B.A. Dar et al. / European Journal of Medicinal Chemistry 111 (2016) 26e32
27
design and synthesize a library of ursolic acid benzylidine de-
rivatives. The benzylidine derivatives were synthesized using
Claisen Schmidt condensation protocol at the position 2.
the cancer treatment strategies involve induction of apoptosis. The
effect of compound 3b on the induction of apoptosis in HCT-
116 cells was investigated using fluorescence staining. The
morphological alterations induced by compound 3b in HCT-
116 cells were studied under fluorescence microscopy using acri-
dine orange (AO)/ethidium bromide (EB), Hoechst 33258, JC-1
mitochondrial membrane potential staining.
2.1. Synthesis of benzylidine derivatives
The synthetic protocol for the ursolic acid benzylidine derivative
3 from the parent ursolic acid 1 involves two steps, including Jones
oxidation and Claisen Schmidt condensation (Scheme 1). The
compound 1 in acetone at 0 ꢀC was treated with Jones reagent to
form the C-3 oxidized derivative 2 in almost quantitative yield. The
benzylidine derivative 3a was prepared by Claisen Schmidt
condensation of compound 2 with benzaldehyde in the presence of
ethanolic potassium hydroxide at room temperature in excellent
yields (Scheme 1). A sharp IR peak at around 1675 cmꢁ1, C-13 signal
2.2.2.1. AO/EB staining. The results from AO/EB staining revealed
that HCT-116 cells exposed to compound 3b stained yellow green
after 24 h. The morphological examination showed the appearance
of pycnosis, membrane blebbing and cell budding. However, the
control cells stained green with no alterations in the morphological
appearance (Fig. 1). These findings indicate that the treatment of
HCT-116 cells with compound 3b induced apoptotic cell death.
at
d
207.9, 137.7 & 133.8 for
a
,
b
unsaturated ketone and 1H NMR
7.52 for olefinic proton confirmed the product formation
signal at
d
2.2.2.2. Hoechst 33258 staining. HCT-116 cells treated with com-
of 3a
pound 3b at a concentration of 20 mM for 24 h showed strong blue
The above set optimized condition was then used for the
condensation of various aromatic aldehydes with compound 2 to
prepare a library of ursolic acid benzylidine derivatives. All the
condensation reactions of aromatic aldehydes with compound 2
worked smoothly at room temperature to deliver the desired
product in excellent yields (Table 1).
fluorescence on Hoechst 33258 staining and typical apoptotic
morphology. However, the nuclei of the control HCT-116 cells
stained light green on exposure to Hoechst 33258 stain. These
observations further confirmed that compound 3b induced
apoptosis in HCT-116 cells (Fig. 2).
2.2.3. Mitochondrial membrane potential staining
The HCT-116 cells treated with 20 mM concentration of com-
2.2. Biological evaluation
pound 3b for 24 h stained with JC-1, however, no JC-1 staining was
observed in the control HCT-116 cells (Fig. 3). The compound 3b
treated cells exhibited strong green fluorescence and showed
typical apoptotic morphology after 24 h whereas the control cells
were normally red. Therefore, the above findings confirm that the
treatment of HCT-116 cells with compound 3b for 24 h induces
apoptosis.
2.2.1. In vitro cytotoxicity
MTT assay was used to study the cytotoxicity of all the synthe-
sized compounds against cultured A-549 (lung), MCF-7 (Breast),
HCT-116 (colon) and THP-1 (leukemia) carcinoma cell lines and FR-
2 (normal epithelial cell line). 5-fluorouracil along with compound
2 were taken as reference standards in this study. Preliminary
cytotoxicity screening of the ursolic acid derivatives was carried out
at 50 mM concentration for 48 h and the percentage cell death was
2.2.4. Cell cycle analysis
determined. Compounds that exhibited significant cytotoxicity
were further evaluated at different concentrations to get their IC50
values (Table 2). The values are the average of the triplicate analysis.
All the compounds exhibited better cytotoxicity compared to the
parent ursolic acid. It is clear from the IC50 values that almost all the
active compounds exhibited less cytotoxicity toward the normal
epithelial cell line (FR-2) compared to their anticancer potential
against carcinoma cell lines, which justifies the role of these de-
rivatives (3a-3p) as anti-cancer agents. Compound 3b with 2,5-
dihydroxy substitution at aromatic ring was found to be the most
promising candidate. The compound 3b exhibited potent cytotox-
icity against all the tested carcinoma cell lines.
Investigation of the effect of compound 3b on cell cycle distri-
bution in HCT-116 cells was performed by flow cytometric analysis
using propidium iodide (PI). The results revealed that treatment of
HCT-116 cells with compound 3b caused a significant increase in
the percentage of cells in G1 phase (65.58%) compared to the
control cells (41.51%). However, the percentage of cells in the G2
phase was significantly decreased in compound 3b treated cells
(11.65%) compared to control cells (32.43%) compared with the
control cells (Fig. 4).
2.2.5. Alterations in mitochondrial protein expression
The results from western blot analysis revealed that compound
3b induced significant increase in the expression of Bax and
decrease in expression of Bcl-2 compared to the control cells. It also
caused accumulation of cytochrome c in the cytosol, most probably
due to the release of mitochondrial cytochrome c (Fig. 5). Treatment
of HCT-116 cells with compound 3b caused a significant increase in
the expression levels of caspase-9 and caspase-3 proteins
2.2.2. Effect of compounds 3b on apoptosis in HCT-116 cells
Apoptosis is the programmed cellular process that takes place
during physiological and pathological conditions. However, in car-
cinoma cells the process of apoptosis is disrupted resulting in the
overgrowth and proliferation of malignant cells. Therefore, most of
Scheme 1. Reagents and conditions (a) CrO3, H2SO4, acetone, 0 ꢀC, 98%; b) Ar-CHO, KOH, Ethanol, rt, 92%.