Design, synthesis and biological evaluation of substituted 2-(thiophen-2-yl)-1,3,5-triazine derivatives as potential dual PI3Kα/mTOR inhibitors

Article abstract of DOI:10.1016/j.bioorg.2019.103525

The phosphoinositide 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) have been regarded as promising targets for the treatment of cancer. Herein, we synthesized a new series of substituted 2-(thiophen-2-yl)-1,3,5-triazine derivatives as novel PI3

Full text of DOI:10.1016/j.bioorg.2019.103525

Journal Pre-proofs
Design, Synthesis, and Biological Evaluation of substituted 2-(thiophen-2-
yl)-1,3,5-triazine derivatives as Potential dual PI3Kα/mTOR inhibitors
Binliang Zhang, Qian Zhang, Zhen Xiao, Xin Sun, Zunhua Yang, Qi Gu,
Ziqin Liu, Ting Xie, Qingqing Jin, Pengwu Zheng, Shan Xu, Wufu Zhu
PII:
S0045-2068(19)31695-5
DOI:
https://doi.org/10.1016/j.bioorg.2019.103525
YBIOO 103525
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
9 October 2019
23 November 2019
18 December 2019
Please cite this article as: B. Zhang, Q. Zhang, Z. Xiao, X. Sun, Z. Yang, Q. Gu, Z. Liu, T. Xie, Q. Jin, P. Zheng,
S. Xu, W. Zhu, Design, Synthesis, and Biological Evaluation of substituted 2-(thiophen-2-yl)-1,3,5-triazine
derivatives as Potential dual PI3Kα/mTOR inhibitors, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/
j.bioorg.2019.103525
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Design, Synthesis, and Biological Evaluation of substituted 2-(thiophen-2-yl)-1,3,5-triazine derivatives as Potential
dual PI3Kα/mTOR inhibitors
a
a
a
a
b
a
a
a
a
Binliang Zhang , Qian Zhang , Zhen Xiao , Xin Sun , Zunhua Yang , Qi Gu , Ziqin Liu , Ting Xie , Qingqing Jin ,
Pengwu Zheng , Shan Xu a,*, Wufu Zhu a,*
a
a
Jiangxi Provincial Key Laboratory of Drug Design and Evaluation, School of Pharmacy, Jiangxi Science & Technology
Normal University, 605 Fenglin Road, Nanchang, Jiangxi 330013, China
b
College of Pharmacy, Jiangxi University of Traditional Chinese Medicine, Nanchang, 330004, China
*
Corresponding author.
Tel. & Fax: +86 791 8380-2393;
E-mail: zhengpw@126.com (P. Zheng), zhuwufu-1122@163.com (W. Zhu)
Abstract：
The phosphoinositide 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) have been regarded as promising
targets for the treatment of cancer. Herein, we synthesized a new series of substituted 2-(thiophen-2-yl)-1,3,5-triazine
derivatives as novel PI3Kα/mTOR dual inhibitors for cancer therapy. All compounds were evaluated for the IC50 values
against three cancer cell lines (A549, MCF-7 and Hela). Most of the target compounds exhibited moderate to excellent
anti-tumor activities against these three tested cancer cell lines especially against A549 and Hela cancer cell lines. Among
them, the most promising compound 13g showed excellent anti-tumor potency for A549, MCF-7 and Hela cell lines with
IC50 values of 0.20±0.05 µM, 1.25±0.11 µM and 1.03±0.24 µM, respectively. Notably, according to the result of enzymatic
activity assay, compound 13g was identified as a novel PI3Kα/mTOR dual inhibitor, which had an approximately 10-fold
improvement in mTOR inhibition, compared to the class I PI3K inhibitor 1 (pictilisib, GDC-0941), with IC50 values of 525
nM to 48 nM. And western blot analysis indicated compound 13g could efficiently suppress the phosphorylation of AKT
at the dose of 0.1 µM, which further demonstrated compound 13g had significant inhibitory effect on the PI3K/Akt/mTOR
pathway. Furthermore, compound 13g could stimulate A549 cells arrest at G0/G1 phase in a dose-dependent manner, and
induced apoptosis at a low concentration.
Keywords: 2-(Thiophen-2-yl)-1,3,5-triazine; PI3Kα/mTOR inhibitors; Anti-tumor activity; Scaffold hopping

Corresponding author. Tel./fax: +86 791 83802393.
E-mail address: shanxu9891@126.com (S. Xu), zhuwufu-1122@163.com (W. Zhu).

1
. Introduction
The phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/the mammalian target of rapamycin (mTOR)
signaling pathway plays a critical role in a diverse set of cellular functions, including cell growth, proliferation, motility,
differentiation, and survival, which has been identified as promising target for the treatment of cancer, providing validated
therapeutic targets associated with malignancies [1-4]. At present, some PI3K inhibitor drugs had been approved by FDA,
such as Alpelisib (BYL719) [5], Copanlisib (BAY 80-6946) [6], Idelalisib (GS-1101) [7] and so on.
Along this critical signaling pathway, a variety of kinase inhibitors were discovered, such as allosteric mTOR inhibitor,
ATP-competitive mTOR inhibitor, pan-PI3K inhibitor, PI3K/mTOR dual inhibitor and a more selective inhibitor of the
class I PI3K family. Among them, dual PI3K/mTOR inhibitors appear to increase efficacy and reduce the likelihood of
inducing drug resistance as they target the pathway at two nodal points. In recent years, many PI3K/mTOR dual inhibitors
were discovered including PI-103 [8], GSK2126458 [9], compoud 3j [10], Apitolisib (GDC-0980) [11] and so on (Figure 1).
Among them, GSK2126458 and Apitelisib (GDC-0980) are now in clinical phase I.
It has been reported that most of these PI3K inhibitors share similar structural and chemical features, i.e., a central
six-membered heterocycle substituted by a morpholine with a hydrogen bond donor group [12] (Figure 1). Among them, 1
(
pictilisib, GDC-0941) [13] was the first potent class I PI3K inhibitor developed by Genentech, which was served as a useful
reference to explore novel PI3K inhibitors. Starting with 1 (pictilisib, GDC-0941), considerable structure−activity
relationship (SAR) investigations have been focused on the substituents of the thienopyrimidine core, and some potential
compounds were developed such as apitolisib (GDC-0980). However, these explorations are restricted to the replacement
of the indazole with aryl or heteroaryl group, and modification of the thieno[3,2-d]pyrimidine core of 1 have barely been
reported. It is worth mentioning that several series of PI3K inhibitors were designed by incorporating 1,3,5-triazine, a
symmetrical structure to improve the solubility and potency, such as ZSTK474 [14] (Figure 1). Therefore, thieno[3,2-
d]pyrimidine and 1,3,5-triazine had been proven to be an effectual scaffold with anti-tumor.
In this study, we applied scaffold hopping method to develop several series of 2-(thiophen-2-yl)-1,3,5-triazine
derivatives as novel PI3Kα/mTOR dual inhibitors. As a result, three series of substituted 2-(thiophen-2-yl)-1,3,5-triazine
derivatives (11a-11g, 12a-12g and 13a-13g) were designed and synthesized. In addition, the bioactivities against three
cancer cell lines (human lung adenocarcinoma cells A549, human breast cancer cells MCF-7 and human cervical cancer
cells Hela) and the normal human fetal lung fibroblast (WI-38) cell line were evaluated.

F
S
O
N
O
N
F
O
N
N
HN
N
O
S
N
N
NH
O
O
N
N
N
OH
N
N
N
N
O
O S
1
(Pictilisib,GDC-0941)
PI-103
GSK2126458
HO
O
O
O
O
N
N
N
N
N
N
N
S
N
N
N
N
N
N
N
HO
N
N
O
N
HF C
N
NH2
2
O
Compoud 3j was published
in Bioorg. Med. Chem.
Aptitolisib(GDC-0980)
ZSTK474
N
F
O
N
N
N
N
O
S
F
HN
O
N
N
N
N
F
H
O
O
F
O
HN
N
N
N
N
NH2
O
N
N
H N
2
NH
Alpelisib (BYL-719)
Copanlisib (BAY 80-6946)
Idelalisib (GS-1101)
Figure 1. Examples of PI3K inhibitors.
2
. Results and discussion
2
.1 Design strategy
To guide our design, we docked GDC-0941 and ZSTK474 into PI3Kα protein (PDB code 4L23). As depicted in
Figure 2A, the whole GDC-0941 skeleton embedded into hydrophobic pocket of PI3Kα kinase and closely combined with
PI3Kα kinase. In this cocrystal structure, the morpholine oxygen of GDC-0941 formed a crucial hydrogen bond interaction
with the hinge region of PI3Kα kinase through the amide of Val851, and the indazole moiety points toward the affinity
pocket where two nitrogen atoms on the indazole make key interactions with Asp810 and Tyr836, respectively. In addition,
the 4-methanesulfonylpiperazin-1-ylmethyl group extended out to solvent, but the sulfonyl oxygen atom could not form
any specific hydrogen bond with the solvent front of PI3Kα kinase. As illustrated in Figure 2B, the orientation of the whole
molecular skeleton of ZSTK474 into hydrophobic pocket of PI3Kα protein was basically the same as that of GDC-0941.
ZSTK474 also combined well with PI3Kα kinase and formed two strong hydrogen bonds with Val851 and Lys802.
According to the molecular simulation results and previous research of PI3K inhibitors, a variety of core can be

accommodated in PI3K kinase cavity, which further indicated that sufficient space was available within the ATP binding
site allowing for the possibility of modifying the thieno[3,2-d]pyrimidine core of GDC-0941.
In this research, we investigated the possibility of scaffold hopping from the thieno[3,2-d]pyrimidine core of GDC-
0
941 to 2-(thiophen-2-yl)-1,3,5-triazine, which was the principal breakthrough and innovation of our research, as illustrated
in Figure 3. Through this strategy, the spatial volume and length of 2-(thiophen-2-yl)-1,3,5-triazine could be further
expanded to occupy the protein cavity. We suspected that substituted 2-(thiophen-2-yl)-1,3,5-triazine derivatives would
better embed into the deep ATP pocket, occupying space adjacent to Asp810 side chain. A morpholine portion was
introduced as the hinge region binding moiety, heterocyclic aromatic or substituted aryl group at the C-6 position of the 2-
(thiophen-2-yl)-1,3,5-triazine core as the possible affinity element, which interacted with the deeper hydrophobic pocket.
Compared with the thieno[3,2-d]pyrimidine core of GDC-0941, the conformation of the new core had changed, so our
strategy was to introduce appropriate substituents on thiophene ring to explore potential interactions with the residue
Gln859 nearby. Herein, we disclosed the preparation and biological evaluation of a series of substituted 2-(thiophen-2-yl)-
1
,3,5-triazine derivatives, which demonstrated potent inhibition of the PI3K/AKT/mTOR pathway, culminating in the
discovery of 13g.
Figure 2. The docking result of GDC-0941 and ZSTK474 bound to PI3Kα protein (PDB code 4L23)
Figure 3. Design of series derivatives based on 1.

2
.2. Chemistry
The synthetic route of the intermediate compounds 8a-8b were shown in Scheme 1. Under the catalysis of
bis(triphenylphosphine)palladium(II) dichloride, compounds 2a-2b were reacted with bis(pinacolato)diboron through
Suzuki coupling reaction to generate compounds 3a-3b. In acetone solution, the nucleophilic reaction between cyanuric
chloride and morpholine yielded compound 5. Subsequently, coupling compound 5 with 3a-3b via Suzuki reaction afforded
corresponding target compounds 6a-6b. Treatment of 6a-6b with NaBH
alcohol, which reacted with thionyl chloride to give compounds 8a-8b. The synthetic route of the target compounds 11a-
1h, 12a-12h, 13a-13h, 14a-14b, 15a-15b and 16a-16b were shown in Schemes 2 and 3. Compound 8a and various
secondary amines were reacted to give compounds 9a-9h and then coupled with 1H-indazole-4-boronic acid pinacol ester,
-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol and 2-aminopyrimidine-5-boronic acid pinacol ester to obtain
4
in MeOH in ice bath provided 7a-7b as a primary
1
3
compounds 11a-11h, 12a-12h and 13a-13h. At the same way, compound 8b substituted with 1-(methylsulfonyl) piperazine
and morpholine to obtain compounds 10a-10b. Finally, three kinds of aromatic nucleus were introduced to compounds
1
0a-10b by Suzuki coupling reaction to obtain compounds 14a-14b, 15a-15b and 16a-16b.
R
R
a
O
B
S
Br
S
O
O
O
3a-3b
2
a-2b
O
N
O
N
O
N
O
N
Cl
b
c
d
e
N
N
N
N
N
N
N
N
N
N
Cl
N
Cl
Cl
N
Cl
N
Cl
N
Cl
N
Cl
R
O
R
R
S
S
S
4
5
6a-6b
7a-7b
8a-8b
HO
Cl
2
a,3a,6a,7a,8a R=H
b,3b,6b,7b,8b R=CH3
2
Scheme 1. Synthetic route of the compounds 8a-8b.
Reagents and conditions: (a) Bis(pinacolato)diboron, KAc, Pd(PPh
3
)Cl
2
, 1,2-dimethoxyethane, 80 ºC, 6 h; (b) Morpholine,
, 0 ºC, 0.5 h; (e) CH Cl , SOCl , DMF,
Acetone, 0 ºC, 1 h; (c) H
r.t., 2 h.
2
O, K
2
CO
3
, Pd(PPh
3
)Cl
2
, 75 ºC, 12 h; (d) MeOH, NaBH
4
2
2
2

O
HN
HN
HN
HN
HN
HN
HN
N S
O
OHN
O
N
N
O
N
O
N
N
NH
N

N
N
N
1
1a-11h Ar=
2a-12h Ar=
N
OH
N
O
N
N
N
N
N
N
N
a
b
1
N
Cl
N
Cl
N
Ar
N
N
N
N
S
S
S
N
8a
9a-9h
11a-11h,12a-12h,13a-13h
13a-13h Ar=
O
N S
Cl
N
N
N
NH2
O
O
B
N
O
B
O
NH
OH O B
N
O
O
N
N
NH2
Scheme 2. Synthetic route of the compounds 11a-11h, 12a-12h and 13a-13h.
Reagents and conditions: (a) K
2
CO
3
, Isopropyl alcohol, 55 ºC, 1.5 h; (b) H
2
O, K
2
CO
3
, Pd(PPh
3
)Cl
2
, 130 ºC, 8 h.
N
NH
O
B
O
B
O
B
O
HN
N S
HN
O
O
O
OH
O
N
N
O
NH
N

N
O
N
NH2
14a-14b Ar=
15a-15b Ar=
16a-16b Ar=
O
N
O
N
O
O
OH
N
N S
N
N
O
N
N
a
N
N
b
N
N
N
N
Cl
N
Cl
Ar
N
NH2
S
S
S
8b
10a-10b
14a-14b,15a-15b,16a-16b
Cl
N
N
Scheme 3. Synthetic route of the compounds 14a-14b, 15a-15b and 16a-16b.
2 3 3 2
Reagents and conditions: (a) K CO , Isopropyl alcohol, 55 ºC, 1.5 h; (b) H O, K CO , Pd(PPh )Cl , 130 ºC, 8 h.
2
3
2
2
.3. Biological evaluation
Three cancer cell lines A549, MCF-7 and Hela were selected for the anti-tumor activities of the target compounds
11a-11h, 12a-12h, 13a-13h, 14a-14b, 15a-15b and 16a-16b) by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium
(
bromide (MTT) cell proliferation assay, using PI3K inhibitor GDC-0941 as positive control. Furthermore, the preferred
target compounds (11c, 11e, 11g, 12c, 12e, 12g, 13c, 13e and 13g) were evaluated for the IC50 values against PI3Kα and
mTOR kinases by the mobility shift assay, together with reference compounds PI-103 and GDC-0941, and according to
the results of kinase inhibitory activity, the compounds 13e and 13g were further evaluated for other tyrosine kinases to
test the enzyme-based selectivity. As shown in Table 1 and Table 2, the results expressed as half-maximal inhibitory
concentration (IC50) values and the values were the average of three independent experiments. Moreover, in order to
investigate the mode of cell proliferation inhibition by compound 13g, cell cycle distribution analysis (Figure 5) and
apoptosis analysis (Figure 6) were performed on A549 cells. In addition, AO single staining (Figure 7) and western blot
(Figure 8) were carried out in this study.
As can be seen, most of compounds displayed moderate to excellent anti-tumor activities against three cancer cells
lines (A549, MCF-7 and Hela) with potency from single-digit µM to nM range in Tables 1 and 2. In general, the target

compounds had more potent anti-tumor activities against both A549 and Hela cell lines than that of MCF-7 cell line. What's
more, we could easily see that the third series of compounds 13a-13h were much more active than the other series of
compounds 11a-11h and 12a-12h against the three cancer cell lines, which suggested that introduction of 2-
aminopyrimidine group to the compounds were preferable to the anti-tumor activity. We speculated that the nitrogen atom
on 2-aminopyrimidine can provide appropriate hydrogen bond donor based on the above results, which may contribute to
high binding affinity. Among these compounds, six of them were more potent than GDC-0941 against one or more cell
lines. The most promising compound 13g exhibited the best activity against A549, MCF-7 and Hela cell lines with the IC50
values of 0.20±0.05 µM, 1.25±0.11 µM and 1.03±0.24 µM, respectively, which were better than that of lead drug GDC-
0
941.
As showed in Table 1, it is worth mentioning that the substituents of the secondary amines had an important influence
on anti-tumor activity. Compared with 11a and 11b, 12a and 12b, 13a and 13b, it was found that the compounds containing
dimethylamine groups were much more active than compounds containing diethylamine groups against the three cancer
cell lines. Similarly, the same trend was observed between pyrrolidine unit and piperidine unit. What's more, introduction
of morpholine and 1-(methylsulfonyl)piperazine to the compounds (11e and 11g, 12e and 12g, 13e and 13g) were more
beneficial to the anti-tumor activity than that of other secondary amines. We speculated that morpholine and 1-
(methylsulfonyl)piperazine groups were preferred toward the solvent front region of PI3Kα or mTOR, which can provide
appropriate hydrogen bond donor to form hydrogen bonds with amino acid residues to enhance anti-tumor activity.
Inspired by Apitolisib (GDC-0980), the electron-donating group methyl was introduced into thiophene ring to change
the electron cloud distribution of the compounds in order to improve the anti-tumor activity of the compounds. Therefore,
a methyl group was introduced into the thiophene ring of compounds 11e, 11g, 12e, 12g, 13e and 13g with excellent anti-
tumor activity. The results were shown in Table 2, these six compounds displayed moderate anticancer activity against
three cancer cells lines (A549, MCF-7 and Hela). However, introducing a methyl group on thiophene ring resulted in
decreasing anti-tumor activity, which was not as expected.
The most promising compound 13g showed only a slight improvement in anti-tumor activity compared to the class I
PI3K inhibitor GDC-0941. Therefore, their cytotoxic activity against normal cells were further compared. In this study,
compounds 13e and 13g were selected for cytotoxic activity against WI-38 normal cell line in vitro. The results of the test
were presented in Table 3, which shown that compound 13g exhibited lower cytotoxic activity against WI-38 normal cell
line compared to Class I PI3K inhibitor GDC-0941. Furthermore, the cytotoxic activity of compound 13e on WI-38 normal
cell line was similar to that of compound 13g, and their IC50 values were greater than 20 µM. These data indicated that
three cancer cells lines (A549, MCF-7 and Hela) were more sensitive than the normal cells.
Table 1

Structures and activity of target compounds 11a-11h, 12a-12h and 13a-13h.
O
N
N
N
N
Ar
R
N
S

a
IC50 (μM)
Compd.
R
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Ar
N
A549
MCF-7
Hela
1
1a
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
5.58±0.74
8.21±0.91
6.38±0.80
1
1b
8.47±0.92
4.69±0.67
21.86±1.34
2.32±0.78
8.43±0.92
1.11±0.83
51.67±1.71
13.26±1.02
15.7±1.19
8.83±0.94
23.57±1.37
1.44±0.08
21.27±1.32
0.32±0.02
12.85±1.10
5.71±0.75
12.93±1.11
3.68±0.69
12.79±1.10
0.33±0.89
20.77±1.31
6.46±0.81
8.36±0.92
4.15±0.61
22.56±1.35
2.47±0.09
6.96±0.84
3.50±0.38
6.81±0.83
4.44±0.64
17.69±1.38
3.62±0.55
5.12±0.71
3.98±0.69
49.63±1.69
5.13±0.71
13.63±1.13
3.60±0.55
7.24±0.86
0.38±0.07
8.82±0.94
2.36±0.55
1
1c
1
1d
N
1
1e
O
N
N
N
NH
1
1f
O
O
1
1g
S
1
1h
1
2a
1
2b
1
2c
1
2d
1
2e
OH
O
N
1
2f
O
1
2g
N
S
O
1
2h
H
N
N
21.27±1.02
31.87±0.92
47.26±1.43
1
3a
H
H
H
H
H
N
N
N
N
N
4.84±0.16
7.06±0.32
1.15±0.22
15.11±1.02
1.50±0.14
5.21±0.28
10.12±0.31
1.21±0.18
20.45±1.21
2.65±0.08
4.73±0.19
11.34±0.51
1.07±0.09
11.46±0.43
1.84±0.11
1
3b
1
3c
N
N
1
3d
NH2
1
3e
O

1
3f
H
H
N
N
7.83±2.21
15.69±1.28
8.52±0.23
O
O
1
3g
N
N
S
0.20±0.05
1.25±0.11
1.03±0.24
1
3h
H
-
N
N
18.38±1.72
1.21±0.13
40.07±1.92
1.47±0.10
27.46±1.35
3.72±0.26
GDC-0941b
-
-
The bold values showed excellent anti-tumor activity (0.2-2.65 µM).
aThe values are an average of three separate determinations.
bUsed as the positive control.
Table 2
Structures and activity of target compounds 14a-14b, 15a-15b and 16a-16b.
a
IC50 (μM)
Compd.
R
CH
CH
Ar
N
A549
MCF-7
Hela
1
4a
3
3
N
N
N
N
N
N
O
9.89±1.90
11.81±1.52
13.97±1.14
NH
O
O
1
4b
N
S
S
6.55±0.81
11.85±1.07
4.26±0.52
5.34±0.16
5.57±0.74
6.38±0.80
8.36±0.92
6.11±0.18
4.00±0.60
9.61±0.98
5.13±0.71
5.85±0.15
1
5a
CH3
CH3
O
OH
O
O
1
5b
N
1
6a
CH
3
3
O
N
N
O
N
1
6b
CH
-
NH2
N
S
3.86±0.84
1.20±0.13
7.04±0.84
1.47±0.10
3.52±0.14
3.72±0.26
O
GDC-0941b
-
-
aThe values are an average of three separate determinations.
bUsed as the positive control.
Table 3
Cytotoxic activity of compounds 13e and 13g against WI-38 normal cell line in Vitro
IC50 (μM)
Compd.
R
Ar
N
WI-38
1
3e
H
N
N
O
N
>20
N
O
O
N
1
3g
H
-
NH2
S
>20
GDC-0941a
-
-
8.4±1.93
aUsed as the positive control.

2
.4. PI3Kα and mTOR enzymatic assays in vitro
Experiments against PI3Kα and mTOR kinases of nine selected compounds (11c, 11e, 11g, 12c, 12e, 12g, 13c, 13e
and 13g) as well as the lead compounds GDC-0941 and PI-103 were carried out in this paper for further study. In Table 4,
obviously, the overall activity against PI3Kα kinase of compounds 13e and 13g were equal to the reference compound
GDC-0941, with the IC50 values of 9.5 nM and 7.0 nM against PI3Kα kinase. As can be seen, the overall activity against
mTOR kinase of the selected compounds were better than that of the reference compound GDC-0941. Notably, compounds
1
3e and 13g showed excellent inhibitory activity to mTOR with the IC50 values of 69 nM and 48 nM, which had an
approximately 7-fold to 10-fold improvement in mTOR inhibition, compared to the class I PI3K inhibitor GDC-0941.
What’s more, the overall activity against PI3Kα and mTOR kinases of compounds 13e and 13g were slightly less than the
reference compound PI-103. In a word, the results prompted us that compounds 13e and 13g may become promising
PI3Kα/mTOR dual inhibitors.
Table 4
PI3Kα and mTOR kinases inhibitory activity of compounds 11c, 11e, 11g, 12c, 12e, 12g, 13c, 13e, 13g, GDC-0941 and
PI-103.
IC50 (nM)
Compd.
R
Ar
N
PI3Ka
mTOR
1
1
1c
1e
H
H
H
H
H
H
H
H
H
N
N
N
N
N
N
N
N
N
747±36.29
>1000
N
O
N
731±40.65
716±33.62
395±24.32
194±13.91
55±4.20
485±19.21
439±21.09
864±56.74
183±15.87
195±11.69
483±17.63
69±6.35
NH
O
O
1
1g
S
S
S
1
1
2c
2e
OH
O
N
O
O
1
2g
1
1
3c
3e
151±8.12
9.5±1.14
O
N
N
O
O
N
1
3g
NH2
7.0±0.99
48±2.47
GDC-0941a
PI-103a
-
-
6.0±1.12
5.1±1.03
525±28.33
21±3.73
-
-
-
-
aUsed as the positive control.

2
.5. Enzymatic selectivity assays
In order to examine whether compounds 13e and 13g were selective PI3Kα/mTOR dual inhibitors, compounds 13e
and 13g were evaluated against other tyrosine kinases. The obtained results were presented in Figure 4, which suggested
that compound 13g exhibited low inhibitory effects against EGFR (8.3%), c-Met (2.0%), VEGFR-2 (0.5%), and EGFR
T790M/L858R
(2.3%) at the concentration of 1 µM. However, compound 13g inhibited mTOR and PI3Kα kinases by more than
9
3% at the dose of 1 µM, especially PI3Kα kinase, up to 100%. The selectivity of compound 13e for kinases was similar
to that of compound 13g. These data demonstrated that compounds 13e and 13g were selective PI3Kα/mTOR dual
inhibitors.
100
13e
13g
80
60
40
20
0
L858R/T790M
EGFR
c-Met
VEGFR-2 EGFR
mTOR
PI3K
Figure 4. Inhibition of tyrosine kinases by compounds 13e and 13g at 1 µM
2
.6. Effect on cell cycle progression.
To explore the way of cell proliferation inhibition by compound 13g, cell cycle distribution analysis was carried out
on A549 cells. The results were shown in Figure 5. As can be seen, compared with the control group, compound 13g can
induce cell cycle arrest of A549 cells at the G0/G1 checkpoint at low concentration, in a dose-dependent manner. As the
concentration of compound 13g increasing, compound 13g significantly increased the G0/G1 phase population from 69.44%
to 77.70%, while the S phase was decreased, while no significant change in G2/M phase was observed. What's more, at a
concentration of 1.6 µM, compound 13g induced cell cycle arrest of A549 cells at the G0/G1 checkpoint with 77.70%,
which was similar to that of GDC-0941 (76.22%).

Figure 5. Cell cycle progression analyses of A549 cells treated with compound 13g and GDC-0941 for 48 h.
.7. Effect on apoptosis
2
To obtain more insight into the growth inhibition mechanism of tumor cells, the influence of compound 13g on
apoptosis was investigated. A549 cells were treated with 0.4 µM, 0.8 µM and 1.6 µM of compound 13g for 48 h,
respectively, and then the flow cytometric apoptosis experiments were carried out. The results of apoptosis analysis were
illustrated in Figure 6, which showed that compound 13g (the total apoptosis rate was 16.05% at 1.6 µM) could induce
1
2.51% increase of apoptotic cells, compared with the control group (the total apoptosis rate was 3.54%). As the
concentration of compound 13g increasing, the total apoptosis rate was also increased, from 7.18% to 16.05%, in a dose-
dependent manner. These findings demonstrated that compound 13g successfully induced apoptosis in a dose-dependent
manner, and the effect was similar to that of GDC-0941.

Figure 6. Apoptosis analyses of A549 cells treated with compound 13g and GDC-0941 for 48 h.
.8. Morphologic changes of A549 cells under inverted microscopy and fluorescence microscopy
2
In order to evaluate whether compound 13g was able to induce apoptotic, the apoptotic experiments were performed
to detect the effect of compound 13g on A549 cells by acridine orange (AO) single staining.
As shown in Figure 7, the control cells (Figure 7A) were showing that the nucleus of A549 cells were stained with
acridine orange (AO) from fluorescence microscopy, and shape of the cell was full, and the edge was clear and the refraction
was great. However, when the cells were treated with 0.2 µM and 0.8 µM of compound 13g, it can be seen in Figures 7B
and 7C that the A549 cells showed significant evidence of apoptosis mediated cell death, such as cell shrinkage. It indicated
that compound 13g could induce apoptosis of A549 cells, which were consistent with the former result of the flow
cytometric apoptosis experiments.
Figure 7. Morphologic changes of A549 cells under inverted microscopy and fluorescence microscopy.

2
.9. Western blot assay in vitro
To further determine whether the target compounds would inhibit the activation of both PI3K/Akt/mTOR signalings
in cancer cell, western blot assay was performed to test the expression of relative proteins. As shown in Figure 8, a dose-
dependent inhibition of the phosphorylation of AKT was found after the treatment of compound 13g. Compound 13g could
efficiently suppress the phosphorylation of AKT at the dose of 0.1 µM. These results were in accordance with the results
of enzymatic assays, which further demonstrated compound 13g had significant inhibitory effect on the PI3K/Akt/mTOR
pathway. Furthermore, western blot analysis indicated that compound 13g significantly reduced the protein expression of
p-GSK-3β in A549 cells, in a dose-dependent, but total GSK-3β protein level remained unchanged.
Figure 8. Effects of compound 13g on the phosphorylation level of AKT in A549 cells after 24 h exposure.
2
.10. Molecular docking study
In order to explore the binding modes of target compounds with the active site of PI3Kα (PDB code: 4L23) and mTOR
(PDB code: 4JT6), molecular docking simulation studies were performed by AutoDock 4.2 software (The Scripps Research
Institute, USA) and the docking results were processed and modified in PyMOL 1.8.x software (https://pymol.org).
According to the above test results, the representative compound 13g as ligand examples was selected, as the best
PI3Kα/mTOR inhibitor in this study. As shown in Figure 9, the binding mode of 13g to PI3Kα was very similar to that of
GDC-0941, which was responsible for similar potency against PI3Kα. The orientation of the whole molecular skeleton of
compound 13g into hydrophobic pocket of PI3Kα protein was basically the same as that of GDC-0941 and the morpholine
formed a key hydrogen bond with Val851 (Val2240 on mTOR) in the hinge region with the hydrogen bond lengths of 1.9
Å (1.8 Å, mTOR). The amino group at the 2-aminopyrimidine engaged in another hydrogen bond with Asp810 and Lys802
(
Tyr2225 and Asp2195 on mTOR) with the hydrogen bond lengths of 2.5 Å and 2.6 Å, respectively, which was the reason
why the three series of compounds 13a-13h were much more active than the other series of compounds 11a-11h and 12a-
2h against the three cancer cell lines. Furthermore, 1-(methylsulfonyl)piperazine group of compound 13g was preferred
1

toward the solvent front region of PI3Kα and an additional hydrogen bond was formed between the oxygen of 1-
methylsulfonyl)piperazine group and the residue GLN859 with the hydrogen bond lengths of 1.8 Å, which verifies our
(
conjecture above. In general, these results of the molecular docking study showed that 2-(thiophen-2-yl)-1,3,5-triazine
derivatives bearing 1-(methylsulfonyl)piperazine group and 2-aminopyrimidine group could act synergistically to interact
with the active binding site of PI3Kα and mTOR, which claimed that compound 13g may be a potential PI3K/mTOR dual
inhibitors. Additionally, based on the results of structure-activity relationships analysis and molecular docking study, more
potential PI3K/mTOR dual inhibitors may allow to be designed rationally.
Figure 9. Docking mode of compound 13g.
(
A) (C) The binding model of representative compound 13g bound to PI3Kα (PDB code: 4L23).
B) (D) The binding model of representative compound 13g bound to mTOR (PDB code: 4JT6).
(
3
. Conclusions
In summary, based on the class I PI3K inhibitor GDC-0941, a new series of substituted 2-(thiophen-2-yl)-1,3,5-
triazine derivatives as potent PI3Kα/mTOR inhibitors were developed and evaluated them for the IC50 values against three
cancer cell lines (A549, MCF-7 and Hela). Most of the compounds showed moderate to excellent anti-tumor activity against
the different cancer cells. Notably, compound 13g showed the best activity against A549, MCF-7 and Hela cancer cell lines
with IC50 values of 0.20±0.05 µM, 1.25±0.11 µM and 1.03±0.24 µM. According to the result of enzymatic activity assay
and western blot, compound 13g was identified as novel PI3Kα/mTOR dual inhibitors, which had an approximately 10-
fold improvement in mTOR inhibition relative to the class I PI3K inhibitor Pictilisib (GDC-0941). Moreover, compound
1
3g could stimulate A549 cells arrest at G0/G1 phase and induced apoptosis at a low concentration. By far, the existing
data indicated that compound 13g may become a potential PI3Kα/mTOR dual inhibitor for oncology indications.

4
. Experimental section
4
.1. Chemistry
Unless otherwise required, all reagents were obtained from commercial analytical grade and were used without further
purification. All melting points were obtained on a Büchi Melting Point B-540 apparatus (Büchi Labortechnik, Flawil,
Switzerland) and were uncorrected. TLC analysis was carried out on silica gel plates GF254 (Qindao Haiyang Chemical,
China) with fluorescent indicator 254 nm. Column chromatography was run on silica gel (200-300 mesh) from Qingdao
Ocean Chemicals (Qingdao, Shandong, China). 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
260 liquid chromatograph fitted with an Inertex-C18 column. HNMR and 13CNMR spectra were recorded on Bruker
1
1
ARX-400, 400 MHz spectrometers (Bruker Bioscience, Billerica, MA, USA) with tetramethylsilane (TMS) as an internal
standard.
4
.2. Preparation of 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)thiophene-2-carbaldehyde (3a) and 3-methyl-5-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)thiophene-2-carbaldehyde (3b)
Compound 3a and 3b were synthesized according to the procedures in previous research [15], and yields are 76.9%
and 68.3%, respectively.
4
.3. Preparation of 4-(4,6-dichloro-1,3,5-triazin-2-yl)morpholine (5)
To a stirred solution of cyanuric chloride (6.5 g, 35 mmol) in acetone (80 mL), a solution of morpholine (2.0 g, 23
mmol) in acetone (20 mL) was added dropwise at 0 ºC. The resulting mixture was quenched with water, filtered, washed
with water and dried to get compound 5 (4.3 g, 79.6%).
4
.4. Preparation of 5-(4-chloro-6-morpholino-1,3,5-triazin-2-yl)thiophene-2-carbaldehyde (6a)
Compound 3a (3.0 g, 12.6 mmol) was combined with bis(triphenylphosphine)palladium(II)chloride (0.21 g) and
compound 5 (3.0 g, 12.7 mmol) in 1,2-dimethoxyethane (100 mL) and 25 mL of 1 M potassium carbonate in water. The
reaction mixture was heated to 75 ºC for 8 h and monitored by TLC. After cooling to room temperature, the reaction was
quenched with saturated aqueous NaCl and then extracted with DCM. The combined organic layer was dried over
anhydrous Na
2 4
SO and concentrated in reduced pressure to obtain the crude product. The crude produc was purified through
a column chromatography on silica with petroleum ether/ethyl acetate as eluent to obtain 6a as yellow solid in 75.9% yield.
4
8
.5. Preparation of 5-(4-chloro-6-morpholino-1,3,5-triazin-2-yl)-3-methylthiophene-2-carbaldehyde (6b)
The synthetic method of compound 6b was the same to compound 6a. Compound 6b was obtained as yellow solid in
1.7% yield.

4
.6. Preparation of (5-(4-chloro-6-morpholino-1,3,5-triazin-2-yl)thiophen-2-yl)methanol (7a)
To a solution of compound 6a (1 g, 3.2 mmol) in methanol (10 mL) was added sodium borohydride (0.4 g, 10.5 mmol)
in small portions at 0 and the reaction was stirred for 1 h. The solvent was evaporated and the residue partitioned between
ethyl acetate (20 mL) and 10% ammonium chloride solution (10 mL). The organic layer was washed with water (10 mL),
dried over sodium sulfate and evaporated to obtain compound (0.9 g, 90.0%) as yellow solid.
4
.7. Preparation of (5-(4-chloro-6-morpholino-1,3,5-triazin-2-yl)-3-methylthiophen-2-yl)methanol (7b)
The synthetic method of compound 7b was the same to compound 7a. Compound 7b was obtained as yellow solid in
0.0% yield.
9
4
.8. Preparation of 4-(4-chloro-6-(5-(chloromethyl)thiophen-2-yl)-1,3,5-triazin-2-yl)morpholine (8a)
2 2
To a stirred solution of compound 7a (2.0 g, 6.3 mmol) in CH C1 (50 mL), thionyl chloride (2.3 mL, 19.3 mmol) and
a few drops of DMF were added. The mixture was stirred at room temperature for 4 hours and monitored by TLC. After
completion, the reaction was quenched with saturated aqueous NaCl and then extracted with DCM. The combined organic
layer was dried over anhydrous Na
2
SO
4
, filtered and concentrated to get yellow solid in 96.4% yield, and then compound
8
a directly used for further reaction.
4
.9. Preparation of 4-(4-chloro-6-(5-(chloromethyl)-4-methylthiophen-2-yl)-1,3,5-triazin-2-yl)morpholine (8b)
The synthetic method of compound 8b was the same to compound 8a. Compound 8b was obtained as yellow solid in
5.7% yield.
9
4
.10. Preparation of 1-(5-(4-chloro-6-morpholino-1,3,5-triazin-2-yl)thiophen-2-yl)-N,N-dimethylmethanamine (9a)
2 3
To a solution of compound 8a (2.0 g, 0.012 mol), Dimethylamine (0.54 g, 0.012 mol) and K CO (2.48 g, 0.018 mol)
in isopropyl alcohol (20 mL) were added. The reaction mixture was heated to 55 ºC for 4 h and monitored by TLC. After
completion, the reaction was quenched with saturated aqueous NaCl and then extracted with DCM. The combined organic
layer was dried over anhydrous Na
2
SO
4
, filtered and concentrated to get yellow solid in 92.9% yield, and then compound
9
a directly used for further reaction.
4
.11. Preparation of compounds 9b-9h
The synthetic method of compounds 9b-9h was the same to compound 9a. Compounds 9b-9h as yellow solid in 80.0-
5.6% yield.
9
4
.12. Preparation of 4-(4-chloro-6-(4-methyl-5-(morpholinomethyl)thiophen-2-yl)-1,3,5-triazin-2-yl)morpholine (10a)
To a solution of compound 8b (2.0 g, 0.012 mol), morpholine (0.54 g, 0.012 mol) and K CO (2.48 g, 0.018 mol) in
isopropyl alcohol (20 mL) were added. The reaction mixture was heated to 55 ºC for 4 h and monitored by TLC. After
2
3

completion, the reaction was quenched with saturated aqueous NaCl and then extracted with DCM. The combined organic
layer was dried over anhydrous Na SO , filtered and concentrated to get yellow solid in 93.7% yield.
2
4
4
.13. Preparation of 4-(4-chloro-6-(4-methyl-5-((4-(methylsulfonyl)piperazin-1-yl)methyl)thiophen-2-yl)-1,3,5-triazin-2 -
yl)morpholine (10b)
The synthetic method of compound 10b was the same to compound 10a. Compound 10b was obtained as yellow solid
in 96.1% yield.
4
.14. General procedure for the preparation of compounds 11a-11h
Compounds 9a-9h was combined with bis(triphenylphosphine)palladium(II)chloride (0.21 g) and 1H-indazole- 4-
boronic acid pinacol ester (1.68 g) in 1,2-dimethoxyethane (20 ml) and 15 mL of 1 M potassium carbonate in water. The
reaction mixture was heated to 130 ºC for 4 h and monitored by TLC. After cooling to room temperature, the reaction was
quenched with saturated aqueous NaCl and then extracted with DCM. The combined organic layer was dried over
anhydrous Na
2 4
SO and concentrated in reduced pressure to obtain the crude product. The crude produc was purified through
a column chromatography on silica with dichloromethane/methanol as eluent to produce 11a-11h as yellow solid.
4
1
.14.1. 1-(5-(4-(1H-indazol-4-yl)-6-morpholino-1,3,5-triazin-2-yl)thiophen-2-yl)-N,N-dimethylmethanamine (11a)
1
Yellow solid in 40.7% yield. M.P.: 106.9-107.8 ºC. H NMR (400 MHz, CDCl
3
) δ 9.07 (s, 1H), 8.44 (d, J = 7.3 Hz,
H), 8.06 (d, J = 3.6 Hz, 1H), 7.69 (d, J = 8.2 Hz, 1H), 7.54-7.52 (m, 1H), 7.03 (s, 1H), 4.08 (d, J = 15.9 Hz, 4H), 3.84 (d,
1
3
J = 2.1 Hz, 5H), 3.74 (s, 2H), 2.37 (s, 6H). C NMR (100 MHz, CDCl
3
) δ 170.04, 168.86, 168.59, 142.00, 138.93, 136.85,
1
28.40, 125.54 (2, C), 122.26 (2, C), 121.85, 118.65, 118.23, 66.31 (2, C), 56.62, 45.38 (2, C), 44.13 (2, C). TOF MS ES
+
+
23 7
(m/z): (M + H) , calcd for C21H N OS: 422.1763, found, 422.1752.
4
.14.2. N-((5-(4-(1H-indazol-4-yl)-6-morpholino-1,3,5-triazin-2-yl)thiophen-2-yl)methyl)-N-ethylethanamine (11b)
1
Yellow solid in 50.5% yield. M.P.: 115.9-117.1 ºC. H NMR (400 MHz, CDCl
3
) δ 9.06 (s, 1H), 8.43 (d, J = 7.2 Hz,
1
3
4
H), 8.05 (d, J = 3.3 Hz, 1H), 7.69 (d, J = 7.9 Hz, 1H), 7.50 (t, J = 7.7 Hz, 1H), 7.02 (s, 1H), 4.06 (s, 4H), 3.90 (s, 2H),
+
.83 (s, 4H), 2.65 (dd, J = 14.0, 6.9 Hz, 4H), 1.13 (t, J = 6.9 Hz, 6H). TOF MS ES + (m/z): (M + H) , calcd for C23
H
27
N
7
OS:
50.2076, found, 450.2060.
4
.14.3. 4-(4-(1H-indazol-4-yl)-6-(5-(pyrrolidin-1-ylmethyl)thiophen-2-yl)-1,3,5-triazin-2-yl)morpholine (11c)
1
Yellow solid in 30.9% yield. M.P.: 128.5-131.3 ºC. H NMR (400 MHz, CDCl
3
) δ 9.03 (s, 1H), 8.42 (s, 1H), 8.03 (s,
1
H), 7.72 (s, 1H), 7.52 (s, 1H), 7.30 (s, 1H), 4.20 (s, 2H), 4.06 (s, 4H), 3.84 (s, 4H), 3.04 (s, 4H), 1.99 (s, 4H). TOF MS
+
ES + (m/z): (M + H) , calcd for C23
H
25
N
7
OS: 448.1920, found, 448.1919.
4
.14.4. 4-(4-(1H-indazol-4-yl)-6-(5-(piperidin-1-ylmethyl)thiophen-2-yl)-1,3,5-triazin-2-yl)morpholine (11d)

1
Yellow solid in 61.2% yield. M.P.: 162.1-163.4 ºC. H NMR (400 MHz, CDCl
3
) δ 9.06 (s, 1H), 8.44 (s, 1H), 8.06 (s,
1
H), 7.71 (s, 1H), 7.52 (s, 1H), 7.10 (s, 1H), 4.08 (s, 4H), 3.85 (s, 6H), 2.61 (s, 4H), 1.71 (s, 6H). 13C NMR (100 MHz,
CDCl
3
) δ 170.43, 166.62, 164.14, 140.25 (2, C), 136.16, 129.63, 126.42 (2, C), 125.67 (2, C), 122.59, 121.12, 112.73,
+
6
4
6.07 (2, C), 56.86, 53.18 (2, C), 43.16 (2, C), 28.95 (2, C), 24.59. TOF MS ES + (m/z): (M + H) , calcd for C24
H
27
N
7
OS:
62.2076, found, 462.2070.
4
.14.5. 4-(4-(1H-indazol-4-yl)-6-(5-(morpholinomethyl)thiophen-2-yl)-1,3,5-triazin-2-yl)morpholine (11e)
1
Light yellow solid in 45.6% yield. M.P.: 110.6-120.3 ºC. H NMR (400 MHz, CDCl
3
) δ 9.09 (s, 1H), 8.45 (s, 1H),
1
3
8
.05 (s, 1H), 7.71 (s, 1H), 7.54 (s, 1H), 7.05 (s, 1H), 4.08 (s, 4H), 3.82 (d, J = 20.0 Hz, 12H), 2.59 (s, 4H). C NMR (100
MHz, CDCl
3
) δ 170.45, 165.70, 164.30, 140.44, 139.41, 137.58, 130.33, 126.35 (2, C), 125.93 (2, C), 123.24, 121.79,
+
1
4
20.22, 66.80 (2, C), 60.45 (2, C), 58.46, 52.21 (2, C), 43.83 (2, C). TOF MS ES + (m/z): (M + H) , calcd for C23
H
25
N
7
O
2
S:
64.1869, found, 464.1870.
4
.14.6. 4-(4-(1H-indazol-4-yl)-6-(5-((4-methylpiperazin-1-yl)methyl)thiophen-2-yl)-1,3,5-triazin-2-yl)morpholine (11f)
1
Light yellow solid in 59.3% yield. M.P.: 127.6-128.3 ºC. H NMR (400 MHz, CDCl
3
) δ 9.07 (s, 1H), 8.44 (d, J = 7.3
Hz, 1H), 8.04 (d, J = 3.7 Hz, 1H), 7.68 (d, J = 8.2 Hz, 1H), 7.51 (t, J = 7.8 Hz, 1H), 7.00 (d, J = 3.6 Hz, 1H), 4.07 (s, 4H),
1
3
3
1
5
3
.85 (d, J = 4.5 Hz, 4H), 3.77 (s, 2H), 2.57 (s, 8H), 2.33 (s, 3H). C NMR (100 MHz, CDCl ) δ 170.14, 166.45, 163.84,
40.91, 139.97, 135.83, 129.23, 126.13 (2, C), 125.34 (2, C), 123.34, 122.26, 120.84, 65.80 (2, C), 56.60, 53.88 (2, C),
+
1.70 (2, C), 44.70 (2, C), 42.86. TOF MS ES + (m/z): (M + H) , calcd for C24
H
28
N
8
OS: 477.2185, found, 477.2180.
4
.14.7. 4-(4-(1H-indazol-4-yl)-6-(5-((4-(methylsulfonyl)piperazin-1-yl)methyl)thiophen-2-yl)-1,3,5-triazin-2-yl)morpho-
line (11g)
Yellow solid in 63.2% yield. M.P.: 124.8-125.5 ºC. H NMR (400 MHz, CDCl
1
3
) δ 9.08 (s, 1H), 8.44 (d, J = 7.4 Hz,
1
H), 8.03 (d, J = 3.5 Hz, 1H), 7.69 (d, J = 8.0 Hz, 1H), 7.52 (dd, J = 8.2, 4.6 Hz, 1H), 7.00 (s, 1H), 4.07 (s, 4H), 3.84-3.80
+
(
m, 6H), 3.29 (s, 4H), 2.79 (s, 3H), 2.66 (s, 4H). TOF MS ES + (m/z): (M + H) , calcd for C24
H
28
N
8
O
3
S2: 541.1804, found,
5
41.1808.
1
1
2
2
4
.14.8. N -((5-(4-(1H-indazol-4-yl)-6-morpholino-1,3,5-triazin-2-yl)thiophen-2-yl)methyl)-N ,N ,N -trimethylethane-1, 2-
diamine (11h)
Yellow solid in 48.7% yield. M.P.: 111.3-113.2 ºC. H NMR (400 MHz, CDCl
1
3
) δ 9.04 (s, 1H), 8.41 (s, 1H), 7.99 (s,
1
H), 7.72 (s, 1H), 7.49 (s, 1H), 6.99 (s, 1H), 4.04 (s, 4H), 3.83 (s, 8H), 2.88 (d, J = 33.5 Hz, 4H), 2.66 (s, 6H), 2.34 (s, 3H).
+
TOF MS ES + (m/z): (M + H) , calcd for C24
H
30
N
8
OS: 479.2342, found, 479.2351.
4
.15. General procedure for the synthesis of compounds 12a-12h and 13a-13h

The synthetic method of compounds 12a-12h and 13a-13h were similar to compounds 11a-11h. The difference was
that we needed to replace 1H-indazole-4-boronic acid pinacol ester with 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan- 2-
yl)phenol and 2-aminopyrimidine-5-boronic acid pinacol ester and the rest of the synthesis conditions were the same.
4
.15.1. 3-(4-(5-((dimethylamino)methyl)thiophen-2-yl)-6-morpholino-1,3,5-triazin-2-yl)phenol (12a)
1
Light yellow solid in 72.5% yield. M.P.: 133.5-134.7 ºC. H NMR (400 MHz, CDCl
3
) δ 8.14–7.89 (m, 3H), 7.36 (d,
J = 6.9 Hz, 1H), 7.03 (d, J = 18.2 Hz, 2H), 4.00 (s, 4H), 3.84 (d, J = 18.8 Hz, 6H), 2.42 (s, 6H). 13C NMR (100 MHz,
CDCl
3
) 170.18, 166.71, 164.22, 156.02, 141.83, 137.50, 129.60, 129.03, 127.39 (2, C), 119.99, 118.81, 114.92, 66.28 (2,
+
C), 57.55, 43.98 (2, C), 43.17 (2, C). TOF MS ES + (m/z): (M + H) , calcd for C20
H
23
N
5
O
2
S: 398.1651, found, 398.1652.
4
.15.2. 3-(4-(5-((diethylamino)methyl)thiophen-2-yl)-6-morpholino-1,3,5-triazin-2-yl)phenol (12b)
1
3
Light yellow solid in 57.2% yield. M.P.: 116.4-117.7 ºC. H NMR (400 MHz, CDCl ) δ 7.87-7.85 (m, 3H), 7.17 (d, J
=
8.0 Hz, 1H), 6.83 (d, J = 12.6 Hz, 2H), 3.82 (s, 6H), 3.64 (s, 4H), 2.51 (d, J = 6.2 Hz, 4H), 1.01–0.98 (m, 6H). TOF MS
+
ES + (m/z): (M + H) , calcd for C22
H
27
N
5
O
2
S: 426.1964 , found, 426.1958.
4
.15.3. 3-(4-morpholino-6-(5-(pyrrolidin-1-ylmethyl)thiophen-2-yl)-1,3,5-triazin-2-yl)phenol (12c)
1
3
Light yellow solid in 56.8% yield. M.P.: 134.5-135.7 ºC. H NMR (400 MHz, CDCl ) δ 8.02 (d, J = 7.7 Hz, 1H), 7.91
(d, J = 10.9 Hz, 2H), 7.33 (d, J = 7.4 Hz, 1H), 7.02 (d, J = 13.6 Hz, 2H), 3.98 (s, 6H), 3.80 (s, 4H), 2.76 (s, 4H), 1.87 (s,
+
4
4
H). TOF MS ES + (m/z): (M + H) , calcd for C22
H
25
N
5
O
2
S: 424.1807, found, 424.1808.
.15.4. 3-(4-morpholino-6-(5-(piperidin-1-ylmethyl)thiophen-2-yl)-1,3,5-triazin-2-yl)phenol (12d)
1
Light yellow solid in 54.7% yield. M.P.: 145.5-146.3 ºC. H NMR (400 MHz, CDCl
3
) δ 8.01 (d, J = 7.6 Hz, 1H), 7.93
(d, J = 3.4 Hz, 2H), 7.35 (t, J = 7.7 Hz, 1H), 7.05 (d, J = 7.1 Hz, 1H), 6.97 (s, 1H), 3.97 (d, J = 20.2 Hz, 6H), 3.82 (s, 4H),
2
1
.66 (s, 4H), 1.70 (s, 4H), 1.45 (s, 2H). 13C NMR (100 MHz, CDCl
) δ 170.20, 166.67, 164.20, 156.31, 141.83, 137.33,
3
29.60, 129.08, 128.06 (2, C), 119.66, 119.05, 114.87, 66.31 (2, C), 57.02, 52.97 (2, C), 43.15 (2, C), 24.93 (2, C), 23.32.
+
TOF MS ES + (m/z): (M + H) , calcd for C23
H
27
N
5
O
2
S: 438.1964, found, 438.1958.
4
.15.5. 3-(4-morpholino-6-(5-(morpholinomethyl)thiophen-2-yl)-1,3,5-triazin-2-yl)phenol (12e)
1
Light yellow solid in 65.7% yield. M.P.: 120.5-122.8 ºC. H NMR (400 MHz, CDCl
3
) δ 8.16 (d, J = 7.8 Hz, 1H), 8.08
1
3
(
d, J = 3.5 Hz, 2H), 7.46-7.43 (m, 1H), 7.14-7.11 (m, 2H), 4.12 (s, 4H), 3.90 (s, 10H), 2.70 (s, 4H). C NMR (100 MHz,
CDCl
3
) δ 170.58, 168.57, 164.73, 157.37, 141.27, 138.20, 130.73, 130.20, 129.59 (2, C), 120.96, 115.26, 117.61, 66.92 (2,
+
C), 66.81 (2, C), 58.50, 53.40 (2, C), 43.69 (2, C). TOF MS ES + (m/z): (M + H) , calcd for C22
H
25
N
5
O
3
S: 440.1756, found,
4
4
40.1761.
.15.6. 3-(4-(5-((4-methylpiperazin-1-yl)methyl)thiophen-2-yl)-6-morpholino-1,3,5-triazin-2-yl)phenol (12f)

1
3
Light yellow solid in 63.8% yield. M.P.: 220.5-221.3 ºC. H NMR (400 MHz, CDCl ) δ 7.98 (s, 1H), 7.89 (d, J = 8.8
Hz, 2H), 7.34 (s, 1H), 7.09 (s, 1H), 7.00 (s, 1H), 3.94 (s, 4H), 3.73 (s, 6H), 2.41 (d, J = 39.0 Hz, 8H), 2.17 (s, 3H). TOF
+
MS ES + (m/z): (M + H) , calcd for C23
H
28
N
6
O
2
S: 453.2073, found, 453.2075.
4
.15.7. 3-(4-(5-((4-(methylsulfonyl)piperazin-1-yl)methyl)thiophen-2-yl)-6-morpholino-1,3,5-triazin-2-yl)phenol (12g)
1
Light yellow solid in 70.8% yield. M.P.: 156.3-157.7 ºC. H NMR (400 MHz, DMSO-d
6
) δ 8.04 (d, J = 19.5 Hz, 1H),
7
6
1
.89 (d, J = 7.6 Hz, 2H), 7.34 (t, J = 7.7 Hz, 1H), 7.14 (s, 1H), 7.00 (d, J = 7.9 H z, 1H), 3.93 (d, J = 13.3 Hz, 4H), 3.73 (s,
1
3
6
H), 3.47 (d, 4H), 3.15 (s, 3H), 2.90 (d, 4H). C NMR (100 MHz, DMSO-d ) δ 171.61, 168.23, 165.68, 159.05, 141.94,
38.74, 131.91, 131.06, 128.67 (2, C), 120.84, 120.64, 116.40, 67.44 (2, C), 58.28, 56.17 (2, C), 53.93 (4, C), 44.92. TOF
+
MS ES + (m/z): (M + H) , calcd for C23
H
28
N
6
O
4
S
2
: 517.1692, found, 517.1690.
4
.15.8. 3-(4-(5-(((2-(dimethylamino)ethyl)(methyl)amino)methyl)thiophen-2-yl)-6-morpholino-1,3,5-triazin-2-yl)phenol
12h)
Yellow solid in 26.8% yield. M.P.: 121.9-213.4 ºC. H NMR (400 MHz, CDCl
(
1
3
) δ 8.10-7.91 (m, 3H), 7.36 (d, J = 6.9
Hz, 1H), 7.03 (d, J = 18.2 Hz, 2H), 3.92 (s, 6H), 3.71 (s, 4H), 2.75 (s, 4H), 2.67 (s, 6H), 2.35 (s, 3H). TOF MS ES + (m/z):
+
(
M + H) , calcd for C23
H
30
N
6
O
2
S: 445.2229, found, 445.2215.
4
.15.9. 5-(4-(5-((dimethylamino)methyl)thiophen-2-yl)-6-morpholino-1,3,5-triazin-2-yl)pyrimidin-2-amine (13a)
1
Yellow solid in 47.8% yield. M.P.: 115.3-116.9 ºC. H NMR (400 MHz, CDCl
3
) δ 9.31 (s, 2H), 7.99 (s, 1H), 7.03 (s,
+
1
3
H), 5.48 (s, 2H), 4.00 (s, 4H), 3.81 (s, 4H), 3.76 (s, 2H), 2.38 (s, 6H). TOF MS ES + (m/z): (M + H) , calcd for C18
22 8
H N OS:
99.1716, found, 399.1719.
4
.15.10. 5-(4-(5-((diethylamino)methyl)thiophen-2-yl)-6-morpholino-1,3,5-triazin-2-yl)pyrimidin-2-amine (13b)
1
Yellow solid in 36.6% yield. M.P.: 127.5-128.8 ºC. H NMR (400 MHz, CDCl
3
) δ 9.33 (s, 2H), 8.01 (s, 1H), 6.99 (s,
+
1
H), 5.49 (s, 2H), 4.02 (s, 4H), 3.85 (d, J = 18.3 Hz, 6H), 2.64 (s, 4H), 1.71 (s, 6H). TOF MS ES + (m/z): (M + H) , calcd
OS: 427.2029, found, 427.2035.
.15.11. 5-(4-morpholino-6-(5-(pyrrolidin-1-ylmethyl)thiophen-2-yl)-1,3,5-triazin-2-yl)pyrimidin-2-amine (13c)
26 8
for C20H N
4
1
3
Yellow solid in 66.7% yield. M.P.: 166.3-167.5 ºC. H NMR (400 MHz, CDCl ) δ 9.29 (s, 2H), 7.97 (s, 1H), 6.97 (s,
1
3
1
1
3
H), 5.57 (s, 2H), 3.99 (s, 4H), 3.83 (d, J = 22.4 Hz, 6H), 2.60 (s, 4H), 1.25 (s, 4H). C NMR (100 MHz, CDCl ) δ 168.30,
67.26, 164.33, 159.45, 149.86 (2, C), 141.13 (2, C), 130.36, 126.28, 120.62, 66.77 (2, C), 55.18, 54.01 (2, C), 43.69 (2,
+
C), 23.60 (2, C). TOF MS ES + (m/z): (M + H) , calcd for C20
H
24
N
8
OS :425.1872, found, 425.1866.
4
.15.12. 5-(4-morpholino-6-(5-(piperidin-1-ylmethyl)thiophen-2-yl)-1,3,5-triazin-2-yl)pyrimidin-2-amine (13d)
1
3
Light yellow solid in 48.9% yield. M.P.: 250.8-251.5 ºC. H NMR (400 MHz, CDCl ) δ 9.32 (s, 2H), 7.99 (s, 1H),

6
.98 (s, 1H), 5.42 (s, 2H), 4.01 (s, 4H), 3.82 (s, 4H), 3.76 (s, 2H), 2.50 (s, 4H), 1.27 (s, 6H). TOF MS ES + (m/z): (M +
+
H) , calcd for, C21
H
26
N
8
OS: 439.2029, found, 439.2019.
4
.15.13. 5-(4-morpholino-6-(5-(morpholinomethyl)thiophen-2-yl)-1,3,5-triazin-2-yl)pyrimidin-2-amine (13e)
1
Light yellow solid in 35.9% yield. M.P.: 270.2-271.4 ºC. H NMR (400 MHz, CDCl
3
) δ 9.19 (s, 2H), 8.00 (s, 1H),
+
7
4
.00 (s, 1H), 5.38 (s, 2H), 3.83 (d, J = 45.6 Hz, 14H), 1.27 (s, 4H). TOF MS ES + (m/z): (M + H) , calcd for C20
H
24
N
8
O
2
S:
41.1821, found, 441.1813.
4
.15.14. 5-(4-(5-((4-methylpiperazin-1-yl)methyl)thiophen-2-yl)-6-morpholino-1,3,5-triazin-2-yl)pyrimidin-2-amine (13f)
1
Yellow solid in 63.2% yield. M.P.: 210.3-211.9 ºC. H NMR (400 MHz, DMSO-d
6
) δ 9.30 (s, 2H), 7.97 (d, J = 3.6
Hz, 1H), 6.98 (s, 1H), 5.62 (s, 2H), 4.00 (s, 4H), 3.84-3.74 (m, 6H), 2.59 (s, 8H), 2.35 (s, 3H). TOF MS ES + (m/z): (M +
+
H) , calcd for C21
H
27
N
9
OS: 454.2138, found, 454.2142.
4
.15.15. 5-(4-(5-((4-(methylsulfonyl)piperazin-1-yl)methyl)thiophen-2-yl)-6-morpholino-1,3,5-triazin-2-yl)pyrimidin-2-
amine (13g)
1
Yellow solid in 30.7% yield. M.P.: 261.1-262.4 ºC. H NMR (400 MHz, CDCl3) δ 9.32 (s, 2H), 7.96 (s, 1H), 6.89 (s,
1
H),5.48 (s, 2H), 4.03-4.00 (m, 4H), 3.84-3.82 (m, 4H), 3.73 (s, 2H), 3.31 (s, 4H), 2.83 (s, 3H), 2.68 (s, 4H). 13C NMR
(
100MHz, CDCl
3
) δ 168.40 (2, C), 165.49, 160.64, 149.79 (2, C), 142.76 (2, C), 131.59, 128.19, 121.72, 67.96 (2, C),
+
5
8.76, 56.08 (2, C), 53.78 (2, C), 46.84 (2, C), 44.89. TOF MS ES + (m/z): (M + H) , calcd for C21
H
27
N
9 3 2
O S : 518.1757,
found, 518.1761.
1
1
2
2
4
.15.16. N -((5-(4-(2-aminopyrimidin-5-yl)-6-morpholino-1,3,5-triazin-2-yl)thiophen-2-yl)methyl)-N ,N ,N -trimethyl-
thane-1,2-diamine (13h)
1
Yellow solid in 49.8% yield . M.P.: 121.3-122.8 ºC. H NMR (400 MHz, CDCl
3
) δ 9.31 (s, 2H), 7.99 (d, J = 3.4 Hz,
1
H), 7.00 (d, J = 3.2 Hz, 1H), 5.63 (s, 2H), 4.01 (s, 4H), 3.83 (s, 6H), 2.71 (s, 4H), 2.45 (s, 6H), 2.36 (s, 3H). 13C NMR
(100 MHz, CDCl
3
) δ 167.22, 166.20, 163.35, 158.45, 147.58 (2, C), 140.55 (2, C), 129.40, 126.09, 119.56, 65.77 (2, C),
+
5
6.20, 55.80, 53.03, 44.20 (2, C), 42.72 (2, C), 41.34. TOF MS ES+ (m/z): (M + H) , calcd for C21
H
29
N
9
OS: 456.2294,
found, 456.2284.
4
.16. General procedure for the synthesis of compounds 14a-14b, 15a-15b and 16a-16b
The synthetic method of compounds 14a-14b, 15a-15b and 16a-16b were similar to compounds 11a-11h. The
difference was that we needed to replace compounds 8a with compounds 8b and and the rest of the synthesis conditions
were the same.
4
.16.1.
4-(4-(1H-indazol-4-yl)-6-(4-methyl-5-((4-(methylsulfonyl)piperazin-1-yl)methyl)thiophen-2-yl)-1,3,5-triazin-2-

yl)morpholine (14a)
Light yellow solid in 64.1% yield. M.P.: 132.4-133.5 ºC. H NMR (400 MHz, CDCl
1
3
) δ 9.10 (s, 1H), 8.47 (s, 1H),
7
.95 (s, 1H), 7.70 (s, 1H), 7.54 (s, 1H), 4.09 (s, 6H), 3.86 (s, 4H),3.31 (s, 4H), 2.83 (s, 3H), 2.69 (s, 4H), 2.32 (s, 3H). TOF
+
MS ES + (m/z): (M + H) , calcd for C24
H
27
N
7
O
2
S: 478.2025, found, 478.2028.
4
.16.2. 4-(4-(1H-indazol-4-yl)-6-(4-methyl-5-(morpholinomethyl)thiophen-2-yl)-1,3,5-triazin-2-yl)morpholine (14b)
1
3
Yellow solid in 43.8% yield. M.P.: 198.9-199.7 ºC. H NMR (400 MHz, CDCl ) δ 9.09 (s, 1H), 8.44 (s, 1H), 7.94 (d,
J = 14.2 Hz, 1H), 7.73 (s, 1H), 7.53 (s, 1H), 4.09 (s, 4H), 3.81 (d, J = 36.2 Hz, 8H), 3.68 (s, 2H), 2.57 (s, 4H), 2.28 (s, 3H).
+
TOF MS ES + (m/z): (M + H) , calcd for C25
H
30
N
8
O
3
S
2
: 555.1961, found, 555.1974.
4
.16.3. 3-(4-(4-methyl-5-((4-(methylsulfonyl)piperazin-1-yl)methyl)thiophen-2-yl)-6-morpholino-1,3,5-triazin-2-yl)phe-
nol (15a)
1
Light yellow solid in 53.4% yield. M.P.: 125.4-127.1 ºC. H NMR (400 MHz, CDCl
3
) δ 8.08 (s, 1H), 7.98 (s, 1H),
7
.87 (s, 1H), 7.35 (s, 1H), 7.02 (s, 1H), 4.03 (s, 4H), 3.81 (s, 4H), 3.72 (s, 2H), 3.30 (s, 4H), 2.79 (s, 3H), 2.67 (s, 4H), 2.23
+
(
s, 3H). TOF MS ES + (m/z): (M + H) , calcd for C23
H
27
N
5
O
3
S: 454.1913, found, 454.1902.
5
.16.4. 3-(4-(4-methyl-5-(morpholinomethyl)thiophen-2-yl)-6-morpholino-1,3,5-triazin-2-yl)phenol (15b)
1
Light yellow solid in 46.3% yield. M.P.: 232.7-233.5 ºC. H NMR (400 MHz, CDCl
3
) δ 8.01 (s, 2H), 7.89 (s, 1H),
7
.35 (s, 1H), 7.04 (s, 1H), 4.02 (s, 8H), 3.81 (s, 4H), 2.30 (d, J = 29.0 Hz, 4H), 1.25 (s, 3H). TOF MS ES + (m/z): (M +
+
H) , calcd for C24
H
30
N
6
O
4
S
2
: 531.1848, found, 531.1853.
4
.16.5. 5-(4-(4-methyl-5-((4-(methylsulfonyl)piperazin-1-yl)methyl)thiophen-2-yl)-6-morpholino-1,3,5-triazin-2-yl)pyri-
midin-2-amine (16a)
Light yellow solid in 60.1% yield. M.P.: 266.1-268.2 ºC. H NMR (400 MHz, CDCl
.48 (s, 2H), 4.02-3.99 (m, 4H), 3.83-3.80 (m, 4H), 3.71 (s, 2H), 3.35 (s, 4H), 2.79 (s, 3H), 2.70 (s, 4H), 2.25 (s, 3H). C
1
3
) δ 9.32 (s, 2H), 7.86 (s, 1H),
1
3
5
NMR (100 MHz, CDCl
3
) δ 168.18, 167.14, 164.31, 159.53, 141.73 (2, C), 139.60, 136.15 (2, C), 133.47, 120.58, 66.83 (2,
+
C), 55.20, 52.40 (2, C), 46.02 (4, C), 43.71, 14.10. TOF MS ES + (m/z): (M + H) , calcd for C21
26 8 2
H N O S: 455.1978,
found, 455.1970.
4
.16.6. 5-(4-(4-methyl-5-((4-(methylsulfonyl)piperazin-1-yl)methyl)thiophen-2-yl)-6-morpholino-1,3,5-triazin-2-yl)pyri-
midin-2-amine (16b)
Light yellow solid in 39.1% yield. M.P.: 279.4-280.6 ºC. H NMR (400 MHz, CDCl
1
3
) δ 9.32 (s, 2H), 7.86 (s, 1H),
1
3
5
.49 (s, 2H), 4.02 (s, 4H), 3.81 (d, J = 25.0 Hz, 8H), 3.67 (s, 2H), 2.57 (s, 4H), 2.26 (s, 3H). C NMR (100 MHz, CDCl
3
)
δ 168.16, 167.21, 164.28, 159.45, 142.02 (2, C), 139.30, 136.00 (2, C), 133.39, 120.73, 67.02 (2, C), 66.77 (2, C), 55.90,

+
5
4
3.64 (2, C), 43.69 (2, C), 14.00. TOF MS ES + (m/z): (M + H) , calcd for C22
H
29
N
9
O
3
S
2
: 532.1913, found, 532.1901.
.17. Anti-tumor assay
The anti-tumor activities of target compounds (11a-11h, 12a-12h, 13a-13h, 14a-14b, 15a-15b and 16a-16b) were
evaluated with A549, MCF-7 and Hela cell lines by the standard MTT assay in vitro, with GDC-0941 as positive control.
Specific operations according to our previous research [16]
.
4
.18. Kinase selectivity assay
The target compounds (11c, 11e, 11g, 12c, 12e, 12g, 13c, 13e and 13g) are tested for their activity against PI3Kα and
mTOR through the mobility shift assay, with GDC-0941 and PI-103 as positive controls. Specific operations according to
our previous research [17]
.
4
.19. Cell cycle progression assay
6
A549 cells were seeded in 6-well plates at a density of 1×10 cells and grown for 24h at 37 ºC in 5% CO
2
, and then
treated with GDC-0941 and compound 13g for 48 h. Specific operations according to our previous research [18]
.
4
.20. Cell apoptosis assay
5
A549 cells were seeded in a 6-well plates at 1×10 cells per well and incubated for 24 h, and treated with compound
1
3g for 48 h. Specific operations according to our previous research [19]
.
4
.21. Western blot analysis
A549 cells were treated with compound 13g at different concentrations of 0.001 µM, 0.01 µM, 0.1 µM, 1 µM and 10
µM for 24 h, and then the protein of A549 cells were extracted by RIPA lysis buffer. The concentration of the cell lysates
were measured by BCA protein Assay (California, USA). The Equal amount of protein was subjected to 10% SDS
polyacrylamide gel electrophoresis and then transferred to PVDF membrane. Next, the PVDF membranes were incubated
with various protein of target proteins (p-AKT (S473), AKT, p-GSK-3β, GSK-3β, GAPDH). Finally, exposure
development was carried out with film in a dark room. The density of the bands were analyzed by Image Lab software.
4
.22. Acridine orange (AO) single staining
The cancer cell apoptotic of target compound 13g was evaluated with A549 cells by acridine orange single staining.
Specific operations according to our previous research [20]
.
4
.23. Molecular docking study

The co-crystal structure of PI3Kα in complex with PI-103 (PDB ID code: 4L23) and the co-crystal structure of mTOR
in complex with PI-103 (PDB ID code: 4JT6) were chosen as the template to generate the docking modes. For the
preparation of ligands, the 3D structures were generated and their energy minimizations were performed by AutoDock 4.2
software (The Scripps Research Institute, USA). The whole PI3Kα and mTOR enzymes were defined as a receptor and the
site sphere were selected based on the PI-103 binding location of PI3Kα and mTOR, after that the molecule PI-103 was
removed and 13g was placed during the molecular docking procedure. Types of interactions of the docked PI3Kα and
mTOR with ligand were analyzed and then the docking conformations were selected and saved based on calculated energy.
All the docking results were processed and modified in PyMOL 1.8.x software (https: // pymol. org).
Acknowledgements
We gratefully acknowledge the generous support provided by The National Natural Science Funds of China (No.
2
1967009), Jiangxi Outstanding Youth Talent Support Program (20171BCB23078), Natural Science Foundation of Jiangxi,
China (20192BAB215061, 20181ACB20025 and 20181BBG70003), Jiangxi Science and Technology Normal University
Innovative Research Team (2017CXTD002).
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Graphical abstract
The preparation and biological evaluation of a series of substituted 2-(thiophen-2-yl)-1,3,5-triazine derivatives
were disclosed, which demonstrated potent inhibition of the PI3K/AKT/mTOR pathway, culminating in the
discovery of 13g.

Research Highlights

A new series of substituted 2-(thiophen-2-yl)-1,3,5-triazine derivatives as potent PI3Kα/mTOR dual inhibitors were
discovered and developed.


Most of the synthesized compounds showed moderate to significant antitumor activity.
Compound 13g showed excellent inhibitory activity to PI3Kα and mTOR kinases with the IC50 values of 7.0 nM and
4
8 nM, respectively.


Compound 13g had an approximately 10-fold improvement in mTOR inhibition (from 525 nM to 48 nM) relative to
GDC-0941.
Compound 13g could stimulate A549 cells arrest at G0/G1 phase, in a dose-dependent manner, and induced apoptosis
at a low concentration.