure 1: Tubulin-targeting agents
Figure 2: General structural formulae of the target compounds.
Figure 3: Active compounds against MCF-7 cell line
Figure 4: Active compounds against Hep G2 cell line
Table 1 Results of in vitro cytotoxic activity of test compounds and colchicine against both MCF-7 and
HepG2 cell lines
Table 2. Percentage inhibition of tubulin-b polymerization of selected target compounds
Table 3 Effect of colchicine and representative target compounds on normal breast cells and normal
hepatic cells
Scheme 1 Synthesis of compounds 2a-g, 3a-d, 4a-f, 5a-b and 6
25

ACCEPTED MANUSCRIPT
Table 1 Results of in vitro cytotoxic activity of test compounds and
colchicine against both MCF-7 and HepG2 cell lines
Cpd. No.
R
R1
R2
MCF-7
(µM)
HepG2
(µM)
C
olchicine
0.1504
0.1500
0.1350
0.1260
0.1239
0.0540
.03400
0.0300
3-OCH3
3-OCH3
4-OCH3
3,4,5-
(OCH3)3
3,4,5-
(OCH3)3
3,4,5-
(OCH3)3
3,4,5-
Cl
Br
Br
Cl
-
-
-
-
2a
2b
2c
0.1200
0.0770
0.1250
0.1030
0.0110
0.0095
0.0619
0.0900
2d
2e
2f
Br
F
-
-
-
OCH3
2g
(OCH3)3
3-OCH3
3-OCH3
4-OCH3
3-OCH3
3-OCH3
4-OCH3
4-OCH3
3-OCH3
3-OCH3
OCH3
Cl
Br
F
Cl
Br
F
OCH3
Cl
Br
Cl
Br
-
-
-
0.0260
0.0130
0.0140
0.0097
0.0110
0.0160
0.0190
0.0490
0.0490
0.0150
0.0230
0.0099
0.0150
0.0300
0.0170
0.0110
0.0099
0.0130
0.0102
0.0353
0.0430
0.0130
0.0110
0.0372
3a
3b
3c
4a
4b
4c
4d
4e
4f
-
C6H5
C6H5
C6H5
C6H5
H
H
-
-
-
5a
5b
6
OCH3
-
1

ACCEPTED MANUSCRIPT
Table 2 Percentage inhibition of tubulin-b polymerization of selected target compounds
Cpd. No.
MCF-7 (µM)
Tubulin %
inhibition
86.2803
92.62997
88.38521
92.29742
87.84939
89.32233
92.16634
Cpd. No.
HepG2
(µM)
Tubulin %
inhibition
82.52266
90.58723
91.43021
92.36795
92.77821
93.02209
84.57606
88.63453
Colchicine
0.1504
0.0130
0.0140
0.0097
0.0110
0.0150
0.0099
Colchicine
0.1239
0.0540
0.0300
0.0110
0.0095
0.0110
0.0099
0.0130
3b
3c
4a
4b
5a
6
2a
2c
2d
2e
4a
4b
5a
0.0110
0.0372
85.71133
88.4942
5b
6
2

ACCEPTED MANUSCRIPT
Table 3 Effect of colchicine and representative target compounds on normal
breast cells and normal hepatic cells
Cpd. No.
MCF-7
(µM)
Hs 371.T
(Breast N
cells) (µM)
0.4434
0.0867
0.4743
Cpd. No.
HepG2
(µM)
AML 12
(Liver N cells)
(µM)
Colchicine
0.1504
0.0097
0.011
Colchicine
0.1239
0.011
0.0095
0.011
0.1381
0.0885
0.0594
0.0741
4a
4b
6
2d
2e
4a
4b
0.0099
0.0902
0.0099
0.0269
3