New 2,9-disubstituted-1,10-phenanthroline derivatives with anticancer activity by selective targeting of telomeric G-quadruplex DNA

Article abstract of DOI:10.1016/j.saa.2020.119318

Fifteen new 1,10-phenanthrolines disubstituted at positions 2 and 9 via amide bonds with different heterocycles have been designed and synthesized as G-quadruplex DNA stabilizers. Ten compounds were evaluated for the in vitro anticancer activity against 60 human tumor cell lines panel, four of them showing a very good inhibitory activity on several cell lines. To assess the ability of the most active compounds to interact with G-quadruplex DNA (G4-DNA), circular dichroism experiments were performed. The potency of the compounds to stabilize the G4-DNA has been shown from the thermal denaturation experiments. The mechanism of compounds binding to DNA and to G4-DNA was theoretically investigated by molecular docking studies. The experimental results demonstrated excellent capacity of the two compounds bearing two pyridin-3-yl residues (methylated and non-methylated) to act as selective G-quadruplex binders with promising anticancer activity.

Full text of DOI:10.1016/j.saa.2020.119318

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 249 (2021) 119318
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
New 2,9-disubstituted-1,10-phenanthroline derivatives with anticancer
activity by selective targeting of telomeric G-quadruplex DNA
Anda-Mihaela Craciun a,b, Alexandru Rotaru b, Corneliu Cojocaru b, Ionel I. Mangalagiu a, Ramona Danac a,
⇑
a Chemistry Department, Faculty of Chemistry, ‘‘Al. I. Cuza” University of Iasi, 11 Carol I, Iasi 700506, Romania
b ‘‘Petru Poni” Institute of Macromolecular Chemistry, 41A, Grigore Ghica Voda Alley, Iasi 700487, Romania
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
ꢀ
ꢀ
ꢀ
ꢀ
New promising anticancer 2,9-disub
stituted-1,10-phenanthroline
derivatives were developed.
Compound 5b displayed GI50
values < 100 nM against fifteen cancer
cell lines.
Cytotoxic effect was associated with
an excellent ability to selectively bind
and stabilize G4-DNA.
CD and molecular docking studies on
the interaction of the most active
compounds with DNA.
a r t i c l e i n f o
a b s t r a c t
Article history:
Fifteen new 1,10-phenanthrolines disubstituted at positions 2 and 9 via amide bonds with different hete-
rocycles have been designed and synthesized as G-quadruplex DNA stabilizers. Ten compounds were
evaluated for the in vitro anticancer activity against 60 human tumor cell lines panel, four of them show-
ing a very good inhibitory activity on several cell lines. To assess the ability of the most active compounds
to interact with G-quadruplex DNA (G4-DNA), circular dichroism experiments were performed. The
potency of the compounds to stabilize the G4-DNA has been shown from the thermal denaturation exper-
iments. The mechanism of compounds binding to DNA and to G4-DNA was theoretically investigated by
molecular docking studies.
Received 25 September 2020
Received in revised form 26 November 2020
Accepted 5 December 2020
Available online 14 December 2020
Keywords:
1,10-Phenanthroline derivatives
Anticancer activity
G-quadruplex ligands
Melting-CD
The experimental results demonstrated excellent capacity of the two compounds bearing two pyridin-
3-yl residues (methylated and non-methylated) to act as selective G-quadruplex binders with promising
anticancer activity.
Heterocycles
Docking
Ó 2020 Elsevier B.V. All rights reserved.
1. Introduction
functional regions of the human genome such as in the promoter
region of some oncogenes that contain G-rich sequences, as well
The drug design for anticancer therapy is based on different
strategies such as: angiogenesis inhibition, DNA intercalation and
groove binding, transcription regulation, enzymes inhibition or
microtubules targeting [1]. Among them, one of the most promis-
ing strategy that developed rapidly over past years is the intercala-
tion of the G4-DNA that has been recognized in the biologically
as in the telomers where may have a role in maintaining the chro-
mosome stability [2–5]. Thus, this anticancer approach relies on
the formation and stabilization of such G4-DNA structures in the
presence of small molecules. In this case, some biological processes
as inhibition of telomerase (present in>85% cancer cells of the dif-
ferent lines [6–8]), downregulation the protein expressions or
avoiding of the telomere elongation could be affected, thus induc-
ing the cell apoptosis [9–13].
⇑
Corresponding author.
E-mail address: rdanac@uaic.ro (R. Danac).
https://doi.org/10.1016/j.saa.2020.119318
1386-1425/Ó 2020 Elsevier B.V. All rights reserved.

Anda-Mihaela Craciun, A. Rotaru, C. Cojocaru et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 249 (2021) 119318
The key step for stabilization the telomeric G4-DNA with small
molecules, is a good selectivity for human telomeric G4-DNA ver-
sus double-stranded DNA (dsDNA) and as well as the necessity to
discriminate different G-quadruplexes structures [2].
Recent literature abounds in molecules with a big variety of
core structures as acridines, actinomycins, anthracyclines, antra-
quinones, quinolones, phenanthridines, phenanthrolines and pyr-
ene, which have been synthesized and proved to have DNA
intercalation properties and/or anticancer activity [14–16]. The
or a Bruker Avance DRX 400 MHz. The following abbreviations
were used to designate chemical shift multiplicities: s = singlet,
d = doublet, t = triplet, q = quartet, m = multiplet, dd = doublet
of doublets, as = apparent singlet. All chemical shifts are quoted
on the d-scale in ppm. Coupling constants are given in hertz (Hz).
Matrix-assisted laser desorption ionization time of flight (MALDI-
TOF) mass spectrometry experiments were carried out on Shi-
madzu AXIMA Performance operated in high-resolution reflection
mode using
a-Cyano-4-hydroxycinnamic acid as matrix. ESI-MS
principal driving forces for intercalation are represented by
p
-
p
experiments were carried out on Agilent 6520 Accurate-Mass Q-
TOF LC/MS. IR spectra were recorded on a FTIR Shimadzu or FTIR
Bruker Vertex 70 spectrophotometer. Analyses indicated by the
symbols of the elements or functions were within 0.4% of the the-
oretical values. Thin layer chromatography (TLC) was carried out
on Merck silica gel 60F254 plates. Visualization of the plates was
achieved using a UV lamp (kmax = 254 or 365 nm). Circular dichro-
ism experiments were recorded by using a Chirascan plus (Applied
Photophysics) equipped with a Peltier temperature controller.
Spectra were recorded at 25 °C in the wavelength range of 240–
340 nm and 240–400 nm.
interactions between G-quartets and the aromatic surface of the
ligands [17]. Also, the charge-transfer interactions, hydrogen bond-
ing and electrostatic forces play an important role in stabilization
of the formed complex [16].
Usually, flat, generally aromatic or heteroaromatic molecules
substituted with groups possessing basic functionalities (typically
amino groups able to get positively charged at physiological pH)
or the presence of the amide NH bond in the molecule which is
preferred to form internal H-bonds with nitrogen atoms from hete-
rocycle are promising compounds for G-quadruplex stabilization
[2,18].
Several 1,10-phenanthroline derivatives were recently reported
as stabilizers of human telomeric G4-DNA, inhibitors of telomerase
and showing cytotoxic activity [19–27]. Among them, bisquinolin-
ium derivative Phen-DC3 (Fig. 1) was the most studied, showing a
high selectivity for G-quadruplexes over dsDNAs [19,20].
As a continuation of our work in the field of developing hetero-
cyclic molecules with biological activity [28–31], we present
herein, the synthesis of several new small molecules as analogs
of Phen-DC3 (Fig. 1) designed to stabilize G4 structures. The new
compounds are derivatives of 1,10-phenanthroline substituted at
positions 2 and 9 with different heterocycles connected via amide
linkages. Thus, we considered the synthesis of 1,10-
phenanthrolines bearing thiazole, pyridine, quinoline or 1,2,3-
triazole as substituents at the amidic groups at positions 2 and 9.
All these heterocycles were found to be part of molecules showing
G4-DNA stabilization properties and/or possessing biological activ-
ity [19–22,32–38]. Subsequently, the capacity of the active com-
pounds to interact with dsDNA and G4-DNA was investigated
experimentally and by means of theoretical calculations.
2.2. Procedure for synthesis of compound 2
Neocuproine (14.4 mmol, 1 equiv., 3 g) was dissolved in dioxane
(50 mL) and water (2 mL), and the resulting solution was stirred for
20 min. at room temperature. A suspension of selenium dioxide
(64.8 mmol, 4.5 equiv., 7.19 g) in a mixture of dioxane (130 mL)
and water (5.2 mL) was then added, and the color changed from
brown to white. The resulting suspension was refluxed for 24 h,
and then the black colored precipitate (selenium metal) was fil-
tered while hot. The resulting solution was cooled and the result-
ing precipitate was filtered to give the desired compound which
was purified by crystallization from acetone.
2.3. Procedure for synthesis of compound 3
1,10-Phenanthroline-2,9-dicarbaldehyde 2 (6.3 mmol, 1.5 g)
was stirred under reflux for 24 h in 65% HNO3 (30 mL). The result-
ing solution was cooled to 0 °C and ice was slowly added to form a
yellow precipitate, which was filtered, washed with water and
dried. The obtained compound was purified by crystallization from
methanol.
2. Experimental section
2.1. Chemistry
2.4. Procedure for synthesis of compound 4
All commercially available reagents and solvents employed
were used without further purification. Melting points were
recorded on an A. Krüss Optronic Melting Point Meter KSPI and
are uncorrected. Proton and carbon nuclear magnetic resonance
(1H, 13C) spectra were recorded on a DRX-500 Bruker (500 MHz)
To a solution of 1,10-phenanthroline-2,9-dicarboxylic acid 3
(0.373 mmol, 1 equiv., 0.1 g) in anhydrous DCM (10 mL), oxalyl
chloride (1.49 mmol, 4 equiv., 0.13 mL) and catalytic anhydrous
DMF were added under the inert atmosphere (using N2). The solu-
tion was refluxed for 24 h. The solvent was removed in vacuo to
Fig. 1. Structure of Phen-DC3 and general structures of target compounds.
2

Anda-Mihaela Craciun, A. Rotaru, C. Cojocaru et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 249 (2021) 119318
afford the corresponding 1,10-phenanthroline-2,9-dicarbonyl
dichloride which was used imediately in the next step without
any purification.
The obtained precipitate was filtered and then washed with
10 mL H2O/NH3 (25%) (1/1, v/v) to give the desired compound.
The product was purified by crystallization from chloroform/
methanol (1:1, v/v).
2.5. General procedure for the synthesis of compounds 5a-f
1,10-phenanthroline-2,9-dicarbaldehyde (2). Yellow solid, 85%
yield; 1H NMR (DMSO d6, 500 MHz): d = 8.27 (2H, s, H-5), 8.29
(2H, d, J = 8.5 Hz, H-3), 8.77 (2H, d, J = 8.5 Hz, H-4), 10.34 (2H, s,
The corresponding amine [propargylamine (0.743 mmol, 2
equiv., 0.05 mL); 2-aminothiazole (0.932 mmol, 2.5 equiv.,
0.0932 g); 2-amino-pyridine (0.932 mmol, 2.5 equiv., 0.0876 g),
3-amino-pydidine (0.932 mmol, 2.5 equiv., 0.0876 g); 8-
CHO); 13C NMR (DMSO d6, 125 MHz): d = 120.1 (C-3), 129.2 (C-
5), 131.4 (C-6), 138.4 (C-4), 145.3 (C-7), 152.2 (C-2), 193.7 (CHO).
Anal. Calcd. for C14H8N2O2: C, 71.18; H, 3.41; N, 11.86. Found: C,
71.15; H, 3.38; N, 11.91.
aminoquinoline (0.746 mmol,
2
equiv., 0.107 g); 5-
aminoquinoline (0.746 mmol, 2 equiv., 0.107 g)] and triethylamine
(TEA) (2.238 mmol, 6 equiv., 0.31 mL) were added to chloroform
(5 mL) for synthesis of compounds 5a-c and 5e or anhydrous ace-
tonitrile (5 mL) for synthesis of compounds 5d-f, and the obtained
solution was stirred under nitrogen at room temperature for
20 min. 1,10-Phenanthroline-2,9-dicarbonyl dichloride 4 was sus-
pended in 5 mL chloroform (for synthesis of compounds 5a-c and
5e) or anhydrous acetonitrile (for synthesis of compounds 5d and
5f) and stirred for 15 min., under nitrogen. The solution of the cor-
responding amine was then added dropwise over the solution of
dichloride 4 under nitrogen atmosphere, and the mixture was stir-
red over night at room temperature. The formed precipitate was
collected by filtration and washed with chloroform (for com-
pounds 5a-c and 5e) or acetonitrile (compounds 5d-f). The product
was purified by crystallization from dichloromethane/methanol
(1:1, v/v).
1,10-phenanthroline-2,9-dicarboxylic acid (3). Pale yellow solid,
90% yield; 1H NMR (DMSO d6, 500 MHz): d = 8.1 (2H, s, H-5),
8.42 (2H, d, J = 8.5 Hz, H-3), 8.62 (2H, d, J = 8.5 Hz, H-4) 9.62
(2H, s, COOH); 13C NMR (DMSO d6, 125 MHz): d = 123.8 (C-3),
128.8 (C-5), 130.5 (C-6), 137.5 (C-4), 142.6 (C-7), 148.3 (C-2),
166.7 (COOH). Anal. Calcd. for C14H8N2O4: C, 62.69; H, 3.01; N,
10.44. Found: C, 62.67; H, 3.98; N, 10.41.
N2,N9-di(pyridin-2-yl)-1,10-phenantroline-2,9-dicarboxamide
(5a) [39]. White solid, 45% yield; mp 252–254 °C; IR (KBr), mmax
2964, 1687, 1537, 1433 cmꢂ1 1H NMR (DMSO d6, 500 MHz):
;
d = 7.33 (2H, t, J = 5.5 Hz, H-13), 8.08 (2H, t, J = 8.5 Hz, H-14),
8.23 (2H, s, H-5), 8.44 (2H, d, J = 8.5 Hz, H-15), 8.50 (2H, d,
J = 5.0 Hz, H-12), 8.59 (2H, d, J = 8.0 Hz, H-3), 8.81 (2H, d,
J
=
8.0 Hz, H-4), 11.51 (2H, s, NH); 13C NMR (DMSO d6,
125 MHz): d = 114.2 (C-15), 120.2 (C-13), 121.3 (C-3), 128.4 (C-
5), 130.9 (C-6), 138.8 (C-4), 140.0 (C-14), 143.4 (C-7), 146.3(C-
12), 148.1 (C-2), 149.9 (C-10), 162.4 (C-8). MALDI-TOF positive
mode, m/z: 421 [M + H+] (100%).
2.6. General procedure for synthesis of compounds 6a-e
N2,N9-di(pyridin-3-yl)-1,10-phenantroline-2,9-dicarboxamide
(5b). White solid, 35% yield, mp 294–297 °C; IR (KBr): mmax 3238,
3058, 2925, 1675, 1654, 1592, 1541, 1485, 1420, 873, 804,
The compound (5a, 5b, 5c or 5d) (1 mmol, 1 equiv.) was sus-
pended in cloroform (10 mL) and stirred under reflux (30 min)
until the compounds are disolved. Over the clear solution the tri-
fluoromethanesulfonate agent was added. The optimized condi-
tions involve the next number of equivalents of the methylating
agent: for 6a – 9 equiv., for 6b and 6e – 4 equiv. and for 6c and
6d – 18 equiv. Immediately after the complete addition of the
agent (aprox. 5 min), the precipitate was obtained. This was filtred,
washed with chloroform and dried, followed by the structural
characterization without any purification.
707 cmꢂ1 1H NMR (DMSO d6, 400 MHz): d = 7.50 (2H, dd,
;
J = 8.0; 6.0 Hz, H-14), 8.29 (2H, s, H-5), 8.41 (2H, d, J = 4.0 Hz, H-
13), 8.52 (2H, d, J = 8.4 Hz, H-15), 8.62 (2H, d, J = 8.4 Hz, H-3),
8.86 (2H, d, J = 8.0 Hz, H-4), 9.28 (2H, bs, H-11), 11.55 (2H, s,
NH); 13C NMR (DMSO d6, 100 MHz): d = 121.6 (C-3), 123.6
(C-14), 127.2 (C-15), 128.4 (C-5), 130.7 (C-6), 135.2 (C-10), 138.7
(C-4), 143.6 (C-7), 142.0 (C-11), 144.9 (C-13), 149.3 (C-2), 163.0
(C-8). Anal. Calcd. for C24H16N6O2: C, 68.56; H, 3.84; N, 19.99.
Found: C, 68.53; H, 3.80; N, 20.05.
2.7. General procedure for synthesis of compounds 8a-e
N2,N9-di(quinolin-5-yl)-1,10-phenanthroline-2,9-dicarboxamide
(5c). Yellow solid, 15% yield; 1H NMR (CDCl3, 500 MHz): d = 7.47
(2H, d, J = 8.0 Hz, H-13), 7.78 (2H, t, J = 8.0 Hz, H-16), 7.93 (2H,
d, J = 8.0 Hz, H-12), 8.10 (2H, s, H-5), 8.20 (2H, bs, H-17), 8.38
(2H, bs, H-11), 8.60 (2H, bs, H-15), 8.63 (2H, d, J = 8.0 Hz, H-3),
8.79 (2H, d, J = 8.0 Hz, H-4), 11.29 (2H, s, NH); 13C NMR (CDCl3,
125 MHz): d = 119.5 (C-11), 120.4 (C-18), 122.0 (C-3), 127.0
(C-12), 127.9 (C-17), 128.3 (C-5), 129.1 (C-13), 129.9 (C-6), 131.5
(C-16), 132.3 (C-10), 138.0 (C-19), 138.6 (C-4), 143.4 (C-7), 149.4
(C-2), 150.0 (C-15), 164.0 (C-8). Anal. Calcd. for C32H20N6O2:
C, 73.84; H, 3.87; N, 16.14. Found: C, 73.88; H, 3.80; N, 16.20.
N2,N9-di(quinolin-8-yl)-1,10-phenanthroline-2,9-dicarboxamide
The corresponding 2-bromoacetophenone 7a-e (1 mmol, 1
equiv.) was dissolved in chloroform (5 mL). Sodium azide (1.1
equiv.), water (2.5 mL) and tetrabutylammonium bromide (0.01
equiv.) were then added. The obtaining reaction mixture was stir-
red at room temperature for 48 h and then extracted with water (3
x15 mL), dried on anhydrous Na2SO4 and concentrated in vacuum.
The residue was triturated with ethanol and the resulting solid
crystallized from ethanol to give the desired compound.
2.8. General procedure for synthesis of compounds 9a-e
N2,N9-di(prop-2-yn-1-yl)-1,10-phenanthroline-2,9-dicarboxa
mide 5f (0.291 mmol, 1 equiv., 0.1 g), CuSO4ꢁ5H2O (0.146 mmol, 0.5
equiv., 0.04 g) and the corresponding azide 8a-e were suspended in
20 mL water/t-butanol mixture (1/1, v/v) for compounds 9a, 9b
and 9e or in 10 mL DMSO/water mixture (4/1, v/v) for compounds
9c and 9d. The resulting mixture was stirred for 20 min under
nitrogen. Sodium ascorbate (0.087 mmol, 0.3 equiv., 0.017 g) was
then added and the obtaining mixture was stirred at 50 °C (for
compounds 9a, 9b and 9e) and at 90 °C (for compounds 9c and
9d) for 24 h. In case of water/t-butanol mixture a precipitate was
obtained, while into the DMSO/water mixture, after the reaction
time, 10 mL of water was added in order to obtain a precipitate.
(5d). Beige solid, 45% yield; mp 303.9–304.5 °C; IR (KBr)
m
max 3551,
3477, 3414, 1681, 1638, 1618, 1537, 1486 cmꢂ1
;
1H NMR
(DMSO d6, 500 MHz): d = 6.55 (2H, dd, J = 8.5; 4.0 Hz, H-15), 7.57
(2H, d, J = 8.0 Hz, H-13), 7.68 (2H, t, J = 8.0 Hz, H-12), 7.83 (2H,
d, J = 3.5 Hz, H-16), 7.85 (2H, d, J = 8.0 Hz, H-14), 8.31 (2H, s, H-
5), 8.65 (2H, d, J = 8.0 Hz, H-3), 8.89 (2H, d, J = 7.5 Hz, H-11),
8.90 (2H, d, J = 8.0 Hz, H-4), 12.73 (2H, s, NH); 13C NMR (DMSO d6,
125 MHz): d = 116.4 (C-11), 120.6 (C-15), 121.1 (C-3), 122.4 (C-13),
126.6 (C-12), 127.4 (C-19), 128.2 (C-5), 130.6 (C-6), 133.9 (C-10),
135.3 (C-14), 138.3 (C-18), 138.8 (C-4), 143.6 (C-7), 147.5 (C-16),
149.4 (C-2), 162.2 (C-8). MALDI-TOF positive mode, m/z: 521
[M
+ +
H+] (25%); 543 [M Na+] (100%). Anal. Calcd. for
3

Anda-Mihaela Craciun, A. Rotaru, C. Cojocaru et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 249 (2021) 119318
C32H20N6O2: C, 73.84; H, 3.87; N, 16.14. Found: C, 73.91; H, 3.82; N,
16.25.
H-130), 7.70 (1H, t, J = 8.0 Hz, H-120), 7.98 (2H, d, J = 4.0 Hz, H-
160), 8.04 (1H, d, J = 8.0 Hz, H-140), 8.18 (1H, t, J = 8.0 Hz, H-12),
8.28 (1H, d, J = 8.0 Hz, H-13), 8.31 (2H, s, H5, H-50), 8.64 (1H, d,
J = 8.5 Hz, H-30), 8.67 (1H, d, J = 8.0 Hz, H-3), 8.73 (1H, d,
J = 5.5 Hz, H-14), 8.83 (1H, d, J = 7.5 Hz, H-11), 8.87 (1H, d,
J = 7.5 Hz, H-110), 8.92 (1H, d, J = 8.0 Hz, H-40), 8.95 (2H, ad,
J = 8.0 Hz, H-4, H-16), 11.73 (1H, s, H-9), 12.53 (1H, s, H-90); 13C
NMR (DMSO d6, 125 MHz): d = 49.9 (CH3), 116.7 (C-110), 121.0
(C-15), 121.2 (C-150), 121.4 (C-3), (C-30), 122.6 (C-130), 127.2 (C-
120), 127.5 (C-190), 128.1 (C-5), 128.2 (C-50), 128.6 (C-13), 129.9
(C-12), 130.7 (C-60), 130.9 (C-6), 131.0 (C-10), 133.2 (C-100),
133.3 (C-11), 133.6 (C-19), 136.6 (C-140), 137.8 (C-180), 139.2 (C-
4), 139.3 (C-40), 143.2 (C-7), 143.3 (C-70), 147.9 (C-16), 148.2 (C-
2), 148.5 (C-160), 149.6 (C-20), 151.9 (C-18), (C-14), 161.9 (C-80),
162.0 (C-8). Anal. Calcd. for C34H23F3N6O5S: C, 59.65; H, 3.39; N,
12.28. Found: C, 59.61; H, 3.32; N, 12.35.
N2,N9-di(thiazol-2-yl)-1,10-phenanthroline-2,9-dicarboxamide
(5e). Yellow solid, 77% yield; mp 348–351 °C; IR (KBr), mmax 3365,
3082, 1671, 1562, 1504 cmꢂ1 1H NMR (DMSO d6, 500 MHz):
;
d = 7.39 (2H, d, J = 3.5 Hz, H-13), 7.65 (2H, d, J = 3.5 Hz, H-12),
8.30 (2H, s, H-5), 8.60 (2H, d, J = 8.0 Hz, H-3), 8.87 (2H, d,
J
=
8.0 Hz, H-4), 12.87 (2H, s, NH); 13C NMR (DMSO d6,
125 MHz): d = 114.6 (C-13), 122.2 (C-3), 128.7 (C-5), 131.0 (C-6),
138.2 (C-12), 138.8 (C-4), 144.1 (C-7), 148.2 (C-2), 157.7 (C-10),
163.1 (C-8). MALDI-TOF positive mode, m/z: 433 [M + H+] (55%);
455 [M + Na+] (100%). Anal. Calcd. for C20H12N6O2S2: C, 55.55; H,
2.78; N, 19.45. Found: C, 55.58; H, 2.74; N, 19.51.
N2,N9-di(prop-2-yn-1-yl)-1,10-phenanthroline-2,9-dicarboxamide
(5f). Brown solid, 40% yield; mp 250–252 °C; IR (KBr) mmax 3405,
;
3276, 3194, 2925, 1682, 1523, 1492 cmꢂ1 1H NMR (CDCl3,
500 MHz): d = 2.34 (2H, as, H-12), 4.36 (4H, as, H-10), 7.96 (2H,
s, H-5), 8.47 (2H, d, J = 8.0 Hz, H-4), 8.60 (2H, d, J = 8.0 Hz, H-3),
8.82 (2H, s, NH); 13C NMR (CDCl3, 125 MHz): d = 29.6 (C-10),
72.0 (C-12), 79.6 (C-11), 121.8 (C-3), 128.1 (C-5), 130.9 (C-6),
138.1 (C-4), 144.4 (C-7), 149.5 (C-2), 164.1 (C-8). Anal. Calcd. for
1-methyl-2-(9-(pyridin-2-ylcarbamoyl)-1,10-phenanthroline-2-ca
rboxamido)pyridin-1-ium trifluoromethanesulfonate (6e). White
solid, 90% yield, mp 236–242 °C; 1H NMR (DMSO d6, 500 MHz):
d = 4.79 (3H, s, CH3), 7.29 (1H, t, J = 6.5 Hz, H-13), 7.89–7.98 (2H,
m, overlapped signals, H-130, H-14), 8.17 (2H, s, H-5), 8.26–8.30
(2H, m, H-50, H-15), 8.49 (1H, d, J = 8.0 Hz, H-150), 8.59 (2H, d,
J = 3.5 Hz, H-12), 8.61–8.66 (3H, m, overlapped signals, H-3, H-30,
H-140), 8.75 (1H, d, J = 8.5 Hz, H-4), 8.88 (1H, d, J = 8.0 Hz, H-40),
9.13 (1H, d, J = 6.0 Hz, H-120), 10.89 (1H, s, NH); 13C NMR (DMSO d6,
125 MHz): d = 44.3 (Me), 113.3 (C-15), 120.4 (C-13), 122.0 (C-30),
121.2 (C-150), 122.2 (C-3), 122.8 (C-130), 128.4 (C-5), 129.2 (C-50),
130.8 (C-60), 131.5 (C-6), 139.2 (C-4), 139.4 (C-40), 138.8 (C-14),
143.2 (C-7), 143.6 (C-70), 145.2 (C-120), 146.2 (C-14), 148.8 (C-
12), 147.4 (C-20), 148.1 (C-2), 148.5 (C-100), 150.6 (C-10), 162.8
(C-8), 162.8 (C-80). MALDI-TOF positive mode, m/z: 435 [M+]
(100%).
C
20H14N4O2: C, 70.17; H, 4.12; N, 16.37. Found: C, 70.19; H, 4.00;
N, 16.40.
2,20-((1,10-phenanthroline-2,9-dicarbonyl)bis(azanediyl))bis(1-m
ethylpyridin-1-ium) trifluoromethanesulfonate (6a). White solid, 70%
yield, mp 260–264 °C; 1H NMR (DMSO d6, 400 MHz): d = 4.38 (6H,
s, 2 ꢃ CH3), 7.99 (2H, t, J = 6.4 Hz, H-13), 8.40 (2H, s, H-5), 8.51 (2H,
d, J = 8.0 Hz, H-15), 8.66–8.70 (4H, overlapped, H-12, H-3), 8.97–
8.99 (4H, overlapped, H-14, H-4); 13C NMR (DMSO d6, 100 MHz):
d = 44.0 (CH3), 123.6 (C-15), 123.8 (C-13), 122.4 (C-3), 129.0 (C-
5), 131.5 (C-6), 139.4 (C-4), 143.5 (C-7), 145.9 (C-14), 146.8 (C-
12), 147.2 (C-10), 147.7 (C-2), 163.7 (C-8). ESI-MS positive mode,
m/z: 449 [MꢂH]+ (100%).
N2,N9-bis((1-(2-(4-bromophenyl)-2-oxoethyl)-1H-1,2,3-triazol-4-
yl)methyl)-1,10-phenanthroline-2,9-dicarboxamide (9a). Brown
powder, 41% yield; mp 181–183 °C; IR (KBr) mmax 3550, 3473,
3,30-((1,10-phenanthroline-2,9-dicarbonyl)bis(azanediyl))bis(1-m
ethylpyridin-1-ium) trifluoromethanesulfonate (6b). White solid, 98%
yield, mp 302–307 °C; IR (KBr):
m
max 3266, 3106, 1691, 1599, 1554,
3414, 1658. 1619, 1585, 1540, 1495 cmꢂ1 1H NMR (DMSO d6,
;
1512, 1460, 1268, 1151, 1030, 638 cmꢂ1
;
1H NMR (DMSO d6,
500 MHz): d = 4.80 (4H, d, J = 5.5 Hz, H-10), 6.11 (4H, s, H-16),
7.56 (4H, d, J = 8.5 Hz, H-20), 7.93 (4H, d, J = 8.5 Hz, H-19), 8.12
(2H, s, H-12), 8.19 (2H, s, H-5), 8.48 (2H, d, J = 8.5 Hz, H-3), 8.75
(2H, d, J = 8.0 Hz, H-4), 9.99 (2H, t, J = 6.0 Hz, NH); 13C NMR
(DMSO d6, 125 MHz): d = 34.8 (C-10), 55.8 (C-16), 121.1 (C-3),
125.1 (C-12), 128.0 (C-5), 128.3 (C-21), 130.1 (C-19), 130.3 (C-6),
132.00 (C-20), 133.1 (C-18), 138.3 (C-4), 143.7 (C-7), 144.8 (C-
11), 149.4 (C-2), 163.8 (C-8), 191.5 (C-17). MALDI-TOF positive
500 MHz): d = 4.49 (6H, s, 2 ꢃ CH3), 8.28 (2H, bs, H-14), 8.36
(2H, s, H-5), 8.70 (2H, d, J = 7.5 Hz, H-3), 8.87 (2H, bs, H-13), 8.94
(2H, d, J = 8.0 Hz, H-4), 9.12 (2H, d, J = 8.0 Hz, H-15), 9.80 (2H, s,
H-11), 12.05 (2H, s, NH); 13C NMR (DMSO d6, 125 MHz): d = 48.7
(CH3), 122.2 (C-3), 127.8 (C-14), 129.0 (C-5), 131.4 (C-6), 135.4
(C-15), 137.2 (C-11), 138.4 (C-10), 139.3 (C-4), 140.7 (C-13),
143.8 (C-7), 148.2 (C-2), 163.6 (C-8). ESI-MS positive mode, m/z:
435 [Mꢂ15]+ (100%), 449 [MꢂH]+ (10%).
mode, m/z: 821/823/825 [M + Na+]. Anal. Calcd. for C36H26Br2N10
-
5,50-((1,10-phenanthroline-2,9-dicarbonyl)bis(azanediyl))bis(1-m
ethylquinolin-1-ium) trifluoromethanesulfonate (6c). Yellow solid,
95% yield, mp 294–299 °C; IR (KBr): mmax 3439, 3286, 3106,
O4: C, 52.57; H, 3.19; N, 17.03. Found: C, 52.63; H, 3.12; N, 17.20.
N2,N9-bis((1-(2-(4-methoxyphenyl)-2-oxoethyl)-1H-1,2,3-triazol-
4-yl)methyl)-1,10-phenanthroline-2,9-dicarboxamide (9b). Beige
solid, 60% yield; mp 240–244 °C; IR (KBr) mmax 3327, 3140, 3070,
1689, 1523, 1275, 1159, 1030, 795 cmꢂ1 1H NMR (DMSO d6,
;
500 MHz): d = 4.64 (6H, s, 2 ꢃ CH3), 7.96 (2H, dd, J = 8.5; 5.5 Hz,
H-16), 8.29–8.32 (4H, m, H-11, H-13), 8.37 (2H, s, H-5), 8.40 (2H,
at, J = 8.0 Hz, H-12), 8.67 (2H, d, J = 8.0 Hz, H-3), 8.94 (2H, d,
J = 8.0 Hz, H-4), 9.38 (2H, d, J = 8.5 Hz, H-17), 9.42 (2H, d,
2930, 1676. 1600, 1239, 1173 cmꢂ1 1H NMR (CDCl3, 500 MHz):
;
d = 3.87 (6H, s, OMe), 4.99 (4H, s, H-10), 5.80 (4H, s, H-16), 6.94
(4H, d, J = 8.0 Hz, H-20), 7.80 (2H, s, H-12), 7.92 (6H, m, H-5, H-
19), 8.42 (2H, d, J = 8.0 Hz, H-4), 8.56 (2H, d, J = 8.0 Hz, H-3),
9.45 (2H, s, NH); 13C NMR (CDCl3, 125 MHz): d = 35.6 (C-10),
55.4 (C-16), 55.7 (OMe), 114.4 (C-20), 121.7 (C-3), 124.3 (C-12),
127.1 (C-18), 127.9 (C-5), 130.7 (C-19, C6), 137.8 (C-4), 143.9 (C-
11), 144.4 (C-7), 149.9 (C-2), 164.7 (C-21, C = Oamide), 189.1
(C = Oketone). Anal. Calcd. for C38H32N10O6: C, 62.98; H, 4.45;
N, 19.33. Found: C, 63.00; H, 4.41; N, 19.36.
J
=
5.5 Hz, H-15), 11.96 (2H, s, NH); 13C NMR (DMSO d6,
125 MHz): d = 46.0 (CH3), 117.1 (C-12), 121.2 (C-16), 122.13
(C-3), 125.4 (C-11), 126.6 (C-18), 128.76 (C-5), 131.1 (C-6), 134.5
(C-10), 135.1 (C-13), 138.9 (C-4), 143.5 (C-7), 143.9 (C-17), 149.0
(C-2), 150.4 (C-15), 154.8 (C-19), 164.1 (C-8). Anal. Calcd. for
C36H26F6N6O8S2: C, 50.94; H, 3.09; N, 9.90. Found: C, 50.89; H,
3.04; N, 9.92.
N2,N9-bis((1-(2-(3,4-dimethoxyphenyl)-2-oxoethyl)-1H-1,2,3-tria
zol-4-yl)methyl)-1,10-phenanthroline-2,9-dicarboxamide (9c). White
solid, 90% yield, mp 248–250 °C; IR (KBr): mmax 3164, 3142, 2936,
1-methyl-8-(9-(quinolin-8-ylcarbamoyl)-1,10-phenanthroline-2-c
arboxamido)quinolin-1-ium trifluoromethanesulfonate (6d). Yellow
solid, 95% yield, mp 273–275 °C; IR (KBr): mmax 3508, 3120, 1682,
1539, 1491, 1278, 1261, 1163, 1030 cmꢂ1; H NMR (DMSO d6,
500 MHz): d = 4.86 (3H, s, CH3), 6.71 (1H, dd, J = 8.0; 4.0 Hz, H-
150), 7.48 (1H, dd, J = 8.0; 5.5 Hz, H-15), 7.62 (2H, d, J = 8.0 Hz,
1675. 1591, 1518, 1269, 1160, 1019, 871, 629 cmꢂ1 1H NMR
;
(CDCl3, 500 MHz): d = 3.89 (3H, s, OMe), 3.90 (3H, s, OMe), 3.93
(3H, s, OMe), 3.94 (3H, s, OMe), 4.96 (4H, bs, H-10), 5.81 (4H, s,
H-16), 6.88 (2H, bs, H-22), 6.46 (2H, bs, H-19), 7.61 (2H, bs,
4

Anda-Mihaela Craciun, A. Rotaru, C. Cojocaru et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 249 (2021) 119318
H-23), 7.83 (2H, s, H-12), 7.91 (2H, s, H-5), 8.42 (2H, d, J = 8.0 Hz, H-
4), 8.56 (2H, d, J = 8.0 Hz, H-3), 9.66 (2H, s, NH); 13C NMR (CDCl3,
125 MHz): d = 35.7 (C-10), 55.4 (C-16), 56.2 (OMe), 56.3 (OMe),
110.2 (C-19), 110.4 (C-22), 121.9 (C-3), 123.1 (C-23), 124.5 (C-
12), 127.2 (C-18), 127.9 (C-5), 129.5 (C-11), 130.7 (C-6), 137.9 (C-
4), 144.4 (C-7), 149.6 (C-2), 150.1 (C-20), 154.6 (C-21), 164.8
(C = Oamide), 189.3 (C = Ocetone). Anal. Calcd. for C40H36N10O8: C,
61.22; H, 4.62; N, 17.85. Found: C, 61.33; H, 4.51; N, 17.96.
N2,N9-bis((1-(2-oxo-2-(3,4,5-trimethoxyphenyl)ethyl)-1H-1,2,3-tri
determined (DTm = Tmwl-TmG4, where Tmwl representing the Tm
value with ligand and TmG4 representing the Tm of the
G-quadruplex without ligand [41–43].
2.11. Docking study
For molecular docking simulations, the AutoDock VINA method
[44] included in the YASARA-Structure program was used [45]. The
molecular structure of phenanthroline compounds 5b, 5d, 6b, 6d
and 9e (ligands) were built and optimized at PM3 semi-empirical
level of theory using Hyperchem software [46]. Two receptors were
considered in this study, namely, dsDNA oligonucleotide (receptor-
1) and a G-quadruplex structure (receptor-2). Details about these
receptors are given in the following. First, to simulate DNA oligonu-
cleotide (receptor-1) we considered the Drew-Dickerson dodecamer
d(CGCGAATTCGCG) containing 24 nucleotides, which was built and
optimized using YASARA tools. Characteristics of Drew-Dickerson
dsDNA dodecamer are as follows: (1) molecular weight, 7.43 kDa;
(2) radius of gyration, 13.543 Å; (3) molecular surface area,
3551 Å2 and (4) solvent accessible surface area, 4531 Å2.
Second, the configuration of G-quadruplex (receptor-2) was taken
from protein data bank site (rcsb.org) by downloading the structure
with PDB ID: 2F8U (NMR solution). This structure (PDB ID: 2F8U)
is related to G-quadruplex formed in human Bcl-2 promoter (hybrid
form) [47]. The second receptor (2F8U) was chosen for this study,
because it has similar characteristics with the first receptor (dsDNA).
More exactly, characteristics of 2F8U (receptor-2) are as follows: (1)
content of 23 nucleotides with the sequence (GGGCGCGGGAG-
GAATTGGGCGGG); (2) molecular weight, 7.27 kDa; (2) radius of
gyration, 10.647 Å; (3) molecular surface area, 3225 Å2 and (4) sol-
vent accessible surface area, 3876 Å2. By comparing values for radii
of gyration and molecular surface areas, it can be inferred that the
G-quadruplex (2F8U) is a more condensed receptor than the Drew-
Dickerson dsDNA dodecamer.
Next, molecular structures of ligands (5b, 5d, 6b, 6d and 9e) and
both receptors (dsDNA and 2F8U) were prepared in YASARA environ-
ment for molecular docking simulations in implicit water solvent at
pH 7.4. Note that, an automatic parameterization procedure (termed
‘‘AutoSMILES”) for the unknown structures is implemented in the
YASARA program. Therefore, this algorithm was employed to gener-
ate force field parameters for receptors and the ligands 5b, 5d, 6b, 6d
and 9e. Simulations were performed using the self-parameterizing
knowledge-based YASARA force field [48,49].
During molecular docking simulations, receptors were treated as
rigid structures, whereas the ligand was treated as a flexible mole-
cule. The computations (by AutoDock VINA) were done using a num-
ber of 100 docking runs followed by the cluster analysis. Generally,
docking results are grouped around certain hot spot conformations,
and the lowest energy complex in each cluster is saved by the
YASARA program. Two complexes belong to different clusters if the
ligand RMSD (root-mean-square-deviation) is greater than an
imposed minimum value. In this study, we considered the default
RMSD limit (for heavy atoms) equal to 5 Å. After clustering all 100
runs, particular complex conformations were found and grouped into
12 clusters and 10 clusters for the systems dsDNA-Ligand and 2F8U-
Ligand, respectively. Thus, molecular docking and clustering results
suggested multiple hotspots for binding. For each generated complex,
the binding energy (Eb, kcal/mol) as well as dissociation constant
(Kd, nM) was determined.
azol-4-yl)methyl)-1,10-phenanthroline-2,9-dicarboxamide
(9d).
White solid, 80% yield, mp 259–260 °C; IR (KBr): mmax 3440,
2924, 2853, 1671, 1586. 1542, 1504, 1457, 1415, 1323, 1127,
1001 cmꢂ1
; d = 3.90 (6H, s,
1H NMR (CDCl3, 500 MHz):
2 ꢃ OMe), 3.92 (3H, s, OMe), 5.01 (4H, bs, H-10), 5.84 (4H, s, H-
16), 7.21 (2H, s, H-19, H-23), 7.81 (2H, s, H-12), 7.93 (2H, s, H-5),
8.44 (2H, d, J = 8.0 Hz, H-4), 8.59 (2H, d, J = 8.0 Hz, H-3), 9.46
(2H, s, NH); 13C NMR (CDCl3, 125 MHz): d = 35.7 (C-10), 55.6 (C-
16, 56.6 (2 ꢃ OMe), 61.2 (OMe), 105.9 (C-19), (C-23), 121.7 (C-3),
124.3 (C-12), 128.0 (C-5), 129.1 (C-11), 130.8 (C-6), 137.1, (C-18),
137.9 (C-4), 143.9 (C-21), 144.4 (C-7), 149.8 (C-2), 153.5 (C-20),
(C-22), 164.7 (C = Oamide), 189.9 (C = Ocetone). Anal. Calcd. For
C42H40N10O10: C, 59.71; H, 4.77; N, 16.58. Found: C, 59.78; H,
4.69; N, 16.66.
N2,N9-bis((1-(anthracen-9-ylmethyl)-1H-1,2,3-triazol-4-yl)methy
l)-1,10-phenanthroline-2,9-dicarboxamide (9e). Yellow solid, 40%
yield, mp 202–204 °C; IR (KBr): mmax 3368, 3050, 2926, 1665,
;
1528, 1493, 1170, 1049, 726 cmꢂ1 1H NMR (DMSO d6,
500 MHz): d = 4.53 (4H, d, J = 4.5 Hz, H-10), 6.54 (4H, s, H-16),
7.45–7.53 (8H, m, H-ar), 7.92 (2H, s, H-12), 8.07 (2H, d,
J = 8.5 Hz, H-ar), 8.14 (2H, s, H-5), 8.39 (2H, d, J = 8.0 Hz, H-3),
8.52 (2H, d, J = 8.5 Hz, Har), 8.63 (2H, s, H-ar), 8.69 (2H, d,
J = 8.5 Hz, H-4), 9.67 (2H, t, J = 5.5 Hz, NH); 13C NMR (DMSO d6,
125 MHz): d = 34.7 (C-10), 45.4 (C-16), 121.0 (C-3), 122.8 (C-12),
124.0 (2 ꢃ CH-ar), 125.8 (Cq-ar), 125.3 (2 ꢃ CH-ar), 127.0
(2 ꢃ CH-ar), 127.9 (C-5), 128.9 (CH-ar), (Cq-ar), 129.0 (2 ꢃ CH-
ar), 130.2 (C-6), 131.0 (Cq-ar), 138.2 (C-4), 143.6 (C-7), 144.5 (C-
11), 149.2 (C-2), 163.7 (C-8). Anal. Calcd. for C52H36N10O4: C,
72.21; H, 4.20; N, 16.19. Found: C, 72.28; H, 4.16; N, 16.26.
2.9. Cell proliferation assay
The in vitro biological tests were performed to the National Can-
cer Institute (NCI, USA), under the Developmental Therapeutics
Program (DTP). The cytotoxicity experiments were performed
using a 48 h exposure protocol which consisted of a sulphorho-
damine B assay [40].
2.10. General protocol for experiments using circular dichroism
The utilised DNA sequences were: G-quadruplex (G_telomer) –
50-TTGGGATTGGGATTGGGATTGGGATT- 30 and dsDNA – G_telo
mer + G_telomer complement (50-AATCCCAATCCCAATCCCAATCC
CAA ꢂ 30).
Circular dichroism experiments were recorded by using a Chi-
rascan plus (Applied Photophysics) equipped with a Peltier tem-
perature controller. Spectra were recorded at 25 °C in the
wavelength range of 240–340 nm and 240–400 nm.
A scan parameters used were: 1.5 s response time and 0.5 nm
bandwidth. The solutions used for the CD experiments: 100
lM
G-quadruplex with 2 mM KCl or 100 M duplex DNA in the TAE
l
solution with pH = 7.4, incubated with 1.5 mM in DMSO of the each
ligand. CD melting experiments were performed by using the G4-
DNA solutions, in the presence and absence of ligands, using
3. Results and discussion
3.1. Chemistry
60
ature range between 25 and 80 °C, with an heating rate of
1 °C/min. By using Origin 7.0 software the Tm values were
lL of the ligands (1.5 mM in DMSO) at 290 nm in the temper-
The synthesis of the new derivatives started with the two steps
oxidation of neocuproine 1 to 1,10-phenanthroline-2,9-dicar
D
5

Anda-Mihaela Craciun, A. Rotaru, C. Cojocaru et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 249 (2021) 119318
boxylic acid 3 [50,51]. The key intermediate in the synthesis of the
ture, we were not able to send these two compounds for testing,
therefore only ten compounds have been tested. The representa-
tive results are summarized in Table 1.
1,10-phenanthroline derivatives was dicarbonyl dichloride
4
obtained by treating the acid 3 with oxalyl chloride and catalytic
dimethylformamide (Scheme 1).
Interestingly, only four compounds, 5b, 5d, 6b and 6d exhibited
a promising growth inhibitory activity against cancer cells. The
best growth inhibitory activity was shown by compounds 5b and
6b, both of them containing two pyridin-3-yl rings as substituents
at the amide groups from positions 2 and 9 of 1,10-phenathroline.
Both compounds showed also cytotoxic activity against several
cancer cells, the most important being shown against UACC-62
melanoma cells for compound 6b and against RXF 393 and A498
renal cancer cells for compound 5b. We observed that double
methylation of 5b changed the profile of the anticancer activity
on the entire panel of cancer cells, being difficult to draw conclu-
sions. It worths also to note, that in Phen-DC3 (Fig. 1), the position
of amino group is also meta relative to the heterocyclic nitrogen
atom.
Compound 5d bearing two quinolin-8-yl rests at the two N-
amide groups showed also a good inhibition effect especially on
leukemia and lung cancer cells. Compound 6d obtained by
mono-N-methylation of compound 5d showed also a different pro-
file regarding the inhibitory activity of the cancer cells, but overall,
the potency appear to be diminished by comparison with 5d.
Interestingly, the substitution of N-amide with more flexible
substituents containing 1,2,3-triazoles in compounds 9 resulted
in the loss of the activity, compounds 9a and 9d presenting almost
no inhibition effect on the tested cell lines. As an exception, com-
pound 9c showed a very selective moderate activity only against
HCT-116 colon cancer cells, M14 melanoma cells and 786–0 renal
cancer cells.
Compounds 5a-f based on amide bond, have been synthesized
in moderate yields (15–77%) by the direct reaction of dichloride
4 with the 2-aminothiazole, 2-aminopyridine, 3-aminopyridine,
5-aminoquinoline, 8-aminoquinoline and propargylamine, respec-
tively (scheme 2).
The attempt to methylate compounds 5a-e using methyl iodide
failed, therefore we used more reactive methyl trifluoromethane-
sulfonate as methylation reagent (scheme 3). Various molar ratios
(from 1:2 to 1:40) of methyl trifluoromethanesulfonate were
added to the compounds 5a-e in chloroform at reflux in order to
obtain the corresponding bis-methylated compounds. Regardless
of the molar ratio used, in the case of compounds 5b and 5c, we
obtained only the corresponding N,N’-dimethylated compounds
6b and 6c, while for the compound 5d, only the N-methylated
compound 6d. In the case of methylation of compound 5a, we
obtained different ratio mixtures of both mono- and dimethylated
compounds 6a and 6e, depending on the molar ratio of trifluo-
romethanesulfonate used. The separation of the two compounds
proved to be very difficult and only very small quantities of pure
compounds could be isolated. Also, in the above conditions, no cor-
responding methylated compound of 5e was obtained.
In order to obtain the triazole substituted target compounds
(Fig. 1), first the azides 8a-e were synthesized by substitution of
halogenated derivatives 7a-e by using sodium azide in a biphasic
system and tetrabutylamonium bromide (TBAB) as phase transfer
catalyst [52] (Scheme 4).
Then, compounds 9a-e were synthesized via a ‘‘click” cycloaddi-
tion of azides 8a-e to the dipropargylated derivative 5f (Scheme 5).
Reactions were carried out using classical conditions (copper (II)
sulphate and sodium ascorbate) in a tert-butanol/water at 50 °C
for the compounds 9a, 9b, 9f and 9e, while for the compounds
9c and 9d we used DMSO/water as solvent at 90 °C and 1,10-
phenanthroline as copper ligand.
All synthesized compounds were fully characterized, including
the already reported compound 5a [39]. The shifting of the amide
proton over a moderately wide range in NMR spectra of com-
pounds 5 (8.82 – 12.87 ppm) suggests their involvement in H-
bonding with the adjacent nitrogen atom of 1,10-phenanthroline
or other atoms.
Due to the promising shown growth inhibition results, com-
pounds 5b and 6b were selected for evaluation against 60 cell lines
at five different concentrations. Selected results from the NCI-60 5-
dose screen are shown in Table 2.
The results presented in Table 2 confirmed the excellent
antiproliferative capacity of the compound 5b that showed GI50
values much lower than the corresponding dimethylated com-
pound 6b. Thus, compound 5b displayed GI50 values < 100 nM
against fifteen cell lines, the best being registered against colon
cancer HCC-2998, HCT-116, HCT-15, HT29, KM12 and SW-620 cell
lines, lung cancer HOP-62 and NCI-H460 cell lines and leukemia K-
562 and HL-60(TB) cell lines.
The best GI50 values for compound 6b were obtained against
For the N-methylated compounds 6a-e, the most relevant sig-
nals observed in NMR spectra to confirm the methylation, were
the ones furnished by the protons and carbon of methyl group
(the signal of methyl protons appear at 4.38–4.86 ppm, while the
signal of carbon at 44–50 ppm).
lung cancer NCI-H226 (0.121
lines and renal cancer SN12C (0.126
6b also displayed selective cytotoxic activity on MDA-MB-468
breast cancer cells (LC50 = 6.83 M), RXF 393 renal cancer
M) and SNB-75 CNS cancer cells
l
M) and A549/ATCC (0.169
lM) cell
lM), respectively. Compound
l
cells (LC50
(LC50 = 17.7
=
16.9 l
l
M).
The lethal action shown by compound 6b on several cancer cells
might be explained by its structural particularities leading to pos-
sible specific interactions and stabilization of G4-DNA structure. In
order to confirm this supposition, circular dichroism experiments
were used and the results discussed further. These interactions
may induce the inhibition mechanism of telomerase which is up
regulated in most cancer subtypes.
3.2. Biological assay
Twelve of the new compounds (5a-c, 5e, 5f, 6a, 6b, 6d, 6e, 9a, 9c
and 9d) were selected by National Cancer Institute (NCI) for
screening against 60 human tumor cell lines panel at a single dose
of 10
lM [40], but unfortunately, due to the difficulties we faced
during separation of compounds 6a and 6e from the reaction mix-
Scheme 1. Oxidation of neocuproine to 1,10-phenanthroline-2,9-dicarboxylic acid 3.
6

Anda-Mihaela Craciun, A. Rotaru, C. Cojocaru et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 249 (2021) 119318
Scheme 2. Synthetic pathway for the 2,9-disubstituted 1,10-phenathrolines 5a-f.
Scheme 3. Synthetic pathway for the methylated heterocyclic compounds 6a-e.
Scheme 4. Synthetic pathway for azides 8a-e.
3.3. Circular dichroism experiments
was formed from human telomere sequence in the presence of
K+, when the topology adopted furnished a major positive band
at 240 nm and two minor negative bands around 290 nm and
270 nm, this being known as the hybrid [3 + 1] structure of
G4-DNA [53].
In order to evaluate the ability of 5b, 5d, 6b, 6d and 9e
ligands to interact with DNA (telomeric or duplex), circular
dichroism (CD) experiments were used. G-quadruplex structure
7

Anda-Mihaela Craciun, A. Rotaru, C. Cojocaru et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 249 (2021) 119318
Scheme 5. Synthetic route for 1,2,3- triazole appended 1,10-phenanthrolines 9a-e.
Fig. 2 presents the profiles of the G4–ligand complexes while
titrated with solutions of the investigated compounds. Modifying
the concentration in case of ligands 5d and 9e, respectively,
appears to not produce substantial modification of G-quadruplex
structures (Fig. 2b,e). Titration with solutions of ligands 6b and
6d, respectively, led to significant modification of the starting sig-
nal for the G-quadruplex structure (Fig. 2c,d). Thus, for both com-
solution, the capacity to unfold the DNA-complex structure was
registered. Thereby, the melting points between 25 and 80 °C,
using an excess (0.45 mM) of compounds solutions, were evaluated
and the results are shown in Fig. 3.
By comparing the
DTm values obtained for the non-methylated
derivatives with the methylated ones (Table 3), higher values were
obtained in the last cases, indicating a higher ability of 6b and 6d
to stabilize the G4 structures, data being are in agreement to other
similar reported results [55,56].
The most promising result, according to the CD melting exper-
iments, was obtained for 6b, for which the presence of two methyl
residues on pyridin-3-yl induced an even higher stabilizing effect,
pounds,
a tendency towards an antiparallel conformational
structure of G4 could be observed. The antiparallel G4 structure
formed for compound 6d is proved by the 270 nm band that
became negative and the 240 nm band that increased to positive
values. Also, even if the same structural characteristic aspects
could be observed for the compound 6b, the structure in this case
does not fit to a complete antiparallel profile.
the
DTm value reaching 24 °C. Typically, DTm values higher than
15 °C are required for promising G4 intercalators using different
However, a different profile of the G-quadruplex structure dur-
ing the increasing concentration of the ligand 5b can be observed
in Fig. 2a: (i) a slow increasing of the 290 nm and 245 nm bands;
(ii) a slow decreasing of the 270 nm band; (iii) the appearance of a
new band at 330 nm. This continuous modification of the profile
suggests the fact that ligand 5b did not lead to a stabilization of
the G4-DNA structure [19,54].
Slight modifications of the intensities observed in case of all
tested compounds can be the result of physical interactions (elec-
trostatic or intermolecular H-bonding) between G4-DNA and
ligands.
Next, the ability of the same set of compounds to interact with
dsDNA was investigated. dsDNA was prepared by thermal anneal-
ing of the single-stranded G-telomer-complement sequence with
the G-telomer sequence. The dsDNA formation was proved by CD
profile by the presence of characteristic positive band around
270 nm and a negative one around 245 nm. The experimental
results with compounds 5b, 5d, 6b, 6d and 9e did not show any
changes neither in the shape nor the intensity of the original
dsDNA signal. This suggests that the interactions are much lower
than in the case of the corresponding G4-ligand complex (see
Fig. 1S in the supplementary material), thus confering a great
selectivity of investigated 5b, 6b and 6d compounds toward
human telomeric G-quadruplex DNA versus duplex DNA.
G4 DNA sequences [13,22,56–58].
3.5. Docking experiment
To understand the structural basis of the investigated com-
pounds to interact with G-quadruplexes or dsDNA, we performed
a series of molecular docking (MD) studies. The AutoDock Vina
method implemented in the YASARA Structure software packages
[43] was employed for the current MD simulations. Structures of
5b, 5d, 6b, 6d and 9e were first drawn and optimized at PM3 level
of theory using Hyperchem software [44], and subsequently
exported to YASARA program. The structures of G-quadruplex
(pdb ID: 2F8U) and dsDNA (pdb ID: 4C64) were used as receptors,
dsDNA representing the Drew Deckerson dodecamer [59,60]
(assembled in YASARA program). The comparative results of MD
simulations indicated the interaction of compounds 5b, 5d, 6b,
6d and 9e with both the dsDNA (receptor-1) and G-quadruplex
(receptor-2), the selected and optimized molecular docking models
being reported in Figures 4, S2-S7 and Table 4.
In both cases, complexes are stabilized mainly by hydrophobic
interactions and one hydrogen bond between oxygen atom (from
ligand, C = O group) and hydrogen atom (from the receptor, –
NH2 group / A, G residuals) (for details, see ESI). According to these
results, the reported compounds interact with receptor-1 by bind-
ing predominantly to the minor groove of the dsDNA (Fig. 4) with
the theoretically calculated binding energy (Eb) for the optimized
interactions ranging from ꢂ8.24 kcal/mol to ꢂ10.57 kcal/mol.
The values of theoretically calculated dissociation constant (Kd)
3.4. CD melting experiments
The ability of the studied compounds to stabilize telomeric G4-
DNA was further confirmed by using a CD melting assay. In this
study, by increasing the temperature (1 °C/min) of the components
for the same interactions vary in
a larger span, between
8

Anda-Mihaela Craciun, A. Rotaru, C. Cojocaru et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 249 (2021) 119318
Table 1
Percentage growth inhibition (PGI)a data of compounds 5a-c, 5e-f, 6b, 6d, 9a and 9c-d at a single concentration (10-5 M) against an NCI 60 human tumor cell lines (selection).b
Panel/Cell line ;
Compound/Percent growth inhibition (PGI, %)
5a
5b
5d
5e
5f
6b
6d
9a
9c
9d
Leukemia
CCRF-CEM
K-562
MOLT-4
RPMI-8226
SR
0
0
0
0
8
72
83
68
81
57
62
44
60
90
61
11
13
0
12
23
12
6
11
6
29
65
28
38
25
55
31
45
56
32
0
0
8
0
4
0
0
0
3
0
2
0
0
3
0
14
Non-Small Cell Lung Cancer
A549/ATCC
HOP-62
3
8
0
4
0
6
0
0
78
91
76
95
77
89
98
70
58
68
21
72
39
14
63
51
18
17
0
19
11
4
13
11
0
11
2
9
0
17
100b (3)
78
43
95
67
15
17
1
12
13
0
7
0
0
0
0
10
0
0
3
0
13
0
3
0
9
0
10
0
0
1
0
0
5
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
Colon Cancer
COLO 205
HCC-2998
HCT-116
HCT-15
63
23
7
100b (7)
100b (19)
19
35
16
0
0
9
0
0
2
6
87
97
95
80
93
94
89
43
15
62
50
48
45
28
7
20
7
37
22
20
27
2
0
7
1
8
2
2
25
58
57
39
66
58
44
18
15
13
0
12
0
0
0
1
0
3
0
0
0
3
78
0
3
0
4
8
0
0
0
0
HT29
KM12
SW-620
0
0
3
CNS cancer
SF-268
SF-295
SF-539
SNB-19
0
1
5
0
7
4
84
95
39
35
5
51
0
0
9
8
8
20
5
1
10
6
0
10
6
47
94
62
86
87
87
6
4
8
30
26
52
0
0
0
0
0
2
0
0
6
3
8
0
0
0
1
8
10
0
100b (7)
87
SNB-75
U251
59
88
70
Melanoma
LOX IMVI
M14
0
7
0
0
0
4
1
2
86
85
75
74
82
74
70
71
60
42
19
24
10
66
15
47
23
16
6
12
0
19
3
12
13
1
0
3
0
13
2
1
35
46
32
43
68
98
31
4
2
16
15
33
1
0
0
0
11
0
0
5
0
0
63
5
0
0
4
0
9
0
0
0
1
0
2
0
0
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian cancer
IGROV1
OVCAR-3
OVCAR-5
OVCAR-8
SK-OV-3
6
100b (54)
25
7
0
8
0
0
85
77
63
77
52
28
65
0
50
34
16
0
7
2
1
12
0
3
7
6
67
37
21
38
92
53
8
0
45
12
6
0
3
2
0
0
3
0
4
0
0
0
5
25
16
Renal cancer
786–0
A498
ACHN
CAKI-1
3
0
10
7
0
80
40
27
44
46
0
0
0
17
39
0
6
0
2
4
0
0
23
5
58
47
0
15
8
14
5
52
2
0
0
4
0
0
57
0
0
13
1
2
0
0
4
10
0
100b (39)
77
89
RXF 393
SN12C
100b (50)
68
67
0
30
1
100b (15)
4
Prostate cancer
PC-3
DU-145
0
0
67
85
63
39
19
0
1
0
45
90
23
5
6
0
7
0
0
0
Breast cancer
MDA-MB-231/ATCC
HS 578 T
MCF7
T-47D
MDA-MB-468
0
0
29
20
0
75
54
71
34
62
26
44
23
0
4
32
23
0
0
0
14
10
11
67
25
54
8
2
0
15
0
0
0
8
3
16
4
17
0
2
11
0
17
39
48
35
63
88
100b (2)
100b (16)
The best values in terms of growth inhibition are highlighted in bold and red.
a
The number reported for the one-dose assay, percent growth inhibition (PGI) is growth inhibition relative to the no-drug control, and relative to the time zero number of
cells.
b
Cytotoxic effect; Lethality percent is reported in brackets.
17.89 nM and 904.35 nM, revealing a stronger binding of 5d and 6d
to receptor-1 when compared to 5b, 6b and 9e.
In case of interaction of the ligands 5b, 5d, 6b, 6d and 9e, with
the receptor-2, the Eb values were comparable to the ones obtained
for receptor-1, while Kd values were dramatically different in case
of ligand 6b. A strong decrease in Kd value from 609.27 nM
(receptor-1) to 405.65 nM (receptor-2) shows stronger affinity of
ligand 6b to G-quadruplex, whereas for other ligands, the Kd values
9

Anda-Mihaela Craciun, A. Rotaru, C. Cojocaru et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 249 (2021) 119318
Table 2
Results of the 5-dose in vitro human cancer cell growth inhibition for compounds 11a, 15a and 15j (selection).a
Panel/Cell line ;
Compound/Percent growth inhibition (GI50, lM)
5b
6b
GI50
(lM)
LC50
(l
M)
GI50
(lM)
LC50 (lM)
Leukemia
CCRF-CEM
K-562
HL-60(TB)
RPMI-8226
SR
0.364
0.051
0.074
0.162
0.180
>100
>100
>100
>100
>100
6.860
2.100
2.160
4.040
4.380
>100
>100
>100
>100
>100
Non-Small Cell Lung Cancer
A549/ATCC
HOP-62
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
Colon Cancer
COLO 205
HCC-2998
HCT-116
HCT-15
0.134
0.083
0.128
0.219
0.099
0.062
n.d.
>100
>100
>100
>100
>100
>100
n.d.
0.169
1.210
0.121
1.560
2.260
0.319
0.599
>100
>100
>100
>100
>100
>100
>100
0.135
0.065
0.043
0.053
0.044
0.049
0.070
>100
>100
>100
>100
>100
>100
>100
4.350
3.200
3.070
4.090
2.160
3.200
3.280
>100
>100
>100
>100
>100
>100
>100
HT29
KM12
SW-620
CNS cancer
SF-268
SF-295
SF-539
SNB-19
0.202
0.237
0.187
0.106
0.273
0.066
>100
>100
>100
>100
>100
>100
4.290
3.000
3.300
1.850
0.521
2.440
>100
>100
>100
>100
17.7
SNB-75
U251
>100
Melanoma
LOX IMVI
M14
MDA-MB-435
SK-MEL-28
SK-MEL-5
UACC-62
Ovarian cancer
IGROV1
OVCAR-3
OVCAR-8
Renal cancer
786–0
0.203
0.080
0.259
0.333
0.229
0.267
>100
>100
>100
>100
>100
>100
6.980
4.640
8.690
1.860
0.697
1.450
>100
>100
>100
>100
>100
>100
0.850
0.243
0.147
>100
>100
>100
1.360
4.690
5.610
>100
>100
>100
0.337
0.214
0.155
0.141
0.110
0.300
>100
>100
>100
>100
>100
>100
14.00
20.30
4.510
4.260
1.980
0.126
>100
>100
>100
>100
16.9
A498
ACHN
CAKI-1
RXF 393
SN12C
>100
Prostate cancer
PC-3
DU-145
0.087
0.177
>100
>100
3.620
0.486
>100
>100
Breast cancer
MDA-MB-231/ATCC
MCF7
T-47D
MDA-MB-468
0.511
0.138
0.243
0.093
>100
>100
>100
>100
5.180
2.890
0.395
0.322
>100
>100
>100
6.83
GI50 – the molar concentration of tested compound causing 50% growth inhibition of tumor cells.
LC50 – the molar concentration of tested compound causing 50% death of tumor cells.
n.d. – Not determined.
The best values in terms of GI50 and LC50 are highlighted in bold and red.
a
In vitro 60 cell 5-dose screening (from NCI’s) [40].
are either comparable for both ligands (5b) or higher for G-
quadruplex DNA (5d, 6d and 9e). These data suggest the similar
affinity of 5b to receptor-1 and receptor-2, and stronger affinity
of 5d, 6d and 9e to receptor-1 than to receptor-2. Interestingly,
the theoretical data obtained for ligand 6b are supported by the
melting point experimental data where 6b have shown the highest
4. Conclusions
A new series of fifteen 1,10-phenanthroline based derivatives
designed as analogues of G-quadruplex stabilizer Phen-DC3 have
been synthesized and tested for the anticancer properties and their
capacity to selectively stabilizing the G4-DNA structures. Com-
pound 5b containing two pyridin-3-yl rests as substituents for
value of
DTm.
10

Anda-Mihaela Craciun, A. Rotaru, C. Cojocaru et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 249 (2021) 119318
Fig. 2. CD spectra of the G4-DNA in the absence (black line) and in presence of compounds: (a) 5b; (b) 5d; (c) 6b; (d) 6d; (e) 9e.
the two nitrogen atoms of the amide groups at the positions 2 and
9 of 1,10-phenanthroline, showed excellent antiproliferative activ-
ity. Even if by comparison with 5b, the dimethylated derivative 6b
has a diminished potency in terms of anticancer activity, it demon-
strated the best ability to bind and stabilize selectively the G-
quadruplex DNA structure in the CD and CD melting experiments.
Also mono-N-methylated compound 6d displayed to be a good sta-
bilizing ligand for G4-DNA suggesting that a positive charge is
important for a better interaction with G-quadruplex structures.
Also, the position of amino group on the appended heterocycles
proved very important, all tested compounds showing anticancer
activity having the amino group in position 3 or 8. Increasing the
molecule flexibility due to the four methylene bridges present in
the structure of derivatives 9 proved detrimental for the growth
inhibitory properties on the cancer cells. Computational docking
studies revealed preferential binding affinities of the investigated
compounds toward dsDNA except for compound 6b which demon-
strated lower Kd value for G-quadruplex rather than for dsDNA. In
case of 6b the theoretically determined affinity to G-quadruplex is
also supported by the melting point experimental data. In order to
correlate the cytotoxic properties shown by compounds 6b on
Fig. 3. CD melting profiles of the G4-DNA in the absence (black line) and in
presence of compounds 5b, 5d, 6b, 6d and 9e.
11

Anda-Mihaela Craciun, A. Rotaru, C. Cojocaru et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 249 (2021) 119318
Table 3
D
Tm values obtained by using CD-melting experiments.
Non-Methylated
Methylated
Ligand
Tm (oC)
5b
5d
6b
6d
9e
D
13
5
24
15
2
Fig. 4. MD sticks and balls rendering models of: 5b (ligand)/dsDNA (receptor-1) (a); 6b (ligand)/dsDNA (b); 5b/G-quadruplex 2F8U (receptor-2) (c); 6b/G-quadruplex 2F8U
(d).
Table 4
Acknowledgements
Values of binding energy and dissociation constants.
Ligand
dsDNA-Ligand
Eb (kcal/mol)
2F8U-Ligand
Eb (kcal/mol)
The authors gratefully acknowledge National Cancer Institute
(NCI) for biological evaluation of compounds on their 60-cell
panel: the testing was performed by the Developmental Therapeu-
tics Program, Division of Cancer Treatment and Diagnosis (the URL
to the Program’s website: http://dtp.cancer.gov/). We also thank
the POSCCE-O 2.2.1, SMIS-CSNR 13984-901, Project no.
257/28.09.2010, CERNESIM, for the NMR experiments. This work
was also supported by a grant of the Ministry of Research and Inno-
vation, CNCS – UEFISCDI, project number PN-III-P4-ID-PCCF-2016-
0050, within PNCDI III.
Kd (nM)
Kd (nM)
5b
5d
6b
6d
9e
ꢂ8.24
ꢂ10.57
ꢂ8.48
ꢂ10.57
ꢂ8.70
904.35
17.898
609.27
18.86
ꢂ8.24
ꢂ9.57
ꢂ8.72
ꢂ10.37
ꢂ8.63
905.87
96.46
405.65
24.96
420.29
471.40
some cancer cells and a possible inhibition of telomerase and to
find the exact cellular effect, further studies have to be conducted.
Appendix A. Supplementary material
CRediT authorship contribution statement
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.saa.2020.119318.
Anda-Mihaela Craciun: Investigation, Writing - original draft,
Writing - review & editing. Alexandru Rotaru: Investigation, Writ-
ing - original draft, Writing - review & editing. Corneliu Cojocaru:
Investigation, Writing - original draft, Validation. Ionel I. Man-
galagiu: Validation. Ramona Danac: Conceptualization, Supervi-
sion, Writing - original draft, Writing - review & editing.
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