Synthesis and anticancer effects evaluation of 1-alkyl-3-(6-(2-methoxy-3-sulfonylaminopyridin-5-yl)benzo[d]thiazol-2-yl)urea as anticancer agents with low toxicity

Article abstract of DOI:10.1016/j.bmc.2015.08.013

As a PI3K and mTOR dual inhibitor, N-(2-chloro-5-(2-acetylaminobenzo[d]thiazol-6-yl)pyridin-3-yl)-4-fluorophenylsulfonamide displays toxicity when orally administrated. In the present study, alkylurea moiety replaced the acetamide group in the compound an

Full text of DOI:10.1016/j.bmc.2015.08.013

Accepted Manuscript
Synthesis and anticancer effects evaluation of 1-alkyl-3-(6-(2-methoxy-3-sul-
fonylaminopyridin-5-yl)benzo[d] thiazol-2-yl)urea as anticancer agents with
low toxicity
Xiao-Xiao Xie, Huan Li, Juan Wang, Shuai Mao, Min-Hang Xin, She-Min Lu,
Qi-Bing Mei, San-Qi Zhang
PII:
S0968-0896(15)00680-X
http://dx.doi.org/10.1016/j.bmc.2015.08.013
BMC 12517
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
13 July 2015
12 August 2015
12 August 2015
Please cite this article as: Xie, X-X., Li, H., Wang, J., Mao, S., Xin, M-H., Lu, S-M., Mei, Q-B., Zhang, S-Q.,
Synthesis and anticancer effects evaluation of 1-alkyl-3-(6-(2-methoxy-3-sulfonylaminopyridin-5-yl)benzo[d]
thiazol-2-yl)urea as anticancer agents with low toxicity, Bioorganic & Medicinal Chemistry (2015), doi: http://
dx.doi.org/10.1016/j.bmc.2015.08.013
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Synthesis and anticancer effects evaluation of
1-alkyl-3-(6-(2-methoxy-3-sulfonylaminopyridin-5-yl)benzo[d]
thiazol-2-yl)urea as anticancer agents with low toxicity
a
a
a
c
Xiao-Xiao Xie , Huan Li , Juan Wang b, Shuai Mao a, Min-Hang Xin , She-Min Lu , Qi-Bing
Mei b, San-Qi Zhang a*
a
Department of Medicinal Chemistry, School of Pharmacy, Xian Jiaotong University, Xian,
Shaanxi, 710061, PR China
b
Center for Pharmacological Evaluation and Research, Shanghai Institute of Pharmaceutical
Industry, Shanghai, 200437, PR China
c
Department of Genetics and Molecular Biology, School of Basic Medical Sciences, Xian
Jiaotong University, Xian, Shaanxi, 710061, PR China
* Corresponding author. E-mail address: sqzhang@xjtu.edu.cn (S. Q. Zhang).
Abstract
As a PI3K and mTOR dual inhibitor, N-(2-chloro-5-(2-acetylaminobenzo[d]
thiazol-6-yl)pyridin-3-yl)-4-fluorophenylsulfonamide displays toxicity when orally
administrated. In the present study, alkylurea moiety replaced the acetamide group in
the compound and a series of 1-alkyl-3-(6-(2,3-disubstituted pyridin-5-yl)benzo[d]
thiazol-2-yl)urea derivatives were synthesized. The antiproliferative activities of the
synthesized compounds in vitro were evaluated against HCT116, MCF-7, U87 MG
and A549 cell lines. The compounds with potent antiproliferative activity were tested
for their acute oral toxicity and inhibitory activity against PI3Ks and mTORC1. The
results indicate that the compound attached a 2-(dialkylamino)ethylurea moiety at the
2-position of benzothiazole can retain the antiproliferative activity and inhibitory
activity against PI3K and mTOR. In addition, their acute oral toxicity reduced
dramatically. Moreover, compound 2f can effectively inhibit tumor growth in a mice
S180 homograft model. These findings suggest that 1-(2-dialkylaminoethyl)-3-
(6-(2-methoxy-3-sulfonylaminopyridin-5-yl)benzo[d]thiazol-2-yl)urea derivatives can
serve as potent PI3K inhibitors and anticancer agents with low toxicity.
Key Words benzo[d]thiazole  urea  PI3K inhibitor  anticancer  acute toxicity
1

1. Introduction
Benzothiazole derivatives exhibit a broad spectrum of biological activities such as
antitumor, antimicrobial, anti-inflammatory, anticonvulsant and antidiabetic activity.
The study progress of benzothiazole derivatives in medicinal chemistry has been
reviewed.1-3 There are hydrogen-bond acceptors and hydrogen-bond donor in the
2-aminobenzothiazole. Thus it is considered as a privileged drug scaffold in drug
discovery.
Chemotherapy is one of the major approaches in cancer treatment. Traditional
cytotoxic agents clinically used have been increasingly limited due to their high risk
of toxicity, drug resistance and lack of selectivity. At present, the molecularly targeted
therapies aiming at special targets have become effective approaches in cancer
therapies. Kinases play an important role in tumor cell proliferation, survival and
metastasis. Therefore, inhibitors against key kinases have emerged as novel targeted
anticancer agents. PI3K (phosphoinositide 3-kinase) and mTOR (mammalian target of
rapamycin) are critical nodes of PI3K/Akt/mTOR pathway, which are abnormally
active in many tumor cells. For example, PIK3CA, the gene encoding for p110
(PI3Kα), is often over-expressed or mutate in a wide variety of cancer cell lines. Thus,
targeting PI3K, especially PI3Kα, and / or mTOR has become an appealing strategy
for cancer therapies.4-6
In recent years, a remarkable progress has been made in the design, synthesis and
evaluation of PI3K and mTOR dual inhibitors,7-12 and thereupon the pharmacophore
of the dual inhibitors has been put forward.13 Among the reported PI3K/mTOR dual
inhibitors, N-(5-(quinilin-6-yl)-pyridin-3-yl)phenylsulfonamide is an important class
of active compounds. GlaxoSmithKline identified GSK2126458 as a potent, orally
bioavailable inhibitor of PI3Kα and mTOR.14 Amgen designed, synthesized and
evaluated several classes of N-(2,5-disubstitutedpyridin-3-yl)phenylsulfonamides.
Therefore, they discovered that N-(2-chloro-5-(4-morpholinoquinilin-6-yl)pyridin-3-
yl)-4-fluorophenylsulfonamide,15 N-(2-chloro-5-(2-acetyl aminobenzo[d]thiazol-6-yl)
pyridin-3-yl)-4-fluorophenylsulfonamide (compound A, Fig. 1),16 N-(2-chloro-5-(2-
2

acetylaminoimidazo[1,2-b]pyridazin-6-yl)pyridin-3-yl)-4-fluorophenylsulfonamide17
and AMG 51118 are excellent PI3K/mTOR dual inhibitors or selective PI3K
inhibitor, and orally bioavailable anticancer agents as well. The pharmacophore of
above compounds consists of the two ring nitrogen atoms in pyridine and quinoline.
Another active compound, N-(2-methoxy-5-(acetylamino[1,2,4]triazolo[1,5-a]
pyridin-6-yl)pyridin-3-yl)-4-fluorophenylsulfonlyamide, having a pharmacophore
similar to that of compound A, displays a potent anticancer effect.19 Later, QSAR and
pharmacophore of analogues of compound A were studied.20 BEZ23521 and
PF-0497906422 also possess a pharmacophore similar to compound A. As PI3K and
mTOR dual inhibitors, they are in phase I/II clinical trials for treating solid tumors.
Recently, it has been reported that VS-5584, a PI3K/mTOR dual inhibitor, can
preferentially targets cancer stem cells.23 This discovery may potentially bring a
breakthrough to the treatment of cancer with small molecules. Thus, it is necessary to
develop some new PI3K/mTOR dual inhibitors.
Figure 1. The structures of PI3K and mTOR dual inhibitors
As a potent PI3K/mTOR dual inhibitor, compound A can inhibit tumor growth
against a wide range of tumors with different genetic backgrounds. Its EC50 ranges
from 0.26 mg/kg to 0.53 mg/kg against three established nude mice human cancer cell
xenograft models. However, compound A displays significant peroral toxicity.16 On
the basis of the result, we suspect that the toxicity of compound A is unfavorable to its
development into clinical trials. To overcome this problem, it is of vital importance to
treat seriously the toxicity of compound A.
According to the co-crystal structure of compound A with PI3Kγ,16 we proposed
that the structure of an amide group may take the place of the water molecule bridge.
Thereupon, we synthesized a series of 2-substituted-3-phenylsulfonylamino-5-
3

(quinazolin-6-yl or quinolin-6-yl)benzamides, and discovered that the designed
compounds are novel PI3K inhibitors and anticancer agents.24 In our previous work,
we combined the benzamide moiety with 2-aminobenzothiazole to discover novel
anticancer agents (strategy A in Figure 2.).25 Recently, we discovered that 1-alkyl-3-
(6-(2-methoxy-3-(4-fluorophenylsulfonylamino)pyridine-5-yl)-[1,2,4]triazolo[1,5-a]p
yridin-2-yl)urea derivatives can serve as potent PI3K inhibitors and anticancer agents
with low toxicity.26 In this work, we intend to replace the 2-acetylamino moiety in
compound A with alkylamino or alkylurea moiety to search for the novel anticancer
agents with low toxicity (strategy B in Fig. 2). Herein, we report our studies on the
synthesis, biological activities and acute toxicity of designed compounds.
Figure 2. optimizing strategy
2. Results and discussion
2.1 Synthesis of designed compounds
The synthetic route of compounds 1 is outlined in Scheme 1. Commercially
available 2-amino-6-bromobenzo[d]thiazole was used as starting material to prepare
intermediates 3, 4, 5 and 6. The details were previously described in our work.25 The
sulfonamides 7 were prepared from 5-bromopyridine derivatives according to the
synthetic route reported in our previous work.19 Catalyzed by PdCl2(dppf),
intermediate 7 was reacted with bis(pinacolato)diboron to produce corresponding
arylboronic esters. Without isolation of arylboronic esters, intermediate 5, or 6,
4

PdCl2(dppf), water and potassium carbonate as well were added to the above reaction
mixture. The resultant mixture was refluxed to produce compounds 1a-1d. The
preparation of arylboronic esters and Suzuki coupling were completed in one pot.
Scheme 1 Reagents and conditions: (a) AcOCHO, rt, ether, overnight; (b) BrCH2CO2Et, NaH,
DMF, rt, 4 h; (c) H3PO4, NaNO2, -10C, 1 h, then CuBr, 40% HBr, rt, 1 h, 40C, 2h; (d) n-PrNH2
or c-PrNH2, 1,4-dioxane, 45C, 4 h; (e) bis(pinacolato)diboron, AcOK, PdCl2(dppf), 1,4-dioxane,
reflux, N2, 3 h; (f) 5 or 6, PdCl2(dppf), K2CO3, 1,4-dioxane/water (5:1), reflux, N2, 2 h.
In Scheme 2, alkylurea moiety replaced the acetylamino group in compound A
and compounds 2 were synthesized to probe the structure-activity relationship.
Scheme 2 Reagents and conditions: (a) CDI, DMF; (b) H2NR1; (c): bis(pinacolato)diboron,
PdCl2(dppf), KOAc, 1,4-dioxane, N2, reflux 3 h; (d) 8, PdCl2(dppf), K2CO3, 1,4-dioxane/water
(5:1), N2, reflux, 2 h.
2-Amino-6-bromobenzo[d]thiazole
was
reacted
successively
with
carbonyldimidazole (CDI) and alkylamine to yield compounds 8. In the same way
with the preparation of compounds 1, intermediate 7 was reacted successively with
5

bis(pinacolato)diboron and intermediate 8, catalyzed by PdCl2(dppf), to yield
compounds 2a-2l.
To compare the activities of compounds with different substituents at the
3-position of pyridine ring, compounds 2m-2q were synthesized. Meanwhile,
compound 2r with a thiazolo[5,4-b]pyridine core was also synthesized (Scheme 3).
Scheme 3 Reagents and conditions: (a) CDI, DMF; (b) H2NCH2CH2N(CH2CH2)2O; (c):
bis(pinacolato)diboron, PdCl2(dppf), KOAc, 1,4-dioxane, N2, reflux 3 h; (d) 8, PdCl2(dppf),
K2CO3, 1,4-dioxane/water (5:1), N2, reflux, 2 h.
2.2. Biological evaluations
2.2.1 Antiproliferative assays in vitro
The antiproliferative activities of synthesized compounds were evaluated against
human colon carcinoma cell line (HCT-116, PI3CA mutant: H1047R), human breast
adenocarcinoma carcinoma cell line (MCF-7, PI3CA mutant: E545K), glioma cell line
(U87 MG, PTEN null) and lung adenocarcinoma epithelial cell line (A549, KRAS
mutant) by applying the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl- 2H-tetrazolium
bromide (MTT) colorimetric assay. The PI3K/mTOR dual inhibitors compound A and
BEZ235 were used as the positive controls. The results are summarized in Table 1.
6

Table 1 Antiproliferative activities of compounds 1, 2 (
, n = 3)
x  s
IC50 (μM)
Compds
HCT-116
>20
MCF-7
U87 MG
A549
nt
nt
nt
nt
nt
1a
1b
1c
5.75 ± 1.76
nt
1.56 ± 0.58
0.45 ± 0.08
1.50 ± 0.26
1.33 ± 0.25
1d
>20
nt
nt
nt
1.75±0.26
0.92±0.13
2.96±0.07
1.09±0.13
0.71±0.16
0.95±0.25
1.19±0.22
1.19±0.32
0.32±0.10
0.75±0.06
0.41±0.04
0.45±0.14
0.47±0.15
0.50±0.06
0.17±0.04
>20
1.72±0.36
1.39±0.39
2.39±0.61
1.23±0.28
0.52±0.07
0.39±0.01
0.49±0.07
0.36±0.04
0.75±0.04
0.44±0.08
1.02±0.04
0.36±0.07
7.23±0.62
8.38±2.13
1.67±0.12
1.05±0.29
0.52±0.15
0.48±0.13
0.44±0.08
1.41±0.32
1.03±0.33
5.26±0.59
6.69±0.29
1.56±0.38
0.90±0.15
0.45±0.04
0.43±0.12
0.30±0.07
0.33±0.07
0.55±0.09
0.47±0.01
0.31±0.05
5.16±0.71
4.71±1.97
1.92±0.32
0.53±0.02
0.38±0.03
0.49±0.04
0.41±0.08
0.76±0.08
2a
2b
2c
0.87±0.15
0.47±0.05
2d
2e
0.30±0.10
0.52±0.07
0.31±0.08
0.61±0.03
0.54±0.12
0.24±0.05
0.18±0.02
>20
2f
2g
2h
2i
2j
2k
2l
2m
2n
4.55±0.34
0.64±0.14
0.54±0.15
2.30±0.42
1.80±0.32
0.65±0.06
2o
2p
0.57±0.05
0.25±0.02
0.44±0.05
0.29±0.03
0.21±0.06
0.26±0.04
0.43±0.07
0.48±0.10
2q
2r
A
BEZ235
nt: not tested.
Firstly, the replacement of 2-acetylamino in compound A with akylamino
produced compounds 1a-1d, which showed a significant drop in the cell-based
activity. These results suggest that alkylamino is not a suitable substituent at the
2-position of benzothiazole core. Secondly, compounds 2a-2l displayed potent
antiproliferative activities against the four cancer cell lines and the activities of most
compounds 2 were close to that of positive controls. These results suggest that
7

alkylurea moiety was tolerable at the 2-position of benzothiazole. The data in Table 1
reveal that the activity of compounds 2 is related to the substituent at the 2- and
3-positions of pyridine ring and the 1-position of urea moiety as well. The fact that
compound 2f was more potent than compound 2b against four cancer lines indicates
that the title compound with a methoxy at the 2-position pyridine ring may improve
the antiproliferative activity. Thirdly, the compounds with the arylsulfonamino group
at the 3-position of pyridine ring, such as 4-methylphenylsulfonamino (2l), exhibited
more potent activity than the compounds with the cyclopropylsulfonamino group (2o)
and the cyano group (2n) at the 3-position of pyridine ring. In the case where there
was no a substituent at the 3-position of pyridine ring (2m), the activity dramatically
dropped. Fourthly, to further investigate the structure-activity relationship (SAR) of
compound 2, a diverse range of amines were selected to attach to the 1-position of
urea moiety. We synthesized compounds 2 by modifying substituent R1 (Scheme 2).
As the data in Table 1 indicate, the simple methyl urea 2d and cyclopropyl urea 2e
showed a drop in their cell-based activity against four cancer lines compared with
compound A. However, 2-(N,N-disubstituted amino)ethylureas 2f-2l displayed similar
or improved antiproliferative activity against the four cancer lines compared with the
positive controls. Fifthly, the replacement of benzothiazole core in compound 2g with
thiazolo[5,4-b]pyridine moiety afforded compound 2r, which displayed an improved
activity against HCT-116 and MCF-7 cells and a close activity against U87 MG and
A549 cells. These results reveal that compound 2r is sensitive to PI3K mutant cells.
In the cell-based activity, the IC50 of compound 2f are comparable to that of PI3K and
mTOR dual inhibitor compound A and BEZ235. In view of this, we further
investigated compound 2f.
2.2.2 PI3K and mTOR enzymatic activity assay
The selected compound 2f was evaluated for its PI3K and mTOR enzymatic
activity using an
.27 BEZ235 was used as the positive drug. The
results are listed in Table 2.
8

Table 2 Inhibitory enzymatic activities of compounds ( n = 2)
IC50 (nM)
Compds
mTORC1
PI3K
13
PI3K
90
PI3K
11
PI3K
148
78
44
2f
49
478
72
138
BEZ235
Compound 2f exhibited significant activities against PI3K and mTORC1. The
inhibitory activities against PI3K, PI3K and PI3K are higher than BEZ235. The
benzothiazole derivatives linked a methylurea moiety to C-2 were reported as
effective inhibitors against the wild-type and T315I mutant of Bcr-Abl kinase.28 In
this work, 1-alkyl-3-(2,3-disubstituted pyridin-5-ylbenzo[d]thiazol-2-yl)urea were
identified as PI3K and mTOR dual inhibitors.
2.2.3 Acute oral toxicity in vivo
Compounds A, 2d, 2f, 2h and 2j with potent antiproliferative activity in vitro were
tested for their acute oral toxicity in male mice by employing an “up-and-down
procedure”.29 The mice were administrated orally the prepared drug solution. The
acute oral toxicity of the tested compounds is listed in Table 3.
Table 3. The acute oral toxicity of tested compounds
Compds
R1
R2
LD50 (mg/kg) 95% CI (mg/kg)*
-CH3
Cl
20
40
19-22
38-42
A
2d
2f
-NHCH3
OCH3
OCH3
OCH3
OCH3
-NHCH2CH2NEt2
320
300
300
304-336
285-315
285-315
-NHCH2CH2N(CH2)4
-NHCH2CH2N(CH2CH2)2NMe
2h
2j
* CI: confidence interval
The data in Table 3 indicate that the sequence of acute oral toxicity for tested
9

compounds is compound A > 2d > 2h > 2j > 2f and the toxicity is closely related to
the structure of substitute at the 2-posotion of benzo[d]thiazole moiety. To our
surprise, the acute oral toxicity of compounds 2f, 2h and 2j, produced by the
replacement of 2-acetylamino moiety in compound A with a 2-(N,N-disubstituted
amino)ethylurea moiety, reduced dramatically. Hitherto, we have discovered new
PI3K and mTOR dual inhibitors with low toxicity by optimizing compound A.
2.2.4 Anticancer effect on the mice S180 homograft models in vivo
Lastly, we tested the anticancer effect of compound 2f in the homograft mice
models established for this study. Mice bearing sarcoma (S180) were treated orally
with compound 2f at 1.0 mg/kg, 3.0 mg/kg and 10 mg/kg once a day for 8 days. As it
was difficult to measure the volume of S180 tumor, tumor weights were used as
evaluating indicators as illustrated in Figure 3A. The inhibitory ratios of compound 2f
at the dosage of 1.0 mg/kg, 3.0 mg/kg and 10.0 mg/kg were 27.2%, 44.3% and 56.2%,
respectively. Additionally, the body weights of the tested mice increased during the
treatment (Figure 3B). These results suggest that 2f might be an effective anticancer
agent with low toxicity.
Figure 3. The anticancer effect of compound 2f in S180 homograft model (A) and the change of
tested mice body weights (B). Mice bearing subcutaneous tumors were orally administered vehicle,
compound 2f (1.0, 3.0 and 10.0 mg/kg doses) once daily for 8 days. **P < 0.01.
2.3 Docking studies
In order to further understand the potency of compounds 2 in Table 1, we performed
a docking analysis utilizing the C-DOCKER Program within Discovery Studio 2.5
software package. Docking simulations were carried out on human PI3Kγ (PDB code
10

3QK0)18 with compounds 2f and 2g. The results are depicted in Figure 4.
Figure 4. Docking mode of compound 2f and 2g with PI3Kγ. Selected residues
Val882, Ala885, Tyr867, Lys833 are shown. Green dashed lines indicate hydrogen
bond.
By the analysis of the binding mode of compounds 2f (Figure 4, left) and 2g
(Figure 4, right) with PI3Kγ, we observed that the interaction of 2f or 2g with PI3Kγ
is similar to that of compound A with PI3Kγ. The pyridine ring nitrogen atom can
form two hydrogen bonds via an ordered water molecule located between the central
and the Tyr867 and Asp841 residues. The methoxy at the 2-position of pyridine ring
can form a hydrogen bond with Lys833, which may explain that the compound with a
methoxy at the 2-position of pyridine ring is more potent than the compound with a
chloride at the 2-position of pyridine ring in cell-based activity. As observed in Figure
4, the urea moiety in two compounds forms three hydrogen bonds with Val882. The
binding mode is similar to benzothiazole-based Bcr-Abl inhibitors.27
3. Conclusion
In the present study, a series of 1-alkyl-3-(6-(2,3-disubstitutedpyridin-5-yl)
benzo[d]thiazol-2-yl)urea derivatives were synthesized and characterized. Their
antiproliferative activities in vitro were evaluated via MTT assay against four human
cancer cell lines. Compound 2f was tested for its inhibitory activity against PI3Ks and
mTORC1. In addition, the acute oral toxicity of four compounds was tested by oral
administration. The results indicate that the compound, produced by replacing the
2-acetylamino moiety in compound A with a 2-(dialkylamino)ethylurea moiety, can
retain the antiproliferative activity and inhibitory PI3K and mTOR activity. In
addition, their acute oral toxicity reduced dramatically. Moreover, compound 2f can
11

effectively inhibit tumor growth in a mice S180 homograft model. These findings
suggest that 1-(2-dialkylamino)ethyl-3-(6-(2-methoxy-3-sulfonylaminopyridin-5-yl)
benzo[d]thiazol-2-yl)urea derivatives can serve as potent PI3K inhibitors and
anticancer agents with low toxicity.
4. Experimental
4.1. Chemistry and chemical methods
Unless specified otherwise, all starting materials, reagents and solvents were
commercially available. All reactions were monitored by thin-layer chromatography
on silica gel plates (GF-254) and visualized with UV light. All the melting points
were determined on a Beijing micro melting-point apparatus and thermometer was
uncorrected. NMR spectra were recorded on a 400 Bruker NMR spectrometer with
tetramethylsilane (TMS) as an internal reference. All chemical shifts are reported in
parts per million (ppm). High-resolution exact mass measurements were performed
using electrospray ionization (positive mode) on a quadrupole time-of-flight (QTOF)
mass spectrometer (Maxis Q-TOF, Bruker Inc.).
4.1.1 General procedure for the synthesis of intermediates 8
4.1.1.1 1-(6-Bromobenzo[d]thiazol-2-yl)-3-cyclopropylurea (8a)
The mixture of 6-bromobenzo[d]thiazol-2-amine (2.0 g, 8.73 mmol),
carbonyldimidazole (4.24 g, 26.2 mmol) and dried DMF (20 ml) was stirred at room
temperature for 8 h, added cyclopropanamine (0.76 g, 13.2 mmol), then stirred at
room temperature for another 8 h. The volatile was removed under reduced pressure.
Water (30 ml) was added to the residue. The resulting suspension was stirred, standed.
The solid was collected by filtration, dried to produce 8a (1.77 g, 65%) as white solid.
mp: >250C; 1H NMR(DMSO-d6) 8.15 (s, 1H, Ar-H), 7.54 (d, J = 8.6 Hz, 1H, Ar-H),
7.48 (d, J = 8.6 Hz, 1H, Ar-H), 2.61 (dd, J = 6.8 Hz, 3.5 Hz, 1H, CH), 0.72-0.65 (m,
2H, CH2), 0.51-0.44 (m, 2H, CH2). MS (ESI, m/z): calcd for [M+H] + C11H11BrN3OS:
312, 314, found 312, 314.
Intermediates 8b-8i were synthesized according to the procedure described in 8a.
4.1.1.2 1-(6-Bromobenzo[d]thiazol-2-yl)-3-(2-(diethylamino)ethyl)urea (8b)
12

White solid; Yield 76%; mp: 57-58C; 1H NMR (CDCl3) δ 7.87 (s, 1H, Ar-H),
7.56 (d, 1H J = 8.6 Hz, 1H, Ar-H), 7.48 (d, J = 8.6 Hz, 1H, Ar-H), 3.46 (b, 2H, CH2),
+
2.71 (m, 6H, CH2×3), 1.12 (s, 6H, CH3×2). MS (ESI, m/z): calcd for [M+H]
C14H20BrN4OS: 314, 316, found 314, 316.
4.1.1.3 1-(6-Bromobenzo[d]thiazol-2-yl)-3-(2-morpholinoethyl)urea (8c)
1
White solid; Yield 90%; mp: 99-101C; H NMR (CDCl3) δ 7.85 (s, 1H, Ar-H),
7.54 (d, J = 8.6 Hz, 1H Ar-H), 7.48 (d, J = 8.6 Hz, 1H, Ar-H), 3.77 (s, 2H, CH2), 3.51
+
(s, 4H, CH2×2), 2.63 (m, 6H, CH2×3). MS (ESI, m/z): calcd for [M+H]
C14H18BrN4O2S 385, 387, found 385, 387.
4.1.1.4 1-(6-Bromobenzo[d]thiazol-2-yl)-3-methylurea(8d)
White solid; Yield 74%; mp: >250C; 1H NMR (DMSO-d6) δ 10.94 (s, 1H, NH),
8.14 (s, 1H, Ar-H), 7.54 (d, J = 8.4 Hz, 1H, Ar-H), 7.48 (d, J = 8.5 Hz, 1H, Ar-H),
6.63 (s, 1H, NH), 2.72 (d, J = 3.7 Hz, 3H, CH3). MS (ESI, m/z): calcd for [M+H] +
C9H9BrN3OS: 286, 288, found 286, 288.
4.1.1.5 1-(6-Bromobenzo[d]thiazol-2-yl)-3-(2-(pyrrolidin-1-yl)ethyl)urea (8e)
1
White solid; Yield 85%; mp: 79-81C; H NMR (CDCl3) δ 7.74 (s, 1H, Ar-H),
7.52 (d, 1H J = 8.6 Hz, 1H, Ar-H), 7.40 (d, J = 8.6 Hz, 1H, Ar-H), 3.56 (s, 2H, CH2),
+
2.87 (m, 6H, CH2×3), 1.88 (s, 4H, CH2×2). MS (ESI, m/z): calcd for [M+H]
C14H18BrN4OS: 369, 371, found 369, 371.
4.1.1.6 1-(6-Bromobenzo[d]thiazol-2-yl)-3-(2-(piperidin-1-yl)ethyl)urea (8f)
White solid; Yield 79%; mp: 84-86C; 1H NMR (CDCl3) δ 7.84 (s, 1H, Ar-H),
7.50 (d, J = 8.5 Hz, 1H, Ar-H), 7.45 (d, J = 8.5 Hz, 1H, Ar-H), 3.48 (s, 2H, CH2), 2.67
- 2.49 (m, 6H, CH2×3), 1.65 - 1.44 (m, 6H, CH2×3). MS (ESI, m/z): calcd for [M+H]
+ C15H20BrN4OS: 383, 385, found 383, 385.
4.1.1.7 1-(6-Bromobenzo[d]thiazol-2-yl)-3-(2-(4-methylpiperazin-1-yl)ethyl)urea
(8g)
1
White solid; Yield 64%; mp: 97-99C; H NMR (CDCl3) δ 7.85 (s, 1H, Ar-H),
7.52 (d, J = 8.6 Hz, 1H, Ar-H), 7.47 (d, J = 8.6 Hz, 1H, Ar-H), 3.49 (s, 2H, CH2), 2.78
- 2.41 (m, 10H, CH2 CH2×5), 2.35 (s, 3H, CH3). MS (ESI, m/z): calcd for [M+H] +
C15H21BrN5OS: 398, 400, found 398, 400.
13

4.1.1.8 1-(6-Bromobenzo[d]thiazol-2-yl)-3-(2-(dimethylamino)ethyl)urea (8h)
White solid; Yield 77%; mp: 176-178C; 1H NMR (CDCl3) δ 7.84 (s, 1H, Ar-H),
7.50 (d, J = 8.5 Hz, 1H, Ar-H), 7.45 (d, J = 8.5 Hz, 1H, Ar-H), 3.52 (s, 2H, CH2), 2.65
+
(s, 2H, CH2), 2.39 (s, 6H, CH3 CH2×3). MS (ESI, m/z): calcd for [M+H]
C14H20BrN4OS: 343, 345, found 343, 345.
4.1.1.9 1-(6-Bromothiazolo[5,4-b]pyridin-2-yl)-3-(2-(morpholinoethyl)urea (8i)
1
White solid; Yield 86%; mp: 152-154C; H NMR (DMSO-d6) δ 11.13 (s, 1H,
NH), 8.01 (d, J = 8.3 Hz, 1H, Ar-H), 7.48 (d, J = 8.4 Hz, 1H, Ar-H), 6.81 (s, 1H, NH),
3.60 (s, 4H, OCH2×2), 3.29 (d, J = 5.2 Hz, 2H, CH2), 2.41 (s, 6H, NCH2×3). MS (ESI,
m/z): calcd for [M+H] + C13H17BrN5O2S: 386, 388, found 386, 388.
4.1.2 General procedure for the synthesis of compounds 1a-1d, 2a-2r
4.1.2.1
N-(2-Chloro-5-(2-(propylamino)benzo[d]thiazol-6-yl-pyridin-3-yl)-4-fluoro
phenylsulfonamide (1a)
The preparation of intermediate 6a, N-propyl-6-bromobenzo[d] thiazolyl-2-amine,
was described in our previous work.27 The preparation of intermediate 7a, N-(2-chloro
(or methoxy)-5-bromopyridin-3-yl)-4-fluorophenylsulfonamide, was described in our
previously work.21 The mixture containing 7a (0.20 g, 0.55 mmol),
bis(pinacolato)diboron (0.17 g, 0.66 mmol), KOAc (0.16 g, 1.65 mmol), PdCl2(dppf)
(30 mg, 0.04 mmol) and 1,4-dioxane (10 ml) was reflux for 2 h under nitrogen
atmosphere, cooled to room temperature. To the resulted mixture were added 6a (0.15
g, 0.55 mmol), Na2CO3 (0.64 g, 4.65 mmol), PdCl2(dppf) (30 mg, 0.04 mmol) and
water (2 ml). The mixture was refluxed under nitrogen atmosphere for 2 h. The
volatiles were removed in vacuum and the residue was purified through a column
chromatography on silica with chloroform/methanol (V:V = 50:1) as eluent to produce
1
1a (0.12 g, 46%) as a white foam. mp: 214-215℃; H NMR (DMSO-d6) δ 10.47 (s,
1H, NH), 8.58 (d, J = 2.4 Hz, 1H, Ar-H), 8.24 (t, J = 5.6 Hz, 1H, NH), 8.04 (d, J = 1.6
Hz, 1H, Ar-H), 7.97 (d, J = 2.4 Hz, 1H, Ar-H), 7.83-7.79 (m, 2H, Ar-H), 7.53 (dd,
J=8.4, 2.0Hz, 1H, Ar-H), 7.47 (d, J = 8.4 Hz, 1H. Ar-H), 7.41-7.46 (m, 2H, Ar-H),
2.50 (m, 2H, CH2), 1.62 (m, 2H, CH2), 0.95 (t, J = 7.2 Hz, 3H, CH3); ESI-HRMS m/z:
calc’d for C21H20ClFN4O2S2 [M + H]+: 477.0622; found 477.0628.
14

Compounds 1b-1d, 2a-2l were synthesized according to the procedure described
in 1a.
4.1.2.2
fluorophenylsulfonamide (1b)
N-(2-Chloro-5-(2-(cyclopropylamino)benzo[d]thiazol-6-yl-pyridin-3-yl)-4-
1
White solid; Yield 24%; mp: 213-215C; H NMR (DMSO-d6) δ 10.48 (s, 1H,
NH), 8.59 (s, 1H, Ar-H), 8.56 (s, 1H, NH), 8.11 (s, 1H, Ar-H), 7.97(s, 1H, Ar-H),
7.79-7.83 (m, 2H, Ar-H), 7.55 (d, J = 8.4 Hz, 1H, Ar-H), 7.51 (d, J = 8.4 Hz, 1H,
Ar-H), 7.44 (m, 1H, Ar-H), 2.74 (m, 1H, CH), 0.80 (m, 2H, CH2), 0.60 (m, 2H, CH2);
ESI-HRMS m/z: calc’d for C21H18ClFN4O2S2 [M + H]+: 475.0465; found 475.0472.
4.1.2.3 N-(2-Methoxy-5-(2-(cyclopropylamino)benzo[d]thiazol-6-yl-pyridin-3-yl)-4-
fluorophenylsulfonamide (1c)
1
White solid; Yield 57%; mp: 194-195C; H NMR (DMSO-d6) δ 10.03 (s, 1H,
NH), 8.46 (s, 1H, NH), 8.27 (s, 1H, Ar-H), 8.00 (s, 1H, Ar-H), 7.84 (s, 1H, Ar-H),
7.82 (m, 2H, Ar-H), 7.81 (d, J = 8.4 Hz, 1H, Ar-H), 7.47 (d, J = 8.4 Hz, 1H, Ar-H),
7.42 (m, 2H, Ar-H), 3.64 (s, 3H, OCH3), 2.72 (m, 1H, CH), 0.79 (m, 2H, CH2), 0.59
(m, 2H, CH2); ESI-HRMS m/z: calc’d for C22H21FN4O3S2 [M + H]+: 471.0961; found
471.0976.
4.1.2.4 2-((6-(5-(4-fluorophenylsulfonamido)-6-mothoxypyridin-3-yl)benzo[d]
thiazol-2-yl)amino)acetate (1d)
1
White solid; Yield 20%; mp: 214-215C; H NMR (DMSO-d6) δ 10.07 (s, 1H,
NH), 8.99 (s, 1H, Ar-H), 8.35 (s, 1H, Ar-H), 7.94 (s, 1H, Ar-H), 7.81 (d, J = 8.4 Hz,
2H, Ar-H), 7.73 (d, J = 8.8 Hz, 1H, Ar-H), 7.51 (d, J = 9.2 Hz, 1H, Ar-H), 7.41(t, J =
8.8 Hz, 2H, Ar-H), 7.12 (t, J = 6.4 Hz, 1H, NH), 4.11 (q, J = 7.2 Hz, 2H, OCH2),
4.01(d, J = 6.4 Hz, 2H, CH2), 3.63(s, 3H, OCH3), 1.20 (t, J = 6.8 Hz, 3H, CH3);
ESI-HRMS m/z: calc’d for C23H23FN4O5S2 [M + H]+: 517.1016; found 517.1026.
4.1.2.5
1-(6-(2-Chloro-3-(4-fluorophenylsulfonamino)pyridin-3-yl)benzo[d]
thiazol-2-yl)-3-cyclopropylurea (2a)
White solid; Yield 39%; mp: >250C; 1H NMR (DMSO-d6) δ 10.71 (s, 1H, NH),
10.51 (s, 1H, NH), 8.62 (s, 1H, Ar-H), 8.28 (s, 1H, Ar-H), 8.02 (s, 1H, Ar-H), 7.82 (d,
J = 8.8 Hz, 2H, Ar-H), 7.73 (d, J = 8.4 Hz, 1H, Ar-H), 7.66 (d, J = 8.4 Hz, 1H, Ar-H),
15

7.44 (t, J = 8.8 Hz, 2H, Ar-H), 6.98 (s, 1H, NH), 2.63 (m, 1H, CH), 0.70 (m, 2H, CH2),
0.50 (m, 2H, CH2); ESI-HRMS m/z: calc’d for C22H17ClFN5NaO3S2 [M+Na]+:
540.0343; found 540.0338.
4.1.2.6 1-(6-(2-Chloro-3-(4-fluorophenylsulfonamino)pyridin-3-yl)benzo[d]
thiazol-2-yl)-3-(2-diethylamino)ethylurea (2b)
White solid; Yield 36%; mp: 164-166C; 1H NMR (DMSO-d6) δ 11.23 (s, 1H,
NH), 9.49 (s, 1H, NH), 8.04 (d, J = 8.0 Hz, 2H, Ar-H), 7.82 (s, 1H, Ar-H), 7.79 (d, J =
5.5 Hz, 1H, Ar-H), 7.77 (s, 1H, Ar-H), 7.69 (d, J = 8.4 Hz, 1H, Ar-H), 7.48 (d, J = 8.4
Hz, 1H, Ar-H), 7.29 (t, J = 8.8 Hz, 2H, Ar-H), 6.92 (s, 2H s, 1H, NH), 3.45 (d, J = 5.6
Hz, 2H, CH2), 3.00-3.05 (m, 6H, CH2×3), 1.16 (t, J = 7.1 Hz, 6H, CH3×2);
ESI-HRMS m/z: calc’d for C25H27ClFN6O3S2 [M + H]+: 577.1259; found 577.1253.
4.1.2.7 1-(6-(2-Chloro-3-(4-fluorophenylsulfonamino)pyridin-3-yl)benzo[d]thiazol-
2-yl)-3-(2-morpholinoethyl)ethyl)urea (2c)
White solid; Yield 38%; mp: 208-210C; 1H NMR(DMSO-d6) δ 11.01 (s, 1H,
NH), 10.22 (s, 1H, NH), 8.50 (s, 1H, Ar-H), 8.22 (s, 1H, Ar-H), 7.97 (s, 1H, Ar-H),
7.82 (d, J = 8.5 Hz, 2H, Ar-H), 7.72 (d, J = 8.4 Hz, 1H, Ar-H), 7.62 (d, J = 8.4 Hz, 1H,
Ar-H), 7.41 (t, J = 8.7 Hz, 2H, Ar-H), 6.86 (s, 1H, NH), 3.66-3.63 (m, 4H, OCH2×2),
3.32-3.30 (m, 2H, NCH2), 2.56- 2.51 (m, 6H, NCH2×3); ESI-HRMS m/z: calc’d for
C25H26ClFN6O4S2 [M+H]+: 591.1051; found 591.1046.
4.1.2.8 1-(6-(2-Methoxy-3-(4-fluorophenylsulfonamino)pyridin-3-yl)benzo[d]
thiazol-2-yl)-3-methylurea (2d)
White solid; Yield 31%; mp: >250C; 1H NMR (DMSO-d6) δ 10.89 (s, 1H, NH),
10.05 (s, 1H, NH), 8.31 (s, 1H, Ar-H), 8.15 (s, 1H, Ar-H), 7.88 (s, 1H, Ar-H), 7.82
(dd, J = 8.8, 5.2 Hz, 2H, Ar-H), 7.69 (d, J = 8.4 Hz, 1H, Ar-H), 7.57 (d, J = 8.4, 1H,
Ar-H), 7.42 (t, J = 8.8 Hz, 2H, Ar-H), 6.65 (s, 1H, NH), 3.65 (s, 3H, OCH3), 2.74 (d, J
= 4.2 Hz, 3H, CH3). ESI-HRMS m/z: calc’d for C21H20FN5O4S2 [M+H]+: 488.0862;
found 488.0857.
4.1.2.9 1-(6-(2-Methoxy-3-(4-fluorophenylsulfonamino)pyridin-3-yl)benzo[d]
thiazol-2-yl)-3-cyclopropylurea (2e)
White solid; Yield 46%; mp: >250C; 1H NMR(DMSO-d6) δ 10.64 (s, 1H, NH),
16

10.05 (s, 1H, NH) , 8.31 (s, 1H, Ar-H), 8.16 (s, 1H, Ar-H), 7.89 (s, 1H, Ar-H), 7.83 (d,
J = 4.5 Hz, 1H, Ar-H), 7.81 (d, J = 4.6 Hz, H, Ar-H), 7.69 (d, J = 8.2 Hz, 1H, Ar-H),
7.58 (d, J = 8.1 Hz, 1H, Ar-H), 7.42 (t, J = 8.5 Hz, 2H, Ar-H), 6.98 (s, 1H, NH), 3.65
(s, 3H, OCH3), 2.64 (m, 1H, CH), 0.71 (s, 2H, CH2), 0.50 (s, 2H, CH2); ESI-HRMS
m/z: calc’d for C23H20FN5O4S2 [M+H]+: 536.0838, found 536.0833.
4.1.2.10 1-(2-Diethylamino)ethyl-3-(6-(2-methoxy-3-(4-fluorophenylsulfonamino)
pyridin-3-yl)benzo[d]thiazol-2-yl)urea (2f)
Off-White solid; Yield 52%; mp: 193-195C; 1H NMR(DMSO-d6) δ 10.96 (s, 1H,
NH), 9.93 (s, 1H, NH), 8.26 (s, 1H, Ar-H), 8.13 (s, 2H, Ar-H), 7.85 (s, 1H, Ar-H),
7.84-7.79 (m, 2H, Ar-H), 7.68 (d, J = 8.4 Hz, 1H, Ar-H), 7.56 (d, J = 8.4 Hz, 1H,
Ar-H), 7.40 (t, J = 8.8 Hz, 2H, Ar-H), 6.80 (s, 1H), 3.66 (s, 3H, OCH3), 3.26 (d, J =
5.7 Hz, 2H, NCH2), 2.57 (m, 4H, CH2×2), 1.01 (t, J = 7.0 Hz, 6H, CH3×2).
ESI-HRMS m/z: calc’d for C26H31FN6O4S2 [M+H]+: 573.1754; found 573.1748.
4.1.2.11 1-(6-(2-Methoxy-3-(4-fluorophenylsulfonamino)pyridin-3-yl)benzo[d]thiazol-
2-yl)-1-(2-morpholino)ethylurea (2g)
1
White solid; Yield 42%; mp: 209-211C; H NMR (DMSO-d6) δ 10.90 (s, 1H,
NH), 10.04 (s, 1H, NH), 8.30 (s, 1H, Ar-H), 8.15 (s, 1H, Ar-H), 7.88 (s, 1H, Ar-H),
7.82 (dd, J = 8.8, 5.2 Hz, 2H, Ar-H), 7.69 (d, J = 8.4 Hz, 1H, Ar-H), 7.57 (d, J = 8.4
Hz, 1H, Ar-H), 7.42 (t, J = 8.8 Hz, 2H, Ar-H), 6.83 (s, 1H, NH), 3.65 (s, 3H, OCH3),
3.61 (s, 4H, OCH2×2), 3.31 (b, 2H, NCH2), 2.44 (m, 6H, NCH2×3). ESI-HRMS m/z:
calc’d for C26H29FN6O5S2 [M+H]+ 587.1547; found 587.1540.
4.1.2.12
1-(6-(2-Methoxy-3-(4-fluorophenylsulfonamino)pyridin-3-yl)benzo[d]
thiazol-2-yl)-1-(2-pyrrolidin-1-yl)ethylurea (2h)
White solid; Yield 34%; mp: 144-146C; 1H NMR(DMSO-d6) δ 11.21 (s, 1H,
NH), 10.12 (s, 1H, NH), 8.31 (d, J = 1.8 Hz, 1H, Ar-H), 8.16 (s, 1H Ar-H), 7.88 (s, 1H,
Ar-H), 7.82 (dd, J = 8.5, 5.2 Hz, 2H, Ar-H), 7.70 (d, J = 8.3 Hz, 1H, Ar-H), 7.59 (d, J
= 8.4 Hz, 1H, Ar-H), 7.42 (t, J = 8.7 Hz, 2H, Ar-H), 7.22 (s, 1H, NH), 3.65 (s, 3H,
OCH3), 3.49 (s, 2H, NCH2), 3.13 (s, 6H, NCH2×3), 1.89 (s, 4H, CH2×2). ESI-HRMS
m/z: calc’d for C26H29FN6O4S2 [M+H]+: 571.1597; found 571.1592.
4.1.2.13
1-(6-(2-Methoxy-3-(4-fluorophenylsulfonamino)pyridin-3-yl)benzo[d]
17

thiazol-2-yl)-1-(2-piperidin-1-yl)ethylurea (2i)
Pale yellow solid; Yield 37%; mp: 160-162C; 1H NMR(DMSO-d6) δ 11.15 (s,
1H, NH), 9.79 (s, 1H, NH), 8.31 (d, J = 2.2 Hz, 1H, Ar-H), 8.16 (s, 1H, Ar-H), 7.88 (d,
J = 2.2 Hz, 1H, Ar-H), 7.82 (dd, J = 8.8, 5.2 Hz, 2H, Ar-H), 7.70 (d, J = 8.4 Hz, 1H,
Ar-H), 7.58 (dd, 1H, Ar-H), 7.42 (t, J = 8.8 Hz, 2H, Ar-H), 7.04 (s, 1H, NH), 3.65 (s,
3H, OCH3), 3.45 (s, 2H, NCH2), 2.87 (m, 6H, NCH2×3), 1.67 (s, 4H, CH2×2), 1.49 (s,
2H, CH2). ESI-HRMS m/z: calc’d for C27H31FN6O4S2 [M+H]+: 585.1754; found
585.1748.
4.1.2.14
1-(6-(2-Methoxy-3-(4-fluorophenylsulfonamino)pyridin-3-yl)benzo[d]
thiazol-2-yl)-1-(2-(4-methylpiperazin-1-yl))ethylurea (2j)
1
White solid; Yield 34%; mp: 214-216C; H NMR(DMSO-d6) δ 11.06 (s, 1H,
NH), 8.24 (d, J = 2.1 Hz, 1H, Ar-H), 8.11 (d, J = 1.5 Hz, 1H, Ar-H), 7.83(d, J = 2.2
Hz, 1H, Ar-H), 7.81 (d, J = 5.2 Hz, 1H), 7.68 (d, J = 8.3 Hz, 2H), 7.54 (dd, 1H, Ar-H),
7.40 (t, J = 8.9 Hz, 2H, Ar-H), 7.08 (s, 1H, NH), 3.66 (s, 3H, OCH3), 3.27 (d, J = 5.8
Hz, 2H, CH2), 2.48-2.38 (m, 10H, NCH2×5), 2.25 (s, 3H, CH3). ESI-HRMS m/z:
calc’d for C27H32FN7O4S2 [M+H]+: 600.1863; found 600.1857.
4.1.2.15
1-(6-(2-Methoxy-3-(4-fluorophenylsulfonamino)pyridin-3-yl)benzo[d]
thiazol-2-yl)-3-(2-dimethylamino)ethylurea (2k)
1
White solid; Yield 33%; mp: 136-138C; H NMR(DMSO-d6) δ 10.84 (s, 1H,
NH), 8.29 (s, 1H, Ar-H), 8.14 (s, 1H, Ar-H), 7.87 (s, 1H, Ar-H), 7.84-7.80 (dd, J = 7.8,
5.4 Hz, 2H, Ar-H), 7.69 (d, J = 8.3 Hz, 1H, Ar-H), 7.57 (d, J = 7.9 Hz, 1H, Ar-H),
7.41 (t, J = 8.6 Hz, 2H, Ar-H), 6.86 (s, 1H, NH), 3.65 (s, 2H, OCH3), 3.30-3.26 (m,
2H, NCH2), 2.29 (s, 6H, CH3×2). ESI-HRMS m/z: calc’d for [M+H]+ C24H26FN6O4S2:
545.1441, found 545.1435.
4.1.2.16
1-(2-Diethylamino)ethyl-3-(6-(2-methoxy-3-(4-methylphenylsulfonamino)
pyridin-5-yl)benzo[d]thiazol-2-yl)urea (2l)
White solid; Yield 51%; mp: 115-117C; 1H NMR(CDCl3) δ 8.02 (s, 1H, Ar-H),
7.97 (s, 1H, Ar-H), 7.75 (s, 1H, Ar-H), 7.70 (d, J = 8.3 Hz, 2H, Ar-H), 7.67 (s, 1H,
Ar-H), 7.43 (d, J = 8.2 Hz, 1H, Ar-H), 7.23 (d, J = 8.0 Hz, 2H, Ar-H), 3.84 (s, 3H,
OCH3), 3.79 (s, 2H, CH2), 3.20 (d, J = 3.4 Hz, 6H, NCH2×3), 2.36 (s, 3H, CH3), 1.33
18

(t, J = 6.6 Hz, 6H, CH3×2). ESI-HRMS m/z: calc’d for C27H33N6O4S2 [M+H]+:
569.2005; found 569.1999.
4.1.2.17
1-(6-(6-methoxypyridin-3-yl)benzo[d]thiazol-2-yl)-3-(2-morpholino)
ethylurea (2m)
White solid; Yield 47%; mp: 100-102C; 1H NMR(DMSO-d6) δ 10.83 (s, 1H,
NH), 8.47 (s, 1H, Ar-H), 8.15 (s, 1H, Ar-H), 8.01 (d, J = 8.5 Hz, 1H, Ar-H), 7.63 (d, J
= 7.9 Hz, 1H, Ar-H), 7.61 (s, 1H, Ar-H), 6.87 (d, J = 8.4 Hz, 1H, Ar-H), 6.79 (s, 1H,
NH), 3.85 (s, 3H, OCH3), 3.56 (s, 4H, OCH2×2), 3.25 (d, J = 4.8 Hz, 2H, NCH2), 2.38
(m, 6H, NCH2×3). ESI-HRMS m/z: calc’d for C20H24N5O3S [M+H]+: 414.1600;
found 414.1594
4.1.2.18 1-(6-(3-Cyano-2-methoxypyridin-5-yl)benzo[d]thiazol-2-yl)-3-(2-morpholino)
ethylurea (2n)
White solid; Yield 42%; mp: 200-202C; 1H NMR(DMSO-d6) δ 10.93 (s, 1H,
NH), 8.83 (s, 1H, Ar-H), 8.65 (s, 1H, Ar-H), 8.28 (s, 1H, Ar-H), 7.74 (dd, J = 8.5, 1.5
Hz, 1H, Ar-H), 7.68 (s, 1H, Ar-H), 6.82 (s, 1H, NH), 4.05 (s, 3H, OCH3), 3.61 (s, 4H,
OCH2×2), 3.320 (m, 2H, NCH2), 2.43 (d, J = 5.6 Hz, 6H, NCH2×3). ESI-HRMS m/z:
calc’d for C21H23N6O3S [M+H]+: 439.1552; found 439.1547.
4.1.2.19 1-(6-(3-cyclopropanylsulfonamino-2-methoxypyridin-5-yl)benzo[d]thiazol-
2-yl)-3-(2-morpholino)ethylurea (2o)
White solid; Yield 40%; mp: 191-193C; 1H NMR (DMSO-d6) δ 10.89 (s, 1H,
NH), 9.38 (s, 1H, NH), 8.34 (s, 1H, Ar-H), 8.20 (s, 1H, Ar-H), 7.93 (s, 1H, Ar-H),
7.69 (d, J = 8.4 Hz, 1H, Ar-H), 7.62 (dd, J = 8.4, 1.8 Hz, 1H, Ar-H), 6.83 (s, 1H, NH),
3.97 (s, 3H, OCH3), 3.61 (m, 4H, OCH2×2), 3.30 (dd, J = 11.7, 6.0 Hz, 2H, NCH2),
2.78 (m, 1H, CH), 2.44 (m, 6H, NCH2×3), 0.99-0.89 (m, 4H, CH2×2). ESI-HRMS m/z:
calc’d for C23H29N6O5S2 [M+H]+: 533.1641; found 533.1635.
4.1.2.20
1-(6-(3-chlorophenylsulfonamino-2-methoxypyridin-5-yl)benzo[d]thiazol-
2-yl)-3-(2-morpholino)ethylurea (2p)
1
Pale yellow solid; Yield 41%; mp: 153-155C; H NMR(DMSO-d6) δ 10.92 (s,
1H, NH), 10.12 (s, 1H, NH), 8.32 (s, 1H, Ar-H), 8.15 (s, 1H, Ar-H), 7.88 (s, 1H,
Ar-H), 7.77 (s, 1H, Ar-H), 7.75 (s, 1H, Ar-H), 7.69 (d, J = 8.6 Hz, 1H, Ar-H), 7.66 (d,
19

J = 8.6 Hz, 2H, Ar-H), 7.58 (d, J = 8.4 Hz, 1H, Ar-H), 6.84 (s, 1H, NH), 3.64 (s, 3H,
OCH3), 3.62 (s, 4H, OCH2×2), 3.30 (m, 2H, NCH2), 2.45 (m, 6H, NCH2×3).
ESI-HRMS m/z: calc’d for C26H28ClN6O5S2 [M+H]+: 603.1251; found 603.1246.
4.1.2.21
1-(6-(2-methoxy-3-(4-methylphenylsulfonamino)pyridin-5-yl)benzo[d]
thiazol-2-yl)-3-(2-morpholino)ethylurea (2q)
1
White solid; Yield 31%; mp: 195-197C; H NMR(DMSO-d6) δ 10.90 (s, 1H,
NH), 9.90 (s, 1H, NH), 8.26 (s, 1H, Ar-H), 8.11 (s, 1H, Ar-H), 7.83 (d, J = 2.2 Hz, 1H,
Ar-H), 7.69 (d, J = 8.6 Hz, 2H, Ar-H), 7.65 (s, 1H, Ar-H), 7.54 (d, J = 8.4, 1H, Ar-H),
7.37 (d, J = 8.1 Hz, 2H, Ar-H), 6.83 (s, 1H, NH), 3.68 (s, 3H, OCH3), 3.62 (s, 4H,
OCH2×3), 3.30 (m, 2H, NCH2), 2.44 (m, 6H, NCH2×3), 2.36 (s, 3H, CH3).
ESI-HRMS m/z: calc’d for C27H31N6O5S2 [M+H]+: 583.1797; found 583.1792.
4.1.2.22 1-(6-(3-(4-fluorophenylsulfonamino)-2-methoxypyridin-5-yl)thiazolo[5,4-b]
pyridin-2-yl)-3-(2-morpholino)ethylurea (2r)
1
White solid; Yield 58%; mp: 192-194C; H NMR(DMSO-d6) δ 11.05 (s, 1H,
NH), 10.06 (s, 1H, NH), 8.67 (s, 1H, Ar-H), 8.28 (s, 1H, Ar-H), 8.02 (d, J = 8.5 Hz,
1H, Ar-H), 7.95 (d, J = 8.5 Hz, 1H, Ar-H), 7.82 (d, J = 5.2 Hz, 1H, Ar-H), 7.80 (d, J =
5.2 Hz, 1H, Ar-H), 7.42 (t, J = 8.8 Hz, 2H, Ar-H), 6.84 (s, 1H, NH), 3.68 (s, 3H,
OCH3), 3.61 (s, 4H, OCH2×2), 3.32 (d, J = 5.7 Hz, 2H, NCH2), 2.45 (d, J = 6.5 Hz,
6H, NCH2×3). ESI-HRMS m/z: calc’d for C25H27N7O5S2 [M+H]+: 588.1499; found
588.1494.
4.2. Biology
4.2.1. Cell Culture
The human cell lines HCT-116, MCF-7and U-87 MG were maintained as a
monolayer culture in DMEM, supplemented with 10% FBS, while A549 was cultured
in RPMI-1640 with 10% calf serum in a humidified atmosphere (5% CO2) at 37 C,
respectively.
4.2.2 Antiproliferative assays
Compound A was synthesized in our laboratory (Purity: 96%, HPLC). BEZ235
was purchased from Shanghai Biochempartner Company (Purity: 99%, HPLC). 3-[4,
20

5-dimethylthiazol-2-yl]-2, 5-diphenyl-2H-tetrazolium bromide (MTT) was purchased
from Sigma (St. Louis, MO, USA). Cellular chemosensitivity was determined by
using a modified MTT method assay in vitro. In brief, HCT-116, MCF-7, U-87 MG or
A549 cells in 200 μl culture medium were seeded into 96-well microplates at
3000-5000 cells per well respectively and cultured in DMEM with 10% FBS or
RPMI-1640 with 10% calf serum, incubated at 37 °C for 24 h prior to drug exposure.
Cell numbers were titrated to keep control cells growing in the exponential phase
throughout the 72 h incubation period. Cells were treated with final concentrations of
100.0, 10.0, 1.0, 0.1 and 0.01 M of tested compounds simultaneously and incubated
for 72 h and then 20 μl of MTT solution (5 mg/ml in PBS) was added to each well at
lucifugal condition and incubated for 4 h at 37°C. The formed purple formazan
crystals were pelleted at the bottom of the well, separated from the supernatant, and
dissolved in 200 μl of DMSO. The optical density at 570 nm was determined by
Varioskan Flash Multimode Reader (Thermo scientific). Three separate experiments
with triplicate data were performed to obtain mean cell viability. The IC50 value, that
is, the concentration (M) of a compound was able to cause 50% cell death with
respect to the control culture, was calculated according to the inhibition ratios.
PI3K and mTOR enzymatic activity assay
PI3K and mTORC1 enzymatic activity assay was performed according to
process described in reference.28 Briefly, compounds 2f and BEZ235 were dissolved
in DMSO and diluted to a series of concentrations. Different concentrations of
compounds were added to the enzyme reaction buffer containing 40 mM Tris-HCl,
pH 7.4, 10 mM MgCl2, 0.1 mg/mL BSA, 1 mM DTT, 2.5 μM ATP, PI3K
(p110α/p85α, p110β/p85α, p110γ/PIK3R5 or p110δ/p85α), mTORC1 and
L-α-phosphatidylinositol. The final reaction volume was 50 μL. After incubation for
40 min at 30 °C, the reaction was terminated by addition of stop solution. The amount
of ADP was then detected via luciferase assay. After incubation for 5 min, the
luminescence signal was determined by the multimode reader (MD-SpectraMax M5).
The signal intensity is proportional to the enzyme activity.
The percentage of inhibition was calculated based on the following equation
21

% inhibition rate = [1 – (Lu compound – Lu min)/(Lu max – Lu min)] × 100%,
Where Lu compound is the signal at a given compound concentration, Lu max is the
signal of PI3Ks without compound and Lu
is the signal of background in the
min
absence of enzyme and compound. The IC50 values were calculated according to the
fit of the dose -response curves by using GraphPad Prism5.
4.2.3 Acute oral toxicity in vivo
Mice (20.1 ± 1.9 g) were purchased from Experiment Animal Center of Xian
Jiaotong University and fed in the same place. The experimental protocol was
approved by Ethic Committee of Xian Jiaotong University.
The tested compounds were, respectively, dissolved in NMP, diluted with
PEG400 and H2O to prepare proper solutions with different concentrations
(NMP/PEG400/H2O is 1:6:3 in volume ratio). A mouse was dosed orally once a day,
and then observed for 7 days. The dosage was started at 10 mg/kg at a volume of 20
ml/kg. If the mouse exercised normally, the dosage was increased to 1.5-fold up to a
mouse died in a single dose. The software AOT425StatPgm was employed to estimate
LD50.
4.2.4 In vivo anticancer effect on established mice S180 homograft models
Mice (19.6 ± 1.9 g) were purchased from Experiment Animal Center of Xian
Jiaotong University Health Science Center and fed in the same place. The
experimental protocol was approved by Ethic Committee of Xian Jiaotong
University.
3  106 S180 cells were injected subcutaneously into the flank of the mice. All
tumor-bearing mice were randomly divided into four groups, with 8 mice in each
group. The next day, compound 2f was dissolved in NMP/PEG400/H2O (1:6:3) and
dosed orally at 1.0 mg/kg, 3.0 mg/kg and 10.0 mg/kg for the low, middle and high
dosage groups once a day for 8 days, respectively. In the solvent group, the same
volume of solvent was administered orally. Body weights were recorded per day. The
mice were anesthetized and sacrificed on Day 9. The weights of the body and the
neoplasm were measured and inhibitory ratios of tumor weight were calculated.
22

4.2.5 Molecular modeling
The protein-ligand complex crystal structure of compound A bound to PI3Kγ was
chosen as the template to compare the docking mode between compound 2f bound to
PI3Kγ and 2g bound to PI3Kγ. The molecular docking procedure was performed by
using C-DOCKER protocol within Discovery Studio 2.5. For enzyme preparation, the
hydrogen atoms were added. The whole PI3Kγ enzyme was defined as a receptor and
the site sphere was selected on the basis of the ligand binding location of compound A.
Compound A was removed and compound 2f or 2g was placed. After end of
molecular docking, ten docking poses was scored and selected based on calculated
C-DOCKER energy.
Acknowledgment: Financial support from National Natural Science Foundation of
China (Grant No. 81272448) is gratefully acknowledged.
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26

Figure and Scheme Legend
Figure 1. The structures of PI3K and mTOR dual inhibitors
Figure 2. optimizing strategy
Figure 3. The anticancer effect of compound 2f in S180 homograft model (A) and the change of
tested mice body weights (B). Mice bearing subcutaneous tumors were orally administered vehicle,
compound 2f (1.0, 3.0 and 10.0 mg/kg doses) once daily for 8 days. **P < 0.01.
Figure 4. Docking mode of compound 2f and 2g with PI3Kγ. Selected residues Val882, Ala885,
Tyr867, Lys833 are shown. Green dashed lines indicate hydrogen bond.
Scheme 1 Reagents and conditions: (a) AcOCHO, rt, ether, overnight; (b) BrCH2CO2Et, NaH,
DMF, rt, 4 h; (c) H3PO4, NaNO2, -10C, 1 h, then CuBr, 40% HBr, rt, 1 h, 40C, 2h; (d) n-PrNH2
or c-PrNH2, 1,4-dioxane, 45C, 4 h; (e) bis(pinacolato)diboron, AcOK, PdCl2(dppf), 1,4-dioxane,
reflux, N2, 3 h; (f) 5 or 6, PdCl2(dppf), K2CO3, 1,4-dioxane/water (5:1), reflux, N2, 2 h.
Scheme 2 Reagents and conditions: (a) CDI, DMF; (b) H2NR1; (c): bis(pinacolato)diboron,
PdCl2(dppf), KOAc, 1,4-dioxane, N2, reflux 3 h; (d) 8, PdCl2(dppf), K2CO3, 1,4-dioxane/water
(5:1), N2, reflux, 2 h.
Scheme 3 Reagents and conditions: (a) CDI, DMF; (b) H2NCH2CH2N(CH2CH2)2O; (c):
bis(pinacolato)diboron, PdCl2(dppf), KOAc, 1,4-dioxane, N2, reflux 3 h; (d) 8, PdCl2(dppf),
K2CO3, 1,4-dioxane/water (5:1), N2, reflux, 2 h.
Table 1 Antiproliferative activities of compounds 1, 2 ( x  s , n = 3)
Table 2 Inhibitory enzymatic activities of compounds ( n = 2)
Table 3. The acute oral toxicity of tested compounds
27

Graphical abstract
28