Synthesis and the interaction of 2-(1H-pyrazol-4-yl)-1H-imidazo[4,5-f][1,10]phenanthrolines with telomeric DNA as lung cancer inhibitors

Article abstract of DOI:10.1016/j.ejmech.2017.03.030

A novel series of 2-(1H-pyrazol-4-yl)-1H-imidazo[4,5-f][1,10]phenanthrolines were designed, synthesized and evaluated for their antitumor activity against lung adenocarcinoma by CCK-8 assay, electrophoretic mobility shift assay (EMSA), UV-melting study, wound healing assay and docking study. These compounds showed good inhibitory activities against lung adenocarcinoma. Especially compound 12c exhibited potential antiproliferative activity against A549?cell line with the half maximal inhibitory concentration (IC₅₀) value of 1.48?μM, which was a more potent inhibitor than cisplatin (IC₅₀?=?12.08?μM) and leading compound 2 (IC₅₀?=?1.69?μM), and the maximum cell inhibitory rate being up to 98.40%. Moreover, further experiments demonstrated that compounds 12a–d can strongly interact with telomeric DNA to stabilize G-quadruplex DNA with increased ΔTm values from 12.44 to 20.54?°C at a ratio of DNA to compound 1:10. These results implied that growth inhibition of A549?cells mediated by these phenanthroline derivatives is possibly positively correlated to the fact their interaction with telomeric G-quadruplexs.

Full text of DOI:10.1016/j.ejmech.2017.03.030

European Journal of Medicinal Chemistry 133 (2017) 36e49
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Synthesis and the interaction of 2-(1H-pyrazol-4-yl)-1H-imidazo[4,5-f]
[1,10]phenanthrolines with telomeric DNA as lung cancer inhibitors
,
**
Jiachun Liu a, Mei Chen a, Yanli Wang a, Xiaoyin Zhao a, Sijia Wang a, Yanling Wu b
,
,
*
Wen Zhang a
a Lab of Chemical Biology and Molecular Drug Design, College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou, 310014, China
b Lab of Molecular Immunology, Virus Inspection Department, Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou, 310051, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of 2-(1H-pyrazol-4-yl)-1H-imidazo[4,5-f][1,10]phenanthrolines were designed, synthe-
sized and evaluated for their antitumor activity against lung adenocarcinoma by CCK-8 assay, electro-
phoretic mobility shift assay (EMSA), UV-melting study, wound healing assay and docking study. These
compounds showed good inhibitory activities against lung adenocarcinoma. Especially compound 12c
exhibited potential antiproliferative activity against A549 cell line with the half maximal inhibitory
Received 8 December 2016
Received in revised form
4 February 2017
Accepted 15 March 2017
Available online 18 March 2017
concentration (IC50) value of 1.48
and leading compound 2 (IC50 ¼ 1.69
Moreover, further experiments demonstrated that compounds 12aed can strongly interact with telo-
meric DNA to stabilize G-quadruplex DNA with increased
Tm values from 12.44 to 20.54 ꢀC at a ratio of
m
M, which was a more potent inhibitor than cisplatin (IC50 ¼ 12.08
mM)
m
M), and the maximum cell inhibitory rate being up to 98.40%.
Keywords:
Phenanthroline
Lung adenocarcinoma
Antiproliferative activity
Telomeric G-quadruplex
D
DNA to compound 1:10. These results implied that growth inhibition of A549 cells mediated by these
phenanthroline derivatives is possibly positively correlated to the fact their interaction with telomeric G-
quadruplexs.
© 2017 Elsevier Masson SAS. All rights reserved.
1. Introduction
year survival from the time of diagnosis [7].
Despite advances in various treatment strategies including
Cancer is a serious public health problem resulting in high
morbidity, mortality, and previously incogitable losses in the cur-
rent world [1]. Lung cancer, a highly invasive and rapidly metas-
tasizing cancer, is the top killer in both men and women, which is
classified into two broad histologic classes in terms of their growth
and spread differently: small-cell lung carcinomas and non-small
cell lung carcinomas (NSCLCs). NSCLC is the most common type
that accounts for 80% of lung cancers and includes mainly
squamous-cell carcinoma, adenocarcinoma, and large-cell carci-
noma [2,3]. At present, the mortality rate remains high for NSCLC
patients with relapse occurring within 5 years in 35e50% of pa-
tients [4e6], and approximately 15% of patients will have only a 5-
surgery, chemotherapy, radiotherapy, even target therapy over
recent decades, some serious problems affect the treatment
outcome. Gamma radiotherapy plays a crucial role, but clinical
outcome is limited because of radio-resistance resulting from tu-
mor heterogeneity. Conventional chemotherapy plays a major role
in the treatment of lung cancer, unfortunately such chemotherapy
is limited by the risk of severe toxicity to nearby normal paren-
chyma, which may result in paralyzed lung function [8]. Cisplatin
(DDP) is a well-known chemotherapeutic drug which has been
used for treatment of numerous human cancers including bladder,
head and neck, lung, ovarian, and testicular cancers, but numerous
undesirable effects can't be ignored, such as severe kidney prob-
lems, gastrointestinal disorders, allergic reactions, hearing loss,
hemorrhage, and lowered immunity leading to infections espe-
cially in younger patients [9]. Additionally, NSCLCs tend to be
intrinsically resistant to multiple anticancer agents and present
acquired resistance to epidermal growth factor receptor tyrosine
kinase inhibitors, which is an important problem in the treatment
of NSCLC [10,11]. Drug resistance has been observed in many pa-
tients who have relapsed from DDP treatment. The DDP-resistant
* Corresponding author. Lab of Chemical Biology and Molecular Drug Design,
College of Pharmaceutical Science, Zhejiang University of Technology, 18 Chaowang
Road, Hangzhou, 310014, China.
** Corresponding author. Lab of Molecular Immunology, Virus Inspection
Department, Zhejiang Provincial Center for Disease Control and Prevention, 630
Xincheng Road, Hangzhou, 310014, China.
E-mail addresses: ylwu@cdc.zj.cn (Y. Wu), wzhang63@zjut.edu.cn (W. Zhang).
http://dx.doi.org/10.1016/j.ejmech.2017.03.030
0223-5234/© 2017 Elsevier Masson SAS. All rights reserved.

J. Liu et al. / European Journal of Medicinal Chemistry 133 (2017) 36e49
37
A549 variant cells have been undermining the therapeutic effect of
drugs because of drug resistance [12]. Thus, there is an urgent need
to develop novel anticancer agents that are highly effective and
preferentially induce apoptosis of lung cancers rather than normal
cells [13].
become a rational and promising approach in anticancer strategies.
Many nitrogen-containing polycyclic systems are present in a
variety of natural and synthetic biologically active agents. Marine
compounds with pyridoacridine skeletons have been reported to
exhibit interesting antitumor activities. Of which, ascididemin is
one of the important examples of these compounds that can
damage DNA oxidatively via a thiol-dependent conversion of oxy-
gen to DNA-cleaving radicals [32,33]. As shown in Scheme 1,
compound 1, a substitution product of ascididemin bearing 2,2'-
bipyridine moiety, showed good antitumor activity against
Human telomeres, special sequences containing (TTAGGG)n re-
peats [14e16], are essential structures at the ends of all eukaryotic
chromosomes to maintain genome stability and cell proliferation
by protecting chromosome ends [17,18]. Nevertheless, telomeres
will be shortened gradually in the process of cell differentiation and
proliferation, which suggests that cells are destined to senesce and
die. Telomerase is a cellular reverse transcriptase which can
maintain and repair chromosome integrity and stability through
adding telomere repeats to telomere DNA [19], as shown in Fig. 1. In
normal cells, the activity of telomerase appears to be quite strictly
regulated, except that it can only be detected in stem cells, he-
matopoietic cells and reproductive cells. However, telomerase is
activated and displays vigorous activity in 85e90% cancer cells
[20e23], which indicates that telomerase may maintain the length
of telomeric DNA and promote the process of cell cycle, so that the
cancer cells endow more vitality and inhibit apoptosis in process of
proliferation. Hence, inhibiting the activity of telomerase may be a
novel strategy for cancer chemotherapy. DNA is the principal ge-
netic molecule in biological systems, especially right handed
duplex structural B-DNA. However, DNA structure is polymorphic, a
number of non-B DNA secondary structures, such as Z-DNAs,
hairpins, cruciforms, and G-quadruplexes, have been found and
identified to be unstable and mutagenic [24e26]. G-quadruplexes
are important DNA secondary structures formed by stacking G-
tetrads in specific G-rich sequences with biological significance,
such as human telomeres and oncogene-promoter regions (bcl-2,
A549 cells with IC50 value of
8 nM [34]. Likewise, 1,10-
phenanthrolines bearing 2,2'-bipyridine moiety are applied
importantly in biological sciences, as well as widely in chemistry,
physics, and material, which implied that 2,2'-bipyridine moiety
seems to have some untapped properties [35e38]. Several mech-
anisms have elucidated the anticancer activities of 1,10-
phenanthrolines, such as the inhibition of DNA synthesis, and G-
quadruplex DNA binding property because of the conjugate struc-
ture with rigid plane of 1,10-phenanthrolines that can provide a
basis for efficient
pꢁp stacking interactions with the terminal G-
tetrads [39e41]. In addition, imidazole heterocycles are receiving
growing attention with a wide range of applications due to the
potency of the imidazole moiety that can be attributed to its
hydrogen bond donor-acceptor capability as well as its high affinity
for metals, which are presented in many protein active sites [42]. As
seen in Scheme 1, compound 2 possessing 1H-imidazo [4,5-f] [1,10]
phenanthroline core showed a surprising antitumor activity against
A549 cells with IC50 value of 0.85
expression in A549 cells via NF-
m
M, which inhibited c-myc gene
k
B pathway [43]. Similarly, as the
bioisostere of imidazole, pyrazole represents a key structural motif
in heterocyclic chemistry and occupies a prominent position in
antitumor agents, compound 3 (Scheme 1) showed good activity
against A549 cells with the most important GI50 (the concentration
for 50% of maximal inhibition of cell proliferation) value of
c-myc, c-kit, k-RAS, PDGF-A, HIF-1a, VEGF and TK1) [27]. Telomere
can fold into G-quadruplex structures through four guanines con-
nected by cyclic Hoogsteen hydrogen bonding of guanine bases in
the presence of several cations, which can effectively inhibit the
activity of telomerase and promote apoptosis of cancer cells
[28e30]. G-quadruplexes play an important role in the cell cycle
and control diverse processes, such as telomere maintenance,
which suggests that G-quadruplexes have a big potential as targets
to develop anticancer agents. However, none of the reported G-
quadruplex-interactive small molecules have entered past phase II
clinical trials [31]. Therefore, design and discovery of such small
molecules that can induce the formation of G-quadruplexes and
stabilize the G-quadruplex structures in human telomere has
13.20 mM [44].
With these results in mind, we designed and synthesized
twenty-one novel compounds using a structure-based design
strategy using compounds 1, 2 and 3 as the lead compounds. Based
on our previous and other groups' studies [45e47], these small
molecules with an extended planar aromatic system can facilitate
the stacking action on the G-quartet through
p-p interactions,
which are similar with a G-quartet in size and shape. 1H-imidazo
[4,5-f][1,10]phenanthroline moiety possesses an extended planar
aromatic system, suggesting that the special structure can provide
great advantages for the development of small molecules to spe-
cifically target G-quadruplexes. Hence, 1H-imidazo[4,5-f][1,10]
phenanthroline moiety was used as key structure skeleton in pre-
sent work. Furthermore, 1,3-disubstituted pyrazoles were intro-
duced into the imidazole ring at C-2 position to make the
synthesized compounds possessing both anti-proliferation activ-
ities and stabilized abilities to G-quadruplexes. Detailed designs are
shown in Scheme 1. As computer-aided molecular simulation
technique is currently regarded as a very useful tool in design of
small molecular drugs to expose the interaction mechanism be-
tween drugs and biomacromolecules, we, herein, utilized LibDock
in Discovery Studio to investigate interactions of compounds (2 and
12aed) with the target G-quadruplex (PDB ID: 3CE5). As shown in
Fig. S1, the results illustrated that the phenanthroline skeleton of
compounds 12a-d could mainly stack on the end G-quartet through
intermolecular
p-p interactions (yellow solid line) and hydrogen
bond interaction (green dash line), and the benzyl groups at the N-1
position of pyrazole moiety extended respectively to the groove or
loop region of G-quadruplex, both of which could contribute to the
stabilization of the G-quadruplex structure. Compounds 12a-
d were mainly adjacent to the residues, such as DG15, DG21, DA2,
Fig. 1. Telomerase maintains and repairs the length of telomeric DNA. While telomeric
DNA forms G-quadruplex structures, the activity of telomerase was inhibited
effectively.

38
J. Liu et al. / European Journal of Medicinal Chemistry 133 (2017) 36e49
Scheme 1. Structure of the leading compounds and design of the target compounds.
DA14, DT13, which were shown in Table 1. However, the p-bro-
mophenyl moiety of compound 2 embedded into the groove of G-
quadruplex and the imidazole moiety interacted with the base
residues (DA2, DG3 and DG21) by hydrogen bonding, not the
phenanthroline skeleton interacting with G-quartets, suggesting
that the 1,3-disubstituted pyrazole moiety seems to be more ad-
vantageous than the p-bromophenyl moiety to make the phenan-
throline skeleton to stabilize the G-quadruplex in present docking
study. These results provided certain theoretical evidence for the
following researches.
previous paper [48e51]. Compounds 6, 7, 8 and 15c were synthe-
sized through the substitution reaction by heating a mixture of
compound
4
and iodomethane, (chloromethyl)benzene, 3-
dimethylaminopropyl chloride and 3,6-dichloropyridazine under
different inorganic base condition in DMF, respectively. Subse-
quently compound 9 was subjected to a condensation reaction with
4, 5, 6, 7, 8 and 15c in glacial acetic acid (90e100 ꢀC) for 6 h to give
the target compounds 10e15, respectively. The structures of the
desired compounds were confirmed by 1H-NMR, a part of 13C-NMR,
and mass spectral analysis.
2. Chemistry
3. Results
The synthetic route of the 2-(1H-pyrazol-4-yl)-1H-imidazo[4,5-
f][1,10]phenanthrolines 10e15 is depicted in Scheme 2. Compounds
4, 5, 9 and 3,6-dichloropyridazine had already been reported in a
3.1. In vitro anti-tumor activity evaluation by CCK-8 assay [52].
To evaluate the antiproliferative abilities of novel synthetic
Table 1
The LibDock scores and docking interactions of compounds (2 and 12a-d) with human telomeric G-quadruplex (PDB ID: 3CE5).
Compound
LibDock score (kcal/mol)
89.82
Interacting residues
Interacting atoms
2
A:DA2:O3’;
A:DG3:OP2;
B:DG21
Molecule:N15- DG21:H21;
Molecule:H32- DG3:OP2;
Molecule:H32- DA2:O30
12a
12b
12c
12d
100.14
84.10
B:DG21;
Phenanthroline - DG21, DG15
DG21:O50-Molecule-H44; DG15:N2-Molecule:N15
DG21-Molecule:N17
B:DG15;
B:DG21:O50
B:DA14;
B:DT13;
A:DA2
Phenanthroline - DA2, DA14, DT13;
97.89
B:DG21;
Phenanthroline - DG21, DG15;
DG15-Molecule:N17;
DG21:O50-Molecule-H43;
Pyrazole - DG21;
B:DG15;
B:DG21:O50
B:DG21;
100.28
B:DG15;
B:DG21:O40
Phenanthroline - DG21;
DG21-Molecule-N17
DG21:O40-Molecule-H43

J. Liu et al. / European Journal of Medicinal Chemistry 133 (2017) 36e49
39
Scheme 2. Synthetic routes of compounds 10e15.
compounds 10e15 to human lung adenocarcinoma lines
(A549 cells) by Cell Counting Kit-8 technique, firstly, A549 cells
were treated with 10 mM of compounds 10e15 for 36 h, results of
treated with two-fold serial dilution of cisplatin, leading compound
2, and 12aed for 36 h, respectively. As shown in Fig. 3 and Table 2,
compounds 12aed presented excellent activity against A549 cells
progressive cell growth inhibition were shown in Fig. 2. All com-
pounds exhibited different degrees of antiproliferative activity
against A549 cells. Especially, compounds 12aed represented the
with IC50 values in the range of 1.48e2.75
possessed outstanding antiproliferative activity with IC50 value of
mM. The compound 12c
1.48 0.06
(1.69
(12.08
m
M, which was almost the same as leading compound 2
0.07 M), but stronger than that of cisplatin
0.41 M). Compounds 12aec showed stronger activity
most potent activity at 10
mM, which was stronger than that of
m
cisplatin and leading compound 2. Secondly, A549 cells were
m

40
J. Liu et al. / European Journal of Medicinal Chemistry 133 (2017) 36e49
Fig. 2. A549 cells were treated with 10
mM of compounds 10e15 for 36 h. Cell viability was determined using Cell Counting Kit-8 assay. Cisplatin and compound 2: positive controls;
DMSO is 1% DMSO solution in RPMI 1640 as a reference.
Fig. 3. A549 cells were treated using compounds 12aed with 0, 1.25, 2.5, 5, 10 and 20 mM for 36 h. Cell viability was determined using Cell Counting Kit-8 assay. Cisplatin and
compound 2: positive control; 0 mM is 1% DMSO solution in RPMI 1640 as a reference.
Table 2
IC50 values (mM) for the A549 and MRC-5 cell lines treated with the desired compounds 12aed and the positive control drugs (2 and cisplatin). The values are expressed as the
mean SD (triplicates).
Cell line
12a
12b
12c
12d
2
cisplatin
A549
MRC-5
SIb
1.78 0.06a
3.80 0.05a
2.13
2.08 0.03
4.17 0.03a
2.00
1.48 0.06
3.83 0.07a
2.58
2.75 0.09
4.76 0.04a
1.73
1.69 0.07a
6.88 0.01a
4.07
12.08 0.41a
21.55 0.26a
1.78
a
Statistically different to lung cancer cell line (A549 and MRC-5) (P < 0.05).
Selectivity-Index.
b
than leading compound 2 at 5, 10 and 20
tunately, the IC50 values of 12a, 12b and 12d were slightly higher
m
M, respectively. Unfor-
than that of leading compound 2.
Also, we have carried out the cytotoxicity experiments with

J. Liu et al. / European Journal of Medicinal Chemistry 133 (2017) 36e49
41
normal human fetal lung fibroblast cells (MRC-5 cells). MRC-5 cells
were treated with both positive controls (cisplatin and leading
compound 2) and the desired compounds 12aed, under the same
conditions. Results were showed in Table 2 and Fig. 4. Compounds
12aed presented moderate cytotoxicity against normal MRC-5 cells
stabilize the DNA-compound complexes. The sequences of newly
designed 26nt telomeric DNAs for novel DNA secondary structure
formation assay were listed in Table 3, including guanine-rich
single stranded-DNA (G-rich DNA), cytosine-rich single stranded-
DNA (C-rich DNA) complementary to G-rich DNA, double-
stranded DNA (ds-DNA) and Mutated single stranded-DNA (Mut-
DNA) with a single G-to-C mutation in the middle of chains of three
guanines, impossibly to form any G-quadruplex.
As shown in Fig. 5AeD, EMSA images indicated the interaction
of compounds 12aed with the G-rich DNA sequences. On gels there
are mainly two bands of DNA secondary conformations obviously
observed in G-rich DNA: the bright band migrated faster and the
fainter band migrated slower roughly at the 20 bp marker level
which both may be ascribed to intermolecular G-quadruplexes
relative to a double-stranded Marker on the left lane in each gel
[53,54]. Based on the EMSA results, 12c exhibited strongest
concentration-dependent interaction with G-quadruplexes, with
with IC50 values in the range of 3.80e4.76 mM, which are 1.73e2.58-
fold higher than those of A549 cells. Among them, 12c exhibited the
2.58-fold cytotoxicity against A549 cells over against normal MRC-
5 cells, indicating it has definite selectivity for cancer cells, but poor
selectivity compared to leading compound 2 with 4.07-fold dif-
ference. Collectively, these results suggested that compounds
12aed showed excellent cytotoxicity against A549 cells and
selectivity to some extent for A549 cells, implying that they may be
further explored as leading compounds for cancer inhibitors.
3.2. Preliminary analysis of interaction mechanism by
electrophoretic mobility shift assay (EMSA) [45]
the bright bands fading away up to 20 mM, but not compounds 12a,
12b and 12 d at the identical concentration level. Likewise, we
completed the same investigation with Mut-DNA and ds-DNA se-
quences as negative controls in the presence of 12c. Mut-DNA
approximately migrated to 15 bp position and ds-DNA displayed
normal mobility behaviors (nearly 25 bp) relative to the Marker. No
In order to explore the cell growth inhibition mechanism, based
on our previous study [45] and structural property of compounds
with large conjugated and planar aromaticity, we examined the
interaction of a serial dilution of compounds (2 and 12aed) with
telomeric DNA in Tris buffer (10 mM TriseHCl, 10 mM KCl, 0.1 mM
EDTA, pH ¼ 7.4) by EMSA assay to detect if these compounds have
the ability to induce the formation of DNA secondary structures,
such as the G-quadruplexes, from the 26nt telomeric G-rich single
stranded-DNA sequence containing (GGGTTA)4 repeats, and
effects of 12c, even at high concentration levels (40 mM), on both
negative control DNAs were observed in Fig. 5EeF, suggesting that
12c has strongly specificity for the G-quadruplexes formed in G-
rich DNA, a (GGGTTA)4TT sequence. Likewise, EMSA images of
Fig. 4. MRC-5 cells were treated using compounds 12aed with 1.25, 2.5, 5, 10 and 20 mM for 36 h. Cell viability was determined using Cell Counting Kit-8 assay. Cisplatin and
compound 2: positive control; 0 mM is 1% DMSO solution in DMEM as a reference.
Table 3
The sequences of DNA oligomers used in this study.
Oligomer
Sequence
Tel26nt-G-rich-strand DNA (G-rich DNA)
Tel26nt-C-rich-strand DNA (C-rich DNA)
Tel26nt-Mutated-strand DNA (Mut-DNA)
Tel26nt-double-stranded DNA (ds-DNA)
5’-ttaGGGttaGGGttaGGGttaGGGtt-30
5’-aaCCCtaaCCCtaaCCCtaaCCCtaa-30
5’-ttaGCGttaGCGttaGCGttaGCGtt-30
5’-ttaGGGttaGGGttaGGGttaGGGtt-30
3’-aatCCCaatCCCaatCCCaatCCCaa-50

42
J. Liu et al. / European Journal of Medicinal Chemistry 133 (2017) 36e49
Fig. 5. The ability of phenanthroline derivatives (2 and 12aed) to promote the formation of G-quadruplex structure in the 26nt telomeric DNA sequences. (A-D) EMSA images of
Tel26nt G-rich DNA in the presence and absence of 12aed at the concentration of 0, 1.25, 2.5, 5, 10, and 20 M, increasing from left to right. (E) Tel26nt ds-DNA and (F) Tel26nt Mut-
DNA were used as negative controls, in which 12c concentration was ranged from 0 to 40 M. (G-I) EMSA images of G-rich DNA (G), ds-DNA (H) and Mut-DNA (I) in the presence and
absence of 2 concentration was ranged from 0 to 40 M. Base pairs for used Marker are put on the left side.
m
m
m
leading compound 2 were observed in Fig. 5GeI. In terms of the G-
rich DNA sequence, no effects were observed in gels, implied that
compound 2 seems unlikely to induce the formation of new DNA
secondary structures (Fig. 5G). Similarly, no effects of 2 on both
negative control DNAs (ds-DNA and Mut-DNA), even at high con-
DNA could form the G-quadruplexes under our experiment con-
ditions. The
Tm values, relative to the Tm (39.34 ꢀC) of the G-rich
DNA alone, increased greatly from 10.96 ꢀC to 20.54 ꢀC in the
presence of compounds (20 M). Especially, the Tm values of
D
m
D
12aed increased higher than that of 2 (10.96 ꢀC) to the G-rich DNA
G-quadruplexes. Combined with the present EMSA analysis and
previous research [42], no new secondary conformations were
observed in the control DNAs, including C-rich DNA, Mut-DNA, ds-
DNA, suggesting that the control DNAs were unlikely to form G-
quadruplexes under the present experimental conditions.
centration levels (40 mM), were observed in Fig. 5HeI.
3.3. Binding of phenanthroline derivatives to telomeric G-
quadruplexes [45]
UV-melting study is widely used to determine thermodynamic
properties of the folded nucleic acid structures including their
stability and interaction with ligands [55,56]. The UV-melting curve
of duplex nucleic acids is generally closely related to a hyper-
chromic shift at 260 nm, while the melting curve of G-quadruplex,
i.e. the absorbance of the G-rich DNA decreases with the temper-
ature increasing, with a standard inverted “S” curve presented, is
associated with a hypochromic shift at 295 nm [57e58]. Thus, in
order to explore binding affinity and mechanism of phenanthroline
derivatives with the 26nt telomeric DNAs, the UV absorbance of the
nucleic acids was monitored as a function of temperature and then
obtained the melting temperature (Tm,ꢀC) that is the mid-point of a
melting curve at which the complex is 50% dissociated for
quadruplex-forming oligonucleotides in the presence and absence
of compounds (2 and 12aed). The tested 26nt telomeric G-rich
DNAs for the specific binding assay were listed in Table 3. As shown
in Fig. 6AeE and Table 4, the melting curve of the G-rich DNA
presented a standard inverted “S” type at 295 nm, which is char-
acteristic of a G-quadruplex structure, suggesting that the G-rich
3.4. Wound healing assay [45].
Cell mobility plays a vital role in tumor metastasis and prognosis
of cancer. Therefore, we assessed the effect of compounds (2 and
12a-d) on the migration of A549 cells in an in-vitro wound healing
model. As seen in Fig. S2, an empty space was created with a pipet
tip streaked in cell culture, sequentially to culture for 48 h in the
presence and absence of compounds (2 and 12a-d) and observe the
space. The empty space was reduced seriously in size in the absence
of compounds because of cell proliferation and migration. The re-
sults in Fig. 7 and Table 5 showed that cell inhibitory rates for
A549 cells are greater than 16.09% after 48 h treatment with all
compounds, while all novel synthesized compounds 12a-d gave
rise to higher cell inhibitory rates for A549 than 2 for target cells at
the experimental concentrations. In particular, we observed the fact
that compound 12c was strongest inhibitor to the cell migration
with inhibitory rates of 98.40% ( 2.03) for A549 cells. These data
are in accord with foregoing EMSA and UV-melting study results

J. Liu et al. / European Journal of Medicinal Chemistry 133 (2017) 36e49
43
Fig. 6. Tm (ꢀC) values for Tel26nt-G-rich-strand DNA (G-rich DNA, 2
(E) at the concentration of 0 and 20
M, respectively. The ordinate shows the relative absorbance of the samples. The abscissa shows the temperature inꢀC. The melting profiles for
mM) interaction with phenanthroline derivatives 12a (A), 12b (B), 12c (C), 12d (D) and a reference compound 2
m
the quardruplex formed by the telomeric DNA were acquired at 295 nm.
Table 4
D
Tm (ꢀC) values for Tel-26nt G-rich DNA interaction with phenanthroline derivatives 12aed.
D
Tm (ꢀC)a
2
12a
12b
12c
12d
G-rich DNA/compound (1:10)
10.96 0.06
12.44 0.07
13.52 1.11
20.54 0.09
16.31 0.05
a
Standard deviation is given.

44
J. Liu et al. / European Journal of Medicinal Chemistry 133 (2017) 36e49
5. Conclusion
Taken together, these findings provided reasonable evidences
that all the new synthesized compounds possessed potent anti-
proliferative activity against A549 cells. Compounds 12aed showed
stronger inhibitory activities with IC50 values in the range of
1.48e2.75
A549 cell line with IC50 value of 1.48
potent than cisplatin (IC50 ¼ 12.08 M) and leading compound 2
(IC50 ¼ 1.69 M). Moreover, compounds 12aed exhibited signifi-
mM, especially compound 12c did the strongest against
mM, which was even more
m
m
cant interactions with telomere 26nt G-rich DNA to stabilize the G-
quadruplexes based on docking and UV-melting study results,
hinting that these evaluated compounds can inhibit the prolifera-
tion of A549 cells greatly possibly through interacting with the 26nt
telomeric G-quadruplexes, but further mechanism need to be
further explored. Similarly, the inhibitory effects of 12a-d on the
mobility of A549 cells were outstanding, indicating that phenan-
throlines bearing 1-benzyl-3-phenyl-1H-pyrazole moiety could
inhibit effectively lung cancer invasion and metastasis. All in all, the
study has demonstrated that the imidazo[4,5-f][1,10]phenanthro-
lines containing the 1,3-substituted pyrazol moiety could be further
developed as new inhibitors against A549 cells and provide theory
basis supporting for phenanthroline drug development.
Fig. 7. The bar chart of Cell inhibitory rate (%) for the migration of A549 cells treated
with compounds (2 and 12a-d) for 48 h, which showed mean results of three inde-
pendent experiments in triplicates. The % cell inhibitory rate was calculated by the
equation based on the results in Fig. S2: cell inhibitory rate (%)
¼
(1-Ddrug/
D
control) ꢂ 100%, where Ddrug is mean distance of cell migration in drug group, Dcontrol is
mean distance of cell migration in control group; The % cell inhibitory rate for control
is zero. Values are the means S.D. (P < 0.05).
and clearly demonstrate that compounds 12a-d suppressed the
mobility of tumor cells.
6. Experimental protocols
6.1. Chemistry
4. Discussion
All solvents and chemicals were analytic pure, used without
further purification, and obtained from commercial suppliers.
However, phosphorus oxychloride and dimethyl formamide (DMF)
were further dried by vacuum distillation based on the conditions
of Vilsmeier-Haack reaction. The melting points of compounds
were observed on the melting point detector (XT-5A). Reaction
processes were tracked by TLC on silica gel-precoated F254 Merck
plates and the thin layer plates were examined under the UV lamps
(254 nm and 365 nm). 1H-NMR spectra were recorded in pure
DMSO-d6 on Bruker NMR spectrometers (AVANCEIII500MHz) using
tetramethylsilane (TMS) as internal standard. Chemical shifts were
Through evaluation of antiproliferative activities of synthesized
compounds 10e15 to A549 cells and human normal MRC-5 cells,
the results revealed following several structure-activity relation-
ships. Firstly, the antiproliferative activities of compounds 10e15
are not significantly influenced by the R1 substituent groups on the
phenyl ring. Secondly, compounds 12aed presented more potent
activity than other synthesized compounds, suggesting that N-
substituted benzyl group at pyrazol moiety seems to play a major
role in antiproliferative activity against A549 cells. Compound 12c
showed the best antitumor activity against A549 cells, but had not
big difference in the selectivity to MRC-5 cells (SI ¼ 2.58), which
was better than that of cisplatin (SI ¼ 1.78) and weaker than that of
compound 2 (SI ¼ 4.07). Thirdly, it is particularly encouraging that
compounds 12a-d had better stability ability to human telomere
DNA G-quadruplexes based on UV-melting study results, compared
to leading compound 2, which are consistent with docking studies,
suggesting that the 1-benzyl-3-phenyl-1H-pyrazole moiety could
be more advantageous than the p-bromophenyl moiety to make the
expressed in d, ppm. Mass spectra were measured on an Agilent
6210 TOF LC/MS (USA).
6.1.1. General synthetic procedure for the key intermediates 6aed
To a solution of compounds 4aed (1.0 mmoL) and iodomethane
(0.14 g, 1.0 mmol) in 10.0 Ml DMF were added potassium carbonate
(0.14 g, 1.0 mmol). The reaction mixture was stirred at 50e60 ꢀC for
4e6 h until the completion of the reaction checked by thin-layer
chromatography (TLC). The resulting reaction mixture was
poured into 50 mL water, stirred for 5 min, extracted with ethyl
acetate (3 ꢂ 40 mL), dried with Na2SO4, filtered, concentrated and
finally purified by silica gel chromatography (dichloromethane/n-
hexane, 60:1) to afford pure white crystals 6aed. The yield, melting
point and spectral data of each compound are given below.
phenanthroline skeleton, and also
p-p interactions seem to be
more effective than hydrogen bonding interaction to stabilize DNA
G-quadruplexs. Similarly, the inhibitory effects of 12a-d on the
mobility of A549 cells were more significant than that of compound
2 under the same condition, implied that the introduction of 1-
benzyl-3-phenyl-1H-pyrazole moiety to phenanthroline skeleton
was successful.
Table 5
Cell inhibitory rate (%) for the migration of A549 cells treated with compounds (2 and 12a-d) for 48 h. Values are evaluated with the mean of triplicates.
Concentration
Cell inhibitory rate (%)a
2
12a
12b
12c
12d
0.05
m
M
M
M
16.09 0.86
23.67 1.15
56.11 1.66
34.46 0.99
86.57 3.04
93.32 2.10
24.72 1.19
54.18 1.26
94.41 2.86
48.31 2.03
78.81 1.07
98.40 2.03
16.34 1.02
54.65 2.34
85.77 2.16
0.1
0.2
m
m
a
Standard deviation is given.

J. Liu et al. / European Journal of Medicinal Chemistry 133 (2017) 36e49
45
6.1.1.1. 3-(4-fluorophenyl)-1-methyl-1H-pyrazole-4-carbaldehyde
(6a). White crystal; yield: 66.6%; m.p. 98e100 ꢀC. 1H-NMR (CDCl3-
hydrochloride (1.73 g, 11.0 mmol) was added to the reaction
mixture. Further, the mixture was stirred at 70 ꢀC for 5e8 h in the
presence of the catalyst potassium iodide. Once the reaction
finished, the mixture was poured into ice water and extracted with
ethyl acetate (3 ꢂ 40 mL), dried with Na2SO4, filtered, concentrated
and finally purified by silica gel chromatography (dichloro-
methane/methanol, 40:1) to afford pure yellow liquid 8aed. The
yield, melting point and spectral data of each compound are given
below.
d1, 500 MHz, ppm):
d9.909 (s, 1H, CHOeH), 7.990 (s, 1H, CH),
7.767e7.727 (m, 2H, Ph-H), 7.179e7.133 (m, 2H, Ph-H), 3.993 (s, 3H,
CH3).
6.1.1.2. 3-(4-chlorophenyl)-1-methyl-1H-pyrazole-4-carbaldehyde
(6b). White crystal; yield: 68.4%; m.p.87e89 ꢀC. 1H-NMR (DMSO-
d6, 500 MHz, ppm):
d9.857 (s, 1H, CHOeH), 8.548 (s, 1H, CH),
7.903e7.876 (m, 2H, Ph-H), 7.536e7.509 (m, 2H, Ph-H), 3.961 (s, 3H,
CH3).
6.1.3.1. 1-(3-(dimethylamino)propyl)-3-(4-fluorophenyl)-1H-pyr-
azole-4-carbaldehyde (8a). Yellow liquid; yield: 44.2%. 1H-NMR
6.1.1.3. 3-(4-bromophenyl)-1-methyl-1H-pyrazole-4-carbaldehyde
(CDCl3-d1, 500 MHz, ppm): d9.909 (s, 1H, CHOeH), 8.057 (s, 1H, CH),
(6c). White crystal; yield: 70.1%; m.p.89e91 ꢀC. 1H-NMR (CDCl3-d1,
7.790e7.750 (m, 2H, Ph-H), 7.173e7.126 (m, 2H, Ph-H), 4.281e4.254
(t, J1 ¼ 6.75 Hz, 2H, CH2), 2.325e2.297 (t, J1 ¼7.0 Hz, 2H, CH2), 2.251
(s, 6H,CH3), 2.131e2.076 (m, 2H, CH2).
500 MHz, ppm):
d9.915 (s, 1H, CHOeH), 7.995 (s, 1H, CH),
7.666e7.641 (m, 2H, Ph-H), 7.608e7.583 (m, 2H, Ph-H), 3.996 (s, 3H,
CH3).
6.1.3.2. 3-(4-chlorophenyl)-1-(3-(dimethylamino)propyl)-1H-pyr-
6.1.1.4. 1-Methyl-3-(p-tolyl)-1H-pyrazole-4-carbaldehyde
White crystal; yield: 70.1%; m.p. 86e88 ꢀC. 1H-NMR (CDCl3-d1,
500 MHz, ppm): 9.931 (s, 1H, CHOeH), 7.986 (s, 1H, CH),
(6d).
azole-4-carbaldehyde (8b). Yellow liquid; yield: 46.8%. 1H-NMR
(CDCl3-d1, 500 MHz, ppm):
7.750e7.724 (m, 2H, Ph-H), 7.450e7.423 (m, 2H, Ph-H),
4.351e4.324 (t, J1 6.75 Hz, 2H, CH2), 2.539e2.514 (t,
d9.921 (s, 1H, CHOeH), 8.125 (s, 1H, CH),
d
7.609e7.593 (d, J1 ¼ 8.0 Hz, 2H, Ph-H), 7.286e7.270 (d, J1 ¼ 8.0 Hz,
¼
2H, Ph-H), 3.988 (s, 3H, CH3), 2.411 (s, 3H, CH3).
J1 ¼ 6.25 Hz, 2H, CH2), 2.417 (s, 6H,CH3), 2.268e2.214 (m, 2H, CH2).
6.1.2. General synthetic procedure for the key intermediates 7aed
A solution of compounds 4aed (1.0 mmol) in DMF (10 mL) was
added potassium carbonate (0.15 g, 1.1 mmol) and (chloromethyl)
benzene (0.14 g, 1.1 mmol), respectively. The reaction was catalyzed
with potassium iodide with stirring at 50 ꢀC for 5e6 h. The reaction
was monitored by TLC for completion. The resulting reaction
mixture was poured into 50 mL water, stirred for 5 min, extracted
with ethyl acetate (3 ꢂ 40 mL), dried with Na2SO4, filtered,
concentrated and finally purified by silica gel chromatography
(dichloromethane/n-hexane, 15:1) to afford pure colorless liquid
7aed. The yield, melting point and spectral data of each compound
are given below.
6.1.3.3. 3-(4-bromophenyl)-1-(3-(dimethylamino)propyl)-1H-pyr-
azole-4-carbaldehyde (8c). Yellow liquid; yield: 47.7%. 1H-NMR
(CDCl3-d1, 500 MHz, ppm): d9.817 (s, 1H, CHOeH), 7.970 (s, 1H, CH),
7.605e7.579 (m, 2H, Ph-H), 7.501e7.474 (m, 2H, Ph-H), 4.183e4.155
(t, J1 ¼ 7.0 Hz, 2H, CH2), 2.213e2.186 (t, J1 ¼ 6.75 Hz, 2H, CH2), 2.146
(s, 6H,CH3), 2.024e1.970 (m, 2H, CH2).
6.1.3.4. 1-(3-(dimethylamino)propyl)-3-(p-tolyl)-1H-pyrazole-4-
carbaldehyde (8d). Yellow liquid; yield: 39.5%. 1H-NMR (CDCl3-d1,
500 MHz, ppm):
d9.858 (s, 1H, CHOeH), 7.973 (s, 1H, CH),
7.546e7.530 (m, 2H, Ph-H), 7.195 (br. s, 2H, Ph-H), 4.206e4.178 (t,
J1 ¼ 7.0 Hz, 2H, CH2), 2.338 (s, 3H, CH3), 2.262e2.234 (t, J1 ¼ 7.0 Hz,
2H, CH2), 2.185 (s, 6H,CH3), 2.066e2.011 (m, 2H, CH2).
6.1.2.1. 1-Benzyl-3-(4-fluorophenyl)-1H-pyrazole-4-carbaldehyde
(7a). Colorless liquid; yield: 73.7%. 1H-NMR (DMSO-d6, 500 MHz,
ppm): d9.868 (s, 1H, CHOeH), 8.695 (s, 1H, CH), 7.916e7.875 (m, 2H,
Ph-H), 7.407e7.357 (m, 4H, Ph-H), 7.350e7.316 (m, 1H, Ph-H),
7.307e7.259 (m, 2H, Ph-H), 5.455 (s, 2H, CH2).
6.1.4. General synthetic procedure for the key intermediate 15c
Firstly, a mixture of maleic hydrazide (8.9 mmol, 1.0 g) and
phosphorus oxychloride (15 mL) was heated at 90 ꢀC for 6 h to
afford 3,6-dichloropyridazine, purified by silica gel chromatog-
raphy (dichloromethane/n-hexane, 15:1) to afford white crystals.
Secondly, a solution of compound 4c (2.50 g, 10.0 mmol) in DMF
(10 mL) was added sodium hydride (0.24 g, 10.0 mmol) and newly
synthetic 3,6-dichloropyridazine (1.63 g, 11.0 mmol), respectively.
The reaction was stirred at 70 ꢀC for 10 h in the presence of a
catalyst potassium iodide and monitored by TLC for completion.
The resulting reaction mixture was poured into 50 mL water and
stirred for 5 min, extracted with ethyl acetate (3 ꢂ 40 mL), dried
with Na2SO4, filtered, concentrated and finally purified by silica gel
chromatography (dichloromethane as the mobile phase) to afford
white product 15c. The yield, melting point and spectral data of
each compound are given below.
6.1.2.2. 1-Benzyl-3-(4-chlorophenyl)-1H-pyrazole-4-carbaldehyde
(7b). Colorless liquid; yield: 76.6%. 1H-NMR (DMSO-d6, 500 MHz,
ppm): d9.886 (s, 1H, CHOeH), 8.717 (s, 1H, CH), 7.900e7.872 (m, 2H,
Ph-H), 7.528e7.501 (m, 2H, Ph-H), 7.410e7.363 (m, 4H, Ph-H),
7.358e7.318 (m, 1H, Ph-H), 5.466 (s, 2H, CH2).
6.1.2.3. 1-Benzyl-3-(4-bromophenyl)-1H-pyrazole-4-carbaldehyde
(7c). Colorless liquid; yield: 85.29%. 1H-NMR (DMSO-d6, 500 MHz,
ppm): d9.890 (s, 1H, CHOeH), 8.721 (s, 1H, CH), 7.834e7.807 (m, 2H,
Ph-H), 7.665e7.637 (m, 2H, Ph-H), 7.408e7.371 (m, 4H, Ph-H),
7.352e7.321 (m, 1H, Ph-H), 5.468 (s, 2H, CH2).
6.1.2.4. 1-Benzyl-3-(p-tolyl)-1H-pyrazole-4-carbaldehyde
Colorless liquid; yield: 69.2%. 1H-NMR (DMSO-d6, 500 MHz, ppm):
9.863 (s, 1H, CHOeH), 8.649 (s, 1H, CH), 7.703e7.687 (m, 2H, Ph-
(7d).
6.1.4.1. 3-(4-bromophenyl)-1-(6-chloropyridazin-3-yl)-1H-pyrazole-
4-carbaldehyde (15c). White product; yield: 59.6%. 1H-NMR
d
(DMSO-d6, 500 MHz, ppm): d 10.082 (s, 1H, CHOeH), 9.648 (s, 1H,
H), 7.400e7.328 (m, 5H, Ph-H), 7.268e7.252 (m, 2H, Ph-H), 5.438
(s, 2H, CH2), 2.345 (s, 3H, CH3).
CH), 8.447e8.429 (d, J1 ¼ 9.0 Hz, 1H, Pyridazine-H), 8.211e8.192 (d,
J1 ¼ 9.5 Hz, 1H, Pyridazine-H), 8.004e7.978 (m, 2H, Ph-H),
7.749e7.722 (m, 2H, Ph-H).
6.1.3. General synthetic procedure for the key intermediates 8aed
A solution of compounds 4aed (10.0 mmol) in DMF (15 mL) was
added sodium hydride (0.72 g, 30.0 mmol) with stirring at room
temperature for 0.5 h. Then, 3-dimethylaminopropyl chloride
6.1.5. General procedure for the target compounds 10e15
A
mixture of 1,10-phenanthroline-5,6-dione
9
(0.10 g,
0.48 mmol), ammonium acetate (0.4 g, 5.19 mmol), 0.50 mmol

46
J. Liu et al. / European Journal of Medicinal Chemistry 133 (2017) 36e49
(4aed, 5aed, 6aed, 7aed, 8aed and 15c, respectively) in glacial
acetic acid (20 mL) was heated and stirred at 90 ꢀC under purified
nitrogen atmosphere for 6 h. The reaction was monitored by TLC for
completion. The resultant solution was concentrated and neutral-
ized with concentrated aqueous ammonia. The yellow precipitate
was collected, washed with water and purified by silica gel chro-
matography (dichloromethane/methanol, 15:1) to afford pure yel-
low products (10aed, 14aed and 15), white crystals (11aed, 12aed
and 13aed), respectively. The yield, melting point, purity and
spectral data of each compound are given below.
J2 ¼ 2.0 Hz, 2H, Phenanthroline-H), 8.835e8.821 (d, J1 ¼ 7.0 Hz, 1H,
Phenanthroline-H), 8.781e8.766 (d, J1 7.5 Hz, 1H,
Phenanthroline-H), 8.409 (s, 1H, CH), 8.032e8.004 (m, 2H, Ph-H),
7.847e7.799 (m, 2H, Phenanthroline-H), 7.490e7.463 (m, 2H, Ph-
H), 4.046 (s, 3H, CH3). MS (ESI) calcd for C23H15ClN6: 410.10,
found: 411.1 (M þ Hþ).
¼
6.1.5.7. 2-(3-(4-bromophenyl)-1-methyl-1H-pyrazol-4-yl)-1H-imi-
dazo[4,5-f][1,10]phenanthroline (11c). White crystal; yield: 86.4%;
m.p. >310 ꢀC; purity by HPLC: 96.9%. 1H-NMR (DMSO-d6, 500 MHz,
ppm):
d
9.014e9.010 (d, J2 ¼ 2.0 Hz, 2H, Phenanthroline-H),
6.1.5.1. 2-(3-(4-fluorophenyl)-1H-pyrazol-4-yl)-1H-imidazo[4,5-f]
[1,10]phenanthroline (10a). Yellow solid; yield: 78.8%; m.p.
272e274 ꢀC; purity by HPLC: 97.2%. 1H-NMR (DMSO-d6, 500 MHz,
8.838e8.823 (d, J1 ¼ 7.5 Hz, 2H, Phenanthroline-H), 8.498 (s, 1H,
CH), 8.022e8.006 (d, J1 ¼ 8.0 Hz, 2H, Ph-H), 7.824e7.801 (m, 2H,
Phenanthroline-H), 7.623e7.607 (d, J1 ¼ 8.0 Hz, 2H, Ph-H), 4.024 (s,
3H, CH3). MS (ESI) calcd for C23H15BrN6: 454.05, found: 455.1
(M þ Hþ).
ppm):
d
13.534 (br. s, 2H, NH), 9.041e9.029 (dd, J1 ¼ 4.5 Hz,
J2 ¼ 2.0 Hz, 2H, Phenanthroline-H), 8.837e8.822 (d, J1 ¼ 7.5 Hz, 2H,
Phenanthroline-H), 8.380 (br. s, 1H, CH), 8.096e8.068 (dd,
J1 ¼ 8.0 Hz, J2 ¼ 6.0 Hz, 2H, Ph-H), 7.853e7.828 (dd, J1 ¼ 8.0 Hz,
J2 ¼ 4.5 Hz, 2H, Phenanthroline-H), 7.312 (br. s, 2H, Ph-H). MS (ESI)
calcd for C22H13FN6: 380.12, found: 381.1 (M þ Hþ).
6.1.5.8. 2-(1-Methyl-3-(p-tolyl)-1H-pyrazol-4-yl)-1H-imidazo[4,5-f]
[1,10]phenanthroline (11d). White crystal; yield: 81.1%; m.p.
>310 ꢀC; purity by HPLC: 97.4%. 1H-NMR (DMSO-d6, 500 MHz,
ppm):
d
13.525 (s, 1H, NH), 9.034e9.022 (dd, J1 ¼ 4.5 Hz, J2 ¼ 2.0 Hz,
6.1.5.2. 2-(3-(4-chlorophenyl)-1H-pyrazol-4-yl)-1H-imidazo[4,5-f]
[1,10]phenanthroline (10b). Yellow solid; yield: 81.2%; m.p.
285e287 ꢀC; purity by HPLC: 96.6%. 1H-NMR (DMSO-d6, 500 MHz,
2H, Phenanthroline-H), 8.805e8.802 (m, 2H, Phenanthroline-H),
8.360 (s, 1H, CH), 7.823e7.790 (m, 4H, Ar-H), 7.195e7.179 (d,
J1 ¼ 8.0 Hz, 2H, Ph-H), 4.024 (s, 3H, CH3), 2.309 (s, 3H, CH3).MS (ESI)
calcd for C24H18N6: 390.16, found: 391.2 (M þ Hþ).
ppm):
d
13.551e13.531 (d, J1 ¼ 10 Hz, 2H, NH), 9.039e9.027 (dd,
J1 ¼ 4.0 Hz, J2 ¼ 1.5 Hz, 2H, Phenanthroline-H), 8.824 (br. s, 2H,
Phenanthroline-H), 8.486 (br. s, 1H, CH), 8.067 (br. s, 2H, Ph-H),
7.830 (br. s, 2H, Phenanthroline-H), 7.622 (br. s, 1H, Ph-H),
7.493e7.481 (d, J1 ¼ 6.0 Hz, 1H, Ph-H). MS (ESI) calcd for
6.1.5.9. 2-(1-Benzyl-3-(4-fluorophenyl)-1H-pyrazol-4-yl)-1H-imi-
dazo[4,5-f][1,10]phenanthroline (12a). White crystal; yield: 79.6%;
m.p. 208e210 ꢀC; purity by HPLC: 97.4%. 1H-NMR (DMSO-d6,
500 MHz, ppm):d d13.543 (s, 1H, NH), 9.029e9.017 (dd, J1 ¼ 4.0 Hz,
J2 ¼ 1.5 Hz, 2H, Phenanthroline-H), 8.823e8.807 (d, J2 ¼ 8.0 Hz, 1H,
C
22H13ClN6: 396.09, found: 397.1 (M þ Hþ).
6.1.5.3. 2-(3-(4-bromophenyl)-1H-pyrazol-4-yl)-1H-imidazo[4,5-f]
[1,10]phenanthroline (10c). Yellow solid; yield: 84.6%; m.p.
279e281 ꢀC; purity by HPLC: 95.8%. 1H-NMR (DMSO-d6, 500 MHz,
Phenanthroline-H), 8.764e8.749 (d, J2
¼
7.5 Hz, 1H,
Phenanthroline-H), 8.508 (s, 1H, CH), 8.029e7.987 (m, 2H, Ph-H),
7.817e7.804 (m, 2H, Phenanthroline-H), 7.450e7.413 (m, 4H, Ph-
H), 7.390e7.346 (m, 1H, Ph-H), 7.264e7.216 (m, 2H, Ph-H), 5.539
ppm): d13.722 (br. s, 1H, NH), 13.614 (br. s, 1H, NH), 9.052e9.041
(dd,J1 ¼ 4.0 Hz, J2 ¼ 1.5 Hz, 2H, Phenanthroline-H), 8.848 (br. s, 2H,
Phenanthroline-H), 8.533 (br. s, 1H, CH), 8.045e8.030 (d, J1 ¼7.5 Hz,
2H, Ph-H), 7.851e7.826 (dd, J1 ¼ 8.0 Hz, J2 ¼ 4.5 Hz, 2H,
Phenanthroline-H), 7.750 (br. s, 1H, Ph-H), 7.646 (br. s, 1H, Ph-H).
MS (ESI) calcd for C22H13BrN6: 440.04, found: 440.1 (M þ Hþ).
(s, 2H, CH2). 13C NMR (DMSO-d6,125 MHz, ppm):
d162.94, 160.99,
148.02, 147.67, 145.13, 143.39, 136.89, 132.81, 130.22, 130.15, 129.31,
129.29, 128.74, 127.97, 127.90, 123.16, 115.00, 114.83, 110.39, 55.31.
MS (ESI) calcd for C29H19FN6: 470.17, found: 471.2 (M þ Hþ).
6.1.5.10. 2-(1-Benzyl-3-(4-chlorophenyl)-1H-pyrazol-4-yl)-1H-imi-
dazo[4,5-f][1,10]phenanthroline (12b). White crystal; yield: 83.7%;
m.p. 214e216 ꢀC; purity by HPLC: 99.7%. 1H-NMR (DMSO-d6,
6.1.5.4. 2-(3-(p-tolyl)-1H-pyrazol-4-yl)-1H-imidazo[4,5-f][1,10]phe-
nanthroline (10d). Yellow solid; yield: 78.3%; m.p. 264e266 ꢀC;
purity by HPLC: 96.3%. 1H-NMR (DMSO-d6, 500 MHz, ppm):
500 MHz, ppm):
d
13.543 (s, 1H, NH), 9.032e9.020 (dd, J1 ¼ 4.0 Hz,
d13.549 (br. s, 2H, NH), 9.036 (br. s, 2H, Phenanthroline-H),
J2 ¼ 1.5 Hz, 2H, Phenanthroline-H), 8.828e8.810 (dd, J2 ¼ 8.0 Hz,
J ¼ 1.0 Hz, 1H, Phenanthroline-H), 8.763e8.745 (dd, J1 ¼ 8.0 Hz,
J2 ¼ 1.0 Hz, 1H, Phenanthroline-H), 8.521 (s, 1H, CH), 8.012e7.985
(m, 2H, Ph-H), 7.837e7.794 (m, 2H, Phenanthroline-H),
7.486e7.458 (m, 2H, Ph-H), 7.449e7.412 (m, 4H, Ph-H),
7.390e7.346 (m, 1H, Ph-H), 5.543 (s, 2H, CH2). 13C NMR (CDCl3-
8.846e8.832 (d, J1 ¼ 6.0 Hz, 2H, Phenanthroline-H), 8.237 (br. s,
1H, CH), 7.887 (br. s, 2H, Ph-H), 7.835e7.820 (m, 2H,
Phenanthroline-H), 7.268 (br. s, 2H, Ph-H), 2.345 (s, 3H, CH3). MS
(ESI) calcd for C23H16N6: 376.14, found: 377.1 (M þ Hþ).
6.1.5.5. 2-(3-(4-fluorophenyl)-1-methyl-1H-pyrazol-4-yl)-1H-imi-
dazo[4,5-f][1,10]phenanthroline (11a). White crystal; yield: 85.3%;
m.p. >310 ꢀC; purity by HPLC: 98.4%. 1H-NMR (DMSO-d6, 500 MHz,
d1þCD3OD-d4, 125 MHz, ppm):
d148.72, 147.59, 145.08, 143.57,
135.37, 134.04, 132.13, 130.93, 130.03, 130.01, 129.98, 129.96, 129.21,
128.90, 128.45, 128.39, 128.06, 123.05, 110.78, 56.36. MS (ESI) calcd
for C29H19ClN6: 486.14, found: 487.1 (M þ Hþ).
ppm):
J2 ¼ 2.0 Hz, 2H, Phenanthroline-H), 8.825e8.809 (d, J1 ¼ 8.0 Hz, 1H,
Phenanthroline-H), 8.779e8.763 (d, J1 8.0 Hz, 1H,
d13.514 (br. s, 1H, NH), 9.034e9.022 (dd, J1 ¼ 4.5 Hz,
¼
6.1.5.11. 2-(1-Benzyl-3-(4-bromophenyl)-1H-pyrazol-4-yl)-1H-imi-
dazo[4,5-f][1,10]phenanthroline (12c). White crystal; yield: 84.3%;
m.p. 208e209 ꢀC; purity by HPLC: 99.6%. 1H-NMR (DMSO-d6,
Phenanthroline-H), 8.394 (s, 1H, CH), 8.037e7.996 (m, 2H, Ph-H),
7.843e7.798 (m, 2H, Phenanthroline-H), 7.268e7.220 (m, 2H, Ph-
H), 4.039 (s, 3H, CH3). MS (ESI) calcd for C23H15FN6: 394.13,
found: 395.2 (M þ Hþ).
500 MHz, ppm):
d
13.565 (s, 1H, NH), 9.032e9.020 (dd, J1 ¼ 4.5 Hz,
J2 ¼ 2.0 Hz, 2H, Phenanthroline-H), 8.820 (br. s,1H, Phenanthroline-
H), 8.768 (br. s, 1H, Phenanthroline-H), 8.525 (s, 1H, CH),
7.952e7.925 (m, 2H, Ph-H), 7.829e7.805 (dd, J1 ¼8.0 Hz, J2 ¼ 4.0 Hz,
2H, Phenanthroline-H), 7.621e7.594 (m, 2H, Ph-H), 7.440e7.413 (m,
4H, Ph-H), 7.382e7.355 (m, 1H, Ph-H), 5.543 (s, 2H, CH2). 13C NMR
6.1.5.6. 2-(3-(4-chlorophenyl)-1-methyl-1H-pyrazol-4-yl)-1H-imi-
dazo[4,5-f][1,10]phenanthroline (11b). White crystal; yield: 84.7%;
m.p. >310 ꢀC; purity by HPLC: 97.1%. 1H-NMR (DMSO-d6, 500 MHz,
ppm):
d13.526 (br. s, 1H, NH), 9.037e9.025 (dd, J1 ¼ 4.5 Hz,
(CDCl3-d1þCD3OD-d4, 125 MHz, ppm):
d149.18, 148.74, 147.67,

J. Liu et al. / European Journal of Medicinal Chemistry 133 (2017) 36e49
47
147.56, 145.01, 143.54, 143.43, 135.37, 132.19, 131.42, 130.04, 129.96,
C
28H27N7: 461.23, found: 462.2 (M þ Hþ).
129.56, 128.92, 128.47, 128.08, 123.09, 122.38, 110.75, 56.39. MS
(ESI) calcd for C29H19BrN6: 530.09, found: 531.1 (M þ Hþ).
6.1.5.17. 2-(1-(2,4-dinitrophenyl)-3-(4-fluorophenyl)-1H-pyrazol-4-
yl)-1H-imidazo[4,5-f][1,10]phenanthroline (14a). Yellow solid;
yield: 86.8%; m.p. >310 ꢀC; purity by HPLC: 97.1%. 1H-NMR (DMSO-
d6, 500 MHz, ppm): 13.917 (s, 1H, NH), 9.243 (s, 1H, Ph-H),
9.072e9.060 (dd, J1 ¼ 4.0 Hz, J2 ¼ 1.5 Hz, 2H, Phenanthroline-H),
8.992e8.987 (d, J1 2.5 Hz, 1H, Ph-H), 8.862 (br. s, 1H,
Phenanthroline-H), 8.827 (br. s, 1H, Phenanthroline-H),
8.759e8.736 (dd, J1 ¼ 9.0 Hz, J2 ¼ 2.5 Hz, 1H, Ph-H), 8.332e8.314
(d, J1 ¼ 9.0 Hz, 1H, CH), 7.942e9.901 (m, 2H,Ph-H), 7.893e7.868 (m,
2H, Phenanthroline-H), 7.340e7.292 (m, 2H, Ph-H). MS (ESI) calcd
for C28H15FN8O4: 546.12, found: 547.1 (M þ Hþ).
6.1.5.12. 2-(1-Benzyl-3-(p-tolyl)-1H-pyrazol-4-yl)-1H-imidazo[4,5-f]
[1,10]phenanthroline (12d). White crystal; yield: 77.3%; m.p.
206e208 ꢀC; purity by HPLC: 99.9%. 1H-NMR (DMSO-d6, 500 MHz,
d
ppm):
d13.545 (s, 1H, NH), 9.026e9.018 (m, 2H, Phenanthroline-H),
¼
8.830e8.814 (d, J1 ¼8.0 Hz, 1H, Phenanthroline-H), 8.766e8.750 (d,
J1 ¼ 8.0 Hz, 1H, Phenanthroline-H), 8.468 (s, 1H, CH), 7.810e7.772
(m, 4H, Ar-H), 7.438e7.410 (m, 4H, Ph-H), 7.376e7.342 (m, 1H, Ph-
H), 7.185e7.169 (d, J1 ¼ 8.0 Hz, 2H, Ph-H), 5.522 (s, 2H, CH2), 2.300
(s, 3H, CH3). 13C NMR (CDCl3-d1þCD3OD-d4, 125 MHz, ppm):
d149.68, 147.77, 145.39, 143.66, 138.45, 135.46, 132.00, 129.80,
129.72, 129.67, 129.30, 128.90, 128.41, 128.17, 128.12, 128.08, 128.01,
123.05, 110.63, 56.35, 21.19. MS (ESI) calcd for C30H22N6: 466.19,
found: 467.2 (M þ Hþ).
6.1.5.18. 2-(3-(4-chlorophenyl)-1-(2,4-dinitrophenyl)-1H-pyrazol-4-
yl)-1H-imidazo[4,5-f][1,10]phenanthroline
yield: 88.6%; m.p. >310 ꢀC; purity by HPLC: 98.18%. 1H-NMR
(DMSO-d6, 500 MHz, ppm): 13.867 (s, 1H, NH), 9.238 (s, 1H, Ph-H),
(14b). Yellow
solid;
d
6.1.5.13. 3-(3-(4-fluorophenyl)-4-(1H-imidazo[4,5-f][1,10]phenan-
throlin-2-yl)-1H-pyrazol-1-yl)-N,N-dimethylpropan-1-amine (13a).
White crystal; yield: 65.3%; m.p. 284e286 ꢀC; purity by HPLC:
9.058e9.050 (d, J1 ¼ 4.0 Hz, 2H, Phenanthroline-H), 8.994e8.989
(d, J1 ¼ 2.5 Hz, 1H, Ph-H), 8.868e8.853 (d, J1 ¼ 7.5 Hz, 1H, Ph-H),
8.767e8.738 (m, 2H, Phenanthroline-H), 8.332e8.314 (d,
J1 ¼ 9.0 Hz, 1H, CH), 7.899e9.882 (d, J1 ¼ 8.5 Hz, 2H, Ph-H),
7.864e7.824 (m, 2H, Phenanthroline-H), 7.551e7.534 (d,
J1 ¼ 8.5 Hz, 2H, Ph-H). MS (ESI) calcd for C28H15ClN8O4: 562.09,
found: 563.3 (M þ Hþ).
96.2%. 1H-NMR (DMSO-d6, 500 MHz, ppm):
d13.527 (s, 1H, NH),
9.034e9.022 (dd, J1 ¼ 4.0 Hz, J2 ¼ 2.0 Hz, 2H, Phenanthroline-H),
8.802 (br. s, 2H, Phenanthroline-H), 8.438 (s, 1H, CH),
8.036e8.001 (m, 2H, Ph-H), 7.830e7.808 (m, 2H, Phenanthroline-
H), 7.269e7.222 (m, 2H, Ph-H), 4.320e4.292 (t, J1 ¼ 7.0 Hz, 2H,
CH2), 2.325e2.298 (t, J1 ¼ 6.75 Hz, 2H, CH2), 2.187 (s, 6H, CH3),
2.085e2.030 (m, 2H, CH2). MS (ESI) calcd for C27H24FN7: 465.21,
found: 466.2 (M þ Hþ).
6.1.5.19. 2-(3-(4-bromophenyl)-1-(2,4-dinitrophenyl)-1H-pyrazol-4-
yl)-1H-imidazo[4,5-f][1,10]phenanthroline (14c). Yellow Solid; yield:
89.1%; m.p. >310 ꢀC; purity by HPLC: 96.4%. 1H-NMR (DMSO-d6,
500 MHz, ppm):
9.055e9.044 (dd, J1 ¼ 4.0 Hz, J2 ¼ 1.5 Hz, 2H, Phenanthroline-H),
8.989e8.984 (d, J1 2.5 Hz, 1H, Ph-H), 8.867e8.852 (d,
d13.866 (s, 1H, NH), 9.232 (s, 1H, Ph-H),
6.1.5.14. 3-(3-(4-chlorophenyl)-4-(1H-imidazo[4,5-f][1,10]phenan-
throlin-2-yl)-1H-pyrazol-1-yl)-N,N-dimethylpropan-1-amine (13b).
White crystal; yield: 68.9%; m.p. 264e265 ꢀC; purity by HPLC:
¼
J1 ¼ 7.5 Hz, 1H, Ph-H), 8.763e8.731 (m, 2H, Phenanthroline-H),
8.330e8.312 (d, J1 ¼ 9.0 Hz, 1H, CH), 7.846e7.816 (m, 4H, Ar-H),
7.861e7.664 (m, 2H, Ph-H). MS (ESI) calcd for C28H15BrN8O4:
606.04, found: 607.3 (M þ Hþ).
96.2%. 1H-NMR (DMSO-d6, 500 MHz, ppm):
d13.550 (s, 1H, NH),
9.036e9.024 (dd, J1 ¼ 4.5 Hz, J2 ¼ 2.0 Hz, 2H, Phenanthroline-H),
8.807 (br. s, 2H, Phenanthroline-H), 8.458 (s, 1H, CH),
8.033e8.006 (m, 2H, Ph-H), 7.825 (br. s, 2H, Phenanthroline-H),
7.493e7.466 (m, 2H, Ph-H), 4.327e4.299 (t, J1 ¼ 7.0 Hz, 2H, CH2),
2.334e2.307 (t, J1 ¼ 6.75 Hz, 2H, CH2), 2.193 (s, 6H, CH3),
2.089e2.033 (m, 2H, CH2). MS (ESI) calcd for C27H24ClN7: 481.18,
found: 482.2 (M þ Hþ).
6.1.5.20. 2-(1-(2,4-dinitrophenyl)-3-(p-tolyl)-1H-pyrazol-4-yl)-1H-
imidazo[4,5-f][1,10]phenanthroline (14d). Yellow solid; yield: 82.3%;
m.p. >310 ꢀC; purity by HPLC: 98.3%. 1H-NMR (DMSO-d6, 500 MHz,
ppm): d13.860 (s, 1H, NH), 9.210 (s, 1H, Ph-H), 9.061e9.049 (dd,
J1 ¼ 4.5 Hz, J2 ¼ 2.0 Hz, 2H, Phenanthroline-H), 8.981e8.975 (d,
J1 ¼ 3.0 Hz, 1H, Ph-H), 8.870e8.856 (d, J1 ¼ 7.0 Hz, 1H, Ph-H),
8.775e8.759 (d, J1 ¼ 7.0 Hz, 1H, Phenanthroline-H), 8.741e8.718
(dd, J1 ¼ 9.0 Hz, J2 ¼ 2.5 Hz, 1H, Phenanthroline-H), 8.322e8.304 (d,
J1 ¼ 9.0 Hz, 1H, CH), 7.860e7.827 (m, 2H, Phenanthroline-H),
6.1.5.15. 3-(3-(4-bromophenyl)-4-(1H-imidazo[4,5-f][1,10]phenan-
throlin-2-yl)-1H-pyrazol-1-yl)-N,N-dimethylpropan-1-amine (13c).
White crystal; yield: 69.1%; m.p. 237e238 ꢀC; purity by HPLC:
97.6%. 1H-NMR (DMSO-d6, 500 MHz, ppm):
d13.546 (s, 1H, NH),
9.034e9.028 (dd, J1 ¼ 3.0 Hz, J2 ¼ 2.0 Hz, 2H, Phenanthroline-H),
8.833e8.818 (d, J1 ¼ 4.5 Hz, 1H, Phenanthroline-H), 8.795e8.780
(d, J1 ¼ 4.5 Hz, 1H, Phenanthroline-H), 8.459 (s, 1H, CH),
7.966e7.949 (d, J1 ¼ 8.5 Hz, 2H, Ph-H), 7.824 (br. s, 2H,
Phenanthroline-H), 7.624e7.607 (d, J1 ¼ 8.5 Hz, 2H, Ph-H),
4.329e4.301 (t, J1 ¼ 7.0 Hz, 2H, CH2), 2.365e2.341 (t, J1 ¼ 6.0 Hz,
2H, CH2), 2.217 (s, 6H, CH3), 2.098e2.043 (m, 2H, CH2). MS (ESI)
calcd for C27H24BrN7: 525.13, found: 526.1 (M þ Hþ).
7.701e7.684 (d, J1
¼
8.5 Hz, 2H, Ph-H), 7.242e7.226 (d,
J1 ¼ 8.0 Hz, 2H, Ph-H), 2.314 (s, 3H, CH3). MS (ESI) calcd for
C
29H18N8O4: 542.15, found: 543.2 (M þ Hþ).
6.1.5.21. 6-(3-(4-bromophenyl)-4-(1H-imidazo[4,5-f][1,10]phenan-
throlin-2-yl)-1H-pyrazol-1-yl)pyridazin-3-amine (15). Yellow solid;
yield: 77.4%; m.p. >310 ꢀC; purity by HPLC: 96.9%. 1H-NMR (DMSO-
d6, 500 MHz, ppm): d9.174 (s, 1H, Pyridazine-H), 9.055 (br. s, 2H,
Phenanthroline-H), 8.827 (br. s, 2H, Phenanthroline-H), 8.221 (s,
1H, CH). 8.197e8.182 (d, J1 ¼ 7.5 Hz, 2H, Ph-H), 7.901 (br. s, 2H,
Phenanthroline-H), 7.727e7.712 (d, J1 ¼ 7.5 Hz, 2H, Ph-H),
7.215e7.196 (d, J1 ¼ 9.5 Hz, 1H, Pyridazine-H). MS (ESI) calcd for
6.1.5.16. 3-(4-(1H-imidazo[4,5-f][1,10]phenanthrolin-2-yl)-3-(p-
tolyl)-1H-pyrazol-1-yl)-N,N-dimethylpropan-1-amine
White crystal; yield: 62.3%; m.p. 232e234 ꢀC; purity by HPLC:
96.6%. 1H-NMR (DMSO-d6, 500 MHz, ppm):
13.504 (s, 1H, NH),
(13d).
d
C
26H16BrN9: 533.07, found: 535.1 (Mþ2Hþ).
9.034e9.022 (dd, J1 ¼ 4.5 Hz, J2 ¼ 2.0 Hz, 2H, Phenanthroline-H),
8.819e8.784 (m, 2H, Phenanthroline-H), 8.380 (s, 1H, CH),
7.830e7.804 (m, 2H, Phenanthroline-H), 7.799e7.783 (d, J1 ¼8.0 Hz,
2H, Ph-H), 7.193e7.177 (d, J1 ¼ 8.0 Hz, 2H, Ph-H), 4.306e4.278 (t,
J1 ¼ 7.0 Hz, 2H, CH2), 2.323e2.296 (t, J1 ¼ 6.75 Hz, 5H, CH2, CH3),
2.184 (s, 6H, CH3), 2.078e2.022 (m, 2H, CH2). MS (ESI) calcd for
6.2. Cytotoxicity assay
The cytotoxicity in vitro of synthesized compounds 10e15 was
evaluated by Cell Counting Kit-8 assay (DOJINDO, Kumamoto,
Japan) [47] on human lung adenocarcinoma and human fetal lung

48
J. Liu et al. / European Journal of Medicinal Chemistry 133 (2017) 36e49
fibroblast cell lines: A549 and MRC-5. Cisplatin and leading com-
pound 2 were used as positive controls. Initially, cells were
routinely cultured in RPMI 1640 (Invitrogen Gibco) supplemented
with 10% FBS (Invitrogen Gibco) and penicillin-streptomycine
(Sigma-Aldrich, 100 U/mL) at 37.0 ꢀC in a humidified atmosphere
Representative images of three independent experiments were
shown.
Acknowledgment
containing 5% CO2. Moreover, 95
at 5000 cells per well in 96 well plates for 24 h to culture. After that,
L solution of compounds (1 L of a solution of compounds in
DMSO was diluted with 4 L culture medium) was mixed into the
L CCK8
m
L cell suspensions were seeded
We gratefully acknowledge the financial support from National
Natural Science Foundation of China (21572207), Program of In-
ternational S & T Cooperation (2015DFG32530), Zhejiang Provincial
Natural Science Foundation of China (LY16160003).
5
m
m
m
96 well plates sequentially to culture for 36 h. Then 10
m
reagents were added to each well and incubated at 37 ꢀC for 1 h.
Finally, the optical density of each sample was measured at 450 nm
using a microplate reader (Molecular Devices M4).
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2017.03.030.
6.3. Electrophoretic mobility shift assay (EMSA)
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