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HCC. However, anti-cancer therapy for HCC is still limited by
intrinsic drug resistance [73]. Sorafenib, an oral multikinase in-
hibitor with anti-proliferation and anti-angiogenic properties [74],
is an U.S. Food and Drug Administration (FDA)-approved drug for
advanced renal cancer and is also the only FDA-approved targeted
therapy for advanced HCC [75]. Sorafenib has also been demon-
strated to inhibit the proliferation of many other human cancer
cells such as non-small cell lung cancer, breast, colon, and pancreas
cancers [76,77]. Inhibition of serine/threonine kinase c-Raf and b-
naturally occurring anti-cancer compound found in turmeric, a
common ingredient in curry [96,97], increased 3y-induced cyto-
toxicity on HCC cells. Using gene expression signatures of 3y to
query the C-Map, a small molecule, AR-A014418, an inhibitor of
glycogen synthase kinase-3 (GSK-3) [98], was predicted to act
similarly to 3y. Further experiments confirmed that 3y exhibited a
cytotoxic effect by the induction of autophagy, and 3y also behaved
like AR-A014418 to suppress expression of phospho-GSK-3b.
Raf, platelet-derived growth factor receptor-a and b, cytokine re-
2. Results and discussion
ceptor c-KIT, and the receptor tyrosine kinases Flt-3 by Sorafenib
has been observed [78e80]. Nevertheless, Sorafenib only improved
overall survival by nearly 3 months in patients with advanced HCC
[81], and the improvement seems to be poor for those with hepa-
titis B [82]. Moreover, it seems to have a distinct adverse effect
profile and reduces patients' quality of life. A meta-analysis of
clinical trials shows that patients who took Sorafenib had many
undesirable side effects, including increases in the risk of hyper-
tension, bleeding, and arterial thromboembolism [83e86]. Major
side effects, such as diarrhea, fatigue, and hand-foot syndrome,
increased in patients with advanced cirrhosis and in those who
received combination therapy of Sorafenib and 5-fluorouracil [82].
Therefore, there is an urgent need to develop novel therapeutic
agents for treatment of HCC.
2.1. Design and synthesis of compounds
More recently, we reported a simple and facile protocol for the
synthesis of 2H-1,4-benzoxazin-3(4H)-ones and their derivatives
[99]. Owing to the biological importance of 1,2,3-triazoles and 2H-
1,4-benzoxazin-3(4H)-ones scaffolds, we were primarily interested
in the synthesis of 4-((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)-2H-
benzo[b] [1,4] oxazin-3(4H)-one (3a) from 4-(prop-2-ynyl)-2H-
benzo[b] [1,4]oxazin-3(4H)-one (alkyne 1a) and benzylazide via
1,3-dipolar cycloaddition in the presence of suitable metal catalysts.
First, we performed a model reaction between alkyne 1a with
benzyl azide in the presence of CuI (0.3 equiv) in 1,4-dioxane at
room temperature for 13 h leading to the formation of 4-((1-
Due to the shortage of new drugs for treating cancers and the
rising expense of drug development, methodologies that allow the
exploration of associations among diseases, genes, and chemical
expression profiles may yield potential therapeutic targets. The
Connectivity Map (C-Map) is a database containing the gene
expression profiles of 4 different cancer cells treated with 1309
drugs, including FDA-approved drugs and other small molecules
[87,88]. Thus, C-Map provides information indicating which genes
were up- or down-regulated by FDA-approved drugs and/or other
small molecules. We hypothesized that if a compound displays a
similar gene expression profile to that of an FDA-approved drug on
the C-Map, the compound may have similar molecular mechanisms
to those of the FDA-approved drug. In our recent publications, we
demonstrated that, using gene expression signatures to query C-
Map, a small molecule (Trifluoperazine, an anti-psychotic agent;
Antimycin A, an anti-fungal agent) exhibited an anti-tumor effect
and enabled lung cancer stem-like cells to overcome drug resis-
tance in vitro and in vivo [89,90]. The target identification of new
compounds based on 2H-1,4-benzoxazin-3-(4H)-one and triazoles
moiety is an ideal example by applying C-Map.
benzyl-1H-1,2,3-triazol-4-yl)methyl)-2H-benzo[b]
[1,4]oxazin-
3(4H)-one (3a) in 93% yield (Table 1, entry 1). Next, we investigated
the effect of solvents on the formation of 4-((1-benzyl-1H-1,2,3-
triazol-4-yl)methyl)-2H-benzo[b] [1,4]oxazin-3(4H)-one. Later
performing the reactions in dimethyl sulfoxide (DMSO), DMF as
solvents at room temperature produced 73% and 69% low product
yields with several unidentified side products, respectively (Table 1,
entries 2 and 3). We conducted same reaction in the absence of
copper catalyst in DMSO at 100 ꢀC for 24 h, the reaction did not lead
to the formation of the required product (3a), and therefore we
recovered the starting materials (Table 1, entry 4). Next we
Programmed cell death can be divided into two main categories:
apoptosis (Type I) and autophagy (Type II). Insufficient apoptosis
contributes to the pathogenesis of cancer, and induction of
apoptosis has been suggested to be a promising strategy for treat-
ing cancers [91]. Typical morphological features of apoptotic cell
death are condensed chromatin in the nucleus, DNA fragmentation,
phosphatidylserine externalization, and generation of apoptotic
bodies [92]. Autophagy is also a normal physiological process. The
dynamic process of autophagy involves protein degradation and
recycling of injured organelles during nutrient starvation and stress
[93]. Recent studies on autophagy-induced death of breast, colon,
prostate, and brain cancer cells in various anti-cancer therapies,
such as chemotherapy, irradiation, and hyperthermia, have
renewed interest in autophagy in the field of oncology [94,95].
In the present study, the newly synthesized novel compounds
were examined for their anti-cancer potential. Induction of
apoptosis and autophagy was examined. Our results indicate that
3y at a lower concentration exhibited a better cytotoxic effect than
that of Sorafenib on human HCC cells via induction of apoptosis and
autophagy. It is noteworthy that 3y was relatively nontoxic to hu-
man normal cells. Combination of 3y with curcumin, an active,
Fig. 1. Crystal structure of compound (3a) [103].
Su, Chun-Li
