Discovery of novel 2-piperidinol-3-(arylsulfonyl)quinoxalines as phosphoinositide 3-kinase α (PI3Kα) inhibitors

Article abstract of DOI:10.1016/j.bmc.2012.03.026

A series of novel 2-aliphatic cyclic amine-3-(arylsulfonyl)quinoxalines was synthesized based on the structural features of a previously identified lead, WR1. The 2-piperidinol-3-(arylsulfonyl)quinoxalines, which showed excellent antitumor activities against five human cell lines, with inhibitory activities ranging from 0.34 to 2.32 μM, proved to be a promising class of novel PI3Kα inhibitors. The most potent compound 10d (WR23) showed an inhibitory IC₅₀ value of 0.025 μM against PI3Kα and significant pAkt suppression effect. Molecular docking analysis was performed to determine possible binding modes between PI3Kα and target compounds.

Full text of DOI:10.1016/j.bmc.2012.03.026

Bioorganic & Medicinal Chemistry 20 (2012) 2837–2844
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of novel 2-piperidinol-3-(arylsulfonyl)quinoxalines
as phosphoinositide 3-kinase
a (PI3Ka) inhibitors
Peng Wu a, Yi Su b, Xiaowen Liu b, Bo Yang b, Qiaojun He b, Yongzhou Hu a,
⇑
a ZJU-ENS Joint laboratory of Medicinal Chemistry, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, People’s Republic of China
b Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel 2-aliphatic cyclic amine-3-(arylsulfonyl)quinoxalines was synthesized based on the
structural features of a previously identified lead, WR1. The 2-piperidinol-3-(arylsulfonyl)quinoxalines,
which showed excellent antitumor activities against five human cell lines, with inhibitory activities rang-
Received 6 February 2012
Revised 10 March 2012
Accepted 12 March 2012
Available online 17 March 2012
ing from 0.34 to 2.32
pound 10d (WR23) showed an inhibitory IC50 value of 0.025
suppression effect. Molecular docking analysis was performed to determine possible binding modes
between PI3K and target compounds.
lM, proved to be a promising class of novel PI3K
a
inhibitors. The most potent com-
l
M against PI3K
a
and significant pAkt
Keywords:
a
2-Piperidinol-3-(arylsulfonyl)quinoxaline
Ó 2012 Elsevier Ltd. All rights reserved.
PI3K
a
pAkt
Antitumor activity
1. Introduction
substituents including pyrrolidine, 1,2,3,4-tetrahydroisoquinoline,
piperidin-3-ol, and piperidin-4-ol were introduced at the 2-posi-
tion of the quinoxaline ring in this study to investigate correspond-
ing impacts on potency. Molecular parameter based virtual
As a family of lipid kinases, phosphoinositide 3-kinases (PI3Ks)
are critical regulators in a wide range of cellular activities such as
growth, transformation, proliferation, motility and differentia-
tion.1,2 PI3Ks are classified into I, II, and III based on sequence
analysis of druglikeness was conducted, and the most potent PI3K
a
inhibitor, 2-(piperidin-4-ol)-3-(4-bromophenylsulfonyl)quinoxa-
line 10d (WR23) was tested for its suppression against levels of
phosphorylated Akt (pAkt). Besides, molecular docking analysis
was performed to determine possible binding modes between
homology and substrate preferences.3 Among class I PI3Ks, PI3K
a
has been well established as a promising target for cancer therapy
due to the prevalent mutation and amplification of PIK3CA, the
gene encoding the catalytic subunit of PI3K
a, in a diverse set of
PI3Ka and target compounds.
human cancers, and the fact that PI3K signaling pathway is one
of the most commonly mutated pathways contributing to tumori-
genesis and progression.4–8
2. Chemistry
We have previously reported 2-(3,5-dimethoxyphenylamino)-
Based on the reported approach for the synthesis of arylsulfonyl-
quinoxalines,9 compounds 7a–e with a pyrrolidine group, 8a–e
with a tetrahydroisoquinoline group, 9a–e with a piperidin-3-ol
3-(phenylsulfonyl)quinoxaline 1 (WQ2) as a potent PI3K
a inhibi-
tor,9 while unfavorable pharmacokinetic properties such as poor
solubility discouraged its further progression. In screening our
chemical library using a pharmacophore-based approach, 2-mor-
pholino-3-phenylsulfonylquinoxaline 2 (WR1) was identified as a
promising lead because it shares the morpholinoaryl motif with
3 (LY294002), an extensively studied PI3K inhibitor with an IC50
value of 0.63
of a series of novel 2-piperidinol-3-(arylsulfonyl)quinoxalines as
PI3K inhibitors as a continuation of our work in developing
l
M (Fig. 1).10–12 Herein, we reported the discovery
a
PI3K inhibitors for cancer therapy. Different aliphatic cyclic amine
⇑
Corresponding author. Tel./fax: +86 571 8820 8460.
E-mail address: huyz@zju.edu.cn (Y. Hu).
Figure 1. Structures of quinoxaline PI3K inhibitors and LY294002.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.03.026

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P. Wu et al. / Bioorg. Med. Chem. 20 (2012) 2837–2844
Scheme 1. The synthetic route for target compounds 7–10. Reagents and conditions: (a) hydrazine hydrate, H2O, 0 °C to rt; (b) ethanol, reflux; (c) iso-propanol, microwave
irradiation, 80 °C, pyrrolidine (for 7a–e), 1,2,3,4-tetrahydroisoquinoline (for 8a–e), 3-hydroxypiperidine (for 9a–e), or 4-hydroxypiperidine (for 10a–e).
Table 1
In vitro cytotoxicity of 2-aliphatic cyclic amine-3-(arylsulfonyl)quinoxalines against five cancer cell lines
OH
N
N
N
S
N
N
N
S
N
N
N
S
N
N
N
S
OH
O
O
O
O
O
O
O
O
R
R
R
9a-e
R
10a-e
7a-e
8a-e
a
Index
Compound
R
Cytotoxicity IC50
HCT116
(lM)
PC3
A549
HL60
KB
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
2
—
H
18.88
4.98
9.52
9.73
2.02
2.01
2.10
5.33
3.41
3.69
2.13
5.01
22.39
>50
12.55
2.06
4.02
5.11
0.95
20.23
0.31
1.35
2.18
2.10
8.17
19.83
>50
>50
>50
22.85
34.82
>50
>50
>50
31.06
89.65
5.35
1.87
5.44
4.69
1.65
1.34
1.39
3.27
3.52
2.11
5.50
18.73
38.95
22.13
>50
4.47
2.90
4.05
3.23
0.34
1.45
3.02
6.08
3.05
3.43
2.62
0.88
3.61
3.69
2.61
4.08
3.46
9.72
16.03
6.07
12.78
9.94
NTb
10a
10b
10c
10d
10e
9a
9b
9c
9d
9e
8a
8b
8c
8d
8e
7a
7b
7c
5.36
6.14
8.06
2.32
9.58
3.59
6.08
2.12
3.72
18.30
26.26
32.06
>50
Methyl
Methoxy
Bromo
Fluoro
H
Methyl
Methoxy
Bromo
Fluoro
H
Methyl
Methoxy
Bromo
Fluoro
H
Methyl
Methoxy
Bromo
Fluoro
—
>50
>50
9.23
20.81
>50
>50
>50
16.60
24.17
>50
35.36
35.75
>50
>50
>50
>50
7d
7e
LY294002
37.68
20.56
56.01
12.42
61.35
30.06
44.76
a
IC50, the mean value of at least two separate determinations.
NT, not tested.
b
group, and 10a–e with a piperidin-4-ol group were prepared using
the synthetic route as shown in Scheme 1. Treatment of arylsulfonyl
chloride (4a–e) with 85% hydrazine hydrate solution gave
phenylsulfonylhydrazines (5a–e), whose reaction with 2,3-dic-
holoroquinoxaline in refluxing ethanol yielded 2-chloro-3-(aryl-
sulfonyl)quinoxalines (6a–e). Reaction of 6a–e with pyrrolidine,
1,2,3,4-tetrahydroisoquinoline, 3-hydroxypiperidine, and 4-hydro-
xypiperidine in iso-propanol under microwave irradiation afforded
compounds 7a–e, 8a–e, 9a–e, and 10a–e, respectively.
3. Results and discussion
3.1. Cytotoxicity
The synthesized 2-aliphatic cyclic amine-3-(arylsulfonyl)quin-
oxalines 7–10 were tested for their cytotoxicities in vitro against
five human cancer cell lines including human prostate cancer cell
(PC3), human lung adenocarcinoma epithelial cell (A549), human
colon cancer cell (HCT116), human promyelocytic leukemia cell

P. Wu et al. / Bioorg. Med. Chem. 20 (2012) 2837–2844
2839
Table 2
Molecular parameters of 2-aliphatic cyclic amine-3-(arylsulfonyl)quinoxalines
N
N
R2
O
S
O
R1
Compound
R1
R2
Molecular parameters
HBAb
0
MW
ClogPa
HBDc
tPSA (ÅA2)a
1
2
H
H
H
3,5-Dimethoxy-phenylamine
Morpholine
Pyrrolidine
Pyrrolidine
Pyrrolidine
421
355
339
384
400
448
388
402
416
462
480
419
369
383
399
448
387
369
383
399
448
387
5.73
3.19
4.02
4.52
4.03
4.90
4.18
5.24
5.74
5.25
6.12
5.40
3.24
3.74
3.26
4.12
3.40
2.49
2.99
2.50
3.37
2.65
7
6
5
5
6
5
5
5
5
6
5
5
6
6
7
6
6
6
6
7
6
6
1
0
0
0
0
0
0
0
0
0
0
0
1
1
1
1
1
1
1
1
1
1
89.35
71.33
62.1
62.1
71.33
62.1
62.1
62.1
62.1
71.33
62.1
7a
7b
7c
7d
7e
8a
8b
8c
8d
8e
9a
9b
9c
9d
9e
10a
10b
10c
10d
10e
Methyl
Methoxy
Bromo
Fluoro
H
Methyl
Methoxy
Bromo
Fluoro
H
Methyl
Methoxy
Bromo
Fluoro
H
Methyl
Methoxy
Bromo
Fluoro
Pyrrolidine
Pyrrolidine
1,2,3,4-Tetrahydro-isoquinoline
1,2,3,4-Tetrahydro-isoquinoline
1,2,3,4-Tetrahydro-isoquinoline
1,2,3,4-Tetrahydro-isoquinoline
1,2,3,4-Tetrahydro-isoquinoline
3-Hydroxypiperidine
3-Hydroxypiperidine
3-Hydroxypiperidine
3-Hydroxypiperidine
3-Hydroxypiperidine
4-Hydroxypiperidine
4-Hydroxypiperidine
4-Hydroxypiperidine
4-Hydroxypiperidine
4-Hydroxypiperidine
62.1
82.33
82.33
91.56
82.33
82.33
82.33
82.33
91.56
82.33
82.33
a
b
c
Calculated by ChemBioDraw Ultra 12.0.
Counted as the sum of Ns and Os.
Counted as the sum of NHs and OHs.
(HL60), and human nasopharyngeal carcinoma cell (KB). LY294002
and compound 2 that share the morpholinoaryl scaffold were
used as the positive controls.10,13 The results are summarized in
Table 1.
As shown in Table 1, replacing the 2-morpholine group of com-
pound 2 with pyrrolidine (7a–e) or tetrahydroisoquinoline (8a–e)
resulted in decreased cytotoxicities against most cell lines in com-
parison with that of 2, for example, 7a with a 3-pyrrolidine group,
Table 3
Inhibition of PI3K
a by 2-aliphatic cyclic amine-3-(arylsulfonyl)quinoxalines
N
N
R2
O
S
O
R1
8a with
a
3-tetrahydroisoquinoline group, and compound
2
showed IC50 values of 24.17, 18.73 and 5.35
l
M against HCT116,
a
Compound
R1
R2
PI3K
0.44
21.83
>50
0.079
0.025
0.63
a IC50 (lM)
respectively. While the presence of either a 3-hydroxypiperidine
(compounds 9a–e) or a 4-hydroxypiperidine (compounds 10a–e)
at the 2-position of the quinoxaline scaffold led to significant in-
crease in cytotoxicities in low micromolar level against the five
tested human cancer cell lines, which are much better than that
2
7a
8a
10a
H
H
H
H
Morpholine
Pyrrolidine
Tetrahydroisoquinoline
4-Hydroxypiperidine
4-Hydroxypiperidine
10d
Bromo
—
LY294002b
of compound
2 and LY294002, especially against the PI3K-
a
activated PC3 cell lines. For example, 9a with a 3-hydroxypiperi-
dine and 10a with a 4-hydroxypiperidine on the 3-position of the
IC50, the mean value of at least two separate determinations.
Report value, Ref. 13
b
quinoxaline scaffold showed IC50 values of 2.10 and 4.98
lM
against PC3, respectively, and the corresponding IC50 values for
compound 2 and LY294002 were 18.88 and 61.35 lM, respectively.
identified quinoxaline PI3K inhibitor 1. ClogP and tPSA values
were calculated by using ChemDrawUltra 12.0.
3.2. Druglikeness analysis
As summarized in Table 2, compounds with a 3-hydroxypiper-
idine (9a–e) or a 4-hydroxypiperidine (10a–e) showed favorable
values for the involved molecular parameters that fit well with
the Lipinski’s Rule of Five.14 It is noteworthy to mention that the
presence of the piperidinol group significantly reduced ClogP val-
ues compared with that of compound 2 with a 3,5-dimethoxylphe-
nylamine, that is, 2 had a ClogP value of 5.73, while 9a with a
3-hydroxypiperidine and 10a with a 4-hydroxypiperidine had
ClogP values of 3.24 and 2.49, respectively.
Analysis of molecular parameters that connected with druglike-
ness, including molecular weight (MW), calculated partition coeffi-
cient for n-octanol/water (ClogP), number of hydrogen bond
acceptor (HBA) and hydrogen bond donor (HBD), and total polar
surface area (tPSA),14,15 was conducted to indicate whether the
potent piperidinol derivatives bear improved solubility and over-
all pharmacokinetic property as compared with the previously

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P. Wu et al. / Bioorg. Med. Chem. 20 (2012) 2837–2844
second interaction with Lys802 in the affinity pocket (length:
1.8 Å); compound 7a retains the hydrogen bond interaction involv-
ing Lys802 (length: 2.3 Å) while fails to form hydrogen bond with
hinge Val 851 (Fig. 3E); compound 10d forms two hydrogen bond
interactions with Val 851 (length: 2.0 Å) and Ser854 (length:
3.1 Å), respectively, through its hydroxy group on the piperidine
ring (Fig. 3G). The lack of the hydrogen bond interaction with
Val851, which is believed to be of crucial importance for PI3K inhi-
bition,5,11,21 might partly explain the poor activity of 7a. Compari-
son of the surface shows of the binding modes between PI3Ka and
LY294002, 7a, or 10d (Fig. 3D, F, and H, respectively) revealed that
10d has a better fit into the ATP binding site than that of LY294002
and 7a, especially in the ribose binding pocket which accommo-
dates the 4-bromophenylsulfonyl moiety of 10d. Binding-mode
study of compounds with similar arylsulfonylquinoxaline scaffold
revealed that the sulfonyl oxygen might be potential hydrogen-
bond acceptor to interact with residues of PI3Ks.9 Overall, the
molecular docking analysis suggested the potential of potent inhi-
Figure 2. pAktSer473 inhibition of 9a and 10d in PC3 cells.PC3 cells were treated
with 10
lM of either 9a or 10d for 3 h. After treatment, the cell lysate was subjected
to SDS–PAGE and Western blot analysis. Actin was used as the internal control.
bition of compound 10d against PI3Ka.
4. Conclusions
3.3. PI3Ka enzymatic activity
A series of novel 2-aliphatic cyclic amine-3-(arylsulfonyl)quin-
oxalines was designed and synthesized as PI3K inhibitors based
2-Piperidinol-3-(arylsulfonyl)quinoxalines 7a, 8a, 10a, and 10d
a
were then selected to test their PI3Ka inhibition using a competi-
tive fluorescence polarization (CFP) assay.16 LY294002 was used as
on a morpholinoaryl lead WR1, which was identified through phar-
macophore-based virtual screening. Structural modification re-
vealed that replacement of the morpholine group with either a
smaller pyrrolidine group or a larger tetrahydroisoquinoline group
reduced in vitro cytotoxicities against five human tumor cell lines,
while incorporation of a peridinol group significantly improved
in vitro cytotoxicities. From the 2-piperidinol-3-(arylsulfo-
nyl)quinoxaline series, compound 10d (WR23) that showed signif-
icant pAktSer473 inhibition was discovered as the most potent
the positive control. As shown in Table 3, compounds 7a with a
pyrrolidine group showed poor PI3K
IC50 value of 21.83 M, compound 8a with a tetrahydroisoquino-
line group did not inhibit PI3K with an IC50 value >50 M, while
compound 10a with a 4-hydroxypiperidine exhibited a value of
79 nM against PI3K , which was an eightfold improvement than
that of LY294002 (IC50 value of 0.63 M against PI3K ), and a six-
fold improvement than that of compound 2 (IC50 value of 0.44
against PI3K ). More encouragingly, 2-(piperidin-4-ol)-3-(4-bro-
a inhibitory potency with an
l
a
l
a
l
a
lM
compound with an IC50 value of 0.025
lM against PI3Ka. Molecular
a
docking analysis further suggested PI3K
a
inhibitory activity of 10d.
mophenylsulfonyl)quinoxaline 10d, the most potent compound
in this series in both the PI3K enzymatic assay and cellular assay,
showed an IC50 value of 25 nM against PI3Ka.
This study might be useful in future development of novel PI3K
a
inhibitors.
3.4. pAkt suppression
5. Experimental
5.1. Chemistry
Akt, or Protein kinase B (PKB), is a key downstream signaling
effector of PI3K.17 Inhibition of pAkt would be strong evidence to
support the PI3K inhibitory activity of target compounds. Thus,
compound 10d with a 4-hydroxypiperidine was further tested for
Melting points were determined with a B-540 Büchi apparatus
and are uncorrected. NMR spectra were recorded on a Brüker 500
(500 MHz) spectrometer at room temperature (chemical shifts are
given in ppm(d) relative to TMS as internal standard, coupling con-
stants (J) are in hertz (Hz), and signals are designated as follows: s,
singlet; d, doublet; t, triplet; m, multiplet; br s, broad singlet, etc.)
Mass spectra (MS), ESI (positive) were recorded on an Esquire-LC-
00075 spectrometer. Thin layer chromatography was carried out
using plate silica gel F254 Merck, Damstadt, Germany. All yields
are unoptimized and generally represent the result of a single
experiment.
its suppression against pAkt. GDC-0941, one of the most potent
18,19
and advanced PI3K inhibitors revealed so far
was used as
the positive control to indicate the suppressive potency of com-
pound 10d. As shown in Figure 2, the result of pAktSer473 inhibition
in PC3 cells revealed that the most potent compound 10d led to
significant pAkt suppression at both low and high concentrations,
the result of which was consistent with cellular PI3Ka inhibition.
3.5. Docking study
To determine possible binding modes and give better guidance
for further SAR studies, molecular docking study using co-crystal
5.1.1. General procedure for the synthesis of 2-pyrrolidinyl-3-
(arylsulfonyl)quinoxalines (7)
structure of mutant PI3K
conducted using the C-Dock protocol within DiscoveryStudio
2.1.20 The binding modes of PI3K
with LY294002, 7a, or 10d,
a
(PDB ID: 3HHM) (Fig. 3A and B) was
To a microwave vial (2–5 mL) were added 2-choloro-3-(aryl-
sulfonyl)quinoxaline
6
(0.1 mmol), pyrrolidine (0.05 mL,
a
0.6 mmol), and isopropyl alcohol (2 mL). The sealed vial was
heated at 80 °C for 10 min by microwave irradiation in a Biotage™
Initiator Synthesizer using a fixed hold time. The mixture was then
cooled to room temperature and the residue obtained after evapo-
rating under vacuum was subjected to purification over silica gel
chromatography eluting with PE/EtOAc (4:1, v/v) to afford target
compounds 7 as yellow solid.
are illustrated in Figure 3. As shown in the simplified docking
graphics with selected amino acid residues around the ATP binding
site, LY294002 forms a hydrogen bond interaction (length: 2.4 Å)
with the hinge residue of Val851 (Fig. 3C), which corresponds to
the widely existed hydrogen bonds formed between Val882 and
PI3K inhibitors in most PI3K
c
co-crystal structures,5,11,21 and a

P. Wu et al. / Bioorg. Med. Chem. 20 (2012) 2837–2844
2841
Figure 3. Molecular docking analysis of LY294002, 7a, and 10d with PI3K
a
. (A) An overall view of the crystal structure of PI3K
a
. The kinase catalytic domain which
accommodates the ATP binding site is shown in yellow to light orange. (B) A close-up view of the ATP binding site of PI3Ka. Selected amino acid residues around the ATP
binding site are shown in cyan sticks. (C) LY294002 (green backbone) bound to PI3K
backbone) bound to PI3K . (E) 7a (pink backbone) bound to PI3K , orange dashed line stands for hydrogen bond. (F) Surface show of 7a (pink backbone) bound to PI3K
10d (blue backbone) bound to PI3K , orange dashed lines stand for hydrogen bonds. (H) Surface show of 10d (blue backbone) bound to PI3K . (PDB ID: 3HHM).
a
, orange dashed lines stand for hydrogen bonds. (D) Surface show of LY294002 (green
a
a
a
. (G)
a
a
5.1.1.1. 2-Pyrrolidinyl-3-(phenylsulfonyl)quinoxaline (7a).
Yield: 91%, mp: 130–133 °C. 1H NMR (500 MHz, CDCl3): d 7.97 (d,
2H, J = 7.0 Hz, aromatic H), 7.66 (d, 1H, J = 8.5 Hz, aromatic H),
7.57–7.54 ()m, 3H, aromatic H), 7.35 (d, 2H, J = 8.0 Hz, aromatic
H), 7.23–7.20 (m, 1H, aromatic H), 4.03 (t, 4H, J = 6.5 Hz, pyrroli-
dine H), 2.08 (t, 4H, J = 6.5 Hz, pyrrolidine H). ESI-MS (m/z): 340
[M+1]+.
matic H), 7.59 (t, 1H, J = 7.5 Hz, aromatic H), 7.40 (d, 1H,
J = 8.0 Hz, aromatic H), 7.35 (d, 2H, J = 8.0 Hz, aromatic H), 7.24
(t, 1H, J = 7.5 Hz, aromatic H), 4.02 (t, 4H, J = 6.5 Hz, pyrrolidine
H), 2.48 (s, 3H, CH3), 2.07 (t, 4H, J = 6.5 Hz, pyrrolidine H). ESI-MS
(m/z): 354 [M+1]+.
5.1.1.3.
2-Pyrrolidinyl-3-(4-methoxyphenylsulfonyl)quinoxa-
Yield: 92%, mp: 177–179 °C. 1H NMR (500 MHz,
CDCl3): d 7.90 (d, 2H, J = 8.5 Hz, aromatic H), 7.65 (d, 1H,
line (7c).
5.1.1.2. 2-Pyrrolidinyl-3-(4-methylphenylsulfonyl)quinoxaline
(7b).
d 7.85 (d, 2H, J = 8.0 Hz, aromatic H), 7.65 (d, 1H, J = 8.5 Hz, aro-
Yield: 96%, mp: 152–154 °C. 1H NMR (500 MHz, CDCl3):
J = 8.5 Hz, aromatic H), 7.59–7.53 (m, 1H, aromatic H), 7.42 (d,
1H, J = 8.5 Hz, aromatic H), 7.25 (t, 1H, J = 7.5 Hz, aromatic H),

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P. Wu et al. / Bioorg. Med. Chem. 20 (2012) 2837–2844
7.03 (d, 2H, J = 8.5 Hz, aromatic H), 4.02 (t, 4H, J = 6.5 Hz, pyrroli-
dine H), 3.91 (s, 3H, OCH3), 2.06 (t, 4H, J = 6.5 Hz, pyrrolidine H).
ESI-MS (m/z): 370 [M+1]+.
H), 4.92 (s, 2H, hydroisoquinoline H), 4.12 (t, 2H, J = 6.0 Hz, hydro-
isoquinoline H), 3.24 (t, 2H, J = 6.0 Hz, hydroisoquinoline H). ESI-
MS (m/z): 482 [M+1]+.
5.1.1.4. 2-Pyrrolidinyl-3-(4-bromophenylsulfonyl)quinoxaline
(7d).
5.1.2.5. 2-(3,4-Dihydroisoquinolin-2(1H)-yl)-3-(4-fluorophenyl-
sulfonyl)quinoxaline (8e).
Yield: 76%, mp: 134–136 °C. 1H NMR (500 MHz, CDCl3):
Yield: 79%, mp: 171–175 °C. 1H
d 7.83 (d, 2H, J = 8.5 Hz, aromatic H), 7.70 (d, 2H, J = 8.5 Hz,
aromatic H), 7.66 (d, 1H, J = 8.5 Hz, aromatic H), 7.59–7.55 (m,
1H, aromatic H), 7.40 (d, 1H, J = 8.0 Hz, aromatic H), 7.25 (d, 1H,
J = 8.0 Hz, aromatic H), 4.01 (t, 4H, J = 6.5 Hz, pyrrolidine H), 2.07
(t, 4H, J = 6.5 Hz, pyrrolidine H). ESI-MS (m/z): 420 [M+1]+.
NMR (500 MHz, CDCl3): d 8.02–7.99 (m, 2H, aromatic H), 7.80 (d,
1H, J = 8.5 Hz, aromatic H), 7.68 (t, 1H, J = 7.5 Hz, aromatic H),
7.61 (d, 1H, J = 8.0 Hz, aromatic H), 7.44 (t, 1H, J = 7.5 Hz, aromatic
H), 7.71–7.16 (m, 6H, aromatic H), 4.92 (s, 2H, hydroisoquinoline
H), 4.12 (t, 2H, J = 5.5 Hz, hydroisoquinoline H), 3.25 (t, 2H,
J = 5.5 Hz, hydroisoquinoline H). ESI-MS (m/z): 420 [M+1]+.
5.1.1.5. 2-Pyrrolidinyl-3-(4-fluorophenylsulfonyl)quinoxaline
(7e).
Yield: 71%, mp: 94–97 °C. 1H NMR (500 MHz, CDCl3): d
5.1.3. General procedure for the synthesis of 2-(piperidin-3-ol)-
3-(arylsulfonyl)quinoxalines (9)
7.98–7.97 (m, 2H, aromatic H), 7.65 (d, 1H, J = 7.5 Hz, aromatic
H), 7.58 (d, 1H, J = 6.5 Hz, aromatic H), 7.38 (d, 1H, J = 7.5 Hz, aro-
matic H), 7.24–7.23 (m, 3H, aromatic H), 4.00 (m, 4H, pyrrolidine
H), 2.06 (m, 4H, pyrrolidine H). ESI-MS (m/z): 358 [M+1]+.
To a microwave vial (2–5 mL) were added 2-choloro-3-(aryl-
sulfonyl)quinoxaline
6
(0.1 mmol), piperidin-3-ol (30 mg,
0.3 mmol), and isopropyl alcohol (2 mL). The sealed vial was
heated at 80 °C for 10 min by microwave irradiation in a Biotage™
Initiator Synthesizer using a fixed hold time. The mixture was then
cooled to room temperature and the residue obtained after evapo-
rating under vacuum was subjected to purification over silica gel
chromatography eluting with PE/EtOAc (3:1, v/v) to afford target
compounds 9 as yellow solid.
5.1.2. General procedure for the synthesis of 2-(3,4-dihydro-
isoquinolin-2(1H)-yl)-3-(arylsulfonyl)quinoxalines (8)
To a microwave vial (2–5 mL) were added 2-choloro-3-(aryl-
sulfonyl)quinoxaline 6 (0.1 mmol), 1,2,3,4-tetrahydroisoquinoline
(40 mg, 0.3 mmol), and isopropyl alcohol (2 mL). The sealed vial
was heated at 80 °C for 10 min by microwave irradiation in a Bio-
tage™ Initiator Synthesizer using a fixed hold time. The mixture
was then cooled to room temperature and the residue obtained
after evaporating under vacuum was subjected to purification over
silica gel chromatography eluting with PE/EtOAc (8:1, v/v) to afford
target compounds 8 as yellow solid.
5.1.3.1. 2-(Piperidin-3-ol)-3-(phenylsulfonyl)quinoxaline (9a).
Yield: 42%, mp: 118–120 °C. 1H NMR (500 MHz, CDCl3): d 8.01
(d, 2H, J = 7.5 Hz, aromatic H), 7.79 (d, 1H, J = 7.5 Hz, aromatic H),
7.70–7.64 (m, 3H, aromatic H), 7.56–7.55 (m, 2H, aromatic H),
7.48–7.46 (m, 1H, aromatic H), 4.14 (m, 1H, piperidine H), 3.90–
3.89 (m, 2H, piperidine H), 3.68 (br s, 1H, hydroxy H), 3.61–3.58
(d, 1H, J = 13.0 Hz, piperidine H), 3.52 (t, 1H, J = 10.0 Hz,
piperidine H), 2.16–2.15 (m, 1H, piperidine H), 1.87–1.82 (m, 2H,
piperidine H), 1.73–1.71 (m, 1H, piperidine H). ESI-MS (m/z): 370
[M+1]+.
5.1.2.1.
nyl)quinoxaline (8a).
2-(3,4-Dihydroisoquinolin-2(1H)-yl)-3-(phenylsulfo-
Yield: 69%, mp: 149–150 °C. 1H NMR
(500 MHz, CDCl3): d 7.99 (d, 2H, J = 7.5 Hz, aromatic H), 7.79 (d,
1H, J = 8.5 Hz, aromatic H), 7.67 (t, 1H, J = 6.0 Hz, aromatic H),
7.62 (t, 2H, J = 8.0 Hz, aromatic H), 7.52 (t, 2H, J = 8.0 Hz, aromatic
H), 7.42 (t, 1H, J = 7.5 Hz, aromatic H), 7.23–7.21 (m, 4H, aromatic
H), 4.92 (s, 2H, hydroisoquinoline H), 4.13 (t, 2H, J = 5.5 Hz, hydro-
isoquinoline H), 3.25 (t, 2H, J = 5.5 Hz, hydroisoquinoline H). ESI-
MS (m/z): 402 [M+1]+.
5.1.3.2. 2-(Piperidin-3-ol)-3-(4-methylphenylsulfonyl)quinoxa-
line (9b).
CDCl3):
Yield: 45%, mp: 139–142 °C. 1H NMR (500 MHz,
7.90 (d, 2H, J = 8.5 Hz, aromatic H), 7.79 (d, 1H,
d
J = 8.5 Hz, aromatic H), 7.70–7.67 (m, 2H, aromatic H), 7.49 (t,
1H, J = 8.0 Hz, aromatic H), 7.34 (d, 2H, J = 8.5 Hz, aromatic H),
4.14–4.13 (m, 1H, piperidine H), 3.93–3.91 (m, 2H, piperidine H),
3.69 (br s, 1H, hydroxy H), 3.57 (d, 1H, J = 13.5 Hz, aromatic H),
3.49 (dt, 1H, J = 10.0 and 2.5 Hz, piperidine H), 2.46 (s, 3H, CH3),
2.19–2.12 (m, 1H, piperidine H), 1.91–1.87 (m, 1H, piperidine H),
1.83–1.78 (m, 1H, piperidine H), 1.72–1.69 (m, 1H, piperidine H).
ESI-MS (m/z): 384 [M+1]+.
5.1.2.2.
2-(3,4-Dihydroisoquinolin-2(1H)-yl)-3-(4-methyl-
Yield: 63%, mp: 170–
phenylsulfonyl)quinoxaline (8b).
172 °C. 1H NMR (500 MHz, CDCl3): d 7.87 (d, 2H, J = 8.0 Hz,
aromatic H), 7.79 (d, 1H, J = 8.5 Hz, aromatic H), 7.66–7.62 (m,
2H, aromatic H), 7.42 (t, 1H, J = 8.5 Hz, aromatic H), 7.29 (d, 2H,
J = 8.0 Hz, aromatic H), 7.23–7.20 (m, 4H, aromatic H), 4.91 (s,
2H, hydroisoquinoline H), 4.12 (t, 2H, J = 6.0 Hz, hydroisoquinoline
H), 3.25 (t, 2H, J = 6.0 Hz, hydroisoquinoline H), 2.43 (s, 3H, CH3).
ESI-MS (m/z): 416 [M+1]+.
5.1.3.3. 2-(Piperidin-3-ol)-3-(4-methoxyphenylsulfonyl)quinox-
aline (9c).
Yield: 50%, mp: 91–94 °C. 1H NMR (500 MHz,
CDCl3): d 7.96 (d, 2H, J = 8.5 Hz, aromatic H), 7.78 (dd, 1H, J = 9.0
and 1.5 Hz, aromatic H), 7.69–7.66 (m, 2H, aromatic H), 7.49 (dt,
1H, J = 8.5 and 1.5 Hz, aromatic H), 7.00 (d, 2H, J = 8.5 Hz, aromatic
H), 4.14–4.13 (m, 1H, piperidine H), 3.92 -3.90 (m, 2H, piperidine
H), 3.88 (s, 1H, OCH3), 3.79 (br s, 1H, hydroxy H), 3.56 (dd, 1H,
J = 13.0 and 1.5 Hz, aromatic H), 3.48 (dt, 1H, J = 10.0 and 2.5 Hz,
piperidine H), 2.17–2.11 (m, 1H, piperidine H), 1.90–1.87 (m, 1H,
piperidine H), 1.83–1.76 (m, 1H, piperidine H), 1.72–1.68 (m, 1H,
piperidine H). ESI-MS (m/z): 400 [M+1]+.
5.1.2.3.
2-(3,4-Dihydroisoquinolin-2(1H)-yl)-3-(4-methoxy-
Yield: 58%, mp: 148–
phenylsulfonyl)quinoxaline (8c).
150 °C. 1H NMR (500 MHz, CDCl3): d 7.93 (d, 2H, J = 9.0 Hz,
aromatic H), 7.79 (d, 1H, J = 9.0 Hz, aromatic H), 7.65 (d, 1H,
J = 8.0 Hz, aromatic H), 7.43 (dt, 1H, J = 8.0 and 1.5 Hz, aromatic
H), 7.28–7.18 (m, 4H, aromatic H), 6.96 (d, 2H, J = 9.0 Hz, aromatic
H), 4.91 (s, 2H, hydroisoquinoline), 4.11 (t, 2H, J = 6.0 Hz, hydroiso-
quinoline H), 3.87 (s, 3H, methoxy H), 3.25 (t, 2H, J = 6.0 Hz, hydro-
isoquinoline H). ESI-MS (m/z): 432 [M+1]+.
5.1.3.4. 2-(Piperidin-3-ol)-3-(4-bromophenylsulfonyl)quinoxa-
5.1.2.4. 2-(3,4-Dihydroisoquinolin-2(1H)-yl)-3-(4-bromophenyl-
sulfonyl)quinoxaline (8d).
line (9d).
CDCl3):
Yield: 57%, mp: 108–112 °C. 1H NMR (500 MHz,
d 7.87 (d, 2H, J = 8.5 Hz, aromatic H), 7.76 (d, 1H,
Yield: 59%, mp: 184–185 °C. 1H
NMR (500 MHz, CDCl3): d 7.85 (d, 2H, J = 8.5 Hz, aromatic H),
7.79 (d, 1H, J = 8.0 Hz, aromatic H), 7.69–7.60 (m, 4H, aromatic
H), 7.44 (t, 1H, J = 8.0 Hz, aromatic H), 7.22–7.20 (m, 4H, aromatic
J = 8.5 Hz, aromatic H), 7.69–7.66 (m, 3H, aromatic H), 7.64 (d,
1H, J = 8.5 Hz, aromatic H), 7.46 (t, 1H, J = 8.0 Hz, aromatic H),
4.11–4.08 (m, 2H, piperidine H), 4.03–4.00 (m, 1H, piperidine H),

P. Wu et al. / Bioorg. Med. Chem. 20 (2012) 2837–2844
2843
3.46 (dt, 2H, J = 13.0 and 3.0 Hz, piperidine H), 2.16–2.12 (m, 2H,
piperidine H), 1.88–1.81 (m, 2H, piperidine H). ESI-MS (m/z): 450
[M+1]+.
5.1.4.5. 2-(Piperidin-4-ol)-3-(4-fluorophenylsulfonyl)quinoxa-
line (10e).
Yield: 67%, mp: 89–93 °C. 1H NMR (500 MHz,
CDCl3): d 8.03–8.00 (dd, 2H, J = 9.0 and 2.0 Hz, aromatic H), 7.75
(dd, 1H, J = 8.0 and 1.0 Hz, aromatic H), 7.67 (dt, 1H, J = 8.5 and
1.5 Hz, aromatic H), 7.63 (dd, 1H, J = 8.0 and 1.0 Hz, aromatic H),
7.45 (dt, 1H, J = 8.0 and 1.0 Hz, aromatic H), 7.22 (dt, 2H, J = 9.0
and 2.0 Hz, aromatic H), 4.13–4.08 (m, 2H, piperidine H), 4.03–
3.98 (m, 1H, piperidine H), 3.45 (dt, 2H, J = 10.0 and 3.5 Hz, piper-
idine H), 2.16–2.11 (m, 2H, piperidine H), 1.88–1.82 (m, 2H, piper-
idine H). ESI-MS (m/z): 388 [M+1]+.
5.1.3.5 2-(Piperidin-3-ol)-3-(4-fluorophenylsulfonyl)quinoxaline
(9e).
Yield: 68%, mp: 121–124 °C. 1H NMR (500 MHz, CDCl3): d 8.04–
8.01 (dd, 2H, J = 8.5 and 2.0 Hz, aromatic H), 7.79 (d, 1H, J = 8.0 Hz,
aromatic H), 7.71 (dt, 1H, J = 8.5 and 1.5 Hz, aromatic H), 7.64 (d,
1H, J = 8.5 Hz, aromatic H), 7.49 (dt, 1H, J = 8.5 and 1.5 Hz, aromatic
H), 7.22 (t, 2H, J = 8.5 Hz, aromatic H), 4.14–4.12 (m, 1H, piperidine
H), 3.92–3.89 (m, 2H, piperidine H), 3.61–3.59 (m, 2H, piperidine H),
3.52 (dt, 1H, J = 10.0 and 3.0 Hz, piperidine H), 2.19–2.12 (m, 1H,
piperidine H), 1.90–1.79 (m, 2H, piperidine H). ESI-MS (m/z): 388
[M+1]+.
5.2. Pharmacology
Five human cancer cell lines, PC3, A549, HCT116, HL60, and KB,
were purchased from the cell bank of Chinese Academy of Sciences,
Shanghai, China. PI3-Kinase fluorescence polarization activity as-
say kit (catalog No. K-1100) and recombinant human PI3K
(catalog No. E-2000) were commercially available from Echelon
Biosciences (Salt Lake City, UT, USA).
a
5.1.4. General procedure for the synthesis of 2-(piperidin-4-ol)-
3-(arylsulfonyl)quinoxalines (10)
To a microwave vial (2–5 mL) were added 2-choloro-3-(aryl-
sulfonyl)quinoxaline
6
(0.1 mmol), piperidin-4-ol (30 mg,
5.2.1. Cytotoxicity assay
0.3 mmol), and isopropyl alcohol (2 mL). The sealed vial was
heated at 80 °C for 10 min by microwave irradiation in a Biotage™
Initiator Synthesizer using a fixed hold time. The mixture was then
cooled to room temperature and the residue obtained after evapo-
rating under vacuum was subjected to purification over silica gel
chromatography eluting with PE/EtOAc (3:1, v/v) to afford target
compounds 10 as yellow solids.
The cytotoxic activity in vitro was measured using the MTT as-
say. PC3, A549, HCT116, HL60, and KB cell lines were cultured in
RPMI-1640 (Invitrogen Corp., Carlsbad, CA) medium with heat-
inactivated 10% fetal bovine serum, penicillin (100 units/mL) and
streptomycin (100 lg/mL) and incubated in normoxic atmosphere
with 20% O2, 5% CO2 at 37 °C. All tested compounds were dissolved
in DMSO at concentrations of 10.0 mg/mL and diluted to appropri-
ate concentrations. Cells were plated in 96-well plates for 24 h and
subsequently treated with different concentrations of all tested
compounds for 72 h. Viable cells were determined using 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay kit
(MTT, Sigma) according to operation instructions provided by the
manufacturer. The concentration of drug causing 50% inhibition
in absorbance compared with control cells (IC50) was calculated
using the software of dose-effect analysis with microcomputers.
5.1.4.1.
(10a).
2-(Piperidin-4-ol)-3-(phenylsulfonyl)quinoxaline
Yield: 69%, mp: 164–167 °C. 1H NMR (500 MHz, CDCl3):
d 8.00 (d, 2H, J = 8.0 Hz, aromatic H), 7.76 (d, 1H, J = 8.5 Hz, aro-
matic H), 7.68–7.62 (m, 3H, aromatic H), 7.54 (t, 2H, J = 8.0 Hz, aro-
matic H), 7.45 (t, 1H, J = 7.5 Hz, aromatic H), 4.13–4.08 (m, 2H,
piperidine H), 4.02–3.99 (m, 1H, piperidine H), 3.45 (dt, 2H,
J = 10.0 and 2.5 Hz, aromatic H), 2.15–2.13 (m, 2H, piperidine H),
1.88–1.81 (m, 2H, piperidine H). ESI-MS (m/z): 370 [M+1]+.
5.2.2. PI3K
a
a
kinase assay
inhibitory test was determined using a competitive
5.1.4.2. 2-(Piperidin-4-ol)-3-(4-methylphenylsulfonyl)quinoxa-
The PI3K
line (10b).
CDCl3):
Yield: 78%, mp: 130–133 °C. 1H NMR (500 MHz,
7.89 (d, 2H, J = 8.0 Hz, aromatic H), 7.76 (d, 1H,
fluorescence polarization kinase activity assay based on the fact
that PI3K phosphorylates PI(3,4)P2 and converts it to PI(3,4,5)P3.16
PI3K reactions were performed in 5 mM HEPES, pH 7, 2.5 mM
d
J = 8.5 Hz, aromatic H), 7.68–7.64 (m, 2H, aromatic H), 7.45 (t,
1H, J = 7.0 Hz, aromatic H), 7.32 (d, 2H, J = 8.0 Hz, aromatic H),
4.10–4.08 (m, 2H, piperidine H), 4.01–3.99 (m, 1H, piperidine H),
3.43 (t, 2H, J = 10.0 Hz, piperidine H), 2.44 (s, 3H, CH3), 2.15–2.13
(m, 2H, piperidine H), 1.86–1.84 (m, 2H, piperidine H). ESI-MS
(m/z): 384 [M+1]+.
MgCl2, 10 mM DTT and 50
strate, and the final reaction volumes were 10
PI3K inhibitors, 50 ng of enzyme and 10 M of substrate were used
per 10 l reaction volume with the concentrations of inhibitors
ranging from 3.2 nM to 50 M. After incubating for 3 h at room
l
M ATP, using diC8-PI(4,5)P2 as the sub-
ll. For evaluation of
l
l
l
temperature, reactions were quenched by adding chelators. A
mixture of phosphoinositide binding protein was added and mixed,
followed by the addition of a fluorophore-labeled phosphoinositide
tracer. Samples were then mixed in 384-well black Corning
nonbinding plates and incubated in a dark environment for 1 h to
equilibrate. Finally, polarization values were measured using red
fluorophores with appropriate filters to determine the extent of
enzyme activity in the reaction.
5.1.4.3. 2-(Piperidin-4-ol)-3-(4-methoxyphenylsulfonyl)quinox-
aline (10c).
CDCl3):
Yield: 53%, mp: 148–150 °C. 1H NMR (500 MHz,
d
7.94 (d, 2H, J = 7.5 Hz, aromatic H), 7.75 (d, 1H,
J = 7.5 Hz, aromatic H), 7.67 (m, 2H, aromatic H), 7.44 (m, 1H, aro-
matic H), 6.99 (d, 2H, J = 7.0 Hz, aromatic H), 4.11 (d, 2H,
J = 12.0 Hz, piperidine H), 4.00 (m, 1H, piperidine H), 3.88 (s, 3H,
OCH3), 3.43 (t, 2H, J = 10.0 Hz, morpholine H), 2.13 (m, 2H, piperi-
dine H), 1.86–1.85(m, 2H, piperidine H). ESI-MS (m/z): 400 [M+1]+.
5.2.3. Docking study
5.1.4.4. 2-(Piperidin-4-ol)-3-(4-bromophenylsulfonyl)quinoxa-
X-ray co-crystal structure of PI3Ka-wortmannin downloaded
line (10d).
CDCl3):
Yield: 74%, mp: 129–133 °C. 1H NMR (500 MHz,
7.86 (d, 2H, J = 8.5 Hz, aromatic H), 7.76 (d, 1H,
from the RCSB Protein Data Bank (PDB ID: 3HHM) was used as
the template to perform molecular docking of LY294002, com-
pounds 7a and 10d using C-Dock protocol within DiscoveryStudio
2.1.20 For ligand preparation, the 3D structures of LY294002, 7a,
and 10d were generated and minimized using DiscoveryStudio
2.1. For protein preparation, the hydrogen atoms were added,
water was removed, and the CHARMm force field was employed.
d
J = 8.5 Hz, aromatic H), 7.67–7.66 (m, 3H, aromatic H), 7.64 (d,
1H, J = 9.0 Hz, aromatic H), 7.46 (t, 1H, J = 7.5 Hz, aromatic H),
4.12–4.09 (m, 2H, piperidine H), 4.02–4.00 (m, 1H, piperidine),
3.46 (dt, 2H, J = 12.5 and 2.5 Hz, piperidine H), 2.15–2.13 (m, 2H,
piperidine H), 1.88–1.82 (m, 2H, piperidine H). ESI-MS (m/z): 450
[M+1]+.
The whole PI3Ka enzyme was defined as a receptor and the site

2844
P. Wu et al. / Bioorg. Med. Chem. 20 (2012) 2837–2844
12. Wu, P.; Zhang, L.; He, Q. J.; Yang, B.; Hu, Y. Z. AAPS J. 2009, 11(S2). Abstract of
Papers, 2009 American Association of Pharmaceutical Scientists Annual
Meeting, Los Angeles, USA, 2009; Abstract 3179.
13. Hayakawa, M.; Kaizawa, H.; Moritomo, H.; Koizumi, T.; Ohishi, T.; Okada, M.;
Ohta, M.; Tsukamoto, S.-i.; Parker, P.; Workman, P.; Waterfield, M. Bioorg. Med.
Chem. 2006, 14, 6847.
sphere was selected based on the ligand binding location of wort-
mannin. LY294002, 7a, or 10d was placed in the binding site during
the docking procedure. All graphical pictures were made using
Pymol.
14. Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Adv. Drug Deliv. Rev.
2001, 46, 3.
Acknowledgment
15. Vistoli, G.; Pedretti, A.; Testa, B. Drug Discov. Today 2008, 13, 285.
16. Drees, B.; Weipert, A.; Hudson, H.; Ferguson, C.; Chakravarty, L.; Prestwich, G.
D. Comb. Chem. High Throughput Screen. 2003, 6, 321.
We thank Ms Jianyang Pan for doing the NMR tests.
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