Design, synthesis and biological evaluation of novel phenylsulfonylurea derivatives as PI3K/mTOR dual inhibitors

Article abstract of DOI:10.3390/molecules23071553

Five series of novel phenylsulfonylurea derivatives, 19a–d, 20a–d, 21a–d, 22a–d and 23a–d, bearing 4-phenylaminoquinoline scaffold were designed, synthesized and their IC₅₀ values against four cancer cell lines (HepG-2, A549, PC-3 and MCF-7) were evaluated. Most compounds showed moderate cytotoxicity activity against the cancer cell lines. Structure–activity relationships (SARs) and pharmacological results indicated that introduction of 4-aminoquinoline scaffold and phenylsulfonylurea scaffold were beneficial for anti-tumor activity. Moreover, para-methoxyl substitution of 4-anilino moiety and para-halogen substitution of phenylsulfonylurea have different impacts on different series of compounds. Furthermore, the micromolecule group substitution in the 6-position of the quinoline ring have a slight impact on the cellular activity of the target compounds.

Full text of DOI:10.3390/molecules23071553

molecules
Article
Design, Synthesis and Biological Evaluation of
Novel Phenylsulfonylurea Derivatives as
PI3K/mTOR Dual Inhibitors
Bingbing Zhao 1, Fei Lei 1, Caolin Wang 1, Binliang Zhang 1, Zunhua Yang 2, Wei Li 3,
Wufu Zhu 1,
*
and Shan Xu 1,
*
ID
1
School of Pharmacy, Jiangxi Science & Technology Normal University, Nanchang 330013, Jiangxi, China;
zhaobb_0628@163.com (B.Z.); m13767032532@163.com (F.L.); wangcllw@163.com (C.W.);
zbl1045762244@163.com (B.Z.)
School of Pharmacy, Jiangxi University of Traditional Chinese Medicine, Nanchang 330004, Jiangxi, China;
mtdzcool@163.com
2
3
School of Chemistry and Bioengineering, Yichun University, Yichun 336000, Jiangxi, China;
nobel2025@163.com
*
Correspondence: zhuwf@jxstnu.edu.cn (W.Z.); xush@jxstnu.edu.cn (S.X.); Tel.: +86-791-8380-2393 (S.X.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 30 May 2018; Accepted: 19 June 2018; Published: 27 June 2018
Abstract: Five series of novel phenylsulfonylurea derivatives, 19a–d, 20a–d, 21a–d, 22a–d and
23a–d, bearing 4-phenylaminoquinoline scaffold were designed, synthesized and their IC50 values
against four cancer cell lines (HepG-2, A549, PC-3 and MCF-7) were evaluated. Most compounds
showed moderate cytotoxicity activity against the cancer cell lines. Structure–activity relationships
(SARs) and pharmacological results indicated that introduction of 4-aminoquinoline scaffold and
phenylsulfonylurea scaffold were beneficial for anti-tumor activity. Moreover, para-methoxyl
substitution of 4-anilino moiety and para-halogen substitution of phenylsulfonylurea have different
impacts on different series of compounds. Furthermore, the micromolecule group substitution in the
6-position of the quinoline ring have a slight impact on the cellular activity of the target compounds.
Keywords: 4-phenylaminoquinoline; phenylsulfonylurea; PI3K/mTOR; inhibitor
1. Introduction
PI3K (phosphatidylinositol-3-kinase) is a kind of cellular signaling molecule, which is closely
associated with human cancers. According to the primary structures, regulatory mechanisms and
distribution positions, the PI3K families [
Type III and Type IV. The type I PI3K enzymes are further divided into PI3K
PI3K ]. PI3K/mTOR signaling pathway plays a crucial role in regulating various cellular
physiological activities such as proliferation, growth, proliferation and apoptosis [ ]. With the
1
–
3] are divided into four different types: Type I, Type II,
α
, PI3K , PI3K and
β
δ
γ [4,5
6
development of cytobiology, accumulating studies have shown that the PI3K/mTOR signaling pathway
is frequently disregarded in most tumor cells, leading to malignant proliferation of normal organism
cells [7–9]. In addition, research has also shown that PI3K/mTOR signaling pathway can promote
the occurrence of cancer by promoting angiogenesis, accelerating cell growth cycle and promoting
tumor cell metastasis [10]. Therefore, the development of double inhibitors targeting PI3K/mTOR has
become a hotspot in this field. Thus far, numerous PI3K/mTOR double target inhibitors have been
developed into clinical trial, such as NVPBEZ235 [11], GSK2126458 [12], GDC0980 [13], PKI587 [14],
BGT226 [15] and PF06491502 [16] (Figure 1). Furthermore, most of these compounds shared quinoline
core and exhibited remarkable potency in both enzymatic and cellular inhibitory activity [17]. Research
Molecules 2018, 23, 553; doi:10.3390/molecules23070553
www.mdpi.com/journal/molecules

Molecules 2018, 23, 553
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data further demonstrated that PI3K/mTOR double target inhibitors have a great potential in the
process of anti-tumor therapy [18,19].
4-Amino quinazoline derivatives have been widely used in small molecule antitumor drugs for
their multiple biological activities, especially EGFR inhibitors [17]. Compounds containing 4-amino
quinazoline core account for a substantial proportion of therapeutic drugs [20], such as Gefitinib,
Erlotinib, Afatinib and Vandetanib. The potency of these drugs has been widely recognized in clinical
treatment. Accordingly, we introduced 4-amino quinazoline structure into PI3K/mTOR inhibitor to
investigate antiproliferative activity of 4-amino quinazoline structure on tumor cells.
We firstly introduced 4-aminoquinoline structure by ring-opening the imidazolone structure of
NVPBEZ235. To guide our design, we carried out the molecular simulation to find the differences
between the binding mode of GSK2126458 and NVPBEZ235 with PI3Kγ protein. From the docking
results in Figure 2, we can see that both drugs can form the hydrogen bond between the N-atom in
quinoline rings and valine. The -CN of substituted aniline side chain in the NVPBEZ235 points to the
hydrophobic cavity and forms a hydrogen bond with SER806. This suggests that the para position
of 4-aniline side chain is a key active site. Therefore, we introduced electron-withdrawing group
–Br, electron-donating group -OCH3, 3-Cl-4-F-aniline active group, 4-Br-2-F-aniline active group and
retained -CN to change benzene ring electron cloud density. Through this design strategy, we expect
to obtain desirable inhibitors with better PI3K/mTOR inhibition activity. In addition, benzene
sulfonamide structure of GSK2124658 buried into hydrophobic pockets, and the O-atom of sulfonyl
group formed a hydrogen bond with residue LYS833. Therefore, we secondly introduced benzene
sulfonamide group into NVPBEZ235 to explore potential interactions between the PI3Kγ protein and
target compounds, and also improve the water solubility. Based on the above, we used the same
design strategy to introduce different substituent groups into the para-position of phenylsulfonylurea.
On the one hand, we changed the electron cloud density of benzene ring. On the other hand,
we appropriately increased the space structure of benzene ring to enhance the affinity of small
molecule ligand with receptor protein. Finally, series of 4-aniline quinoline derivatives containing
phenylsulfonylurea structure were designed, synthesized and all compounds were evaluated for their
anti-proliferative effects.
Figure 1. Structures of representative clinical PI3K/mTOR dual inhibitors.

Molecules 2018, 23, 553
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Figure 2. Structure based drug design strategy based on the molecular simulation.
2. Results and Discussion
2.1. Chemistry
Based on the structure based drug design (SBDD) strategy, taking NVPBEZ235 as reference
compound, five series of PI3K/mTOR inhibitors were designed and synthesized. The synthetic routes
of target compounds 19a–d, 20a–d, 21a–d, 22a–d and 23a–d are outlined in Schemes 1–3.
Scheme 1. The synthetic route for target compounds 8a
–
d
. Reagents and c◦onditions: (a) Triethoxy
◦
methane; (b) 4-Bromoanilines, C2H5OH, reflux, 105 C. 6◦h; (c) Ph2O, 200 C, Microwave, 15 min;
◦
(d) HNO3, Propionic acid, 125 C, 2 h; (e) POCl3, DMF, 100 C, 3 h; (f) Substituted aniline, Acetic acid,
◦
◦
100 C, 5 h; and (g) FeCl3, N2H4·H2O, C2H5OH, 80 C, 3 h.
◦
Scheme 2. The synthetic route for target compounds 13a–d. Reagents and conditions: (a) PPA, 190 C,
6 h; (b) HNO3,◦Propionic acid, 125 ◦C, 2 h; (c) POCl3, DMF, ◦100 ◦C, 3 h; (d) Substituted aniline,
Acetic acid, 100 C, 5 h; and (e) FeCl3, N2H4·H2O, C2H5OH, 80 C, 3 h.

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Scheme 3. The synthetic route for target compounds 19a–d, 20a–d, 21a–d, 22a–d and 23a–d. Reagents
◦
and conditions: (a) HSO3Cl, reflux, 6 h; (b) C2H5OH, 0 C, 1 h; (c) DIPEA, CH2Cl2, r.t., 0.5 h; and (d)
◦
Toluene, 120 C, 6 h.
As shown in Schemes 1–3, all target compounds were synthesized by resolution synthetic
method. Target compounds were separately split into 4-anilinoquinoline fragment in the left half and
phenylsulfonylurea fragment in the right half, and finally the two fragments were connected to obtain
target compounds. As shown in Scheme 1, we used commercially available isopropyl malonate (
triethyl orthoformate as starting materials to obtain segments 8a by seven steps of condensation,
cyclization, substitution, nitration, chlorination, amination and reduction. Then, we used as the
starting material to get fragments 14a by a method similar to that of synthesizing 8a . Detailed
synthetic route of 14a is shown in Scheme 2. Next, as show in Scheme 3, we used different substituted
aromatic rings as starting materials to obtain fragments 18a by chlorosulfonation, amminolysis and
substitution reaction. Finally, target compounds 19a 20a 21a 22a and 23a were obtained
by linking fragments 18a with fragments 8a and 14a , respectively. The structural information
1) and
–
d
9
–
d
–d
–
d
–d
–d
,
–
d,
–d
,
–
d
–d
–d
–d
–d
of all target compounds was confirmed by 1H-NMR, 13C-NMR and TOF MS (ES+), which were in
agreement with the structures depicted.
2.2. Biological Discussion
Four cells (HepG-2, A549, PC-3, MCF-7) were selected to evaluate the in vitro antiproliferative
activity of all target compounds. As shown in Table 1, phenylsulfonylurea derivatives (19a
–d,
20a 21a and 22a ) with bromine substitution at 6-position of quinoline ring showed moderate
–
d,
–d
–d
antiproliferative activity against HepG-2 cell, A549 cell and PC-3 cell, and some compounds (19a and
19c) showed strong sensitivity to MCF-7 cell. In addition, the introduction of different substituents
into 4-position of aniline in the quinoline ring can significantly improve anti-proliferative activity and
cell selectivity of target compounds, and when aniline was substituted with -CN or -OCH3, target
compounds (19a–d and 20a–d) showed stronger anti-proliferative activity to MCF-7 cell. Compounds
with -CN or -OCH3 substituted at the 4-position of aniline structure in the quinoline ring showed better
anti-proliferative activity against MCF-7 cell than that of the other three cells. When -CH3 or Cl-atom
were introduced into the phenylsulfonylurea structure, target compounds (20c and 22b) showed good
anti-proliferative activity on MCF-7 cell, but when aniline was substituted with F-atom or Cl-atom,
the anti-proliferative activity of target compound (21a
three cells. Overall, 4-anilino side chain para-methoxy substituted compounds 20a showed better
MCF-7 cell anti-proliferation activity than other compounds (19a 21a and 22a–d). We speculate
–d) on MCF-7 cell was weaker than that of other
–d
–d
,
–d
that electron-donating group -OCH3 increased the electron cloud density of aniline side chain. Thus,
the force between aniline side chain and hydrophobic cavity of protein was enhanced.

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Table 1. Structures and cytotoxicity of compounds 19a–d, 20a–d, 21a–d, 22a–d and 23a–d.
IC50 (µM) a
A549
Compound
R1
R2
HepG-2
PC-3
MCF-7
19a
19b
19c
19d
20a
20b
20c
20d
21a
21b
21c
21d
22a
22b
22c
22d
23a
pivalonitrile
pivalonitrile
pivalonitrile
pivalonitrile
-OCH3
-OCH3
-OCH3
-OCH3
-Br
-F
-Cl
-CH3
Tert-Butyl
-F
-Cl
-CH3
Tert-Butyl
-F
-Cl
-CH3
Tert-Butyl
-F
-Cl
-CH3
Tert-Butyl
33.28 ± 1.52
30.22 ± 1.45
31.25 ± 1.50
>100
38.52 ± 1.04
66.62 ± 1.28
34.31 ± 1.32
40.59 ± 1.11
68.25 ± 1.32
>100
3.88 ± 0.58
15.10 ± 1.1
3.96 ± 0.59
9.95 ± 0.99
7.65 ± 0.88
10.65 ± 0.97
4.94 ± 0.64
10.46 ± 0.98
37.59 ± 1.57
13.78 ± 1.14
16.63 ± 1.22
52.67 ± 1.72
22.83 ± 1.35
6.84 ± 0.83
16.81 ± 1.22
46.30 ± 1.67
6.30 ± 0.79
6.55 ± 0.81
25.44 ± 1.43
57.03 ± 1.75
0.14 ± 0.10
4.21 ± 0.15
>100
28.96 ± 1.31
30.58 ± 1.45
26.34 ± 1.13
30.22 ± 1.23
23.35 ± 1.12
32.12 ± 1.18
58.98 ± 1.76
71.11 ± 1.85
22.74 ± 1.35
16.16 ± 1.20
12.02 ± 0.91
>100
20.34 ± 1.04
22.56 ± 1.12
26.83 ± 1.22
33.24 ± 1.26
45.68 ± 1.66
11.13 ± 1.04
31.05 ± 1.49
16.97 ± 1.22
19.63 ± 0.18
6.443 ± 0.89
>100
>100
>100
>100
>100
>100
>100
-Br
-Br
-Br
-3-Cl-4-F
-3-Cl-4-F
-3-Cl-4-F
-3-Cl-4-F
-OCH3
-OCH3
-Br
52.52 ± 1.03
17.72 ± 0.95
16.74 ± 0.88
29.27 ± 2.23
>100
11.274
±
0.84
>100
28.43 ± 0.89
15.98 ± 1.10
2.71 ± 0.43
32.12 ± 1.12
6.08 ± 1.04
0.54 ± 0.83
3.97 ± 0.13
22.40 ± 1.21
39.76 ± 1.59
7.47 ± 0.87
12.96 ± 0.84
38.14 ± 0.96
0.36 ± 0.86
6.53 ± 0.23
-Cl
Tert-Butyl
23b
23c
23d
>100
-F
-F
-
39.76 ± 1.25
57.85 ± 0.99
0.20 ± 0.11
3.03 ± 0.11
-3-Cl-4-F
NVP-BEZ235 b
Sorafenib b
-
-
-
a
The values are an average of two separate determinations; b used as a positive control.
Phenylsulfonylurea derivatives (23a–d) with methoxyl substitution at 6-position of the quinoline
ring showed moderate antitumor activity against HepG-2 cell, A549 cell and PC-3 cell. Similarly,
4-anilino side chain para-methoxy substituted compounds 23a and 23b showed better MCF-7 cell
anti-proliferation activity than that of other compounds (23c and 23d). It is noteworthy that the activity
of compounds 23a
compounds 23b and 23d (with an IC50 value of 2.71
–
d
to HepG-2 cell was generally superior to that of the other three cells, especially
M and 6.08 M, respectively). We supposed that
µ
µ
the quinoline ring and the 4-phenylamino side chain of compound 23b are not only both located in the
hydrophobic cavity of the protein but also all substituted by electron-donating group -OCH3 group,
which greatly increased the electron cloud density of the structure in the left half of compound 23b.
Thus, the affinity between compound 23b and the target protein was enhanced. In addition, from the
docking results (Figure 3), we can see that the -OCH3 group on the quinoline ring of compound 23b
points to inside of the protein cavity and fully occupies the space of this part, thus resulting in better
antiproliferative activity of compounds 23a–d against HepG-2 cell.

Molecules 2018, 23, 553
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A
C
B
Figure 3.
(A
) The binding mode of compound 19a with PI3K
γ
(3L08) kinase; (B) the binding mode
of compound 23b with PI3Kγ kinase; and (C) the contrast of binding mode between compound 19a
(green) and 23b (red).
Finally, the inhibitory rate against PI3Kα kinase and mTOR kinase of selected compounds 19a
and 23b at 10 M was further examined. As shown in Table 2, enzymatic activity results of compounds
µ
19a and 23b exhibited a moderate inhibitory rate against PI3Kα kinase and mTOR kinase.
Table 2. Enzymatic activities of compounds 19a and 23b against PI3Kα and mTOR (IC50, µM).
IC50(µM)
Compound
PI3Kα
mTOR
19a
23b
NVP-BEZ235
PI101
0.72
>10
2.62
>10
0.004 ± 0.002
0.006 ± 0.003
0.011 ± 0.002
0.019 ± 0.002
PI3Kα: Phosphatidylinositol-3-kinase alpha subunit; mTOR: Mammalian target of rapamycin.
2.3. Molecular Docking Study
To explore the binding modes of target compounds (19a and 23b) with the active site of
PI3K , molecular docking simulation studies were carried out by the AutoDock 4.2 software.
γ
The docking tutorial we used and the detailed AutoDock basic operational methods can be found
at: http://autodock.scripps.edu/faqs-help/tutorial. Based on in vitro inhibition results, we selected
compounds 19a and 23b as ligand examples, and the structures of PI3Kγ (PDB code: 3L08) were
selected as the docking models. Only the best-scoring ligand–protein complex (Figure 3) was used for
the binding site analysis.
The binding mode of compounds 19a and 23b with the active site of PI3K
in Figure 3, which depicts that the oxygen atom on benzene sulfonamide group of compounds 19a
and 23b both formed a hydrogen bond with PI3K residue LYS833 and the nitrogen atom in quinoline
ring formed a hydrogen bond with hinge residue VAL882. In the docking model of compound 19a
with PI3K , we can see the cyano group on the aniline group in the 4-position of quinoline ring
γ molecular is shown
γ
γ
formed a hydrogen bond with the residue SER806. All these factors above contribute to the excellent
antiproliferative activity and selectivity of the compound 19a. The whole 4-aminoquinoline ring

Molecules 2018, 23, 553
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structure of the selected compounds extended into ATP hydrophobic pocket, which is basically
the same as our previous prediction binding mode (show in Figure 2). Furthermore, binding
modes of compounds 19a and 23b were almost completely overlapped. The abovementioned SAR
(Structure–Activity Relationship) analysis and molecular docking study results may allow the rational
design of potential PI3K and mTOR inhibitors.
3. Experimental Section
3.1. General Information
Unless otherwise required, all reagents used in the experiment were purchased as commercial
analytical grade and used without further purification. Frequently used solvents (Ethanol, petroleum
ether, ethyl acetate, dichloromethane, etc.) were absolutely anhydrous. All actions were monitored
through GF254 thin-layer chromatography plate and spots were visualized with iodine or light (in
254 nm or 365 nm). The structure of the target compound was confirmed by 1H-NMR and 13C-NMR
spectra at room temperature on Bruker 400 MHz spectrometer (Bruker Bioscience, Billerica, MA, USA)
with tetramethylsilane (TMS) as an internal standard. Mass spectrometry (MS) was performed on
Waters High Resolution Quadrupole Time of Flight Tandem Mass Spectrometry (QTOF). The purity
of the compound was determined by Agilent 1260 liquid chromatograph fitted with an Inertex-C18
column. All target compounds had the purity of ≥95%.
3.2. Chemistry
3.2.1. General Procedure for the Preparation of Compounds 8a–d, 14a–d and 18a–d
We using isopropyl malonate (
intermediate . Next, intermediate
4-hydroxy-6-bromoquinoline ( ), and then 3-nitro-4-chloro-6-bromoquinoline (
nitration and chlorination. Intermediate reacted with para-substituted anilines (
intermediates 7a , and then underwent a reduction reaction to obtain intermediates 8a
we used 4-methoxyaniline ( ) as the starting material to obtain intermediates 14a through a similar
) to obtain (16a ),
1
2
) and triethyl orthoformate as the starting material to get
was condensed and cyclized with p-bromoaniline to obtain
) was generated by
) to obtain
. Similarly,
2
5
6
6
a–d
–d
–d
9
–d
reaction. Chlorosulfonic acid was reacted with different substituted anilines (15a
and then 18a–d were obtained by amination reaction and acylation reaction.
–
d
–d
3.2.2. General Procedure for Preparation of Target Compounds 19a
A solution of ethyl (phenylsulfonyl) carbamate (1.2 mmol, 18a
dropwise to a solution of 8a or 14a
–
d
,
20a
–d, 21a–d, 22a–d and 23a–d
–
d) in toluene (5 mL) was added
–d
–d
(1 mmol) in toluene (15 mL). Upon completion of the addition,
the reaction mixture was stirred at 120 ◦C for 6 h and monitored by thin-layer chromatography (TLC).
The reaction mixture was cooled to room temperature and concentrated under a reduced pressure.
Then, ethyl acetate was added and the insoluble materials were collected by filtration and dried
to yield the target compounds 19a–d, 20a–d, 21a–d, 22a–d and 23a–d, which were recrystallized
from isopropanol.
N-((6-Bromo-4-((4-(2-cyanopropan-2-yl)phenyl)amino)quinolin-3-yl)car◦bamoyl)-4-fluorobenzenesulfonamide
carboxamide (19a) A pale white solid; Yield: 52.6%; m.p.: 315.2–316.4 C; 1H-NMR (400 MHz, DMSO-d6)
δ
(ppm): 12.01 (s, 1H), 8.95 (s, 1H), 8.08 (d, J = 9.0 Hz, 1H), 8.05–7.94 (m, 4H), 7.82 (dd, J = 16.8, 8.6 Hz,
3H), 7.59–7.50 (m, 3H), 7.11 (s, 1H), 1.95 (s, 6H). 13C NMR (400 MHz, DMSO-d6)
165.41, 153.89,
143.83, 142.85, 134.90, 132.57, 130.12, 129.89 2, 129.42, 129.06, 128.97, 127.26 2, 124.80, 122.84,
122.66, 119.37, 116.55 2, 116.40, 116.33 2, 37.32, 28.86
2. TOF MS ES+ (m/z): [M + H]+, calcd for
C26H21BrFN5O3S: 581.0533, found: 582.0611.
δ
×
×
×
×
×
N-((6-Bromo-4-((4-(2-cyanopropan-2-yl)phenyl)amino)quinolin-3-yl)carbamoyl)-4-chlorobenzenesulfonamide
19b). A pale white solid; Yield: 50.1%; m.p.: 279.2–280.1 ◦C; 1H-NMR (400 MHz, DMSO-d6)
(ppm):
(
δ

Molecules 2018, 23, 553
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11.87 (s, 1H), 8.81 (s, 1H), 8.23(d, J = 9.8Hz, 2H), 7.94 (d, J = 9.0 Hz, 1H), 7.89 (s, 1H), 7.88 (d, J = 3.1 Hz,
1H), 7.85 (s, 1H), 7.83 (s, 1H), 7.70 (d, J = 8.2 Hz, 2H), 7.66 (d, J = 9.1 Hz, 1H), 7.45–7.38 (m, 3H), 6.97
(s, 1H), 1.81 (s, 6H), 13C-NMR (400 MHz, DMSO-d6)
δ
153.88, 143.79, 143.12, 135.17
2, 126.09, 124.80, 122.83, 122.62 2, 122.62, 119.30, 116.45,
2, 21.37. TOF MS ES+ (m/z): [M + H]+, calcd for C26H21BrClN5O3S: 597.0237,
×
2, 134.93, 132.86
×
2, 129.95, 129.90
×
2, 129.15, 127.24
×
×
102.00, 37.32, 28.86
found: 598.0315.
×
N-((6-Bromo-4-((4-(2-cyanopropan-2-yl)phenyl)amino)quinolin-3-yl)carbamoyl)-4-methylbenzenesulfonamide
19c). A pale white solid; Yield: 51.4%; m.p.: 289.2–290.4 ◦C; 1H-NMR (400 MHz, DMSO-d6)
(ppm):
(
δ
12.01 (s, 1H), 8.95 (s, 1H), 8.25(d, J = 9.5 Hz), 8.08 (d, J = 9.0 Hz, 1H), 7.98 (d, J = 8.4 Hz, 3H), 7.84
(d, J = 8.2 Hz, 2H), 7.80 (d, J = 9.1 Hz, 1H), 7.59–7.51 (m, 3H), 7.11 (s, 1H), 6.23 (s, 1H), 2.30 (s, 3H) 1.95
(s, 6H). 13C-NMR (400 MHz, DMSO-d6)
129.90 2, 129.15, 127.24
δ
153.88, 143.79, 143.12, 135.17
×
2, 134.93, 132.86
×
2, 129.95,
×
×
2, 126.09, 124.80, 122.83, 122.62
28.86 × 2, 21.37. TOF MS ES+ (m/z): [M + H]+, calcd for C27H24BrN5O3S: 577.0783, found: 578.0861.
×
2, 122.62, 119.30, 116.45, 102.00, 37.32,
N-((6-Bromo-4-((4-(2-cyanopropan-2-yl)phenyl)amino)quinolin-3-yl)carbamoyl)-4-(tert-butyl)
◦
1
benzenesulfonamide (19d). A pale white solid; Yield: 45.6%; m.p.: 287.2–288.4 C; H-NMR (400 MHz,
DMSO-d6) (ppm): 12.06 (s, 1H), 9.00 (s, 1H), 8.23 (s, 1H), 8.13 (d, J = 9.0 Hz, 1H), 8.03 (d, J = 8.4 Hz, 4H),
7.89 (d, J = 8.2 Hz, 2H), 7.85 (d, J = 9.1 Hz, 1H), 7.65–7.57 (m, 3H), 7.16 (s, 1H), 2.00 (s, 1H), 1.63 (s, 9H),
13C-NMR (400 MHz, DMSO-d6)
156.15, 153.88, 143.77, 143.09, 135.17, 134.91, 132.87, 129.96, 129.92, 128.97
3, 127.25 2, 125.32 2, 121.18, 121.09, 120.15 2, 119.72, 118.38, 116.44, 37.32, 29.72, 28.84 3, 25.62
2. TOF MS ES+ (m/z): [M + H]+, calcd for C30H30BrN5O3S: 619.1253, found: 620.1331.
δ
δ
×
×
×
×
×
×
N-((6-Bromo-4-((4-methoxyphenyl)amino)quinolin-3-yl)carbamoyl)-4-fluorobenzenesulfonamide (20a). A pale
white solid; Yield: 47.9%; m.p.: 289.1–290.2 ◦C; 1H-NMR (400 MHz, DMSO-d6)
(ppm): 11.93 (s, 1H),
δ
8.93 (s, 1H), 8.08 (d, J = 9.0 Hz, 1H), 7.86 (d, J = 8.0 Hz, 2H), 7.82–7.77 (d, J = 8.6 Hz, 1H), 7.68 (d,
J = 8.7 Hz, 2H), 7.52 (d, J = 8.0 Hz, 2H), 7.43 (s, 2H), 7.37 (d, J = 8.7 Hz, 2H), 7.28 (s, 1H), 4.05 (s, 3H),
13C-NMR (400 MHz, DMSO-d6)
129.92, 128.81, 128.26, 121.98, 121.61
ES+ (m/z): [M + H]+, calcd for C23H18BrFN4O4S: 544.0216, found: 545.0294.
δ
165.72, 153.88, 152.52, 143.78, 138.17, 136.91, 132.86, 130.98
×
2,
×
2, 119.57, 118.32, 117.44, 116.02 2, 115.18 2, 59.84. TOF MS
×
×
N-((6-Bromo-4-((4-methoxyphenyl)amino)quinolin-3-yl)carbamoyl)-4-chlorobenzenesulfonamide (20b). A pale
white solid; Yield:48.2%; m.p.: 282.2–283.1 ◦C; 1H-NMR (400 MHz, DMSO-d6)
(ppm): 10.83 (s, 1H),
δ
8.87 (s, 1H), 8.68 (s, 1H), 8.48 (s, 1H), 8.08-7.89 (d, J = 8.0 Hz, 2H), 7.84 (d, J = 8.0 Hz, 2H), 7.58 (d,
J = 8.7 Hz, 2H), 7.51 (d, J = 8.0 Hz, 2H), 7.43 (s, 2H), 7.37 (d, J = 8.7 Hz, 2H), 7.07 (s, 1H), 3.97 (s, 3H),
13C-NMR (400 MHz, DMSO-d6)
128.92 2, 128.81, 128.02, 121.88, 121.76
(m/z): [M + H]+, calcd for C23H18BrClN4O4S: 559.9921, found: 560.9999.
δ
153.72, 153.03, 144.05, 138.95, 138.47, 137.41, 132.86, 130.03
×
3,
×
×
2, 119.86, 118.91, 117.84, 115.11
×
2, 59.64. TOF MS ES+
N-((6-Bromo-4-((4-methoxyphenyl)amino)quinolin-3-yl)carbamoyl)-4-methylbenzenesulfonamide (20c). A pale
white solid; Yield: 46.6%; m.p.: 279.2–280.6 ◦C; 1H-NMR (400 MHz, DMSO-d6)
(ppm): 11.77 (s, 1H),
δ
8.77 (s, 1H), 7.93 (d, J = 9.0 Hz, 1H), 7.70 (d, J = 8.0 Hz, 2H), 7.67–7.60 (m, 1H), 7.53 (d, J = 8.7 Hz,
2H), 7.36 (d, J = 8.0 Hz, 2H), 7.28 (s, 2H), 7.21 (d, J = 8.7 Hz, 2H), 7.13 (s, 1H), 3.89 (s, 3H), 2.37 (s, 3H).
13C-NMR (400 MHz, DMSO-d6)
129.91, 129.77, 129.49 2, 127.81, 126.08
TOF MS ES+ (m/z): [M + H]+, calcd for C24H21BrN4O4S: 540.0467, found: 541.0545.
δ
160.46, 154.20, 143.08, 142.33, 141.89, 135.04, 132.80, 130.57
×
2,
×
×
2, 122.63, 122.59, 119.24, 116.57, 115.52 2, 56.13, 21.38.
×
N-((6-Bromo-4-((4-methoxyphenyl)amino)quinolin-3-yl)carbamoyl)-4-(tert-butyl)benzenesulfonamide (20d).
A pale white solid; Yield: 40.6%; m.p.: 263.3–264.2 ◦C; 1H-NMR (400 MHz, DMSO-d6)
(ppm): 11.93
δ
(s, 1H), 8.93 (s, 1H), 8.08 (d, J = 9.0 Hz, 1H), 7.86 (d, J = 8.0 Hz, 2H), 7.82–7.77 (d, J = 8.6 Hz, 1H),
7.68 (d, J = 8.7 Hz, 2H), 7.52 (d, J = 8.0 Hz, 2H), 7.43 (s, 2H), 7.37 (d, J = 8.7 Hz, 2H), 7.28 (s, 1H),
4.05 (s, 3H),1.72(s, 9H), 13C-NMR (400 MHz, DMSO-d6)
δ
154.87, 153.23, 152.12, 143.27, 138.54, 137.24,
132.48, 129.45, 128.36, 128.97
×
2, 129.87, 125.32
×
2, 121.89, 121.43 2, 119.58, 118.65, 118.47, 115.52
×
×

Molecules 2018, 23, 553
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2, 55.98, 35.12, 31.25
583.1015.
×
3. TOF MS ES+ (m/z): [M + H]+, calcd for C27H27BrN4O4S: 582.0936, found:
N-((6-Bromo-4-((4-bromophenyl)amino)quinolin-3-yl)carbamoyl)-4-fluorobenzenesulfonamide (21a). A pale
white solid; Yield: 45.8%; m.p.: 278.7–279.5 ◦C; 1H-NMR (400 MHz, DMSO-d6)
(ppm): 12.04 (s, 1H),
8.95 (s, 1H), 8.84 (s, 2H), 8.76 (s, 1H), 8.34 (d, J = 12.0 Hz, 4H), 8.19 (s, 1H), 7.76 (d, J = 8.5 Hz, 2H),
7.42 (d, J = 8.5 Hz, 2H), 6.60 (s, 1H). 13C-NMR (400 MHz, DMSO-d6)
144.98, 138.52, 135.18, 133.35,
131.99, 131.92, 131.73, 131.54, 131.05, 130.95, 129.06, 128.97, 128.45, 125.57, 123.84, 120.28, 118.54 2,
2. TOF MS ES+ (m/z): [M + H]+, calcd for C22H15Br2FN4O3S: 591.9216, found:
δ
δ
×
117.76, 116.51, 116.36
592.9294.
×
N-((6-Bromo-4-((4-bromophenyl)amino)quinolin-3-yl)carbamoyl)-4-chlorobenzenesulfonamide (21b). A pale
white solid; Yield: 42.6%; m.p.: 284.1–285.4 ◦C; 1H-NMR (400 MHz, DMSO-d6)
(ppm): 11.88 (s, 1H),
8.79 (s, 1H), 8.60 (s, 1H), 8.18 (d, J = 12.0 Hz, 4H), 8.03 (s, 1H), 7.94 (d, J = 8.2 Hz, 1H), 7.93–7.90 (m, 2H),
7.45 (s, 1H), 7.26 (d, J = 8.5 Hz, 2H), 6.44 (s, 1H). 13C-NMR (400 MHz, DMSO-d6)
144.63, 138.07,
134.75, 132.89, 132.46, 131.54 2, 131.34, 131.05, 129.56, 129.07 2, 128.04, 127.62 2, 127.02, 123.39,
2. TOF MS ES+ (m/z): [M + H]+, calcd for C22H15Br2ClN4O3S: 607.8920,
δ
δ
×
×
×
122.07, 119.82, 118.96, 116.03
found: 608.8998.
×
N-((6-Bromo-4-((4-bromophenyl)amino)quinolin-3-yl)carbamoyl)-4-methylbenzenesulfonamide (21c). A pale
white solid; Yield: 41.9%; m.p.: 279.6–280.2 ◦C; 1H-NMR (400 MHz, DMSO-d6)
(ppm): 12.01 (s, 1H),
8.92 (s, 1H), 8.76 (d, J = 11.1 Hz, 4H), 8.13 (s, 1H), 7.94 (s, 2H), 7.74 (d, J = 8.3 Hz, 1H), 7.49 (d, J = 7.8 Hz,
3H), 7.25 (s, 1H), 6.58 (s, 1H), 2.49 (s, 3H). 13C-NMR (400 MHz, DMSO-d6)
144.71, 144.21, 141.87,
141.46, 138.11, 132.89, 131.55 2, 131.41, 131.07, 129.30 2, 128.08, 126.99, 125.64 2, 123.37, 120.88,
119.82, 116.00
2, 108.86, 20.91. TOF MS ES+ (m/z): [M + H]+, calcd for C23H18Br2N4O3S: 587.9466,
found: 588.9545.
δ
δ
×
×
×
×
N-((6-Bromo-4-((4-bromophenyl)amino)quinolin-3-yl)carbamoyl)-4-(tert-butyl)benzenesulfonamide
(
21d).
11.88
(s, 1H), 8.68 (s, 2H), 8.60 (s, 1H), 8.18 (d, J = 12.0 Hz, 3H), 8.03 (s, 1H), 7.26 (d, J = 8.5 Hz, 2H), 7.18-7.09
(m, 4H), 6.44 (s, 1H), 1.63(s, 9H). 13C-NMR (400 MHz, DMSO-d6)
155.20, 145.17, 144.65, 141.80, 141.03,
138.58, 133.35, 132.00 2, 131.88, 131.54, 128.53, 127.44, 126.18 2, 125.98 2, 123.80, 121.28, 120.28,
116.43 2, 109.28, 35.23, 31.32
2. TOF MS ES+ (m/z): [M + H]+, calcd for C26H24Br2N4O3S: 629.9936,
found: 631.0014.
◦
A pale white solid; Yield: 39.3%; m.p.: 259.4–260.3 C; 1H-NMR (400 MHz, DMSO-d6)
δ (ppm): δ
δ
×
×
×
×
×
N-((6-Bromo-4-((3-chloro-4-fluorophenyl)amino)quinolin-3-yl)carbamoyl)-4-fluorobenzenesulfonamide (22a).
A pale white solid; Yield: 46.5%; m.p.: 295.1–296.3 ◦C; 1H-NMR (400 MHz, DMSO-d6)
(ppm): 11.87
δ
(s, 1H), 8.34 (s, 1H), 7.81 (d, J = 5.8 Hz, 1H), 7.77–7.59 (m,2H), 7.50 (d, J = 8.5 Hz, 2H), 7.18 (q, J = 9.1 Hz,
2H), 7.01 (dd, J = 18.2, 8.8 Hz, 2H), 6.95–6.90 (m, 1H), 6.04 (d, J = 2.2 Hz, 1H). 13C-NMR (400 MHz,
DMSO-d6)
122.37, 122.01
C22H14BrClF2N4O3S: 565.9627, found: 566.9705.
δ
156.47, 153.39, 142.65, 134.77
×
×
2, 132.51
×
2, 132.10, 131.50
×
2, 130.15, 129.56
×
2, 128.44,
2, 120.65, 119.02, 118.15, 117.92, 115.98. TOF MS ES+ (m/z): [M + H]+, calcd for
N-((6-Bromo-4-((3-chloro-4-fluorophenyl)amino)quinolin-3-yl)carbamoyl)-4-chlorobenzenesulfonamide (22b).
A pale white solid; Yield: 42.6%; m.p.: 282.3–283.7 ◦C; 1H-NMR (400 MHz, DMSO-d6)
(ppm): 11.91
δ
(s, 1H), 10.48 (s, 1H), 7.84 (d, J = 8.0 Hz, 3H), 7.76 (d, J = 7.5 Hz, 2H), 7.68 (d, J = 8.3 Hz, 1H), 7.60 (dd,
J = 18.5, 10.8 Hz, 2H), 7.54 (d, J = 7.9 Hz, 2H), 7.28 (d, J = 9.1 Hz, 1H), 6.29 (s, 1H). 13C-NMR (400 MHz,
DMSO-d6)
δ
153.83, 143.10, 135.27, 132.96, 132.55, 131.93
×
2, 130.58, 130.50, 130.01, 129.51, 128.89,
128.13
×
2, 128.08, 127.96, 122.86, 122.46, 119.46, 118.59, 118.37, 116.45. TOF MS ES+ (m/z): [M + H]+,
calcd for C22H14BrCl2FN4O3S: 581.9331, found: 582.9409.
N-((6-Bromo-4-((3-chloro-4-fluorophenyl)amino)quinolin-3-yl)carbamoyl)-4-methylbenzenesulfonamide (22c).
A pale white solid; Yield: 43.2%; m.p.: 278.7–279.9 ◦C; 1H-NMR (400 MHz, DMSO-d6)
δ
(ppm): 11.77
(s, 1H), 10.34 (s, 1H), 7.69 (d, J = 8.0 Hz, 3H), 7.61 (d, J = 7.5 Hz, 2H), 7.54 (d, J = 8.3 Hz, 1H), 7.46

Molecules 2018, 23, 553
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(d, J = 7.7 Hz, 2H), 7.39 (d, J = 7.9 Hz, 2H), 7.13 (d, J = 9.1 Hz, 1H), 6.14 (s, 1H), 2.68 (s, 3H). 13C-NMR
(400 MHz, DMSO-d6)
δ
153.84, 143.08
×
2, 135.23
×
2, 132.96
×
2, 131.95
×
2, 130.62, 130.54, 130.04
×
2,
128.90, 122.81, 122.45, 121.10, 120.91, 119.48, 118.61, 118.39, 116.43, 21.4. TOF MS ES+ (m/z): [M + H]+,
calcd for C23H17BrClFN4O3S: 561.9877, found: 562.9956.
N-((6-Bromo-4-((3-chloro-4-fluorophenyl)amino)quinolin-3-yl)carbamoyl)-4-(tert-butyl)benzenesulfonamide
(
22d). A pale white solid; Yield: 38.7%; m.p.: 290.1–291.6 ◦C; 1H-NMR (400 MHz, DMSO-d6)
δ (ppm):
11.83 (s, 1H), 10.40 (s, 1H), 7.90 (d, J = 7.3 Hz, 3H), 7.75 (d, J = 8.0 Hz, 3H), 7.67 (d, J = 7.5 Hz, 2H), 7.52
(d, J = 7.7 Hz, 1H), 7.45 (d, J = 7.9 Hz, 1H), 6.66 (s, 1H), 2.65 (s, 3H), 1.86 (s, 9H). 13C-NMR (400 MHz,
DMSO-d6)
121.5 2, 119.49, 118.61
C26H23BrClFN4O3S: 604.0347, found: 605.0425.
δ
153.85, 143.08, 135.23, 132.96, 132.51, 131.95, 130.62, 128.41, 128.01
×
3, 122.82, 122.46,
×
×
2, 118.39 2, 116.43, 34.3, 31.4
×
×
3. TOF MS ES+ (m/z): [M + H]+, calcd for
4-Chloro-N-((6-methoxy-4-((4-methoxyphenyl)amino)-2-methylquinolin-3-yl)carbamoyl)benzenesulfonamide
23a). A pale white solid; Yield: 42.6%; m.p.: 290.8–291.2 ◦C; 1H-NMR (400 MHz, DMSO-d6)
(ppm):
(
δ
11.86 (s, 1H), 8.31(d, J = 10.8 Hz, 2H), 8.25(s, 1H), 7.98 (d, J = 9.2 Hz, 1H), 7.78 (d, J = 8.5 Hz, 1H), 7.67
(d, J = 8.8 Hz, 2H), 7.55 (d, J = 8.4 Hz, 1H), 7.35 (t, J = 10.7 Hz, 2H), 7.33–7.16 (m, 2H), 6.44 (s, 1H),
4.04 (s, 3H), 3.58 (s, 3H), 2.83 (s, 3H), 13C-NMR (400 MHz, DMSO-d6)
δ
154.08, 153.92, 152.32, 148.63,
2, 128.55, 127.21, 121.98 2, 121.08, 118.98, 117.23,
2, 20.05. TOF MS ES+ (m/z): [M + H]+, calcd for C25H23ClN4O5S: 526.1078,
139.21, 138.67, 138.05, 137.56, 129.21
115.18 2, 97.86, 54.77
found: 527.1156.
×
2, 128.87
×
×
×
×
4-(tert-Butyl)-N-((6-methoxy-4-((4-methoxyphenyl)amino)-2-methylquinolin-3-yl)carbamoyl)
benzenesulfonamide (23b). A pale white solid; Yield: 52.6%; m.p.: 291.2–292.7 ◦C; 1H-NMR
(400 MHz, DMSO-d6)
δ (ppm): 11.75 (d, J = 3.7 Hz, 1H), 7.89 (dd, J = 9.0, 6.4 Hz, 1H), 7.78 (d, J = 9.2 Hz,
2H), 7.62–7.53 (m, 2H), 7.47–7.40 (m, 2H), 7.33 (d, J = 8.2 Hz, 2H), 7.24 (s, 2H), 7.23–7.16 (m, 1H), 6.30
(s, 1H), 3.94 (s, 3H), 3.47 (s, 3H), 2.74 (d, J = 4.0 Hz, 3H), 1.55 (s, 9H), 13C-NMR (400 MHz, DMSO-d6)
δ
156.28, 154.11, 153.94, 153.23, 148.73, 138.07, 137.94, 137.34, 130.28, 128.53
121.9 2 121.30, 119.02, 118.12, 114.99 2, 98.72, 54.74 2, 37.18, 28.63
3, 20.74. TOF MS ES+ (m/z):
[M + H]+, calcd for C29H32N4O5S: 548.2093, found: 549.2172.
×
2, 127.00, 124.86
×
2,
×
×
×
×
N-((4-((4-Bromophenyl)amino)-6-methoxy-2-methylquinolin-3-yl)carbamoyl)-4-fluorobenzenesulfonamide
23c). A pale white solid; Yield: 40.3%; m.p.: 356.1–357.3 ◦C; 1H-NMR (400 MHz, DMSO-d6)
(ppm):
(
δ
11.83 (s, 1H), 7.93 (d, J = 8.4 Hz, 3H), 7.81 (d, J = 8.4 Hz, 2H), 7.32 (d, J = 9.4 Hz, 3H), 7.22 (dd, J = 9.2,
2.7 Hz, 2H), 7.07 (dd, J = 9.0, 2.5 Hz, 1H), 7.00 (d, J = 2.4 Hz, 1H), 6.29 (d, J = 2.5 Hz, 1H), 3.76 (s, 3H),
2.71 (s, 3H). 13C-NMR (400 MHz, DMSO-d6)
δ 159.82, 155.81, 153.97, 152.07, 140.16, 139.41, 136.48,
132.45
×
2, 130.61, 130.44
×
2, 128.60, 127.75, 120.61, 118.52
×
2, 118.16, 114.79
×
2, 114.72, 98.94, 55.67,
20.15. TOF MS ES+ (m/z): [M + H]+, calcd for C24H20BrFN4O4S: 558.0373, found: 559.0451.
N-((4-((3-Chloro-4-fluorophenyl)amino)-6-methoxy-2-methylquinolin-3-yl)carbamoyl)-4-
fluorobenzenesulfonamide (23d). A pale white solid; Yield: 39.5%; m.p.: 285.3–286.1 C; H-NMR
◦
1
(400 MHz, DMSO-d6)
7.50 (d, J = 8.5 Hz, 2H), 7.40(s, 1H) 7.18 (q, J = 9.1 Hz, 2H), 7.01 (dd, J = 18.2, 8.8 Hz, 1H), 6.96–6.88
(m, 1H), 6.04 (d, J = 2.2 Hz, 1H), 3.23 (s, 3H), 2.26 (s, 3H), 13C-NMR (400 MHz, DMSO-d6)
156.33,
154.04, 140.82, 140.04, 135.10, 133.00, 131.80, 131.42, 129.37 2, 128.67, 128.23, 121.34, 120.85, 118.87
2, 118.56, 117.05, 115.91
2, 115.01, 99.35, 54.95, 20.73. TOF MS ES+ (m/z): [M + H]+, calcd for
δ (ppm): 11.87 (s, 1H), 8.34 (s, 1H), 7.81 (d, J = 5.8 Hz, 1H), 7.77–7.59 (m, 2H),
δ
×
×
×
C24H19ClF2N4O4S: 532.0784, found: 533.0862.
1H-NMR spectra of all the target compounds, 13C-NMR spectra of representative target
compounds (19a
, 19c, 20c, 21b, 21c, 21d and 22b) and TOF-MS spectra of representative target
compounds (19a, 19c, 22b and 23b) can be seen in the Supplementary Materials.

Molecules 2018, 23, 553
11 of 13
3.3. PI3Kα Kinase Assay
The potent compounds 19a and 23b were tested for their activities against PI3K
α
enzyme using
Kinase-Glo Luminescent Kinase Assay (Promega, Madison, WI, USA), with NVPBEZ-235 and PI101 as
positive control. The kinase reaction was done in a 384-well black plate. Each well was loaded with
50
µL of test items (in 90% DMSO) and 5
µL reaction buffer containing 10 µg/mL PI substrate
(L-
α-phosphatidylinositol); Avanti Polar Lipids (Avanti Polar Lipids, Inc., Alabaster, AL, USA);
prepared in 3% octyl-glucoside) and the PI3Ka protein 10 nM was then added to it. The reaction
was started by the addition of 5 L of 1 M ATP prepared in the reaction buffer (50 mM HEPES pH
7.5, 1 mM EGTA, 3 mM MnCl2, 10 mM MgCl2, 2 mM DTT and 0.01% Tween-20) and was incubated
for 60 min. It was terminated by the addition of 10 L Kinase-Glo buffer. The plates were then
µ
µ
µ
read in Synergy 2 readers (BioTek, Winooski, VT, USA) for luminescence detection. The assay was
repeated two times and the results expressed as IC50 (inhibitory concentration 50%) were the averages
of two determinations.
3.4. mTOR Kinase Assay
The potent compounds 19a and 23b were tested for their activities against mTOR enzyme
using Kinase-Glo Luminescent Kinase Assay (Promega, Madison, WI, USA), with NVPBEZ-235
and PI101 as positive control. The kinase reaction was done in a 384-well black plate. Each well was
loaded with 50
substrate (L- -phosphatidylinositol); Avanti Polar Lipids (Avanti Polar Lipids, Inc., Alabaster, AL,
USA); prepared in 3% octyl-glucoside) and the mTOR protein 2.5 nM was then added to it. The reaction
was started by the addition of 5 L of 10 M ATP prepared in the reaction buffer (50 mM HEPES pH
7.5, 1 mM EGTA, 3 mM MnCl2, 10 mM MgCl2, 2 mM DTT and 0.01% Tween-20) and was incubated
for 60 min. It was terminated by the addition of 10 L Kinase-Glo buffer. The plates were then
µL of test items (in 90% DMSO) and 5 µL reaction buffer containing 10 µg/mL PI
α
µ
µ
µ
read in Synergy 2 readers (BioTek, Winooski, VT, USA) for luminescence detection. The assay was
repeated two times and the results expressed as IC50 (inhibitory concentration 50%) were the averages
of two determinations.
3.5. Cytotoxicity Assay In Vitro
The in vitro cytotoxic activities of all compounds 19a–d, 20a–d, 21a–d, 22a–d and 23a–d were
evaluated with HepG-2, A549, PC-3 and MCF-7 cell lines by the standard MTT assay, with NVPBEZ235
and Sorafenib as positive control. The cancer cell lines were cultured in minimum essential medium
(MEM) supplement with 10% fetal bovine serum (FBS). Approximately 4
×
103 cells, suspended in
MEM medium, were plated onto each well of a 96-well plate and incubated in 5% CO2 at 37 ◦C for
24 h. The test compounds at indicated final concentrations were added to the culture medium and the
cell cultures were continued for 72 h. Fresh MTT was added to each well at a terminal concentration of
5 µ µL
g/mL and incubated with cells at 37 ◦C for 4 h. The formazan crystals were dissolved in 100
DMSO each well, and the absorbency at 492 nm (for absorbance of MTT formalin) and 630 nm (for the
reference wavelength) was measured with an ELISA reader. All of the compounds were tested three
times in each of the cell lines. The results were expressed as inhibition rates or IC50 (half-maximal
inhibitory concentration) were the averages of two determinations and calculated by using the Bacus
Laboratories Inc. Slide Scanner (Bliss) software (the Bacus Laboratories Inc. Slide Scanner (BLISS)
system, Lombard, II, USA).
3.6. Docking Studies
Molecular docking simulation studies were carried out by the AutoDock 4.2 software (The Scripps
Research Institute, USA). The docking tutorial we used and the detailed AutoDock basic operational
methods can be found at: http://autodock.scripps.edu/faqs-help/tutorial. The protein preparation
process of flexible docking mainly includes fixing the exact residues, adding hydrogen atoms, removing

Molecules 2018, 23, 553
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irrelevant water molecules, adding charges, etc. The potent compounds were selected as ligand
examples, and the structures of PI3K (PDB code: 3L08, http://www.pdb.org/) were selected as
γ
the docking models. Only the best-scoring ligand–protein complexes were used for the binding site
analyses. All the docking results were processed and modified in Open-Source PyMOL 1.8. x software
(https://pymol.org).
4. Conclusions
In summary, five series of phenylsulfonylurea derivatives, 19a
bearing 4-phenylaminoquinoline scaffolds were designed and synthesized. In addition, we evaluated
them for antitumor activity against four cancer cell lines and both PI3K kinase and mTOR kinase
activity (only compounds 19a and 23b in vitro. The pharmacological results indicated that most of
–d, 20a–d, 21a–d, 22a–d and 23a–d,
α
)
the compounds showed moderate cytotoxicity activity against the four cancer cell lines. In particular,
the cellular activity of the potent compounds 19a and 23b against MCF-7 cell was equal to the positive
control Soraffenib, with the IC50 value of 2.88
±
0.58 and 6.55
±
0.81, respectively. Structure–activity
relationships (SARs) indicated that the introduction of the methoxyl scaffold into 4-anilinoquinoline
scaffold was more favorable than the introduction of halogen to the cellular activity. In general,
the methoxy substitution and halogen substitution at 6-position of the quinoline ring played no
significant impact on the activity. The para-methoxyl substitution of 4-anilino moiety and para-halogen
substitution of phenylsulfonylurea have different impacts on different series of compounds. Moreover,
the oxygen atom of phenylsulfonylurea structure can form a hydrogen bond with the residue LYS833
of PI3Kγ protein. Further studies will be carried out in the near future.
Supplementary Materials: The Supplementary Materials are available online at http://www.mdpi.com/1420-
3049/23/7/553/s1.
Author Contributions: W.Z., S.X. and Z.Y. conceived and designed the experiments; C.W., B.Z. and F.L. performed
the experiments; W.L. ran the bioassay evaluation and statistics analysis; and B.Z. started the project and wrote
the paper.
Funding: We gratefully acknowledge the generous support provided by The National Natural Science Funds
(No. 81460527); The Project Supported by Natural Science Foundation of Jiangxi, China (20171BAB215073
and 20171BAB205101); Natural Science Funds for Distinguished Young Scholar of Jiangxi Province, China
(20171BCB23078); Key projects of the youth fund, Natural Science Funds of Jiangxi Province (20171ACB21052);
and Jiangxi Provincial Key Laboratory of Drug Design and Evaluation (20171BCD40015).
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 19a–d, 20a–d, 21a–d, 22a–d and 23a–d are available from
the authors.
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