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increased their stemness. Complexes 5 and 6 inhibit the CSCs, a similar aniline-imidazole RuII-p-cymene complex recently
DOI: 10.1039/D0CC00472C
similar to that of salinomycin but at least at a four times lower reported shows much poorer IC50 (ca. 15 µM) against a similar
dosage.
panel of cancer cells24. The use of mesalazine in the ligand
Two sets of chelating ligands were prepared with mesalazine seems to induce a positive effect also in enhancing the
and its methyl ester, one set contained a pyridine (L1-L2) and cytotoxicity of the Ru (II) complexes. 5 & 6 when treated with
another set contained an imidazole (L3-L4) in the Schiff base normal foreskin fibroblast (HFF-1) cells the IC50 ranges ca. 5-6
moiety. Six new Ru(II) complexes (1-6) with the respective µM, thus the complexes are marginally less toxic to normal cells.
ligands (Figure 1) were obtained in high yields (60-70%) by
Table 1. In vitro cytotoxicity profile of complexes 1-6 in various cancer cell lines under
reacting the respective ligands with 0.5 mol equivalent of
normoxic condition in comparison to oxaliplatin.
[RuII(p-cymene)X2]2 (X = Cl, I) in dry MeOH at 27 C for 8-12 h
IC50 (µM) ± SDa
(Supporting information, Scheme S1). The complexes were
1
characterized by H NMR (nuclear magnetic resonance), 13C-
Complexes
HT-29
>200
82 ± 6
56 ± 2
>200
3.2 ± 0.3
2.6 ± 0.3
8.9 ± 0.5
MIA PaCa-2
>200
90 ± 6
HepG2
>200
92 ± 9
MDA-MB-231
>200
1
2
3
4
5
6
NMR, ESI-HRMS (Electron spray ionization High Resolution Mass
Spectrometry), FT-IR (Fourier-transform infrared spectroscopy)
and UV-Vis (Ultraviolet-visible spectroscopy) studies. The bulk
purity was confirmed by elemental analysis (Supporting
information, Figure S1-S24).
97 ± 14
42 ± 5
>200
2.9 ± 0.4
2.2 ± 0.2
NDb
54 ± 6
51 ± 8
>200
>200
2.8 ± 0.1
2.3 ± 0.2
5.7 ± 0.2
2.9 ± 0.3
2.4 ± 0.4
9.8 ± 0.3
The stability of the complexes in the physiological condition
was investigated by ESI-HRMS in 1:99 v/v MeOH and phosphate
buffer (pH 7.4, containing 4 mM NaCl). All the complexes (1-6)
formed aquated species, to various extents during the 24 h
period (Figure S25-S36). The extent of hydrolysis is greater in
the case of chlorido coordinated 4 and 5 compared to their
respective iodido bound 3 and 6. The difference is more
Oxaliplatin
aIC50 ± SD are determined by MTT assay in normoxia (~15% O2). SD = standard
deviation. The statistical significance (P) of the data is > 0.001 to <0.05. bNot
done. Plots are in Figure S47. See experimental section for full detail.
The distribution coefficient between octanol and water (log D)
values of the free acid derivatives (1 & 4) are in the range of -
0.1 to -0.3 whereas esterification enhances the log D to 0.2 - 0.7
(Figure 2A) making the ester-based complexes more lipophilic.
Lipophilicity is as an important parameter to optimize passage
through the lipophilic cell membrane and promote hydrophobic
interaction with protein targets to enhance the cytotoxic
efficacy of a compound. The ester derivatives with greater
lipophilicity show better cytotoxicity than free acid derivatives.
HT-29 cells, even upon treatment with higher concentration of
the free acid-based complexes compared to their ester
analogues (25 µM for 1,4 and 10 µM for 2-3,5-6) showed multi-
fold higher accumulation of the ester derivatives inside the cell
(Figure 2B). The imidazole derivatives (5, 6) showed higher
accumulation than the pyridine analogue (2, 3) besides, iodido
analogues accumulates more than their respective chlorido
analogues (3>2 & 6>5) thus supporting their toxicity.
1
prominent among the imidazole derivatives 5 and 6. H NMR
data shows that hydrolyzed species of 5 starts to appear within
3 h and by 24 h most of the complex convert to its aquated
form; 6 showed no hydrolysis even after 24 h (Figure S37-S38),
which correlated well with their ESI-MS results. The cellular
thiol-based tripeptide glutathione (GSH) binds with metal
complexes leading to their deactivation. Therefore, we
investigated the binding affinity of the complexes in the
presence of 2.5 equivalents of GSH by ESI-MS using the afore-
mentioned solution condition. The pyridine analogues (2 & 3)
completely bound to GSH within 24 h whereas the imidazole
derivatives (5 & 6) are more reluctant to form GSH adduct
(Figure S41-S46).
The in vitro efficacy of our newly designed complexes (1-6)
were tested against the human CRC HT-29 and also screened for
cancer cells derived from other digestive organs viz. Hep G2
(liver carcinoma) and MIA PaCa-2 (pancreatic ductal
adenocarcinoma). We have also extended the investigation
towards a highly aggressive triple-negative human metastatic
breast adenocarcinoma, MDA-MB-231. In vitro, cytotoxic data
under 2D culture conditions showed that the complexes with
free mesalazine groups (1 & 4) are non-toxic up to the tested
range (IC50 > 200 µM) (Table 1). The non-toxicity may be due to
the low pKa of the free -CO2H group, which leads to its
deprotonation at pH 7.4, preventing the complex to traverse
the cell membrane. Esterification of the carboxylic group
enhanced the membrane traversing capability and therefore
increasing the cytotoxicity. IC50 of the pyridine derivatives 2 and
3 were in the range 50-100 µM and for imidazole analogues 5
and 6 (with low GSH reactivity) it is around 2-3 µM (Table 1).
Complexes 5 and 6 are most potent in the series and are three
times more toxic than oxaliplatin in HT 29 cells, a drug used to
treat the advanced level of colon cancer. Comparison of 6 with
Figure 2. (A) Distribution coefficient of the metal complexes (1-6) in a 1:1 (v/v)
octanol/water mixture at 37C (B) Total Ru content measured after 6 h of incubation
with complexes (1-6) in HT 29 cells.
Cellular effluxing proteins, and various stemness genes, are
highly expressed in CSCs, which help them to survive under drug
treatment condition. Promising in vitro cytotoxicity profile as
well as the GSH resistivity of the imidazole derivatives (5 & 6),
lead us to investigate the effect of the complexes on HT-29 stem
cell-derived 3D-spheroids. The capacity of a compound to
2 | J. Name., 2012, 00, 1-3
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Acharya, Sourav
