Identification of novel thiazolo[5,4-b]pyridine derivatives as potent phosphoinositide 3-kinase inhibitors

Article abstract of DOI:10.3390/molecules25204630

A series of novel 2-pyridyl, 4-morpholinyl substituted thiazolo[5,4-b]pyridine analogues have been designed and synthesized in this paper. These thiazolo[5,4-b]pyridines were efficiently prepared in seven steps from commercially available substances in mo

Full text of DOI:10.3390/molecules25204630

molecules
Article
Identification of Novel Thiazolo[5,4-b]Pyridine
Derivatives as Potent Phosphoinositide
3-Kinase Inhibitors
Liang Xia 1,2, Yan Zhang 1,2, Jingbo Zhang 1,2, Songwen Lin 1,2, Kehui Zhang 1,2, Hua Tian 1,2
,
Yi Dong 1,2,
*
and Heng Xu 1,2,
*
1
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;
xialiang@imm.ac.cn (L.X.); zhangyan@imm.ac.cn (Y.Z.); zhangjingbo@imm.ac.cn (J.Z.);
linsongwen@imm.ac.cn (S.L.); kehuizhang@imm.ac.cn (K.Z.); tianh@imm.ac.cn (H.T.)
Beijing Key Laboratory of Active Substances Discovery and Druggability Evaluation, Institute of Materia
Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China
Correspondence: dongyi@imm.ac.cn (Y.D.); xuheng@imm.ac.cn (H.X.)
2
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editors: Panayiotis A. Koutentis and Andreas S. Kalogirou
Received: 10 September 2020; Accepted: 1 October 2020; Published: 12 October 2020
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: A series of novel 2-pyridyl, 4-morpholinyl substituted thiazolo[5,4-b]pyridine analogues
have been designed and synthesized in this paper. These thiazolo[5,4-b]pyridines were efficiently
prepared in seven steps from commercially available substances in moderate to good yields. All of
these N-heterocyclic compounds were characterized by nuclear magnetic resonance (NMR) and
high-resolution mass spectrometry (HRMS) analysis and tested for phosphoinositide 3-kinase
(PI3K) enzymatic assay. The results indicated that these N-heterocyclic compounds showed
potent PI3K inhibitory activity, and the IC50 of a representative compound (19a) could reach
to 3.6 nm. The structure
was important for PI3K
−
activity relationships (SAR) study showed that sulfonamide functionality
inhibitory activity, and 2-chloro-4-florophenyl sulfonamide (19b),
α
or 5-chlorothiophene-2-sulfonamide (19c) showed potent inhibitory activity with a nanomolar
IC50 value. The pyridyl attached to thiazolo[5,4-b]pyridine was another key structural unit for
PI3K
Enzymatic Inhibition results showed that compound 19a inhibited PI3K
a nanomolar IC50 value, but its inhibitory activity on PI3K was approximately 10-fold reduced.
α
inhibitory potency, and replacement by phenyl lead to a significant decrease in activity.
α
, PI3K , or PI3K with
γ
δ
β
Further docking analysis revealed that the N-heterocyclic core of compound 19a was directly involved
in the binding to the kinase through the key hydrogen bonds interaction.
Keywords: heterocycle; thiazolo[5,4-b]pyridine; phosphoinositide 3-kinase; inhibitory potency;
docking analysis
1. Introduction
Heterocycles are important organic chemical structures commonly found in a large number
of agrochemicals, materials, and pharmaceutical molecules [
especially prevalent in synthetic drug molecules because they can improve the interaction between
drug molecules and proteins and regulate the physicochemical properties of drugs [ ]. It is estimated
1–3]. Heterocyclic structural units are
4–6
that about 70% of pharmaceutical products contain heterocyclic substructures, which clearly shows
that heterocycles play an extremely important role in drug development and discovery [7,8].
Over the past few decades, many biological compounds containing bicyclic heterocycles have
been found with potent pharmacological activities; moreover, some of them have also been successfully
developed into drugs approved for marketing [9,10]. As a typical [5,6]-fused bicyclic scaffold,
Molecules 2020, 25, 4630; doi:10.3390/molecules25204630
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thiazolo[5,4-b]pyridine has been regarded as an important privileged structure in medicinal chemistry
because of its structural similarity to thiazolo[4,5-d]pyrimidine, which is a classic biologically useful
skeleton [11]. Furthermore, thiazolo[5,4-b]pyridine analogues have been found to exhibit a range of
biological activities, such as S1p1 and S1p5 agonist (
1) [12], H3 receptor antagonist (2) [13], DNA gyrase
B inhibitor ( ) [14], anticancer agent ( ) [15], and glucokinase activator (5
3
4
) [16], among others (Figure 1).
A few of them show promising drug-like properties, which has led to increased interest in the design,
synthesis, and bioactivity evaluation of thiazolo[5,4-b]pyridine analogues. In this paper, the novel
2-pyridyl, 4-morpholinyl substituted thiazolo[5,4-b]pyridine has been designed and utilized as a
template to afford multi-heterocyclic phosphoinositide 3-kinase (PI3K) inhibitors with high potency.
Figure 1. Selected biological compounds with thiazolo[5,4-b]pyridine skeleton.
The PI3Ks signaling pathway plays a crucial biological function in the process of cell growth,
survival, proliferation, and differentiation, which has been proven to be an important target for
tumor-targeted therapy [17]. In many tumor cells, the amplification, overexpression, or activating
mutation of PI3Kα can lead to abnormal activation of the PI3K signaling pathway, which is a key driving
force for tumor occurrence. Given that the PI3K signaling pathway is closely related to tumorigenesis,
chemotherapy by inhibiting the PI3K signaling pathway has become an important means of tumor
treatment [18], and some potent PI3K inhibitors have been discovered in recent years. It is worth
noting that heteroaryl morpholines are common structural cores of PI3K inhibitor molecules—such as
PI-103 (
the treatment for cancer. Another important core is the 2-methoxyl pyridine unit, which has been
seen in PF-04691502 ( ) [22] and GSK2126458 (10) [23] as PI3K/mTOR dual inhibitors. In this paper,
6) [19], BKM120 (7) [20], and GDC-0980 (8) [21]—and are representative PI3K inhibitors for
9
we designed a new structural template consisting of morpholinyl and 2-methoxyl pyridine to obtain a
series of thiazolo[5,4-b]pyridines with high PI3K inhibitory activity. Further analysis of the isoform
inhibitory selectivity showed that compound 19a showed highly potent inhibition of PI3k
and PI3k with an IC50 of 3.4 nM, 1.8 nM, and 2.5 nM, respectively. Furthermore, the IC50 value of
PI3kβ was approximately 10-fold higher than the other three (Figure 2).
α, PI3kγ,
δ

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Figure 2. Design of 2-pyridyl, 4-morpholinyl substituted thiazolo[5,4-b]pyridine PI3K inhibitor.
2. Results
2.1. Synthesis
An effective and economical method for preparing the thiazolo[5,4-b]pyridine analogues is shown
in Figure 3. A commercially available and inexpensive 2,4-dichloro-3-nitropyridine (11) was used
as a starting material, and the designed seven-step synthetic route was simple and easy to handle.
2,4-dichloro-3-nitropyridine (11) could be smoothly transformed into 4-morpholinyl pyridine derivative
12 via selective substation with morpholine in the presence of triethylamine. Molecule 12 underwent
another substitution with a thiocyanate by treatment with KSCN in acetic acid at 80 ◦C to successfully
afford 4-(3-nitro-2-thiocyanatopyridin-4-yl)morpholine (13). The nitro group was reduced by treatment
with Fe powder in acetic acid at 60 ◦C; the subsequent intramolecular cyclization occurred in one-pot
to construct the thiazolo[5,4-b]pyridine skeleton, and the corresponding amino thiazolo[5,4-b]pyridine
derivative (14) was obtained in moderate yield. A copper bromide mediated bromination effectively
gave the bromothiazolo[5,4-b]pyridine derivative (15) at room temperature, which underwent a Suzuki
reaction with aryl borates at 100 ◦C to afford the desired heterocycle substituted thiazolo[5,4-b]pyridine
analogues (19a–19f) in good yields. Sulfonamide substituted aryl borates were prepared from the
corresponding amino-substituted aryl bromide through sulfonamidation and boronization in two steps.
2.2. Enzymatic Assay
All of the N-heterocycle substituted thiazolo[5,4-b]pyridine analogues were evaluated for their
PI3K
are summarized in Table 1. We firstly designed and synthesized an N-heterocyclic compound
19a) composed of methoxypyridine and morpholinyl thiazolo[5,4-b]pyridine. As expected,
this thiazolo[5,4-b]pyridine compound exhibited extremely strong PI3k inhibitory activity with
α
inhibitory activity through enzymatic assay. The structure
−
activity relationships (SAR)
(
α
an IC50 of 3.6 nM. In further SAR studies, the sulfonamide functionality was proved to be
a key structural unit that affected activity. For example, despite a 2–3 fold drop in potency
compared with that of 2,4-difluorophenyl sulfonamide, 2-chloro-4-florophenyl sulfonamide (19b),
or 5-chlorothiophene-2-sulfonamide (19c) showed high inhibitory activity with an IC50 of 4.6 nM and
8.0 nM, respectively. This activity was attributed to the fact that the electron-deficient aryl group
resulted in a more acidic sulfonamide NH proton being able to make a stronger charged interaction with
Lys802 in PI3K
19d), the PI3K
group attached to the pyridine ring lead did not significantly affect the potency (IC50 = 4.0 nM).
Replacement of pyridyl with phenyl resulted in an apparent decrease in PI3K inhibitory activity
α
[24]. Consistent with this finding, when 2,4-difluorophenyl was replaced with methyl
(
α inhibitory potency dropped over 10-fold (IC50 = 53 nM). Removal of the methoxyl
α

Molecules 2020, 25, 4630
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(IC50 = 501 nM), demonstrating that the pyridyl attached to thiazolo[5,4-b]pyridine was a necessary
moiety for enzymatic potency. We also evaluated the inhibitory selectivity of compound 19a against
four isoforms of class I PI3K (Table 2). It potently inhibited PI3K
α
, PI3K
γ, and PI3Kδ with nanomolar
IC50 values, which is approximately 10-fold higher than that of PI3Kβ, clearly proving that it is a novel
and potent PI3K inhibitor.
Figure 3. Synthesis of thiazolo[5,4-b]pyridine analogues. (
HOAc, 80 ◦C. ( ) Fe powder, HOAc, 60 ◦C. (
) CuBr2, tert-butyl nitrite, CH3CN, rt. (
DCM, rt. ( ) PdCl2(dppf), K2CO3,
) PdCl2(dppf), KOAc, bis(pinacolato)diboron, 1,4-dioxane, 100 ◦C. (
a
) Morpholine, TEA, THF, 0 ◦C. (
) DMAP, pyridine,
b) KSCN,
c
d
e
f
g
1,4-dioxane/H2O, aryl borate, 100 ◦C. dppf = 1,10-Bis(diphenylphosphino)ferrocene.
Table 1. SAR studies of 2-substitutions on the thiazolo[5,4-b]pyridine.
Compd
19a
X
R1
R2
cLogP a
PSA a
PI3Kα (nm) b
N
OMe
4.1
105.0
3.6
19b
N
OMe
4.4
105.0
4.6
19c
N
N
OMe
OMe
4.3
2.1
105.0
105.0
8.0
53
19d
Me
19e
19f
CH OMe
4.4
3.3
92.6
95.7
501
4.0
N
H
a
b
Calculated from ChemBioDraw Ultra 14.0. Mean of at least three separate experiments.

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Table 2. Enzymatic inhibition by compound 19a a.
Enzymatic Assay
IC50 (nM)
PI3Kα
PI3Kβ
PI3Kγ
PI3Kδ
3.6
34
1.6
2.9
a
Mean of at least three separate experiments.
2.3. Molecular Docking Study
Because of its superior PI3K potency, thiazolo[5,4-b]pyridine 19a was selected for a molecular
docking study. As shown in Figure 4, the 2-pyridyl thiazolo[5,4-b]pyridine scaffold fits well into the
ATP binding pocket of the PI3Kα kinase, forming a hydrogen bond interaction with the Val851 residue
of the hinge region and a water bridge with Typ836 and Asp810. It is worth noting that another
hydrogen bond interaction was observed between the sulfonamide group and Lys802. These interaction
networks are the key factors for the PI3K bonding of these pyridyl thiazolo[5,4-b]pyridine analogues.
Figure 4. Predicted binding mode of 19a with PI3Kα.
3. Materials and Methods
3.1. General
Acetic acid, 1,4-dioxane, acetonitrile and dried THF were purchased from domestic corporations
and used without further purification. Aryl borates were synthesized according to our previous
work [24]. Nuclear magnetic resonance spectroscopy (1H NMR and 13C NMR) was performed on
Bruker Advance 400M NMR spectrometers, and high-resolution LC-MS was carried out by Agilent
LC/MSD TOF. Purity of all compounds tested for PI3K activity were determined to be >95% by LCMS
analysis. The kinase inhibitory activity assay was performed by Shanghai ChemPartner Co., Ltd.
(Shanghai, China). For details, see our previously published protocols [24]. Molecular docking study
was conducted in the Schrodinger software.
3.2. Synthesis
3.2.1. Synthesis of 4-(2-chloro-3-nitropyridin-4-yl)morpholine (12)
Morpholine (50 mmol, 4.36 mL) was added dropwise to the mixture of 2,4-dichloro-3-nitropyridine
(
11, 50 mmol, 9.6 g) and trimethylamine (50 mmol, 7.0 mL) in THF (100 mL) at 0 ◦C, then the reaction

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mixture was stirred at 0 ◦C for 1 h, and the reaction was monitored by LC-MS. When the starting
material was consumed completely, solvent was removed under reduced pressure, and the residue
was purified with flash column chromatography on silica gel to afford compound
Yellow solid, Rf: 0.5 (EtOAc/Petroleum ether = 1:1), m.p.: 138–140 ◦C. 1H NMR (400 MHz, DMSO-d6)
8.22 (d, J = 6.0 Hz, 1H), 7.25 (d, J = 6.1 Hz, 1H), 3.69–3.64 (m, 4H), 3.25–3.20 (m, 4H). 13C NMR
(101 MHz, DMSO-d6)
149.9, 149.7, 142.3, 128.0, 113.9, 65.5, 48.6. HRMS (ESI): m/z (M + H+) calcd for
7 (9.1 g, 75% yield).
δ
δ
C9H11ClN3O3, 244.0483, found: 244.0492. IR (ATR): 3155, 2965, 2865, 1524, 1356, 1215, 1117, 964, 848,
719 cm−1. The data is consistent with the data reported in the literature. [25]
3.2.2. Synthesis of 4-(3-nitro-2-thiocyanatopyridin-4-yl)morpholine (13)
A mixture of KSCN (48 mmol, 4.7 g) and compound 12 (37 mmol, 9 g) in acetic acid was stirred at
80 ◦C for 2h. Solvent was removed under reduced pressure, and the residue was purified with flash
column chromatography on silica gel to afford compound
(EtOAc/Petroleum ether = 1:1), m.p.: 177–179 ◦C. 1H NMR (400 MHz, DMSO-d6)
1H), 7.32 (d, J = 6.2 Hz, 1H), 3.72–3.64 (m, 4H), 3.35–3.29 (m, 4H). 13C NMR (101 MHz, DMSO-d6)
8
(5.1 g, 52% yield). Yellow solid, Rf: 0.4
δ
8.29 (d, J = 6.1 Hz,
δ
151.5, 150.2, 149.8, 130.5, 113.4, 109.4, 65.5, 50.2. HRMS (ESI): m/z (M + H+) calcd for C10H11N4O3S,
267.0546, found: 267.0541. IR (ATR): 3109, 2985, 2861, 1585, 1504, 1312, 1209, 1108, 961, 822 cm−1
.
3.2.3. Synthesis of 7-morpholinothiazolo[5,4-b]pyridin-2-amine (14)
A mixture of Fe powder (75.2 mmol, 4.2 g) and compound 13 (18.8 mmol, 5 g) in acetic acid was
stirred at 60 ◦C for 2h and filtered. The filtrate was concentrated under reduced pressure, and the
residue was purified with flash column chromatography on silica gel to aff◦ord compound
9 (2.44 g,
1
55% yield). Colorless solid, Rf: 0.4 (DCM/MeOH = 20:1). m.p.: 230–232 C. H NMR (400 MHz,
DMSO-d6)
(m, 4H). 13C NMR (101 MHz, DMSO-d6)
m/z (M + H+) calcd for C10H13N4OS, 237.0805, found: 237.0807. IR (ATR): 3346, 3121, 2961, 1659, 1558,
1297, 1113, 973, 799, 700 cm−1
δ
7.89 (d, J = 5.5 Hz, 1H), 7.55 (s, 2H), 6.65 (d, J = 5.6 Hz, 1H), 3.78–3.70 (m, 4H), 3.56–3.50
δ
162.1, 155.6, 146.0, 143.0, 135.3, 107.0, 66.0, 48.3. HRMS (ESI):
.
3.2.4. Synthesis of 4-(2-bromothiazolo[5,4-b]pyridin-7-yl)morpholine (15)
A mixture of CuBr2 (15 mmol, 3.4 g), tert-butyl nitrite (20 mmol, 2.0 g) and compound 14 (10 mmol,
2.4 g) in acetonitrile (20 mL) was stirred at room temperature for 2h. Water (20 mL) was added,
and aqueous layer was extracted by DCM (20 mL × 2). The organic layer was combined and washed
with brine and dried over anhydrous MgSO4. DCM was then removed under reduced pressure and
the residue was purified with flash column chromatography on silica gel to afford the compound 10
(1.2 g, 40% yield. Colorless solid, Rf: 0.4 (EtOAc/Petroleum ether = 1:1). m.p.: 168–170 ◦C. 1H NMR
(400 MHz, DMSO-d6)
(101 MHz, DMSO-d6)
δ
8.23 (d, J = 5.8 Hz, 1H), 6.88 (d, J = 5.9 Hz, 1H), 3.82–3.74 (m, 8H). 13C NMR
161.1, 148.8, 148.2, 134.2, 132.7, 106.9, 65.8, 48.3. HRMS (ESI): m/z (M + H+)
δ
calcd for C10H11BrN3OS, 299.9801, found: 299.9802. IR (ATR): 3014, 2948, 2865, 1568, 1463, 1259, 1120,
1008, 956, 803, 669 cm−1
.
3.2.5. General Procedure for the Synthesis of Aryl Borates 18a–18f
Sulfonyl chloride (1.2 equiv.) was added to a mixture of compound 16 (20 mmol), DMAP (2 mmol)
and pyridine (30 mmol) in the DCM (50 mL), and the reaction; mixture was stirred at room temperature
overnight. Solvent was then removed under reduced pressure and the residue was purified with flash
column chromatography on silica gel to give the compound 17a
–17f. KOAc (25 mmol) was added to
a mixture of PdCl2(dppf) (0.63 mmol), compound 17a 17f (12.5 mmol) and bis(pinacolato)diboron
–
(15 mmol) in 1,4-dioxane (25 mL), and the reaction mixture was stirred at 100 ◦C for 5 h. Solvent was
removed under reduced pressure, and DCM (20 mL) was added. Then the mixture was filtered, and the
filtrate was concentrated under reduced pressure and the residue was purified with flash column
chromatography on silica gel to give the compound 18a–18f.

Molecules 2020, 25, 4630
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2,4-Difluoro-N-(2-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-3-yl)benzenesulfonamide (18a).
Colorless solid (3.5 g, 66% yield), m.p.: 163–165 ◦C. 1H NMR (400 MHz, DMSO-d6)
10.18 (s, 1H), 8.21
(s, 1H), 7.72 (s, 1H), 7.71–7.65 (m, 1H), 7.60–7.52 (m, 1H), 7.22–7.16 (m, 1H), 3.62 (s, 3H), 1.29 (s, 12H).
13C NMR (101 MHz, DMSO-d6).
165.0 (dd, J = 255.2, 12.1 Hz), 160.2, 159.3 (dd, J = 258.6, 14.1 Hz),
δ
δ
150.9, 141.0, 131.8 (d, J = 11.1 Hz), 125.0 (dd, J = 15.2, 4.0 Hz), 119.2, 116.7, 111.8 (dd, J = 22.2, 4.0 Hz),
105.7 (t, J = 26.3 Hz), 84.0, 53.4, 24.6. HRMS (ESI): m/z (M + H+) calcd for C18H22BF2N2O5S, 427.1305,
found: 427.1314. IR (ATR): 3264, 2989, 2883, 1600, 1406, 1340, 1139, 1019, 969, 850, 670 cm−1
.
2-Chloro-4-fluoro-N-(2-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-3-yl)benzenesulfonamide
◦
1
(
18b). Colorless solid (3.4 g, 61% yield), m.p.: 138–140 C. H NMR (400 MHz, DMSO-d6)
δ
10.07
(
s, 1H), 8.18 (d, J = 1.6 Hz, 1H), 7.85 (dd, J = 8.8, 6.0 Hz, 1H), 7.73 (dd, J = 8.7, 2.5 Hz, 1H), 7.68
(d, J
DMSO-d6)
=
1.5 Hz, 1H), 7.34 (td, J = 8.5, 2.5 Hz, 1H), 3.63 (s, 3H), 1.28 (s, 12H). 13C NMR (101 MHz,
163.8 (d, J = 255.9 Hz), 159.9, 150.6, 140.1, 134.5 (d, J = 3.4 Hz), 133.2 (d, J = 11.6 Hz), 132.9
δ
(d, J = 10.3 Hz), 119.5, 119.2 (d, J = 24.7 Hz), 117.1, 114.5 (d, J = 21.9 Hz), 84.0, 53.4, 24.6. HRMS (ESI):
m/z (M + H+) calcd for C18H22BClFN2O5S, 443.1010, found: 443.1001. IR (ATR): 3288, 3103, 2981, 1586,
1341, 1154, 908, 850, 679 cm−1
.
5-Chloro-N-(2-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-3-yl)thiophene-2-sulfonamide (18c).
Colorless solid (3.6 g, 67% yield), m.p.: 156–158 ◦C. 1H NMR (400 MHz, DMSO-d6)
10.21 (s, 1H),
8.24 (s, 1H), 7.73 (s, 1H), 7.29 (d, J = 4.1 Hz, 1H), 7.20 (d, J = 4.1 Hz, 1H), 3.71 (s, 3H), 1.29 (s, 12H).
13C NMR (101 MHz, DMSO-d6)
160.4, 151.2, 140.4, 139.4, 135.5, 132.5, 128.2, 119.9, 117.2, 84.5, 54.1,
25.1. HRMS (ESI): m/z (M + H+) calcd for C16H21BClN2O5S2, 431.0668, found: 431.0680. IR (ATR):
3251, 3088, 2924, 1599, 1397, 1344, 1253, 1153, 992, 852, 679 cm−1
δ
δ
.
N-(2-Methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-3-yl)methanesulfonamide (18d).
Colorless solid (2.4 g, 58% yield), 1H NMR (400 MHz, DMSO-d6)
9.23 (s, 1H), 8.20 (s, 1H), 7.77 (s, 1H),
3.93 (d, J = 3.6 Hz, 3H), 3.01 (s, 3H), 1.29 (s, 12H). 13C NMR (101 MHz, DMSO-d6)
158.8, 149.24,
137.0, 121.0, 116.6, 83.9, 73.5, 53.8, 40.4, 24.9, 24.6. HRMS (ESI): m/z (M + H+) calcd for C13H22BN2O5S,
329.1337, found: 329.1318. IR (ATR): 3263, 2979, 1603, 1497, 1392, 1256, 1138, 970, 850, 771, 670 cm−1
δ
δ
.
2,4-Difluoro-N-(2-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)benzenesulfonamide (18e).
◦
1
Colorless solid (3.3 g, 62% yield), m.p.: 150–152 C. H NMR (400 MHz, DMSO-d6)
7.64 (dd, J = 14.9, 8.4 Hz, 1H), 7.57–7.47 (m, 3H), 7.16 (td, J = 8.6, 2.1 Hz, 1H), 6.92 (d, J = 8.2 Hz,
1H), 3.49 (s, 3H), 1.29 (s, 12H). 13C NMR (101 MHz, DMSO-d6)
164.8 (dd, J = 254.5, 12.1 Hz), 159.4
δ 9.83 (s, 1H),
δ
(
dd, J = 258.6, 13.1 Hz), 156.5, 134.8, 134.5, 131.8 (d, J = 11.1 Hz), 125.3 (dd, J = 14.1, 4.0 Hz), 123.7, 120.1,
111.4 (dd, J = 22.2, 3.0 Hz), 111.3, 105.5 (t, J = 30.3 Hz), 83.6, 55.2, 24.7. HRMS (ESI): m/z (M + H+) calcd
for C19H23BF2NO5S, 426.1353, found: 426.1369. IR (ATR): 3300, 2925, 2850, 1601, 1340, 1258 1167, 1129,
850, 669 cm−1
.
3.2.6. General Procedure for the Synthesis of Target Compounds 19a–19f
K2CO3 aqueous solution (2N, 0.39 mL) was added to a mixture of PdCl2(dppf) (0.03 mmol),
compound 15 (0.26 mmol) and Aryl borate 18 (0.31 mmol) in 1,4-dioxane (2.5 mL), and the reaction
mixture was stirred at 100 ◦C for 5h. Solvent was removed under reduced pressure, and DCM (20 mL)
was added. Then the mixture was filtered, and the filtrate was concentrated under reduced pressure
and the residue was purified with flash column chromatography on silica gel to give compound 19
.
1H NMR and 13CNMR spectra of the compounds 19a
–19f, and the HPLC spectra of compound 19a can
be found in the Supplementary Materials.
2,4-Difluoro-N-(2-methoxy-5-(7-morpholinothiazolo[5,4-b]pyridin-2-yl)pyridin-3-yl)benzenesulfonamide (19a).
Colorless solid (103 mg, 76% yield), Rf: 0.5 (DCM/MeOH = 30:1), m.p.: 175–177 ◦C. 1H NMR (400 MHz,
DMSO-d6)
7.80 (dd, J = 14.9, 8.5 Hz, 1H), 7.64–7.56 (m, 1H), 7.25 (td, J = 8.6, 2.1 Hz, 1H), 6.86 (d, J = 5.7 Hz, 1H),
3.88–3.78 (m, 8H), 3.75 (s, 3H). 13C NMR (101 MHz, DMSO-d6)
165.1 (dd, J = 255.5 Hz, 12.1 Hz), 159.4,
δ 10.52 (s, 1H), 8.62 (d, J = 2.1 Hz, 1H), 8.21 (d, J = 5.7 Hz, 1H), 8.11 (d, J = 2.1 Hz, 1H),
δ

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159.3 (dd, J = 258.6 Hz, 14.1 Hz), 158.9, 157.2, 149.3, 147.9, 142.7, 135.2, 131.8 (d, J = 11.1 Hz), 131.2, 124.8
(dd, J = 14.1 Hz, 3.0 Hz), 123.4, 120.5, 112.1 (dd, J = 22.2 Hz, 3.0 Hz), 106.4, 105.9 (t, J = 26.3 Hz), 65.9,
54.0, 48.5. HRMS (ESI): m/z (M + H+) calcd for C22H20F2N5O4S2, 520.0919, found: 520.0909. IR (ATR):
3183, 2922, 2854, 1602, 1423, 1261, 1149, 971, 864, 715, 668 cm−1
.
2-Chloro-4-fluoro-N-(2-methoxy-5-(7-morpholinothiazolo[5,4-b]pyridin-2-yl)pyridin-3-yl)benzenesulfonamide
19b). Colorless solid (102 mg, 73% yield), Rf: 0.5 (DCM/MeOH = 30:1), m.p.: 188–190 ◦C. 1H NMR
(400 MHz, DMSO-d6) 10.45 (s, 1H), 8.60 (d, J = 2.1 Hz, 1H), 8.20 (d, J = 5.7 Hz, 1H), 8.07 (d, J 2.1 Hz,
1H), 7.97 (dd, J = 8.9, 5.9 Hz, 1H), 7.77 (dd, J = 8.7, 2.4 Hz, 1H), 7.40 (td, J = 8.5, 2.5 Hz, 1H),
6.86 (d, J = 5.7 Hz, 1H), 3.88–3.78 (m, 8H), 3.77 (s, 3H). 13C NMR (101 MHz, DMSO-d6)
163.9
(
δ
=
δ
(d, J = 255.5 Hz), 159.3, 158.6, 157.2, 149.3, 148.0, 142.4, 135.2, 134.3 (d, J = 3.0 Hz), 133.1 (d, J = 6.1 Hz),
133.0 (d, J = 4.0 Hz), 130.3, 123.3, 120.9, 119.4 (d, J = 26.3 Hz), 114.7 (d, J = 22.2 Hz), 106.4, 65.9, 54.0,
48.5. HRMS (ESI): m/z (M + H+) calcd for C22H20ClFN5O4S2, 536.0624, found: 536.0612. IR (ATR):
3376, 3008, 2921, 1568, 1471, 1341, 1253, 1157, 977, 803, 661 cm−1
.
5-Chloro-N-(2-methoxy-5-(7-morpholinothiazolo[5,4-b]pyridin-2-yl)pyridin-3-yl)thiophene-2-sulfonamide (19c).
Colorless solid (95 mg, 70% yield), Rf: 0.5 (DCM/MeOH = 30:1), m.p.: 116–118 ◦C. 1H NMR (400 MHz,
DMSO-d6)
(d, J = 4.1 Hz, 1H), 7.25 (d, J = 4.1 Hz, 1H), 6.87 (d, J = 5.7 Hz, 1H), 3.91–3.80 (m, 11H). 13C NMR (101
MHz, DMSO-d6) 159.3, 158.5, 157.4, 149.3, 148.0, 142.3, 138.8, 135.4, 135.3, 132.4, 129.4, 128.1, 123.4,
121.0, 106.5, 66.0, 54.2, 48.5. HRMS (ESI): m/z (M + H+) calcd for C20H20ClN5O4S3, 524.0282, found:
524.0285. IR (ATR): 3350 3015 2921, 1566, 1406, 1341, 1256, 1149, 976, 802, 676 cm−1
δ 10.56 (s, 1H), 8.63 (d, J = 1.9 Hz, 1H), 8.21 (d, J = 5.6 Hz, 1H), 8.14 (d, J = 2.0 Hz, 1H), 7.44
δ
.
N-(2-Methoxy-5-(7-morpholinothiazolo[5,4-b]pyridin-2-yl)pyridin-3-yl)methanesulfonamide
Colorless solid (73 mg, 67% yield), Rf: 0.5 (DCM/MeOH = 30:1),m.p.: 230–232 ◦C. 1H NMR (400 MHz,
DMSO-d6) 9.56 (s, 1H), 8.63 (d, J = 2.2 Hz, 1H), 8.24–8.20 (m, 2H), 6.89 (d, J = 5.7 Hz, 1H), 4.02 (s, 3H),
(19d).
δ
3.92–3.80 (m, 8H), 3.13 (s, 3H). 13C NMR (101 MHz, DMSO-d6)
δ 159.4, 157.7, 157.6, 149.3, 148.0, 140.9,
135.3, 128.1, 123.5, 122.4, 106.5, 65.9, 54.3, 48.5, 40.6. HRMS (ESI): m/z (M + H+) calcd for C17H20N5O4S2,
422.0951, found: 422.0944. IR (ATR): 3215, 2998, 2882, 1585, 1464, 1393, 1287, 1137, 1003, 841, 771,
684 cm−1
.
2,4-Difluoro-N-(2-methoxy-5-(7-morpholinothiazolo[5,4-b]pyridin-2-yl)phenyl)benzenesulfonamide
(
19e).
Colorless solid (101 mg, 75% yield), Rf: 0.5 (DCM/MeOH = 30:1), m.p.: 195–197 C. H NMR
(400 MHz, DMSO-d6) 10.15 (s, 1H), 8.19 (d, J = 5.6 Hz, 1H), 7.84–7.80 (m, 2H), 7.74 (dd, J = 14.9, 8.5
Hz, 1H), 7.61–7.54 (m, 1H), 7.21 (t, J = 7.5 Hz, 1H), 7.11 (d, J = 8.4 Hz, 1H), 6.86 (d, J = 5.7 Hz, 1H),
3.89–3.79 (m, 8H), 3.62 (s, 3H). 13C NMR (101 MHz, DMSO-d6)
165.0 (dd, J = 253.5 Hz, 14.1 Hz),
◦
1
δ
δ
160.0, 159.3, 159.2 (dd, J = 258.6 Hz, 15.2 Hz), 155.6, 149.3, 147.7, 135.6, 131.8 (d, J = 1.1 Hz), 126.6,
125.5, 125.3, 125.1 (dd, J = 14.1 Hz, 4.0 Hz), 124.7, 112.5, 111.8 (dd, J = 23.2 Hz, 4.0 Hz), 106.5,105.8
(t, J = 26.3 Hz), 65.9, 55.8, 48.5. HRMS (ESI): m/z (M + H+) calcd for C23H21F2N4O4S2, 519.0967, found:
519.0970. IR (ATR): 3268, 2922, 2844, 1567, 1426, 1336, 1272, 1122, 969, 816, 670 cm−1
.
2,4-Difluoro-N-(5-(7-morpholinothiazolo[5,4-b]pyridin-2-yl)pyridin-3-yl)benzenesulfonamide (19f).
Colorless solid (57 mg, 45% yield), Rf: 0.3 (DCM/MeOH = 30:1). m.p.: 99–101 ◦C. 1H NMR (400 MHz,
DMSO-d6)
(dd, J = 14.8, 8.5 Hz, 1H), 7.61–7.54 (m, 1H), 7.32 (t, J = 8.4 Hz, 1H), 6.89 (d, J = 5.7 Hz, 1H), 3.92–3.80
(m, 8H). 13C NMR (101 MHz, DMSO-d6)
165.5 (dd, J = 256.5, 12.1 Hz), 159.6, 159.0 (dd, J = 258.6,
δ 11.35 (s, 1H), 8.87 (s, 1H), 8.47 (d, J = 2.0 Hz, 1H), 8.23 (d, J = 5.7 Hz, 1H), 8.08 (s, 1H), 8.00
δ
14.1 Hz), 156.9, 149.6, 148.5, 142.9, 142.5, 135.3, 134.6, 132.4 (d, J = 10.1 Hz), 129.1, 123.4 (dd, J = 14.1,
3.0 Hz), 123.2, 112.7 (dd, J = 22.2, 4.0 Hz), 106. (t, J = 25.3 Hz), 65.9, 48.5. HRMS (ESI): m/z (M + H+)
calcd for C21H18F2N5O3S2, 490.0814, found: 490.0805. IR (ATR): 3000, 2928, 1557, 1476, 1425, 1343,
1266, 1068, 961, 846, 669 cm−1
.

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4. Conclusions
In summary, we have designed and synthesized a series of 2-pyridyl, 4-morpholinyl substituted
thiazolo[5,4-b]pyridines by pharmacophore splicing of methoxyl pyridine and morpholinyl heterocyclic
inhibitors. These molecules were evaluated for the inhibitory activity of kinases. It was found that
compound 19a exhibited nanomolar inhibitory activity against the three isoforms of PI3K
and PI3K . Further docking study demonstrated that compound 19a fit well into the ATP binding
pocket of the PI3K kinase. Overall, these novel thiazolo[5,4-b]pyridines may be potentially used as
α, PI3Kγ,
δ
α
the potent PI3K inhibitors for the treatment of disease mediated by the PI3K signaling pathway after
further activity evaluation in vivo and optimization of their pharmacological properties.
Supplementary Materials: 1H NMR and 13CNMR spectra of the compounds 19a
compound 19a.
–19f, and the HPLC spectra of
Author Contributions: L.X., Y.Z., J.Z., S.L., K.Z., and H.T. performed the experiments and analyzed the data;
H.X. and Y.D. conceived and designed the study; all authors contributed to writing and editing of the paper.
All authors have read and agreed to the published version of the manuscript.
Funding: This research was Funded by the National Natural Science Foundation of China (81703329 and 21702234),
the CAMS Innovation Fund for Medical Sciences (2017-I2M-3-011 and 2019-I2M-1-005), the Drug Innovation
Major Project (2018ZX09711-001-005), the Non-profit Central Research Institute Fund of Chinese Academy of
Medical Sciences (2018PT35003 and 2019RC-HL-008), and the Fundamental Research Funds for the Central
Universities (3332020041).
Acknowledgments: We thank Xudong Wu for his help on molecular modeling.
Conflicts of Interest: The authors declare no conflict of interest.
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2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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