Synthesis and in vitro screening of phenylbipyridinylpyrazole derivatives as potential antiproliferative agents

Article abstract of DOI:10.3390/molecules20011031

A series of phenylbipyridinylpyrazoles was synthesized through the reaction of 2-(4-(2-chloropyridin-4-yl)-3-(3-methoxy-5-methylphenyl)-1H-pyrazol-1-yl)acetonitrile (4) with different 6-substituted pyridine-3-ylboronic acids. The final compounds 5a-j were screened at 10 μM against over 60 tumor cell lines at the U.S. National Cancer Institute (NCI). In light of the NCI results, compounds 5c and 5h showed a broad spectrum of activity against NCI cell lines with mean growth of 53% and 58%, respectively. Compound 5e behaved differently as it showed high degree of selectivity and potency by inhibiting 96% of growth of leukemia SR cell line at 10 μM. Standard COMPARE analyses were performed at the GI 50 level and the results exhibit high correlation in the form of pairwise correlation coefficient (PCC) of more than 0.6 between three of the current compounds and three standard known anticancer agents. Compound 5e demonstrated high correlation levels with merbarone (NSC S336628) with a PCC value of 0.631. Compound 5h showed a considerably high PCC value of 0.626 with dichloroallyl lawsone, while compound 5i, showed PCC values of 0.601 and 0.604 with both dichloroallyl lawsone and N,N-dibenzyldaunomycin (NSC S268242), respectively. These three standard agents have anticancer activity via two major mechanism of actions, inhibition of topoisomerase II and inhibition of biosynthesis of pyrimidine nucleotides, therefore, compounds 5a-j are promising therapeutic agents for targeting different human malignancies. Prediction of drug-likeness and toxicity of these newly synthesized derivatives were also considered.

Full text of DOI:10.3390/molecules20011031

Molecules 2015, 20, 1031-1045; doi:10.3390/molecules20011031
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and in Vitro Screening of Phenylbipyridinylpyrazole
Derivatives as Potential Antiproliferative Agents
Mohammad M. Al-Sanea 1,2,*, Ahmed Elkamhawy 1,2,†, Ahmed Zakaria 1,2,†, Byung Sun Park 1,
Youngjoo Kwon 3, So Ha Lee 1, Sang Woo Lee 4 and In Tae Kim 4
1
Chemical Kinomics Research Center, Korea Institute of Science and Technology,
Hwarangno 14-gil 5, Seongbuk-gu, Seoul 136-791, Korea;
E-Mails: a.elkamhawy@gmail.com (A.E.); aznagi2003@gmail.com (A.Z.);
yangwenli1@nate.com (B.S.P); lsh6211@kist.re.kr (S.H.L.)
2
Department of Biological Chemistry, Korea University of Science and Technology,
113 Gwahangno, Yuseong-gu, Daejeon 305-333, Korea
3
College of Pharmacy and Graduate School of Pharmaceutical Sciences, Ewha Womans University,
Seoul 120-750, Korea; E-Mail: ykwon@ewha.ac.kr
4
Department of Chemistry, Kwangwoon University, Seoul 139-701, Korea;
E-Mails: lsw1018@kw.ac.kr (S.W.L.); itkim@kw.ac.kr (I.T.K.)
†
These authors contributed equally to this work.
* Author to whom correspondence should be addressed; E-Mail: mohmah80@gmail.com;
Tel.: +82-2-958-6795; Fax: +82-2-958-5189.
Academic Editor: Derek J. McPhee
Received: 10 December 2014 / Accepted: 4 January 2015 / Published: 9 January 2015
Abstract: A series of phenylbipyridinylpyrazoles was synthesized through the reaction
of 2-(4-(2-chloropyridin-4-yl)-3-(3-methoxy-5-methylphenyl)-1H-pyrazol-1-yl)acetonitrile
(4) with different 6-substituted pyridine-3-ylboronic acids. The final compounds 5a–j were
screened at 10 µM against over 60 tumor cell lines at the U.S. National Cancer Institute
(NCI). In light of the NCI results, compounds 5c and 5h showed a broad spectrum of
activity against NCI cell lines with mean growth of 53% and 58%, respectively. Compound
5e behaved differently as it showed high degree of selectivity and potency by inhibiting
96% of growth of leukemia SR cell line at 10 µM. Standard COMPARE analyses were
performed at the GI50 level and the results exhibit high correlation in the form of pairwise
correlation coefficient (PCC) of more than 0.6 between three of the current compounds and
three standard known anticancer agents. Compound 5e demonstrated high correlation

Molecules 2015, 20
levels with merbarone (NSC S336628) with a PCC value of 0.631. Compound 5h showed
1032
a considerably high PCC value of 0.626 with dichloroallyl lawsone, while compound 5i,
showed PCC values of 0.601 and 0.604 with both dichloroallyl lawsone and
N,N-dibenzyldaunomycin (NSC S268242), respectively. These three standard agents have
anticancer activity via two major mechanism of actions, inhibition of topoisomerase II and
inhibition of biosynthesis of pyrimidine nucleotides, therefore, compounds 5a–j are
promising therapeutic agents for targeting different human malignancies. Prediction of
drug-likeness and toxicity of these newly synthesized derivatives were also considered.
Keywords: cancer; cell lines; phenylbipyridinylpyrazole; antiproliferative agents;
COMPARE analysis
1. Introduction
Cancer has been the leading worldwide cause of death, and accounted for 7.6 million deaths in 2008
according to WHO reports [1]. The conventional cancer treatments such as surgery, radiation and
cytotoxic chemotherapy seem to be no longer efficient neither for the complete abolition of disease nor
for the improvement of cancer patients’ quality of life. This has driven the development of a variety of
new anticancer agents with more potent, high specific and low cytotoxic properties.
The term anti-metabolite is used to cover a number of cytotoxic agents that act by masquerading as
purines or pyrimidines, the building blocks of DNA. Inhibition of biosynthesis of pyrimidine
nucleotides has become a new strategy in cancer treatment. Dichlorallyl lawsone [2-hydroxy-3-(3,3-
dichloroallyl)-1,4-naphthoquinone] is of interest as an cytotoxic agent through inhibition of
biosynthesis of pyrimidine nucleotides. It stimulates the incorporation of [14C]uridine into
macromolecules, decreases the pool sizes of uridine triphosphate, prevents the pyrazofurin-induced
accumulation of orotidine and orotic acid, and exerts cytotoxicity that could be prevented or reversed
almost completely by uridine [2].
Another cancer treatment strategy is the inhibition of DNA-topoisomerases. This enzyme are
omnipresent nuclear enzymes which induce transient breaks in the DNA allowing DNA strands or
double helices to pass through each other and play essential roles in DNA replication and
transcription [3,4]. DNA topoisomerases fall into two major classes: the type I enzymes that induce
single stranded cuts in DNA, and the type II enzymes that cut and pass double stranded DNA [5,6].
Therefore, tumor cell multiplication and growth can be controlled by interfering with topoisomerases
and then tumor control could be achievable [7,8]. In particular, different compounds with a pyrazole
core show an interesting antiproliferative activity [9–12].
Herein, a new series of phenylbipyridinylpyrazole scaffold-based compounds has been synthesized
and biologically screened against over 60 cancerous cell lines in order to check their in vitro
anti-proliferative activity. In order to gain insight into the mechanism of the cytotoxic action of
some final compounds, matching mean graph fingerprint (Compare Analysis) has been applied.
The Compare Analysis results have suggested two possible mechanisms of action for these

Molecules 2015, 20
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phenyl-bipyridinylpyrazole scaffold-based compounds: (i) topoiomerase IIα inhibition and
(ii) pyrimidine nucleotide synthesis inhibition.
2. Results and Discussion
2.1. Chemistry
The preparation of bipyridinylpyrazole compounds 5a–j is outlined in Scheme 1. Substituted
benzoic acid ester 1 is subjected to a nucleophilic attack on its trigonal carbonyl carbon by an activated
methylene group of 2-chloro-4-methylpyrimidine as reported in the literature [13–15].
Scheme 1. Synthesis of target compounds: (i) LHMDS, 2-chloro-4-methylpyrimidine,
THF, N2, rt, 18 h, 85%; (ii) DMF-DMA, 90 °C, 12 h; (iii) hydrazine hydrate, abs. ethanol,
rt, 2 h, 80%; (iv) K2CO3, iodoacetonitrile, acetone, reflux, 4 h, 90%; (v) 6-substituted
pyridine-3-boronic acid, Pd(PPh3)2Cl2, K2CO3, N2, CH3CN/H2O (4:1), 78 °C, 4 h.
The methylene activation was achieved by dropwise addition of lithium hexamethyldisilazide
(LHMDS) in dry THF at room temperature. The resulting tautomeric α,β-unsaturated ketone 2 was
then converted into the pyrazole derivative 3 through two sequential steps. First compound 2 was
heated with an excess of N,N-dimethylformamide dimethylacetal for 12 h, and the resulting product
was then cyclized into the pyrazole derivative 3 with hydrazine monohydrate in absolute ethanol. The
reaction of the resulting pyrazole 3 with iodoacetonitrile in the presence of excess potassium carbonate

Molecules 2015, 20
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produced two regioisomers; isomer 4 having a lower Rf value of 0.84 (ethyl acetate/hexane 1:1, v/v)
and another isomer having a higher Rf value of 0.74 [13]. The location of the acetonitrile moiety on the
pyrazole nitrogens was assigned and confirmed using 2D-NOESY NMR as addressed before in the
literature [16]. The two isomers were separated chromatographically using a mixed solvent of ethyl
acetate and hexane (1:1, v/v) and isomer 4 is taken to the next Suzuki coupling step with a series of
4-substituted pyridineboronic acids to furnish final compounds 5a–j.
2.2. Biological Evaluation
2.2.1. In Vitro Anticancer Screening
Compounds 5a–j were selected for evaluation of their antiproliferative activity by the National
Cancer Institute (NCI, Bethesda, MD, USA), on the basis of their degree of structural variation and
computer modeling techniques. The compounds were subjected to in vitro anticancer assays against
tumor cells in a full panel of 60 different human tumor cell lines representing nine different tissues
(leukemia, melanoma and cancers of the lung, colon, brain, ovary, breast, prostate, and kidney). The
compounds were tested at a single dose concentration of 10 µM and the cultures were incubated for
48 h. The results for each compound are reported as a mean graph of the percent growth of the treated
cells when compared to the untreated control cells. The percentage growth of the tested compounds
against the full 60-cell lines panel is illustrated in Table 1.
The mean growth percentages of all of the tested compounds over full 60-cell lines are illustrated in
Table 2. Compounds 5c and 5h exhibited the lowest mean percentage growth against the full 60-cell
line panel, which means they had a broad spectrum of activity against different cancer cell lines. It is
worth mentioning that compound 5c showed strong inhibitory activity (>90%) against three cell lines
(OVCAR-3, NCI/ADR-RES, MDA-MB-468) with a lethal effect rather than growth inhibition on
MDA-MB-435 melanoma cell line (this appears as a negative column as shown in Figure 1).
Compound 5h, in particular, exhibited considerable efficacy against all leukemia cell lines with a
percentage growth range of 17%–40% (Figure 2). Regarding the sensitivity against individual cell
lines, compound 5e showed observed single strong cell growth inhibition of one leukemia cell line,
SR, with a growth percentage of 4.04%. As the mean percentage growth of Compound 5e is 85%, high
degree of selectivity profile has become obvious and it could be useful in targeting specific leukemia
SR cell line.
By referring to the results of the inhibitory effects of the tested compounds summarized in
Tables 1 and 2, and correlating between the structural variations and the resulted anticancer activities,
we cannot establish precise structure activity relationships (SAR) between the bipyridinylpyrazole
scaffold and the current NCI screening results. However, some clues for understanding the currently
available screening results could be deduced, which perhaps could be useful for our future work in
developing this scaffold. Generally, substitution at the terminal pyridinyl group with a hydrophobic
bulky moiety is more favorable than small groups like methoxy or ethoxy. Deep hydrophobic
interaction with the complementary hydrophobic pocket at the target macromolecule(s) could be
achieved if 4-pyridinyl position has been substituted with a phenyl moiety carried on a spacer like
piperazine or oxymethylene bridge groups, as revealed by compounds 5h and 5i.

Molecules 2015, 20
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Table 1. Percentage of cell growth for the tested compounds against 60 tumor cell lines.
Percentage of Cell Growth a
Cell Lines
CCRF-CEM
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
56.06
93.71
44.4
48.23
88.74
29.76
54.13
58.94
49.8
40.94
63
95.08
98.85
104.4
88.68
98.94
89.52
85.83
85.9
62.47
95.75
58.46
69.09
75.11
4.04
70.45
90.3
92.95
102.6
109.7
100.7
94.57
91.36
86.18
99.97
52.65
86.04
96.39
101.0
96.78
100.4
106.2
103.9
93.73
94.72
102.7
92.91
96.35
95.6
38.31
38.58
17.62
31.55
20.91
40.73
60.74
110.8
52.61
74.88
72.15
95.42
57.17
41.02
67.09
43.2
90.4
57.97
91.84
40.04
55.14
67.43
61.59
74.32
110.1
84.65
81.26
86.9
HL-60(TB)
K-562
97.47
99.8
13.4
50.75
65.33
77.93
55.17
87.01
88.22
46.04
88.58
92.09
85.69
101.6
100.9
99.21
86.09
68.16
77.25
79.26
94.36
94.35
56.53
79.27
90.84
63.06
96.88
Leukemia
MOLT-4
RPMI-8226
SR
56.34
60.48
67.87
87.9
51.91
29.52
35.47
61.47
73.86
45.34
83.09
51.85
100.2
94.09
73.72
85.57
24.5
81.16
92.35
83.31
88.37
87.28
60.87
92.03
92.21
93.24
98.8
A549/ATCC
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
91.94
95.83
43.6
102.7
104.2
44.52
92.64
94.98
89.82
102.4
79.75
100.0
61.7
107.0
69.97
88.11
85.56
91.42
94.42
82.6
49.16
87.54
99.59
113.6
102.9
96.46
98.48
91.03
94.81
104.3
104.7
102.5
100.6
88.62
95.06
91.29
66.83
93.01
NSCLC
74.6
83.56
77.33
98.84
76.8
97.23
94.88
82.76
92.82
69.84
53.49
37.66
87.17
77.97
96.09
90.11
88.07
88.35
85.68
88.99
102.3
99.95
85.8
88.15
67.45
57.32
37.1
84.24
60.72
52.74
48.01
54.28
76.97
98.85
84.01
78.11
85.25
44.53
91.11
Colon Cancer
CNS Cancer
44.88
19.08
54.32
65.02
76.61
18.24
37.87
82.27
30.49
86.66
70.42
51.26
95.36
82.84
104.1
95.43
88.77
89.56
55.01
95.17
24.6
88.06
92.24
100.5
97.48
90.2
11.94
43.97
60.55
76.17
63.38
30.21
79.89
65.89
68.64
KM12
86.66
92.45
100.2
89.92
95.97
92.1
SW-620
SF-268
SF-295
97.89
92.94
94.61
73.17
93.18
SF-539
96.35
92.17
76.9
SNB-19
SNB-75
U251
95.38
94.47
93.53

Molecules 2015, 20
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Table 1. Cont.
Percentage of Cell Growth a
Cell Lines
LOX IMVI
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
69.37
107.5
90.13
96.21
105.3
101.5
93.55
107.7
73.42
109.7
98.29
87.46
91.13
81.22
83.72
98.39
91.57
91.0
82.25
82.27
78.84
109.8
91.95
61.14
93.7
76.85
60.44
84.41
57.58
68.1
61.68
77.19
92
32.53
76.41
74.58
−8.52
82.05
48.94
42.91
69.05
52.73
85.16
6.29
97.91
102.9
93.6
96.15
121.0
94.86
97.01
97.1
87.66
110.2
104.3
90.49
87.78
89.33
92.72
95.22
91.19
103.4
87.6
83.76
90.66
107.4
82.51
85.21
112.5
98.85
89.4
89.63
93.51
107.8
96.69
123.4
97.45
81.82
114.1
94.86
105.7
97.5
82.78
91.08
92.19
91.54
106.3
81.84
80.4
81.27
90.18
78.88
113.3
67.72
87.85
90.3
76.91
79.16
91.78
72.63
63.44
65.72
92.92
98.58
59.14
96.91
86.64
73.07
95.97
80.73
93.48
92.24
81.73
90.84
84.12
66.54
101.1
86.41
93.82
55.86
84.89
85.35
115.5
71.4
97.2
32.95
79.55
64.79
68.22
83.3
69.36
70.71
83.45
65.51
87.36
55.41
60.1
91.89
97.85 104.28
120.8 100.25
106.4
120.9
102.6 107.17
100.4 93.82
102.2 105.51
85.59
101.5
94.35
90.31
74.9
89.73
84.09
98.24
92
87.49
75.87
86.4
87.72
86.43
69.35
86.85
111.1
89.1
67.44
MALME-3M
M14
104.1
104.3
102.0
101.4
108.0
96.02
105.4
87.72
95.63
105.0
100.8
84.93
88.68
95.56
104.1
98.75
97.71
91.97
87.76
90.12
96
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
786-0
46.59
117.9
90.11
77.97
114.0
72.26
69.24
77.61
63.79
72.62
84.54
77.68
91.79
76.97
110.2
63.48
72.03
72.33
100.5
61.17
65.35
84.57
26.85
52.64
81.85
26.9
95.83
112.4
Melanoma
87.91
96.37
91.6
90.57
91.76
79.8
75.16
92.76
83.91
98.66
95.16
81.52
79.58
111.5
80.58
69.44
93.49
63.72
47.6
39.58
94.75
55.05
7.25
Ovarian
Cancer
77.7
63.03
60.92
81.49
67.64
87.81
51.64
61.49
51.19
90.95
57.74
34.01
66.92
28.28
31.94
65.96
28.42
40.28
70.56
67.03
50.46
29.86
54.56
68.88
112.1
41.05
48.35
86.53
18.15
48
A498
ACHN
CAKI-1
SN12C
TK-10
UO-31
PC-3
DU-145
Renal Cancer
70.5
Prostate
Cancer
67.24
96.15
43.18
69.26
89.77
72.37
76.26
83.77
109.2
102.5
80.57
85.33
80.58
86.7
MCF7
MDA-MB-231/ATCC
HS 578T
77.26
82.47
68.23
69.27
Breast
Cancer
82.21
42.13
1.31
109.4
70.07
87.94
79.81
40.82
61.8
T-47D
MDA-MB-468
19.33
Note: a screened at 10 µM concentration.

Molecules 2015, 20
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Table 2. Screening data of the tested compounds with the most sensitive cell lines and
mean percentage growth.
Cell Lines
K-562
5a
5b
5c
5d
-
5e
5f
-
5g
-
5h
5i
-
5j
-
a
-
29.76
13.4
35.47
19.08
−8.52
6.29
7.25
18.15
1.31
53
-
17.62
SR
-
-
-
4.04
-
-
-
-
-
HT29
37.1
-
-
-
-
-
-
11.94
-
37.66
MDA-MB-435
OVCAR-3
NCI/ADR-RES
MCF7
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
28.28
-
-
-
-
26.85
19.33
72
-
-
-
-
-
-
MDA-MB-468
Mean % growth
-
-
-
-
-
-
-
84
94
85
81
94
58
82
91
Note: a Values which exceed 40% growth.
120
100
80
60
40
20
0
-20
Figure 1. Inhibitory effects of compound 5c at 10 uM concentration over 60 cell lines.
120
100
80
60
40
20
0
LeukemiaCCRF-CEM
LeukemiaHL-60(TB)
LeukemiaK-562
LeukemiaMOLT-4
LeukemiaRPMI-8226
LeukemiaSR
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
Figure 2. Inhibitory effects of the tested target compounds at 10 μM concentration over all
six tested leukemia cell lines.
COMPARE analyses was applied to all the final compounds in order to investigate the similarity of
their cytotoxicity patterns (mean graph fingerprints) with data from known anticancer standard agents,
NCI active synthetic compounds and natural extracts, which are available in public available
databases. Such an analysis is based on comparing the differential growth inhibition patterns for
cultured cell lines and can potentially provide insight into the mechanism of the cytotoxic action. If the
data pattern correlates well with those of compounds found in a standard agent database (pairwise
correlation coefficient (PCC > 0.6), the compound of interest may have the same mechanism of

Molecules 2015, 20
1038
action [17,18]. On the other hand, if the activity pattern does not correlate with any standard agent, it is
possible that the compound has a novel mechanism of action. Standard COMPARE analyses were
performed at the GI50 level.
The results of that screening exhibit high correlation (PCC > 0.6) between three of our compounds
and three known anticancer standard agents. Compound 5e demonstrated high correlation levels with
merbarone (NSC S336628) with a PCC value of 0.631. Compound 5h showed a considerably high
PCC value of 0.626 with dichloroallyl lawsone (NSC S126771), while compound 5i had PCC values
of 0.601 and 0.604 with both dichloroallyl lawsone and N,N-dibenzyldaunomycin (NSC S268242),
respectively. Such similarity in COMPARE results could indicate a resemblance in the corresponding
mechanisms of action. Merbarone and N,N-dibenzyldaunomycin share the same mechanism of action
as anti-cancer agents by inhibiting the topoisomerase II enzyme. The DNA-topoisomerases are
omnipresent nuclear enzymes that play vital roles in DNA metabolism events, such as replication and
transcription. Because of its critical role in the maintenance and replication of DNA during
proliferation, cells become highly vulnerable when these functions are disrupted [7,8].
2.2.2. Topoisomerase IIα Inhibitory Activity
From the topoisomerase IIα screening results, we found that compounds 5e and 5i, with inhibitory
activity of 1.2%, 0% and 0% did not show topoisomerase IIα inhibitory activity at three different
concentrations, 100, 25 and 5 μM (as compared to 78.8%, 50.3% and 15.0% of etoposide at 100, 25
and 5 μM, respectively).
Figure 3 shows the results of topoisomerase IIα inhibitory activities of the two evaluated
compounds 5e and 5i. It means that the mechanism of action of these two promising antiproliferative
compounds is not topoisomerase IIα inhibition as suggested by the Compare Analysis correlation study.
D
T
E
5i 5e
Relaxed
100 µM
25 µM
Supercoiled
Relaxed
Supercoiled
Relaxed
5 µM
Supercoiled
Figure 3. Topoisomerase IIα inhibitory activities of the compounds, 5e and 5i. The
compounds were examined in a final concentration of 5, 25 and 100 µM, respectively, as
designated. Lane D: pBR322 only, Lane T: pBR322 + topoisomerase IIα, Lane E: pBR322
+ topoisomerase IIα + etoposide, Lane 5e: pBR322 + topoisomerase IIα + compound 5e in
designated concentrations, Lane 5i: pBR322 + topoisomerase IIα + compound 5i in
designated concentrations.

Molecules 2015, 20
1039
2.3. Assessment of Toxicity Profiles and Lipinski’s Rule of Five
The OSIRIS program is generally used for prediction of various drug-relevant properties and
overall toxicity and some undesired effects like mutagenicity or a poor intestinal absorption. Herein,
OSIRIS was applied for predicting those issues for our designed compounds. None of the target
compounds showed any predicted toxicity risks.
The total polar surface area (TPSA) was calculated as it is a key property that has been linked to
drug bioavailability. Thus, passively absorbed molecules with a TPSA > 140 are thought to have low
oral bioavailability [19]. As all the final compounds have TPSA value ranging from 76 to 89 (Table 3),
they hypothetically should show good passive oral absorption. Furthermore, the Lipinski’s rule of five
was reviewed as it clearly estimates the ADME properties which are considered important in the drug
development stage and clinical trials. Predictions of ADME properties for the studied compounds are
given in Table 3. The results showed that most of the target compounds obey these rules suggesting
that the synthesized compounds would be possible drug molecules.
Table 3. Total polar surface area, calculated Lipinski’s rule of five, and toxicity risks for
final compounds.
Comp No. TPSA a
MW b nHBA c nHBD d V e RB f Volume g Clog P h Toxicity i
j
5a
5b
5c
5d
5e
5f
85.87
425.492
7
8
8
7
8
6
7
7
6
7
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
0
0
2
0
1
7
6
6
6
8
6
6
8
5
8
389.283
445.429
425.069
372.481
517.079
381.625
416.084
464.491
351.867
444.13
3.34
2.78
2.68
2.94
4.2
-
83.112 479.588
89.108 466.545
-
-
-
-
-
-
-
-
-
85.87
411.465
83.112 555.686
76.636 427.533
79.874 450.546
79.874 500.606
76.636 399.429
3.23
3.5
5g
5h
5i
4.32
3.11
4.35
5j
85.87
487.563
a
b
c
d
Notes: Total polar surface area; Molecular weight; Number of hydrogen bond acceptor; Number of
e
f
g
hydrogen bond acceptor; Number of violation to Lipinski’s rule of five; Rotatable bonds; molecular
volume; h calculated lipophilicity; i no toxicity risks (mutagenicity, tumorigenicity, irritancy, reproductive effects).
3. Experimental Section
3.1. Chemistry
3.1.1. General
Commercially available reagents and anhydrous solvents were commercially available and used
without further purification. Thin layer chromatography (TLC) analyses were performed with
precoated silica gel 60 F254 plates. NMR spectra were recorded with a Bruker 300 MHz spectrometer
at ambient temperature with the residual solvent peaks as internal standards. The line positions of
multiplets were given in ppm (δ) and the coupling constants (J) were given in hertz. The Liquid
Chromatography high-resolution mass spectra (LC-HRMS, electron spray ionization) experiments
were performed with a Synapt G2 Q-TOF MS (Waters MS Technology, Manchester, UK) mass

Molecules 2015, 20
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analyzer. Data were acquired in the positive ion mode. Calibration was performed with an external
calibration mixture immediately prior to analysis. Melting points were taken on a Thomas-Hoover
Capillary Melting Apparatus and were uncorrected. Flash chromatography was performed on columns
of Merck silica gel (230–400 mesh, 40–63 μm) with ethyl acetate/hexane or methanol/dichloromethane as
eluents. The synthesis and analytical data of compounds 2, 3 and 4 were published in the literature [13].
3.1.2. General Procedure for Synthesis of Compounds 5a–j
A mixture of compound 4 (100 mg, 0.29 mmol), aryl boronic acid (0.35 mmol), bis(triphenylphosphine)
palladium(II) dichloride (9 mg, 0.013 mmol) and K2CO3 (71 mg, 0.52 mmol) was placed in a mixed
solvent of THF and water (4:1, v/v, 10 mL). N2 gas was bubbled into this mixture for 10 min, and then the
mixture was heated at 70 °C while stirring under N2 for 12 h. The reaction mixture was left to cool at room
temperature, and extracted with ethyl acetate (100 mL × 3). The combined organic extracts were dried
over anhydrous MgSO4 and evaporated under vacuum. The target compounds 5a–j were separated in
pure form by column chromatography (silica gel) using the proper ratio of ethyl acetate and n-hexane.
2-(3-(3-Methoxy-5-methylphenyl)-4-(2-(6-(benzyl(methyl)amino)pyridin-3-yl)pyridin-4-yl)-1H-pyrazole-
1-yl)acetonitrile (5a). Flash column chromatography was carried out using hexane/ethyl acetate, 1:1
(v/v) to give the title compound as a yellow solid. Yield: 65%. mp 59–61 °C. 1H-NMR (CDCl3) δ 2.23
(s, 3H), 3.05 (s, 3H), 3.62 (s, 3H), 4.79 (s, 2H), 5.08 (s, 2H), 6.51 (d, J = 8.96 Hz, 1H), 6.68 (s, 1H),
6.73 (s, 1H), 6.86 (s, 1H), 6.97 (d, J = 5.08 Hz, 1H), 7.29 (m, 5 H), 7.47 (s, 1H), 7.73 (s, 1H), 7.98 (dd,
13
J = 8.96, 2.08 Hz, 1H), 8.45 (d, J = 5.08, 1H), 8.58 (d, J = 2.08, 1H). C-NMR (CDCl3) δ 24.89,
36.41, 39.92, 53.32, 55.26, 105.63, 110.78, 113.52, 115.73, 118.16, 120.40, 120.53, 121.67, 127.11,
128.63, 130.08, 132.75, 136.14, 138.18, 139.97, 140.96, 146.59, 149.55, 151.49, 155.79, 159.70;
positive ion ESI-MS m/z 523.2220 (M+Na)+.
2-(3-(3-Methoxy-5-methylphenyl)-4-(2-(6-(methylthio)pyridin-3-yl)pyridin-4-yl)-1H-pyrazole-1-
yl)acetonitrile (5b). Flash column chromatography was carried out using hexane/ethyl acetate, 1:1
(v/v) to give the title compound as a white solid. Yield: 88.7%. mp 71–73 °C. 1H-NMR (CDCl3) δ 2.24
(s, 3H), 2.53 (s, 3H), 3.65 (s, 3H), 5.12 (s, 2H), 6.70 (s, 1H), 6.72 (s, 1H), 6.84 (s, 1H), 7.11 (d,
J = 5.08 Hz, 1H), 7.19 (s, 1H), 7.54 (s, 1H), 7.82 (s, 1H), 7.99 (dd, J = 8.88, 2.08 Hz, 1H), 8.52 (d,
J = 5.08 Hz, 1H), 8.78 (d, J = 2.08 Hz, 1H). 13C-NMR (CDCl3) δ 11.31, 19.45, 37.93, 53.24, 108.99,
111.43, 113.61, 117.87, 118.5, 119.2, 119.3, 127.48, 128.26, 130.56, 132.19, 138.08, 139.84, 145.64,
149.59, 152.41, 157.75, 159.46; positive ion ESI-MS m/z 450.1364 (M+Na)+.
2-(3-(3-Methoxy-5-methylphenyl)-4-(2-(6-(1-pyrrolidinyl)pyridin-3-yl)pyridin-4-yl)-1H-pyrazole-1-
yl)acetonitrile (5c). Flash column chromatography was carried out using hexane/ethyl acetate, 1:1
1
(v/v) to give the title compound as a white solid. Yield: 92%. mp 123–125 °C. H-NMR (CDCl3) δ
1.97 (s, 4H), 2.23 (s, 3H), 3.47 (s, 4H), 3.63 (s, 3H), 5.1 (s, 2H), 6.38 (d, J = 8.92 Hz, 1H), 6.68 (s, 1H),
6.73 (s, 1H) , 6.87 (s, 1H), 6.96 (d, J = 5.00 Hz, 1H), 7.46 (s, 1H), 7.74 (s, 1H) , 8.02 (dd, J = 8.92,
2.12 Hz, 1H), 8.45 (d, J = 5.00 Hz, 1H), 8.56 (d, J = 2.12 Hz, 1H). 13C-NMR (CDCl3) δ 21.51, 25.51,
39.90, 47.10, 110.73, 113.49, 115.77, 117.8, 118.04, 120.61, 121.64, 122.53, 129.7, 129.9, 132.75,
136.04, 139.91, 140.73, 149.9, 151.44, 159.67; positive ion ESI-MS m/z 473.2060 (M+Na)+.

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2-(3-(3-Methoxy-5-methylphenyl)-4-(2-(6-ethoxypyridin-3-yl)pyridin-4-yl)-1H-pyrazole-1-yl)acetonitrile
(5d). Flash column chromatography was carried out using hexane/ethyl acetate, 1:2 (v/v) to give the
1
title compound as a white solid. Yield: 48%. mp 58–60 °C. H-NMR (CDCl3) δ 1.32 (t, J = 6.92 Hz,
3H), 2.21 (s, 3H), 3.62 (s, 3H), 4.29 (q, J = 6.92 Hz, 2H), 4.81 (s, 2H), 5.21 (d, J = 8.96 Hz, 1H), 6.27
(s, 1H), 6.54 (s, 1H), 6.67 (s, 1H), 6.7 (s, 1H), 6.73 (s, 1H), 6.84 (s, 1H), 7.04 (s, 1H), 7.48 (s, 1H),
13
7.71 (s, 1H), 8.02 (d, J = 7.96 Hz, 1H), 8.46 (s, 1H), 8.48 (d, J = 7.96 Hz, 1H). C-NMR (CDCl3) δ
14.67, 21.51, 54.96, 55.28, 62.08, 110.91, 111.09, 115.29, 115.38, 118.80, 118.94, 119.08, 121.98,
127.99, 133.14, 137.24, 139.92, 141.48, 151.17, 155.11, 159.69, 164.46, 169.19, 172.89.
2-(3-(3-Methoxy-5-methylphenyl)-4-(2-(6-(4-methylpiprazine-1-yl)pyridin-3-yl)pyridin-4-yl)-1H-pyrazole-
1-yl)acetonitrile (5e). Flash column chromatography was carried out using hexane/ethyl acetate, 1:5
(v/v) to give the title compound as a white solid. Yield: 25%. mp 84–86 °C. 1H-NMR (CDCl3) δ 2.23
(s, 4H) 2.34 (s, 3H), 2.55 (s, 4H), 3.62 (s, 3H), 5.11 (s, 2H), 6.62 (d, J = 8.96 Hz, 1H), 6.68 (s, 1H),
6.73 (s, 1H), 6.86 (s, 1H), 6.99 (d, J = 5.08 Hz, 1H), 7.46 (s, 1H), 7.74 (s, 1H), 7.98 (dd, J = 8.96, 2.24
13
Hz, 1H), 8.46 (d, J = 5.08 Hz, 1H), 8.56 (d, J = 2.24 Hz, 1H). C-NMR (CDCl3) δ 21.51, 39.91,
44.64, 45.82, 54.55, 55.29, 110.82, 113.57, 115.63, 115.70, 118.37, 120.49, 121.68, 124.54, 130.09,
132.79, 136.07, 139.93, 140.72, 146.74, 150.01, 151.42, 155.69, 159.22, 159.68; positive ion ESI-MS
m/z 480.2509 (M+H)+.
2-(3-(3-Methoxy-5-methylphenyl)-4-(2-(6-morpholinopyridin-3-yl)pyridin-4-yl)-1H-pyrazole-1-yl-
)acetonitrile (5f). Flash column chromatography was carried out using hexane/ethyl acetate, 1:5 (v/v)
1
to give the title compound as a yellow solid. Yield: 73%. mp 85–87 °C. H-NMR (CDCl3) δ 2.21 (s,
3H), 3.48 (t, J = 4.68 Hz,4H), 3.6 (s, 3H), 3.72 (t, J = 4.68 Hz, 4H), 5.07 (s, 2H), 6.58 (d, J = 8.84 Hz,
1H), 6.66 (s, 1H) 6.72 (s, 1H), 6.85 (s, 1H), 6.97 (d, J = 5.04 Hz, 1H), 7.44 (s, 1H), 7.71 (s, 1H), 7.98
13
(dd, J = 8.84, 2.08, 1H), 8.45 (d, J = 5.04 Hz, 1H), 8.56 (d, J = 2.08 Hz, 1H). C-NMR (CDCl3) δ
21.52, 39.85, 45.41, 55.18, 66.67, 106.44, 110.90, 113.76, 115.63, 118.23, 118.38, 120.26, 121.69,
124.67, 128.56, 132.06, 132.87, 136.05, 139.91, 140.87, 146.50, 151.33, 155.52, 159.51, 159.68;
positive ion ESI-MS m/z 467.2186 (M+H)+.
2-(3-(3-Methoxy-5-methylphenyl)-4-(2-(6-methoxypyridin-3-yl)pyridin-4-yl)-1H-pyrazole-1-yl)-
acetonitrile (5g). Flash column chromatography was carried out using hexane/ethyl acetate, 1:2 (v/v)
to give the title compound as a white solid. Yield: 90.6%. mp 176–178 °C. 1H-NMR (CDCl3) δ 2.21 (s,
3H), 3.61 (t, J = 4.68 Hz, 4H), 3.89 (s, 3H), 4.81 (t, J = 4.68 Hz, 4H), 6.26 (s, 1H), 6.55 (brs, 1H),
6.67 (s, 1H), 6.69 (s, 1H), 6.72 (s, 1H), 6.84 (s, 1H), 7.03 (d, J = 4.8 Hz, 1H), 7.47 (s, 1H), 7.69 (s,
1H), 8.03 (d, J = 8.56 Hz, 1H), 8.46 (d, J = 4.8 Hz, 1H), 8.5 (s, 1H). 13C-NMR (CDCl3) δ 21.52,
53.45, 54.96, 55.18, 110.82, 115.30, 115.38, 115.65, 118.80, 118.95, 119.13, 121.68, 128.39, 133.15,
137.27, 139.92, 141.29, 150.04, 151.16, 155.18, 159.69, 164.66, 169.19.
2-(3-(3-Methoxy-5-methylphenyl)-4-(2-(6-(4-benzyl-1-piprazinyl)pyridin-3-yl)pyridin-4-yl)-1H-
pyrazole-1-yl)acetonitrile (5h). Flash column chromatography was carried out using hexane/ethyl acetate,
1:3 (v/v) to give the title compound as a yellow solid. Yield: 70%. mp 71–73 °C. 1H-NMR (CDCl3) δ
2.23 (s, 3H), 2.52 (brs, 4H), 3.53 (s, 2H), 3.58 (s, 3H), 3.62 (s, 3H), 5.09 (s, 2H), 6.60 (d, J = 8.8 Hz),
6.68 (s, 1H), 6.73 (s, 1H), 6.86 (s, 1H), 6.98 (brs, 1H), 7.21 (m, 5H), 7.45 (s, 1H), 7.72 (s, 1H), 7.98 (d,

Molecules 2015, 20
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13
J = 8.8 Hz, 1H), 8.46 (brs, 1H), 8.55 (s, 1H). C-NMR (CDCl3) δ 21.51, 39.90, 45.5, 52.72, 55.29,
63.5, 106.54, 110.74, 113.48, 115.68, 115.75, 118.18, 118.33, 120.61, 121.66, 128.39, 129.48, 129.99,
132.77, 135.99, 139.93, 140.67, 146.77, 150.04, 151.45, 155.81, 159.39, 159.69; positive ion ESI-MS
m/z 556.2825 (M+H)+.
2-(3-(3-Methoxy-5-methylphenyl)-4-(2-(6-benzyloxypyridin-3-yl)pyridin-4-yl)-1H-pyrazole-1-yl)-
acetonitrile (5i). Flash column chromatography was carried out using hexane/ethyl acetate, 1:1 (v/v) to
give the title compound as a white solid. Yield: 95%. mp 58–60 °C. 1H-NMR (CDCl3) δ 2.18 (s, 3H),
3.58 (s, 3H), 4.99 (s, 2H), 5.31 (s, 2H), 6.65 (s, 1H) , 6.7 (s, 1H), 6.75 (d, J = 8.67 Hz, 1H), 6.82 (s,
1H), 6.99 (d, J = 5.16 Hz, 1H), 7.22 (m, 3H), 7.35 (s, 1H) , 7.37 (s, 1H), 7.45 (s, 1H), 7.64 (s, 1H),
13
8.02 (dd, J = 8.67, 2.46 Hz, 1H), 8.44 (d, J = 5.16 Hz, 1H), 8.52 (d, J = 2.46 Hz, 1H). C-NMR
(CDCl3) δ 21.58, 39.88, 55.28, 67.96, 111.02, 111.22, 113.77, 115.57, 118.95, 120.17, 121.19, 121.73,
127.98, 128.05, 128.60, 128.71, 130.27, 132.85, 137.18, 137.43, 140.04, 140.99, 145.55, 150.00,
151.41, 155.21, 159.74, 164.16; positive ion ESI-MS m/z 510.1909 (M+Na)+.
2-(3-(3-Methoxy-5-methylphenyl)-4-(2-(6-fluoropyridin-3-yl)pyridin-4-yl)-1H-pyrazole-1-yl)-
acetonitrile (5j). Flash column chromatography was carried out using hexane/ethyl acetate, 1:1 (v/v) to
give the title compound as a white solid. Yield: 81%. mp 64–66 °C. 1H-NMR (CDCl3) δ 2.19 (s, 3H),
3.61 (s, 3H), 5.12 (s, 2H), 6.67 (s, 1H), 6.7 (s, 1H), 6.82 (s, 1H), 6.9 (dd, J = 8.55, 2.49 Hz, 1H), 7.09
(d, J = 5.08 Hz, 1H), 7.49 (s, 1H), 7.79 (s, 1H), 7.98 (dt, J = 8.55, 2.49 Hz, 1H), 8.48 (s, 1H), 8.49 (s,
1H). 13C-NMR (CDCl3) δ 21.52, 39.97, 55.27, 109.32, 109.82, 111.21, 113.76, 115.43, 119.41, 119.83,
121.73, 130.33, 133.05, 140.08, 141.22, 146.00, 146.20, 150.20, 151.49, 153.84, 159.75, 162.38,
165.57; positive ion ESI-MS m/z 422.1386 (M+Na)+.
3.2. In Vitro Anticancer Screening
Cell line screening was performed at the National Cancer Institute (NCI, Bethesda, MD, USA)
applying the following procedure: the human tumor cell lines of the cancer screening panel are grown
in RPMI 1640 medium containing 5% fetal bovine serum and 2 mM L-glutamine. For a typical
screening experiment, cells are inoculated into 96 well microtiter plates in 100 µL at plating densities
ranging from 5000 to 40,000 cells/well depending on the doubling time of individual cell lines. After
cell inoculation, the microtiter plates are incubated at 37 °C, 5% CO2, 95% air, and 100% relative
humidity for 24 h prior to addition of experimental drugs. After 24 h, two plates of each cell line are
fixed in situ with TCA, to represent a measurement of the cell population for each cell line at the time
of drug addition (Tz).
Experimental drugs are solubilized in dimethyl sulfoxide at 400-fold the desired final maximum test
concentration and stored frozen prior to use. At the time of drug addition, an aliquot of frozen
concentrate is thawed and diluted to twice the desired final maximum test concentration with complete
medium containing 50 µg/mL gentamicin. Additional four, 10-fold or ½log serial dilutions are made to
provide a total of five drug concentrations plus control. Aliquots of 100 µL of these different drug
dilutions are added to the appropriate microtiter wells already containing 100 µL of medium, resulting
in the required final drug concentrations. Following drug addition, the plates are incubated for an
additional 48 h at 37 °C, 5% CO2, 95% air, and 100% relative humidity. For adherent cells, the assay is

Molecules 2015, 20
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terminated by the addition of cold TCA. Cells are fixed in situ by the gentle addition of 50 µL of cold
50% (w/v) TCA (final concentration, 10% TCA) and incubated for 60 min at 4 °C. The supernatant is
discarded, and the plates are washed five times with tap water and air dried. Sulforhodamine B (SRB)
solution (100 µL) at 0.4% (w/v) in 1% acetic acid is added to each well, and plates are incubated for
10 min at room temperature. After staining, unbound dye is removed by washing five times with 1%
acetic acid and the plates are air dried. Bound stain is subsequently solubilized with 10 mM Trizma
base, and the absorbance is read on an automated plate reader at a wavelength of 515 nm. For
suspension cells, the methodology is the same except that the assay is terminated by fixing settled cells
at the bottom of the wells by gently adding 50 µL of 80% TCA (final concentration, 16% TCA). Using
the seven absorbance measurements [time zero, (Tz), control growth, (C), and test growth in the
presence of drug at the five concentration levels (Ti)], the percentage growth is calculated at each of
the drug concentrations levels. Percentage growth inhibition is calculated as:
[(Ti − Tz)/(C − Tz)] × 100 for concentrations for which Ti ≥ Tz.
[(Ti − Tz)/Tz] × 100 for concentrations for which Ti < Tz.
Three dose response parameters are calculated for each experimental agent. Growth inhibition of
50% (IC50) is calculated from [(Ti − Tz)/(C − Tz)] × 100 = 50, which is the drug concentration
resulting in a 50% reduction in the net protein increase (as measured by SRB staining) in control cells
during the drug incubation. The drug concentration resulting in total growth inhibition (TGI) is calculated
from Ti = Tz. The LC50 (concentration of drug resulting in a 50% reduction in the measured protein at
the end of the drug treatment as compared to that at the beginning) indicating a net loss of cells following
treatment is calculated from [(Ti − Tz)/Tz] × 100 = −50. Values are calculated for each of these three
parameters if the level of activity is reached; however, if the effect is not reached or is exceeded, the
value for that parameter is expressed as greater or less than the maximum or minimum concentration tested.
3.3. DNA Topoisomerase IIα Inhibition Screening in Vitro
The DNA topoisomerase IIα inhibitory activity of the compounds was measured as follows [20]:
a mixture containing 200 ng of supercoiled pBR322 plasmid DNA and 1 unit of human DNA
topoisomerase IIα (USB Corp., Cleverland, OH, USA) was incubated with and without the prepared
compounds in the assay buffer (10 mM Tris HCl (pH 7.9) containing 50 mM NaCl, 50 mM KCl,
5 mM MgCl2, 1 mM EDTA, 1 mM ATP, and 15 mg/mL bovine serum albumin) for 30 min at 30 °C.
The reaction in a final volume of 20 mL was quenched by adding 3 mL of 7 mM EDTA. The reaction
products were analyzed on 1% agarose gel at 25 V for 4 h with TAE as the running buffer. Gels were
stained for 30 min in an aqueous solution of ethidium bromide (0.5 μg/mL). DNA bands were
visualized by transillumination with UV light and supercoiled DNA was quantitated using
AlphaImagerTM (Alpha Innotech Corporation, San Leandro, CA, USA).
4. Conclusions
The preliminary screening of this new set of compounds shed light on the effect of substitution on
the distal pyridinyl moiety. Compounds 5c and 5h revealed a broad spectrum of activity while 5e
showed a high degree of selectivity toward several NCI cancerous cell lines. On that basis, we could

Molecules 2015, 20
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say that 5c and 5h react with common cancer-related macromolecules while 5e acts more specifically
on some macromolecule responsible for driving the tumorigenicity of the SR leukemia cell line.
COMPARE analysis has suggested one of these macromolecules is topoisomerase IIα enzyme, but on
applying the in vitro screening assay for topoisomerase IIα enzyme, 5e and 5i have not shown any
topoisomease IIα activity. In sum, these compounds still have an unknown mechanism of action and
finding out the molecular target for this activity will be our next challenge.
Acknowledgments
This research was supported by the Korea Institute of Science and Technology and a research grant
of Kwangwoon University (2014). We would like to express our gratitude and thanks to the National
Cancer Institute (NCI), Bethesda MD, USA for performing the anticancer testing of our new compounds.
We would like to thank Hyun-Mee Park from Advanced Analysis Center of Korea Institute of Science
and Technology (KIST) for Mass Spectroscopy analysis funded by Grant No. KIST2V03612.
Author Contributions
Mohammad M. Al-Sanea: Chemical Synthesis and manuscript preparation. Ahmed Elkamhawy and
Ahmed Zakaria: equally performed the Experimental analysis data. Byung Sun Park: Participate in the
Experimental work. Youngjoo Kwon: Topoisomerase II α enzyme assay. So Ha Lee, Sang Woo Lee
and In Tae Kim: work design.
Conflicts of Interest
The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are available from the authors.
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