Kumar et al.
Structure-activity relationships
involvement of apoptosis in MCF-7 cells. RNA was isolated from
MCF-7 cells with and without treatment with 1a 2 and 4 lM. Prim-
ers specific for Bax and Bcl-2 were used to confirm the involvement
of apoptosis. RT-PCR results showed upregulation of Bax expression
and downregulation of Bcl-2 expression when compared with that
of untreated MCF-7 cells. It has been reported that there is a
strong relationship in apoptosis and farnesyl transferase inhibition
(16–18). Hence, this finding supports that molecule 1a may be a
farnesyl transferase inhibitor.
To explore the structure-activity relationships (SAR) of substituted
N-(4-methyl-2-oxo-1,2 dihydroquinolin-7-yl) amides, functional modi-
fications have been carried out at the amino group.
The compound 1a with haloalkyl chain attached to the parent
nucleus through an amide linkage at amino group was considered
as a reasonable starting point for SAR studies. It showed signifi-
cant anticancer activity of CTC50 = 31.83 lM against EAC cells in
Trypan blue assay and CTC50 = 3.433 lM against MCF-7 cell line in
MTT assay. Encouraged with this result,
a
more lipophilic
Ehrlich tumor is a hurriedly growing carcinoma with very aggressive
behavior (19). It can grow in almost all strains of mice. The tumor
implantation induces a local inflammatory reaction, with increasing
vascular permeability, resulting in an intense edema formation, cel-
lular migration, and progressive ascitic fluid formation (20). The as-
citic fluid is essential for tumor growth, as it constitutes a direct
nutritional source for tumor cells. Drug treatment was given on 1st,
3rd, 5th, 7th, 9th, 11th, and 13th day of inoculation and checked
for anticancer activity at these time points. Compound 1a has been
found to be significantly effective in reducing the body weight and
also improved MST significantly compared to control EAC injected
mice. Prolongation in the life span of tumor inoculated mice is a
reliable criterion for judging the anticancer activity of any drug (13).
In untreated mice, EAC inoculation causes 100% mortality within
20 days, which is supported by our present data. An enhancement
of life span by 25% or more over that of control was considered as
effective antitumor response (13). Treatment with compound 1a at
50 and 100 mg ⁄ kg has delayed the onset of mortality and
increased the life span of EAC inoculated mice by 49.48% and
70.1%, respectively.
(ClogP = 2.663) '4-toluyl' substituent was introduced in place of
haloalkyl substituent of compound 1a to afford compound 1b. To
our surprise, this modification drastically lowered the anticancer
activity in multifold against both the cancer cells. Because the elec-
tron-donating substituent on phenyl ring was inactive, analogs of
1b in which the 4-methyl substituent on phenyl ring was altered
with electron-withdrawing group like 4-fluorine (compound 1c) and
4-chlorine (compound 1d) to investigate the cytotoxicity. Remark-
ably, compound 1c exhibited an excellent improvement in CTC50 of
1.289 lM against MCF-7 cell line where as compound 1d showed
a little improvement in CTC50 of 98.91 lM against the proliferation
of EAC cells. The promising results of compound 1c and 1d
prompted us to probe the effect of a charged –NO2 group in place
of halogens on the phenyl ring. Nevertheless, it was failed to
improve the anticancer activity. Introduction of dimethoxy functional-
ity in the phenyl ring of our moiety also resulted in equally frustrat-
ing activity (CTC50 h 1000 lM against both the cells). Discouraged
with the insensitivity of electron-donating functionality on antican-
cer activity, strong electron-withdrawing functional groups like –Cl
and –Br were placed twice in the phenyl to afford compounds 1g
and 1h, respectively. Interestingly, with this simple modification,
compound 1h showed a CTC50 of 6.360 lM against MCF-7 cell
lines, whereas compound 1g was moderately active against both
the cancer cells. However, introduction of a spacer in between
amide functionality and the phenyl ring of our moiety obliterated
the anticancer activity. On the contrary, our SAR study reveals that
nature and position of functional groups on the phenyl ring influ-
ence the anticancer activity of N-(4-methyl-2-oxo-1,2 dihydroquino-
lin-7-yl) amides. In addition, we speculate that the functional group
that increases the acidity of our parent nucleus is essential for the
anticancer activity. However, in our SAR study, we could not estab-
lish a strong correlation between anticancer activity and Log P of
the compounds.
Significant decrease in hemoglobin and RBC (owing to hypoxic con-
dition) with parallel increase in WBC [may be owing to the influ-
ence of tumor cell (antigen) on the immune system] has been
observed in EAC inoculated control mice. Compound 1a at
100 mg ⁄ kg significantly reduced the elevated WBC count and
increased RBC number compared to EAC inoculated control mice.
The reversal of WBC indicates protective action on the hemopoietic
system and supports the antitumor activity of the compound. Usu-
ally, in cancer chemotherapy, the major problems that are encoun-
tered include myelosuppression and anemia (21). The results had
clearly shown that compound 1a at 100 mg ⁄ kg was found to be
significantly active in improving the hemoglobin content and RBC
count to normal levels.
As compound 1a has shown better cytotoxicity than other com-
pounds (CTC50 = 3.433 € 0.39 lM), it has been screened further on
four cell lines including MCF-7 for 24 and 48 h. It has also shown
promising result on the remaining cell lines at both time intervals
with CTC50 <10 lM. This compound has been further studied for
DNA fragmentation assay on MCF-7 cell line. It has shown a band
with laddering, which indicates its action responsible for DNA
damage. Nuclear staining experiments were performed using acri-
dine orange staining dye. Compound 1a at 2 and 4 lM concentra-
tion was tested against MCF-7 cells, and nuclear staining images
showed induction of apoptosis such as membrane blebbing,
cytoplasmic condensation, and nuclear fragmentation. Further
reverse transcriptase PCR (RT-PCR) was performed to confirm the
Conclusion
The study shows that 2-quinolone without 3-aryl substitution can
also be a potential moiety for anticancer research. Upregulation
of Bax expression and downregulation of BCl-2 expression in
cancer cells is a very important event in apoptosis. Hence, we
propose Bax-induced apoptosis as
a
potential alternative
mechanism of 2-quinolone derivatives other than farnesyl transfer-
ase inhibition. Further, they have the significant cytotoxic poten-
tial and are safe as indicated by acute toxicity activity.
Compound 1a merits further investigation to explore in molecular
mechanisms.
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Chem Biol Drug Des 2012; 80: 291–299