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M.Y. Alfaifi et al. / Journal of Molecular Structure 1191 (2019) 118e128
2.3.3. N,N0-Bis[3-ethyl-5-((1-nbutylimidazol-3-ium)methylene)-
salicylidene)-R,R-1,2-cyclohex- anediamine bis-(tetrafluoroborate)
(4c)
2.4.3. R,R-[[2,2`-][(1,2-cyclohexanediyl)-bis(nitrilomethylidyne)]-
bis[4-((1-nbutylimidaz-olium)-methylene-6-(ethyl-phenolato)]-
[N,N',O,O'] palladium(II) bis-(tetrafluoro-borate) (5c)
Faint yellow powder, (87%); mp 77ꢂ78 ꢁC. FT-IR (KBr, cmꢂ1):
3426 (m, br, n(O-H)), 3098 (m, sh), 2936 (m, sh), 1623 (vs, sh), 1538,
1463, 1389 (s, sh), 1281 (s, sh),1158 (s, sh), 1060 (vs, sh), 865 (m, sh),
Reddish-brown powder (62%). FT-IR (KBr, cmꢂ1): 1634 (vs, sh,
n(C
]
N)), 1267 (s, sh), 1058 (vs, sh), 615(m, sh), 493 (w, br). 1H NMR
(200 MHz, DMSO‑d6)
d
(ppm): 8.35 (s, 2H), 7.80 (d, J ¼ 2.01 Hz, 2H),
771 (m, sh). 1H NMR (200 MHz, DMSO‑d6)
d
(ppm): 9.22 (s, 2H), 8.43
7.74 (d, J ¼ 2.01 Hz), 7.36 (d, J ¼ 1.89 Hz, 2H), 7.26 (d, J ¼ 2.00 Hz,
2H), 5.83 (s, 4H), 4.21 (t, J ¼ 7.0 Hz, 4H), 3.42 (m, 2H), 2.73 (q,
J ¼ 7.1 Hz, 4H), 1.81 (m, 4H), 1.53 (m, 4H), 1.27 (t, J ¼ 7.1 Hz, 6H), 1.16
(m, 4H), 0.87 (t, J ¼ 6.3 Hz, 3H). 19F NMR (470 MHz, DMSO‑d6):
(s, 2H), 7.81 (d, J ¼ 2.05 Hz, 2H), 7.73 (d, J ¼ 2.05 Hz), 7.37 (d,
J ¼ 1.86 Hz, 2H), 7.25 (d, J ¼ 2.01 Hz, 2H), 5.82 (s, 4H), 4.20 (t,
J ¼ 7.1 Hz, 4H), 3.41 (m, 2H), 2.73 (q, J ¼ 7.3 Hz, 4H), 1.79 (m, 4H),
1.52 (m, 4H), 1.28 (t, J ¼ 7.2 Hz, 6H), 1.19 (m, 4H), 0.87 (t, J ¼ 6.0 Hz,
148.72 ppm
(singlet).
ESI-MS:
844.1
and
378.1
3H). 13C NMR (125 MHz, DMSO‑d6)
d
(ppm): 165.58, 151.03, 138.85,
([C40H54BF4N6O2Pd]þ and [C40H54N6O2Pd]2þ
,
[M ꢂ BFꢂ4 ]þ and
138.02, 133.35, 129.94, 129.52, 123.91, 122.88, 71.24, 55.89, 48.52,
34.31, 30.87, 29.76, 28.41, 25.94, 23.68, 22.34, 13.86. 19F NMR
(470 MHz, DMSO‑d6): 148.72 ppm (singlet). ESI-MS: 739.4 and
326.0 ([C40H56BF4N6O2]þ and [C40H56N6O2]2þ, [M ꢂ BF4ꢂ]þ and
[M ꢂ 2 BFꢂ4 ]2þ, respectively). Anal. Calcd. for C40H54B2F8N6O2Pd
(M ¼ 930.92): C, 51.61; 5.85; N, 9.03; Found: C, 51.47; H, 5.91; N,
8.94.
[M ꢂ 2 BFꢂ4 ]2þ
,
respectively). Anal. Calcd. for C40H56B2F8N6O2
2.5. Stability of complexes under physiological conditions
(M ¼ 826.52): C, 58.13; H, 6.83; N, 10.17; Found: C, 57.99; H, 6.86; N,
10.05.
The stability of Pd(II) complexes was investigated by recording
UVeVis spectroscopy for a solution (1 ꢀ 10ꢂ3 M) of complex in a
DMSO/phosphate buffer of pH 7.4 in time intervals (t ¼ 0 h, 1 week,
2 weeks) at 25 ꢁC. Buffer solution was prepared by adding 70 mL
0.1 M aqueous NaOH solution to 0.1 M aqueous KH2PO4 solution.
The pH of the obtained DMSO:PBS buffer was checked with the
HANA instrument 8519 digital pH-meter.
2.4. Preparation of {Pd(II)(Et)2saldach(nBu-Imþ-Xe)2} complexes
(5a-c)
A solution of palladium(II) chloride (0.126 g, 1 mmol/5 mL EtOH)
was added dropwise to an ethanolic solution (10 mL) containing
the free ligand (4a-c) (1 mmol) and 1 mL of conc HCl. Thereafter, the
reaction mixture was refluxed under constant stiring for 6 h. Then
the solvent was evaporated under vacuum to leave an oily residue,
which was solidified by addtion of petroleum ether (40e60) and
keeping overnight in a refrigerator. The isolated products were
filtered off and washed with ice-cold mixed-solvent of MeOH/Et2O
(1 : 2) (3 ꢀ 3 mL) to yield Pd(II) saldach complexes (5aec).
2.6. In vitro anticancer (cytotoxicity)
2.6.1. Cell cultures
Human tumor cell lines MCF-7 (breast adenocarcinoma) have
been acquired from the VACSERA Tissue Culture Unit, and cultured
using RPMI-1640 media with and 10% FBS, 1%
L-glutamine, HEPES
buffer with the addition of 50
m
g/mL gentamycin. Cells have been
sustained at 37 ꢁC in 5% CO2. Toxicity has been measured according
to the activity of cell morphology and cell viability. Control cells
were treated with 0.5% DMSO.
2.4.1. R,R-[[2,2`-][(1,2-cyclohexanediyl)-bis(nitrilomethylidyne)]-
bis[4-((1-nbutylimidaz- olium)-methylene-6-(ethyl-phenolato)]-
[N,N',O,O'] palladium(II) dichloride (5a)
Dark yellow powder (68%). FT-IR (KBr, cmꢂ1): 1633 (vs, sh), 1267
2.6.2. Cytotoxic effect assay
Cytotoxicity effects of R,R-{H2(Et)2saldach(nBu-Imþ-Xe)2} (4a-c)
and their Pd(II) complexes [Pd(II){(Et)2saldach(nBu-Imþ-Xe)2}] (5a-
c) were assessed by the in-vitro cytotoxicity assay according to the
adopted methods instruction of the Regional Center for Mycology &
(s, sh), 615 (m, sh), 496 (w, br). 1H NMR (200 MHz, CDCl3)
d (ppm):
8.37 (s, 2H), 7.75 (d, J ¼ 2.00 Hz, 2H), 7.71 (d, J ¼ 1.99 Hz, 2H), 7.39 (d,
J ¼ 1.63 Hz, 2H), 7.24 (d, J ¼ 1.85 Hz, 2H), 5.83 (s, 4H), 4.18 (t,
J ¼ 7.0 Hz, 4H), 3.35 (m, 2H), 2.72 (q, J ¼ 7.3 Hz, 4H), 1.77 (m, 4H),
1.50 (m, 4H), 1.26 (t, J ¼ 7.5 Hz, 6H), 1.19 (m, 4H), 0.91 (t, J ¼ 7.1 Hz,
3H). ESI MS: m/z 792.6 and 378.3 ([C40H54ClN6O2Pd]þ and
[C40H54N6O2Pd]2þ, [M ꢂ Clꢂ]þ and [M ꢂ 2Cle]2þ, respectively).
Anal. Calcd. for C40H54Cl2N6O2Pd (M ¼ 828.22): C, 58.01; H, 6.57; N,
10.15; Found: C, 57.96; H, 6.63; N, 10.09.
Biotechnology, Egypt. In brief, 1 ꢀ 104 cells per well in 100
mL of
growth medium were seeded in 96-well plates. Moreover, serial
dilutions of the compounds have been added to the confluent cell
mono layers plates (Falcon, NJ, USA) using a multichannel pipette;
followed by incubation at 37 ꢁC using humid 5% CO2 incubator for
48 h plate wells have been divided into three wells for each sample
concentration and control cells have been incubated with and
without DMSO. Different sample concentrations were used as fol-
2.4.2. R,R-[[2,2`-][(1,2-cyclohexanediyl)-bis(nitrilomethylidyne)]-
bis[4-((1-nbutylimidaz-olium)-methylene-6-(ethyl-phenolato)]-
[N,N',O,O'] palladium(II) bis-(hexafluoro-phosphate) (5b)
lows; 50, 25, 12.5, 6.25, 3.125, 1.56 mg for 24 h and continued in-
cubation for time for 48 h. Finally, the viable cells yield was
determined by a colorimetric method followed by the addition of
crystal violet solution (1%) to each well for at least 30 min. The
crystal violet stain excess has been detached using running water
followed by addition of glacial acetic acid (30%) to all wells and
mixed thoroughly by gently shaking to measure their absorbance
using Micro plate reader (TECAN, Inc.) at wavelength of 490 nm.
Additionally, the cytotoxic effects of the used compounds have
been measured through comparison of the treated samples with
the control cell [28].
Faint brown powder (67%). FT-IR (KBr, cmꢂ1): 1633 (vs, sh), 1266
(s, sh), 840 (vs, sh), 612 (m, sh), 559 (m, sh), 492 (w, br). 1H NMR
(200 MHz, DMSO‑d6)
d
(ppm): 8.36 (s, 2H), 7.64 (d, J ¼ 2.03 Hz, 2H),
7.59 (d, J ¼ 2.00 Hz, 2H), 7.36 (s, 2H), 7.23 (d, J ¼ 1.86 Hz, 2H), 5.28 (s,
4H), 4.18 (t, J ¼ 7.0 Hz, 4H), 3.37 (m, 2H), 2.73 (q, J ¼ 7.1 Hz, 4H), 1.80
(m, 4H), 1.53 (m, 4H), 1.28 (t, J ¼ 7.4 Hz, 6H), 1.18 (m, 4H), 0.89 (t,
J ¼ 7.0 Hz, 3H). 31P NMR (202 MHz, DMSO‑d6): e142.96 ppm
(septet, 2JPF ¼ 711.23 Hz). 19F NMR (470 MHz, DMSO‑d6):
e70.59 ppm (doublet, 1JFP ¼ 715.68 Hz). ESI MS: m/z 901.4 and 378.1
([C40H54F4N6O2PPd]þ and [C40H54N6O2Pd]2þ, [M ꢂ PF6ꢂ]þ and [M ꢂ
2PFꢂ6 ]2þ
,
respectively). Anal. Calcd. for C40H54F12N6O2P2Pd
2.7. DNA-binding study
(M ¼ 1047.24): C, 45.88; H, 5.20; N, 8.02; Found: C, 45.63; H, 5.22;
N, 7.98.
The DNA-binding studies for the free ligand (4a) and its Pd(II)