Discovery of benzenesulfonamide derivatives as potent PI3K/mTOR dual inhibitors with in vivo efficacies against hepatocellular carcinoma

Article abstract of DOI:10.1016/j.bmc.2016.01.008

The phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway is related to cellular activities. Abnormalities of this signaling pathway were discovered in various cancers, including hepatocellular carcinoma (HCC). The PI3K/mTOR dual inhibitors were proposed to have enhanced antitumor efficacies by targeting multiple points of the signaling pathway. We synthesized a series of propynyl-substituted benzenesulfonamide derivatives as PI3K/mTOR dual inhibitors. Compound 7k (NSC781406) was identified as a highly potent dual inhibitor, which exhibited potent tumor growth inhibition in the hepatocellular carcinoma BEL-7404 xenograft model. Compound 7k may be a potential therapeutic drug candidate for HCC.

Full text of DOI:10.1016/j.bmc.2016.01.008

Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of benzenesulfonamide derivatives as potent PI3K/mTOR
dual inhibitors with in vivo efficacies against hepatocellular
carcinoma
Ying Chen y, Ling Zhang y, Chao Yang y, Jinsong Han, Chongqing Wang, Canhui Zheng, Youjun Zhou,
⇑
⇑
Jiaguo Lv, Yunlong Song , Ju Zhu
Department of Medicinal Chemistry, School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433, China
a r t i c l e i n f o
a b s t r a c t
Article history:
The phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR)
signaling pathway is related to cellular activities. Abnormalities of this signaling pathway were discov-
ered in various cancers, including hepatocellular carcinoma (HCC). The PI3K/mTOR dual inhibitors were
proposed to have enhanced antitumor efficacies by targeting multiple points of the signaling pathway.
We synthesized a series of propynyl-substituted benzenesulfonamide derivatives as PI3K/mTOR dual
inhibitors. Compound 7k (NSC781406) was identified as a highly potent dual inhibitor, which exhibited
potent tumor growth inhibition in the hepatocellular carcinoma BEL-7404 xenograft model. Compound
7k may be a potential therapeutic drug candidate for HCC.
Received 6 November 2015
Revised 6 January 2016
Accepted 6 January 2016
Available online xxxx
Keywords:
Phosphoinositide 3-kinase (PI3K)
Dual inhibitors
Hepatocellular carcinoma
Benzenesulfonamide derivatives
Antitumor
Ó 2016 Elsevier Ltd. All rights reserved.
Mammalian target of rapamycin (mTOR)
1. Introduction
Deregulation of this signaling pathway, due to mutation and
amplification of genes encoding PI3K isoforms, loss of expression
Lipid kinase PI3K phosphorylates 3-hydroxyl group of phos-
phatidylinositol 4,5-bisphosphate (PIP2) to produce phosphatidyli-
nositol 3,4,5-triphosphate (PIP3).1 PIP3 phosphorylates and
activates AKT, and then leads to several downstream effects, such
as activating cAMP response element binding protein (CREB),2
localizing Forkhead box O (FoxO) in the cytoplasm, and activating
mTOR.3 The PI3K/AKT/mTOR signaling pathway regulates various
biological processes, which are involved in multiple essential
cellular functions, including cell growth, survival, proliferation,
migration and metabolism.1,4–6
of the gene encoding suppressor phosphatase and tensin homolog
(PTEN), and hyperactivity of receptor tyrosine kinases, was
reported in advanced hepatocellular carcinoma (HCC).8–10
Activation of the PI3K/AKT/mTOR pathway has been observed in
30–50% of HCC patients.11 The overexpression of the AKT gene
and phosphorylation of mTOR have been reported in 63% and
15% of HCC patients.12 These observations suggested that inhibi-
tion of PI3K/AKT/mTOR signaling pathway may be a promising
therapeutic target for HCC. The multikinase inhibitor, sorafenib,
which exerts its beneficial effects via inhibition of several kinases
related to tumor cell proliferation and angiogenesis, has become
the standard first-line treatment for patients with inoperable
advanced HCC.13 However, sorafenib treatment showed limited
overall survival outcomes, poor toleration, and was associated with
increased incidence of several side effects, including hypertension,
gastrointestinal and dermatological problems.14 Therefore, identi-
fying novel therapeutic agents with better efficacies and safeties
for HCC treatment is urgently needed.
The PI3K/AKT/mTOR pathway is one of the most frequently up-
regulated signaling pathways associated with numerous cancers.1,7
Abbreviations: AKT, protein kinase B; CREB, cAMP response element binding
protein; FoxO, Forkhead box O; GI50, drug concentration yielding 50% growth
inhibition; HCC, hepatocellular carcinoma; IC50, drug concentration yielding 50%
inhibition; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide;
PI3K, phosphoinositide 3-kinase; mTOR, mammalian target of rapamycin;
PIP2,
phosphatidylinositol
4,5-bisphosphate;
PIP3,
phosphatidylinositol
The deregulation of PI3K/AKT/mTOR signaling pathway in HCC
and lack of effective therapeutic approaches for HCC patients led
to the search for novel inhibitors targeting the key kinases of this
signaling pathway. Small molecular inhibitors targeting PI3K and
3,4,5-trisphosphate.
⇑
Corresponding authors. Tel.: +86 21 81871232 (Y.S.), +86 21 81871238 (J.Z.).
E-mail addresses: ylsong@smmu.edu.cn (Y. Song), zhuju@smmu.edu.cn (J. Zhu).
The first three authors contributed equally to this work.
y
http://dx.doi.org/10.1016/j.bmc.2016.01.008
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Chen, Y.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.01.008

2
Y. Chen et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
mTOR have been suggested as potential new candidates for tar-
geted HCC therapy.8 Dual PI3K/mTOR inhibitors targeting the four
2. Results and discussion
isozymes of class I PI3K (PI3Ka, PI3Kb, PI3Kd, and PI3Kc) and mTOR
2.1. Molecular design
have been designed and generated. Several dual PI3K/mTOR
inhibitors have advanced into preclinical development, while
some other inhibitors have entered clinical trials (Fig. 1), such as
PKI-587 (Wyeth),15 PF-04691502 (Pfizer),16 BEZ235 (Novartis),17
GDC-0980 (Genentech),18 BGT226 (Novartis)19 and PKI-402
(Wyeth).20
Several compounds with benzenesulfonamide scaffold were
reported as potent PI3K inhibitors (Fig. 2). These benzenesulfon-
amide inhibitors exhibit excellent antitumor activities in cytotoxi-
city assays or xenograft models.21,22 Herein, we designed and
synthesized a series of propynyl-substituted benzenesulfonamide
derivatives. We synthesized and evaluated compound 7k
(NSC781406), an analog with potent tumor growth inhibition
against HCC.
As a consequence of close homology between PI3Ka and PI3Kc,
it is not surprising that the predictive binding pose for pan PI3K
inhibitor in PI3K was very similar to that with PI3K . According
to the cocrystal structure of GSK2126458 with PI3K (PDB ID:
a
c
c
3L08), the difluorophenyl group was located in a hydrophobic
region of the enzyme, while the quinoline nitrogen atom formed
a hydrogen bond interaction with Val882 in the hinge region of
the PI3Kc kinase domain. The oxygen and nitrogen atoms of sul-
fonamide formed two interactions with Lys833 in the affinity
pocket.22
Our molecular modeling studies suggested that long side chains
with hydrogen bond acceptors projecting from the 4-position of
the quinoline ring into the ribose pocket might interact favorably
HO
O
O
N
NC
O
N
N
O
N
H2N
N
N
O
N
N
N
O
N
N
N
O
N
N
H
N
H
N
N
O
PKI-587
BEZ235
PF-04691502
OH
N
O
N
HN
N
F
F
HO
O
F
O
O
OH
N
N
O
N
N
N
O
NH
O
N
S
O
O
N
N
NH
N
N
N
N
N
N
NH2
N
N
N
GDC-0980
BGT226
PKI-402
Figure 1. Examples of dual PI3K/mTOR inhibitors in clinical trials.
O
Cl
N
S
HO
HN
H
N
O
N
O
Br
S
O
N
O
O
N
N
S
NO2
O
S
N
N
NH
N
HCl
O
O
HS-173
PIK-75
PIK-93
Cl
N
N
O
O
N
HN
S
N
N
OMe
N
N
NH
O
O
S
O
O
H
O
N
S
N
O
NH HN
F
N
H
NH2
Me
MeO
O
O
HN
OMe
F
XL 147
XL765
Figure 2. PI3K inhibitors of benzenesulfonamide scaffold.
GSK2126458
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Y. Chen et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
3
L
y
s
8
0
2
Ribose Pocket
Al
a
8
0
5
R
Affinity Pocket
O
N
Lys 833
O
S
O
R1
N
H
N
F
Val882
Hinge Region
Figure 3. Generic structure revealing potential interactions with PI3Kc.
with the ribose pocket of PI3K
c
(Fig. 3). In order to find a suitable
2.3. Structure–activity relationships
replacement for the pyridazine group, we searched a library which
was composed of fragments with 3–4 carbon atom chains. A
propynyl group was identified to have a suitable shape and length
fitting the ribose pocket. We synthesized a series of propynyl-
substituted benzenesulfonamides, and evaluated their activities
against PI3K and mTOR, and further evaluated them on the efficacy
in the HCC xenograft model.
All the compounds were assayed for cell proliferation inhibition
in an MTT assay with several tumor cell lines, including human
non-small cell lung cancer cell (A549), human colon cancer cell
(HCT116), human breast cancer cell (MDA-MB-231) and human
hepatocellular carcinoma cell (BEL-7404). The IC50 values for PI3Ks
and mTOR activities were measured using PI103 and GSK2126458
as reference inhibitors. Since the overexpression of PI3K
mostly observed in various cancers, we discussed the structure–
activity relationship (SAR) based on the IC50 data against PI3K
a was
2.2. Chemistry
a
.
In order to establish the structure–activity relationship of the
propynyl substituted compounds, we prepared the compounds as
shown in Scheme 1. Substitution of the 2-chloro group of
5-bromo-2-chloro-3-nitropyridine 1 yielded 5-bromo-2-methoxy-
3-nitropyridine 2, followed by treatment with benzenesulfonyl
chloride 4 to afford sulfonamides 5. The key intermediate 6 was
synthesized through one-pot palladium-catalyzed Suzuki coupling
Estimation of the oxidative metabolism liability for the compounds
was based on the in vitro rat microsomal stability data in Table 3,
which was used to guide the selection of compounds for the in vivo
rodent studies.
We examined the effects of replacement at R1 position on
PI3Ka inhibition and cytotoxicity. As shown in Table 1, the
2,4-difluoro substituted compound 7b was more potent than the
4-fluoro substituted compound 7a in cell proliferation inhibitory
activities against A549 cells, HCT116 cells, MDA-MB-231 cells
and BEL-7404 cells. The fluorine substitution of 7b gave
of compound
5 with 6-bromo-4-chloroquinoline. The target
compounds were obtained by Sonogashira carbon–carbon coupling
reaction of intermediate 6 with substituted propargyl amines.
R1
O
O
N
Cl
S
O
HN
S
Br
F
4a: R1 = H
O
O
N
N
Cl
O
O
N
b
: R1 = F
a
b
R1
c
Br
Br
O2N
O2N
H2N
Br
5a: R1 = H
3
2
1
b
: R1 = F
F
R
O
N
Cl
O
N
HN
S
HN
O
O
N
O
S
O
R1
N
d
e
R1
6a
: R1 = H
b
: R1 = F
F
7
F
Scheme 1. Reagents and conditions: (a) MeONa, MeOH, 0 °C–rt. (b) Fe, NH4Cl, EtOH, H2O, 90 °C. (c) Pyridine, 0 °C–rt. (d) 6-Bromo-4-chloroquinoline, bis(pinacolato)diboron,
Pd(dppf)Cl2-CH2Cl2, potassium acetate, 1,4-dioxane, 100 °C, 2 h; then compound 5, Pd(dppf)Cl2-CH2Cl2, 2 M aqueous K2CO3, 110 °C, 20 h. (e) Pd(OAc)2,
( )-2,20-bis(diphenylphosphino)-1,10-binaphthyl, propargyl amines, K2CO3, DMF, 100 °C, 16 h.
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Y. Chen et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
Table 1
Inhibition of PI3K
a and antiproliferative activities
R
O
N
HN
S
O
O
N
R1
F
Compd
R1
H
R
PI3K
a
A549
0.97
HCT116
1.34
MDA-MB-231
IC50 (lM)
BEL-7404
1.16
IC50 (nM)
N
N
7a
32
2.08
7b
7c
F
F
3.7
0.11
2.43
1.08
2.27
1.67
2.68
0.23
0.30
N
33.0
N
7d
7e
F
F
3.9
1.01
1.15
2.80
0.08
6.10
OH
>100
11.99
11.70
27.66
N
N
7f
F
F
37
70
1.81
2.90
3.61
4.02
10.30
7.99
2.30
1.55
N
7g
7h
7i
F
F
F
61
1.35
0.24
0.11
4.52
0.51
1.39
4.73
1.99
3.81
1.14
0.04
0.48
N
N
O
2.1
5.5
O
N
N
N
7j
O
O
S
O
N
7k
F
2.0
1.1
1.78
0.60
0.93
0.01
1.44
0.13
0.02
0.01
GSK2126458
approximately 9-fold improvement in PI3K
a
enzymatic activity
in PI3Ka inhibition. In addition, 7i exhibited over 5-fold improve-
relative to the 2-unsubstituted compound. As both compounds dis-
played nanomolar level of PI3K inhibitory activities, substituting
with propynyl group at the quinoline 4-position was a feasible
strategy for jointing these functional groups. We then synthesized
the 2,4-difluoro substituted derivatives to investigate the effects of
ment in potency on cellular antiproliferation of three cancer
cell lines compared to 7f. For hepatocellular cancer cell line
BEL-7404, 7i showed 57-fold more potency than 7f on cellular
antiproliferation. The improvement was also observed as the
hydrogen-binding receptor compound 7j displayed more potency
on both enzyme inhibition and cellular antiproliferation compared
to 7f. Methyl sulfonamide-substituted piperazine 7k demonstrated
various side chains at the propynyl linker on PI3K
cytotoxicity.
a inhibition and
Table 1 shows the enzymatic and cellular inhibition data for
propargyl amines. Four aliphatic amines substituted compounds
showed that N,N-dimethyl of 7b and the N-methyl-N-hydroxyethyl
of 7d were comparable in terms of enzyme inhibition. However,
the N,N-diethyl substituted 7c demonstrated roughly a 10-fold
potent PI3K inhibition (PI3Ka IC50 = 2.0 nM) that translated into
improvement in BEL-7404 proliferation inhibition (IC50 = 20 nM)
relative to 7i and 7j.
2.4. Molecular modeling studies
drop in PI3Ka inhibition activity, which indicated that introduction
of bulky aliphatic chains were unfavorable for enzymatic and cel-
lular inhibition. This observation was supported by the loss of
PI3K inhibitory activity of compound 7e. Substitution of the
propargyl amines with heterocyclic groups was effective in
improving activities against PI3K enzyme, with 7i, 7j and 7k
inhibiting PI3K with IC50 values <10 nM. Heterocyclic compounds
7f, 7g and 7h showed modest improvement in both enzyme and
cell inhibitory activities relative to 7e. Of the three compounds,
non-substituted heterocyclic 7f showed comparatively stronger
Molecular docking studies of compound 7k (NSC781406) with
crystal structure of PI3Ka (PDB ID: 3ZIM) indicated that the quino-
line hinge binder interacted with Val851 through a hydrogen bond,
while the sulfonamide group interacted in the affinity pocket, as
indicated in the PI3Kc model (Fig. 3). Molecular docking studies
also suggested that a propynyl linker at the 4-quinoline position
would provide a favorable orientation for substituents to penetrate
and interact in the ribose pocket. Of the series of aliphatic hetero-
cycles, the piperazine group of 7k was of high potency on PI3Ka.
inhibition against PI3K
a
. A further increase in enzyme inhibition
Modeling suggested that substitution from the propargyl position
with hydrogen bond acceptors was not only able to maintain the
interactions with the hinge region and affinity pocket, but also
interacted with the side chains of Ser773 and Arg770 in the ribose
was observed when the groups with hydrogen bond receptors
were introduced to propynyl linker. Morpholine compound 7i
was over 10-fold more potent than the piperidine compound 7f
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Y. Chen et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
5
lung cancer, colon cancer, central nervous system cancer, mela-
noma, ovarian cancer, renal cancer, prostate cancer, and breast
cancer. Compound 7k was potent against 60 cancer cell lines with
a mean GI50 value of 65 nM, and with a GI50 value less than 10 nM
against four cancer cell lines (Fig. S1) indicating that 7k may be a
potential broad-spectrum inhibitor against multiple cancers.
2.7. Cellular mechanism analysis
Since compound 7k showed potent enzymatic and cellular
activities, the molecular mechanism underlying the cytotoxicity
was investigated. We performed Western blot to validate how
these analogs regulated activation of PI3K signaling pathway on
lung cancer cell lines A549. After treatment with four concentra-
tions of compound 7k for 30 min, 7k showed remarkable suppres-
sion of p-AKT at 0.1
M (Fig. 5).
lM, and almost completely blocked p-AKT at
1
l
2.8. In vivo efficacy studies
Considering the potency in vitro cytotoxic activities and favor-
able microsomal stability of compound 7k, the in vivo efficacy
was further evaluated in BEL-7404 xenograft model (Fig. 6). After
incubation of BEL-7404 cells for 13 days in the BALB/C nude mice,
suspension of 7k in Tween-80/water was orally administered once
a day for 14 consecutive days. The relative tumor volume to
vehicle-treated control mice was monitored. The clinically
Figure 4. Molecular docking of compound 7k in the ATP binding site of PI3Ka (PDB
code: 3ZIM). Hydrogen bonds are represented in red dotted lines.
pocket of PI3Ka, boosting the affinity with the enzyme by making
additional hydrogen bonding interactions (Fig. 4).
2.5. In vitro liver microsomal stability
This series of compounds maintained high potencies against
PI3K isoforms and mTOR at the nanomolar level (Table 2), so we
tested half-life time values of five selected potent compounds by
incubating them with rat liver microsome. The data in Table 3 pro-
vided an estimate of oxidative metabolism liability aiding the
selection of the chemical entities for the in vivo anti-cancer
studies.
Compounds 7d, 7i, 7j and 7k displayed reasonable liver micro-
some stability, while compound 7b with a short sidechain showed
less favorable stability.
Figure 5. Inhibition of PI3K signaling in A549. PI3K signaling pathway was
activated by treatment of 1 lg/mL insulin for 2 h, then A549 cells were treated
with four concentrations of 7k for 30 min. 7k suppressed phosphorylation of AKT
2.6. NCI-60 cancer cell screening
and mTOR in a dose-dependent manner.
Given that PI3K/mTOR pathway is abnormally activated in
many human cancers, 7k was tested at the National Cancer Insti-
tute (USA) for cytotoxicity against 60 human cancer cell lines
(NCI-60 cytotoxic assay). As shown in Figure S1, compound 7k
demonstrated cytotoxic activities against leukemia, non-small cell
Table 2
Biochemical inhibition of PI3K isoforms and mTOR
Compd
mTOR
PI3K
a
PI3Kb
PI3K
c
PI3Kd
IC50 (nM)
7j
7k
9.9
5.4
0.3
5.3
5.5
2.0
1.1
6.8
23
4.0
2.7
0.6
63
19
14
2.5
13
9.4
1.2
11
GSK2126458
PI-103
Table 3
In vitro liver microsomal stability of potent compounds
Figure 6. In vivo efficacy of 7 k against BEL-7404 hepatic cancer xenografts. BALB/C
nude male mice bearing BEL-7404 tumors were dosed orally with 7k at the
indicated concentration 30 mg/kg for 14 days or with sorafenib at 50 mg/kg. Data
represent the mean SE (for vehicle group n = 8, for treatment groups n = 6).
Compd
7b
7d
7i
7j
46.45
7k
t1/2 (min)
18.79
28.44
36.85
28.96
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Y. Chen et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
approved drug sorafenib for HCC treatment was used as a positive
control.
4.2. Synthesis
In the xenograft models, treatment with 30 mg/kg of compound
7k resulted in statistically significant antitumor activity, with a
mean reduction in relative tumor volume ratio of 52%. Sorafenib
displayed an inhibition ratio of 44% at 50 mg/kg. Compound 7k
was well tolerated at 30 mg/kg, with no observed mortality or sig-
nificant reduction of body weight. The data indicate that antitumor
efficacy of compound 7k can be achieved in this xenograft model. It
is noted that the molecular weight of 7k is much larger than
500 Daltons, which violates the Linpski’s Rule of Five. It might indi-
cate that the compound does not display good pharmacokinetic
profile by oral administration.
4.2.1. General procedure for the synthesis of N-(5-bromo-2-
methoxypyridin-3-yl)benzenesulfonamides 5a–b
To a solution of 3 (10 g, 49.25 mmol) in pyridine (50 mL) was
added benzenesulfonyl chloride 4 (64.03 mmol) slowly under ice
bath. After adding 4, the mixture was stirred for 18 h at room tem-
perature. The resulting solution was quenched with iced water
(500 mL), and stirred at room temperature for 1 h. The mixture
was filtered, and the precipitate was washed with water and
diethyl ether, and dried overnight to afford 5a–b.
4.2.1.1. N-(5-Bromo-2-methoxypyridin-3-yl)-4-fluorobenzene-
sulfonamide (5a). The compound 5a was obtained according
to general procedure using 4-fluorobenzene-1-sulfonyl chloride
4a. Yield: 60%; Light yellow powder; 1H NMR (300 MHz, CDCl3) d
7.90 (d, J = 2.0 Hz, 1H), 7.84–7.81 (m, 2H), 7.20–7.13 (m, 2H),
6.97–6.87 (m, 1H), 3.81 (d, J = 8.5 Hz, 3H). MS (ESI, positive ion)
m/z: 361 (M+H+).
3. Conclusions
In summary, a series of potent and efficacious dual PI3K and
mTOR inhibitors were discovered based on
a scaffold of
6-quinolinyl-3-pyridinyl-benzenesulfonamide. The pyridazine at
the 4-position of quinoline was replaced by propynyl-substituted
sidechains to afford a series of analogs, which accessed the ribose
pocket and thus increased affinities to target enzyme. Within the
series of propynyl derivatives, compound 7k possessed excellent
activities against PI3K isoforms and mTOR. This compound also
demonstrated potent activities against several cancer cell lines.
Compound 7k was found to be efficacious in BEL-7404 xenograft
models at 30 mg/kg administered orally. Western blot of
compound 7k showed that AKT phosphorylation was significantly
inhibited in A549 cells. It also possessed a reasonable half-life of
hepatic microsomal stability in vitro. Moreover, compound 7k
showed antitumor activity in human BEL-7404 HCC xenograft
model. This data suggest that compound 7k acts as a novel effica-
cious dual PI3K/mTOR inhibitor and may be a potential therapeutic
drug candidate for HCC.
4.2.1.2. N-(5-Bromo-2-methoxypyridin-3-yl)-2,4-difluoroben-
zenesulfonamide (5b). The compound 5b was obtained accord-
ing to general procedure using 2,4-fluorobenzene-1-sulfonyl
chloride 4b. Yield: 70%; Light yellow powder; 1H NMR (300 MHz,
CDCl3) d 7.96–7.86 (m, 2H), 7.83 (d, J = 2.2 Hz, 1H), 7.21 (s, 1H),
7.04–6.91 (m, 2H), 3.91 (s, 3H). MS (ESI, positive ion) m/z: 379.17
(M+H+, 79Br), 381.45 (M+H+, 81Br).
4.2.2. General procedure for the synthesis of 6a–b
A mixture of 6-bromo-4-chloroquinoline (2.00 g, 8.25 mmol),
bis(pinacolato)diboron (2.11 g, 8.25 mmol), Pd(dppf)Cl2-CH2Cl2
(0.34 g, 0.40 mmol) and potassium acetate (1.62 g, 16.50 mmol)
in anhydrous 1,4-dioxane (30 mL) was heated at 100 °C under
argon for 3 h. The resulting mixture was added corresponding 5
(8.25 mmol), 2.0 M aqueous Na2CO3 (10 mL) and another portion
of Pd(dppf)Cl2-CH2Cl2 (0.34 g, 0.40 mmol), then the mixture was
heated at 110 °C for 20 h under argon. The reaction mixture was
cooled to room temperature, filtered, and concentrated. The mix-
ture was purified by MPLC (ISCO CombiFlash purification system)
(MeOH/DCM, eluted from 0% to 8%). The fractions were collected,
concentrated to afford 6a–b.
4. Experimental section
4.1. Materials and measurements
All solvents and reagents were analytically pure and used as
purchased without further purification unless specified otherwise.
The progress of reactions was monitored on pre-coated plates
HSGF-254 using thin layer chromatography analysis (Qingdao
Haiyang Chemicals Co., Ltd, China). Melting points were measured
on a RK-Z melting point apparatus and were uncorrected. Chemical
shifts were given in parts per million (tetramethylsilane, internal
standard). Bruker Avance 300 MHz or 600 MHz instrument was
used for routine 1H NMR and 13C NMR spectra with CDCl3 or
DMSO-d6 as solvents (Bruker Company, Germany). Agilent liquid
chromatography–mass spectroscopy (LC–MS) mass spectrometer
was used to record mass spectra (MS) data using the ESI method
in positive mode. Low-resolution MS data were recorded at the
time of purity determination. High resolution mass spectrometry
(HRMS) analysis was performed on an Agilent Q-TOF system.
Agilent 1100 series HPLC was used to determine the purity of each
compound (purity P95%). Purity of the final compounds was
analyzed at 25 C using an Agilent 1100 HPLC system with a
detector (SPD-M20A, Shimadzu), an ACCHROM Unitary C18
4.2.2.1. N-(5-(4-Chloroquinolin-6-yl)-2-methoxypyridin-3-yl)-4-
fluorobenzenesulfonamide (6a). The compound 6a was
obtained according to general procedure using N-(5-bromo-2-
methoxypyridin-3-yl)-4-fluorobenzenesulfonamide 5a. Yield:
35%; White solid; 1H NMR (300 MHz, CDCl3) d 8.81 (d, J = 4.2 Hz,
1H), 8.30 (s, 1H), 8.23 (d, J = 8.7 Hz, 2H), 8.13 (d, J = 1.9 Hz, 1H),
7.94 (d, J = 7.6 Hz, 1H), 7.91–7.85 (m, 2H), 7.56 (d, J = 4.2 Hz, 1H),
7.16 (t, J = 8.5 Hz, 2H), 7.07 (s, 1H), 3.91 (s, 3H). 13C NMR
(300 MHz, CDCl3)
d 167.17, 163.78, 154.35, 150.03, 148.44,
142.78, 140.80, 136.52, 134.97, 130.77, 130.12, 129.99, 129.89,
129.51, 126.88, 121.90, 121.63, 121.06, 116.63, 116.33, 54.15. MS
(ESI, positive ion) m/z: 444.37 (M+H+).
4.2.2.2. N-(5-(4-Chloroquinolin-6-yl)-2-methoxypyridin-3-yl)-
2,4-difluorobenzenesulfonamide (6b). The compound 6b was
obtained according to general procedure using N-(5-bromo-2-
methoxypyridin-3-yl)-2,4-difluorobenzenesulfonamide 5b. Yield:
30%; off-white solid; mp: 171–173 °C. 1H NMR (300 MHz, CDCl3)
(4.6 ꢀ 250 mm)
column,
and
a
Waters
HILIC
C18
(100 mm ꢀ 3.0 mm, 3.0
l
m) column; Method A: solvent A, CH3OH,
d
8.82 (d, J = 4.8 Hz, 1H), 8.28 (d, J = 2.0 Hz, 1H), 8.24
solvent B, H2O, A/B = 80:20; or Method B: solvent A, acetonitrile,
(d, J = 2.2 Hz, 1H), 8.09 (d, J = 2.2 Hz, 1H), 7.93 (m, 2H), 7.58 (d,
J = 4.8 Hz, 1H), 7.32 (s, 1H), 6.98 (m, 2H), 4.00 (s, 3H). MS (ESI,
positive ion) m/z: 462.56 (M+H+).
solvent B, 0.1% Methanoic acid in H2O, A/B = 80:20; flow rate
1.0 mL/min; injection volume 5
254, 280 nm.
lL; monitored at wavelengths of
Please cite this article in press as: Chen, Y.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.01.008

Y. Chen et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
7
4.2.3. General procedure for the synthesis of 7a–j
A mixture of 6 (0.65 mmol), ( )-2,20-bis(diphenylphosphino)-
1,10-binaphthyl
(41 mg, 0.065 mmol), Pd(OAc)2 (8 mg,
(151 MHz, DMSO)
d
158.01, 150.52, 147.28, 136.83, 131.96,
131.88, 130.88, 129.33, 129.14, 128.88, 127.80, 124.81, 122.28,
111.69, 111.56, 106.00, 105.83, 105.65, 96.93, 81.00, 67.73, 59.53,
58.29, 53.61, 46.92, 42.56, 20.46. HRMS (ESI): [M+H]+: 539.3100.
HPLC: (Method B) tR = 5.61 min.
0.0325 mmol), K2CO3 (0.18 g, 1.30 mmol) and corresponding
propargyl amino compound (0.78 mmol) in anhydrous DMF
(10 mL) was heated at 100 °C under argon for 16 h. The reaction
mixture was cooled to room temperature, filtered, and quenched
with water (100 mL). The mixture was extracted with EtOAc
(4 ꢀ 50 mL), the organic layer was combined and washed with
brine (15 mL). The organic layer was dried over anhydrous Na2SO4,
filtered, and concentrated. The resulting mixture was purified by
MPLC (ISCO CombiFlash purification system) (MeOH/DCM, eluted
from 0% to 10%). The fractions were collected, concentrated to
afford 7a–7k.
4.2.3.5. N-(5-(4-(3-(Dicyclohexylamino)prop-1-yn-1-yl)quinolin-
6-yl)-2-methoxypyridin-3-yl)-2,4-difluorobenzenesulfonamide
(7e). The compound 7e was obtained according to general proce-
dure using 6b and N-cyclohexyl-N-(prop-2-yn-1-yl)cyclohex-
anamine. Yellow solid; mp > 226 °C. 1H NMR (600 MHz, CDCl3) d
8.86 (d, J = 4.1 Hz, 1H), 8.35 (d, J = 1.0 Hz, 1H), 8.23 (d, J = 2.3 Hz,
1H), 8.17 (d, J = 8.5 Hz, 1H), 8.10 (d, J = 2.3 Hz, 1H), 7.92–7.80 (m,
2H), 7.48 (d, J = 4.1 Hz, 1H), 7.05–6.77 (m, 2H), 3. 96 (s, 3H), 3.89
(s, 2H), 2.88 (d, J = 6.3 Hz, 2H), 1.94 (d, J = 10.9 Hz, 4H), 1.76 (d,
J = 11.0 Hz, 4H), 1.63–1.48 (m, 2H), 1.42–1.35 (m, 4H), 1.30–1.23
(m, 4H), 1.17–1.08 (m, 2H). 13C NMR (151 MHz, CDCl3) d 154.48,
149.97, 147.49, 141.12, 135.79, 132.52, 130.63, 130.48, 130.29,
128.86, 128.24, 127.57, 124.24, 123.72, 120.39, 111.91, 111.76,
105.85, 105.68, 105.51, 93.69, 79.36, 57.78, 54.07, 35.99, 31.27,
26.17, 26.06. HRMS (ESI): [M+H]+: 645.2724. HPLC: (Method A)
tR = 10.15 min.
4.2.3.1.
N-(5-(4-(3-(Dimethylamino)prop-1-yn-1-yl)quinolin-
6-yl)-2-methoxypyridin-3-yl)-4-fluorobenzenesulfonamide
(7a). The compound 7a was obtained according to general proce-
dure using 6a and N,N-dimethylprop-2-yn-1-amine. White solid;
mp: 158–162 °C. 1H NMR (300 MHz, CDCl3) d 8.88 (d, J = 4.4 Hz,
1H), 8.41 (d, J = 1.6 Hz, 1H), 8.26 (d, J = 2.0 Hz, 1H), 8.22–8.11 (m,
2H), 7.90 (dd, J = 8.7, 1.8 Hz, 1H), 7.84 (dd, J = 8.7, 5.0 Hz, 2H),
7.54 (d, J = 4.4 Hz, 1H), 7.14 (m, 2H), 3.89 (s, 3H), 3.71 (s, 2H),
2.49 (s, 6H). 13C NMR (151 MHz, CDCl3) d 167.13, 163.73, 154.40,
150.01, 147.57, 140.88, 135.79, 135.12, 130.81, 130.19, 130.00,
129.87, 128.83, 128.11, 127.43, 124.59, 123.47, 120.99, 116.56,
116.26, 95.21, 81.21, 54.10, 48.81, 44.40. HRMS (ESI): [M+H]+:
491.1555. HPLC: (Method A) tR = 7.12 min.
4.2.3.6. 2,4-Difluoro-N-(2-methoxy-5-(4-(3-(piperidin-1-yl)-
prop-1-yn-1-yl)quinolin-6-yl)pyridin-3-yl)benzenesulfonamide
(7f). The compound 7f was obtained according to general proce-
dure using 6b and 1-(prop-2-yn-1-yl)piperidine. White solid; mp:
200–204 °C. 1H NMR (300 MHz, CDCl3) d 8.87 (d, J = 4.5 Hz, 1H),
8.39 (d, J = 2.0 Hz, 1H), 8.25 (d, J = 2.2 Hz, 1H), 8.18 (d, J = 8.7 Hz,
1H), 8.12 (d, J = 2.2 Hz, 1H), 7.93–7.82 (m, 2H), 7.53 (d, J = 4.5 Hz,
1H), 7.01–6.89 (m, 2H), 3.96 (s, 3H), 3.72 (s, 2H), 2.77–2.61 (m,
4H), 1.74–1.65 (m, 4H), 1.53–1.42 (m, 2H). 13C NMR (151 MHz,
CDCl3) d 154.53, 149.97, 147.51, 141.02, 135.72, 132.52, 130.71,
130.14, 129.95, 128.74, 128.12, 127.54, 124.44, 123.58, 120.48,
111.97, 111.83, 105.87, 105.70, 105.54, 95.82, 80.95, 54.09, 53.59,
48.67, 25.89, 23.82. HRMS (ESI): [M+H]+: 549.1772. HPLC: (Method
A) tR = 7.72 min.
4.2.3.2. N-(5-(4-(3-(Dimethylamino)prop-1-yn-1-yl)quinolin-6-
yl)-2-methoxypyridin-3-yl)-2,4-difluorobenzenesulfonamide
(7b). The compound 7b was obtained according to general pro-
cedure using 6b and N,N-dimethylprop-2-yn-1-amine. White
solid; mp: 153–161 °C. 1H NMR (300 MHz, CDCl3) d 8.89 (d,
J = 4.5 Hz, 1H), 8.40 (d, J = 2.0 Hz, 1H), 8.26 (d, J = 2.2 Hz, 1H),
8.20 (d, J = 8.7 Hz, 1H), 8.14 (d, J = 2.2 Hz, 1H), 7.97–7.84 (m, 2H),
7.56 (d, J = 4.5 Hz, 1H), 7.02–6.91 (m, 2H), 3.99 (s, 3H), 3.73 (s,
2H), 2.51 (s, 6H). 13C NMR (151 MHz, CDCl3) d 154.43, 149.98,
147.54, 140.92, 135.79, 132.56, 130.77, 130.13, 129.81, 128.82,
128.08, 127.29, 124.56, 123.45, 120.55, 112.10, 111.75, 106.08,
105.75, 105.40, 95.16, 81.21, 54.15, 48.81, 44.39. HRMS (ESI): [M
+H]+: 509.1460. HPLC: (Method B) tR = 5.69 min.
4.2.3.7. 2,4-Difluoro-N-(2-methoxy-5-(4-(3-(4-methylpiperidin-
1-yl)prop-1-yn-1-yl)quinolin-6-yl)pyridin-3-yl)benzenesulfon-
amide (7g). The compound 7g was obtained according to
general procedure using 6b and 4-methyl-1-(prop-2-yn-1-yl)
piperidine. White solid; mp: 186–188 °C. 1H NMR (300 MHz,
CDCl3) d 8.87 (d, J = 4.5 Hz, 1H), 8.39 (d, J = 2.0 Hz, 1H), 8.25 (d,
J = 2.2 Hz, 1H), 8.18 (d, J = 8.7 Hz, 1H), 8.12 (d, J = 2.3 Hz, 1H),
7.94–7.82 (m, 2H), 7.53 (d, J = 4.5 Hz, 1H), 7.01–6.89 (m, 2H),
3.97 (s, 3H), 3.75 (s, 2H), 3.05 (d, J = 11.2 Hz, 2H), 2.42 (t,
J = 10.6 Hz, 2H), 1.74 (d, J = 9.5 Hz, 2H), 1.36 (m, 3H), 0.95 (d,
J = 5.7 Hz, 3H). 13C NMR (151 MHz, CDCl3) d 154.58, 149.95,
147.49, 141.06, 135.70, 132.52, 130.69, 130.12, 129.95, 128.73,
128.11, 127.64, 124.38, 123.57, 120.47, 111.95, 111.81, 105.86,
105.69, 105.52, 95.88, 80.96, 54.06, 53.03, 48.30, 34.21, 30.27,
21.80. HRMS (ESI): [M+H]+: 563.1931. HPLC: (Method A)
tR = 8.91 min.
4.2.3.3.
N-(5-(4-(3-(Diethylamino)prop-1-yn-1-yl)quinolin-6-
yl)-2-methoxypyridin-3-yl)-2,4-difluorobenzenesulfonamide
(7c). The compound 7c was obtained according to general proce-
dure using 6b and N,N-diethylprop-2-yn-1-amine. White solid;
mp: 103–109 °C. 1H NMR (300 MHz, CDCl3) d 8.81 (d, J = 4.4 Hz,
1H), 8.32 (br s, 1H), 8.11 (d, J = 8.1 Hz, 2H), 7.96–7.69 (m, 3H),
7.47 (d, J = 4.4 Hz, 1H), 6.81 (br s, 2H), 3.91 (s, 3H), 3.82 (s, 2H),
2.69 (q, J = 7.1 Hz, 4H), 1.14 (t, J = 7.1 Hz, 6H). 13C NMR (151 MHz,
CDCl3) d 160.55, 158.93, 155.10, 149.66, 147.27, 136.24, 132.20,
132.14, 130.42, 130.20, 129.99, 128.88, 128.09, 124.36, 123.21,
111.57, 111.43, 105.60, 105.43, 105.25, 95.37, 80.87, 53.97, 47.57,
41.74, 12.65. HRMS (ESI): [M+H]+: 537.1780. HPLC: (Method A)
tR = 7.71 min.
4.2.3.8. 2,4-Difluoro-N-(2-methoxy-5-(4-(3-(2-methylpiperidin-
1-yl)prop-1-yn-1-yl)quinolin-6-yl)pyridin-3-yl)benzenesulfon-
amide (7h). The compound 7h was obtained according to
general procedure using 6b and 2-(methyl(prop-2-yn-1-yl)
amino)ethanol. White solid; mp: 192–196 °C. 1H NMR (300 MHz,
CDCl3) d 8.87 (d, J = 4.5 Hz, 1H), 8.41 (d, J = 2.0 Hz, 1H), 8.25 (d,
J = 2.3 Hz, 1H), 8.18 (d, J = 8.7 Hz, 1H), 8.12 (d, J = 2.3 Hz, 1H),
7.91–7.80 (m, 2H), 7.53 (d, J = 4.5 Hz, 1H), 7.00–6.89 (m, 2H),
4.11 (d, J = 17.8 Hz, 1H), 3.96 (s, 3H), 3.74 (d, J = 17.8 Hz, 1H),
2.98 (d, J = 11.9 Hz, 1H), 2.74–2.49 (m, 2H), 1.81–1.60 (m, 4H),
1.33 (dd, J = 20.6, 12.1 Hz, 2H), 1.22 (d, J = 6.2 Hz, 3H). 13C NMR
4.2.3.4.
2,4-Difluoro-N-(5-(4-(3-((2-hydroxyethyl)(methyl)-
amino)prop-1-yn-1-yl)quinolin-6-yl)-2-methoxypyridin-3-yl)
benzenesulfonamide (7d). The compound 7d was obtained
according to general procedure using 6b and N-cyclohexyl-N-
(prop-2-yn-1-yl)cyclohexanamine. White solid; mp: 102–108 °C.
1H NMR (300 MHz, CDCl3) d 8.85 (d, J = 4.0 Hz, 1H), 8.36 (s, 1H),
8.22–8.13 (m, 2H), 8.07 (s, 1H), 7.86 (d, J = 8.2 Hz, 2H), 7.50 (d,
J = 3.5 Hz, 1H), 6.91 (dd, J = 9.6, 4.2 Hz, 2H), 3.95 (s, 3H), 3.81 (s,
2H), 3.77–3.68 (m, 2H), 2.88–2.74 (m, 2H), 2.51 (s, 3H). 13C NMR
Please cite this article in press as: Chen, Y.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.01.008

8
Y. Chen et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
(151 MHz, CDCl3) d 154.56, 149.99, 147.52, 141.10, 135.77, 132.49,
130.74, 130.21, 130.02, 128.78, 128.16, 127.66, 124.42, 123.64,
120.45, 111.95, 111.80, 105.86, 105.69, 105.53, 95.20, 81.21,
55.26, 54.09, 53.87, 44.43, 34.68, 26.28, 24.56, 20.13. HRMS (ESI):
[M+H]+: 563.1934. HPLC: (Method A) tR = 9.72 min.
(pH = 7.4) for all the compounds. All the ligands conformation
energies were minimized with the modified parameters algorithm
(smart minimizer), max steps (2000), and rms gradient (0.05). The
binding site sphere radius was set at 10 Å.
4.4. Western blotting analysis
4.2.3.9. 2,4-Difluoro-N-(2-methoxy-5-(4-(3-morpholinoprop-1-yn-
1-yl)quinolin-6-yl)pyridin-3-yl)benzenesulfonamide (7i).
The
A549 human lung cancer cell bought from the Cell Bank of the
Chinese Academy of Sciences (Shanghai, China) was cultured in
recommended media supplemented with 10% FBS (Invitrogen) in
a humidified environment with 5% CO2. A549 cells were treated
compound 7i was obtained according to general procedure using
6b and 4-(prop-2-yn-1-yl)morpholine. White solid; mp: 202–
206 °C. 1H NMR (300 MHz, CDCl3) d 8.88 (d, J = 4.5 Hz, 1H), 8.37
(d, J = 1.9 Hz, 1H), 8.26 (d, J = 2.3 Hz, 1H), 8.19 (d, J = 8.8 Hz, 1H),
8.12 (d, J = 2.2 Hz, 1H), 7.93–7.82 (m, 2H), 7.53 (d, J = 4.5 Hz, 1H),
7.35 (s, 1H), 7.03–6.86 (m, 2H), 3.95 (s, 3H), 3.86 (t, J = 4.5 Hz,
4H), 3.76 (s, 2H), 2.78 (t, J = 4.9 Hz, 4H). 13C NMR (151 MHz, CDCl3)
d 154.71, 149.93, 147.50, 141.04, 135.75, 132.45, 130.76, 129.98,
129.60, 128.71, 128.05, 127.75, 124.49, 123.37, 120.53, 111.94,
111.77, 105.88, 105.71, 105.54, 94.70, 81.43, 66.82, 54.08, 52.45,
48.22. HRMS (ESI): [M+H]+: 551.1570. HPLC: (Method A)
tR = 7.30 min.
with various concentrations of compound 7k (0–1
lM) for
30 min, followed with 1 g/mL insulin treatment for 2 h (Gibco,
l
NY, USA). Cells were lysed in ice-cold radio immunoprecipitation
assay (RIPA) buffer (10–1 v/v) for 10 min before centrifuging at
4 °C for 5 min (10,000g). Total protein concentration of super-
natants was assayed using the bicinchoninic acid (BCA) method.
Proteins of samples were separated by 10% sodium dodecyl sul-
fate–polyacrylamide gel electrophoresis (SDS–PAGE) and trans-
ferred to nitrocellulose membranes. Membranes were incubated
with primary and secondary antibodies at the dilution and time
recommended by the manufacturers, and visualized by Odyssey
Infrared Imaging System (LI-COR Biosciences, Inc.). Beta-actin
was used as a loading control for all blots. The RIPA buffer, BCA
kit and SDS–PAGE kit were purchased from the Beyotime Institute
of Biotechnology (Shanghai, China). The anti-beta-actin (Catalog
No. 5779-1), anti-phospho-AKT (Ser473) (Catalog No. 2118-1),
anti-AKT (pan) (Catalog No. 1081-1), and anti-mTOR (Catalog No.
1612-1) antibodies (Epitomics, CA, USA) were used in this experi-
ment. IRDye 800CW secondary antibody (Lot No. C20906-02) was
purchased from LI-COR Biosciences (Lincoln, NE, USA).
4.2.3.10. Ethyl 4-(3-(6-(5-((2,4-difluorophenyl)sulfonamido)-6-
methoxypyridin-3-yl)quinolin-4-yl)prop-2-yn-1-yl)piperazine-
1-carboxylate (7j). The compound 7j was obtained according to
general procedure using 6b and ethyl 4-(prop-2-yn-1-yl)piper-
azine-1-carboxylate. White solid; mp: 142–148 °C. 1H NMR
(300 MHz, CDCl3) d 8.85 (d, J = 4.5 Hz, 1H), 8.30 (d, J = 1.4 Hz, 1H),
8.14 (d, J = 8.7 Hz, 1H), 8.08 (br s, 1H), 7.95 (br s, 1H), 7.86 (m,
2H), 7.50 (d, J = 4.5 Hz, 1H), 6.89–6.72 (m, 2H), 4.08 (q, J = 7.1 Hz,
2H), 3.92 (s, 3H), 3.73 (s, 2H), 3.57 (s, 4H), 2.68 (s, 4H), 1.19 (t,
J = 7.1 Hz, 3H). 13C NMR (151 MHz, CDCl3) d 157.03, 155.52,
155.22, 149.70, 147.37, 136.45, 132.10, 132.04, 130.52, 130.19,
129.49, 128.90, 128.02, 124.47, 123.21, 111.55, 111.40, 105.58,
105.42, 105.24, 94.52, 81.39, 61.53, 53.96, 51.93, 48.00, 43.56,
20.39, 14.56. HRMS (ESI): [M+H]+: 622.1937. HPLC: (Method B)
tR = 3.50 min.
4.5. In vitro PI3K enzyme assay
4.5.1. PI3K
a
, PI3Kd Kinase-Glo luminescent kinase assay
, PI3Kd were measured
IC50 values for inhibition of the PI3K
a
using a Kinase-Glo luminescent kinase assay with ATP concentra-
tion in vitro. PI-103 was used as the reference compound. The com-
pounds were tested in duplicate for 10 concentrations, 100 nM or
500 nM as the top concentration. All reagents were diluted in
kinase buffer (50 mM HEPES, pH 7.5, 3 mM MgCl2, 1 mM EGTA,
100 mM NaCl, 0.03% CHAPS, and 2 mM DTT). Three-fold, ten-point
serial compound dilutions were performed in kinase buffer. The
4.2.3.11.
2,4-Difluoro-N-(2-methoxy-5-(4-(3-(4-(methylsul-
fonyl)piperazin-1-yl)prop-1-yn-1-yl)quinolin-6-yl)pyridin-3-yl)
benzenesulfonamide (7k). The compound 7k was obtained
according to general procedure using 6b and 1-(methylsulfonyl)-
4-(prop-2-yn-1-yl)piperazine. Yellow powder; mp: 113–117 °C.
1H NMR (300 MHz, CDCl3) d 8.85 (d, J = 4.5 Hz, 1H), 8.30 (d,
J = 1.8 Hz, 1H), 8.19–8.10 (m, 2H), 8.00 (br s, 1H), 7.92–7.82 (m,
2H), 7.51 (d, J = 4.5 Hz, 1H), 6.92–6.86 (m, 2H), 3.94 (s, 3H), 3.77
(s, 2H), 3.35 (t, J = 4.7 Hz, 4H), 2.85 (t, J = 4.7, 4H), 2.80 (s, 3H).
13C NMR (151 MHz, CDCl3) d 155.21, 149.77, 147.36, 139.37,
136.19, 132.37, 132.23, 130.59, 129.97, 129.39, 128.84, 128.01,
126.82, 124.46, 123.17, 111.87, 111.53, 105.94, 105.60, 105.26,
94.22, 81.54, 54.11, 51.55, 47.72, 45.75, 34.48. HRMS (ESI): [M
+H]+: 628.1502. HPLC: (Method B) tR = 2.42 min.
final concentration of kinase solution: PI3K
5.7 nM. Kinase solution (2.5 L) was added to each well of the
assay plate, except for control wells (add 2.5
L of 1ꢀ kinase buffer
instead). The final concentration of substrate solution: PIP2 50 M,
ATP 25 M. Substrate solution (5 L) was added to each well of the
a 1.65 nM PI3Kd
l
l
l
l
l
assay plate, shake the plate. Cover the assay plate and incubate at
room temperature for 1 h. Kinase-Glo reagent was equilibrated to
room temperature. Then 10 ll of Kinase-Glo reagent was added
to each well of the assay plate. Mix briefly with centrifuge. Shake
slowly for 15 min before reading on a plate reader for lumines-
cence. Copy values of RLU from Flexstation program. Convert RLU
values to percent inhibition values. Graphpad 5.0 was used to
determine the way of fitted curves.
4.3. Molecular docking study
The Discovery Studio 3.0 software was used for computational
studies, and co-crystal structure of human PI3K
a was obtained
from the RCSB Protein Data Bank (PDB: 3ZIM). The protein and
ligands were prepared step by step with the Guide-Docking ligands
tool in Discovery Studio 3.0 software. We performed dockings with
the CDOCKER module, and analyzed results with other relevant
modules in the DS 3.0 Base.
Based on a scaffold of 6-quinolinyl-3-pyridinyl-benzenesulfon-
amide extracted from the crystal structure of GSK2126458, we
built a series of propynyl-substituted benzenesulfonamide deriva-
tives. The protonation state was set at physiological environment
Percent inhibition ¼ 100ꢁðsample RLUꢁminÞ=ðmaxꢁminÞ ꢂ 100
4.5.2. PI3Kb, PI3K
IC50 values of targeted compounds for inhibition of the PI3Kb,
PI3K were measured using an ADP-Glo Luminescent kinase Assay
c ADP-Glo luminescent kinase assay
c
with ATP concentration at Km in vitro and used PI-103 as the
reference compound. All reagents were diluted in kinase buffer
Please cite this article in press as: Chen, Y.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.01.008

Y. Chen et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
9
for testing kinase PI3Kb, PI3K
MgCl2, 1 mM EGTA, 100 mM NaCl, 0.03% CHAPS, 2 mM DTT). The
final concentration: PI3Kb 4.8 nM, PI3K 7.6 nM. ADP-Glo reagent
was used stop the reaction. Equilibrate for 40 min, 10 L Kinase
Detection Reagent was added to each wells, shake 1 min. Equili-
c
(50 mM HEPES, pH 7.5, 3 mM
4.8. Nude mouse xenograft in vivo efficacy
c
Compound 7k was evaluated in vivo antitumor activities; sora-
fenib was used as a reference drug. Four-week-old male BALB/c
nude mice (Certificate SCXK2007-0005, 18–20 g) were obtained
from Shanghai SLAC Laboratory Animal Co., Ltd (Shanghai, China).
The animals were maintained under specific pathogen-free condi-
tions in Shanghai Institute of Pharmaceutical Industry. BEL-7404
cancer cell line was obtained from Cell Bank of the Chinese Acad-
emy of Sciences (Shanghai, China). After implanted tumors of nude
mice had reached a volume of 100–300 mm3 (after 12 days), ani-
mals were randomized into groups (6 animals per treatment and
8 animals for the control group). Compound 7k and sorafenib were
formulated in 5% polysorbate 80 in water. Tumor-bearing mice
were dosed orally once daily for 14 consecutive days from day
13 after implantation of the cells. Tumor volumes and bodyweights
were monitored over the course of treatment. Tumor volumes
were calculated using the formula of TV = 1/2 ꢀ a ꢀ b ꢀ b, where
a is the tumor length and b is the width. Mice were sacrificed on
day 29 after cell implantation, and tumors were removed and
recorded for analysis. The study was approved by Shanghai Insti-
tute of Pharmaceutical Industry.
l
brating for 60 min before reading on
luminescence.
a plate reader for
Percent inhibition ¼ ðsample RLU ꢁ minÞ=ðmax ꢁ minÞ ꢂ 100:
4.5.3. mTOR lance ultra assay
All reagents were diluted in kinase buffer (50 mM HEPES, pH
7.5, 3 mM MgCl2, 1 mM EGTA, 3 mM MnCl, 0.01% Tween-20,
2 mM DTT) for testing IC50 of inhibition of kinase mTOR. Serial
dilution prepared in kinase buffer with a top concentration of
500 nM. The final concentration: mTOR 2.5 nM. Add 2.5 ll of
kinase solution to each well of the assay plate, shake the plate.
Preparation of 2ꢀ substrate solution of ULight-4E-BP1 peptide
and ATP in 1ꢀ kinase reaction buffer at 2-fold of the final concen-
tration was desired in the assay. The final concentration: ULight-
4E-BP1 peptide 50 nM, ATP 10.8 lM. Substrate solution (5 ll)
was added to each well of the assay plate to start reaction. Cover
the assay plate and incubate at room temperature for 1 h. Prepare
detection solution of kinase quench buffer (EDTA) and Eu-anti-
phospho-4E-BP1 antibody at 2-fold the final concentrations in
Lance detection buffer. The final concentration: EDTA 8 mM,
Eu-anti-phospho-4E-BP1 antibody 2 nM. Detection solution
Acknowledgments
The authors thank Anna Sromek and John Neumeyer at McLean
Hospital, Harvard Medical School for the help during the prepara-
tion of this manuscript. We also wish to acknowledge Qilin Wang
at Shanghai ChemPartner Co., Ltd for her support on biochemical
assays, Juan Wang and Xiuhua Chen at Shanghai Institute of Phar-
maceutical Industry on cellular and xenograft model, Hai Zhang on
liver microsomal stability and Yi Zhang for support with western
blotting. This work was supported by Shanghai Pujiang Talent Pro-
gram (No. 09PJ1400300). This research work was financially sup-
ported by the Natural Science Foundation of China (No. 81172928).
(10 ll) was added to each well of the assay plate. Mix briefly with
Centrifuge. Cover the assay plate and equilibrate for 60 min at
room temperature. Collect data on Envision. Copy values of Lance
signal (665 nm) from Envision program, and convert Lance signal
(665 nm) values to percent inhibition values. IC50 values were
determined by way of curves fitted by Graphpad 5.0.
Percent inhibition ¼ ðLance signal ꢁ minÞ=ðmax ꢁ minÞ ꢂ 100:
4.6. In vitro cytotoxic assays
Supplementary data
Cytotoxic effects were tested in the human lung adenocarci-
noma cells A549, human colon cancer cells HCT-116, human breast
cancer cells MDA-MB-231 and human hepatocellular carcinoma
cells BEL-7404. These four tumor cells were diluted to a density
of 40,000–50,000 cells/mL in logarithmic phase. The culture med-
ium contained Dulbecco modified eagle medium (DMEM) supple-
mented with 10% fetal bovine serum (FBS) and also supplement
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2016.01.008.
These data include MOL files and InChiKeys of the most important
compounds described in this article.
References and notes
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