Discovery of new thienopyrimidine derivatives as potent and orally efficacious phosphoinositide 3-kinase inhibitors

Article abstract of DOI:10.1016/j.bmc.2017.12.025

A series of new thienopyrimidine derivatives has been discovered as potent PI3K inhibitors. The systematic SAR studies for these analogues are described. Among them, 8a and 9a exhibit nanomolar enzymatic potencies and sub-micromolar cellular anti-prolifer

Full text of DOI:10.1016/j.bmc.2017.12.025

Accepted Manuscript
Discovery of New Thienopyrimidine Derivatives as Potent and Orally Effica-
cious Phosphoinositide 3-Kinase Inhibitors
Songwen Lin, Chunyang Wang, Ming Ji, Deyu Wu, Yuanhao Lv, Li Sheng,
Fangbin Han, Yi Dong, Kehui Zhang, Yakun Yang, Yan Li, Xiaoguang Chen,
Heng Xu
PII:
S0968-0896(17)32142-9
https://doi.org/10.1016/j.bmc.2017.12.025
BMC 14130
DOI:
Reference:
Bioorganic & Medicinal Chemistry
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Revised Date:
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17 December 2017
19 December 2017
Please cite this article as: Lin, S., Wang, C., Ji, M., Wu, D., Lv, Y., Sheng, L., Han, F., Dong, Y., Zhang, K., Yang,
Y., Li, Y., Chen, X., Xu, H., Discovery of New Thienopyrimidine Derivatives as Potent and Orally Efficacious
Phosphoinositide 3-Kinase Inhibitors, Bioorganic & Medicinal Chemistry (2017), doi: https://doi.org/10.1016/
j.bmc.2017.12.025
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Bioorganic & Medicinal Chemistry
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Discovery of New Thienopyrimidine Derivatives as Potent and Orally Efficacious
Phosphoinositide 3-Kinase Inhibitors
Songwen Lin a,1, Chunyang Wang a,1, Ming Ji a, Deyu Wu b, Yuanhao Lv a, Li Sheng c, Fangbin Han d, Yi
Dong b, Kehui Zhang b, Yakun Yang c, Yan Lic, Xiaoguang Chen a, ∗ , Heng Xu a, ∗
a State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and
Peking Union Medical College, Beijing 100050, China.
b Beijing Key Laboratory of Active Substances Discovery and Drugability Evaluation, Institute of Materia Medica, Chinese Academy of Medical Sciences and
Peking Union Medical College, Beijing 100050, China.
c Beijing Key Laboratory of Non-Clinical Drug Metabolism and PK/PD study, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing
100050, China
d PKUCare Pharmaceutical R&D Center, Beijing 102206, China
ARTICLE INFO
ABSTRACT
Article history:
Received
A series of new thienopyrimidine derivatives has been discovered as potent PI3K inhibitors. The
systematic SAR studies for these analogues are described. Among them, 8a and 9a exhibit
nanomolar enzymatic potencies and sub-micromolar cellular anti-proliferative activities. 8a
displays favorable pharmacokinetic profiles, while 9a easily undergoes deacetylation to yield a
major metabolite 8a. Furthermore, 8a and 9a potently inhibit tumor growth in a dose-dependent
manner in the NCI-H460 xenograft model with an acceptable safety profile.
Received in revised form
Accepted
Available online
Keywords:
2009 Elsevier Ltd. All rights reserved.
Phosphoinositide 3-kinase inhibitors
anti-tumor activities
thienopyrimidine
acetylation
1. Introduction
and proliferation.4 Moreover, phosphatase and tensin homologue
(PTEN), a lipid phosphatase that catalyzes the dephosphorylation
Phosphoinositide 3-kinases (PI3Ks) are a family of lipid
kinases that are involved in many essential cellular functions
including cell growth, proliferation, differentiation, motility,
survival, and intracellular trafficking.1-3 Depending upon their
sequence homology and substrate preferences, PI3Ks are divided
into three distinct classes (I, II & III).3 The class I PI3Ks are most
intensively studied to date and consist of a regulatory subunit and
a catalytic subunit. The catalytic subunit occurs in four isoforms
designated as p110α, p110β, p110γ and p110δ. Signaling from
receptor tyrosine kinases and G-protein-coupled receptors, the
class I PI3Ks catalyze the phosphorylation of the 3'-hydroxyl
group of phosphatidylinositol 4,5-biphosphate (PIP2) to generate
phosphatidylinositol 3,4,5-triphosphate (PIP3). PIP3 functions as
a potent secondary cellular messenger to initiate a signal
transduction cascade leading to activation of the serine/threonine
kinase AKT (also known as protein kinase B or PKB).3 The
activated AKT in turn triggers the downstream signaling events
that ultimately stimulates mammalian target of rapamycin
of PIP3, is identified as a major negative regulator of the PI3K
pathway.5
The PI3K pathway is one of the most frequently aberrantly
6
activated signaling pathways in human cancers.3, Molecular
alterations in this pathway are significantly associated with
tumorigenesis.7 According to a recent analysis on 19,784
consecutive tumor samples (> 40 cancer types), 38% of patients
had at least one alternation in the PI3K pathway components.8
Inhibition of this pathway therefore provides a promising
approach to discover novel therapeutics for cancer treatment. A
number of dual PI3K/mTOR, pan-PI3K and isoform selective
PI3K inhibitors have entered into clinical trials, alone or in
combination, in both solid tumors and hematologic
malignancies.9-12 In 2014, FDA approved the first-in-class PI3Kδ
inhibitor Idelalisib for the treatment of patients with three types
of hematologic cancers (CLL, FL and SLL).13 In 2017, the class I
PI3K inhibitor copanlisib received an accelerated approval from
FDA for the treatment of relapsed follicular lymphoma based on
a single-arm trial that included only 104 patients (Figure 1).14
(mTOR) that plays a key role in regulating cell growth, survival
———
∗ Corresponding author. Email addresses: chxg@imm.ac.cn; xuheng@imm.ac.cn
1 These authors contributed equally to this work.

Copanlisib showed preferential activity against PI3Kα and p110δ
as compared with PI3Kβ and p110γ.15 Extensive efforts to
develop PI3K inhibitors to treat solid tumors are still ongoing.
However, emerging clinical data showed those PI3K inhibitors
had limited single-agent activities in solid tumors and none has
yet reached a regulatory approval.16 Clearly, challenges remain
for PI3K inhibitors to become a significant component of the
anticancer portfolio. To address these challenges, future
directions for clinical development of PI3K inhibitors are getting
a greater focus on patient selection, rational combination as well
as understanding of immune modulation.16-18 From medicinal
chemistry point of view, identification of more structurally
diversified PI3K inhibitors would provide opportunities to differ
their pharmacological and safety profiles, thereby facilitating
discovery of inhibitors targeting PI3K pathway with better
clinical outcomes.
Scheme 1. Synthesis of compounds 8a-h and 9a-g. Reagents and
conditions. (a) methylboronic acid, 2M K2CO3, PdCl2(dppf), 1,4-
dioxane, 105 °C, 3 h, Ar, 42%; (b) NBS, DMF, rt, 4 h, 76%; (c)
2M NaOH, EtOH, 90 °C, 1.5 h, 82%; (d) acyl chloride, pyridine,
DMF, rt, 4 h, 52-60%; (e) MeI, NaH, DMF, rt, 4 h, 28%; (f) aryl
boronic ester, PdCl2(dppf), 2M K2CO3, dioxane, 100 °C, Ar, 4-6
h, 20-59%.
2.2. Enzymatic and cellular assays
Table 1. SAR of pyridopyrimidine replacements.
Fig. 1. Selected PI3K inhibitors (1 - 4).
PI3K
IC50 (nM)a
U87MG
IC50 (µM)b
Compd.
bicycles
As previously reported, we disclosed a series of 2-amino-4-
methylpyrido[2,3-d]pyrimidine derivatives as potent PI3K
inhibitors, in which the key aminopyridopyrimidine, sulfonamide
and pyridyl nitrogen moieties interacts with Val 882, Lys 833,
and a conserved water molecule respectively in the ATP binding
pocket.19 Among them, the representative compound 4 provided
potent inhibition of PI3Kα with an IC50 of 2.0 nM and a
reasonable U87 cell IC50 of 0.63 µM (Fig. 1). However, 4 could
not achieve tumor growth inhibition in in vivo xenograft models
at various doses, probably due to its undesirable ADME
properties originated from poor physicochemical properties. In
this paper, we explored various 6,6- and 6,5- bicycles (5-9) as
alternatives to the pyridopyrimidine core in the previous scaffold
and further optimization led to the discovery of a series of new
thienopyrimidine derivatives (8a and 9a) that demonstrated
excellent enzymatic, cellular and in vivo antitumor activities.
2.0
6.1
2.4
1.3
1.3
0.63
>50
0.96
0.63
2.4
4
5
6
7
8a
PI-103
--
--
7.4
--
--
0.34
PF-04691502
a
The IC50s are mean values of duplicates.
The IC50s are mean values of triplicates.
b
2. Results and discussion
Our initial structural modification focused on replacement of
the pyridopyrimidine core of compound 4 with different 6,6- and
6,5- bicycles as these bicycles were envisioned to adopt similar
binding topologies that could maintain major interactions with
the PI3K kinase (Table 1). Pyridopyrimidinone 5 and 6 were
firstly synthesized for evaluation. As shown in Table 1, both
compounds displayed effective kinase inhibition with single-digit
nanomolar potencies against PI3Kα, comparable to 4. Notably,
compound 5 was inactive in a U87 cellular assay (IC50 > 50 µM).
We reasoned that the NH proton of the pyridopyrimidinone in 5
deleteriously affected its membrane permeability. Pteridin-7(8H)-
one 7 demonstrated similar PI3Kα and cellular potencies relative
to 4 and 6, indicating 6,6-bicylics was generally a good scaffold
to project the key peripheral moieties to access the key
interactions. It is important to note that the 6,5-bicyclic
thienopyrimidine 8a, structurally distinct from above 6,6-
2.1. Chemistry
The representative synthetic approach for thienopyrimidine
derivatives 8a-h and 9a-g was described in Scheme 1. The
starting material 10 was prepared according to the reported
methods.20 Treatment of 10 with methylboronic acid afforded 11,
which was brominated with NBS and then deprotected to afford
the key intermediate 13. 13 was then converted to 14a-e by either
acylation or methylation. Finally, 13 and 14a-e were coupled
with various aryl boronic esters to yield target compounds 8a-h
and 9a-g.

bicycles, potently inhibit PI3Kα kinase with an IC50 of 1.3 nM,
while its U87 cellular proliferation activity was considerably
lower than those of pyridopyrimidinone 6 and pteridin-7(8H)-one
7. Encouraged by its differentiated structure bearing nanomolar
PI3Kα potency, thienopyrimidine 8a was further derivatized to
improve its cellular activity along with other drug-like properties.
could also boost cellular potency for 5-chlorothiophene-2-
sulfonamide (8c vs 9f), but, in contrast, it could not make
improvement on methanesulfonamide (8d vs 9g).
Table 3. SAR of the pyrimidine amine and sulphonamide.
Sulfonamide functionality in 8a was subsequently targeted to
modify given that the deprotonated sulfonamide NH at
physiological conditions could form charge interactions with
Lys833 to increase the potency (Table 2).21, 22 4-fluorobenzene-
sulfonamide 8b exhibited the similar level of both PI3K and U87
cellular potencies in comparison to 8a. When 5-chlorothiophene
was introduced, U87 cellular potency dropped ~3 fold while
PI3Kα potency maintained (8c vs 8a). Methanesulfonamide 8d
and cyclopropanesulfonamide 8e displayed decreased potencies
for both PI3Kα and U87 cellular growth inhibition relative to 8a.
To explore the SAR, methoxy group adjacent to the pyridine
nitrogen was changed to other small substituents (e.g. Cl or Me)
and the corresponding 8f, 8g and 8h showed a significant
decrease in cellular activity (~2-6 fold decrease relative to 8a).
Overall, above variations on sulfonamide functionality failed to
yield compounds with improved cellular potencies.
PI3Kα
U87MG
IC50 (µM)b
Compd.
R2
R3
IC50 (nM)a
9a
1.1
2.2
0.30
9b
9c
1.5
1.6
2.3
9d
9e
2.3
1.6
6.5
2.1
CH3
9f
1.0
4.5
0.70
3.0
Table 2. SAR of pyridine substituents.
9g
CH3
PI-103
PF-04691502
--
--
--
--
7.4
--
--
0.34
a The IC50s are mean values of duplicates.
b The IC50s are mean values of triplicates.
PI3Kα
U87MG
IC50 (µM)b
Compd.
R1
R2
IC50 (nM)a
2.3. In vitro profile of 8a and 9a
8a
OMe
1.3
2.4
On the basis of the above SAR exploration, 8a and its
acetylated counterpart 9a were then selected for further profiling
(Table 4). 8a and 9a potently inhibited all four different class I
PI3K isoforms (α, β, δ and γ). While they are least potent against
PI3Kγ with an IC50 of ~10 nM, 8a and 9a did not show an
appreciated selectivity for those isoforms. As anticipated, 8a and
9a also had mTOR inhibitory activity since it shares a high
degree of sequence homology with class I PI3Ks (mTOR IC50: 10
nM for 8a and 9a, respectively). For a panel of 5 selected cancer
cell lines, 9a displayed sub-micromolar potencies. In this cell line
panel, 9a was most sensitive to the PIK3CA mutated T47D
breast cancer line (IC50 = 0.009 µM) and least sensitive to the
PTEN null U87 glioblastoma cell line (IC50 = 0.30 µM). Relative
to 9a, 8a showed 3-14 fold lower cellular activity across this cell
line panel for the aforementioned permeability reason.
8b
8c
OMe
OMe
2.2
1.5
2.9
8.1
8d
8e
OMe
OMe
CH3-
2.8
5.6
3.0
6.5
8f
Cl
2.2
9.3
5.8
15
8g
Me
8h
Me
3.0
8.7
PI-103
PF-04691502
a The IC50s are mean values of duplicates.
--
--
--
--
7.4
--
--
0.34
b The IC50s are mean values of triplicates.
The challenge of translating its enzymatic inhibition into
potent cellular activity for these thienopyrimidine derivatives still
remains to be tackled. Finally, we turned our attention to the
polar pyrimidylamino group. To mask the amino group, we
carried out various amidation as well as methylation reactions.
As shown in Table 3, in comparison to their free amine form 8a,
acetamide 9a, propionamide 9b and butyramide 9c more or less
maintained its enzymatic potency. More importantly, the
amidation can give rise to enhanced cellular potency (9a and 9b
vs 8a, respectively). In particular, acetamide 9a had an IC50 of
0.3 µM in a U87 cell proliferation assay, around 8 fold
improvement over that of 8a. This improvement was mainly
attributed to its enhanced permeability as observed in a PAMPA
assay (Table 4, 8a vs 9a, 4.8×10-6 cm/s vs 9.6×10-6 cm/s). It is
also worth noting that methylation of 8a yielded less potent
compound 9e compared to acetylation. Further structural
modification was to vary sulfonamide functionality while
retaining the acetamide moiety. It was found that acetylation
Table 4. In vitro profile of 8a and 9a.
Assay
8a
1.3
1.2
2.7
9a
1.1
3.4
4.2
PI3Kα
PI3Kβ
PI3Kδ
Enzymatic
activity
IC50a (nM)
PI3Kγ
10
10
11
10
mTOR
U87MG
T47D
2.4
0.30
0.0090
0.052
0.062
0.10
9.6
0.13
0.29
0.21
0.50
4.8
Cell Growth
IC50b (µM)
SKOV3
H460
A549
(10-6 cm/s)
PAMPA
Mouse liver
microsome
stability
T1/2 (min)
139
53
CL (µL/min/mg)
7.1
19
a The IC50s are mean values of duplicates.
b The IC50s are mean values of triplicates..

Table 5. Mouse pharmacokinetic parameters of 8a and 9a.
IV (2 mg/kg)a
Compd
PO (10 mg/kg)a
T1/2 (h)
7.7
CL (mL/h/kg)
37
Vss (mL/kg)
407
AUC(0-∞) (h*ng/mL)
54617
Cmax (ng/mL)
10912
AUC(0-∞) (h*ng/mL)
186143
F (%)
67
8a
9ab
8ab
0.80
6.9
351
22
407
217
5693
294
1544
6.4
/
91410
1717
34461
a Three animals per study for IV and five animals per study for PO.
b 8a was a major metabolite of 9a and blood concentrations of 8a and 9a were measured simultaneously to determine their PK parameters after administration of
9a.
Western blot analysis revealed the effect of 8a and 9a on PI3K
downstream effectors in NCI-H460 cells (Fig. 2). After 3 hour
treatment, 8a and 9a inhibited phosphorylation of AKT at S473
and T308, the mTORC1 substrate p70S6K at T389 and the
p70S6K substrate S6RP at S235/236 in a dose-dependent manner
respectively. 8a started to show the suppression on
phosphorylation from the concentration of 100 nM, while 9a
produced the similar effect from a lower concentration of 30 nM.
The data clearly demonstrated 9a was more potent to inhibit
phosphorylation of the PI3K downstream effectors than 8a,
which is consistent with their inhibitory effects on cell
proliferation.
significantly lower than those of 8a. As depicted in
supplementary Figure S1, 9a was metabolically unstable and
readily underwent deacetylation in vivo, generating a major
active metabolite 8a. Noteworthy, the overall AUC exposure of
metabolite 8a by oral dosing of 9a is about 20% of that by orally
administrating 8a itself, while the exposure of metabolite 8a by
intravenous dosing of 9a is 1.5 fold higher compared to that by
dosing of 8a through the same administration route. These
pharmacokinetic studies suggest that 9a could be considered as a
pro-drug of 8a.
2.5. In vivo efficacy study
Furthermore, 8a and 9a were progressed into the established
NCI-H460 non-small cell lung cancer (NSCLC) xenograft model.
After once-daily oral dosing for 14 consecutive days, 8a and 9a
exhibited robust and dose-dependent antitumor activity as
evidenced by the decrease in tumor volume and tumor weight
(Figure 3A and 3B). At the same dose level of 10 mg/kg, 9a
produced a more pronounced in vivo efficacy than 8a (TGI:
85.2% vs 56.7%). The effect of 8a and 9a on phosphorylation of
PI3K signaling components was examined by Western blot on
day 14. A significant decrease in phosphorylation of AKT at
S473 and S6RP at S235/236 was observed in tumor lysates
collected 3 hours after last doses (Fig. 3D). 8a and 9a were well
tolerated over the course of the study. Except for the 10 mg/kg
dose of 9a (14.8% body weight drop), the body weight drop for
all the other treatment groups was below 10% compared to their
initial weight (Fig. 3C), demonstrating an encouraging safety
profile.
DMSO 10 30 100 300 1000 (nM)
DMSO 10 30 100 300 1000 (nM )
p-AKT
(T308)
p-AKT
(S473)
AKT
p-p70S6K
p70S6K
p-S6RP
S6RP
β-actin
8a
9a
Fig. 2. The PI3K pathway was inhibited by 8a and 9a in NCI-H460 cells. The
cells were treated with the compounds for 3 hours. Cell lysates were analyzed
by Western blot with indicated antibodies. β-actin was used as a loading
control.
3
A
B
3000
2500
2000
1500
1000
500
Vehicle
8a 10mg/kg qd, TGI 56.7%
8a 20mg/kg qd, TGI 73.1%
2
1
0
9a 5mg/kg qd, TGI 75.9%
9a 10mg/kg qd, TGI 85.2%
***
***
2.4. Pharmacokinetic study
***
***
***
***
***
***
To measure their in vitro intrinsic clearance, 8a and 9a were
tested in a mouse microsomal stability assay. 9a was determined
to be moderately stable (CL = 19 µL/min/mg), while 8a had a
much lower clearance rate (CL = 7.1 µL/min/mg). As was also
observed in other series,23 9a easily underwent metabolic N-
deacetylation to generate the corresponding 8a, explaining the
difference between the microsomal stability in these two
compounds.
0
0
2
4
6
8
10
12
14
D
16
Days after treatment
Vehicle
8a (20mg/kg)
9a (10mg/kg)
C
24
1#
2#
3#
1#
2#
3#
1#
2# 3#
20
16
12
8
p-AKT(473)
AKT
Vehicle
8a 10mg/kg qd
8a 20mg/kg qd
9a 5mg/kg qd
p-S6RP
4
9a 10mg/kg qd
S6RP
0
In vivo pharmacokinetic profile of 8a and 9a was examined in
mice (Table 5). When 8a was administrated intravenously at a
dose of 2 mg/kg, it exhibited a low clearance (CL = 37 mL/h/kg)
and moderate to long half life (T1/2 = 7.7 h). After an oral
administration at a dose of 10 mg/kg, 8a demonstrated a very
high exposure (AUC(0-∞) = 186143 h*ng/mL) and good oral
bioavailability (F% = 67%). 9a, however, showed a much higher
clearance (CL = 351 mL/h/kg) and shorter half life (T1/2 = 0.80 h)
in comparison to 8a. Its AUC(0-∞) and bioavailability were
determined to be 1544 h*ng/mL and 6.4% respectively,
0
2
4
6
8
10 12 14 16
β-actin
Days after treatment
Fig. 3. Antitumor activity of 8a and 9a in the NCI-H460 xenograft model.
(A) Tumor volume changes. Results are expressed as the mean tumor volume
SEM (n = 6 for each group). *** p<0.001, vs vehicle. (B) Tumor weight
changes at day 14. Results are expressed as tumor weight (n = 6 for each
group). *** p<0.001, compared to Vehicle. (C) Body weight changes. Results
are expressed as Body Weight SEM (n = 6 for each group). (D)
Phosphorylation of AKT at S473 and S6RP at S235/236 was significantly

inhibited in the same xenograft model. Tumor lysates were collected 3 hours
post doses on the last day.
Method A: A mixture of aryl bromide derivatives (1.0
equiv.), 2M aqueous K2CO3 solution (4.0 equiv.) and aryl
boronic ester (1.2-1.4 equiv.) in dioxane or DMF was degassed,
and then Pd(PPh3)2Cl2 or PdCl2(dppf) (0.05-0.1 equiv.) was
added. The mixture was degassed and back-filled with argon
(three cycles), and then stirred at 80-100 °C for 4-6 h. The
reaction mixture was diluted with water (50 mL) and extracted
with EtOAc (30 mL×3). The combined organic layers were
washed with water and brine, dried over Na2SO4, filtered and
concentrated. The residue was purified by column
chromatography (silica gel) to afford the target compounds.
In a separate in vivo study, nude mice bearing NCI-H460
tumors received a single 10mg/kg oral dose of 9a and pAKT
levels in tumor tissues were then determined over the course of
24 hours. As depicted in Fig. 4, 9a showed a sustained PD
response over the 12-hour period with pAKT levels returning to
those of control by 24 hour, correlated with the efficacy observed
in the NCI-H460 xenograft tumor model.
Method B: A mixture of 2,3-disubstituted-5-bromopyridine
derivatives (1.2 equiv.), bis(pinacolato)diboron (1.32 equiv.) and
potassium acetate (2.4 equiv.) in 1,4-dioxane was degassed, and
then PdCl2(dppf) (0.06 equiv.) was added. The reaction mixture
was degassed and back-filled with argon (three cycles), stirred at
100 °C under argon atmosphere for 4 h, and then cooled to room
temperature. To the resulting mixture was added 13 (1.0 equiv.),
2M aqueous K2CO3 solution (3.0 equiv.) and PdCl2(dppf) (0.1
equiv.). The resulting mixture was degassed and back-filled with
argon (three cycles) and then stirred at 100 °C under argon
atmosphere for 4 h. The reaction mixture was cooled to room
temperature, diluted with water (30 mL), and extracted with ethyl
acetate (30 mL×3). The combined organic layers were washed
with water and brine, dried over anhydrous Na2SO4, filtered and
concentrated. The residue was purified with column
chromatography (silica gel) to afford the target compounds.
Fig. 4. PD effect of 9a in the NCI-H460 xenograft model following a single
oral dose of 10mg/kg. pAKT (Ser473) levels and compound concentrations
(the parent compound 9a and its metabolite 8a) in plasma and tumor tissues
were measured at 2, 4, 8, 12 and 24 hours post-dose. Error bars denote the
standard deviation of the mean (n=3 per group at each time point).
N-(5-(2-amino-4-methyl-7-oxo-7,8-dihydropyrido[2,3-
d]pyrimidin-6-yl)-2-methoxypyridin-3-yl)-2,4-
difluorobenzenesulfonamide (5)
1
Yellow solid (method A, 33% yield). mp: >300 °C. H NMR
3. Conclusion
(400 MHz, DMSO-d6) δ 11.93 (s, 1H), 10.19 (s, 1H), 8.34 (d, J =
2.2 Hz, 1H), 8.02 (s, 1H), 8.00 (d, J = 2.2 Hz, 1H), 7.73 (td, J =
8.4, 2.0 Hz, 1H), 7.62 – 7.56 (m, 1H), 7.20 (td, J = 8.4, 2.0 Hz,
1H), 7.08 (s, 2H), 3.62 (s, 3H), 2.54 (s, 3H). 13C NMR (101 MHz,
DMSO-d6) δ 167.5, 165.0 (dd, JC-F = 255, 11.8 Hz), 162.5, 162.3,
159.4 (dd, JC-F = 259, 13.5 Hz), 157.3, 155.5, 143.9, 136.3, 134.0,
131.8 (d, JC-F = 11.2 Hz), 126.0, 125.1 (dd, JC-F = 14.5, 3.6 Hz),
121.6, 118.5, 111.7 (dd, JC-F = 22.2, 3.0 Hz), 105.7 (t, JC-F = 26.2
Hz), 103.0, 53.2, 20.6, 20.7. HRMS (ESI): m/z [M + H]+ calcd
for C20H17O4N6F2S, 475.0995, found: 475.0992.
In summary, we have described the discovery of a series of
new thienopyrimidines as potent PI3K inhibitors. 8a and 9a with
distinct structures from the series demonstrated nano-molar
enzymatic potencies against the Class I PI3K subtypes and sub-
micromolar cellular anti-proliferative activities across several
cancer cell lines. They could potently suppress phosphorylation
of the PI3K downstream effectors including AKT, p70S6K and
S6RP. 8a also displayed favorable pharmacokinetic properties,
while 9a readily underwent deacetylation to afford 8a as a major
active metabolite. Both compounds were efficacious in a dose-
dependent manner with an acceptable tolerable safety profile in
the NCI-H460 xenograft model. The above findings support
further advancement of this new thienopyrimidine series, in
particular, 8a and 9a into preclinical evaluation.
N-(5-(2-amino-4,8-dimethyl-7-oxo-7,8-dihydropyrido[2,3-
d]pyrimidin-6-yl)-2-methoxypyridin-3-yl)-2,4-
difluorobenzenesulfonamide (6)
1
Yellow solid (method A, 69% yield). mp: 243 – 244 °C. H
NMR (400 MHz, DMSO-d6) δ 10.21 (s, 1H), 8.32 (d, J = 2.2 Hz,
1H), 8.05 (s, 1H), 8.01 (d, J = 2.2 Hz, 1H), 7.72 (td, J = 8.6, 6.4
Hz, 1H), 7.63 – 7.53 (m, 1H), 7.26 (s, 2H), 7.20 (td, J = 8.4, 2.2
Hz, 1H), 3.62 (s, 3H), 3.58 (s, 3H), 2.57 (s, 3H). 13C NMR (101
MHz, DMSO-d6) δ 168.0, 165.0 (dd, JC-F = 255, 11.8 Hz), 162.0,
161.7, 159.4 (dd, JC-F = 259, 13.5 Hz), 157.3, 155.3, 144.1, 136.6,
132.7, 131.9 (d, JC-F = 10.7 Hz), 126.4, 125.2 (dd, JC-F = 14.5, 3.5
4. Experimental Section
Unless otherwise noted, all reagents and solvents were from
commercial sources. 1H and 13C NMR spectra were recorded on a
Varian 300 MHz NMR spectrometer or a Bruker 400 MHz NMR
spectrometer. LC-MS spectra were measured with an Agilent
LC-MS 6120-1260 instrument. HRMS spectra were measured
with Thermo LCQ Deca XP Max mass spectrometer. Melting
points were determined on a Yanaco MP-J3 micro melting point
apparatus and the maximum temperature was 300 °C. Purity of
all compounds tested in biological assays were determined to be
>95% by LCMS.
Hz), 120.5, 118.3, 111.7 (dd, JC-F = 22.3, 3.2 Hz), 105.8 (t, JC-F
=
26.3 Hz), 103.3, 53.2, 28.0, 20.7. HRMS (ESI): m/z [M + H]+
calcd for C21H19O4N6F2S, 489.1151, found: 489.1146.
N-(5-(2-amino-4,8-dimethyl-7-oxo-7,8-dihydropteridin-6-yl)-
2-methoxypyridin-3-yl)-2,4-difluorobenzenesulfonamide (7)
1
Yellow solid (method A, 66% yield). mp: 269 – 271 °C. H
NMR (400 MHz, DMSO-d6) δ 10.32 (s, 1H), 8.99 (d, J = 2.1 Hz,
1H), 8.40 (d, J = 2.1 Hz, 1H), 7.76 (td, J = 8.6, 6.4 Hz, 1H), 7.64
– 7.54 (m, 1H), 7.43 (s, 2H), 7.23 (td, J = 8.4, 2.4 Hz, 1H), 3.71
(s, 3H), 3.54 (s, 3H), 2.60 (s, 3H). 13C NMR (101 MHz, DMSO-
General procedure for the preparation of target
compounds

d6) δ 168.6, 165.1 (dd, JC-F = 255, 11.8 Hz), 161.6, 159.4 (dd, JC-F
= 259, 13.5 Hz), 158.2, 156.0, 149.5, 144.9, 141.6, 134.8, 131.9
(d, JC-F = 10.7 Hz), 126.0, 125.1 (dd, JC-F = 14.3, 3.7 Hz), 119.0,
117.8, 111.9 (dd, JC-F = 22.3, 2.9 Hz), 105.9 (t, JC-F = 26.2 Hz),
53.6, 27.1, 18.9. HRMS (ESI): m/z [M + H]+ calcd for
C20H18O4N7F2S, 490.1104, found: 490.1096.
N-(5-(2-amino-4-methylthieno[2,3-d]pyrimidin-6-yl)-2-
chloropyridin-3-yl)-4-fluorobenzenesulfonamide (8f)
White solid (method B, 46% yield). mp: 280 – 281 °C. 1H
NMR (400 MHz, DMSO-d6) δ 10.52 (s, 1H), 8.53 (s, 1H), 7.92
(s, 1H), 7.84 – 7.78 (m, 3H), 7.42 (t, J = 8.8 Hz, 2H), 6.92 (s,
2H), 2.58 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 170.0,
164.6 (d, JC-F = 253 Hz), 163.7, 160.8, 144.5, 143.3, 136.3 (d, JC-F
= 2.8 Hz), 132.2, 130.7, 129.91, 129.90 (d, JC-F = 9.8 Hz), 127.7,
122.2, 119.6, 116.7 (d, 2JC-F = 23 Hz), 21.8. HRMS (ESI): m/z[M
+ H]+ calcd for C18H14O2N5ClFS2, 450.0256, found: 450.0248.
N-(5-(2-amino-4-methylthieno[2,3-d]pyrimidin-6-yl)-2-
methoxypyridin-3-yl)-2,4-difluorobenzenesulfonamide (8a).
White solid (method A, 48% yield). mp: 229 – 230 °C. 1H
NMR (400 MHz, DMSO-d6) δ 10.33 (s, 1H), 8.32 (d, J = 2.4 Hz,
1H), 7.91 (d, J = 2.4 Hz, 1H), 7.82 – 7.71 (m, 2H), 7.58 (ddd, J =
10.4, 9.2, 2.4 Hz, 1H), 7.22 (td, J = 8.4, 2.0 Hz, 1H), 6.81 (s, 2H),
3.63 (s, 3H), 2.57 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ
169.6, 165.1 (dd, JC-F = 255, 11.8 Hz), 162.9, 160.6, 159.4 (dd,
JC-F = 259, 13.6 Hz), 157.6, 141.1, 132.8, 131.9 (d, JC-F = 10.9
Hz), 129.5, 125.0 (dd, JC-F = 14.5, 3.6 Hz), 124.0, 122.4, 119.8,
117.0, 111.9 (dd, JC-F = 22.2, 3.3 Hz), 105.8 (t, JC-F = 26.2 Hz),
N-(5-(2-amino-4-methylthieno[2,3-d]pyrimidin-6-yl)-2-
methylpyridin-3-yl)-4-fluorobenzenesulfonamide (8g)
White solid (method B, 40% yield). mp: >300 °C. 1H NMR
(300 MHz, DMSO-d6) δ 10.05 (s, 1H), 8.70 (d, J = 2.0 Hz, 1H),
7.82 (s, 1H), 7.76 (dd, J = 8.8, 5.2 Hz, 2H), 7.49 (d, J = 2.0 Hz,
1H), 7.46 (t, J = 8.8 Hz, 2H), 6.87 (s, 2H), 2.57 (s, 3H), 2.17 (s,
3H). HRMS (ESI): m/z [M + H]+ calcd for C19H17O2N5FS2,
430.0802, found: 430.0795.
53.5, 21.7. HRMS (ESI): m/z [M
C19H16O3N5F2S2, 464.0657, found: 464.0649.
+
H]+ calcd for
N-(5-(2-amino-4-methylthieno[2,3-d]pyrimidin-6-yl)-2-
methylpyridin-3-yl)-2,4-difluorobenzenesulfonamide (8h)
N-(5-(2-amino-4-methylthieno[2,3-d]pyrimidin-6-yl)-2-
methoxypyridin-3-yl)-4-fluorobenzenesulfonamide (8b)
Pale yellow solid (method B, 28% yield). mp: 275 – 277 °C.
1H NMR (400 MHz, DMSO-d6) δ 10.49 (br s, 1H), 8.68 (s, 1H),
7.85 – 7.74 (m, 2H), 7.65 – 7.56 (m, 2H), 7.26 (td, J = 8.6, 2.1
Hz, 1H), 6.87 (s, 2H), 2.57 (s, 3H), 2.29 (s, 3H). HRMS (ESI):
m/z [M + H]+ calcd for C19H16O2N5F2S2, 448.0708, found:
448.0702.
White solid (method A, 38% yield). mp: 259 – 261 °C. 1H
NMR (400 MHz, DMSO-d6) δ 10.07 (s, 1H), 8.30 (d, J = 2.4 Hz,
1H), 7.85 (d, J = 2.4 Hz, 1H), 7.82 (dd, J = 8.8, 5.2 Hz, 2H), 7.72
(s, 1H), 7.42 (t, J = 8.8 Hz, 2H), 6.81 (s, 2H), 3.64 (s, 3H), 2.57
(s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 169.5, 164.4 (d, JC-F
=
253 Hz), 162.8, 160.5, 156.6, 140.3, 136.5 (d, JC-F = 2.8 Hz),
130.6, 129.8 (d, JC-F = 9.7 Hz), 123.8, 122.4, 120.5, 116.8, 116.2
(d, JC-F = 22.9 Hz), 53.6, 21.8. HRMS (ESI): m/z[M + H]+ calcd
for C19H17O3N5FS2, 446.0751, found: 446.0745.
N-(6-(5-(2,4-difluorophenylsulfonamido)-6-methoxypyridin-
3-yl)-4-methylthieno[2,3-d]pyrimidin-2-yl)acetamide (9a).
White solid (method A, 42% yield). mp: 254 – 256 °C. 1H
NMR (400 MHz, DMSO-d6) δ 10.67 (s, 1H), 10.39 (s, 1H), 8.44
(d, J = 2.4 Hz, 1H), 8.03 (d, J = 2.4 Hz, 1H), 8.02 (s, 1H), 7.78
(td, J = 8.6, 6.4 Hz, 1H), 7.59 (ddd, J = 10.6, 9.2, 2.4 Hz, 1H),
7.23 (td, J = 8.4, 2.4 Hz, 1H), 3.65 (s, 3H), 2.74 (s, 3H), 2.20 (s,
3H). 13C NMR (101 MHz, DMSO-d6) δ 168.8, 167.9, 165.1 (dd,
JC-F = 255, 11.8 Hz), 163.0, 159.4 (dd, JC-F = 258, 13.3 Hz),
158.1, 153.5, 141.8, 135.3, 133.1, 131.9 (d, JC-F = 10.9 Hz),
126.9, 125.0 (dd, JC-F = 14.5, 3.5 Hz), 123.3, 120.0, 116.7, 111.9
(dd, JC-F = 22.6, 2.7 Hz), 105.8 (d, JC-F = 26.2 Hz), 53.6, 24.6,
21.8. HRMS (ESI): m/z [M + H]+ calcd for C21H18O4N5F2S2,
506.0763, found: 506.0754.
N-(5-(2-amino-4-methylthieno[2,3-d]pyrimidin-6-yl)-2-
methoxypyridin-3-yl)-5-chlorothiophene-2-sulfonamide (8c)
Yellow solid (method A, 43% yield). mp: 239 – 240 °C. 1H
NMR (400 MHz, DMSO-d6) δ 10.37 (s, 1H), 8.37 (d, J = 2.4 Hz,
1H), 7.89 (d, J = 2.4 Hz, 1H), 7.76 (s, 1H), 7.40 (d, J = 4.0 Hz,
1H), 7.24 (d, J = 4.0 Hz, 1H), 6.81 (s, 2H), 3.73 (s, 3H), 2.57 (s,
3H). 13C NMR (101 MHz, DMSO-d6) δ 169.6, 162.9, 160.6,
157.2, 140.9, 138.9, 135.4, 132.4, 131.6, 129.5, 127.9, 124.0,
122.4, 120.0, 116.9, 53.7, 21.8. HRMS (ESI): m/z[M + H]+ calcd
for C17H15O3N5ClS3, 468.0020, found: 468.0019.
N-(5-(2-amino-4-methylthieno[2,3-d]pyrimidin-6-yl)-2-
methoxypyridin-3-yl)methanesulfonamide (8d)
N-(6-(5-((2,4-difluorophenyl)sulfonamido)-6-methoxypyridin-
3-yl)-4-methylthieno[2,3-d]pyrimidin-2-yl)propionamide (9b)
White solid (method A, 53% yield). mp: 278 – 280 °C. 1H
NMR (400 MHz, DMSO-d6) δ 9.40 (s, 1H), 8.32 (d, J = 2.3 Hz,
1H), 7.92 (d, J = 2.3 Hz, 1H), 7.75 (s, 1H), 6.81 (s, 2H), 3.96 (s,
3H), 3.10 (s, 3H), 2.57 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ
169.5, 162.8, 160.5, 156.4, 139.5, 129.9, 129.4, 124.0, 122.4,
121.6, 116.7, 53.9, 40.8, 21.8. HRMS (ESI): m/z [M + H]+ calcd
for C14H16O3N5S2, 366.0689, found: 366.0686.
Pale yellow solid (method A, 40% yield). mp: 173 – 175 °C.
1H NMR (400 MHz, DMSO-d6) δ 10.63 (s, 1H), 10.39 (s, 1H),
8.44 (d, J = 2.4 Hz, 1H), 8.03 (d, J = 2.4 Hz, 1H), 8.01 (s, 1H),
7.78 (dd, J = 8.4, 2.4 Hz, 1H), 7.64 – 7.54 (m, 1H), 7.23 (td, J =
8.0, 1.6 Hz, 1H), 3.65 (s, 3H), 2.74 (s, 3H), 2.50 (q, J = 7.4 Hz,
2H), 1.07 (t, J = 7.4 Hz, 3H). 13C NMR (101 MHz, DMSO-d6) δ
172.2, 167.9, 165.1 (dd, JC-F = 255, 11.8 Hz), 162.9, 159.4 (dd,
JC-F = 258, 13.5 Hz), 158.1, 153.5, 141.7, 135.2, 133.1, 131.9 (d,
JC-F = 11.0 Hz), 126.9, 125.0 (dd, JC-F = 14.4, 3.6 Hz), 123.3,
120.0, 116.6, 111.9 (dd, JC-F = 22.4, 3.3 Hz), 105.8 (d, JC-F = 26.2
Hz), 53.6, 29.7, 21.8, 9.2. HRMS (ESI): m/z[M + H]+ calcd for
C22H20O4N5F2S2, 520.0919, found: 520.0915.
N-(5-(2-amino-4-methylthieno[2,3-d]pyrimidin-6-yl)-2-
methoxypyridin-3-yl)cyclopropanesulfonamide (8e)
White solid (method A, 59% yield). mp: 254 – 255 °C. 1H
NMR (400 MHz, DMSO-d6) δ 9.43 (s, 1H), 8.32 (d, J = 2.3 Hz,
1H), 7.93 (d, J = 2.3 Hz, 1H), 7.74 (s, 1H), 6.81 (s, 2H), 3.96 (s,
3H), 2.80 – 2.72 (m, 4H), 2.57 (s, 3H), 1.00 – 0.87 (m, 4H). 13C
NMR (101 MHz, DMSO-d6) δ 169.6, 162.8, 160.5, 156.7, 139.6,
129.9, 129.7, 123.9, 122.4, 121.7, 116.7, 53.9, 30.5, 21.8, 5.1.
HRMS (ESI): m/z [M + H]+ calcd for C16H18O3N5S2, 392.0846;
found: 392.0840.
N-(6-(5-((2,4-difluorophenyl)sulfonamido)-6-methoxypyridin-
3-yl)-4-methylthieno[2,3-d]pyrimidin-2-yl)butyramide (9c)
Pale yellow solid (method A, 49% yield). mp: 178 – 179 °C.
1H NMR (400 MHz, DMSO-d6) δ 10.64 (s, 1H), 10.39 (s, 1H),
8.44 (d, J = 2.0 Hz, 1H), 8.03 (d, J = 2.0 Hz, 1H), 8.02 (s, 1H),
7.78 (dd, J = 8.0, 2.8 Hz, 1H), 7.63 – 7.54 (m, 1H), 7.23 (td, J =

8.4, 1.2 Hz, 1H), 3.65 (s, 3H), 2.74 (s, 3H), 2.46 (t, J = 7.2 Hz,
2H), 1.68 – 1.53 (m, 2H), 0.92 (t, J = 7.4 Hz, 3H). 13C NMR (101
MHz, DMSO-d6) δ 171.2, 167.9, 165.1 (dd, JC-F = 255, 11.8 Hz),
163.0, 159.4 (dd, JC-F = 259, 13.7 Hz), 158.1, 153.4, 141.7, 135.3,
133.2, 131.9 (d, JC-F = 10.8 Hz), 126.9, 125.0 (dd, JC-F = 14.3, 3.6
Hz), 123.3, 120.0, 116.7, 111.9 (dd, JC-F = 22.2, 3.2 Hz), 105.8
(d, JC-F = 26.2 Hz), 53.6, 38.3, 21.8, 18.2, 13.6. HRMS (ESI): m/z
[M + H]+ calcd for C23H22O4N5F2S2, 534.1076, found: 534.1072.
Kinase-Glo™ kinase detection kit was from Promega. PI3Kα
kinase was and the substrate PIP2 was from Invitrogen. All
assays were performed on a 384-well plate at room temperature.
The kinase buffer contained 50 mM Hepes (pH 7.5), 3 mM
MgCl2, 100 mM NaCl, 1 mM EGTA, 0.03% CHAPS and 2 mM
DTT. PI3Kα kinase solution was prepared by diluting PI3Kα in
the kinase buffer to 6.6 nM. The ATP/substrate solution
contained 100 µM PIP2 and 50 µM ATP. Compounds for testing
were diluted into 10 mM in 100% DMSO then serially diluted 3-
fold in 100% DMSO at 10 different concentrations. Then the
diluted compounds in 100% DMSO were 25-fold diluted in the
kinase buffer. 2.5 µL of diluted compound solution and 2.5 µL of
PI3Kα kinase solution were added to individual wells of 384-
well assay plates. The reactions were started by adding 5 µL of
ATP/substrate solution per well. The final volume for the
reaction was 10 µL, ATP concentration was 25 µM, PIP2
concentration was 50 µM, and PI3Kα kinase concentration was
1.65 nM. The assay plates were covered and reactions were
allowed to proceed for 1 hour, after which 10 µL of Kinase-
Glo™ reagent per well was added. The plates were incubated for
15 minutes, after which luminescence was measured using an
Envision plate reader. IC50 values were determined by curve
fitting using XLfit program of Excel software (Microsoft, USA).
N-(6-(5-((2,4-difluorophenyl)sulfonamido)-6-methoxypyridin-
3-yl)-4-methylthieno[2,3-d]pyrimidin-2-
yl)cyclopropanecarboxamide (9d)
White solid (method A, 37% yield). mp: 138 – 139 °C. 1H
NMR (400 MHz, DMSO-d6) δ 11.03 (s, 1H), 10.42 (s, 1H), 8.45
(d, J = 2.4 Hz, 1H), 8.04 (s, 1H), 8.03 (d, J = 2.4 Hz, 1H), 7.77
(td, J = 8.4, 6.4 Hz, 1H), 7.64 – 7.56 (m, 1H), 7.23 (td, J = 8.4,
2.0 Hz, 1H), 3.65 (s, 3H), 2.75 (s, 3H), 2.17 – 2.07 (m, 1H), 0.88
– 0.76 (m, 4H). 13C NMR (101 MHz, DMSO-d6) δ 171.6, 167.9,
165.1 (dd, JC-F = 255, 11.8 Hz), 163.0, 159.4 (dd, JC-F = 256, 13.3
Hz), 158.1, 153.3, 141.8, 135.3, 133.2, 131.9 (d, JC-F = 10.8 Hz),
127.0, 125.0 (dd, JC-F = 14.4, 3.6 Hz), 123.3, 120.0, 116.7, 111.9
(dd, JC-F = 22.4, 3.3 Hz), 105.8 (d, JC-F = 26.3 Hz), 53.6, 21.8,
14.4, 8.0. HRMS (ESI): m/z[M + H]+ calcd for C23H20O4N5F2S2,
532.0919, found: 532.0917.
Proliferation assays
N-(2-methoxy-5-(4-methyl-2-(methylamino)thieno[2,3-
d]pyrimidin-6-yl)pyridin-3-yl)2,4-
difluorobenzenesulfonamide (9e)
Compounds were evaluated for antiproliferative potency
against U87MG, T47D, SKOV3, H460 and A549 tumor cell lines
using a CellTiter-Glo® Luminescent Cell Viability Assay from
Promega. The human tumor cell lines used were obtained from
the ATCC. All the mediums and FBS were Gibco. T47D and
H460 tumor cells were cultured in RPMI1640 medium
supplemented with 10% FBS. SKOV3 ovarian cancer cells were
cultured in McCoy's 5a 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.Tumor cells were seeded at a
volume of 80 µL/well and a density of 3000 cells/well in 96-well
plates and subsequently incubated at 37 °C and 5% CO2 for 2
hours. Compounds for testing were diluted into 10 mM in 100%
DMSO then serially diluted 3.16-fold in 100% DMSO at 10
different concentrations. Then the diluted compounds in 100%
DMSO were 20-fold diluted in 100% DMSO. The tumor cells
were then treated with serially diluted compound or DMSO
control in the incubator at 37 °C and 5% CO2 for 4 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).
Yellow solid (method A, 40% yield). mp: 272 – 273 °C. 1H
NMR (400 MHz, DMSO-d6) δ 10.34 (s, 1H), 8.32 (d, J = 2.2 Hz,
1H), 7.91 (d, J = 2.2 Hz, 1H), 7.82 – 7.72 (m, 2H), 7.64 – 7.53
(m, 1H), 7.30 (br s, 1H), 7.22 (td, J = 8.7, 2.5 Hz, 1H), 3.63 (s,
3H), 2.85 (d, J = 4.8 Hz, 3H), 2.58 (s, 3H). 13C NMR (101 MHz,
DMSO-d6) δ 169.5, 165.1 (dd, JC-F = 255, 11.8 Hz), 162.6, 160.0,
159.4 (dd, JC-F = 259, 13.4 Hz), 157.5, 141.0, 132.8, 131.9 (d, JC-F
= 10.7 Hz), 129.3, 125.0 (dd, JC-F = 14.4, 3.6 Hz), 124.0, 122.2,
119.8, 117.0, 111.9 (dd, JC-F = 22.3, 3.2 Hz), 105.8 (t, JC-F = 26.3
Hz), 53.5, 28.0, 21.9. HRMS (ESI): m/z [M + H]+ calcd for
C20H18O3N5F2S2, 478.0814, found: 478.0808.
N-(6-(5-(5-chlorothiophene-2-sulfonamido)-6-
methoxypyridin-3-yl)-4-methylthieno[2,3-d]pyrimidin-2-
yl)acetamide (9f)
Pale yellow solid (method A, 40% yield). mp: 223 – 224 °C.
1H NMR (400 MHz, DMSO-d6) δ 10.69 (s, 1H), 10.45 (s, 1H),
8.48 (d, J = 2.4 Hz, 1H), 8.02 (s, 1H), 8.01 (d, J = 2.4 Hz, 1H),
7.42 (d, J = 4.1 Hz, 1H), 7.25 (d, J = 4.1 Hz, 1H), 3.75 (s, 3H),
2.74 (s, 3H), 2.20 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ
168.8, 167.9, 163.0, 157.6, 153.4, 141.6, 138.8, 135.4, 132.4,
131.9, 127.9, 126.9, 123.3, 120.1, 116.6, 53.8, 24.6, 21.9. HRMS
(ESI): m/z[M + H]+ calcd for C19H17O4N5ClS2, 510.0126, found:
510.0123.
Parallel artificial membrane permeation assay (PAMPA)
The pre-coated PAMPA plate system (BD Gentest) was used
to evaluate the effective permeabilities (Pe) of compounds. The
working solutions of compounds were added in the receiver plate
(donor plate) while the blank buffer added in the filter plate
(acceptor plate). The filter plate was assembled on the receiver
plate by slowly lowering the pre-coated PAMPA plate, which
was then incubated at room temperature for 5 hours. After the
incubation, aliquots from donor and acceptor plates were
delivered to LC/MS/MS analysis. The Waters UPLC system
coupling to an API 4000 MS instrument was used for the
LC/MS/MS analysis with a Waters UPLC AQURITY BEH C18
column.
N-(6-(6-methoxy-5-(methylsulfonamido)pyridin-3-yl)-4-
methylthieno[2,3-d]pyrimidin-2-yl)acetamide (9g)
Pale yellow solid (method A, 20% yield). mp: 225 – 227 °C.
1H NMR (400 MHz, DMSO-d6) δ 10.67 (s, 1H), 9.46 (s, 1H),
8.43 (d, J = 2.3 Hz, 1H), 8.01 (d, J = 2.3 Hz, 1H), 7.99 (s, 1H),
3.98 (s, 3H), 3.11 (s, 3H), 2.74 (s, 3H), 2.20 (s, 3H). 13C NMR
(101 MHz, DMSO-d6) δ 168.8, 167.9, 162.9, 157.0, 153.4, 140.2,
135.8, 129.7, 127.0, 123.3, 121.8, 116.5, 54.0, 40.9, 24.6, 21.9.
HRMS (ESI): m/z [M + H]+ calcd for C16H18O4N5S2, 408.0795,
found: 408.0789.
Immunoblotting
PI3Kα Biochemical Assay
NCI-H460 cancer cells (ATCC) were exposed to compounds
for 3 hours. Cells were collected and then lysed in a buffer

containing 1% sodium dodecyl sulfate (SDS), 10 mM
ethylenediaminetetraacetic acid (EDTA), 50 mM Tris–HCl (pH
8.1), 1% proteinase inhibitor cock tails and 1% protease inhibitor
mixture. Lysates were sonicated and then centrifuged at 13,000g
for 10 min. Protein concentrations in the supernatants were
determined (Micro BCA Protein Assay Kit, Pierce
Biotechnology, Rockford, IL). Equal amounts of proteins were
resolved in an SDS–polyacrylamide gel and transferred to a
polyvinylidene fluoride membrane (Merck Millipore). The
membrane was washed twice with Tris-buffered saline
containing 0.1% Tween-20 (TBST), blocked with TBST
containing 5% non-fat milk for 30 min, and then incubated with
primary antibody (1:500–1:4000 dilution) in TBST at 4 °C
overnight. AKT, phosphor-Akt (S473), phosphor-Akt (T308),
p70S6K, phosphor-p70S6K, S6RP, phosphor-S6RP antibodies
were obtained from CST and actin antibody was from Santa
Cruz. After washing with TBST, the membrane was incubated
with goat anti-rabbit or anti-mouse IgG–HRP conjugates (1:5000
dilution, Origene ZB-2301 or ZB-2305) for 1 h at room
temperature. The immunoblots were visualized by enhanced
chemiluminescence.
RTVtreatment/RTVvehicle
performed by GraphPad Prism5 software and the significance
levels were evaluated using one-way ANOVA model.
)
×100%. Statistical analyses were
PK-PD study
Female athymic BALB/c nude mice (8–10 week old) were
implanted with NCI-H460 tumor cells. When the tumor volumes
reached to 500-600 mm3, mice were administered orally (via
gavage) at a single dose of 10mg/kg. At different time points as
indicated, animals were anaesthetized and sacrificed. Blood and
tumor samples were collected. Blood samples were centrifuged
to obtain the plasma for PK analysis. Tumor tissues were
homogenized for PK analysis. For Akt (Ser 473) phosphorylation
analysis, it follows the protocol of phospho-AKT (S473) ELISA
Kit (Abcam, ab176657). The data was analyzed by Graphpad
Prism 7.
Acknowledgments
This work was supported in part by CAMS Innovation Fund
for Medical Sciences (grant no. 2017-I2M-3-011).
Mouse liver microsome stability Assay
Mouse liver microsomes were purchased from Xenotech.
Compounds with a final concentration of 1.11 µM were
incubated with NADP co-factor solution and mouse liver
Supplementary Material
o
The procedures for the preparation of synthetic intermediates
and biological evaluations are reported in the supplementary data.
microsome (final concentration 0.7 mg protein/mL) at 37 C for
0min, 10min, 30min, 60min and 90min respectively. Samples
were then quenched with acetonitrile and centrifuged for 20min
at 4000rpm. The supernatants were analyzed by LC/MS/MS.
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Graphical Abstract
Discovery of New Thienopyrimidine
Derivatives as Potent and Orally Efficacious
Phosphoinositide 3-Kinase Inhibitors
Leave this area blank for abstract info.
Songwen Lina,1, Chunyang Wang a,1, Ming Jia, Deyu Wub, Yuanhao Lva, Li Shengc, Fangbin Hand, Yi Dongb,
Kehui Zhangb, Yakun Yangc, Yan Li c, Xiaoguang Chena,*, Heng Xua,*
NCI-H460 Xenograft Model
3000
2500
2000
1500
1000
500
Vehicle
8a 10mg/kg qd, TGI 56.7%
8a 20mg/kg qd, TGI 73.1%
9a 5mg/kg qd, TGI 75.9%
9a 10mg/kg qd, TGI 85.2%
8a R = H
IC50
1.3 nM
0.21 µM
0.13 µM
9a R = Ac
IC50
1.1 nM
0.062 µM
0.009 µM
***
***
In Vitro
***
***
PI3K alpha
NCI-H460
T47D
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0
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16
Days after treatment