B.A. Babgi, K.H. Mashat, M.H. Abdellattif et al.
Polyhedron 192 (2020) 114847
CuBr(PPh
CuBr(PPh (0.203 g, 0.218 mmol) and 3-(2-pyridyl)-5,6-diphe-
nyl-1,2,4-triazine (0.071 g, 0.229 mmol) were reacted to yield 6
3
)(3-[2-pyridyl]-5,6-diphenyl-1,2,4-triazine)
(5).
investigations were carried out at pH 7.4 (using a phosphate buf-
fer), keeping the molarity of the compounds at 20 mM while succes-
sively changing the concentration of ct-DNA. The photometric
responses were followed after incubating the solutions for 2 min.
From the absorbance values, the binding constants (K ) of the com-
b
pounds with ct-DNA were extracted from the Benesi-Hildebrand
equation (Eq. (1)):
3 3
)
79
63
+
as a brown powder (0.127 g, 81%). HR ESI MS [C38
H
29Br CuN
4
P] :
P]+: calcd
29BrCuN P: C,
3.74; H, 4.08; N, 7.82%; found: C, 63.98; H, 3.91; N, 7.46%. IR
81
63
calcd 712.0804, found 712.0910; [C42
H31Br CuN
4
7
6
14.0784, found 714.0890. Anal. Calcd for C38
H
4
ꢀ1
ꢀ1
31
(
solid): 525 cm
s, P(C ).
CuBr(PPh
Diphenyl-1,10-phenanthroline (0.106 g, 0.319 mmol) and CuBr
PPh (0.296 g, 0.318 mmol) were reacted to yield 6 as an orange
powder (0.202 g, 86%). HR ESI MS [C42
m
(P-Cu), 487 cm
m
(Cu–N). P NMR d: 30.4
Â
À
ÁÃ ÈÂ
À
ÁÃ
É
½
A
o
=ðA ꢀꢀ A
o
Þꢁ¼
e
g
=
e
hꢀg
ꢀ
e
g
þ
e
g
=
e
hꢀg
ꢀ
e
g
ꢂ1=ðK ½DNAꢁÞ
b
(
6 5 3
H )
3
)(4,7-diphenyl-1,10-phenanthroline)
(6).
4,7-
ð1Þ
A
o
/A ꢀ A
o o
(where A and A are the absorbance values of the com-
(
3 3
)
7
9
63
+
pounds in the absence and presence of ct-DNA, respectively) was
H
31Br CuN
2
P] : calcd
P] : calcd 738.0708,
2
P: C, 68.34; H, 4.23;
8
1
63
+
plotted against 1/[DNA] and the K
the ratio of intercept to slope [29].
b
values were calculated from
7
36.0804, found 736.1240; [C42
found 738.1005. Anal. Calcd for C42
N, 3.80%; found: C, 68.09; H, 3.91; N, 3.36%. IR (solid): 522 cm
H
31Br CuN
2
H
31BrCuN
ꢀ1
ꢀ1
31
2.6. Anticancer studies
m
(P-Cu), 489 cm
CuBr(PPh )(5-nitro-1,10-phenanthroline) (7). 5-Nitro-1,10-
phenanthroline (0.107 g, 0.475 mmol) and CuBr(PPh (0.439 g,
.472 mmol) were reacted to yield 8 as a bright orange powder
6 5 3
m (Cu–N). P NMR d: 30.2 (s, P(C H ) ).
3
The cells were delivered by the Egyptian Holding Company for
Biological Products and Vaccines (VACSERA), Giza, Egypt, and then
reserved in the tissue culture unit. The cells were grown in RBMI-
1640 medium, supplemented with 10% heat inactivated FBS, 50
units/mL of penicillin, and 50 mg/mL of streptomycin, and reserved
in a humidified atmosphere containing 5% CO2 [30,31]. The cells
were maintained as monolayer cultures by serial sub-culturing.
Cell culture reagents were sourced from Lonza (Basel, Switzerland).
The anticancer activities of the compounds were evaluated against
MCF-7 cells (breast cancer), HEPG-2 cells (liver cancer), PC-3 cells
(prostate cancer), and MOLT-4 cells (leukemia cancer).
The sulforhodamine B (SRB) assay method was used to deter-
mine the cytotoxicity, as described by Skehan et al. [32]. Exponen-
tially-growing cells were collected using 0.25% Trypsin-EDTA and
seeded in 96-well plates at 1000-2000 cells/well in RBMI-1640
supplemented medium. After 24 h, cells were incubated for 72 h
with various concentrations of the tested compounds. Following
3 3
)
0
7
9
63
(
0.274 g, 92%). HR ESI MS [C30
H
22Br CuN
22Br CuN P]: calcd 631.0009, found
22BrCuN P: C, 57.11; H, 3.51; N,
3 2
O P]: calcd 629.0029,
81
63
found 629.0114; [C30
H
3 2
O
6
6
31.0072. Anal. Calcd for C30
.66%; found: C, 56.58; H, 3.28; N, 5.92%. IR (solid): 522 cm
H
3 2
O
ꢀ1
m
ꢀ1
31
(
P-Cu), 501 cm
CuBr(PPh
0.104 g, 0.409 mmol) and CuBr(PPh
m
(Cu–N). P NMR d: 30.1 (s, P(C
6
H
5 3
) ).
0
0
3
)(dppz)
(8).
Dipyrido[3,2-a:2 ,3 -c]phenazine
(
3
)
3
(0.380 g, 0.409 mmol) were
reacted to yield 9 as an orange powder (0.301 g, 84%). HR ESI MS
7
9
63
81 63
[
C
36
H
25Br CuN
4
P]: calcd 686.0396, found 686.0146; [C36
H
25Br
-
-
CuN
BrCuN
.92%. IR (solid): 522 cm
d: 30.6 (s, P(C ).
4 25
P]: calcd 688.0376, found 688.0137. Anal. Calcd for C36H
4
P: C, 62.84; H, 3.66; N, 8.14%; found: C, 62.71; H, 3.48; N,
ꢀ1
ꢀ1
31
7
m
(P-Cu), 496 cm
m(Cu–N). P NMR
6
5 3
H )
2.4. Crystallographic studies
7
2 h treatments, the cells were fixed with 10% trichloroacetic acid
for 1 h at 4 °C. Wells were stained for 10 min at room temperature
with 0.4% SRBC dissolved in 1% AcOH. The plates were air dried
for 24 h and the dye was dissolved in Tris-HCl for 5 min with
shaking at 1600 rpm. The optical density (OD) of each well was
assessed spectrophotometrically at 564 nm with an ELISA micro-
plate reader (ChroMate-4300, FL, USA). The measurements for each
compound were undertaken three times. The IC50 values were
calculated from a Boltzman sigmoidal concentration response
curve using nonlinear regression fitting models (Graph Pad, Prism
Version 5).
Crystals of 5 and 7 suitable for analysis were obtained by vapor
diffusion of hexane into solutions of the compounds in dichloro-
methane at 7 °C. Crystals were examined under a microscope
and suitable samples were selected and mounted on an Agilent
SuperNova (dual source) Agilent Technologies Diffractometer,
equipped with microfocused Cu/Mo K
tion. The data collections were accomplished using CrysAlisPro
software [23] at 296 K with Mo K radiation. The structure
a radiation for data collec-
a
solutions were performed using SHELXS–97 [24] and refined by
2
full–matrix least–squares methods on F using SHELXL–97 [24],
interfaced with WinGX [25]. All non–hydrogen atoms were refined
anisotropically by full–matrix least-squares methods [24]. The
figures were generated through PLATON [26] and ORTEP [27]
interfaced with WinGX. All hydrogen atoms were positioned
geometrically and treated as riding atoms with CAH = 0.93 Å and
2.7. Molecular docking Studies:
Molecular docking studies were conducted with Molecular
Operating Environment (MOE) 2008.10 (Chemical Computing
Group Inc., Quebec, Canada, 2008). The docking scores were first
attained utilizing the London dG scoring function in the MOE soft-
ware, and were then improved using two unrelated refinement
methods. Grid-Min pose and Force-field were employed to confirm
that the refined poses of the coordination compounds were geo-
metrically correct. Bond rotations were allowed, and the best five
binding poses were then examined. To assess the binding free
energy of the compounds toward DNA, the docking poses of the
compounds and the co-crystallized structure of the B-DNA were
docked (RSCP PDB code: 1BNA). RMSD values were used to assess
the best binding pose. To evaluate the interaction between each
compounds and MDM2 protein binding site, the docking poses of
the compounds and the crystal structure of MDM2 bound to the
transactivation domain of p53 (RSCP PDB code: 1YCR) were used
for the docking calculation.
U
iso(H) = 1.2Ueq(C) for all carbon atoms. For 7, disorder correspond-
ing to solvent could not been modelled satisfactorily, so was
removed using Solvent Mask in Olex2. The crystal data were
deposited at the Cambridge Crystallographic Data Centre with
deposition numbers 1,892,006 (5) and 2,007,025 (7 with solvent
suppression using Solvent Mask). Crystal data can be obtained free
of charge from CCDC, 12 Union Road, Cambridge CB21 EZ, UK (Fax:
(
+44) 1223 336–033; e-mail: data_request@ccdc.cam. ac.uk).
2.5. DNA binding studies
An aqueous solution of ct-DNA was prepared and the concen-
tration was obtained from the absorbance values at 260 nm using
ꢀ1
ꢀ1
the reported e value of 6600 M cm , with the ratio of absor-
bances at 260 nm to that at 280 nm being 1.8 [28]. Spectroscopic
3