L.A. Ismail, M.Y. Alfaifi, S.E.I. Elbehairi et al.
Journal of Organometallic Chemistry 949 (2021) 121960
relative abundance %): 287.1 (M+, 15.12), 286.1 (basic peak, 100);
as indicative of the purity of this solution and its devoid of pro-
tein [35]. The concentration of CT-DNA was obtained from its ab-
sorption value at 260 nm. On the other hand, the Ru(II) complex
was dissolved in a mixture of DMSO and Tris–HCl (5:95) to obtain
a stock solution of concentration (2.5 × 10−5 M) which was used
for all following experiments. The absorption titration experiments
were carried out by the addition of gradually increasing concen-
tration of DNA (0 – 25 μM) to a constant concentration of com-
plex (2.5 mL, 2.5 × 10−5 M) in the range of 250–700 nm, Tris–HCl
buffer was used as a reference solution. All absorption data were
recorded 25 min after each addition for attaining equilibration of
titration.
CI(NH3)-MS (m/z, relative abundance %): 305.2 ([M
+
NH4]+,
5.5), 288.1 ([M + H]+, 24.2) and 287.1 (M+, 100). Anal. Calcd. for
C14 H9NO2S2 (287.35 g/mol): C, 58.52; H, 3.16; N, 4.87; S, 22.31%.
Found: C, 58.48; H, 3.19; N, 4.625; S, 22.19%.
2.2. Synthesis of thiophene-benzazoles-Ru(II)(η6-p-cymene) hybrids
To a suspension of Na2CO3 (0.6 mg, 5.66 mmol) in acetone
(50 mL), thiophene-benzazoles hybrid (2.29 mmol) and [Ru(η6-p-
cymene)Cl2]2 (0.7 g, 1.14 mmol) were added under vigorous stir-
ring at ambient temperature. Afterward, the reaction mixture was
stirred at the temperature for 3 h. Then, the solvent was evap-
orated in vacuo and the residue was re-dispersed in CH2Cl2 (20
mL), followed by filtration through a short pad of Celite. The re-
dispersion of the residue in CH2Cl2 (10 mL) and filtration were
repeated a further four times to ensure the almost complete ex-
traction of the product. The collected filtrates were concentrated
in vacuo to yield the desired product.
DNA
DNA
]
1
[
]
[
ꢀ
ꢁ
ꢀ
ꢁ
ꢀ
ꢁ
=
+
(1)
εa − εf
εb − εf
Kb εb − εf
Where [DNA] is the concentration of DNA, εa, εb, and εf cor-
respond to the extinction coefficients of the apparent, bound and
free complex, respectively. The apparent extinction coefficient (εa)
defended as the ratio between the observed absorbance of the
complex and its concentration (Aobs/[complex]). The plot of [DNA]/
(εa - εf) versus [DNA] exhibits a slope and intercept equal to 1/ (εa
- εf) and 1/ Kb(εb – εf), respectively. The Kb values were calculated
from the ratio of the slope to the intercept.
Chloro (p-cymene) ruthenium(II) (benzoxazol-2-yl)-3-oxo-3-
(thiophen-2-yl)propen-1-olate ([η6-(p-cymene)Ru(3a)Cl], 4a):
Orange-red powder (0.97 g, 1.74 mmol, 76%). ATR-IR (cm−1): 1727
(s, sh, ν(C=O)), 1612 (w, sh, ν(C=N)oxazol), 1551 (m, sh, ν(C=C)Ar),
1444 (m, sh), 1405 (m, sh, ν(C=C)Ar), 1345 (w, sh, ν(C-S-C)thiophen),
1117 (m, sh, ν(C-O-C)oxazol), 1055 (m, sh), 994 (m, sh), 867 (m, sh,
Oxazol ring), 853 (m, sh, ν(Ru-O=C)), 747 (s, sh, ν(C-S)thiophen),
631 (s, sh, ν(Ru-C)), 557 (m, sh, ν(Ru-O-C)), 495 (m, sh), 443 (m,
sh, ν(Ru-O-Cl)). 1H NMR (200 MHz, DMSO-d6) δ (ppm): 8.09-7.93
(m, 2H, Ar-H), 7.66-7.52 (m, 1H, Ar-H), 7.27 (s, 4H, Ar-H), 6.79 (s,
1H, Olefinic-H), 5.77 (d, J = 6.8 Hz, 2 H, p-Cymene-H), 5.56 (d,
J = 6.8 Hz, 2 H, p-Cymene-H), 2.97 (sept, 1 H, isoPr-H, CH(CH3)2),
1.91(s, 3 H, Me, CH3), 1.21 (d, J =6.9 Hz, 6 H, Me, CH(CH3)2). 13C
NMR (200 MHz, DMSO-d6) δ (ppm): 183.28, 161.92, 152.96, 146.02,
140.02, 136.88, 129.49, 127.96, 126.91, 126.24, 125.78, 124.86,
124.51, 123.99, 122.39, 118.50, 110.00, 98.81, 32.61, 23.28, 21.11.
EI-MS (m/z, relative abundance %): 555.9 (M+, 11.52). Anal. Calcd.
for C25H25ClNO3RuS (556.06 g/mol): C, 54.00; H, 4.53; N, 2.52; S,
5.77%. Found: C, 53.97; H, 4.55; N, 2.42; S, 5.69%.
2.3.2. Competitive binding of EB vs. complex–DNA
The competitive binding of each Ru(II) complex versus
3,8-diamino-5-ethyl-6-phenyl-phenanthridinium bromide (EB) was
studied using fluorescence spectral technique in order to inves-
tigate the capacity of complex to displace EB from its DNA-EB
complex and consequently estimate the apparent binding constant
(Kapp) of the complex. The CT-DNA–EB complex was initially pre-
pared by mixing CT-DNA solution (15 μM) and EB solution (10
μM) in a buffer (10 mM Tris-HCl and 150 mM NaCl) (pH 7.2). All
fluorescence measurements were recorded at 605 nm (545 nm ex-
citation) of EB bound to DNA upon addition of increasing concen-
trations of the complex (0–100 μM).
Chloro (p-cymene) ruthenium(II) (benzthiazol-2-yl)-3-oxo-3-
(thiophen-2-yl)propen-1-olate ([η6-(p-cymene)Ru(3b)Cl], 4b):
yellowish-brown solid (0.96 g, 1.67 mmol, 73%). ATR-IR (cm−1):
3030 (m, sh, ν(C-H)Ar), 2915 (w, sh), 1733 (s, sh, ν(C=O)), 1603 (w,
sh, ν(C=N)Thiazol), 1532 (m, sh, ν(C=C)Ar) 1510 (m, sh) 1448 (m,
sh), 1407 (m, sh, ν(C=C)Ar), 1355 (m, sh, ν(C-S-C)Thiophen), 1288
(m, sh, ν(C-S-C)Thiazol), 1124 (m, sh), 1029 (m, sh), 940 (m, sh), 846
(m, sh, ν(Ru-O=C)), 748 (s, sh, ν(C-S)thiophen), 632 (m, sh, ν(Ru-C)),
580 (m, sh, ν(Ru-O-C)), 507 (m, sh), 438 (m, sh, ν(Ru-O-Cl)). 1H
NMR (200 MHz, DMSO-d6) δ (ppm): 8.01 (dd, J = 6.5, 3.3 Hz, 2 H,
Ar-H), 7.59 (d, J = 6.9 Hz, 1 H, Ar-H), 7.26 (t, J = 4.4 Hz, 1 H, Ar-H),
7.20-7.10 (m, 3 H, Ar-H), 6.70 (s, 1 H, Olefinic-H), 5.67 (d, J = 5.9
Hz, 2 H, p-Cymene-H), 5.45 (d, J = 5.9 Hz, 2 H, p-Cymene-H),
2.95 (sept, 1 H, isoPr-H, CH(CH3)2), 1.83 (s, 3 H, Me, CH3), 1.37 (d,
J = 6.9 Hz, 6 H, Me, CH(CH3)2). 13C NMR (200 MHz, DMSO-d6)
δ (ppm): 182.99, 161.97, 151.99, 146.59, 141.23, 136.56, 129.36,
128.01, 127.04, 126.17, 125.61, 124.70, 124.44, 124.05, 122.37, 118.51,
110.04, 98.16, 32.42, 23.21, 21.09. EI-MS (m/z, relative abundance
%): 572.1 (M+, 7.48). Anal. Calcd. for C25H25ClNO2RuS2 (572.12
g/mol): C, 52.48; H, 4.40; N, 2.45; S, 11.21%. Found: C, 52.51; H,
4.43; N, 2.38; S, 11.19%.
The classical Stern–Volmer equation (Eq. (2)) was used to cal-
culate the quenching constant (Kq) [37];
I / I = 1 + K
Q
q[ ]
(2)
0
where I and I0 are the emission intensities in the presence and ab-
sence of the quencher (Ru(II) complex), respectively, and [Q] is the
quencher concentration. The slope of a plot I0/ I vs. [Q] is quan-
tified as Kq. On the other hand, Kapp values have been estimated
using the following equation (Eq. (3));
kEB EB = 1 + Kapp Complex
(3)
[
]
[
]
where [EB] = 10 μM and KEB = 1 × 107 M−1
.
2.4. MTT assay
The cytotoxic effects of ruthenium(II) complexes toward two
human carcinoma cell lines (breast carcinoma (MCF-7) and lung
carcinoma (A549)) and normal human cells (Hela) as well, were
assessed using the standard MTT protocol as described in our ear-
lier work [38]. In brief, the cancer cells were cultivated in a 96-
well plate (2 × 103 cells/well) containing Dulbecco’s Modified Ea-
gle Medium (DMEM, Gibco) and 10% fetal bovine serum (FBS; TBD
Science) FBS under a moist carbon dioxide atmosphere (5%) at 37°C
for 20 hr. Thereafter, the culture mediums were replaced with so-
lutions of serial concentrations (200 μL) of the treating samples
in DMSO and incubated overnight. After treatment, 20 μL of MTT
solution (5 mg/mL, Sigma) was added to each well and then in-
cubated for 4 h after which the medium was carefully replaced
2.3. Ru(II) complexes–DNA interaction studies
2.3.1. Electronic absorption titration
Initially, fresh stock solutions of CT-DNA in a Tris–HCl buffer (5
mM, pH 7.2) were prepared and kept at 4°C. The ratio of UV ab-
sorbance of DNA solution at 260 and 280 nm was about 1:1.8–1.9
3