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RSC Advances
Page 5 of 7
DOI: 10.1039/C5RA08733C
Journal Name
ARTICLE
myeloid leukaemia) and K562-TAX (acute myeloid leukaemia, corresponding methylesters (16a,b) provided approximately
paclitaxel resistant), A549 (human lung adenocarcinoma), the same results. On the other hand, cytotoxicity of 3HQs-5-
HCT116 (human colorectal cancer), HCT116p53-/- (human phenacylesters 14 was in general significantly higher, with
colorectal
cancer,
p53
deficient),
U2OS
(human compound 14b being the most active derivative from the
osteosarcoma). The therapeutic index (TI) was evaluated using whole set. IC50 of aminophthalate 14b for A-549, K56A, CEM,
one representative of non-malignant cell lines – BJ (human K562-TAX and CEM-DNR was comparable to isomeric
fibroblasts). The obtained results are summarized as the IC50 aminoterephthalate.2 In contrast, 4-methylphenyl phthalate
values in Table 3.
derivative 14c was inactive compared to isomeric
aminoterephthalate that exhibited micromolar IC50 for all
tested lines.2 The reversed dependence was observed for the
unsubstituted phenacylester 14a (Figure 6). The majority of
active compounds were less active against the CEM-DNR
multidrug resistant cell line overexpressing the multidrug
resistance protein
1
(MRP-1), than against highly
chemosensitive parental CCRF-CEM cell line. However, the
opposite pattern was identified in the case of P glycoprotein
(Pgp-1) overexpressing multidrug resistant K562-TAX cell line
which was more sensitive than parental K562 cell line.
Interestingly, CEM-DNR cells also lack topoisomerase IIα gene,
which has been previously reported as a molecular target for
quinolone derivatives. It is suggesting either the
topoisomerase II
α as a molecular target for 3HQs-5-
phenacylesters 14 or involvement of MPR-1 but not Pgp-1 in
drug efflux and resistance mechanisms.26 The growth
inhibitory activity of compounds against human colorectal
cancer cell line HCT116 and its p53 deficient counterpart
(HCT116p53-/-) were similar, thus indicating independence of
cell death mechanism on the p53 gene. The therapeutic index
of the most active compound 14b ranged from 15-30 for
majority of cell lines, suggesting preferential activity against
malignant cells.
Finally, two representative compounds were subjected to the
fluorescent microscopy assay. We selected the most cytotoxic
compound 14b along with the most active carboxamide
derivative 15i. The results are depicted in Picture 2. The
microscopy imaging is showing that the fluorescent
compounds are penetrating cellular membranes of living cells.
The highest intensity of the fluorescence and accumulation of
the compound was observed in the cytoplasm, with discrete
nuclear spots. Maximum cytoplasmic positivity was seen in
perinuclear region, the staining pattern was overlapping in
both emission wavelengths. Detailed subcellular localization is
to be determined in future studies.
Figure 5: Illustrative emission spectra of 13c (red, solid line pH 5.75, dashed
line pH 7.69 and dotted line pH 12.49), 14a (green, solid line pH 6.43,
dashed line pH 2.48 and dotted line pH 11.53), 15d (black, solid line pH 3.60,
dashed line pH 6.49 and dotted line pH 12.49) and 16a (blue, solid line pH
5.75, dashed line pH 8.58 and dotted line pH 11.63) and fluorescence
intensity at different pH.
As in the case of bisphenacyl-2-aminoterephthalates,2 the
corresponding bisphenacyl-3-aminophthalates 13 did not
exhibit significant cytotoxic activity. However, an exception
has been observed for compound 13g which showed a
medium cytotoxicity against K562-TAX, U2OS and CEM cells.
Within the group of HQ-5-carboxamides 15, the similar SAR
pattern as for analogical 6-8 carboxamides was observed: The
highest activity was detected for 2-(3,5-dichloro-4-
Conclusions
We have developed a synthetic procedure for 3HQs-5-
carboxamides 15 to enable the study of their properties
compared to analogical 6-8 isomers. In the field of
fluorescence properties, the synthesized 5-carboxamides
exhibited similar results to their 7-isomers that have previously
been studied. In this regard, some derivatives showed a
potential to serve as pH indicators and/or molecular probes.
Based on the fluorescence microscopy imaging of U2OS cells
treated with two representative compounds 14b and 15i it was
demonstrated, that compounds enter live cells and show
preferential cytoplasmic staining. The cytotoxicity of 3HQs-5-
aminophenyl)-3HQs with unpolar
propyl and 15i -benzyl), whereas the unsubstituted
carboxamide 15j and compounds with polar 15g:N-
N-alkyl substituents (15b:N-
:N
(
hydroxyethyl) or basic (15h:N-piperidinyl-ethyl) ligands were
not cytotoxic. Structural change of propylamides (15a,b) to the
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