Design, synthesis and screening of 4,6-diaryl pyridine and pyrimidine derivatives as potential cytotoxic molecules

Article abstract of DOI:10.1248/cpb.c18-00269

A new series of pyridine and pyrimidine derivatives is designed and synthesized as potential antitumor molecules. The tested compounds show promising in vitro cytotoxic activity against HL-60 cell line as eight compounds: 4, 6, 11, 13, 14, 15, 18 and 21 exhibit potent cytotoxic activity in sub-micromolar concentration higher than the combretastatin A4 (CA-4). Compound 21 shows a cytotoxic activity 5-fold more potent than CA-4 on HL-60 cells. DNA-Flow cytometry cell cycle analysis and annexin-V assay on HL-60 cells show that compounds 4, 18 and 21 exhibit potent cell growth inhibition, cell cycle arrest at G₂/M phase and proapoptotic inducing activities. The percentage inhibition assay of β-tubulin polymerization on HL-60 cells shows that the antitumor activity of the tested compounds appears to correlate well with its ability to inhibit β-tubulin polymerization. In addition, enzyme-linked immunosorbene assay (ELlSA) measurement for compound 21 shows apoptotic inducing activities through significant up regulation of p53, Bax/Bcl-2 ratio and caspase-3 proteins parallel to down regulation of the level of survivin proteins.

Full text of DOI:10.1248/cpb.c18-00269

Chemical and Pharmaceutical Bulletin Advance Publication by J-STAGE
DOI:10.1248/cpb.c18-00269
Advance Publication
August 14, 2018
Chem Pharm Bull.
Regular Article
Design, Synthesis and Screening of 4, 6-Diaryl Pyridine and Pyrimidine
Derivatives As Potential Cytotoxic Molecules
Mohammed K. Abd elhameida*, Noha Ryadb, Al-Shorbagy MYc,d, Manal R. mohammede,
Mohammed M. Ismaila,b, Salwa El Meligiea
aPharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Cairo University, Cairo11562,
Egypt ,bPharmaceutical Organic Chemistry Department, College of Pharmaceutical Sciences and
Drug Manufacturing , Misr University for Science and Technology, Giza, Egypt, cPharmacology and
Toxicology Department, Faculty of Pharmacy, Cairo University, Cairo, Egypt, dSchool of pharmacy,
Newgiza University, Egypt and eDepartment of Radiation Biology, National Center for Radiation
Research and Technology, Cairo, Egypt.
(present address: 33 Kasr El-Aini Street, Cairo, Egypt.)
Correspondence e-mail: mohorganic@hotmail.com
Ⓒ 2018 The Pharmaceutical Society of Japan

Abstract:
A new series of pyridine and pyrimidine derivatives is designed and synthesized as potential
antitumor molecules. The tested compounds show promising in vitro cytotoxic activity
against HL-60 cell line as eight compounds: 4, 6, 11, 13, 14, 15, 18 and 21 exhibit potent
cytotoxic activity in sub-micromolar concentration higher than the combretastatin A4 (CA-4).
Compound 21 shows a cytotoxic activity 5- fold more potent than CA-4 on HL-60 cells.
DNA- Flow cytometry cell cycle analysis and annexin-V assay on HL-60 cells show that
compounds 4, 18 and 21 exhibit potent cell growth inhibition, cell cycle arrest at G2/M phase
and pro-apoptotic inducing activities. The percentage inhibition assay of β-tubulin
polymerization on HL-60 cells shows that the antitumor activity of the tested compounds
appears to correlate well with its ability to inhibit β-tubulin polymerization. In addition,
ELlSA measurement for compound 21 shows apoptotic inducing activities through
significant up regulation of p53, Bax/Bcl-2 ratio and caspase-3 proteins parallel to down
regulation of the level of survivin proteins.
Keywords: pyridine, pyrimidine, leukemia, survivin, tubulin, combretastatin A4
Chemical and Pharmaceutical Bulletin Advance Publication

1. Introduction:
Cancer is a major health dilemma all over the world.1) Leukemia is a type of cancer that
affects the blood and bone marrow and considered as one of the most life threatening agent to
human.2) In the last decade, target based chemotherapy drugs are developed for treatment of
cancer to overcome the drawbacks of conventional chemotherapy.3) Microtubules synthesis is
considered one of the main target for the natural lead cytotoxic compounds as colchicine and
etoposide (Fig. 1). These drugs act by inhibiting tubulin polymerization into microtubules.
They act as catalytic inhibitors on the binding sites of tubulin leading to inhibition of
microtubules assembly and prevent the formation of the mitotic spindle in the mitosis and
consequently, mitotic cell death (MCD).4-6) The classical antimitotic drugs are highly
successful in the control of cancer cells growth, especially tumor cells with high mitotic
index.7) The different binding sites of antimitotic drugs are known as colchicine and vinca
alkaloids binding sites.8,9) Resistance of cancer cells to apoptosis induction by antimitotic
drugs leads to increase in the used dose of the drugs which also, increase side effects and
drawbacks of these drugs.10,11)
Combretastatin-A4 (CA-4) (Fig. 1) is one of the naturally potent newer MCD targeting drugs.
The main clinical drawback of CA-4 that limit its use is cis-trans isomerization.12) Several
reported studies published to solve the structural problem based on the synthesis of rigid
isosteric analogues containing pyridine and pyrimidine rigid core rings I- V, as structural
13-15)
modification instead of ethylene bridge (Fig. 1).
Our research group is interested in
synthesis of different rigid analogues of CA-4.15-18) The reported 2, 6-diarylpyridines I-III
and 4, 6-diaryl pyrimidines IV and V show a remarkable anti-proliferative activity against
different cancer cell lines, such as breast cancer MCF-7 cells and colon cancer HCT-116 cells.
The promising cytotoxicity is explained by keeping the common pharmacophoric structural
as colchicine and CA-4.19) The structural basis for the cytotoxic action of these antimitotic
compounds I-V are a triarylated ring system represented by two aryl rings (A) and (C)
decorated with methoxy/hydroxyl groups similar to the aryl rings of CA-4 and colchicine
linked via rigid core rings (B). Furthermore, survivin protein is a member of inhibitors of
apoptosis proteins (IAPs), it has a dual biological actions in the resistance of cancer cells to
antitumor agents by suppressing induction of cancer cells to apoptosis and promoting cell
growth as mitotic facilitator.20,21) Survivin is highly expressed in leukemia and almost all
types of cancer cells.22-24) Interestingly, by literature searching for chemical scaffolds acting
as survivin inhibitors, it is found that 3-cyano-4,6-diaryl-2-pyridone derivatives VI and VII
Chemical and Pharmaceutical Bulletin Advance Publication

(Fig. 1) possess anticancer activity due to their ability to act as survivin inhibitors.25)
Undoubtedly, the need to bridge the gap between minimizing side effects and the mode of
action of antimitotic drugs encourage us for further investigations towards the synthesis of
novel potent cytotoxic compound.26, 27)
Fig. 1: Chemical structures of some natural mitosis inhibitors (colchicine and etoposide), CA-
4, diaryl pyridine I-III, VI, VII and diaryl pyrimidine derivatives IV, V as antimitotic agents
and survivin inhibitors. First aryl rings A, Core rings B represented by pyridine and
pyrimidine moieties, the third aryl rings C, and pharmacophoric groups responsible for
survivin inhibition.
Structural analysis for both mentioned compounds, as antimitotic I-V and survivin inhibitors
VI and VII, shows, regardless the positions of coupled two aryl groups (A) and (C) either at
C-2, C-6 or at C-2, C-4 of the core rings (B), the structural skeleton as non-fused triarylated
ring system is similar, as both pharmacophores are composed of pyridine or pyrimidine core
Chemical and Pharmaceutical Bulletin Advance Publication

rings and two other aryl rings directly connected without spacer (Fig. 2). Based on
aforementioned findings, three pharmacophoric models A, B and C are generated from
common template formed from tricyclic ring system (Fig. 2). The required three essential
pharmacophoric features of colchicine site inhibitors (CSI) are present in the main structural
template.28-30) The structural variation between the three models is the change in the core
rings either as -2-oxopyridine and 2-thioxopyridine, as in model A or 3-pyrimidinthione, as in
model B. In addition, a second variation is present in the structural extension of the model A
with hydrophobic hydrogen bond acceptor-donner pair fragments, as in model C. The model
A (Fig. 2) is designed as the prototype structure with pyridine core ring representing a
common part in the reported survivin and β-tubulin polymerization inhibitors. Furthermore,
in model A, the pyridine ring is reinforced with ortho-oxo or thioxo-cyano functional groups
present in survivin inhibitors. Both aryl arms of the core ring are decorated with 4-
methoxyphenyl, hydroxy methoxyphenyl and 3, 4, 5-TMP groups similar to the ones present
in the CA-4, and colchicine. Moreover, isosteric replacement of the pyridine ring core by
pyrimidine ring as in the reported cytotoxic compounds IV and V that show promising
antitumor activities lead to generation of pharmacophoric model B (Fig. 2). In recent reports,
the docking study results of colchicine binding site inhibitors (CSI) show that there are 7-
pharmacophoric structural features for the binding between the receptor site and CSI drugs
and to the best of our knowledge, none of the present CSI compounds have all of the 7-
pharmacophoric features.28-30) This finding motivate us to design a third model C. The
pharmacophoric model C is designed by grafting the structure of model A with N-aryl
acetamide groups at C-2 of pyridine ring, as hydrophobic and hydrogen bond acceptor-
donner pair groups, to increase pharmacophoric binding features towards binding site (Fig. 2).
The therapeutic target of this rational design is to increase cytotoxic and to enhance pro-
apoptotic activities of the novel compounds towards tumor cells. This tactic is the most
efficacious therapeutic strategy to increase cytotoxic properties of the chemotherapeutic
agents and decrease their side effects.31)
Chemical and Pharmaceutical Bulletin Advance Publication

Fig. 2: Diagrammatic sketch for design strategy of the elucidated lead template and the
generated three hybrid structural model A, model B, model C, where triaryl ring system
represent as core ring (ring B), aryl ring present in C-6 of the core (ring A) and C-4 aryl (ring
C). N-Aryl acetamide groups as hydrophobic hydrogen bond donner acceptor moieties that
increased binding affinity.
2. Results and Discussion:
2.1. Chemistry:
Sustainable development in organic chemistry is a growing target for all chemists in order to
improve synthetic protocols for building up novel synthetic compound libraries. Microwave-
assisted organic synthesis (MWAOS) is a branch of green chemistry which used in
sustainable synthesis and has many advantages over conventional heating methods, such as
being pollution free and eco-friendly.32) Recently, two synthetic microwave-assisted synthesis
methodologies are established, open vessel microwave-assisted organic synthesis
(OVMAOS), and closed vessel microwave-assisted organic synthesis (CVMAOS). The
Chemical and Pharmaceutical Bulletin Advance Publication

former has the advantages of being safe and could be used in a large scale, which is suitable
for industrial chemistry.33) (OVMAOS) methodology is used herein in the synthesis of final
compounds 4-23. The synthetic steps included in the preparation of starting materials,
intermediates and the target compounds 4-23 are illustrated in (Charts 1 and 2). The required
starting materials 1-3 are prepared by condensation of aromatic aldehydes with substituted
acetophenones under basic conditions (ethanolic sodium hydroxide solution), as reported.34,
35)
Chemical and Pharmaceutical Bulletin Advance Publication

Chart 1: Synthesis of 3-cyano-4, 6-diaryl -2-pyridinthione derivatives 4-10, 3-cyano- 4,6-
diaryl 2-pyridinone derivatives11-16 and 4,6-diaryl-3-pyrimidinthione derivatives 17-19.
Literature reports show that pyridine derivatives could be prepared using two synthetic
pathways either by cyclization in one pot reaction via reaction of respective aldehydes,
ketones, active methylene derivatives (ethylcyanoacetate or cyanoacetamide) and excess
ammonium acetate, or, in two steps, via heating aldehydes and ketones to form chalcone
derivatives then reacting the prepared chalcones with active methylenes in basic medium.36-38)
The 2-pyridinthiones 4-10 and isosteric 2-pyridinones 11-16 are prepared herein by one pot
four component-reaction, by reacting equimolar amount of appropriate aromatic aldehydes,
substituted acetophenones, cyanothioacetamide or ethyl cyanoacetate and excess of
ammonium acetate (Chart 1). The reaction mixture is irradiated in high boiling solvent, such
as n-butanol. The cyclization of the reacting materials into corresponding 3-cyano-2-
pyridinthione derivatives 4-10 is confirmed by its 1H-NMR spectrum that displayed a singlet
at δ 6.88 ppm for the CH proton of pyridine, while its 13C-NMR spectrum also revealed
signals at δ 117.6 and 161.8 ppm for nitrile and thiocarbonyl groups, respectively. The
isomeric cyano-2-pyridinones 11-16 are prepared at low temperature and short reaction time
than corresponding cyano-2-pyridinhiones. Its 1H-NMR spectrum displays a singlet at δ 6.97
ppm for the CH proton of pyridine ring and a broad D2O exchangeable singlet at δ 12.69 ppm
for the NH proton. Compounds 13 and 15 are reported39) under conventional heating in low
yields compared with that of microwave-assisted synthesis method. Furthermore, the reported
data characterized compounds 13 and 15 only without any biological screening.
Condensation of chalcones with thiourea under basic conditions, to afford the corresponding
3-pyrimidinthione is reported.15,40-42) In the present work, chalcone derivatives 1-3, when
heated with thiourea in presence of sodium ethoxide as basic catalyst for 5 minutes, under
microwave- assisted conditions, give the corresponding 3,4-dihydropyrimidine-2-(1H)
thiones. Proton spectra of compounds 17 and 18 show two doublets in the regions 5.04, 5.41
ppm correlated to the neighboring methine and methylene protons of pyrimidine.
Condensation of chalcone derivative 3 with thiourea require a longer reaction time (15 min)
to obtain the pyrimidine derivative 19. The structure of the obtained product is 2-
pyrimidinhione and not 3, 4-dihydropyrimidine-2- (1H) thiones (Chart 1). The structure of
the pyrimidine derivative 19 is confirmed by1H-NMR spectra that display a singlet peak of
Chemical and Pharmaceutical Bulletin Advance Publication

methylene protons of pyrimidine at 7.74 ppm and devoid the two doublets correlated to the
neighboring methine and methylene protons of pyrimidine. Compound 17 is previously
prepared under conventional heating with the same physical and spectral data.15) several
synthetic trails are performed to prepare 3-pyrimidinone derivatives, using urea, however,
unfortunately, all cyclization trails are unsuccessful. Alkylation of the 3-cyano-2-
pyridinones11 and 12 with different N-aryl acetamide derivatives using ethanol as a solvent
and in the presence of anhydrous K2CO3 results in synthesis of ether derivatives 20-23 (Chart
1
2). The Structure of the products is elucidated by H-NMR spectra that showed singlet peak
of 2H of methylene protons of the side chain at 5.19 ppm, and by 13C-NMR that shows peak
at 66.02 ppm, revealed to methylene carbon of the chain. Furthermore, 2D analysis for
compound 23 (HMBC) is carried out and shows a key correlation of the proton signal at δ
5.17 (CH2) to 13C signals at δ 163 (C-2 of 3-cyanopyridine ring) and absence of correlation to
13C signals at δ 157 (C-6 of cyano pyridine ring), indicating that alkylation is performed with
oxygen and not nitrogen atoms. Similar synthetic procedures are performed to alkylate 3-
cyano-2-pyridinthione derivatives 4-10, however, these experimental trails failed to prepare
thioether derivatives.
R4
R3
R5
d
CN
O
11,12
H
R
N
N
O
R1
R6
R2
20-23
Reagents and conditions: d) N-aryl chloroacetamides, anhyrous potasium carbonate,
ethanol,100 0C M.W, 15 min.
22-R , R1, R2 , R3, R4, R5 = OCH3 , R6 =H
23-R , R1, R2, R3, R4 , R5, R6 = OCH3
20-R, R1, R2, R4= OCH3 , R3, R5 , R6 =H
21-R, R1, R2, R4, R6= OCH3 , R3, R5 =H
Chart 2: Synthesis of 2- (3-cyano-4, 6-diarylpyridin-2-yl) oxy)-N-aryl acetamides 20-23
Chemical and Pharmaceutical Bulletin Advance Publication

2.2. Biological results and discussion:
2.2.1. In vitro cytotoxic activity of compounds 4-23 over HL-60, MCF-7 and HCT-116
cancer cell lines:
The cytotoxic activity of the synthesized compounds 4-23 is evaluated against three human
tumor cancer cell lines, leukemia HL- 60 cells, breast cancer MCF-7cells, and colon cancer
HCT-116 cells, using MTT assay, as described.43) The tested cancer cell lines are chosen for
the following reasons :HL-60 leukemic cells are an excellent in vitro models for apoptosis
studying assay.44,45) Further, the reported lead structures IV, V showed a good cytotoxic
activity against MCF-7 lines.15) CA-4 is included in the experiment as potent antimitotic
standard drug for the study against HL-60, MCF-7 and HCT-116 cancer cells (IC50 (0.48µM,
0.04 µM and 1 µM, respectively). The results of the mean values of four experiments
performed, expressed as half maximal inhibitory concentration (IC50) values, are summarized
in (Tables 1-3). Analysis of cytotoxicity results against three cancer cell lines shows the
following experimental observations. Cytotoxicity results over HL-60 cells, compounds 4 and
21 with IC50 (0.19 µM and 0.09 µM) are more potent by 2- and 5 –fold higher than CA-4 (0.
48µM), respectively. Six pyridine derivatives, 4, 6, 11, 13, 14 and 15, pyrimidinthione
derivative 18 and ether derivative 21 (IC50 values ranging from 0.19 µM to 0.47 µM) are
more potent than control drug, meanwhile the seven compounds 5, 12, 17, 19, 20, 22 and 23
show moderate activity (IC50 values ranging from 1.42 µM to 9.24 µM). Furthermore,
compounds 8, 9 and 16 show weak activity (IC50 values ranging from 23 µM to 110 µM). In
respect to MCF-7 cells, Ether derivative 21 is equipotent compound to CA-4 (IC50: 0.06 µM).
The following compounds, pyridinthione derivatives 4, 5, 7, 9, 10, pyridinone derivatives 11,
12, 14, 15, pyrimidinthione derivative 18, and ether derivative 21 show high cytotoxic
activity in sub-micromolar concentrations. Regarding HCT cell line, pyidinethione 10 is the
most cytotoxic compound (IC50: 0.41µM) and is 2- fold higher than CA-4. Pyridinthione
derivatives 6 and 10, pyridinone derivatives 11, 13 and 16, pyrimidinthione derivatives 18
and 19 and ether derivatives 20 and 21 with sub-micromolar IC50 concentration values, show
higher activity than control drug (1 µM). According to the structural modifications of the
designed compounds 4-23, a structural activity relationship (SAR) analysis is performed for
the three models A, B and C over all three cell lines, as follows.
Chemical and Pharmaceutical Bulletin Advance Publication

Table 1: In vitro cytotoxic activity (IC50 µM) of the compounds 4-16 over HL-60, MCF-7
and HCT-116 cancer cell lines.
R4
R3
R5
C
CN
X
B
R
N
H
A
R1
R2
4-16
R
R1
R2
R3
R4
R5
X
(IC50 µM)a
MCF-7c
Comp
No
HL-60b
0.19±0.02
9.21±0.81
0.21±0.03
0.50±0.04
29.61±3.1
23.2±2.1
HCT-116d
3.97±0.38
65.6±6.3
OCH3 OCH3 OCH3
H
OCH3
H
S
S
0.13±0.012
0.30±0.02
2.19±0.17
0.21±0.02
4
5
OCH3 OCH3 OCH3 OCH3 OCH3 OCH3
OCH3 OCH3 OCH3 OH OCH3
OCH3 OCH3 OCH3
OH OCH3
OCH3 OCH3 OCH3
H
S
0.48±0.05
5.91±0.6
6
OCH3
OCH3
OH
H
H
H
H
S
7
H
H
H
H
H
S
19.0±1.8
6.71±0.56
8
S
0.31±0.04 512.14±48.6
9
OH
H
H
OH
OCH3
H
S
3.44±0.41
0.25±0.02
2.66±0.27
0.28±0.03
0.47±0.04
0.41±0.03
0.15±0.03
0.36±0.03
0.39±0.04
15.08±1.8
0.18±0.02
0.19±0.04
0.41±0.02
0.38±0.04
2.31±0.25
0.51±0.05
2.55±0.26
5.13±0.54
0.51±0.06
10
11
12
13
14
15
16
OCH3 OCH3 OCH3
OCH3
O
O
O
O
O
O
OCH3 OCH3 OCH3 OCH3 OCH3 OCH3
OCH3 OCH3 OCH3 OH OCH3
OCH3 OCH3 OCH3
OH OCH3
OCH3 OCH3 OCH3
H
OCH3
OCH3
OH
H
H
H
H
H
H
H
110±12.6 218.2±22.2
a The values given are mean of four experiments ± SEM., HL-60b, leukemia carcinoma, MCF-7c, Human
breast, HCT-116d, human colon cancer cell lines.
The structural variations present among pyridine derivatives 4-16 (model A) are in the core
rings, either, pyridinthione or pyridinone, and the different decorations of C-4 and C-6 diaryl
groups of core ring. Therefore, model A would be further subdivided into 3 sub-models,
according to three different kinds of decoration of aryl groups present at C-6 of the pyridine.
Chemical and Pharmaceutical Bulletin Advance Publication

The first sub-model A1 includes six compounds 4-6 and 11-13 and contain the same 3, 4, 5-
TMP moiety as C-6 aryl group, a common trait in colchicine and CA-4. Two variations are
included in submodel A1, sulphur or oxygen, at C-2 of the core and different aryl rings at C-4
of pyridine. Pyridinthione 4 with 4-methoxyphenyl group as C-4 arylation is the most
cytotoxic agent within A1 group and is more potent than CA-4 over HL-60 and MCF-7 cell
lines. Furthermore, compound 11 that contains 4-methoxyphenyl group as a C-4 aryl of
pyridinone is the most cytotoxic agent and is more potent than standard against HCT-116 cell
line, indicating that 4-methoxyphenyl and TMP as a coupled diaryl groups on the core give
the best activity in this submodel A1. Concerning the presence of oxygen and sulphur,
pyridinthiones 4 and 6 have higher cytotoxic activity than pyridinones 11 and 13 against HL-
L60 and MCF-7 cell lines. On the other hand, pyridinones 11 and 12 are more cytotoxic than
pyridinthiones 4 and 5 against HCT-116 cell line (Table 1). The second sub-model A2 group
includes four pyridine derivatives 7, 8, 14 and 15 contain 4-methoxyphenyl moiety as C-6
aryl group in pyridine. The results show that: pyridinone derivative 14 with 3, 4, 5-TMP
group as C-4 arylation with IC50 (0.18 and 2.55µM) is the most cytotoxic agent against MCF-
7 and HCT-116 cell lines respectively. Pyridinones 14 and 15 are more cytotoxic than
pyridinthiones 7 and 8 against three cell lines, indicating that oxygen is more contributing to
cytotoxicity than sulphur in this submodel A2 (Table1). The third sub-model A3 included
three pyridine derivatives 9, 10 and 16 that have the same 4-hydroxyphenyl moiety at C-6 of
the pyridine. Pyridinthione 10 that contains 4-hydroxy-3-methoxyphenyl group as C-4
arylation is the most cytotoxic agent of this group against MCF-7, HL-60 and HCT-116 cell
lines. Meanwhile, pyridinthione 9 that contains sulphur is more cytotoxic than 2-pyridinone
16 against three cell lines (Table1). In the light of structural analysis of model, A results, it
appeared that the cytotoxic activity of pyridine derivatives 4-16 correlated to the general
skeleton of the decorated diaryl groups, as the presence of TMP and 4-methoxy groups
together, either at C-4 or C-6 of pyridine generated the best cytotoxic activity as in
compounds 4, 11 and 14 (Table1).
Chemical and Pharmaceutical Bulletin Advance Publication

Table 2: In vitro cytotoxic activity (IC50 µM) of the compounds 17-19 over HL-60, MCF-7
and HCT-116 cancer cell lines.
R4
R3
R5
C
N
B
R
N
H
S
A
R1
R2
17-19
Comp
No
R
R1
R2
R3
R4
R5
(IC50 µM)a
MCF-7c
HL-60b
HCT-
116d
H
OCH3
H
OCH3 OCH3 OCH3
OH OCH3
OCH3 OCH3 OCH3 OCH3 OCH3 OCH3
17
4.6±0.39
1.46±0.15 1.65±0.093
OCH3 OCH3 OCH3
H
0.36±0.08 0.32±0.07 0.71±0.07
7.9±0.8 2.14±0.22 0.56±0.051
18
19
a The values given are mean of four experiments ± SEM., HL-60b, leukemia carcinoma, MCF-7c, Human
breast, HCT-116d, human colon cancer cell lines.
In respect to model B, pyrimidinthiones 17 and 18 are more cytotoxic than corresponding
pyridinthiones 5 and 7 against both HCT-116 and HL-60 cell lines, while pyridinthiones are
more cytotoxic than pyrimidinthiones to MCF-7 cells. Compound 18 with IC50: 0.32µM and
0.36 µM over MCF-7 and HL-60 cell lines, respectively is the most potent cytotoxic
derivative of the pyrimidines 17-19 and the pyridine analogue 6. The good cytotoxicity
profile of compound 18 might have structural similarities with the aryl groups of CA-4 (Table
2). Regarding model C, ether derivatives 20 and 21 exhibit a good cytotoxicity higher than
compounds 22 and 23, against HCT-116 and HL60 cells. Also, the obtained data shows that
structural extension of pyridinone derivatives 11 and 12 by ether formation result in
decreasing the potency of cytotoxic activity, as the ether derivatives 20, 22 and 23 are less
cytotoxic than parent pyridine derivatives 11 and 12, except in compound 21 (Table 3). Ether
derivatives 22 and 23 containing TMP group, as coupled C-4 and C-6 diaryl groups, are less
cytotoxic than other derivatives. In terms of the effect of N-aryl groups on cytotoxicity results,
Chemical and Pharmaceutical Bulletin Advance Publication

N-phenyl acetamide derivative 20 is less cytotoxic than N-4-methoxyphenyl acetamide
derivative 21 (Table 3). A brief final conclusion from this results analysis shows that
pyridinthiones are more active than pyridinones over HL-60 and MCF-7 cells. This might be
due to difference in lipid solubility of sulphur and oxygen. Synthesized pyrimidine
derivatives are more active than the corresponding pyridine analogues over HL-60 and HCT-
116 cells. Regarding the decoration of the structure of C-4 and C-6 diaryl groups at the core
ring, 4-methoxyphenyl and TMP groups are the most active cytotoxic coupled diaryl groups
at C-4 and C-6 of the core. Finally, extension of the structure to increase cytotoxicity is
controlled by decoration of the C-4 and C-6 diaryl rings at the core part by either by one or
two TMP groups, as in compounds 21 and 23.
Table 3: In vitro cytotoxic activity (IC50 µM) of the compounds 20-23 over HL-60, MCF-7
and HCT-116 cancer cell lines.
Comp
No
R
R1
R2
R3
R4
R5
R6
(IC50 µM)a
MCF-7c
HL-60b
2.58±0.26
0.09±0.01
1.42±0.13
9.24±0.89
HCT-116d
OCH3 OCH3 OCH
OCH3 OCH3 OCH3
OCH3
OCH3
20
21
22
23
H
H
H
H
H
2.69±0.3
0.70±0.06
OCH3
0.06±0.007 0.54±0.06
OCH3 OCH3 OCH3 OCH3 OCH3 OCH3
H
3.8±0.4
3.73±0.4
52.4±5.3
OCH3 OCH3 OCH3 OCH3 OCH3 OCH3 OCH3
1.79±0.18
a The values given are mean of four experiments ± SEM., HL-60b, leukemia carcinoma, MCF-7c, Human
breast, HCT-116d, human colon cancer cell lines.
Chemical and Pharmaceutical Bulletin Advance Publication

2.2.2. In vitro DNA- flow cytometry cell cycle analysis:
Three compounds: pyridinthione derivative 4, pyrimidinthione 18 and ether derivative 21
over HL-60 cell line (0.19 µM, 0.36 µM and 0.09 µM, respectively) are selected as potent
cytotoxic agents, which represent the three pharmacophoric models A, B and C to be further
studied for their effects on cell cycle progression and induction of apoptosis. The obtained
results after treatment of HL-60 cancer cells with compounds 4, 18 and 21 are shown in (Fig.
3A). The results of cell cycle analysis show that a significant change in cell cycle distribution
is observed compared with untreated HL-60 cells and CA-4.
Pyridine derivative 4 causes a cell cycle at G1 phase by increasing G1 phase by 3- fold and
decreasing S phase by 2- fold than CA-4. Furthermore, pyrimidine derivative 18 increases the
accumulation of cells at G0, G1 and G2/M phase by 1- fold and decreases and S phase by 2-
fold compared to CA-4 (Fig. 3C2). In addition, compound 21 increases the G0 phase and
decreases G1 and S phase about 2- fold compared to CA-4 and increases the accumulation of
cells at G2/M phase by 2 -fold than CA-4, indicating that of the tested compounds, cause cell
cycle block at G1 and G2/M phase and hence cell death of HL-60 cells.
2.2.3. Apoptosis analysis by Annexin V/PI staining:
To ensure the ability of compounds 4, 18 and 21 to induce apoptosis, flow cytometry analysis
is performed using annexin-V and propidium iodide (PI) staining.46) The results of
experiment performed are summarized and presented graphically in (Fig. 3B). After
treatment with compounds 4, 18 and 21 at their IC50 concentration, a decrease in the
percentage of survived cells is observed. Moreover, a significant increase in the percentage of
annexin-V positive cells occurs in compounds 4, 18 and 21 compared untreated cells and CA-
4 indicating that these compounds induced apoptosis in HL-60cells (Fig 3B). Compound 21
give about 10-fold more potent activity as apoptotic inducer than untreated control cells
(Fig.3C2).
Chemical and Pharmaceutical Bulletin Advance Publication

Fig. 3: A) Cell cycle analysis of compounds 4, 18 and 21 on HL-60 cancer cells compared
with CA-4 (0.48 µM) as control drug at their IC50 (0.19 µM, 0.36 µM and 0.09 µM,
respectively) for 24h. B) Annexin V/PI staining analysis for apoptosis percentage of
compounds 4, 18, 21, and CA-4 on HL-60 cancer cells. C1) Statistical analysis of cell cycle
analysis. C2) Statistical analysis of Annexin V/PI staining analysis.
2.2.4. In vitro immunofluorescence localization of tubulin in HL-60 cancer cells:
To explore the cell cycle arrest at G2/M phase induced by the tested compounds 4, 18 and 21,
immunofluorescence labeling assay for tubulin localization under fluorescence microscope is
performed after treatment of HL-60 cells with each compound at its IC50 for 48 h and then
submitted to analysis. The results of assay shows that compound 21 has potent inhibitory
effect on cellular tubulin formation higher than CA-4 compared with untreated cells (Fig. 4).
Meanwhile compound 18 shows good inhibitory activity but less than compound 21, while
compound 4 shows a moderate degree of tubulin polymerization inhibitory activity as
concluded from the inhibition of the fluorescence intensity.
Chemical and Pharmaceutical Bulletin Advance Publication

Fig. 4: Fluorescence intensity (IFU) of tubulin localization in HL-60 cancer cells for the
effect of compounds 4, 18 and 21 compared with CA-4 (0.48 µM) as control drug at their
IC50 (0.19 µM, 0.36 µM and 0.09 µM, respectively) for 24h.
2.2.5. In vitro β-tubulin (TUBβ) polymerization inhibition assay:
Pyridinthione derivatives 4, 6, 11 and 13, pyrimidinthione 18, ether derivatives 21 and 23
represent the designed models A, B and C and contain TMP moiety, as C-6 aryl group of the
core ring are further investigated for inhibition percentage of β-tubulin polymerization in HL-
60 cell line at their IC50 via enzyme linked immunosorbent assay (ELISA) using human-β-
tubulin assay kit.
Chemical and Pharmaceutical Bulletin Advance Publication

Table 4: Percentage inhibition of β- tubulin polymerization on human HL-60 cell line for the
compounds 4, 6, 11, 13, 18, 21, 23 and CA-4 at their IC50 (µM).
Comp
No
% inhibition of tubulin β
Polymerization
78 ±1
HL-60a
IC50 (µM)
0.19
4
6
83±2
71±2
80±2
87±2
94±1
56±3
0.21
0.25
0.28
0.36
0.09
9.24
11
13
18
21
23
CA-4
87±1
0.48
a The values given are mean of two experiments ± SEM.HL-60, leukemia carcinoma cells.
The inhibitory activity of β-tubulin polymerization is given as the percentage inhibition at
IC50 concentration of each compound. The results of performed experiment are summarized
in (Table 4). Structural analysis of the tested compounds shows that pyrimidine derivative 18
shows higher β-tubulin inhibition percentage than corresponding pyridine derivative 13. This
experimental finding correlates the good cytotoxic activity of compound 18 to structural
similarities of diaryl groups with C4-A. Furthermore, pyridine derivatives 6 and 13 contain
sulphur and oxygen, at C-2 of the core ring, show similar inhibition percentage. Ether
derivative of pyridine 21 shows higher tubulin inhibition than similar C4-A and
corresponding pyridine derivative 11. This experimental result supports the aim of the design
of model C, as the additional group (N-aryl acetamide group) might have allowed the
structure to possess bitter affinity towards binding sites. Compound 23 shows moderate
inhibition percentage parallel to cytotoxic activity results. The results of tubulin inhibition
percentage are in agreement of IC50 cytotoxicity.
Chemical and Pharmaceutical Bulletin Advance Publication

2.2.6. In vitro ELISA immunoassay of apoptosis mediators:
Resistance of tumor cells to apoptosis induction occurred by increasing expression of anti-
apoptotic proteins, as Bcl-2 and decreasing pro-apoptotic proteins, as Bax. Both proteins are
regulated with p53.47) Compound 21 is the most potent apoptotic inducer with 12.07 % within
tested compounds as demonstrated by Anexienn V assay Further investigation of apoptosis
inducing activity of compound 21 compared with CA-4 by measuring the concentration of
the cancer biomarkers, p53, Bax, Bcl-2, caspase-3 and survivin, is performed using ELISA
immunoassay in a time-dependent manner. The obtained results are summarized and
graphically represent in (Fig. 5A-E).
Fig. 5: In vitro Elisa measurement showed the effect of compound 21 at its IC50 (0.091 µM)
for (24, 48h) on HL-60 cell line as negative control compared with CA-4 (0.48 µM) as
control drug, A) Effect on p53, B) Effect on Bax levels, C) Effect on Bcl-2, D) Effect on
caspase-3 levels, E) Effect on survivin levels, Data was expressed as mean (n=4 experiments)
± SEM and statistical comparisons were carried out using one-way analysis of variance
(ANOVA) followed by Tukey multiple comparisons test at P < 0.05.
Treatment of HL-60 cells to compound 21 in a time-dependent manner result in a significant
increasing in the level of p53 by 1- and 2-fold more than CA-4, after 24 and 48h treatments,
respectively (Fig. 5A). Consistent with this up-regulation, the level of Bax increases by 2-fold
after 24 and 48 h, compared with CA-4 (Fig. 5B). On the other hand, the level of Bcl-2
decreases by 1- and 2-fold compared with CA-4, for the 24 and 48h treatments, respectively
Chemical and Pharmaceutical Bulletin Advance Publication

(Fig. 5C). As compound 21 significantly elevates the Bax / Bcl-2 ratio, therefore, the
subsequent step is to measure the level of active caspase-3. Activation of caspase -3 is a final
step in mitochondrial induced apoptosis (intrinsic pathway), which result in DNA
fragmentation.48) The results exhibit that the level of caspse-3 increases by 1- and 2-fold, after
24 and 48h, respectively, compared with CA-4. In conclusion, compound 21 shows a
apoptosis inducing activity via mitochondrial pathway, by increasing the level of p53, Bax/
Bcl-2 ratio and caspase-3 (Fig. 5D). Survivin is an IAP that directly acts on caspase-3 and
inhibits its apoptotic actions, even after its activation.49) Compound 21 decreases the survivin
protein level by 1.5- and 2-fold after the 24 and 48h, respectively, compared with CA-4 (Fig.
5E). The obtained data demonstrate that compound 21 is more potent cytotoxic and apoptotic
inducer higher than CA-4. Also, the obtained results rationalized possible mode of cytotoxic
activity and high apoptotic inducing activity induced by compound 21 on HL-60 cells.
3. Conclusion:
The biological rationale of the work is the design and construction of a potential antimitotic
compounds endowed with pro-apoptotic inducing activity. The design approach is likely to
transcend the shortcomings of the existing antimitotic drugs where cancer cells resistance to
apoptosis induction by anti-tumor agents will decrease. In-vitro cytotoxicity results show a
substantial improvement in cytotoxic activity of the synthesized compounds against HL-60,
MCF-7 and HCT-116 cell lines, compared with CA-4. The pyridinthione 4 and ether
analogue 21 are the most cytotoxic derivatives (IC50: 0.19 µM and 0.091 µM) against HL-60
cell line, while, in general, eight of compounds: 4, 6, 11, 13, 14, 15, 18 and 21 (IC50 values
ranging from 0.19 µM to 0.47 µM) are more potent against HL 60 cell line than CA-4
(0.48µM). Regarding the cytotoxicity against MCF-7 cell line, ether derivative 21 is an
equipotent compound to CA-4 (IC50: 0.06µM). In addition, the following nine final
compounds 4, 5, 7, 9, 10, 12, 14, 15 and 18 show high cytotoxic activity in sub-micromolar
values with a similar cytotoxic potency against both HL-60 and MCF-7 cell lines. In terms of
cytotoxic activity against HCT-116 cell line, the most cytotoxic agent to HCT-116 cells is
compound 11 (IC50:0.38 µM). In addition, nine of the tested compounds 6, 10, 11, 13, 16, 18,
19, 20 and 21 (sub-micromolar IC50values ranging from (0.18 µM to 0.71 µM) show higher
cytotoxic activity than control drug (1 µM). The obtained results of cell cycle analysis and
annexin-V test on HL-60 cancer cells for the compounds 4, 18 and 21 show cell cycle arrest
at G2/M phase and a potent apoptotic activity compared with CA-4. The results of β-tubulin
polymerization inhibition percentage of pyridinthione 4, pyridinone 13, pyrimidinthione 18
Chemical and Pharmaceutical Bulletin Advance Publication

and ether 21, compared with CA-4, revealed that the tested compounds 18 and 21 possessed
excellent β-tubulin inhibition activity. Furthermore, the potent apoptotic inducing activity
exhibited by compound 21 higher than CA-4 is rationalized by increasing concentration of
p53, Bax/Bcl-2 ratio and caspase-3 proteins and decreasing survivin protein levels. Finally,
the small compounds library 4-23 represent lead structures for potential antitumor agents
against HL-60 cell line and other solid tumor tumors as MCF-7 and HCT-116 cancer cells.
4. Experimental:
4.1. Synthesis:
4. 1. 1. General
Solvents and reagents are obtained from Aldrich, Fluca or Merck and are used without further
purification unless otherwise indicated. Melting Points are determined on a Griffin apparatus
and are uncorrected. The elemental analysis is performed at The Regional Center for
Mycology and Biotechnology, Al-Azhar University, Egypt. IR spectra are recorded on a
Shimadzu 435 spectrophotometer and Mattson Genesis II FTIR, using KBr discs, Cairo
1
University, and values are represented in cm-1. H NMR spectra were performed Bruker400
MHz&Bruker300 MHz spectrophotometer using TMS as internal standard, chemical shifts
13
(δ) are recorded in ppm on δ scale. C NMR spectra are carried out using Bruker 100 MHz
using TMS as internal standard, chemical shifts (δ) are recorded in ppm on δ scale, Micro
analytical unit Center, Cairo University, Egypt. Mass spectra are run on Hewlett Packard
5988 spectrometer or shimadzu QP-2010 plus at The Regional Center for Mycology &
Biotechnology, Al-Azhar University, Egypt. Progress of the reactions is monitored by TLC
using precoated aluminum sheets silica gel (Merck 60 F 254) and is visualized by UV lamp
and iodine vapor. The solvent system used in TLC is hexane: ethyl acetate [7: 3], Mass-II
microwave Synthesis work station is used with the following specification, operating
frequency 2450 MHz and rated output power range 0-1000 W.
4.1.1.1. 1, 3-Diaryl-2-propen-1-ones Ia-c were prepared as reported.34, 35)
4.1.1.2. General procedure for the synthesis of 4, 6-diaryl-2-thioxo-1, 2-dihydropyridine-
3-carbonitriles (4-10).
A mixture of the required acetophenones (0.001 mol), the appropriate aromatic aldehydes
(0.001 mol), cyanothioacetamide (0.1 g, 0.001 mol) and ammonium acetate (0.62 g, 0.008
mol) in n-butanol (10 ml) is irradiated at 150 0C for 20 min in the microwave oven. After the
reaction mixture is cooled to room temperature, the reaction mixture is diluted with 10 ml
Chemical and Pharmaceutical Bulletin Advance Publication

diethyl ether and the separated solid is filtered, dried and crystallized from ethanol to afford
compounds 4-10.
4.1.1.2.1. 1, 2-Dihydro-6- (3, 4, 5-trimethoxyphenyl)-4- (4-methoxyphenyl)-2-
thioxopyridine-3-carbonitrile (4).
Yellow powder, yield: 0.35g (87 %), mp: 294-296°C. IR (KBr, cm-1): 3421 (NH), 3066 (CH-
1
aromatic), 2843 (CH-aliphatic), 2222 (CN), 1136 (C=S). H-NMR (400 MHz, DMSO-d6),δ
ppm: 3.73 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 3.88 (s, 6H, 2OCH3), 6.88 (s, 1H, Ar-H), 7.13
(d, 2H, J=7.5 Hz, Ar-H), 7.20 (s, 2H, Ar-H), 7.72 (d, 2H, J=7.7 Hz, Ar-H), 12.53 (s, 1H, NH,
D2O exchangeable).13C-NMR (100 MHz, DMSO-d6),δ ppm: 55.9, 56.2 (2C), 56.7, 97.7,
102.3, 105.8 (2C), 114.6 (2C), 117.4, 128.6, 129.5, 130.5 (2C), 140.4, 153.5 (2C), 155.9,
161.5, 167, 169. Anal. Calcd for: C22H20N2O4S (408.47): C, 64.69; H, 4.94; N, 6.86. Found:
C, 64.93; H, 5.07; N, 7.04.
4.1.1.2.2. 1, 2-Dihydro-4, 6-bis (3, 4, 5-trimethoxyphenyl)-2-thioxopyridine-3-
carbonitrile (5).
Yellow powder, yield: 0.41 g (87 %), mp: 190-192°C. IR (KBr, cm-1): 3408 (NH), 3045 (CH-
1
aromatic), 2837 (CH-aliphatic), 2223 (CN), 1120 (C=S). H-NMR (400 MHz, DMSO-d6),δ
ppm: 3.73 (s, 6H, 2OCH3), 3.75 (s, 6H, 2OCH3), 3.83 (s, 6H, 2OCH3), 7.35 (s, 2H, Ar-H),
7.73 (s, 1H, Ar-H), 7.84 (s, 1H, Ar-H), 8.13 (s, 1H, Ar-H), 12.68 (s, 1H, NH, D2O
exchangeable).13C-NMR (100 MHz, DMSO-d6), δ ppm: 56.5 (4C), 57.6 (2C), 95.3, 105.4
(2C), 107.7, 108.4, 115.5, 117.3, 118 (2C), 127.6, 138.3, 141.5, 149.2, 151.3, 152.9, 153.4,
158.5, 163.2. Anal. Calcd for C24H24N2O6S (468.52): C, 61.52; H, 5.16; N, 5.98. Found: C,
61.87; H, 5.22; N, 6.05.
4.1.1.2.3. 1, 2-Dihydro-4- (4-hydroxy-3-methoxyphenyl)-6- (3, 4, 5-trimethoxyphenyl)-2-
thioxopyridine-3-carbonitrile (6).
Yellow powder, yield: 0.33 g (80%), mp: 296-298 °C. IR (KBr, cm-1): 3421-3224 (NH, OH),
1
3056 (CH-aromatic), 2839 (CH-aliphatic), 2220 (CN), 1139 (C=S). H-NMR (400 MHz,
DMSO-d6), δ ppm: 3.73 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 3.88 (s, 6H, 2OCH3), 4.41 (s, 1H,
D2O exchangeable), 6.89 (s, 1H, Ar-H), 6.94 (d, 1H, J=7.3 Hz, Ar-H), 7.19 (s, 2H, Ar-H),
13
7.23 (d, 2H, J= 7.1 Hz , Ar-H+H, D2O exchangeable), 7.32 (s, 1H, Ar-H). C-NMR (100
MHz, DMSO-d6), δ ppm: 56.7, 60.1 (2C), 60.6, 100, 102.1, 105.8 (2C), 116, 117.3, 122.5,
Chemical and Pharmaceutical Bulletin Advance Publication

125.2, 133, 138.7, 141.5, 148, 149.5, 153.5 (2C), 163.2, 167.7, 169.2. Anal. Calcd for
C22H20N2O5S (424.47) : C, 62.25; H, 4.75; N, 6.60. Found: C, 62.39; H, 4.81; N, 6.53.
4.1.1.2.4. 1, 2-Dihydro-4- (3, 4, 5-trimethoxyphenyl)-6- (4-methoxyphenyl)-2-
thioxopyridine-3-carbonitrile (7).
Orange powder, yield: 0.35 g (85 %), mp: 293-295°C. IR (KBr, cm-1): 3425 (NH), 3068 (CH-
1
aromatic), 2843 (CH-aliphatic), 2197 (CN), 1127 (C=S). H-NMR (400 MHz, DMSO-d6),δ
ppm: 2.51 (s, 1H, NH, D2O exchangeable), 3.71 (s, 3H, OCH3), 3.79 (s, 3H, OCH3), 3.83 (s,
6H, 2OCH3), 6.57 (s, 1H, Ar-H), 6.87 (s, 2H, Ar-H), 6.95 (d, 2H, J=7.8 Hz, Ar-H), 7.97 (d,
2H, J=7.6 Hz, Ar-H). 13C-NMR (100 MHz, DMSO-d6), δ ppm: 56.0, 56.6 (2C), 60.6, 105.5,
106.5, 114.8 (2C), 117.4 (2C), 125.5, 130 (2C), 131.8, 139.6, 152.1, 153.3 (2C), 160.1, 162.3,
162.6, 169.7. Anal. Calcd for C22H20N2O4S (408.47): C, 64.69; H, 4.94; N, 6.86. Found: C,
64.98; H, 5.01; N, 6.98.
4.1.1.2.5. 1, 2-Dihydro-4- (4-hydroxy-3-methoxyphenyl)-6- (4-methoxyphenyl)-2-
thioxopyridine-3-carbonitrile (8).
Yellow powder, yield: 0.29 g (83%), mp: 295-297 °C. IR ( KBr, cm-1): 3471-3360 (NH, OH) ,
1
3077 (CH-aromatic), 2833 (CH-aliphatic), 2202 (CN), 1128 (C=S). H-NMR (300 MHz,
DMSO-d6), δ ppm: 3.63 (s, 6H, 2OCH3), 6.12 (d, 2H, J=7.1 Hz, Ar-H), 6.91 (brs, 2H, D2O
exchangeable), 7.17 (d, 2H, J=7.2 Hz, Ar-H), 7.44 (s, 2H, Ar-H), 7.67 (d, 2H, J=6.9 Hz, Ar-
H).13C-NMR (100 MHz, DMSO-d6), δ ppm: 56, 56.3, 98.2, 105.4 (2C), 113, 114.8, 116,
117.7, 122.2, 124.8, 127.3 , 129.9 (2C), 148, 149.5, 150.8, 160.1,162.1, 162.8. Anal. Calcd
for C20H16N2O3S (364.42): C, 65.92; H, 4.43; N, 7.69. Found: C, 66.17; H, 4.51; N, 7.94.
4.1.1.2.6. 1, 2-Dihydro-6- (4-hydroxyphenyl)-4- (3, 4, 5-trimethoxyphenyl)-2-
thioxopyridine-3-carbonitrile (9).
Yellow powder, Yield: 0.34 g (86%), mp: 260-262 °C. IR (KBr, cm-1): 3406-3231 (NH, OH),
1
3060 (CH-aromatic), 2837 (CH-aliphatic), 2223 (CN), 1170 (C=S). H NMR (400 MHz,
DMSO-d6), δ ppm: 3.77 (s, 3H, OCH3), 3.83 (s, 6H, 2OCH3), 6.78 (s, 1H, Ar-H), 6.88 (d, 2H,
J=7.6 Hz, Ar-H), 7.04 (s, 1H, Ar-H), 7.36 (s, 1H, Ar-H), 7.79 (d, 2H, J=7.4 Hz, Ar-H), 10.23
(s, 1H, OH, D2O exchangeable), 12.47 (s, 1H, NH, D2O exchangeable). 13C-NMR (100 MHz,
DMSO-d6), δ ppm: 56.5 (2C), 56.6, 105.5, 106.5, 108.4 (2C), 116.2 (2C), 117.3, 127.6 (2C),
130.1, 131.9, 139.5, 141.5, 151.3, 153.4 (2C), 160.7, 163.2. Anal. Calcd for C21H18N2O4S
(394.44): C, 63.94; H, 4.60; N, 7.10. Found: C, 64.17; H, 4.68; N, 7.26.
Chemical and Pharmaceutical Bulletin Advance Publication

4.1.1.2.7. 1, 2-Dihydro-4- (4-hydroxy-3-methoxyphenyl)-6- (4-hydroxyphenyl)-2-
thioxopyridine-3-carbonitrile (10).
Yellow powder, yield: 0.28 g (80%), mp: >300 °C. IR (KBr, cm-1): 3421-3319 (NH, OH),
1
3021 (CH-aromatic), 2835 (CH-aliphatic), 2218 (CN), 1120 (C=S). H-NMR (400 MHz,
DMSO-d6), δ ppm: 3.86 (s, 3H, OCH3), 6.71-6.72 (m, 2H, Ar-H+ OH, D2O exchangeable),
6.89-6.91 (d, 2H, J=7.4 Hz, Ar-H), 7.23 (d, 1H, J=7.7 Hz, Ar-H), 7.32 (d, 1H, J=7.6 Hz, Ar-
H), 7.77 (s, 1H, Ar-H), 7.84 (s, 1H, Ar-H), 9.88 (s, 1H, OH, D2O exchangeable), 12.03 (s, 1H,
13
NH, D2O exchangeable). C-NMR (100 MHz, DMSO-d6), δ ppm: 56.3, 96.4, 104.9 (2C),
113, 116, 116.2, 117.9, 122.2, 123.2, 127.4 (2C), 130, 148, 149.5, 151.2, 160, 160.9, 162.8.
Anal. Calcd for C19H14N2O3S (350.39): C, 65.13; H, 4.03; N, 7.99. Found: C, 65.41; H, 3.97;
N, 8.21.
4.1.1.3. General procedure for the synthesis of 4,6-diaryl -2-oxo-1,2-dihydropyridine -3-
carbonitriles (11-16).
A mixture of the appropriate acetophenone (0.001 mol), the aromatic aldehydes (0.001 mol),
ethyl cyanoacetate (0.15 g, 0.001 mol), and ammonium acetate (0.62 g, 0.008 mol) in n-
0
butanol (10 ml) is stirred for 5 min at room temperature and then irradiates at 150 C for 10
min in the microwave oven. After cooling to room temperature, the reaction mixture is
diluted with pet. Ether (20 ml). The formed solid is filtered, dried and crystallized from
ethanol to give pyridine-3-carbonitrile derivatives 11-16.
4.1.1.3.1. 1, 2-Dihydro-6- (3, 4, 5-trimethoxyphenyl)-4- (4-methoxyphenyl)-2-
oxopyridine-3-carbonitrile (11).
Yellow powder, yield: 0.33 g (86%), mp: 286-288°C. IR (KBr, cm-1): 3448 (NH), 3050 (CH-
1
aromatic), 2843 (CH-aliphatic), 2214 (CN), 1635 (C=O). H-NMR (400 MHz, DMSO-d6),δ
ppm: 3.73 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 3.88 (s, 6H, 2OCH3), 6.87 (s, 1H, ArH), 7.12
(d, 2H, J=7.6 Hz, Ar-H), 7.20 (s, 2H, Ar-H), 7.72 (d, 2H, J=7.3 Hz, Ar-H), 12.63 (s, 1H, NH,
D2O exchangeable). 13C-NM (100 MHz, DMSO-d6), δ ppm: 55.9, 56.7 (2C), 60.6, 105.8,
106, 114.6 (2C), 117.4 (2C), 127.7, 128.6, 130.5 (2C), 133.7, 140.4, 151, 153.5 (2C), 159.9,
161.5, 162.5. MS: m/z (%) 392M+ (100), 377 (M-15) (47.87), 349 (M-43) (28.27). Anal.
Calcd for C22H20N2O5 (392.4): C, 67.34; H, 5.14; N, 7.14. Found: C, 67.45; H, 5.18; N, 7.04.
4.1.1.3.2. 1, 2-Dihydro-4, 6-bis (3, 4, 5-trimethoxyphenyl)-2-oxopyridine-3-carbonitrile
(12).
Chemical and Pharmaceutical Bulletin Advance Publication

Yellow powder, yield: 0.38 g (85 %), mp: 286-288°C. IR (KBr, cm-1): 3448 (NH), 3046
1
(CH-aromatic), 2839 (CH-aliphatic), 2223 (CN), 1650 (C=O). H-NMR (400 MHz, DMSO-
d6) δ ppm: 3.73 (s, 3H, OCH3), 3.75 (s, 3H, OCH3), 3.86 (s, 6H, 4OCH3), 3.88 (s, 6H,
4OCH3), 6.97 (s, 1H, Ar-H), 7.04 (s, 2H, Ar-H), 7.20 (s, 2H, Ar-H), 12.69 (s, 1H, NH, D2O
13
exchangeable). C-NMR (100 MHz, DMSO-d6), δ ppm: 56.7 (4C), 60.6 (2C), 103.7, 105.9
(2C), 106.6 (2C), 115.3, 117.3, 125.3, 127.7, 131.8, 139.6, 140.4, 151.1, 153.3, 153.5, 157.4,
160.3, 162.4. Anal. Calcd for C24H24N2O7 (452.46): C, 63.71; H, 5.35; N, 6.19. Found: C,
63.95; H, 5.43; N, 6.42.
4.1.1.3.3. 1, 2-Dihydro-4- (4-hydroxy-3-methoxyphenyl)-6- (3, 4, 5-trimethoxyphenyl)-2-
oxopyridine-3-carbonitrile (13).
Orange powder, yield: 0.37 g (91%), mp: 291-293 °C. IR (KBr, cm-1): 3446-3226 (NH, OH),
1
3076 (CH-aromatic), 2843 (CH-aliphatic), 2218 (CN), 1651 (C=O). H-NMR (400 MHz,
DMSO-d6),δ ppm: 3.72 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 3.87 (s, 6H, 2OCH3), 6.39 (s, 1H,
D2O exchangeable), 6.87 (s, 1H, Ar-H), 6.96 (d, 1H, J=7.4 Hz, Ar-H), 7.18 (d, 1H, J=7.3 Hz,
Ar-H), 7.22 (s, 2H, J=7.4 Hz, Ar-H), 7.29 (s, 1H, Ar-H).13C-NMR ( 100 MHz, DMSO-d6),δ
ppm:56.2, 56.6 (2C), 60.6, 96.3, 105.6, 105.7, 113.1 (2C), 116, 118.4, 122.1, 127.6, 129.5,
140, 148, 149.5, 152.4, 153.4 (2C), 159.2, 164.8. Anal. Calcd for C22H20N2O6 (408.4): C,
64.70; H, 4.94; N, 6.86. Found: C, 64.96; H, 5.01; N, 7.03.
4.1.1.3.4. 1, 2-Dihydro-4- (3, 4, 5-trimethoxyphenyl)-6- (4-methoxyphenyl)-2-
oxopyridine-3-carbonitrile (14).
Yellow powder, yield: 0.32 g (85 %), mp: > 300 °C. IR (KBr, cm-1): 3443 (NH), 3032 (CH-
1
aromatic), 2841 (CH-aliphatic), 2216 (CN), 1663 (C=O). H-NMR (300 MHz, DMSO-d6), δ
ppm: 3.74 (s, 3H, OCH3), 3.84 (s, 6H, 2OCH3), 3.86 (s, 3H, OCH3), 4.31 (s, 1H, D2O
exchangeable), 6.83 (s,1H, Ar-H), 7.05 (s, 2H, Ar-H), 6.08 (d, 2H, J=7.3 Hz, Ar-H), 7.90 (d,
13
2H, J=7.4 Hz, Ar-H), 12.60 (s, 1H, NH, D2O exchangeable). C-NMR (100 MHz, DMSO-
d6), δ ppm: 56, 56.6 (2C), 60.6, 105.6, 106.5, 114.8 (2C), 117.4 (2C), 124.8, 130 (2C), 131.8,
139.6, 147.7, 151.3, 153.3 (2C), 160, 162.2, 162.6. Anal. Calcd for C22H20N2O5 (392.4): C,
67.34; H, 5.14; N, 7.14. Found: C, 67.52; H, 5.09; N, 7.28.
4.1.1.3.5. 1, 2-Dihydro-4- (4-hydroxy-3-methoxyphenyl)-6- (4-methoxyphenyl)-2-
oxopyridine-3-carbonitrile (15).
Chemical and Pharmaceutical Bulletin Advance Publication

Yellow powder, yield: 0.3 g (88 %), mp: > 300 °C. IR (KBr, cm-1): 3435-3143 (NH, OH)
1
3071 (CH-aromatic), 2838 (CH-aliphatic), 2215 (CN), 1653 (C=O). H-NMR (300 MHz,
DMSO-d6) δ ppm: 3.83 (s, 3H , OCH3), 3.85 (s, 3H , OCH3), 6.76 (s, 1H, Ar-H), 6.92 (d, 2H,
J=7.6 Hz, Ar-H), 7.05 (d, 2H, J=7.5 Hz, Ar-H), 7.23 (d, 1H, J=7.2 Hz, Ar-H), 7.32 (s, 1H,
Ar-H), 7.87 (d, 1H, J=7.9 Hz, Ar-H), 9.65 (s, 1H, D2O exchangeable), 12.48 (s, 1H, D2O
exchangeable).13C-NMR (100 MHz, DMSO-d6), δ ppm: 56, 56.3, 97, 105.4, 113, 114.8 (2C),
116, 117.7, 122.2, 124.8, 127.3, 129.9 (2C), 148, 147.5, 150.8, 160.1, 162.1, 162.8. Anal.
Calcd for C20H16N2O4 (348.35): C, 68.96; H, 4.63; N, 8.04. Found: C, 68.89; H, 4.68; N, 8.21.
4.1.1.3.6. 1, 2-Dihydro-6- (4-hydroxyphenyl)-4- (3, 4, 5-trimethoxyphenyl)-2-
oxopyridine-3-carbonitrile (16).
Yellow powder, yield: 0.33 g (89%), mp: 298-300 °C. IR (KBr, cm-1): 3421-3228 (NH, OH)
1
3042 (CH-aromatic), 2835 (CH-aliphatic), 2210 (CN), 1631 (C=O). H-NMR (400 MHz,
DMSO-d6), δ ppm: 3.73 (s, 3H, 3OCH3), 3.85 (s, 6H, 2OCH3), 5.85 (s, 2H, D2O
exchangeable), 6.75 (s, 1H, Ar-H), 6.86 (d, 2H, J=7.7 Hz, Ar-H), 6.97 (s, 2H, Ar-H), 7.83 (d,
2H, J=7.5 Hz, Ar-H). 13C- NMR (100 MHz, DMSO-d6), δ ppm: 56.6 (2C), 60.6, 104.6, 106.4
(2C), 116.1 (2C), 118.7, 123.4, 125.1, 129.7, 132.8, 139.1 (2C), 153.2 (2C), 154, 158.5, 160.9,
165.7. Anal. Calcd for C21H18N2O5 (378.38): C, 66.66; H, 4.79; N, 7.40. Found: C, 66.90; H,
4.88; N, 7.57.
4.1.1.4. General procedure for the synthesis of 4, 6- diaryl-3, 4-dihydropyrimidine-2-
(1H) thione (17-19).
The corresponding chalcone 1-3 (0.003 mol) and thiourea (0.03mol) are added successively
to a solution of sodium ethoxide (0.003 mol) in absolute ethanol (15 ml). The reaction
0
mixture is irradiated at 100 C for 10 min. The solvent is evaporated and the residue is
dissolved in water (10 ml). The alkaline solution is acidified with acetic acid (10 ml) and the
precipitated solid is separated by filtration and crystallized from ethanol to produce
compounds 17-19.
4.1.1.4.1. 3, 4-Dihydro-4- (3, 4, 5-trimethoxyphenyl)-6- (4-methoxyphenyl) pyrimidine-2
(1H) thione (17).15)
4.1.1.4. 2. 3, 4-Dihydro-6- (3, 4, 5-trimethoxyphenyl)-4- (4-hydroxy-3-methoxyphenyl)
pyrimidine-2 (1H) thione (18).
Chemical and Pharmaceutical Bulletin Advance Publication

Yellow powder, Yield: 0.81 g (71%), mp: 223-225°C. IR (KBr, cm-1): 3421-3215 (NH, OH),
3030 (CH-aromatic), 2835 (CH-aliphatic), 1120 (C=S). 1H-NMR (400 MHz, DMSO-d6), δ
ppm: 3.77 (s, 3H, OCH3), 3.80 (s, 3H, OCH3), 3.82 (s, 6H, 2OCH3), 4.32 (s, 1H,
D2Oexchangeable), 5.04 (d, 1H, CH pyrimidine), 5.41 (d, 1H, CH pyrimidine), 6.52 (s, 1H,
Ar-H), 6.81 (s, 2H, Ar-H), 6.96 (d, 1H, J=7.7 Hz, Ar-H), 7.24 (d,1H, J=7.5 Hz, Ar-H), 9.02
(s, 1H, D2O exchangeable), 9.83 (s, 1H, D2O exchangeable). 13C-NMR (100 MHz, DMSO-
d6), δ ppm: 55.4, 55.6 (2C), 56.2, 103.2 (2C), 114.5 (2C), 123.7, 129.2 (2C), 134.4, 136.5,
136.7, 138.2, 138.4, 153.1 (2C), 159.3 175.15. Anal. Calcd for C20H22N2O5S (402.12): C,
59.69; H, 5.51; N, 6.96. Found: C, 59.79; H, 5.68; N, 6.84.
4.1.1.4. 3. 4, 6-Bis (3, 4, 5-trimethoxyphenyl) pyrimidine-2 (1H)-thione (19).
Yellow powder, yield: 0.93 g (70 %) mp: 205-207°C. IR (KBr, cm-1): 3423 (NH), 3045 (CH-
aromatic), 2835 (CH-aliphatic), 1120 (C=S). 1H-NMR (400 MHz, DMSO-d6), δ ppm: 3.76 (s,
6H, 2OCH3), 3.92 (s, 12H, 4OCH3), 7.42 (s, 2H, Ar-H), 7.50 (s, 2H, Ar-H), 7.74 (s, 1H, CH
pyrimidine), 13.52 (s, 1H, D2O exchangeable). 13C-NMR (100 MHz, DMSO-d6), δ ppm:
56.7 (2C), 56.8 (2C), 58.7, 60.7, 104.9 (2C), 106.1, 106.5, 108.1 (2C), 126.3, 128.4, 138.6,
141.4, 141.9, 153.5 (2C), 165.4, 175.8, 181. Anal. Calcd for C22H24N2O6S (444.5): C, 59.45;
H, 5.44; N, 6.30. Found: C, 59.39; H, 5.50; N, 6.49.
4.1.1.5. 2-Chloro-N-aryl-acetamides were prepared as reported.50)
4.1.1.6. General procedure for the synthesis of 2- (3-cyano-4, 6-diaryl pyridin-2-yloxy)-
N-aryl acetamide (20-23).
To a solution of the corresponding 3-cyano-2-pyridinones 11 or 12 (0.001 mol) in ethanol
(10 ml) containing anhydrous potassium carbonate (0.56 g, 0.004 mol), the appropriate 2-
chloroacetamide derivatives (0.001 mol) are added. The reaction mixture is stirred with
0
irradiation in the microwave oven at 100 C for 15 min, the reaction mixture is cooled and
poured onto crushed ice and the separated solid is filtered, washed with water, dried and
crystallized from ethanol to furnish compounds 20-23.
3.1.6.1. 2- (3-Cyano-6- (3, 4, 5-trimethoxyphenyl)-4- (4-methoxyphenyl) pyridin-2-
yloxy)-N-phenyl acetamide (20).
Yellowish green powder, yield: 0.37 g (72 %), mp: 174-176°C. IR (KBr, cm-1): 3444 (NH),
3072 (CH-aromatic), 2830 (CH-aliphatic), 2221 (CN), 1687 (C=O).1H-NMR (400 MHz,
DMSO-d6), δ ppm: 3.67 (s, 9H, 3OCH3), 3.87 (s, 3H, OCH3), 5.18 (s, 2H, CH2), 7.06-7.08 (m,
Chemical and Pharmaceutical Bulletin Advance Publication

2H, Ar-H), 7.17-7.19 (d, 2H, J=7.1 Hz, Ar-H), 7.29-7.33 (m, 2H, Ar-H), 7.44 (s, 2H, Ar-H),
7.59 -7.61 (d, 2H, J=7.9 Hz, Ar-H), 7.75 (d, 2H, J=7.4 Hz, Ar-H), 7.88 (s, 1H, Ar-H), 10.36
13
(s, 1H, NH, D2O exchangeable). C-NMR (100 MHz, DMSO-d6), δ ppm: 55.6, 56.4, 56.7,
60.6, 92.5, 105.4 (2C), 114.4, 114.6, 116 (2C), 121 (2C), 127.8, 128.4, 131.6, 131.8, 132.3
(2C), 140.3, 153.3, 153.5, 153.6, 155.8 (2C), 156.6, 156.9, 161.3, 163.7, 166.2. Anal. Calcd
for C30H27N3O6 (525.5), Calcd: C, 68.56; H, 5.18; N, 8.00. Found: C, 68.79; H, 5.26; N, 7.89.
3.1.6.2. 2- (3-Cyano-6- (3, 4, 5-trimethoxyphenyl)-4- (4-methoxyphenyl) pyridin-2-yl
oxy)-N- (4-methoxyphenyl) acetamide (21).
Greyish powder, Yield: 0.38 g (69 %) mp: 170-172 °C. IR (KBr, cm-1): 3257 (NH), 3021
1
(CH-aromatic), 2837 (CH-aliphatic), 2220 (CN), 1672 (C=O). H-NMR (400 MHz, DMSO-
d6), δ ppm: 3.68 (s, 3H, OCH3), 3.70 (s, 6H, 2OCH3), 3.72 (s, 3H, OCH3), 3.87 (s, 3H, OCH3),
5.14 (s, 2H, CH2), 6.88 (d, 2H, J=7.8 Hz, Ar-H), 7.16-7.18 (m, 2H, Ar-H), 7.45-7.51 (m, 4H,
Ar-H), 7.75 (d, 2H, J=7.4 Hz, Ar-H), 7.88 (s, 1H, Ar-H), 10.21 (s, 1H, NH, D2O
exchangeable). 13C-NMR (100 MHz, DMSO-d6), δ ppm: 55.6, 55.9, 56.4 (2C), 60.6, 92,
105.3 (2C), 114.3, 114.8, 116.1 (2C), 121, 121.1, 128.4 (2C), 130.6, 130.7 (2C), 132.3 (2C),
140.3, 153.4, 153.6, 155.8 (2C), 156.6, 156.9, 161.3, 163.7, 166.2. Anal. Calcd for
C31H29N3O7 (555.58): C, 67.02; H, 5.26; N, 7.56. Found: C, 67.23; H, 5.40; N, 7.68.
3.1.6.3. 2- (3-Cyano-4, 6-bis (3, 4, 5-trimethoxyphenyl) pyridin-2-yl oxy)-N-phenyl
acetamide (22).
Green crystals, yield: 0.38 g (65%) mp: 168-170 °C. IR (KBr, cm-1): 3257 (NH), 3012 (CH-
aromatic), 2837 (CH-aliphatic), 2220 (CN), 1672 (C=O). 1H-NMR (400 MHz, DMSO-d6),δ
ppm: 3.67 (s, 6H, 2OCH3), 3.71 (s, 3H, OCH3), 3.75 (s, 3H, OCH3), 3.76 (s, 3H, OCH3), 3.89
(s, 3H, OCH3), 5.19 (s, 2H, CH2), 7.04-7.08 (m, 3H ,Ar-H) , 7.29-7.33 (m, 2H, Ar-H), 7.44 (s,
2H, Ar-H), 7.59-7.61 (d, 2H, J=7.5 Hz, Ar-H), 7.94 (s, 1H, Ar-H), 10.37 (s, 1H, D2O
exchangeable). 13C-NMR (100 MHz, DMSO-d6), δ ppm: 56.3 (2C), 56.7 (2C), 60.6 (2C),
92.4, 105.4 (2C), 107, 114.7 (2C), 116, 119.5, 119.8, 123.9 (2C), 129.3, 130.9, 131.5 (2C),
132.3, 139.2, 139.4, 140.3, 153.3, 153.6, 156.9 (2C), 157.1, 163.6, 166.7. Anal. Calcd for
C32H31N3O8 (585.6): C, 65.63; H, 5.34; N, 7.18. Found: C, 65.85; H, 5.30; N, 7.29.
3.1.6.4. 2- (3-Cyano-4, 6-bis (3, 4, 5-trimethoxyphenyl) pyridin-2-yloxy)-N- (4-
methoxyphenyl) acetamide (23).
Chemical and Pharmaceutical Bulletin Advance Publication

Gray powder, Yield: 0.43 g (71%), m.p: 156-158 °C. IR (KBr, cm-1): 3257 (NH), 3037 (CH-
1
aromatic), 2837 (CH-aliphatic), 2220 (CN), 1672 (C=O). H-NMR (400 MHz, DMSO-d6), δ
ppm: 3.70 (s, 3H, OCH3), 3.73 (s, 6H, 2OCH3), 3.75 (s, 3H, OCH3), 3.86 (s, 6H, 2OCH3),
3.89 (s, 3H, OCH3), 4.66 (s, 2H, CH2), 6.72 (s, 1H, ArH), 6.89-6.91 (m, 3H, ArH), 7.04 (s,
1H, ArH), 7.08 (s, 2H, ArH), 7.46 (d, 2H, J=7.5 Hz, Ar-H), 10.14 (s, 1H, D2O exchangeable).
13C-NMR (100 MHz, DMSO-d6), δ ppm: 55.6, 56.4 (2C), 56.7, 60.6 (2C), 66, 92.5, 105.4,
105.9, 106.6 (2C), 114.4 (2C), 116 (2C), 117.3, 121.1 (2C), 131.6, 132.3 (2C), 139.4, 139.6,
140.3 (2C), 166.2. MS: m/z (%) 615 M+ (5.6). Anal. Calcd for C33H33N3O9 (615.63): C,
64.38; H, 5.40; N, 6.83. Found:C, 64.52; H, 5.47; N, 6.96.
4.2. Biological evaluation:
4.2.1. In vitro cytotoxic activity of compounds 4-23 over HL-60, MCF-7 and HCT-116
cancer cell lines.
4.2.1.1 Materials:
The human leukemia cell line (HL- 60), breast adenocarcinoma cell line (MCF-7) and colon
cancer cell line (HCT-116) were obtained as a gift from NCI, MD, USA. All chemicals and
solvents were purchased from Sigma–Aldrich. CA-4 was purchased from Sigma-Aldrich.
4.2.1.2. Methodology:
The cells are grown on RPMI-1640 medium supplemented with 10% inactivated fetal calf
serum and 50µg/ml gentamycin. The cells are maintained at 37ºC in a humidified atmosphere
with 5% CO2 and are subcultured two to three times a week. For antitumor assays, the tumor
cell lines are suspended in medium at concentration 5x104 cell/well in Corning® 96-well
tissue culture plates, then incubated for 24h. The tested molecules 4-23 are then added into
96-well plates (six replicates) to achieve eight concentrations for each compound. Six vehicle
controls with media or 0.5 % DMSO are run for each 96 well plate as a control. After
incubating for 24 h, the numbers of viable cells are determined by the MTT test.43) Briefly,
the media is removed from the 96 well plate and replaced with 100 µl of fresh culture RPMI
1640 medium without phenol red then 10 µl of the 12 mM MTT stock solution (5 mg of
MTT in 1 mL of PBS) to each well including the untreated controls. The 96 well plates are
then incubated at 37°C and 5% CO2 for 4h. An 85 µl aliquot of the media is removed from
the wells, and 50 µl of DMSO is added to each well and mixed thoroughly with the pipette
and incubated at 37°C for 10 min. Then, the optical density is measured at 590 nm with the
microplate reader (Sun Rise, TECAN, Inc, USA) to determine the number of viable cells and
the percentage of viability is calculated as[1- (ODt/ODc)] x100% where ODt is the mean
Chemical and Pharmaceutical Bulletin Advance Publication

optical density of wells treated with the tested sample and ODc is the mean optical density of
untreated cells. The relation between surviving cells and drug concentration is plotted to get
the survival curve of each tumor cell line after treatment with the specified compound. The
50% inhibitory concentration (IC50), the concentration required to cause toxic effects in 50%
of intact cells, is estimated from graphic plots of the dose response curve for each conc. using
Graph pad Prism software. Each concentration is repeated four times and the mean of the
results.
4.2.2. In vitro DNA- flow cytometry cell cycle analysis
4.2.2.1. Cell culture and lysate collection:
HL-60 cells are cultured in RPMI10 % +5 % FBS penicillin/streptomycin and are incubated
at humidity 5% CO2 and maintained at 37°C. HL-60 cells are treated with the IC50
concentration of 4, 18 and 21 (0.19, 0.36 and 0.091 µM) and CA-4 (0.48uM), HL-60 cells are
cultured as a monolayer in T-25 flasks and are seeded to attain 30% confluency prior to
treatment. The main 100mM stock dissolved in DMSO is diluted with cell culture medium in
order to reach the previously determined IC50 concentration of each of the three preparations.
After 24h of treatment with 4, 18 and 21, HL-60 cells are collected via trypsinization and
centrifuged at 10,000 rpm. The pellet is then rinsed with PBS and lysed in RIPA lysis buffer
at 4°C for 45 min, then centrifuged at 14,000 rpm for 20 min to remove the cellular debris.
Cell lysates are then collected and stored at 80°C for later protein determination and
immunoassays. Cell lysates stained by propidium iodide and analyzed by flow cytometry
using FACS caliber (Becton Dickinson). The experiment is repeated four times and the mean
of the results are statistically analyzed. The cell cycle distributions are calculated using Cell-
Quest software (Becton Dickinson).
4.2.3. Apoptosis analysis by Annexin V/PI staining:
HL-60 cells are seeded into each well of a 6-well plate and treated with compounds 4, 18, 21
and CA-4 at the IC50 concentration for 24h. Staining was performed using FITC Annexin V
Apoptosis Detection Kit II, BD Pharmingen (BD Biosciences, USA) according to the
manufacturer protocol.19) Briefly, cells were washed with PBS at least three times, re-
suspended in 1× binding buffer, and FITC-conjugated annexin V and propidium iodide (PI)
was added for 15 min in the dark. Samples were analyzed by a LSR-II flow cytometer
(Becton-Dickinson, San Jose, CA, USA).
4.2.4. In vitro immunofluorescence localization of tubulin in HL-60 cancer cells:
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