Design, synthesis and biological activity of novel 2,3,4,5-tetra-substituted thiophene derivatives as PI3Kα inhibitors with potent antitumor activity

Article abstract of DOI:10.1016/j.ejmech.2020.112309

Using a rational design strategy for isoform-selective inhibition of PI3Kα, two series of novel 2,3,4,5-tetra-substituted thiophene derivatives containing either diaryl urea or N-Acylarylhydrazone scaffold were designed and synthesized. The most promising compound 12k was demonstrated to bear nanomolar PI3Kα inhibitory potency with 12, 28, 30, 196-fold selectivity against isoforms β, γ, δ and mTOR. Besides, it also showed good developability profiles in cell-based proliferation against a panel of human tumor cells as well as ADME assays. We herein report on their design, synthesis, SAR and potential developability properties.

Full text of DOI:10.1016/j.ejmech.2020.112309

Journal Pre-proof
Design, synthesis and biological activity of novel 2,3,4,5-tetra-substituted thiophene
derivatives as PI3Kα inhibitors with potent antitumor activity
Weike Liao, Zhongyuan Wang, Yufei Han, Yinliang Qi, Jiaan Liu, Juan Xie, Ye Tian,
Qiancheng Lei, Rui Chen, Ming Sun, Lei Tang, Guowei Gong, Yanfang Zhao
PII:
S0223-5234(20)30278-6
DOI:
https://doi.org/10.1016/j.ejmech.2020.112309
EJMECH 112309
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 3 January 2020
Revised Date: 1 April 2020
Accepted Date: 2 April 2020
Please cite this article as: W. Liao, Z. Wang, Y. Han, Y. Qi, J. Liu, J. Xie, Y. Tian, Q. Lei, R. Chen,
M. Sun, L. Tang, G. Gong, Y. Zhao, Design, synthesis and biological activity of novel 2,3,4,5-tetra-
substituted thiophene derivatives as PI3Kα inhibitors with potent antitumor activity, European Journal of
Medicinal Chemistry (2020), doi: https://doi.org/10.1016/j.ejmech.2020.112309.
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Graphical Abstract
Two series of novel 2,3,4,5-tetra-substituted thiophene derivatives containing diaryl urea or
N-Acylarylhydrazone scaffold were designed and synthesized to evaluate their anticancer
activities

Design, synthesis and biological activity of novel 2,3,4,5-tetra-substituted
thiophene derivatives as PI3Kα inhibitors with potent antitumor activity
Weike Liaoa,1, Zhongyuan Wang b,1, Yufei Hanc,1, Yinliang Qic, Jiaan Liue, Juan Xieb, Ye Tianc,
Qiancheng Leia, Rui Chena, Ming Sunc, Lei Tanga***, Guowei Gongd**, Yanfang Zhaoc*
a
State Key Laboratory of Functions and Applications of Medicinal Plants, Guizhou Provincial Engineering
Technology Research Center for Chemical Drug R&D, Guizhou Medical University, Guiyang 550004, PR China.
b Department of Pharmacy, Guizhou Provincial People's Hospital, Guiyang 550002, PR China.
c Key Laboratory of Structure-Based Drug Design and Discovery (Shenyang Pharmaceutical University), Ministry
of Education, 103 Wenhua Road, Shenhe District, Shenyang 110016, PR China
d Department of Bioengineering, Zunyi Medical University, Zhuhai Campus, Zhuhai 519041, Guangdong, PR
China
e Department of Chemistry, University of Massachusetts-Amherst, Massachusetts 01003, United States
Abstract
Using a rational design strategy for isoform-selective inhibition of PI3Kα, two series of novel
2,3,4,5-tetra-substituted thiophene derivatives containing either diaryl urea or
N-Acylarylhydrazone scaffold were designed and synthesized. The most promising compound
12k was demonstrated to bear nanomolar PI3Kα inhibitory potency with 12, 28, 30, 196-fold
selectivity against isoforms β, γ, δ and mTOR. Besides, it also showed good developability
profiles in cell-based proliferation against a panel of human tumor cells as well as ADME assays.
We herein report on their design, synthesis, SAR and potential developability properties.
Keywords: PI3Kα; antiproliferative activities; synthesis; 2,3,4,5-tetra-substituted thiophene
derivatives
1. Introduction
Phosphatidylinositol-3-kinases (PI3Ks) play a distinctly important role in a variety of
downstream effectors mediating cellular processes, including cell growth, proliferation, survival,
and differentiation.[1-4] According to sequence homology and substrate preference, PI3Ks can be
classified into classes I, II and III.[5] The PI3K family in class I consists of four isoforms (α, β, γ,
and δ). Recently, activated PI3Kα, β, and δ signaling pathway has been increasingly related to
cancer, and these isoforms can be activated by inactivation of the tumor suppressor PTEN[6-8] and
direct somatic mutations in PIK3CA which encodes the p110α subunit of PI3K.[9] The human
* Corresponding author.
** Corresponding author.
*** Corresponding author.
E-mail addresses: tlei1974@hotmail.com (L. Tang), sammyld@163.com (G. Gong), yanfangzhao@126.com (Y.
Zhao).
1 These authors contributed equally to this work.

tumor genetic data indicated that mutations in PI3Kα can prompt tumor growth.[10] Furthermore, a
selective inhibitor that does not bind to PI3Kβ, δ, and γ may reduce some treatment side effects
caused by preventing platelet aggregation and activation,[11] inhibiting immune responses
mediated by T, B, and mast cells, [12] and blocking anti-inflammatory signals, [13] etc. Giving these
observations, targeting PI3Kα pathway will be one of the most promising approaches for cancer
treatment.
Recently, notable success for PI3K-isoform selective inhibitors had been achieved, Idelalisib
(1) [14,15], previously called GS-1101 or CAL-101, was a first-in-class, oral, potent, and selective
inhibitor of PI3Kδ. Also, Alpelisib[16,17](2), a selective PI3Kα inhibitor was approved by the FDA
on May 24, 2019, in Combination With Fulvestrant for patients with HR+, HER2-,
PIK3CA-mutated advanced breast cancer. Furthermore, several class I selective PI3K inhibitors
were in various phases of clinical trials, including PF-4989216 (3),[18] and GDC-0032 (4)[19,20] for
PI3Kα/δ and AZD8186 (5)[21] for PI3Kβ (Fig. 1). Among them, PF-4989216 exhibited potent and
selective inhibition against PI3K kinase activity in preclinical small-cell lung cancer (SCLC)
models and was especially effective against the proliferation of SCLCs harboring PIK3CA
mutation. Among all four isoforms of PI3K, the importance and high frequency of PIK3CA
mutation in most of the solid tumors aroused the concern about the selectivity development of
[22]
PI3Kα inhibitors.
Present work aims to design, synthesize, and optimize
2,3,4,5-tetra-substituted thiophene derivatives, which can selectively bind to PI3Kα ATP binding
site.
Fig.1.Representative Structures of PI3K inhibitor
From the docking model of PF-4989216 with PI3Kα protein (PDB code 3ZIM),[23] the key

hydrogen-bonding interactions were formed by the 1,2,4-triazole moiety and the morpholino
group with residues in the ATP binding site (Fig. 2). Through a detailed docking analysis, C-4
phenyl moiety did not generate any hydrogen-bonding interactions with the residues directly or
indirectly. Therefore, appropriate groups in C-4 phenyl moiety were introduced to explore
potential interactions with the residues in solvent zone in order to increase PI3Kα selectivity. In
our previous study, it was observed that the small molecules bearing the diaryl urea scaffold or
N-Acylarylhydrazone moiety showed superior anticancer activity due to the presence of hydrogen
bond donors and acceptors as well as its flexible skeleton.[24, 25] Based on these findings, the diaryl
urea scaffold or N-Acylarylhydrazone moiety was introduced into C-4 phenyl moiety on
PF-4989216. As a result，two series of novel 2,3,4,5-tetra-substituted thiophene derivatives were
designed and synthesized. Compounds were screened for their PI3K/mTOR inhibitory activity and
cytotoxicity in a panel of cancer cell lines. Additionally, in Vitro ADME and Pharmacokinetics,
data for compound 12k was also investigated.
Fig. 2 The design strategy based on the docking study
2. Results and discussion
2.1. Chemistry
The general synthetic routes of the title compounds are illustrated in Schemes 1–3. The key
intermediate 2,3,4,5-tetrasubstituted thiophene 11 was achieved in 11 steps as shown in Scheme 1.
Commercially available malononitrile was condensed with carbon disulfide in the presence of
K2CO3 in DMSO at 0-20 ºC for 2 h to obtain 2-(dimercaptomethylene)malononitrile, followed by
methylated with iodomethane at 20 ºC for another 20
h
to provide
2-(bis(methylthio)methylene)malononitrile 1. Further, intermediate 1 was treated with ethyl

2-mercaptoacetate to generate ethyl 3-amino-4-cyano-5-(methylthio)thiophene-2-carboxylate (2)
[26], which was then iodinated in the presence of isoamyl nitrite in acetonitrile to yield intermediate
3. Oxidation of intermediate 3 with 30% H2O2 and sodium tungstate in acetic acid gave ethyl
4-cyano-3-iodo-5-(methylsulfonyl)thiophene-2-carboxylate(4), which was reacted with
morpholine through an SN2 mechanism to generate intermediate 5. Subsequently, intermediate 5
was
subjected
to
palladium-catalyzed
Suzuki-coupling
reaction
with
4-(N-Boc-amino)phenylboronic acid pinacol ester in hot dioxane/toluene/H2O (16:1:4, 95 ºC) to
get a key intermediate 6, which was hydrolyzed to acid 7 by aqueous LiOH in MeOH:THF (1:2).
Treatment of 7 with methyl chloroformate in the presence of Et3N yielded anhydride intermediate,
which
was
converted
to
tert-butyl
(4-(2-carbamoyl-4-cyano-5-morpholinothiophen-3-yl)phenyl)carbamate
8
by
ammonium
hydroxide. Then, 8 was condensed with N,N-dimethylformamide dimethyl acetal (DMF-DMA) at
110 ºC for 2 h to afford 9, which was treated with hydrazine hydrate in acetic acid at 60 ºC for 2 h
to give intermediate 10. The key intermediate 2,3,4,5-tetrasubstituted thiophene 11 was obtained
by the deprotection of 10 in the presence of trifluoroacetic acid in CH2Cl2 at room temperature for
3 h.
Scheme 1. Reagents and conditions: (i) a) CS2, K2CO3, DMSO, 20 ºC, 2 h; b) iodomethane, 20 ºC, 12 h; (ii) ethyl
2-mercaptoacetate, Et3N, EtOH, 20 ºC, 12 h; (iii) isoamyl nitrite, I2, CH3CN, 20 ºC, 12 h; (iv) 30% H2O2, sodium
tungstate, HOAc, 60 ºC, 2 h; (v) morpholine, THF, r.t., 8 h; (vi) a) 4-(N-Boc-amino)phenylboronic acid pinacol

ester, CsF, 1,4-dioxane:H2O (4:1), r.t., 15 min; b) Pd2(dba)3, P(t-Bu)3, Toluene/dioxane, 95 ºC, 12 h; (vii) LiOH,
MeOH:THF (1:2), r.t., 4 h; (viii) a) methyl chloroformate, Et3N, THF, r.t., 3 h; b) NH3·H2O , r.t., 1 h; (ix)
DMF-DMA, DMF, 110 ºC, 2 h; (x) hydrazine hydrate, HOAc, 60 ºC, 2 h; (xi) CF3COOH, CH2Cl2, r.t., 3 h.
The target compounds 12a-m were prepared, as illustrated in Scheme 2. The commercially
available substituted isocyanatobenzene was treated with the key intermediate 11 in CH2Cl2 at
room temperature for 12 h to afford the target compounds 12a-m.
Scheme 2. Reagents and conditions: (i) isocyanatobenzene, CH2Cl2, r.t., 12 h.
As described in Scheme 3, the key intermediate 11 was converted into amides 13 by the
treatment with phenyl chloroformate in dry acetone at ambient temperature for 3 h; subsequently,
hydrazinolysis of 13 with 80% hydrazine hydrate in refluxing 1,4-dioxane for 2 h provided
semicarbazide 14, followed by condensation with appropriate aldehydes in EtOH at 80 ºC for 5 h
with catalytic amount of acetic acid to give the target compounds 15a-m successfully.
Scheme 3. Reagents and conditions: (i) phenyl chloroformate, dry acetone, r.t., 3 h.; (ii) 80% hydrazine hydrate,
1,4-dioxane, reflux, 2 h; (iii) aldehydes, acetic acid, EtOH, 80 ºC, 5 h.
1
The chemical structures of the target compounds were confirmed with H NMR, 13C NMR,
and MS spectrum. Also, the configuration of the double bond in the imine of 15a-m was
1
determined. In H NMR spectra, the –N=CH- signal was observed as a single peak for each

compound, indicating that only one isomer was generated. To confirm the stereochemistry more
accurately, the NOESY experiment of compound 15d was performed. The result showed that a
single NOE signal was observed between the proton of =N-NH- (δ = 10.80 ppm) and the proton of
–N=CH- (δ = 8.46 ppm), which existed only in the E isomer due to the appropriate intramolecular
H-H distance (Fig.3. and see also Supporting information). Thus, the target compounds were
unequivocally confirmed as the E isomer.
H
H
N
N
N
N
N
N
OH
S
S
O
O
N
N
O
O
N
N
H
N
N
H
N
N
H
H
H
N
H
N
OH
(E)-15d
(Z)-15d
Fig. 3. NOESY effect of the representative compound 15d.
2.2. Bioactivity and discussion
2.2.1. PI3K/mTOR inhibition and SAR study of target compounds
During the course of our SAR investigation, compound 12m with diaryl urea scaffold was
firstly synthesized and evaluated. We found compound 12m exhibited an IC50 of 116 nM in PI3Kα
assay (Table 1), which was around 21-fold less potent compared to PF-4989216. Next, we
assembled a focused collection of diaryl urea analogues that could be readily synthesized; the
effect of substitute group at the 4-position was first assessed. Introduction of electron-donating
group such as -CH3, -OCH3 did not offer the potency benefit (12a to 12c vs 12a). A strong
electron-withdrawing group such as -CN, -CF3, -OCF3 proved to be deleterious to PI3Kα potency,
as a gradual decrease in potency was observed (12d to 12f vs 12a). However, when the small
electron-withdrawing fluoride group was introduced, compound 12g gained back some degree of
potency (3.8-fold increase vs 12a) but still around 6-fold less potent than PF-4989216. Through
scanning the 2-, 3-position of phenyl, it was clearly found that 3-position was not well tolerated
(12i vs 12g); a similar trend was also observed for compound 12j (12j vs 12g), whereas
ortho-fluoro substitute compound 12h showed slightly enhanced PI3Kα activity. Further
replacement of fluoro with the chloro group led to isolation of the potent PI3Kα inhibitor 12k with
an IC50 of 9.2 nM. These data suggested that a proper degree of electron density on the terminal
aromatic ring was beneficial to achieve better inhibitory activity.

In comparison, the SARs of compounds 15a-m bearing N-Acylarylhydrazone moiety were
conducted in the same way. Compound 15a maintained a PI3Kα inhibitive activity similar to that
of 12a. We found phenyl substitution such as halogen (15b, 15c) did not have a profound impact
on PI3Kα potency. However, a noticeable improvement was seen with a hydroxyl group at the
3-position (15d vs 15a). Meanwhile, it was also noted that introduction of a 3,4-dihydroxy 15f or
3,5-dimethyl-4-hydroxy group 15i led to a similar potency improvement as 15d. In contrast, the
introduction of electron-withdrawing trifluoromethoxy group eroded the PI3Kα potency (15g vs.
15a). With SAR expansion, analogs with six or five-membered heterocycles (15k to 15m) were
also prepared. Notably, compound 15m, which exhibited an IC50 of 6.9 nM against PI3Kα, had
been optimized up to the similarly level of PF-4989216.
Table 1. SAR studies of compounds 12a-m and 15a-m
PI3Kα
PI3Kα
Compd. Linker
Ar
Compd.
Linker
Ar
IC50(nM)a
IC50(nM)a
A
A
A
A
A
A
A
A
2.6-(CH3)2-Ph
4-CH3-Ph
2-OCH3-Ph
4-CN-Ph
4-CF3-Ph
4-OCF3-Ph
4-F-Ph
344
95.8
105
290
403
835
35.3
22.3
B
B
B
B
B
B
B
B
Ph
76.6
75.5
89.8
17.7
72.0
21.9
631
12a
12b
12c
12d
12e
12f
15a
15b
15c
15d
15e
15f
4-F-Ph
3-Br-Ph
3-OH-Ph
4-OCH3-Ph
3,4-(OH)2-Ph
4-OCF3-Ph
2,6-(Cl)2-Ph
12g
12h
15g
15h
2-F-Ph
323

A
A
A
A
A
3-F-Ph
3-Cl-4-F-Ph
2-Cl-Ph
3-Br-Ph
Ph
151
407
9.2
B
B
B
B
B
3,5-(CH3)2-4-OH-Ph
3,4,5-(OCH3)3-Ph
20.3
110
9.4
12i
12j
15i
15j
12k
12l
15k
15l
239
116
5.4
13.3
6.5
12m
15m
PF-4989216b
BYL-719(Alpelisib)b
3.9
a The average is indicated (n ≥ 2 independent experiments); b Used as a positive control.
Through the SAR investigation, novel thiophene derivatives were identified to bear decent
potency against PI3Kα. Six representative compounds were selected for further profiling. The
inhibitory activities to other Class I PI3K isoforms (β, γ, and δ) and mTOR were evaluated. As
show in Table 2, all the tested compounds displayed more potent activity against PI3Kα than the
rest of three isoforms as well as mTOR. Additionally, compounds (12g, 12h, 12k) bearing diaryl
urea moiety displayed a higher level of selectivity than that of compounds with
N-Acylarylhydrazone scaffold, especially towards PI3Kδ and mTOR, making it a promising
selective PI3Kα inhibitor. We believed that the overall profiles obtained from the enzyme assays
would contribute significantly to the further design of more potent and selective inhibitors.
Table 2. Inhibitory effects of compounds and PF-4989216, BYL-719 against PI3Ks and mTOR
Compound
IC50 in nMa
PI3Kδ
PI3Kβ
95
PI3Kγ
289
302
258
440
204
274
60
mTOR
1662
1589
1802
489
238
334
275
72
12g
89
12h
108
43
12k
15d
74
74
598
15l
53
49
416
15m
PF-4989216 b
BYL-719(Alpelisib)b
124
1540
15
4064
1461
94
72

a Data reported as the average of at least two runs; b Used as a positive control.
2.2.2. In vitro Antiproliferative Activity
To investigate the functional consequence of inhibiting PI3Kα in cells, compounds were also
tested in five different cancer cell lines for its sensitivity in cell proliferation assays. Cell lines
either harbor PIK3CA mutations or bear the PTEN deletion except for A549 were treated, and
growth was monitored. Interestingly, these selective PI3K inhibitors were more sensitive with
human breast cancer and non-small cell lung cancer cell lines that harbor PI3KCA mutations than
cell lines with the PTEN deletion, as shown in Table 3. Consistent with the potency against the
PI3K, compound 12g, 12h, 12k were moderately potent with at least single-digital micromolar
IC50s across tumor cell originating from different tissues. Notably, compound 12k potently
inhibited the proliferation of human breast cancer line T47D and human lung adenocarcinoma
NCI-H460 with IC50 of 0.31 and 0.20 μM, respectively. However, an apparent disconnection
between the cell activity and the PI3Kα inhibition of compounds 15d, 15l, 15m was observed. The
N-Acylarylhydrazone analogs are active in the PI3K assay but possess weak cytotoxicity in cells
probably due to their low membrane permeability. In view of this, compound 12k was selected for
further profiling.
Table 3. Antiproliferative Effect of Compounds against a Panel of Human Cancer Cell
IC50 in μMa
U87MGc
3.35
T47Dd
0.52
0.41
0.31
2.22
2.68
2.13
0.34
NCI-H1975d NCI-H460d
A549e
5.52
Compound
2.04
1.52
1.06
9.76
12.89
11.20
0.94
0.59
0.40
0.20
1.39
1.22
2.69
0.12
12g
12h
12k
15d
15l
3.98
4.19
2.86
3.84
13.01
12.29
22.14
2.31
12.66
19.36
18.52
3.58
15m
PF-4989216 b
a
Cell viability was measured using a CellTiter-Glo assay 72 h after treatment. Average IC50 values (n = 3) are

c
d
shown. b Used as a positive control. Tumor cell line with PTEN deletion. Tumor cell line harboring PIK3CA
mutation. e Tumor cell line harboring neither PIK3CA mutation nor PTEN deletion.
2.2.3. Kinase selectivity profiling of 12k
Given the fact that 12k exhibited the desirable in vitro biochemical activity against PI3Ks as
well as antiproliferative efficacy in cancer cell lines, 12k was further chosen to examine its
kinome wide selectivity profile containing 97 kinases (scanEDGE KINOMEscan, DiscoverX) at a
compound concentration of 1 μM. 12k proved to be highly selective as PF-4989216 against the
panel, compound 12k did not show any binding activity to this broad panel of kinases except for
the PI3Ks, illustrating excellent kinase selectivity for this thiophene chemotype. The results are
shown in Figure 4 and the complete profiling data of 12k and PF-4989216 on 97 kinases can be
found in Table S2 of the Supporting Information.
Fig. 4. Percent inhibition profile (TREEspot Interaction Map) in the DiscoverX ScanEDGE kinome scan for
compound 12k and PF-4989216. S-score is a quantitative measure of compound selectivity. It is calculated by
dividing the number of kinases that compounds bind to by the total number of distinct kinases tested, excluding
mutant variants.
2.2.4. Western Blot

Activation of the PI3K pathway leads to phosphorylation of the Ser473 of Akt and
subsequently to several downstream substrates. To confirm that the newly synthesized compounds
were inhibiting PI3K signaling in cells, 12k was further profiled for its ability to suppress cellular
biomarkers. After treatment with 12k for 2 h, T47D cells were treated with 1 μg/mL insulin to
activate the PI3K pathway. Consistent with expectations for a potent PI3Kα inhibitor, 12k strongly
suppressed phosphorylation of Akt at the serine 473 in a dose dependent manner. We also
compared the suppression of PI3K signaling by 12k with PF-4989216, as shown in Figure 5.
Compound 12k shows p-Akt reduction starting at 0.1 μM and is complete at 3 μM. We concluded
that the anti-proliferative activity of 12k against T47D cell line was attributed to its remarkable
capability to attenuate the PI3K signaling.
Fig. 5. Effects of 12k and PF-4989216 on p-AktS373, Akt. T47D cells were treated with various concentration of
12k or PF-4989216 for 2 h, and then the PI3K pathway was activated by treatment of 1 μg/mL insulin for 2 h.
Compound 12k decreased the phosphorylation of AktS473 in a dose-dependent manner.
2.2.5. In Vitro ADME and Pharmacokinetics for Compound 12k
In vitro ADME properties and pharmacokinetic parameters of compound 12k were also
determined. Caco-2 bidirectional permeability assay confirmed that 12k had moderate
permeability and was not a substrate for P-glycoprotein (efflux ratio of 1.08). From the
multispecies liver microsomal stability study, compound 12k showed acceptable stability with
clearance rate of 24.8, 32.8 μL/min/mg in human and rat liver microsomes, respectively. Also,
three cytochrome P450 (CYP1A2, 2D6 and 3A4) enzymes commonly metabolizing exogenous
chemicals were used to test the direct inhibition of compound 12k. As listed in Table 4, the
compound showed favorable metabolic properties, as the inhibition ratio for three CYPs was less
than 30%, even at the concentration of 10 μM. With these in vitro effects and preliminary ADMET
profiling, further intravenous pharmacokinetic parameters for 12k were performed by

Sprague−Dawley rats at a dose of 5 mg/kg, 12k exhibited a moderate half-life of 1.98 ± 0.17 h and
achieved a maximum concentration (Cmax) of 2.57 ± 0.32 μg/mL. The area under the curve
(AUC(0−∞)) was 1622 ± 89 ng·h/mL. It is worth noting that compound 12k exhibited relatively
high plasma clearance (52.36 ± 3.32 mL/min/kg). Finally, compound 12k was found to be safe
(IC50 > 30 μM, see Fig. S2) when tested in a hERG Qpatch assay for assessing hERG-associated
cardiotoxicity.
Table 4. In Vitro ADME and Pharmacokinetics for Compound 12k
Permeability assay (10–6 cm/s) a
Caco-2
-
Papp (A to B)
7.8
Papp (B to A)
8.4
Efflux ratio
1.08
Metabolic stability b
Species
T1/2(min)
44.7
CL (μL/min/mg)
Remaining b (T=60min)
RLM
32.8
24.8
42.6%
47.8%
HLM
58.3
CYP450 inhibition c
isozyme
CYP1A2
27.8
CYP2D6
28
CYP3A4
1.5
% inhibition at 10μM
Pharmacokinetics d
Cmax(μg/mL)
2.57±0.32
t1/2 (h)
Vd (L/Kg)
8.6±1.33
CL (mL/min/kg)
52.36±3.32
AUC 0-∞ (ng•h/mL)
1.98±0.17
1622±89
aThe permeation was assessed over 2 h with 2 μM concentration incubation at 37±1°C and 5% CO2 with saturated
b
humidity. No NADPH regenerating system was added to the sample (replaced by buffer) during the 60 min
c
incubation. Performed at 10 μM concentration. α-Naphthoflavone (CYP1A2), squinidine (CYP2D6), and
d
ketoconazole (CYP3A4) were used as the positive controls. Determined from intravenous dosing to male SD
rats at a dose of 5 mg/kg (n=6).

2.2.6. Binding model analysis
The better understand the isoform selectivity of 12k against PI3Ks, the affinity of compound
12k for each PI3K isoform was calculated by applying the Cheng-Prusoff equation. Compound
12k exhibited Ki values of 5.1nM, 89 nM, 193 nM, 245 nM against PI3Kα, PI3Kβ, PI3Kγ, PI3Kδ,
respectively (see Table S1 for details). The binding modes between the 12k and PI3Ks were then
proposed by molecular simulation. The structures of PI3Kα (PDB ID code: 3ZIM), PI3Kβ (PDB
ID code: 2Y3A), PI3Kγ (PDB ID code: 3DBS) and PI3Kδ (PDB ID code: 2WXP) were selected
as the docking models.
Fig. 6. Predicted binding models for 12k with PI3Ks. (A) Predicted binding conformation for 12k in the binding
site cavity of PI3Kα (PDB 3ZIM). (B) Binding to PI3Kβ (PDB 2Y3A). (C) Binding to PI3Kγ (PDB 3DBS). (d)
Binding to PI3Kδ (PDB 2WXP). Hydrogen bonds are indicated by yellow dashed lines. Images generated using
PyMol.
As shown in Fig. 6A, compound 12k occupied the kinase domain in a similar manner to
PF-4989216. Six hydrogen-bond interacted with 12k and PI3Kα: the morpholine moiety formed
one hydrogen bond with Val 851; meanwhile, the 1,2,4-triazole group formed two hydrogen bonds
with Tyr 863 and Asp 810. Besides, the urea part of compound 12k could form three hydrogen
bonds with Asp 805, Asp 933 and Ser 774, validating the rationality of our design. We also
performed the docking analysis of compound 12k binding to β (PDB 2Y3A) (Fig. 6B) and γ (PDB
3DBS) (Fig. 6C) isoforms. Six hydrogen-bond interactions with residues of PI3Kβ were noticed:

Val 848, Asp 807, Asp 931, Lys 799 and Ser 775. For the binding of 12k to PI3Kγ, four
hydrogen-bond interactions with the residues were indicated: Val 882, Asp 836, Asp 950 and Lys
833. The analysis of compound 12k with PI3Kδ showed that this compound formed four
hydrogen-bond interactions with Val 828, Asp 911, Asp 782 and Ser 754. Also, compound 12k
was in a reversed pose in PI3Kδ compared to PF-4989216 (Fig. 7), which indicated compound
12k could not fit well into the cavity of PI3Kδ isoform, thus resulted in a decrease of inhibitory
activity against PI3Kδ. Furthermore, molecular dynamics calculations had been implemented to
verify the stability of ligand-protein complexes in the proposed model. The MD simulation
undertaken for the two compounds bound to PI3Kδ showed the overall stability of the
ligand/target adducts over the tested 25ꢀns (see Fig. S3). In general, the docking results further
confirm the rationality of our design strategy.
Fig 7．Overlapping images of compound 12k (blue) and PF-4989216(green) complexed with PI3Kδ. Hydrogen
bonds are indicated by blue lines.
3. Conclusions
In this study, two series of novel potent PI3Kα inhibitors bearing the diaryl urea scaffold or
N-Acylarylhydrazone moiety based on PF-4989216 were designed. The PI3K enzymatic activity
assays identified compound 12k as a selective PI3Kα inhibitor with 12, 28, 30, 196-fold
selectivity over isoforms β, γ, δ and mTOR. Compound 12k also showed potent cytotoxic
activities with IC50 values of 0.31 μM and 0.20 μM against human breast cancer T47D and
non-small cell lung cancer H460 cell lines, respectively. To evaluate its druggability, compound
12k was submitted to in vitro ADME and in vivo Pharmacokinetics assay. The pharmacokinetic
properties of the compound 12k are suboptimal and are the focus of a medicinal chemistry effort
to improve its oral bioavailability while maintaining its potency and selectivity. This will be the
focus of a future publication.

4. Experimental
4.1. Chemistry
Unless otherwise specified, reagents and solvents were obtained from commercial sources
and used without further purification. Reactions’ time and purity of the products were monitored
by TLC on FLUKA silica gel aluminum cards (0.2 mm thickness) with fluorescent indicator 254
nm. All melting points were obtained on a Büchi Melting Point B-540 apparatus (Büchi
Labortechnik, Flawil, Switzerland) and were uncorrected. Flash chromatography was performed
using silica gel (200–300 mesh) from Qingdao ocean Chemicals (Qingdao, Shandong, China).
PF-4989216 and BYL-719 were purchased from TargetMol (Shanghai, China). Mass spectra (MS)
1
were taken in ESI mode on Waters e2695. H NMR and 13C NMR spectra were recorded on
Bruker ARX-400, 400 MHz spectrometers (Bruker Bioscience, Billerica, MA, USA) with TMS as
an internal standard.
4.2. Synthesis of the key intermediate 11
4.2.1. Preparation of 2-(bis(methylthio)methylene)malononitrile(1)
A suspension of K2CO3 (17.3 g, 125.0 mmol) in DMSO (75 mL) at 20 °C was treated with
malononitrile (7.5 g, 113.7 mmol) in one portion. The reactor was cooled to 0 °C, and CS2 (7.5
mL, 125.0 mmol) was added at 18-22 °C. The mixture was stirred at 20 °C for 2 h and then cooled
to 0 °C. Iodomethane (14.1 mL, 227.3 mmol) was added with pot temperature below 5 °C. The
reaction was warmed to 20 °C and stirred for 12 h. The reaction mixture was poured into ice-water
(75 mL), and the resulting precipitate was filtered, washed with H2O (20 mL) and dried under
1
reduced pressure to afford product 1 as a yellow solid (18.2 g, 93.9%). H NMR (400 MHz,
CDCl3) δ 2.69 (s, 6H). HRMS (ESI) calculated for C6H7N2S2 [M + H]+ m/z 170.2480, found
170.2482.
4.2.2. Preparation of ethyl 3-amino-4-cyano-5-(methylthio)thiophene-2-carboxylate(2)
At 0 °C, 2-(Bis(Methylthio)methylene)malononitrile 1 (15.0 g, 88.2 mmol) was added to a
solution of ethyl 2-mercaptoacetate (9.8 mL, 88.2 mmol) in EtOH (75 mL), then Et3N (12.2 mL,
88.2 mmol) was added dropwise to the solution and stirred for 12 h at 20 °C. The reaction mixture
was filtered, washed with EtOH (20 mL) and dried under reduced pressure to afford product 2 as a

white solid (15.5 g, 72.6%). 1H NMR (400 MHz, CDCl3) δ 5.79 (s, 2H). 4.28 (q, J = 7.2 Hz, 2H)
2.64 (s, 3H) 1.34 (t, J = 7.1 Hz, 3H). HRMS (ESI) calculated for C9H11N2O2S2 [M + H]+ m/z
243.0264, found 243.0266.
4.2.3. Preparation of ethyl 4-cyano-3-iodo-5-(methylthio)thiophene-2-carboxylate(3)
A suspension of ethyl 3-amino-4-cyano-5-(methylthio)thiophene-2-carboxylate 2 (7.5 g, 31.1
mmol) in acetontrile (75 mL) was treated with I2 (6.0 g, 62.0 mmol) then heated to 70 °C, and
isoamyl nitrite (6.3 mL, 62.0 mmol) was added at a rate to maintain the pot temperature between
60 °C and 70 °C. Then the mixture was cooled to 20 °C and stirred for 12 h. Upon cooling to 0 °C,
the reaction mixture was filtered, and washed with acetonitrile (10 mL) , the solid was redissolved
in 60 mL ethyl acetate, washed with saturated sodium thiosulfate (15 mL×2) and then brine. The
organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure and dried
under reduced pressure to afford product 3 as a yellow solid (6.9 g, 63.0%). 1H NMR (400 MHz,
CDCl3) δ 4.31 (q, J = 7.1 Hz, 2H) 2.63 (s, 3H) 1.34 (t, J = 7.1 Hz, 3H). HRMS (ESI) calculated
for C9H9INO2S2 [M + H]+ m/z 353.9119, found: 353.9110.
4.2.4. Preparation of ethyl 4-cyano-3-iodo-5-(methylsulfonyl)thiophene-2-carboxylate(4)
30%
H2O2
(37.5
mL)
was
added
to
a
solution
of
ethyl
4-cyano-3-iodo-5-(methylthio)thiophene-2-carboxylate 3 (3.8 g, 10.7 mmol) and sodium tungstate
(0.3 g, 1.1 mmol) in acetic acid (75 mL). The mixture was stirred at 60 °C for 2 h. After Cooling
to room temperature., the reaction mixture was filtered, washed with H2O (20 mL) and dried under
reduced pressure to afford product 4 as a white solid (3.9 g), which was carried into the next step
whithout further purification.
4.2.5. Preparation of ethyl 4-cyano-3-iodo-5-morpholinothiophene-2-carboxylate(5)
Ethyl 4-cyano-3-iodo-5-(methylsulfonyl)thiophene-2-carboxylate 4 (3.9 g, 10.7 mmol) was
added to THF (60 mL), and then the mixture was cooled to 0 °C, morpholine (4.8 mL, 55.1 mmol)
was added with pot temperature below 10 °C. The reaction was stirred at 25 °C for 8 h. The
reaction mixture was poured into ice-water (30 mL) and the resulting precipitate was filtered and
1
dried under reduced pressure to abtain 5 as a white solid (3.2 g, 75.5%). H NMR (400 MHz,
CDCl3) δ 4.34 (q, J = 7.2 Hz, 2H) 3.96-3.83 (m, 4H), 3.70-3.54 (m, 4H), 1.36 (t, J = 7.1 Hz, 3H).
HRMS (ESI) calculated for C12H14IN2O3S [M + H]+ m/z 392.9770, found: 392.9762.
4.2.6. Preparation of ethyl 3-(4-((tert-butoxycarbonyl)amino)phenyl)-4-cyano-5-morpholin-

othiophene-2-carboxylate(6)
Ethyl 4-cyano-3-iodo-5-morpholinothiophene-2-carboxylate
5
(2.6 g, 6.6 mmol),
4-(N-Boc-amino)phenylboronic acid pinacol ester (2.2 g, 6.6 mmol), Pd2(dba)3 (0.18g, 0.2 mmol)
and CsF (3.0 g, 19.8 mmol) were added to a solution of 1,4-dioxane and H2O (30 mL,
1,4-dioxane:H2O = 4:1). The reactor was flushed with N2, P(t-Bu)3 (0.4 mL of a 1M solution in
Toluene, 0.4 mmol) was added via syringe to the mixture and stirred for 12 h at 95 °C. Upon
completion, Water (30 mL) was added followed by extraction with EtOAc (10 mL×3). The
organic layer was decolored with 5% active carbon and then filtered through a Celite pad. The
filtrate was concentrated to dryness and then triturated with MeOH to obtain 6 as a white solid
(2.4 g, 78.9%). 1H NMR (400 MHz, CDCl3) δ 7.43 (d, J = 8.4 Hz, 2H), 7.36 – 7.29 (d, J = 8.4 Hz,
2H), 6.59 (s, 1H), 4.14 (q, J = 7.1 Hz, 2H), 3.90 – 3.83 (m, 4H), 3.65 – 3.58 (m, 4H), 1.52 (s, 9H),
1.16 (t, J = 7.1 Hz, 3H). HRMS (ESI) calculated for C23H28N3O5S [M + H]+ m/z 458.1750, found:
458.1751.
4.2.7. Preparation of 3-(4-((tert-butoxycarbonyl)amino)phenyl)-4-cyano-5-morpholino-thiophene
-2-carboxylic acid(7)
A solution of LiOH (1.6 g, 66 mmol) in H2O (15 mL) was added to a solution of
3-(4-((tert-butoxycarbonyl)amino)phenyl)-4-cyano-5-morpholin-othiophene-2-carboxylate 6 (6.0
g, 13.2 mmol) in MeOH (30 mL) and THF (60 mL). The mixture was stirred for 4 h at 25 °C. The
reactor was concentrated under reduced pressure and poured into water (30 mL). The solution was
adjusted to pH=5 with 2N HCl, and then the resulting precipitate was filtered, washed with H2O
(20 mL) and a minimal amount of MeOH, dried under reduced pressure to afford product 8 as a
white solid (4.4 g, 71.4%). HRMS (ESI) calculated for C21H22N3O5S [M - H]- m/z 430.1437, found:
430.1442.
4.2.8. Preparation of tert-butyl(4-(2-carbamoyl-4-cyano-5-morpholinothiophen-3-yl)phenyl)
carbamate(8)
A suspension of 3-(4-((tert-butoxycarbonyl)amino)phenyl)-4-cyano-5-morpholino-thiophene
-2-carboxylic acid 7 (5.4 g, 12.6 mmol) in THF (120 mL) was treated with Et3N (2.4 mL, 18.9
mmol) in one portion. The reactor was cooled to 0 °C, and methyl chloroformate (1.8 g, 18.9
mmol) was added drop-wise. The mixture was stirred at 25 °C for 3 h and then cooled to 0 °C.
NH3·H2O (27 mL) was added to the solution. The reaction was warmed to 25 °C and stirred for 1

h. The mixture was concentrated under reduced pressure and poured into water (50 mL), and the
resulting precipitate was filtered, washed with H2O (20 mL) and dried under reduced pressure to
afford product 8 as a yellow solid (4.7 g, 86.1%). 1H NMR (400 MHz, DMSO-d6) δ 9.61 (s, 1H),
7.59 (d, J = 8.7 Hz, 2H), 7.29 (d, J = 8.7 Hz, 2H), 3.80 – 3.72 (m, 4H), 3.56 – 3.50 (m, 4H), 1.49
(s, 9H). HRMS (ESI) calculated for C21H25N4O4S [M + H]+ m/z 429.1597, found: 429.1603.
4.2.9. Preparation of tert-butyl (E)-(4-(4-cyano-2-(((dimethylamino)methylene)carbamoyl)-5-
morpholinothiophen-3-yl)phenyl)carbamate(9)
DMF-DMA (3.0 g, 25.2 mmol) was added to a stirring solution of tert-butyl
(4-(2-carbamoyl-4-cyano-5-morpholinothiophen-3-yl)phenyl)carbamate 8 (5.4 g, 12.6 mmol) in
DMF (40 mL) and heated to 110 °C for 2 h. After being cooled to room temperature, the mixture
was concentrated under reduced pressure and poured into ice-water (30 mL). The precipitate was
filtered, washed with H2O (10 mL) and MeOH (5 mL), dried under reduced pressure to afford
product 9 as a yellow solid (4.4 g, 71.4%). HRMS (ESI) calculated for C24H30N5O4S[M + H]+ m/z
484.2019, found: 484.2020.
4.2.10. Preparation of tert-butyl (4-(4-cyano-5-morpholino-2-(1H-1,2,4-triazol-3-yl)thiophen-3-yl)
phenyl)carbamate(10)
80% hydrazine hydrate (15 mL) was added drop-wise to a solution of tert-butyl
(E)-(4-(4-cyano-2-(((dimethylamino)methylene)carbamoyl)-5-morpholinothiophen-3-yl)phenyl)ca
rbamate 9 (3.0g, 6.2 mmol) in HOAc (30 mL). The mixture was stirred at 60 °C for 2 h. Upon
cooling to room temperature, the solvent was removed under vacuum, and then H2O (30 mL) was
added to the mixture. The resulting precipitate was filtered and dried under reduced pressure to
afford 10 as a white solid (2.4 g, 85.5%). HRMS (ESI) calculated for C22H25N6O3S [M + H]+ m/z
453.1709, found: 453.1710.
4.2.11. Preparation of 4-(4-aminophenyl)-2-morpholino-5-(1H-1,2,4-triazol-3-yl)thiophene-3-
carbonitrile(11)
tert-Butyl(4-(4-cyano-5-morpholino-2-(1H-1,2,4-triazol-3-yl)thiophen-3yl)phenyl)carbamate
10 (3.0 g, 6.6 mmol) was added to CH2Cl2 (60 mL), and then the mixture was cooled to 0 °C,
CF3COOH (3.1 mL, 22.0 mmol) was added to the solution drop-wise. The mixture was stirred at
25 °C for 3 h. The solvent was removed under vacuum, and then H2O (30 mL) was added to the
mixture. The solution was adjusted to pH=8 with Et3N, and then the resulting precipitate was

1
filtered and dried under reduced pressure to afford a white solid (1.7 g, 80.0%). H NMR (400
MHz, DMSO-d6) δ 8.37 (s, 1H), 7.04 (d, J = 8.4 Hz, 2H), 6.58 – 6.48 (d, J = 8.4 Hz, 2H), 5.24 (s,
2H), 3.85 – 3.71 (m, 4H), 3.52 – 3.41 (m, 4H). HRMS (ESI) calculated for C17H17N6OS [M + H]+
m/z 353.1185, found: 353.1170.
4.3. Synthesis of the target compounds 12a-m
4.3.1. General procedure for preparation of the target compounds 12a-m
A mixture of different substituted isocyanate (14.0 mmol), CH2Cl2 (40 mL) and
4-(4-aminophenyl)-2-morpholino-5-(1H-1,2,4-triazol-3-yl)thiophene-3-carbonitrile 11 (9.3 mmol)
was stirred at 25 °C for 12 h. The resulting precipitate was filtered and recrystallization from
CH3CN to afford target products 12a-m.
4.3.1.1.1-(4-(4-cyano-5-morpholino-2-(1H-1,2,4-triazol-5-yl)thiophen-3-yl)phenyl)-3-(2,6-dimeth
ylphenyl)urea(12a)
White solid; Yield: 70.1%; 1H NMR (400 MHz, DMSO) δ 13.99 (s, 1H), 9.25 (s, 1H), 8.45 (s,
1H), 8.08 (s, 1H), 7.47 (d, J = 8.4 Hz, 2H), 7.25 (d, J = 8.0 Hz, 2H), 7.11–7.00 (m, 3H), 3.84 –
o
3.76 (m, 4H), 3.55 – 3.48 (m, 4H). 2.22 (s, 6H). m.p.: 217.8 – 219.3 C. HRMS (ESI) calculated
for C26H26N7O2S [M + H]+ m/z 476.1869, found: 476.1866.
4.3.1.2.
1-(4-(4-cyano-5-morpholino-2-(1H-1,2,4-triazol-5-yl)thiophen-3-yl)phenyl)-3-(p-tolyl)urea (12b)
1
White solid; Yield: 67.8%; H NMR (400 MHz, DMSO-d6) δ 13.99 (s, 1H), 8.95 (s, 1H),
8.81 (s, 1H), 8.46 (s, 1H), 7.46 (d, J = 8.3 Hz, 2H), 7.35 (d, J = 8.3 Hz, 2H), 7.28 (d, J = 8.3 Hz,
2H), 7.09 (d, J = 8.2 Hz, 2H), 3.84 – 3.74 (m, 4H), 3.55 – 3.48 (m, 4H), 2.24 (s, 3H). 13C NMR
(101 MHz, DMSO-d6) δ 175.0, 166.3, 156.3, 153.0, 144.2, 140.2, 137.5, 131.2 (2C), 130.7, 129.6
(2C), 127.3, 118.8 (2C), 117.9 (2C), 117.1, 116.0, 90.9, 65.8 (2C), 51.0 (2C), 20.8. m.p.: 231.4 –
232.3 oC. HRMS (ESI) calculated for C25H24N7O2S [M + H]+ m/z 486.1712, found: 486.1711.
4.3.1.3.1-(4-(4-cyano-5-morpholino-2-(1H-1,2,4-triazol-5-yl)thiophen-3-yl)phenyl)-3-(2-methoxyp
henyl)urea (12c)
1
White solid; Yield: 66.2%; H NMR (400 MHz, DMSO-d6) δ 13.98 (s, 1H,), 9.46 (s, 1H),
8.46 (s, 1H), 8.29 (s, 1H), 8.15 (d, J = 7.8 Hz, 1H), 7.47 (d, J = 8.3 Hz, 2H), 7.29 (d, J = 8.1 Hz,
2H), 7.03 (d, J = 7.4 Hz, 1H), 6.98–6.93 (m, 1H), 6.90 (t, J = 7.7 Hz, 1H), 3.89 (s, 3H), 3.84 –
3.74 (m, 4H), 3.55 – 3.48 (m, 4H). 13C NMR (101 MHz, DMSO-d6) δ 166.3, 153.8, 152.9, 148.2,

140.3, 130.7 (2C), 129.1, 127.2, 123.6, 123.2, 122.3, 121.0, 118.8, 117.7 (2C), 117.1, 116.1, 111.2,
90.9, 65.8 (2C), 56.2, 51.0 (2C). m.p.: 222.3 – 223.9 oC. HRMS (ESI) calculated for C25H24N7O3S
[M + H]+ m/z 502.1661, found:502.1655.
4.3.1.4.1-(4-(4-cyano-5-morpholino-2-(1H-1,2,4-triazol-5-yl)thiophen-3-yl)phenyl)-3-(4-cyanophe
nyl)urea (12d)
1
White solid; Yield: 72.5%; H NMR (400 MHz, DMSO-d6) δ 13.99 (s, 1H), 9.31 (s, 1H),
9.02 (s, 1H), 8.47 (s, 1H), 7.74 (d, J = 8.8 Hz, 2H), 7.66 (d, J = 8.8 Hz, 2H), 7.50 (d, J = 8.7 Hz,
2H), 7.32 (d, J = 8.2 Hz, 2H), 3.83 – 3.78 (m, 4H), 3.55 – 3.48 (m, 4H). 13C NMR (101 MHz,
DMSO-d6) δ 175.0, 166.2, 152.5, 144.6, 144.3, 139.5, 133.8 (2C), 130.7 (2C), 128.0, 123.6, 123.2,
o
119.8, 118.5 (2C), 118.3 (2C), 117.1, 103.7, 90.9, 65.8 (2C), 51.0 (2C). m.p.: 198.7 – 200.3 C.
HRMS (ESI) calculated for C25H21N8O2S [M + H]+ m/z 497.1508, found: 497.1510.
4.3.1.5.1-(4-(4-cyano-5-morpholino-2-(1H-1,2,4-triazol-5-yl)thiophen-3-yl)phenyl)-3-(4-(trifluoro
methyl)phenyl)urea (12e)
1
White solid; Yield: 67.8%; H NMR (400 MHz, DMSO-d6) δ 13.98 (s, 1H), 9.19 (s, 1H),
8.95 (s, 1H), 8.47 (s, 1H), 7.71 –7.62 (m, 4H), 7.53 – 7.47 (d, J = 8.6 Hz, 2H), 7.32 (d, J = 8.3 Hz,
2H), 3.84 – 3.76 (m, 4H), 3.56 – 3.48 (m, 4H). 13C NMR (101 MHz, DMSO-d6) δ 175.0, 166.2,
152.7, 143.9, 134.0, 130.7 (2C), 126.55 (q, JC–F = 3.6 Hz, 2C), 126.4, 123.7, 123.6, 123.2, 122.2
(q, JC–F = 31.0 Hz, 2C), 118.3 (2C), 118.2, 117.1, 116.0, 90.9, 65.8 (2C), 51.0 (2C). m.p.: 228.5 –
230.1 oC. HRMS (ESI) calculated for C25H21F3N7O2S [M + H]+ m/z 540.1430, found: 540.1432.
4.3.1.6.1-(4-(4-cyano-5-morpholino-2-(1H-1,2,4-triazol-5-yl)thiophen-3-yl)phenyl)-3-(4-(trifluoro
methoxy)phenyl)urea (12f)
White solid; Yield: 66.2%; 1H NMR (400 MHz, DMSO-d6) δ 13.98 (s, 1H), 9.32 (s 1H), 9.22
(s, 1H), 8.43 (s, 1H), 7.64 – 7.55 (d, J = 9.3 Hz, 2H), 7.53 – 7.47 (d, J = 8.6 Hz, 2H), 7.35 – 7.25
(m, 4H), 3.85 – 3.76 (m, 4H), 3.55 – 3.48 (m, 4H). 13C NMR (101 MHz, DMSO-d6) δ 166.3,
153.8, 153.0, 143.0, 140.7, 140.0, 139.6, 130.7 (2C), 127.4, 123.6, 123.2, 122.2 (2C), 120.4 (q, JC–
o
F = 253.5 Hz), 119.7 (2C), 117.9 (2C), 117.1, 90.9, 65.8 (2C), 51.0 (2C). m.p.: 208.8 – 210.1 C.
HRMS (ESI) calculated for C25H21F3N7O3S [M + H]+ m/z 556.1379, found: 556.1385.
4.3.1.7.1-(4-(4-cyano-5-morpholino-2-(1H-1,2,4-triazol-5-yl)thiophen-3-yl)phenyl)-3-(4-fluorophe
nyl)urea (12g)
1
White solid; Yield: 65.9%; H NMR (400 MHz, DMSO-d6) δ 13.99 (s, 1H), 9.17 (s, 1H),

9.07 (s, 1H), 8.46 (s, 1H), 7.58 (d, J = 8.9 Hz, 2H), 7.48 (d, J = 8.3 Hz, 2H), 7.29 (d, J = 8.4 Hz,
4H), 3.84 – 3.76 (m, 4H), 3.55 – 3.48 (m, 4H). 13C NMR (101 MHz, DMSO-d6) δ 166.2, 159.0,
156.6, 155.0 (d, JC–F = 247.8 Hz), 153.1, 144.3, 140.1, 139.5, 130.7 (2C), 127.5, 123.6, 123.2,
120.4 (d, JC–F = 7.6 Hz, 2C), 118.0 (2C), 117.1, 115.7 (d, JC–F = 22.0 Hz, 2C), 90.9, 65.8 (2C),
o
51.0 (2C). m.p.: 244.5 – 245.8 C. HRMS (ESI) calculated for C24H21FN7O2S [M + H]+ m/z
490.1461, found:490.1456.
4.3.1.8.1-(4-(4-cyano-5-morpholino-2-(1H-1,2,4-triazol-5-yl)thiophen-3-yl)phenyl)-3-(2-fluorophe
nyl)urea (12h)
1
White solid; Yield: 58.7%; H NMR (400 MHz, DMSO-d6) δ 14.04 (s, 1H), 9.65 (s, 1H),
8.80 (s, 1H), 8.46 (s, 1H), 8.16 (t, J = 7.7 Hz, 1H), 7.49 (d, J = 8.0 Hz, 2H), 7.30 (d, J = 7.9 Hz,
2H), 7.27–7.18 (m, 1H), 7.14 (t, J = 7.6 Hz, 1H), 7.08 – 6.95 (m, 1H), 3.83 – 3.76 (m, 4H), 3.55 –
o
3.48 (m, 4H). m.p.: 244.5 – 245.7 C. HRMS (ESI) calculated for C24H21FN7O2S [M + H]+ m/z
490.1461, found:490.1464.
4.3.1.9.1-(4-(4-cyano-5-morpholino-2-(1H-1,2,4-triazol-5-yl)thiophen-3-yl)phenyl)-3-(3-fluorophe
nyl)urea (12i)
White solid; Yield: 81.2%; 1H NMR (400 MHz, DMSO-d6) 1H NMR (400 MHz, DMSO-d6) δ
13.98 (s, 1H), 8.99 (s, 1H), 8.88 (s, 1H), 8.46 (s, 1H), 7.54 – 7.45 (m, 3H), 7.33 – 7.25 (m, 3H),
7.16 – 7.12 (m, 1H), 6.82 – 6.75 (m, 1H), 3.83 – 3.76 (m, 4H), 3.55 – 3.48 (m, 4H). 13C NMR
(101 MHz, DMSO-d6) δ 175.0, 162.9 (d, JC–F = 239.2 Hz), 153.8, 152.8 144.3, 142.1, 142.0, 130.8,
130.7 (2C), 123.6, 123.2, 118.1 (2C), 117.1, 116.0, 114.4 (d, JC–F = 2.6 Hz), 108.6 (d, JC–F = 21.3
o
Hz), 105.3 (d, JC–F = 26.5 Hz)，90.9, 65.8 (2C), 51.0 (2C). m.p.: 223.2 – 225.0 C. HRMS (ESI)
calculated for C24H21FN7O2S [M + H]+ m/z 490.1457, found:490.1457.
4.3.1.10.1-(3-chloro-4-fluorophenyl)-3-(4-(4-cyano-5-morpholino-2-(1H-1,2,4-triazol-5-yl)thioph
en-3-yl)phenyl)urea (12j)
1
White solid; Yield: 72.1%; H NMR (400 MHz, DMSO-d6) δ 14.02 (s, 1H), 9.57 (s, 1H),
9.41 (s, 1H), 8.46 (s, 1H), 7.81 (d, J = 7.5 Hz, 1H), 7.47 (d, J = 8.4 Hz, 2H), 7.34 (s, 1H), 7.31 (d,
J = 8.6 Hz, 2H), 7.28 (s, 1H), 3.83 – 3.76 (m, 4H), 3.55 – 3.48 (m, 4H). 13C NMR (101 MHz,
DMSO-d6) δ 174.9, 166.3, 153.8, 152.9, 152.8 (d, JC–F = 247.8 Hz), 151.6, 137.5 (d, JC–F = 2.9
Hz), 134.6, 130.7 (2C), 123.4 (d, JC–F = 44.0 Hz), 120.0 (2C), 119.6 (d, JC–F = 18.2 Hz), 119.0 (d,

J = 6.8 Hz), 118.2, 117.3 (d, JC–F = 36.1 Hz), 117.2, 116.0, 90.9, 65.8 (2C), 51.0 (2C). m.p.: 238.7
– 240.4 oC. HRMS (ESI) calculated for C24H20ClFN7O2S [M + H]+ m/z 524.9777, found:524.1204.
4.3.1.11.1-(2-chlorophenyl)-3-(4-(4-cyano-5-morpholino-2-(1H-1,2,4-triazol-5-yl)thiophen-3-yl)p
henyl)urea(12k)
1
White solid; Yield: 77.1%; H NMR (400 MHz, DMSO-d6) δ 14.03 (s, 1H), 9.84 (s, 1H),
8.49 (s, 1H), 8.46 (s, 1H), 8.17 (d, J = 8.2 Hz, 1H), 7.51 (d, J = 8.3 Hz, 2H), 7.46 (d, J = 8.0 Hz,
1H), 7.32 (s, 1H), 7.29 (d, J = 7.0 Hz, 2H), 7.03 (t, J = 7.6 Hz, 1H), 3.84 – 3.76 (m, 4H), 3.55 –
3.48 (m, 4H). 13C NMR (101 MHz, DMSO-d6) δ 166.2, 156.3, 152.6, 144.3, 139.8, 139.4, 136.4,
130.8, 129.7, 128.1 (2C), 127.8, 123.8, 122.5, 121.8, 117.9 (2C), 117.1, 115.0, 90.9, 65.8 (2C),
o
51.0 (2C). m.p.: 232.5 – 233.7 C. HRMS (ESI) calculated for C24H21ClN7O2S [M + H]+ m/z
506.1166, found: 506.1170.
4.3.1.12.1-(3-bromophenyl)-3-(4-(4-cyano-5-morpholino-2-(1H-1,2,4-triazol-5-yl)thiophen-3-yl)p
henyl)urea(12l)
1
White solid; Yield: 68.1%; H NMR (400 MHz, DMSO-d6) δ 13.99 (s, 1H), 9.14 (s, 1H),
9.06 (s, 1H), 8.46 (s, 1H), 7.87 (s, 1H), 7.47 (d, J = 8.4 Hz, 2H), 7.33 (d, J = 8.7 Hz, 1H), 7.30 (d,
J = 8.3 Hz, 2H), 7.24 (t, J = 8.0 Hz, 1H), 7.15 (d, J = 7.9 Hz, 1H), 3.84 – 3.76 (m, 4H), 3.55 – 3.48
o
(m, 4H). m.p.: 245.6 – 247.0 C. HRMS (ESI) calculated for C24H21BrN7O2S [M + H]+ m/z
550.0661, found: 550.0660.
4.3.1.13.1-(4-(4-cyano-5-morpholino-2-(1H-1,2,4-triazol-5-yl)thiophen-3-yl)phenyl)-3-phenylurea
(12m)
1
White solid; Yield: 62.7%; H NMR (400 MHz, DMSO-d6) δ 14.00 (s, 1H), 9.26 (s, 1H),
9.05 (s, 1H), 8.57 (s, 1H), 7.91 (d, J = 8.2 Hz, 2H), 7.58 (d, J = 8.3 Hz, 2H), 7.47-7.36 (m, 3H),
o
7.32 – 7.26 (m, 2H), 3.84 – 3.76 (m, 4H), 3.56 – 3.48 (m, 4H). m.p.: 229.1 – 231.8 C. HRMS
(ESI) calculated for C24H22N7O2S [M + H]+ m/z 471.1467, found: 471.1462.
4.4. Synthesis of the target compounds 15a-o
4.4.1. Preparation of phenyl (4-(4-cyano-5-morpholino-2-(1H-1,2,4-triazol-3-yl)thiophen-3-yl)
phenyl)carbamate(13)
Phenyl chloroformate (2.3 mL, 18.0 mmol) was added drop-wise to a solution of intermediate
11 (4.2 g, 12.0 mmol) in acetone (80 mL). The mixture was stirred at 25 °C for 3 h. The solvent
was evaporated in vacuo. The mixture was diluted with CH2Cl2 (30 mL) and washed with H2O (2

× 10 mL). The organic phase was dried with Na2SO4 and evaporated in vacuo to afford 13 as a
white solid (4.1 g, 73.2%). HRMS (ESI) calculated for C24H21N6O3S [M + H]+ m/z 473.1318,
found: 473.1316.
4.4.2.
Preparation
of
N-(4-(4-cyano-5-morpholino-2-(1H-1,2,4-triazol-3-yl)thiophen-3-yl)phenyl)hydrazinecarboxamid
e(14)
80% NH2NH2·H2O (1.0 mL, 12.0 mmol) was added drop-wise to a solution of intermediate
13 (2.8 g, 6.0 mmol) in 1, 4-dioxane (50 mL). The mixture was stirred at 100 °C for 2 h. The
resulting precipitate was filtered and dried under reduced pressure to afford 14 as a white solid
(2.1 g, 85.3%). HRMS (ESI) calculated for C18H19N8O2S [M + H]+ m/z 411.1273, found:
411.1276.
4.4.3. General procedure for preparation of the target compounds 15a-m
A mixture of intermediate 14 (5.0 mmol) and different substituted aldehydes (6.0 mmol) in
EtOH (10 mL) and HOAc (1 mL) was stirred at 80 °C for 5 h. Upon cooling to room temperature,
the resulting precipitate was filtered, washed with EtOH and dried under reduced pressure to
afford target products 15a-m.
4.4.3.1(E)-2-benzylidene-N-(4-(4-cyano-5-morpholino-2-(1H-1,2,4-triazol-3-yl)thiophen-3-yl)phe
nyl)hydrazine-1-carboxamide (15a)
1
White solid; Yield: 64.5%; H NMR (400 MHz, DMSO-d6) δ 14.00 (s, 1H), 10.80 (s, 1H),
9.03 (s, 1H), 8.46 (s, 1H), 7.97 (s, 1H), 7.86 (d, J = 6.9 Hz, 2H), 7.68 (d, J = 8.3 Hz, 2H),
7.47-7.36 (m, 3H), 7.32 (d, J = 8.1 Hz, 2H), 3.84 – 3.76 (m, 4H), 3.56 – 3.48 (m, 4H). 13C
NMR(101MHz, DMSO-d6) δ 166.2, 156.3, 153.5, 144.3, 141.4, 139.5, 139.4, 134.8 (2C), 130.4,
129.9 (2C), 129.1, 128.2, 127.5 (2C), 119.7 (2C), 117.1, 114.9, 90.8, 65.7 (2C), 51.0 (2C). m.p.:
o
241.8 – 243.6 C. HRMS (ESI) calculated for C25H23N8O2S [M + H]+ m/z 499.1665, found:
499.1659.
4.4.3.2.
(E)-N-(4-(4-cyano-5-morpholino-2-(1H-1,2,4-triazol-3-yl)thiophen-3-yl)phenyl)-2-
(4-fluorobenzylidene)hydrazine-1-carboxamide (15b)
1
White solid; Yield: 74.2%; H NMR (400 MHz, DMSO-d6) δ 13.99 (s, 1H), 10.90 (s, 1H),
9.08 (s, 1H), 8.47 (s, 1H), 8.02 (s, 1H), 7.99 (s, 2H), 7.66 (d, J = 8.3 Hz, 2H), 7.41 (d, J = 8.3 Hz,
2H), 7.32 (d, J = 8.3 Hz, 2H), 3.84 – 3.76 (m, 4H), 3.56 – 3.48 (m, 4H).13C NMR(101MHz,

DMSO-d6) δ 166.2, 163.2 (d, JC–F = 247.2 Hz), 156.3, 153.6, 144.3, 140.2, 139.5, 139.5, 139.4,
131.5, 131.4, 130.4 (2C), 129.6 (d, JC–F = 8.2 Hz), 128.2, 119. 8 (2C), 117.1, 116.0 (d, JC–F = 21.7
o
Hz), 114.9, 90.9, 65.8 (2C), 51.0 (2C). m.p.: 239.1 – 240.7 C. HRMS (ESI) calculated for
C25H22FN8O2S [M + H]+ m/z 517.1570, found: 517.1562.
4.4.3.3.
(E)-N-(4-(4-cyano-5-morpholino-2-(1H-1,2,4-triazol-3-yl)thiophen-3-yl)phenyl)-2-
(3-bromobenzylidene)hydrazine-1-carboxamide (15c)
1
White solid; Yield: 75.2%; H NMR (400 MHz, DMSO-d6) δ 13.99 (s, 1H), 10.91 (s, 1H),
9.14 (s, 1H), 8.45 (s, 1H), 8.18 (s, 1H), 7.93 (s, 1H), 7.78 (d, J = 7.8 Hz, 1H), 7.66 (d, J = 8.5 Hz,
2H), 7.56 (d, J = 8.0 Hz, 1H), 7.38 (t, J = 7.8 Hz, 1H), 7.31 (d, J = 8.3 Hz, 2H), 3.82 – 3.77 (m,
4H), 3.54 – 3.49 (m, 4H). m.p.: 240.1 – 241.5 oC. HRMS (ESI) calculated for C25H22BrN8O2S[M
+ H]+ m/z 577.0770, found: 577.0763.
4.4.3.4.
(E)-N-(4-(4-cyano-5-morpholino-2-(1H-1,2,4-triazol-3-yl)thiophen-3-yl)phenyl)-2-
(3-hydroxybenzylidene)hydrazine-1-carboxamide (15d)
1
White solid; Yield: 61.5%; H NMR (400 MHz, DMSO-d6) δ 13.99 (s, 1H), 10.72 (s, 1H),
9.54 (s, 1H), 8.98 (s, 1H), 8.46 (s, 1H), 7.88 (s, 1H), 7.66 (d, J = 8.5 Hz, 2H), 7.31 (d, J = 8.4 Hz,
2H), 7.27 – 7.19 (m, 3H), 6.81 (d, J = 7.4 Hz, 1H), 3.82 – 3.77 (m, 4H), 3.54 – 3.49 (m, 4H). 13
C
NMR (101MHz, DMSO-d6) δ 166.2, 158.0, 156.3, 153.5, 144.3, 141.6, 139.5, 136.1, 130.4, 130.1
(2C), 127,5, 119.6, 119.2, 118.7, 117.1 (2C), 114.9, 113.8, 90.9, 65.8 (2C), 51.0 (2C). m.p.: 241.7
– 242.5 oC. HRMS (ESI) calculated for C25H23N8O3S [M + H]+ m/z 515.1614, found: 515.1602.
4.4.3.5.
(E)-N-(4-(4-cyano-5-morpholino-2-(1H-1,2,4-triazol-3-yl)thiophen-3-yl)phenyl)-2-
(4-methoxyphenyl)benzylidene)hydrazine-1-carboxamide (15e)
1
White solid; Yield: 77.3%; H NMR (400 MHz, DMSO-d6) δ 14.00 (s, 1H), 10.67 (s, 1H),
8.98 (s, 1H), 8.47 (s, 1H), 7.92 (s, 1H), 7.80 (d, J = 8.7 Hz, 2H), 7.68 (d, J = 8.5 Hz, 2H), 7.32 (d,
J = 8.5 Hz, 2H), 6.99 (d, J = 8.8 Hz, 2H), 3.81 (s, 3H), 3.82 – 3.77 (m, 4H), 3.54 – 3.49 (m, 4H).
13C NMR (101MHz, DMSO-d6) δ 166.2, 160.8, 156.3, 153.6, 144.3, 141.3, 139.5, 130.4 (2C),
129.1 (2C), 128.1, 127.5, 119.6 (2C), 117.1, 114.9, 114.5 (2C), 91.0, 65.8, 55.7 (2C), 51.0 (2C).
o
m.p.: 255.6 – 257.7 C. HRMS (ESI) calculated for C26H25N8O3S [M + H]+ m/z 529.1770, found:
529.1761.
4.4.3.6.
(E)-N-(4-(4-cyano-5-morpholino-2-(1H-1,2,4-triazol-3-yl)thiophen-3-yl)phenyl)-2-
(3,4-dihydroxybenzylidene)hydrazine-1-carboxamide (15f)

1
White solid; Yield: 77.2%; H NMR (400 MHz, DMSO-d6) δ 13.96 (s, 1H), 10.52 (s, 1H),
9.43 (s, 1H), 9.12 (s, 1H), 8.90 (s, 1H), 8.43 (s, 1H), 7.79 (s, 1H), 7.66 (d, J = 8.5 Hz, 2H), 7.30 (d,
J = 8.5 Hz, 2H), 7.26 (d, J = 1.6 Hz, 1H), 7.05 (dd, J = 8.2, 1.7 Hz, 1H), 6.77 (d, J = 8.1 Hz, 1H),
o
3.82 – 3.77 (m, 4H), 3.54 – 3.49 (m, 4H). m.p.: 246.8 – 248.5 C. HRMS (ESI) calculated for
C25H23N8O4S [M + H]+ m/z 531.1563, found: 531.1556.
4.4.3.7.(E)-N-(4-(4-cyano-5-morpholino-2-(1H-1,2,4-triazol-5-yl)thiophen-3-yl)phenyl)-2-(4-(trifl
uoromethoxy)benzylidene)hydrazine-1-carboxamide (15g)
1
White solid; Yield: 85.5%; H NMR (400 MHz, DMSO-d6) δ 13.99 (s, 1H), 10.90 (s, 1H),
9.08 (s, 1H), 8.46 (s, 1H), 8.01 (d, J = 8.8 Hz, 2H), 7.99 (s, 1H), 7.67 (d, J = 8.5 Hz, 2H), 7.41 (d,
J = 8.3 Hz, 2H), 7.32 (d, J = 8.3 Hz, 2H), 3.83 – 3.76 (m, 4H), 3.55 – 3.49 (m, 4H). 13C NMR
(101MHz, DMSO-d6) δ 166.2, 165.2, 153.5, 149.3, 144.3, 139.7, 134.3, 133.9, 130.4, 130.1 (2C),
130.0, 129.3 (2C), 121.6 (2C), 121.3, 120.5 (q, JC–F = 256.4 Hz), 119.8 (2C), 117.1, 115.0, 91.0,
o
65.8 (2C), 51.0 (2C). m.p.: 254.6 – 256.3 C. HRMS (ESI) calculated for C26H22F3N8O3S [M +
H]+ m/z 583.1488, found: 583.1481.
4.4.3.8.(E)-N-(4-(4-cyano-5-morpholino-2-(1H-1,2,4-triazol-5-yl)thiophen-3-yl)phenyl)-2-(2,6-dic
hlorobenzylidene)hydrazine-1-carboxamide (15h)
1
White solid; Yield: 76.5%; H NMR (400 MHz, DMSO-d6) δ 13.98 (s, 1H), 11.16 (s, 1H),
8.79 (s, 1H), 8.45 (s, 1H), 8.19 (s, 1H), 7.59 (t, J = 8.1 Hz, 4H), 7.43 (t, J = 8.1 Hz, 1H), 7.29 (d, J
= 8.2 Hz, 2H), 3.83 – 3.76 (m, 4H), 3.55 – 3.49 (m, 4H). 13C NMR (101MHz, DMSO-d6) δ 166.2,
156.3, 153.1, 152.1, 144.3, 139.4, 139.1, 136.1, 134.4 (2C), 131.3, 130.6 (2C), 129.7, 129.4, 128.3,
o
119.0 (2C), 117.1, 115.0, 90.9, 65.8 (2C), 51.0 (2C). m.p.: 269.7 – 271.4 C. HRMS (ESI)
calculated for C25H21Cl2N8O2S [M + H]+ m/z 567.0885, found: 567.0879.
4.4.3.9.(E)-N-(4-(4-cyano-5-morpholino-2-(1H-1,2,4-triazol-5-yl)thiophen-3-yl)phenyl)-2-(4-hydr
oxy-3,5-dimethylbenzylidene)hydrazine-1-carboxamide (15i)
1
White solid; Yield: 82.4%; H NMR (400 MHz, DMSO-d6) δ 13.99 (s, 1H), 10.55 (s, 1H),
8.90 (s, 1H), 8.63 (s, 1H), 8.47 (s, 1H), 7.81 (s, 1H), 7.68 (d, J = 8.5 Hz, 2H), 7.41 (s, 2H), 7.31 (d,
J = 8.5 Hz, 2H), 3.83 – 3.76 (m, 4H), 3.55 – 3.49 (m, 4H), 2.20(s, 6H). 13C NMR (101MHz,
DMSO-d6) δ 166.2, 156.3, 152.1, 144.3, 139.4, 139.1 (2C), 134.4 (2C), 131.3, 130.6 (2C), 129.7
o
(2C), 129.4, 128.3, 119.1 (2C), 117.1, 115.0, 90.9, 65.8 (2C), 51.0 (2C). m.p.: 258.7 – 260.1 C.
HRMS (ESI) calculated for C27H27N8O3S [M + H]+ m/z 543.1927, found: 543.1920.

4.4.3.10.(E)-N-(4-(4-cyano-5-morpholino-2-(1H-1,2,4-triazol-5-yl)thiophen-3-yl)phenyl)-2-(3,4,5-
trimethoxybenzylidene)hydrazine-1-carboxamide (15j)
1
White solid; Yield: 77.9%; H NMR (400 MHz, DMSO-d6) δ 13.98 (s, 1H), 10.81 (s, 1H),
9.04 (s, 1H), 8.47 (s, 1H), 7.89 (s, 1H), 7.66 (d, J = 8.5 Hz, 2H), 7.32 (d, J = 8.5 Hz, 2H), 7.15 (s,
2H), 3.86 (s, 6H), 3.83 – 3.76 (m, 4H), 3.70 (s, 3H), 3.55 – 3.49 (m, 4H). 13C NMR (101MHz,
DMSO-d6) δ 166.2, 156.3, 153.6 (2C), 144.3, 141.3, 139.5, 139.4 (2C), 139.2, 130.5, 130.4 (2C),
128.2, 119.8 (2C), 117.1, 115.0, 104.9, 91.0, 65.8, 60.6 (3C), 56.5 (2C), 51.0 (2C). m.p.: 274.0 –
275.9 oC. HRMS (ESI) calculated for C28H29N8O5S [M + H]+ m/z 589.1982, found: 589.1976.
4.4.3.11.(E)-2-((1H-pyrrol-2-yl)methylene)-N-(4-(4-cyano-5-morpholino-2-(1H-1,2,4-triazol-5-yl)t
hiophen-3-yl)phenyl)hydrazine-1-carboxamide (15k)
1
White solid; Yield: 59.2%; H NMR (400 MHz, DMSO-d6) δ 14.00 (s, 1H), 11.51 (s, 1H),
10.56 (s, 1H), 9.03 (s, 1H), 8.47 (s, 1H), 7.76 (s, 1H), 7.63 (d, J = 8.4 Hz, 2H), 7.33 (d, J = 8.4 Hz,
2H), 7.01 (s, 1H), 6.38 (s, 1H), 6.12 (dd, J = 5.4, 2.4 Hz, 1H), 3.83 – 3.76 (m, 4H), 3.55 – 3.49 (m,
4H). 13C NMR (101MHz, DMSO-d6) δ 166.3, 165.2, 153.6, 139.6, 134.0, 133.3, 130.5 (2C), 130.1,
130.0, 128.2, 121.7, 117.2, 119.3 (2C), 112.4, 109.5, 70.2, 65.8, 63.3 (2C), 51.0 (2C). m.p.: 258.1
– 259.9 oC. HRMS (ESI) calculated for C23H22N9O2S [M + H]+ m/z 488.1617, found: 488.1611.
4.4.3.12.(E)-N-(4-(4-cyano-5-morpholino-2-(1H-1,2,4-triazol-5-yl)thiophen-3-yl)phenyl)-2-(furan
-2-ylmethylene)hydrazine-1-carboxamide (15l)
White solid; Yield: 66.8%; 1H NMR (400 MHz, DMSO-d6) δ 14.01 (s, 1H), 10.76 (s, 1H), 8.81 (s,
1H), 8.46 (s, 1H), 8.18 (s, 1H), 7.63 (d, J = 5.0 Hz, 1H), 7.61 (d, J = 8.2 Hz, 2H), 7.44 (d, J = 3.1
Hz, 1H), 7.30 (d, J = 7.7 Hz, 2H), 7.12 (dd, J = 4.9, 3.7 Hz, 1H), 3.83 – 3.76 (m, 4H), 3.54 – 3.48
(m, 4H). 13C NMR (101MHz, DMSO-d6) δ 166.2, 156.3, 153.3, 150.1, 144.9, 144.3, 139.4, 139.3,
131.8 (2C), 130.5, 128.1, 119.3, 117.1, 115.0, 112.6 (2C), 112.2, 91.0, 65.8 (2C), 51.0 (2C). m.p.:
o
247.4 – 249.1 C. HRMS (ESI) calculated for C23H21N8O3S [M + H]+ m/z 489.1457, found:
489.1448.
4.4.3.13.(E)-N-(4-(4-cyano-5-morpholino-2-(1H-1,2,4-triazol-5-yl)thiophen-3-yl)phenyl)-2-(pyridi
n-2-ylmethylene)hydrazine-1-carboxamide (15m)
1
White solid; Yield: 59.2%; H NMR (400 MHz, DMSO-d6) 13.99 (s, 1H), 10.97 (s, 1H), 9.10 (s,
1H), 9.01 (s, 1H), 8.57 (d, J = 3.8 Hz, 1H), 8.47 (s, 1H), 8.32 (d, J = 7.9 Hz, 1H), 7.99 (s, 1H),
7.67 (d, J = 8.4 Hz, 2H), 7.45 (dd, J = 7.8, 4.8 Hz, 1H), 7.32 (d, J = 8.4 Hz, 2H), 3.83 – 3.76 (m,

4H), 3.54 – 3.48 (m, 4H). m.p.: 245.8 – 247.2 oC. HRMS (ESI) calculated for C24H22N9O2S [M +
H]+ m/z 500.1617, found: 500.1598.
4.5. Pharmacology
4.5.1. In vitro enzymatic assays
The in vitro enzymatic assays of target compounds on PI3Ks were evaluated by
Kinase-Glo™ assay and ADP-Glo™ Kinase Assay. Briefly, the compound, PI3K enzyme (PI3Kα
from Invitrogen, PIK3Cβ from Millipore, PIK3Cγ from Invitrogen, and PIK3Cδ from Millipore),
the PIP2 (Life) substrate, and ATP (25 μM, Sigma) were diluted in kinase buffer to the indicated
concentrations. The assay plate was covered and incubated at room temperature (PI3Kα, PI3Kβ,
and PI3Kγ for 1 h and PI3Kδ for 2 h). Then, the Kinase-Glo reagent (Promega) was added to the
PI3Kα plate to stop the reaction and shaken for 15 min. For the PI3Kδ, PI3Kβ, and PI3Kγ
inhibition assay, ADP-Glo reagent 1(Promega) was added and shaken slowly for 40 min, followed
by the addition of ADP-Glo reagent 2 (Promega), shaken for 1 min, and equilibrated for 60 min,
while mTOR inhibition of compound were evaluated by Lance Ultra Assay. Briefly, mTOR
enzyme (from Millipore), the ULight-4E-BP1 (Thr37/46) Peptide (Promega), and ATP (13 μM,
Sigma) were diluted in kinase buffer to the indicated concentrations. The assay plate was covered
and incubated at room temperature for 1h. Then, the detection solution of kinase quench buffer
(EDTA, Sigma) and Eu-anti-phospho-4E-BP1 (Promega) was added to the plate to stop the
reaction, shaken for 15 min and equilibrated for 60 min. The data were collected on Envision and
presented in Excel. IC50 values were calculated from the inhibition curves.
4.5.2. Cell proliferation assays
All target compounds were evaluated for antiproliferative potency against U87MG, T47D,
H1975, H460 and A549 tumor cell lines using a CellTiter-Glo® Luminescent Cell Viability Assay.
The human tumor cell lines used were obtained from the ATCC. All the mediums and FBS were
Gibco. T47D, H1975 and H460 tumor cells were cultured in RPMI1640 medium supplemented
with 10% FBS. U87MG were cultured in DMEM medium supplemented with 10%FBS. A549
were cultured in Ham's F12K medium supplemented with 10%FBS. The day before treatment
with compounds, cells were seeded at a density of 3 × 103 in each well of a 96-well plate. The

tumor cells were then treated with 3-fold serial diluted compound or DMSO control in the
incubator at 37 °C and 5% CO2 for 3 days, prior to the addition of CellTiter-Glo reagents
(Promega) and reading of luminescence using a PerkinElmer Envision plate reader. IC50 values
were determined by curve fitting using XLfit program of Excel software (Microsoft, USA).
4.5.3. Kinase Competition Binding Assays
KINOMEscan competition binding assays (www.kinomescan.com) were performed as
described previously.[27] Kinases were produced displayed on T7 phage or by expression in
HEK-293 cells and tagged with DNA. Binding reactions were performed at rt for 1 h, and the
fraction of kinase not bound to test compound was determined by capture with an immobilized
affinity ligand and quantitation by quantitative PCR. Each kinase was tested individually against
each compound.
4.5.4. Western Blot Analysis
Cells were treated with 12k and PF-4989216 at the indicated concentrations for 2 h at 37 °C
before stimulated with 1 μg/mL insulin for 2 h, then the cells were harvested, washed in ice-cold
PBS, and lysed with RIPA bu er, protease inhibitors, phosphatase cocktails A and B, and PMSF
(1 mM). Protein concentration was determined by the BCA Kit. The samples were subjected to
SDS− PAGE and then transferred onto PVDF membranes (Millpore). The membranes were
incubated overnight at 4 °C with the primary antibody (anti-AKT(CST#4691T), anti-pAKTser473
(CST#4060T)in 5% BSA/TBST bu er with gentle shaking, then washed with 1 × TBS/T 3 times,
followed by incubation with secondary antibodies (IRDye 680RD Goat anti-Rabbit antibody; from
Licor, Cat. No. 926-68021, 1:10000 dilution), diluted in PBST, for 1 hour at RT. The target blots
were detected with Li-Cor Odyssey imager system.
4.5.5. Caco-2 permeability assay
Caco-2 cells purchased from ATCC were seeded onto polyethylene membranes (PET) in
96-well Insert plates at 1 x 105 cells/ cm2, and refreshed medium every 4~5 days until to the 21st
to 28th day for confluent cell monolayer formation. Test compound was tested at 2 μM
bi-directionally in duplicate. Digoxin was tested at 10 μM bi-directionally in duplicate, while
nadolol and metoprolol were tested at 2 μM in A to B direction in duplicate. Final DMSO