Preparation of S14161 and its analogues and the discovery of 6-bromo-8-ethoxy-3-nitro-2H-chromene as a more potent antitumor agent in vitro

Article abstract of DOI:10.1016/j.bmcl.2013.03.097

The small chemical compound 8-ethoxy-2-(4-fluorophenyl)-3-nitro-2H-chromene (S14161) was recently identified as an inhibitor of the phosphoinositide 3-kinase (PI3K). In the present study, we designed a novel synthesis of S14161 and prepared a series of its analogues via the oxa-Michael-Henry reaction in the presence of catalytic amounts of l-proline and triethylamine. Further structural simplification led to the identification of 6-bromo-8-ethoxy-3-nitro- 2H-chromene (BENC-511) that exhibited potent antiproliferative activities against a panel of 12 tumor cell lines. Compared with S14161, BENC-511 was more potent in blocking the AKT phosphorylation and inducing cancer cell apoptosis. BENC-511 also displayed more potent effects on human umbilical vein epithelial cells (HUVEC) migration, suggesting its anti-angiogenesis activity.

Full text of DOI:10.1016/j.bmcl.2013.03.097

Bioorganic & Medicinal Chemistry Letters 23 (2013) 3314–3319
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Preparation of S14161 and its analogues and the discovery
of 6-bromo-8-ethoxy-3-nitro-2H-chromene as a more potent
antitumor agent in vitro
Shu-Qiang Yin a, , Min Shi b, , Ting-Ting Kong a, Cheng-Mei Zhang a, Kunkun Han b, Biyin Cao b,
Zubin Zhang b, Xiaolin Du b, Long-Qian Tang a, Xinliang Mao b, , Zhao-Peng Liu a,
⇑
⇑
a Department of Organic Chemistry, Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Shandong University, Jinan 250012,
Shandong Province, PR China
b Cyrus Tang Hematology Center, Soochow University, Suzhou, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
The small chemical compound 8-ethoxy-2-(4-fluorophenyl)-3-nitro-2H-chromene (S14161) was recently
identified as an inhibitor of the phosphoinositide 3-kinase (PI3K). In the present study, we designed a
novel synthesis of S14161 and prepared a series of its analogues via the oxa-Michael–Henry reaction
Received 15 February 2013
Revised 20 March 2013
Accepted 23 March 2013
Available online 4 April 2013
in the presence of catalytic amounts of L-proline and triethylamine. Further structural simplification
led to the identification of 6-bromo-8-ethoxy-3-nitro-2H-chromene (BENC-511) that exhibited potent
antiproliferative activities against a panel of 12 tumor cell lines. Compared with S14161, BENC-511
was more potent in blocking the AKT phosphorylation and inducing cancer cell apoptosis. BENC-511 also
displayed more potent effects on human umbilical vein epithelial cells (HUVEC) migration, suggesting its
anti-angiogenesis activity.
Keywords:
Phosphoinositide 3-kinase pathway (PI3K)
inhibitors
Akt phosphorylation
3-Nitro-2H-chromene
Oxa-Michael–Henry reaction
Antiproliferative
Ó 2013 Elsevier Ltd. All rights reserved.
Apoptosis
Antitumor
The phosphoinositol 3-kinase (PI3K)/AKT signaling pathway is
the central node of many important signals involved in mitogenic
signaling, metabolic control, cell survival, growth, and prolifera-
tion. Among the three classes of PI3K enzymes, the class I PI3Ks
PI3K inhibitors have been advanced into clinical development in
cancer therapy.4–9
Chromenes, or benzopyrans, are an important class of scaffolds
found in numerous naturally occurring and synthetic molecules
possessing a broad spectrum of biological activities.10 8-Ethoxy-
2-(4-fluorophenyl)-3-nitro-2H-chromene (S14161) was discovered
as a potent PI3K inhibitor through high-throughput screening of a
library of 56,000 organic compounds (Maybridge Chemical Com-
pany).11 S14161 inhibited the activity of phosphoinositide 3-kinase
in intact cells and the activity of the phosphoinositide 3-kinases in
a cell-free enzymatic assay thus decreasing the production of PIP3
and blocked AKT translocation to the plasma membrane for activa-
tion. Given its potent antileukemia and antimyeloma activity and
minimal toxicity, S14161 can serve as a new lead for cancer ther-
apy. Because there are no literature reports available on S14161
synthesis, to translate its application and to find more potent
PI3K inhibitors, we reported here a novel synthesis of S14161
and its analogues and the discovery of 6-bromo-8-ethoxy-3-ni-
tro-2H-chromene (BENC-511) that exhibited more potent antipro-
liferative activity than S14161 against a panel of tumor cell lines.
Based on the chemical structure of the compound S14161, we
proposed that one of the useful synthetic methods for the
include four members, PI3Ka, b, d, and c, which catalyze the phos-
phorylation of phosphatidylinositol-4,5-bisphosphonate (PIP2) to
phosphatidylinositol-3,4,5-triphosphonate (PIP3) that acts as a
second messenger to recruit and activate AKT.1–3 Activation of
the PI3K/AKT signaling cascade is critical for cell biological activity,
however aberrant upregulation of this signaling is deleterious,
which is observed in several types of cancers, leading to uncon-
trolled proliferation, enhanced migration, and adhesion-indepen-
dent growth. While knock-down of PI3K/AKT leads to decreased
cancer cell proliferation and induction of apoptosis, therefore,
blocking the PI3K/AKT signaling pathway provides a promising
strategy for cancer therapy. In the past two decades, a number of
⇑
Corresponding authors. Tel.: +86 512 65880877x507; fax: +86 512 55882123
(X.M.); tel.: +86 531 88382006; fax: +86 531 88382548 (Z.-P.L.).
E-mail addresses: xinliangmao@suda.edu.cn (X. Mao), liuzhaop@sdu.edu.cn
(Z.-P. Liu).
These authors contributed equally to this project.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.03.097

S.-Q. Yin et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3314–3319
3315
NO2
NO2
CHO
OH
R
120% mol
catalyst, solvent
2 (S14161): R = F
3: R = H
O
4: R = CN
OEt
OEt
5: R = OMe
R
1
Scheme 1. Preparation of S14161 and its analogues.
preparation of 3-nitro-2-phenyl-2H-chromenes is the domino oxa-
Michael–Henry reactions of salicylaldehyde with b-nitrostyrene.12
Basically this reaction is promoted by an organocatalyst like 1,4-
Table 2
Growth inhibitory activity of S14161 and its analogues against tumor cell lines (GI50
M)a
:
l
diazabicyclo[2.2.2]octane (DABCO), 2-pipecolinic acid, L-proline,
a
Compds
GI50 (lm)
among many others. Therefore, the preparation of S14161 and its
analogues could be achieved via the reaction of 3-ethoxysalicylal-
dehyde 1 with the appropriate b-nitrostyrenes shown in Scheme 1.
To explore the best oxa-Michael–Henry reaction conditions, a
model reaction between 3-ethoxysalicylaldehyde and excess b-
nitrostyrene (120% mol, R = F) was performed in the presence of
DABCO (100% mol) without any solvents. S14161 was isolated with
a best yield of 48% when the reaction was carried out at a temper-
ature of 45 °C (Table 1, entry 1). At the time of 1.5 h, the reaction
was complete as indicated by the disappearance of 1 in a TLC anal-
ysis. In the further optimization studies, other factors that influ-
ence the reaction were thoroughly investigated including solvent,
catalyst loading, and reaction temperature. It was found that a
50% mol catalyst loading was necessary for this domino oxa-Mi-
chael–Henry reaction. A slight improved yield of 53% was obtained
with toluene as a solvent, a reaction temperature of 45 °C and a
reaction time of 24 h (Table 1, entry 2). In the catalyst screens, 2-
LP1
K562
MDA-MB-231
8.53
B16
U87
2 (S14161)
3
4
5
0.57
0.96
0.44
3.16
0.64
0.33
1.24
1.18
1.02
4.86
1.68
0.92
23.28
18.72
7
8 (BENC-511)
2.03
20.38
12.60
a
Dose causing 50% inhibition of growth compared to untreated tumor cell lines
studied in cell culture. Each agent was studied in two sets of triplicate experiments
(N = 6) and 95% confidence limit variation was generally less than 20% of the
determined values.
R1
CHO
OH
R
NO2
phthalic anhydride
NO2
+
HO
O
n-Bu2NH, PhCH3
OEt
OEt
1: R = H
7: R = H
pipecolinic acid was proved to be not the choice. However, L-pro-
6: R = Br
8: R = Br
line was effective in a range of solvents, and S14161 was obtained
in a yield up to 54% (Table 1, entry 10). Next, we examined the
cocatalyst effects on this domino reaction. Acidic cocatalysts like
hydrobromic acid and salicylic acid did not show any benefit ef-
fects, however, a basic cocatalyst had a great influence on this reac-
tion. The presence of 20% mol of DABCO or triethylamine led to an
improved yield of 70% or 87% (Table 1, entries 12 and 13). These
observations suggested that a basic component might contribute
a synergistic effect in the reaction in which nitroolefin was
involved.
Scheme 2. Preparation of 8-ethoxy-3-nitro-2H-chromenes 7 and 8.
position of the 2-phenyl ring in S14161, while compounds 4 and
5 had an electron-withdrawing cyano group and electron-donating
methoxy substituent at the same position, respectively.
In a growth inhibition screening of S14161 and its analogues 3–
5 against human myeloma cell LP1 and leukemia cell K562, com-
pound 4 that has an electron-withdrawing cyano group at the para
position of 2-phenyl ring was more potent than S14161, while
compound 3 that has no substituent at the para position of 2-phe-
nyl ring exhibited a comparable potency as that of S14161. On the
contrary, analogue 5 was less potent than S14161, 3 and 4 (Table
2). Therefore, an electron-donating methoxy substituent at the
After successfully optimizing the catalyst (L-proline), cocatalyst
(triethylamine), solvent (toluene), temperature (90 °C) and reac-
tion time (24 h), we further prepared three analogues of S14161
in good yields (Table 1, entries 14–16). Compound 3 had a replace-
ment of the fluoro substituent with a hydrogen atom at the para
Table 1
Optimization of the oxa-Michael–Henry reaction conditions for the synthesis of S14161 and its analogues
Entry
R
Catalyst/co-catalyst
Solvent
T (°C)
Time (h)
Yield ()
1
2
3
4
5
6
F
F
F
F
F
F
DABCO (100% mol)
DABCO (50% mol)
DABCO (50% mol)
2-Pipecolinic acid (50% mol)
2-Pipecolinic acid (50% mol)
45
45
90
45
90
Reflux
15
24
24
24
24
24
48
53
45
Trace
21
Toluene
Toluene
Toluene
Toluene
THF
50
L
L
L
L
L
L
L
L
L
L
L
-Proline (50% mol)
7
F
MeCN
Reflux
90
24
24
24
24
24
24
24
24
24
24
42
40
35
54
44
70
87
74
81
79
-Proline (50% mol)
8
F
DMF
-Proline (50% mol)
9
F
Toluene
Toluene
Toluene
Toluene
Toluene
Toluene
Toluene
Toluene
45
-Proline (50% mol)
10
11
12
13
14
15
16
F
90
-Proline (50% mol)
F
Reflux
90
-Proline (50% mol)
F
-Proline (50% mol)/DABCO (20% mol)
-Proline (50% mol)/Et3N (20% mol)
-Proline (50% mol)/Et3N (20% mol)
-Proline (50% mol)/Et3N (20% mol)
-Proline (50% mol)/Et3N (20% mol)
F
90
H
90
CN
MeO
90
90

3316
S.-Q. Yin et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3314–3319
Figure 1. Effects of S14161 and BENC-511 on cancer cell proliferation at 20 lM.
para position of 2-phenyl ring disfavored the antitumor activities
of these compounds.
activity than S14161 against a panel of tumor cell lines, including
solid tumor of human breast cancer MDA-MB-231, mouse mela-
noma B16 and human Glioma U87 cell lines. Especially, BENC-
511 was 4-fold more potent than S14161 against human breast
cancer MDA-MB-231 cells.
To further evaluate the effects of BENC-511 on cancer cell pro-
liferation, seven solid tumor cell lines, including the prostate can-
cer (PC3), ovarian cancer (SKVO3), cervical cancer (HeLa), breast
cancer (H47D), and lung cancer (H460, A549 and SPCA-1), were
treated with S14161 or BENC-511 for 72 h, followed by cell prolif-
eration analysis using inverted microscope. As indicated in Figure
1, all the cell lines were inhibited by both S14161 and BENC-511
Since S14161 has one chiral center in its molecule and it was
used as a racemate in its biological studies, we further simplified
its structure by removing the 4-fluorophenyl group at the 2-posi-
tion of the chromene core, prepared compounds 7 and 8 (Scheme
2). The reaction of 3-ethoxysalicylaldehydes 1 or 6 with 2-nitroeth-
anol in the present of 200% mol of phthalic anhydride and 50% mol
of dibutylamine in toluene under reflux conditions gave the corre-
sponding 8-ethoxy-3-nitro-2H-chromene 7 and 6-bromo-8-eth-
oxy-3-nitro-2H-chromene (BENC-511) 8 in 64% and 74% yields,
respectively.
As shown in Table 2, analogue 7 was less potent than S14161
against human myeloma LP1 and leukemia K562 cell lines, while
the 6-bromo derivative BENC-511 (8) exhibited better inhibitory
in 72 h at the concentration of 20 lM.
To further study the inhibitory efficacy of S14161 and BENC-
511 at different concentrations, lung cancer cell line A549 and
Figure 2. Effects of S14161 and BENC-511 on A549 and HeLa cell proliferation.

S.-Q. Yin et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3314–3319
3317
cervical cancer cell line HeLa were treated with a concentration
range from 0 to 20 M for 24 h. As shown in Figure 2, both
S14161 and BENC-511 displayed similar activity for A459 cells,
mark analysis. Poly(ADP-ribose) polymerase (PARP) is a family of
proteins involved in a number of cellular processes involving
mainly DNA repair and programmed cell death. Caspase-3 is one
of the major executive enzymes in apoptosis. As indicated in Fig-
l
but S14161 was less potent than BENC-511 against HeLa cells, even
at 20
dependent manner. It started to decrease cell growth at 5
inhibited HeLa cell growth more than 90% at 20 M. This study
l
M. BENC-511 inhibited HeLa cell growth in a concentration-
ure 3B, BENC-511 activated caspase-3 at 5
creased at 10 M, while S14161 had little effects on caspase-3 in
the same cell line at 10 M. Therefore, this study further demon-
lM and significantly in-
lM, and
l
l
l
suggested that BENC-511 was more potent in anti-cancer prolifer-
strated that BENC-511 was more potent than S14161 in cancer cell
ation at least in cervical cancers.
apoptosis induction.
Because S14161 was an inhibitor of the PI3K/AKT signaling
pathway, to find out whether the inhibitory activity on PI3K was
associated with the anti-proliferation of BENC-511, we next evalu-
ated the effects of S14161 and BENC-511 on PI3K activity. Four
cancer cell lines (A549, SKVO3, –Hela, and H47D) were starved
and treated with S14161 or BENC-511, followed by EGF1 stimula-
tion. Western blotting analysis showed that AKT phosphorylation
was completely blocked by BENC-511 in all four cell lines tested,
while there were not significant changes in AKT protein (Fig. 3A).
In contrast, S14161 had little effects on AKT phosphorylation in
SKVO3 cells and less inhibition in HeLa cells (Fig. 3A). This result
demonstrated that BENC-511 was more potent in suppressing
PI3K activity in terms of AKT activation. Notably, this finding was
consistent with the inhibitory activity of S14161 and BENC-511
on HeLa and A549 cells as indicated in Figure 2, where S14161
had no marked effects on HeLa cell proliferation.
PI3K/AKT signaling pathway is important in regulation vessel
formation and growth. To view the effect of BENC-511 on angio-
genesis, human umbilical vein epithelial cells (HUVEC) were trea-
ted with BENC-511 or S14161. In wounded and scratching test,
HUVEC cells migrated and merged within 24 h. Both BENC-511
and S14161 inhibited HUVEC cell migration (Fig. 4). By measuring
the area of the wounded gap, BENC-511 inhibited HUVEC forma-
tion in a stronger manner than S14161. At the concentration of
5 lM, less than 50% of the gap was filled by the migrated HUVEC
cells treated by S14161, while in BENC-511 treatment group, only
20% of the gap was filled. Therefore, BENC-511 displayed more po-
tent effects on cell migration.
To gain molecular insight into the ligand-binding site interac-
tions responsible for its inhibitory activity on PI3K, BENC-511
and S14161 were docked into the ATP site of PI3Kc
from 3L5413
utilizing Sybyl 7.0. Both molecules could fit well into the adenine
binding pocket, and similar to the N1 nitrogen of ATP, the nitro
group in BENC-511 or S14161 acted as a hinge binder that inter-
acted with the hinge region through the formation of the critical
To find out whether BENC-511 was more potent in inducing
cancer cell apoptosis, A549 cells were treated with S14161 or
BENC-511 at the same concentrations followed by apoptotic hall-
Figure 3. Effects of S14161 and BENC-511 on PI3K activity and induction of cell apoptosis.

3318
S.-Q. Yin et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3314–3319
Figure 4. Effects of S14161 and BENC-511 on HUVEC cells migration.
hydrogen bond with the backbone Val882.14 The 4-fluorophenyl in
S14161 extended into the affinity binding pocket, while the substi-
tuted phenyl ring in BENC-511 occupied the same hydrophobic
pocket and the ethoxy oxygen atom formed additional hydrogen
bond interactions with residues of Try867 and Asp964 (Fig. 5). This
may partly explain why the compound BENC-511 without a 4-fluo-
rophenyl substituent at the 2-position of the chromene ring still
exhibited potent antitumor activities. However, further studies
are needed to identify the molecular targets of BENC-511.
In conclusion, optimization of the oxa-Michael–Henry reaction
conditions in the presence of catalytic amounts of L-proline and tri-
ethylamin led to an efficient preparation of S14161 and its ana-
logues. Further structural simplification of S14161 by removing
the 2-aryl substituent at its chiral center led to the discovery of
BENC-511 as a more potent antitumor agent in vitro. Because
BENC-511 was effective in inducing cancer cell death and inhibit-
ing HUVEC cell migration, it will be a new lead for the development
of novel anticancer and anti-metastatic agents.
Acknowledgments
This work was partially supported by the Shandong Provin-
cial Natural Science Foundation (ZR2011CZ001 to Z.-P.L.), the
National Natural Science Foundation of China (Grant No.
81101795 to B.C.), the Natural Science Foundation of Jiangsu
Province (BK2010218 to X.M. BK2011268 to B.C.), and the Prior-
ity Academic Program Development of Jiangsu Higher Education
Institutions (PAPD).

S.-Q. Yin et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3314–3319
3319
Figure 5. Docking of S14161 (carbon atoms in orange) and BENC-511 (carbon atoms in grey) into the ATP binding site of PI3Kc (left), the interactions between PI3Kc and
BENC-511.
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