Combination of 2-methoxy-3-phenylsulfonylaminobenzamide and 2-aminobenzothiazole to discover novel anticancer agents

Article abstract of DOI:10.1016/j.bmc.2014.04.064

The fragment of 2-substituted-3-sulfonylaminobenzamide has been proposed to replace the fragment of 2-substituted-3-sulfonylaminopyridine in PI3K and mTOR dual inhibitors to design novel anticancer agents based on bioisostere. The combination of the fragm

Full text of DOI:10.1016/j.bmc.2014.04.064

Accepted Manuscript
Combination of 2-methoxy-3-phenylsulfonylaminobenzamide and 2-amino-
benzothiazole to discover novel anticancer agents
Huan Li, Xiao-Meng Wang, Juan Wang, Teng Shao, Yi-Ping Li, Qi-Bing Mei,
She-Min Lu, San-Qi Zhang
PII:
S0968-0896(14)00338-1
DOI:
http://dx.doi.org/10.1016/j.bmc.2014.04.064
BMC 11559
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
26 March 2014
29 April 2014
30 April 2014
Please cite this article as: Li, H., Wang, X-M., Wang, J., Shao, T., Li, Y-P., Mei, Q-B., Lu, S-M., Zhang, S-Q.,
Combination of 2-methoxy-3-phenylsulfonylaminobenzamide and 2-aminobenzothiazole to discover novel
anticancer agents, Bioorganic & Medicinal Chemistry (2014), doi: http://dx.doi.org/10.1016/j.bmc.2014.04.064
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Combination of 2-methoxy-3-phenylsulfonylaminobenzamide and
2-aminobenzothiazole to discover novel anticancer agents
Huan Li a,1, Xiao-Meng Wang a,1, Juan Wang b,Teng Shao a, Yi-Ping Li a, Qi-Bing Mei b, She-Min
Lu c, and San-Qi Zhanga *
a Department of Medicinal Chemistry, School of Pharmacy, Health Science Center, Xi′an Jiaotong
University, Xi′an, 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 Medicine, Health Science Center,
Xi′an Jiaotong University, Xi′an, 710061, PR China
1 H. Li and X. M. Wang contributed equally to this work.
* Corresponding author: San-Qi Zhang, Ph. D., Professor,
E-mail address: sqzhang@mail.xjtu.edu.cn (S. Q. Zhang)
Tel: 86-29-82657040; Fax: 86- 29-82657040
Abstract
The fragment of 2-substituted-3-sulfonylaminobenzamide has been proposed to
replace the fragment of 2-substituted-3-sulfonylaminopyridine in PI3K and mTOR
dual inhibitors to design novel anticancer agents based on bioisostere. The
combination of the fragment of 2-substituted-3-sulfonylaminobenzamide with the
fragment of 2-aminobenzothiazole or 2-aminothiazolo[5,4-b]pyridine, or
2-amino[1,2,4]triazolo[1,5-a]pyridine produced the novel structures of anticancer
agents. As a result, nineteen target compounds were synthesized and characterized.
Their antiproliferative activities in vitro were evaluated via MTT assay against four
human cancer cell lines including HCT-116, A549, MCF-7 and U-87 MG. The SAR
of target compounds was preliminarily discussed. Compound 1g with potent
antiproliferative activity was examined for its effect on the AKT and p-AKT473. The
1

anticancer effect of 1g was evaluated in established nude mice HCT-116 xenograft
model. The results suggested that compound 1g can block PI3K/AKT/mTOR pathway
and significantly inhibit tumor growth. These findings strongly support our
assumption that the fragment of benzamide can replace the pyridine ring in some
PI3K and mTOR dual inhibitor to design novel anticancer agents.
Key Words benzamide ∗ benzothiazole ∗ synthesis ∗ anticancer effect∗ bioisostere
1. Introduction
The chemical and biological study of heterocyclic compounds has been an
interesting field for a long time in medicinal chemistry. Benzothiazole consists of
thiazole ring fused with benzene ring and its derivatives exhibit a broad spectrum of
biological activities such as antitumor, antimicrobial, anti-inflammatory,
anticonvulsant and antidiabetic activity. Khokra et al. reviewed the progress on the
synthesis and biological activities of benzothiazole derivatives in 2011.1 Recently, Shi
et al. reported that pyridinyl-2-amine linked benzothiazole-2-thiol compounds
exhibited potent and broad-spectrum inhibitory activities against human tumor cell
lines.2 Caputo et al. reported benzothiazole derivatives linked arylurea moiety at C-2
also showed significant growth inhibitory activities against several human tumor cell
lines.3 Hong et al. discovered benzothiazole derivatives linked methylurea moiety at
C-2 were effective inhibitors against the wild-type and T315I mutant of Bcr-Abl
kinase.4 Noolvi et al. synthesized a series of 2-arylaminobenzothiazole and evaluated
their antitumor effects in vitro.5 Our group reported that 1-(2-acetylamidobenzothiazol
-6-yl)-3-(4-(1-methylpiperazine-4-carbinyl)phenyl)urea displayed potent cytotoxic
activity against A549.6 There are two hydrogen-bond acceptors and one
hydrogen-bond donor in the 2-acetylaminobenzothiazole. Thus it is a desired
fragment in drug design.
Cancer, as a major cause of death in the world, has posed a great challenge to the
fields of medicine and immunology. Chemotherapy has been widely employed for
2

various cancer treatments. The toxicity and resistance of traditional chemotherapeutic
drugs makes it urgent to develop new targets and novel drugs for the cancer therapy.
Kinases are well-known targets for a variety of diseases and disorders.7 PI3K (a
family of lipid kinase) and mTOR (mammalian target of rapamycin) have been found
to play a regulatory role in many cellular processes, including cell growth,
proliferation, differentiation, motility and survival.8 In many kinds of cancers, the
PI3K/AKT/mTOR signal transduction pathway is dysregulated contributing to
cellular transformation and tumor growth.9 Therefore, PI3K and mTOR have become
the targets of intense research for anticancer drug discovery.10, 11 In recent years, a
significant progress has been made on the design, synthesis and evaluation of PI3K
and mTOR dual inhibitors, and thereupon the pharmacophore of the dual inhibitors
has been proposed.12 Among the reported PI3K/mTOR dual inhibitors,
N-(5-quinilin-6-yl)pyridine-3-yl)phenylsulfonamide is a class of important active
compounds. Two ring nitrogen atoms in pyridine and quinoline are the main
components of pharmacophore. GlaxoSmithKline identified GSK2126458,
N-(2-methoxy-5-(4-pyridazin-4-yl)quinilin-6-yl)pyridine-3-yl)-2,4-difluorobenzenesu
lfonamide, as a potent, orally bioavailable inhibitor of PI3Kα and mTOR.13 Amgen
Inc. designed, synthesized and evaluated several classes of N-(2,
5-disubstitutedpyridin-3-yl)phenylsulfonamides. Thus they discovered that N-(2-
chloro-5-(4-morpholinoquinilin-6-yl)pyridin-3-yl)-4-fluorophenylsulfonamide,14 N-
(2-chloro-5-(2-acetylaminobenzo[d]thiazol-6-yl)pyridin-3-yl)-4-fluorophenyl
sulfonamide (compound A, Figure 1),15 N-(2-chloro-5-(2-acetylaminoimidazo
[1,2-b]pyridazin-6-yl)pyridin-3-yl)-4-fluorophenyl sulfonamide16 and AMG 51117 are
excellent PI3Kα/mTOR dual inhibitors or PI3Kα selective inhibitor, and orally
bioavailable anticancer agents as well. The analogue of compound A,
N-(6-(2-methoxy-3-(4-fluorophenylsulfonlyamino)pyridin-5-yl)[1,2,4]triazolo[1,5-a]
pyridin-2-yl)acetamide, also possesses the same pharmacophore as compound A and
displayed anticancer effect.18 Recently, QSAR and pharmacophore of analogues of
compound A were studied.19 BEZ23520 and PF-0497906421 possess the similar
pharmacophore with GSK2126458. As PI3Kα and mTOR dual inhibitors, they are in
3

phase I/II clinical trials for treating solid tumors.
[Figure 1.]
Compound A exhibited very high antitumor effect in vivo. Its EC50 ranges from
0.26 mg/kg to 0.53 mg/kg against three established nude mice human cancer cell
xenograft models. The interaction of compound A with PI3K was clarified by the
co-crystal of compound A with PI3Kγ protein.15 According to the co-crystal structure,
the N-acetylaminobenzothiazole moiety can form two hydrogen bonds with the NH
and the carbonyl group of backbone Val882. A water molecule bridge is formed
among the pyridine nitrogen, the carboxyl in Asp841 and the hydroxyl in Tyr867. The
interaction is outlined in Figure 2. Based on bioisostere, we propose that the water
molecule located among the pyridine ring, the Tyr867 and Asp841 residues can be
replaced with a small group linked to the pyridine ring. This reasoning was based on
the realization that (a) displacing a ‘trapped’ water molecule may sometimes have an
entropic advantage;22 (b) replacing a water molecule may enable additional favorable
direct interactions between ligand and protein. The amide group is the most favorable
because the amide would project its oxygen and hydrogen to the positions similar to
that of water. Thus, the O-H…O hydrogen bond between water and Asp841 and the
O…H-O hydrogen bond between water and Tyr867 can be replaced by the N-H…O
and O…H-O hydrogen bonds formed between the benzamide and the Asp841 and the
Tyr867
residues
directly.
In
other
words,
2-methoxy-3-
phenylsulfonylamino-5-(acetylaminobenzo[d]thiazol-6-yl) benzamide might interact
with PI3K without the aid of water molecule. In this novel scaffold,
2-methoxy-3-phenylsulfonylamino-5-(acetylaminobenzo[d]thiazol-6-yl) benzamide
shares the same pharmacophore (labeled as red in Figure 2) with compound A. In this
way, benzamide fragment can replace pyridine ring fragment in compound A to
design novel structures of PI3K inhibitors and anticancer agents.
[Figure 2.]
In our previous work, we reported the design, synthesis and evaluation of
4

2-methoxy-3-phenylsulfonylamino-5-(quinazolin-6-yl or quinolin-6-yl)benzamide as
novel PI3K inhibitors and anticancer agents.23 In this work, we combined the
fragment of benzamide with benzothiazole or thiazolo[5,4-b]pyridine, or
[1,2,4]triazolo[1,5-a]pyridine into one molecule to design novel anticancer agents
(Figure 3, compounds 1, 2 and 3). Herein, we report our studies on the design and
synthesis of compounds 1, 2 and 3 and demonstrate the biological activities of these
compounds.
[Figure 3.]
2. Results and discussion
2.1. Chemistry
The synthetic route of compounds 1 is outlined in Scheme 1.
[Scheme 1.]
In the presence of TEA or DMAP, commercially available compound 4a or 4b
was acylated with formic acetic anhydride, acetic anhydride, or cyclopropylcarbonyl
chloride respectively, to produce intermediates 5. The reaction of 2-formylamino-
6-bromobenzothiazole (5a) with ethyl bromoacetate afforded compound 6 in the
presence of sodium hydride. Formyl group in intermediate 5a was removed in
work-up. Conversion of 4 to 7 was completed via a two-step process of diazo-reaction
and bromo-substitution. Without further purification, compound 7 was converted to 8
via nucleophilic substitution of 7a or 7b with cyclopropylamine. The intermediates 10,
sulfonamide, were prepared from 5-bromobenzoic acid derivatives according to the
synthetic route reported in our previous work.23 Catalyzed by PdCl2(dppf),
intermediate 10 was reacted with bis(pinacolato)diboron to produce corresponding
arylboronic esters. Without isolation of arylboronic esters, intermediate 5, or 6, or 8,
PdCl2(dppf), water and potassium carbonate as well were added to the above reaction
mixture. The resulted mixture was refluxed to produce the title compounds 1a-1j. The
preparation of arylboronic esters and Suzuki coupling were completed in one pot.
To expand the structural diversity of title compounds, 6-position or 7-position of
5

2-substitutedamino-[1,2,4]triazolo[1,5-a]pyridine was combined with intermediate 10
to produce compounds 2 or 3. The synthetic routes are shown in Scheme 2. The
acylation of commercially available 6-bromo-[1,2,4]triazolo[1,5-a]pyridin-2-amine
(11) or 7-bromo-[1,2,4]triazolo[1,5-a]pyridin-2-amine (13) gave intermediates 12 or
14. Compounds 2 and 3 were synthesized in a similar approach as compounds 1.
[Scheme 2]
2.2. Antiproliferative assays in vitro
We first evaluated the antiproliferative activities of synthesized compounds
against human colon carcinoma cell line (HCT-116) by applying the MTT
colorimetric assay. The PI3K and mTOR dual inhibitors BEZ235 and compound A
were used as the positive controls. For the active compounds, we next evaluated their
antiproliferative activities against human breast adenocarcinoma carcinoma cell line
(MCF-7), lung adenocarcinoma epithelial cell line (A549) and glioma cell line (U-87
MG). The results are summarized in Table 1.
[Table 1]
As expected, some compounds significantly exhibited antiproliferative activities
against HCT-116. Notably, compounds 1g (IC50 = 1.95 µM against HCT-116) and 1h
(IC50 = 4.25 µM against HCT-116) showed approximate 10 times more potencies than
compounds 2b (IC50 = 27.33 µM against HCT-116) and 3b (IC50 = 32.63 µM against
HCT-116). Similarly, compounds 1c (IC50 = 2.01 µM against HCT-116) and 1i (IC50 =
2.50 µM against HCT-116) were more active than compounds 2f (IC50 = 43.5 µM
against HCT-116) and 3c (IC50 > 50 µM against HCT-116). These data indicated that
the benzothiazole fragment was a little better than thiazolo[5,4-b]pyridine fragment
and much better than [1,2,4]triazolo[1,5-a]pyridine fragment in the structures of target
compounds on antiproliferative activity. To further determine whether benzothiazole
ring was a better selection than thiazolo[5,4-b]pyridine ring, we examined the
antiproliferative activities of compounds 1c, 1g, 1i and 1h against the other three
cancer lines MCF-7, A549 and U-87 MG. The results (shown in Table 1) revealed that
the benzothiazole was optimal fragment in these series. The results were consistent
6

with the activity of similar kinase inhibitors.4 As for R1 substituted group, the
4-fluorophenyl was an optimal fragment for this series (compared 1c with 1d) and our
reported compounds.22 The fact that compound 1c (IC50 =2.87 µM against U-87 MG)
was more potent than compounds 1b (IC50 = 17.93 µM against U-87 MG) and 1a
(IC50> 50 µM against U-87 MG) indicated that the compound with a methoxy group
at 2-position (R2) of benzene ring was important to improve the antiproliferative
activity. From the docking study we found that the oxygen atom in the methoxy group
appeared to establish an additional hydrogen bond with the Lys833 backbone. Thus
an additional hydrogen bond would contribute to strengthening inhibitory activity. We
further attempted to explore the SAR of the side chain linked to the 2-position (R3) of
benzothiazole by replacing the acetyl group in 1g at position of R3 with different
groups. Replacement of the acetyl group with cyclopropylcarbonyl group (1g and 1c)
displayed a negative effect on potency. Replacement of the acetyl with the
cyclopropyl or ethoxycarbonylmethyl (1e and 1f) resulted in a complete loss of
activity, which reveals the indispensable nature of the acetyl group at this position for
maintaining activity in this series. From above mentioned results we could reveal a
SAR that the benzamide fragment combined with benzothiazole fragment with aryl at
R1, methoxy at R2 and acetyl at R3 were favorable for antiproliferative activity. In
addition, the IC50 of compound 1g against MCF-7 was comparable to that of positive
drugs compound A and BEZ235. Therefore, compound 1g was further investigated.
2.3. Western blot assay
To determine whether these compounds inhibit the PI3K, we evaluated the
suppressive effects of compound 1g at 0.1, 1.0 and 10.0 µM and positive BEZ235 at
1.0 µM on AKT and p-AKT473 in HCT-116 cells through Western blot. As shown in
Figure 4, the suppressive effects of compound 1g on p-AKT473 were almost equal to
BEZ235 at 1.0 µM. The results suggest compound 1g can block the
PI3K/AKT/mTOR pathway and thus 1g is a potential PI3K inhibitor.
[Figure 4.]
7

2.4. Anticancer effects in established nude mouse HCT-116 xenograft model in
vivo
Compound 1g, displayed evident activities against the four carcinoma cell lines,
was chosen to evaluate its anticancer effect in the nude mouse HCT-116 colon
adenocarcinoma xenograft model. BEZ235 (30 mg/kg) was used as positive drug. 1g
was dosed orally at 10 mg/kg or 30 mg/kg once a day for 14 days. The control group
was administrated orally the solvent only.
The change in the tumor volumes was observed in Figure 5 (A). In this model,
treatment groups with compound 1g at 10 mg/kg or 30 mg/kg significantly inhibit
tumor growth by 40.06% and 46.87%, compared to the control group, respectively.
BEZ235 group displayed a delay in tumor growth by 24.22% with 30 mg/kg. The low
suppression ratio in positive group may be related to the poor solubility of BEZ235.
These results suggest that 1g display significant inhibitory effect on the tumor growth.
[Figure 5.]
During the experimental period, the weight loss of tested animals was observed in
BEZ235 group, indicating that BEZ235 displays some toxicity at the dosage
administration. But compound 1g was well tolerated at the dose of 10 mg/kg and 30
mg/kg in terms of its effect on body weight (Figure 5, B). This is due to the low
toxicity of compound 1g.
2.5. Docking studies
Docking analysis utilizing the C-DOCKER program within Discovery Studio 2.5
software package was performed to further explore the binding mode of the
compound 1g with PI3K protein. Docking simulations were performed on human
PI3Kγ (PDB code 3QKO). 15
[Figure 6.]
The docking result of compound 1g with PI3Kγ (Figure 6) has the following three
indications: (a) 2-acetylaminobenzothiazole fragment can form two hydrogen bonds
with Val882. (b) The oxygen atom of benzamide can form two hydrogen bonds, one
with Tyr867 and the other with Asp964. The hydrogen atom of benzamide can form a
8

hydrogen bond with Asp841. These interactions revealed that benzamide can replace
the pyridine ring in the structure of compound A. (c) The oxygen atoms of sulfamide
and the methoxy group at 2-position of benzamide can form two hydrogen bonds with
Lys833. These interactions suggested that compound 1g displays rational interaction
with PI3Kγ, and the interaction mode of compound 1g is similar with that of
compound A. As for compound 1b or 1a, the chloride or hydrogen atom at 2-position
of benzamide cannot form hydrogen bond with Lys833, which suggests that the
methoxy group is a more suitable substitute group than chloride and hydrogen in the
target compound 1.
3. Conclusion
In the present study, we proposed that the combination of 2-substituted-3-
sulfonylaminobenzamide with benzothiazole, or thiazolo[5,4-b]pyridine, or
[1,2,4]triazolo[1,5-a]pyridine could produce the novel structures of anticancer agents.
Nineteen compounds were synthesized and characterized. The results of
antiproliferative activities indicated that compound 1g significantly exhibits inhibitory
activity against the four cancer cell lines. The SAR of these compounds indicated that
2-methoxy-3-(4-fluorophenylsulfonylamino) benzamide and 2-acetylamino-5-
benzothiazol-6-yl are optimal fragments. The Western blot assay results of compound
1g suggested that these compounds can block the PI3K/AKT/mTOR pathway.
Compound 1g significantly displayed inhibitory effect on tumor growth in established
nude mice HCT-116 xenograft model. These findings strongly support our
assumption that the fragment of benzamide can replace the pyridine ring in compound
A to design novel anticancer agents.
4. Experimental
4.1. Chemistry
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
9

were determined on a Beijing micro melting-point apparatus and thermometer was
uncorrected. 1H-NMR spectra were recorded on a 400 MHz 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. N-(6-bromobenzo[d]thiazol-2-yl)formamide (5a)
The mixture of acetic anhydride (7.0 g, 69 mmol) and formic acid (85%, 2.5 g,
54 mmol) was heated at 60 °C for 2 h, cooled. 6-Bromobenzothiazole-2-amine (2.0 g,
8.7 mmol) was added portion wise to the resulting anhydride while keeping the
temperature below 40 °C. Then ether (15 ml) was added and the mixture was stirred at
room temperature overnight. The precipitated crystals was collected by filtration and
1
washed with ether to give 5a (1.7 g, 77%) as white solid. Mp: 235.0-236.0 °C; H
NMR (DMSO-d6): δ 12.60 (s, 1H, NH), 8.61 (s, 1H, COH), 8.28 (s, 1H, Ar-H), 7.71
(d, J = 8.4 Hz, 1H, Ar-H), 7.59 (d, J = 8.4 Hz, 1H, Ar-H). ESI-HRMS m/z: calcd for
C8H5BrN2NaOS [M+Na]+: 278.9204; found 278.9203.
4.1.2. N-(6-bromobenzo[d]thiazol-2-yl)acetamide (5b)
6-Bromobenzo[d]thiazol-2-amine (2.3 g, 10.0 mmol) was suspended in
dichloromethane (30 ml), and then 4-dimethylaminopyridine (1.3 g, 11.5 mmol) was
added. The mixture was cooled in an ice-water bath under nitrogen, and acetic
anhydride (1.0 ml, 11.5 mmol) was added. The resulting mixture was allowed to
slowly warm to room temperature while being stirred overnight under nitrogen. Then
hydrochloric acid (10%, 10 ml) was added and 5b (2.3 g, 85%) was obtained by
1
filtration as white solid. Mp: 217.0-218.0 °C; H NMR (DMSO-d6): δ 12.46 (s, 1H,
NH), 8.24 (s, 1H, Ar-H), 7.67 (d, J = 7.2 Hz, 1H, Ar-H), 7.57 (d, J = 6.8 Hz, 1H,
Ar-H), 2.21 (s, 3H, CH3). ESI-HRMS m/z: calcd for C9H7BrN2NaOS [M+Na]+:
292.9360; found 292.9360.
10

4.1.3. N-(6-bromobenzo[d]thiazol-2-yl)cyclopropanecarboxamide (5c)
6-Bromobenzothiazol-2-amine (0.6 g, 2.6 mmol) was suspended in
dichloromethane (15 ml), and then triethylamine (6 ml) was added. The mixture was
cooled in an ice-water bath, and cyclopropanecarbonyl chloride (0.29 ml, 3.2 mmol)
was added dropwise. The mixture was allowed to slowly warm to room temperature,
and stirred at room temperature for 1 h. Then the mixture was heated to reflux for 5 h
under nitrogen, cooled to room temperature and the solvent was evaporated off in
vacuo. The residue was extracted with dichloromethane (100 ml × 1, 50 ml × 2), and
then the organic layer was collected, washed with 2N HCl and saturated salt water,
then dried over anhydrous Na2SO4 and filtered. The solvent was evaporated off to
1
give 5c (0.5 g, 64%) as white solid. Mp: 235.0-236.0 °C; H NMR (DMSO-d6): δ
12.75 (s, 1H, NH), 8.24 (d, J = 2.0 Hz, 1H, Ar-H), 7.67( d, J = 8.4 Hz, 1H, Ar-H),
7.57 (dd, J = 2.0, 8.4 Hz, 1H, Ar-H), 2.00 (m, 1H, CH), 0.96 (m, 4H, 2CH2).
ESI-HRMS m/z: calcd for C11H9BrN2NaOS [M+Na]+: 320.1607; found 320.1608.
4.1.4. N-(6-bromothiazolo[5,4-b]pyridin-2-yl)acetamide (5d)
In a similar manner as for 5b, started with 4b (0.25 g, 1.09 mmol) to produce 5d
1
(0.23 g, 80%). Mp: 240.0-242.0 °C; H NMR (DMSO-d6): δ 12.62 (s, 1H, NH), 8.16
(d, J = 8.8 Hz, 1H, Ar-H), 7.57 (d, J = 8.8 Hz, 1H, Ar-H), 2.24 (s, 3H, CH3).
ESI-HRMS m/z: calcd for C8H6BrN3NaOS [M+Na]+: 293.9313; found 293.9315.
4.1.5. N-(5-bromothiazolo[5,4-b]pyridin-2-yl)cyclopropanecarboxamide (5e)
In a similar manner as for 5c, started with 4b (0.30 g, 1.62 mmol) to produce 5e
(0.35 g, 72%). Mp: >270 °C; 1H NMR (DMSO-d6): δ 12.94 (s, 1H, NH), 8.16 (d, J =
8.8 Hz, 1H, Ar-H), 7.57 (d, J = 8.8 Hz, 1H, Ar-H), 2.03 (m, 1H, CH), 0.99 (m, 4H,
CH2). ESI-HRMS m/z: calcd for C10H8BrN3OS [M+H]+: 297.9650; found 297.9659.
4.1.6. Ethyl 2-(6-bromobenzo[d]thiazol-2-ylamino)acetate (6)
To the suspension of NaH (60%, 0.16 g) in DMF (20 ml) was added 5a (1.7 g，6.6
mmol) in small portions. After stirring for 30 min, the solution of ethyl bromoacetate
11

(0.81 ml, 7.3 mmol) in DMF (4 ml) was added and the resulting mixture was stirred at
room temperature for 4 h, poured into 50 ml of water. Compound 6 (1.5 g, 74%) was
1
obtained by filtration as white solid. Mp: 210.0-211.0 °C; H NMR (DMSO-d6): δ
8.86 (s, 1H, NH), 8.33 (s, 1H, Ar-H), 7.73 (d, J = 8.8 Hz, 1H, Ar-H), 7.60 (d, J = 8.8
Hz, 1H, Ar-H), 4.98 (s, 2H, CH2), 4.18 (q, J = 7.2 Hz, 2H, OCH2), 1.20 (t, J = 7.2 Hz,
3H, CH3). ESI-HRMS m/z: calcd for C11H12BrN2O2S [M+H]+: 314.9803; found
314.9804.
4.1.7. 6-Bromo-N-cyclopropylbenzo[d]thiazol-2-amine (8a)
Compound 4a (0.8 g, 3.5 mmol) was mixed under intensive stirring with 85%
phosphoric acid (8 ml) at 50 °C. The solution was cooled to -20 °C and a solution of
sodium nitrite (0.29 g, 4.2 mmol) in water (2 ml) was added slowly to keep the
temperature below -10 °C. After stirring for 1 h, the resulting diazonium salt was
poured into a solution of active copper (I) bromide (0.65 g, 4.5 mmol) in 48% HBr (5
ml). The mixture was intensively stirred for 1 h at room temperature, at 40 °C for 2 h
and then at room temperature overnight, poured into ice-water (100 ml) then extracted
with ethyl acetate (100 ml × 1, 50 ml × 2). The organic layer was collected and
washed with saturated Na2CO3, saturated salt water, then dried over anhydrous
Na2SO4 and filtered. Then ethyl acetate was evaporated off to give 7a as brown crude
product which was used for the next step directly.
Compound 7a (1.0 g, 3.4 mmol) was suspended in 1,4-dioxane (10 ml), and
cyclopropylamine (3.6 ml, 51.0 mmol) was added. The resulting mixture was heated
to reflux under nitrogen for 6 h, cooled to room temperature. The solvent was
evaporated off to give crude product which was purified through column
1
chromatograph to give 8a (0.44 g, 48%) as off-white solid. Mp: 183.0-184.0 °C; H
NMR (DMSO-d6): δ 8.49 (s, 1H, NH), 7.97 (d, J = 2.0 Hz, 1H, Ar-H), 7.37 (d, J = 8.8
Hz, 1H, Ar-H), 7.33 (m, 1H, Ar-H), 2.68 (m, 1H, CH), 0.77 (m, 2H, CH2), 0.57 (m,
2H, CH2). ESI-HRMS m/z: calcd for C10H10BrN2S [M+H]+: 268.9748; found
268.9749.
12

4.1.8. 5-Bromo-N-cyclopropylthiazolo[5,4-b]pyridin-2-amine (8b)
In a similar manner as for compound 8a, started with compound 4b to produce
compound 7b as crude product which was used for the next step directly, then
following the procedure to prepare 8a and using compound 7b (0.5 g, 2.0 mmol) to
produce 8b (0.35 g, 77%). Mp: 163.0-165.0 °C; 1H NMR (DMSO-d6): δ 8.78 (s, 1H,
NH), 8.16 (d, J = 8.8 Hz, 1H, Ar-H), 7.57 (d, J = 8.8 Hz, 1H, Ar-H), 2.78 (m, 1H, CH),
0.82 (m, 2H, CH2), 0.62 (m, 2H, CH2). ESI-HRMS m/z: calcd for C9H9BrN3S
[M+H]+: 269.9701; found 269.9702.
4.1.9. General procedure for the synthesis of 10a-10f
The compounds were prepared according to previously described methods. 23
4.1.9.1. 3-Bromo-5-(4-fluorophenylsulfonylamino)benzamide (10a)
White solid; yield: 83%; Mp: 238.0-240.0 °C; 1H NMR (DMSO-d6): δ 10.77 (1H,
s, SO2NH), 8.06 (1H, s, Ar-H), 7.84 (2H, dd, J = 8.8, 5.2 Hz, Ar-H), 7.74 (1H, s,
CONH2), 7.56 (1H, s, CONH2), 7.60 (1H, s, Ar-H), 7.44 (2H, t, J = 8.8 Hz, Ar-H),
7.38 (1H, s, Ar-H). ESI-HRMS m/z: calc’d for C13H11BrFN2O3S [M+H]+: 372.9658;
found 372.9655.
4.1.9.2. 5-Bromo-2-chloro-3-(4-fluorophenylsulfonylamino)benzamide (10b)
White solid; yield: 75%; Mp: 228.0-229.0 °C; 1H NMR (DMSO-d6): δ 10.47 (1H,
s, SO2NH), 7.96 (1H, s, CONH2), 7.82 (2H, dd, J = 5.2, 8.8 Hz, Ar-H), 7.45 (2H, t, J
= 8.8 Hz, Ar-H), 7.70 (1H, s, CONH2), 7.47 (1H, s, Ar-H), 7.41 (1H, d, J = 2.0 Hz,
Ar-H). ESI-HRMS m/z: calc’d for C13H10BrClFN2O3S [M+H]+: 406.9268; found
406.9264.
4.1.9.3. 5-Bromo-3-(cyclopropanesulfonylamino)-2-methoxybenzamide (10c)
White solid; yield: 57%; Mp: 175.5-177.0 °C; 1H NMR (DMSO-d6): δ 9.55 (1H,
s, SO2NH), 7.85 (1H, s, CONH2), 7.69 (1H, s, CONH2), 7.64 (1H, d, J = 2.4 Hz,
Ar-H), 7.39 (1H, d, J = 2.4 Hz, Ar-H), 3.77 (3H, s, OCH3), 2.82 (1H, m, CH), 0.99
13

(4H, m, 2CH2). ESI-HRMS m/z: calc’d for C11H14BrN2O4S [M+H]+: 348.9858; found
348.9860.
4.1.9.4. 5-Bromo-3-(4-fluorophenylsulfonamido)-2-methoxybenzamide (10d)
White solid; yield: 85%; Mp: 186.0-188.0 °C; 1H NMR (DMSO-d6): δ 7.88 (2H,
dd, J = 5.2, 8.8 Hz, Ar-H), 7.82 (1H, s, Ar-H), 7.75 (1H, s, SO2NH), 7.19 (3H, t, J =
8.4 Hz, Ar-H), 6.94 (1H, s, NH), 3.51 (3H, s, OCH3), 3.07 (3H, s, NCH3). ESI-HRMS
m/z: calc’d for C14H13BrFN2O4S [M+H]+: 402.9763; found 402.9760.
4.1.9.5. 5-Bromo-3-(4-chlorophenylsulfonylamido)-2-methoxybenzamide (10e)
White solid; yield: 84%; Mp: 177.5-178.5 °C;1H NMR (DMSO-d6): δ 10.26 (1H,
s, SO2NH), 7.82 (2H, d, J = 8.8 Hz, Ar-H), 3.45 (3H, s, OCH3), 7.81 (1H, s, CONH2),
7.70 (2H, d, J = 8.8 Hz, Ar-H), 7.64 (1H, s, CONH2), 7.48 (1H, d, J = 2.4 Hz, Ar-H),
7.34 (1H, d, J = 2.4 Hz, Ar-H). ESI-HRMS m/z: calc’d for C14H13BrClN2O4S [M+H]+:
418.9468; found 418.9466.
4.1.9.6. 5-Bromo-2-methoxy-3-(4-methylphenylsulfonylamino)benzamide (10f)
White solid; yield: 87%; Mp: 169.0-170.5 °C;1H NMR (DMSO-d6): δ 10.11 (1H,
s, SO2NH), 7.81 (1H, s, CONH2), 7.72 (2H, d, J = 8.0 Hz, Ar-H), 7.64 (1H, s,
CONH2), 7.50 (1H, d, J = 2.4 Hz, Ar-H), 7.41 (2H, d, J = 8.4 Hz, Ar-H), 7.29 (1H, d,
J = 2.4 Hz, Ar-H), 3.44 (3H, s, OCH3), 2.36 (3H, s, Ar-CH3). ESI-HRMS m/z: calc’d
for C15H16BrN2O4S [M+H]+: 399.0014; found 399.0010.
4.1.10. General procedure for the preparation of 12a-12b and 14a-14b
The compounds were prepared according to previously described methods.18
4.1.10.1. N-(6-bromo-[1,2,4]triazolo[1,5-a]pyridin-2-yl)acetamide (12a)
White solid; yield: 85%. Mp: 222.0-225.0 °C; 1H NMR (DMSO-d6): δ 10.87 (s,
1H, NH), 9.29 (s, 1H, Ar-H), 7.78 (d, J = 9.2 Hz, 1H, Ar-H), 7.78 (d, J = 9.2 Hz, 1H,
Ar-H), 2.13 (s, 3H, CH3). ESI-HRMS m/z: calc’d for C8H7BrN4NaO [M + Na]+:
14

276.9701; found 276.9699.
4.1.10.2. N-(6-bromo-[1,2,4]triazolo[1,5-a]pyridin-2-yl)cyclopropanecarboxamide
(12b)
White solid; yield: 77%; Mp: 216.0-219.0 °C; 1H NMR (DMSO-d6): δ 11.14 (s,
1H, NH), 9.28 (s, 1H, Ar-H), 7.78 (d, J = 9.6 Hz, 1H, Ar-H), 7.66 (d, J = 9.2Hz, 1H,
Ar-H), 2.03 (m, 1H, CH), 0.83 (d, J = 5.6 Hz, 4H, 2CH2). ESI-HRMS m/z: calc’d for
C10H9BrN4NaO [M + Na]+: 302.9857; found 302.9857.
4.1.10.3. N-(7-bromo-[1,2,4]triazolo[1,5-a]pyridin-2-yl)acetamide (14a)
White solid; yield: 62%; Mp: 228.0-230.0 °C; 1H NMR (DMSO-d6): δ 10.87 (s,
1H, NH), 8.82 (d, J = 5.6 Hz, 1H, Ar-H), 8.05 (s, 1H, Ar-H), 7.30 (d, J = 5.6 Hz, 1H,
Ar-H), 2.14 (s, 3H, CH3). ESI-HRMS m/z: calc’d for C8H7BrN4NaO [M + Na]+:
276.9701; found 276.9699.
4.1.10.4. N-(7-bromo-[1,2,4]triazolo[1,5-a]pyridin-2-yl)cyclopropanecarboxamide
(14b)
1
White solid; yield: 70%; Mp: 212.0-214.0 °C; H NMR (DMSO-d6): δ 11.12 (s,
1H, NH), 8.85 (d, J = 5.6 Hz, 1H, Ar-H), 8.05 (s, 1H, Ar-H), 7.35 (d, J = 5.6 Hz, 1H,
Ar-H), 2.03 (m, 1H, CH), 0.83 (d, J= 5.6 Hz, 4H, CH2). ESI-HRMS m/z: calc’d for
C10H9BrN4NaO [M + Na]+: 302.9857; found 302.9857.
4.1.11. General procedure for the preparation of 1a-1j, 2a-2e and 3a-3c.
To a round bottomed flask were added compound 10 (0.45 mmol),
bis(pinacolato)diboron (0.54 mmol), potassium acetate (1.35 mmol), PdCl2(dppf)
(0.03 mmol) and 1,2-dimethoxyethane (6 ml), then the mixture was heated to reflux
for 4 h under nitrogen atmosphere, cooled to room temperature. Then compound
5a-5e, or 6, or 8a-8b, or 12a-12b, or 14a-14b (0.45 mmol), potassium carbonate
(1.35 mmol), PdCl2(dppf) (0.03 mmol) and water (1 ml) were added. The resulting
mixture was refluxed for another 1-3 h under nitrogen atmosphere. The solvent was
15

evaporated off under vacuum and the residue was purified through column
chromatography (CHCl3: MeOH = 30:1, v/v) to produce 1a-1j, 2a-2f and 3a-3c as
white solid.
4.1.11.1. 3-(2-(Cyclopropanecarboxamido)benzo[d]thiazol-6-yl)-5-(4-
fluorophenylsulfonylamino)benzamide (1a)
Yield: 78%; Mp: >270 °C; 1H NMR (DMSO-d6): δ 12.77 (1H, s, CONH), 10.61
(1H, s, SO2NH), 8.23 (1H, s, Ar-H), 8.12 (1H, s, Ar-H), 7.92 (1H, s, CONH2), 7.87
(2H, dd, J = 8.4, 4.8 Hz, Ar-H), 7.84 (1H, d , J = 8.4 Hz, Ar-H), 7.64 (1H, d, J = 8.4
Hz, Ar-H), 7.57 (1H, s, CONH2), 7.56 (1H, s, Ar-H), 7.49 (1H, s, Ar-H), 7.42 (2H, t, J
= 8.6 Hz, Ar-H), 2.02 (1H, m, CH), 0.98 (4H, m, 2CH2). ESI-HRMS m/z: calcd for
C24H19FN4NaO4S2 [M+Na]+: 533.0729; found 533.0737.
4.1.11.2. 2-Chloro-5-(2-(cyclopropanecarboxamido)benzo[d]thiazol-6-yl)-3-
(4-fluorophenylsulfonylamino)benzamide (1b)
Yield: 73%; Mp: >270 °C; 1H NMR (DMSO-d6): δ 12.79 (1H, s, CONH), 10.35
(1H, s, SO2NH), 8.24 (1H, s, Ar-H), 7.97 (1H, s, CONH2 ), 7.85 (2H, dd, J = 8.4, 5.6
Hz, Ar-H), 7.82 (1H, d , J = 9.2 Hz, Ar-H), 7.67 (1H, s, CONH2), 7.63 (1H, d, J =
10.0 Hz, Ar-H), 7.61 (1H, s, Ar-H), 7.56 (1H, s, Ar-H), 7.45 (2H, t, J = 8.6 Hz), 2.02
(1H, m, CH), 0.98 (4H, m, 2CH2). ESI-HRMS m/z: calcd for C24H18ClFN4NaO4S2
[M+Na]+: 567.034; found 567.0343.
4.1.11.3.
5-(2-(Cyclopropanecarboxamido)benzo[d]thiazol-6-yl)-3-(4-fluoro
phenylsulfonylamino)-2-methoxybenzamide (1c)
Yield: 68%; Mp: 168.0-170.0 °C; 1H NMR (DMSO-d6): δ 12.74 (1H, s, CONH),
10.04 (1H, s, SO2NH), 8.18 (1H, s, Ar-H), 7.92 (2H, dd, J = 8.4, 5.6 Hz, Ar-H ), 7.81
(1H, d, J = 8.4 Hz, Ar-H), 7.80 (1H, s, CONH2), 7.65 (1H, s, CONH2), 7.60 (1H, d , J
= 3.2 Hz, Ar-H), 7.58 (1H, d, J = 9.6 Hz, Ar-H), 7.56 (1H, s, Ar-H), 7.45 (2H, t, J =
8.8 Hz, Ar-H), 3.49 (3H, s, OCH3), 2.02 (1H, s, COCH), 0.98 (4H, m, CH2).
ESI-HRMS m/z: calc’d for C25H21FN4NaO5S2 [M+Na]+: 563.0835; found 563.0847.
16

4.1.11.4.
5-(2-(Cyclopropanecarboxamido)benzo[d]thiazol-6-yl)-3-(cyclo
propanesulfonamido)-2-methoxybenzamide (1d)
Yield: 50%; Mp: 209.5-211.0 °C; 1H NMR (DMSO-d6): δ 12.74 (1H, s, CONH),
9.40 (1H, s, SO2NH), 8.27 (1H, s, Ar-H), 7.87 (1H, s, CONH2), 7.83 (1H, s, Ar-H),
7.80 (1H, d , J = 4.8 Hz, Ar-H), 7.69 (1H, s, Ar-H), 7.67 (1H, s, CONH2), 7.62 (1H, s,
Ar-H), 3.84 (3H, s, OCH3), 2.85 (1H, m, SO2CH), 2.02 (1H, s, COCH), 0.98 (8H, m,
CH2). ESI-HRMS m/z: calcd for C22H22N4NaO5S2 [M+Na]+: 509.0929; found
509.0939.
4.1.11.5.
5-(2-(Cyclopropylamino)benzo[d]thiazol-6-yl)-3-(4-fluorophenyl
sulfonylamino)-2-methoxybenzamide (1e)
1
Yield: 41%; Mp: 173.0-174.0 °C; H NMR (DMSO-d6): δ 10.01 (s, 1H, NH),
8.48 (s, 1H, NH), 7.94 (s, 1H, Ar-H), 7.9.-7.92 (m, 2H, Ar-H), 7.79 (s, 1H, Ar-H),
7.59 (s, 2H, Ar-H), 7.49 (s, 1H, CONH2), 7.47 (s, 1H, CONH2), 7.45 (s, 1H, Ar-H),
7.44 (d, J = 8.8 Hz, 1H, Ar-H), 7.38 (d, J = 8.4 Hz, 1H, Ar-H), 3.47 (s, 3H, OCH3),
2.72 (m, 1H, CH), 0.79 (m, 2H, CH2), 0.59 (m, 2H, CH2). ESI-HRMS m/z: calcd for
C24H22FN4O4S2 [M+H]+: 513.1066; found 513.1082.
4.1.11.6.
Ethyl-2-(6-(3-carbamoyl-5-(4-fluorophenylsulfonylamino)-4-methoxy
phenyl)benzo[d]thiazol-2-ylamino)acetate (1f)
1
Yield: 22%; Mp: 203.0-204.0 °C; H NMR (DMSO-d6): δ 10.01 (s, 1H, NH),
8.55 (t, J = 5.6 Hz, 1H, NH), 7.90 - 7.92 (m, 2H, Ar-H), 7.89(s, 1H, Ar-H), 7.79 (s, 1H,
Ar-H), 7.59 (m, 2H, Ar-H), 7.48 (s, 1H, CONH2), 7.47 (s, 1H, Ar-H), 7.45 (s, 1H,
Ar-H), 7.43 (d, J = 8.8 Hz, 1H, Ar-H), 7.39 (d, J = 7.6 Hz, 1H, Ar-H), 4.21 (d, J = 5.6
Hz, 2H, CH2), 4.15 (q, J = 7.2 Hz, 2H, OCH2), 3.46 (s, 3H, OCH3), 1.22 (t, J = 7.2 Hz,
3H, CH3). ESI-HRMS m/z: calcd for C25H24FN4O6S2 [M+H]+: 559.1121; found
559.1133.
4.1.11.7. 5-(2-Acetamidobenzo[d]thiazol-6-yl)-3-(4-fluorophenylsulfonylamino)-
17

2-methoxybenzamide (1g)
Yield: 31%; Mp: 191.0-192.0 °C; 1H NMR (DMSO-d6): δ 12.44 (s, 1H, CONH),
10.05 (s, 1H, SO2NH), 8.18 (s, 1H, Ar-H), 7.93 (dd, 2H, J = 8.4, 5.2 Hz, Ar-H), 7.81
(d, 1H, J=8.0Hz, Ar-H), 7.80 (s, 1H, Ar-H), 7.65 (s, 1H, CONH2), 7.60 (d, 1H, J = 7.2
Hz, Ar-H), 7.58 (s, 1H, Ar-H), 7.55 (s, 1H, CONH2), 7.45 (t, 2H, J = 8.8 Hz, Ar-H),
3.48 (s, 3H, OCH3), 2.22 (s, 3H, COCH3); HRMS m/z: calcd for C23H19FN4NaO5S2
[M+Na]+: 537.0679; found 537.0691.
4.1.11.8.
5-(2-Acetamidothiazolo[5,4-b]pyridin-5-yl)-3-(4-fluorophenylsulfonyl
amino)-2-methoxybenzamide (1h)
Yield: 26%; Mp: >270 °C; 1H NMR (DMSO-d6): δ 12.56 (s, 1H, NH), 10.05 (s,
1H, NH), 8.16 (d, J = 8.4 Hz, 1H, Ar-H), 8.09 (s, 1H, Ar-H), 7.96 (s, 1H, Ar-H), 7.94
(d, J = 8.4 Hz, 1H, Ar-H), 7.88-7.92 (m, 2H, Ar-H), 7.84 (s, 1H, CONH2), 7.63 (s, 1H,
CONH2), 7.42-7.46 (m, 2H, Ar-H), 3.52 (s, 3H, OCH3), 2.25 (s, 3H, CH3).
ESI-HRMS m/z: calcd for C22H18FN5NaO5S2 [M+Na]+: 538.0631; found 538.0638.
4.1.11.9.
5-(2-(Cyclopropanecarboxamido)thiazolo[5,4-b]pyridin-5-yl)-3-(4-
fluorophenylsulfonylamino)-2-methoxybenzamide (1i)
1
Yield: 33%; Mp: 234.0-235.0 °C; H NMR (DMSO-d6): δ 12.87 (s, 1H, NH),
10.04 (s, 1H, NH), 8.16 (d, J = 8.4 Hz, 1H, Ar-H), 8.08 (s, 1H, Ar-H), 7.97 (s, 1H,
Ar-H), 7.95 (d, J = 8.4 Hz, 1H, Ar-H), 7.88-7.92 (m, 2H, Ar-H), 7.84 (s, 1H, CONH2),
7.63 (s, 1H, CONH2), 7.42-7.46 (m, 2H, Ar-H), 3.52 (s, 3H, OCH3), 2.04 (m, 1H, CH),
1.00 (m, 4H, CH2). ESI-HRMS m/z: calcd for C24H21FN5O5S2 [M+H]+: 542.0968;
found 542.0978.
4.1.11.10.
5-(2-(Cyclopropylamino)thiazolo[5,4-b]pyridin-5-yl)-3-(4-fluoro
phenylsulfonylamino)-2-methoxybenzamide (1j)
Yield: 42%; Mp: 155.0-156.0 °C; 1H NMR (DMSO-d6): δ 9.72 (s, 1H, NH), 8.74
(s, 1H, NH), 8.00 (s, 1H, Ar-H), 7.88-7.91 (m, 2H, Ar-H), 7.87 (s, 1H, Ar-H), 7.80 (s,
1H, CONH2), 7.77 (d, J = 8.4 Hz, 1H, Ar-H), 7.72 (d, J = 8.4 Hz, 1H, Ar-H), 7.59 (s,
18

1H, CONH2), 7.41-7.45 (m, 2H, Ar-H), 3.51 (s, 3H, OCH3), 2.78 (m, 1H, CH), 0.82
(m, 2H, CH2), 0.62 (m, 2H, CH2). ESI-HRMS m/z: calcd for C23H21FN5O4S2 [M+H]+:
514.1019; found 514.1027.
4.1.11.11.
5-(2-Acetamido-[1,2,4]triazolo[1,5-a]pyridin-6-yl)-3-(cyclopropane
sulfonylamino)-2-methoxybenzamide (2a)
1
Yield: 50%; Mp: 257.0-259.0 °C; H NMR (DMSO-d6): δ 10.84 (s, 1H, NH),
9.41 (s, 1H, NH), 9.20 (s, 1H, Ar-H), 7.92 (d, J = 9.2 Hz, 1H, Ar-H), 7.86 (s, 1H,
Ar-H), 7.78 (m, 3H, Ar-H), 7.66 (s, 2H, NH2), 3.85 (s, 3H, OCH3), 2.51 (m, 1H, CH),
2.16 (s, 3H, CH3), 1.00 (d, 4H, J = 6.6 Hz, CH2). ESI-HRMS m/z: calc’d for
C23H22N6O5S [M+H]+: 495.5309; found 495.5312.
4.1.11.12.
5-(2-Acetamido-[1,2,4]triazolo[1,5-a]pyridin-6-yl)-2-methoxy-3-(4-
methylphenylsulfonylamino)benzamide (2b)
1
Yield: 46%; Mp: 224.0-226.0 °C; H NMR (DMSO-d6): δ 10.85 (s, 1H, NH),
9.92 (s, 1H, NH), 9.05 (s, 1H, Ar-H), 7.76 (m, 5H, Ar-H), 7.68 (s, 1H, Ar-H), 7.58 (s,
2H, NH2), 7.39 (d, J = 7.6 Hz, 2H, Ar-H), 3.47 (s, 3H, OCH3), 2.36 (s, 3H, CH3), 2.16
(s, 3H, CH3). ESI-HRMS m/z: calc’d for C23H22N6O5S [M+H]+: 495.5309; found
495.5312.
4.1.11.13.
5-(2-Acetamido-[1,2,4]triazolo[1,5-a]pyridin-6-yl)-3-(4-chlorophenyl
sulfonylamino)-2-methoxybenzamide (2c)
1
Yield: 35%; Mp: 248.0-251.0 °C; H NMR (DMSO-d6): δ 10.85 (s, 1H, NH),
10.12 (s, 1H, NH), 9.13 (s, 1H, Ar-H), 7.82 (m, 5H, Ar-H), 7.72 (s, 1H, Ar-H), 7.67 (d,
J = 8.0 Hz, 2H, Ar-H), 7.62 (s, 2H, NH2), 3.46 (s, 3H, OCH3), 2.16 (s, 3H, CH3).
ESI-HRMS m/z: calc’d for C22H20ClN6O5S [M+H]+: 515.0904; found 515.0902.
4.1.11.14.
5-(2-Acetamido-[1,2,4]triazolo[1,5-a]pyridin-6-yl)-3-(4-fluorophenyl
sulfonylamino)-2-methoxybenzamide (2d)
1
Yield: 49%; Mp: 252.0-254.0 °C; H NMR (DMSO-d6): δ 10.85 (s, 1H, NH),
19

10.06 (s, 1H, NH), 9.12 (s, 1H, Ar-H), 7.92 (m, 2H, Ar-H), 8.03 (s, 1H, Ar-H), 7.80
(m, 3H, Ar-H), 7.73 (s, 1H, Ar-H), 7.61 (s, 2H, NH2), 7.33 (m, 2H, Ar-H), 3.46 (s, 3H,
OCH3), 2.16 (s, 3H, CH3). ESI-HRMS m/z: calc’d for C22H20FN6O5S [M+H]+:
499.1200; found 499.1202.
4.1.11.15.
5-(2-Acetamido-[1,2,4]triazolo[1,5-a]pyridin-6-yl)-2-chloro-3-(4-fluoro
phenylsulfonylamino)benzamide (2e)
1
Yield: 35%; Mp: 264.0-266.0 °C; H NMR (DMSO-d6): δ 10.88 (s, 1H, NH),
10.38 (s, 1H, NH), 9.24 (s, 1H, Ar-H), 7.93 (s, 1H, Ar-H), 7.84 (m, 4H, Ar-H, NH2),
7.72 (d, J = 7.6 Hz, 2H, Ar-H), 7.67 (s, 1H, Ar-H), 7.43 (m, 2H, Ar-H), 2.17 (s, 3H,
CH3). ESI-HRMS m/z: calc’d for C21H17ClFN6O4S [M+H]+: 503.0705; found
503.0701.
4.1.11.16.
5-(2-(Cyclopropanecarboxamido)-[1,2,4]triazolo[1,5-a]pyridin-6-yl)-3-
(4-fluorophenylsulfonylamino)-2-methoxybenzamide (2f)
1
Yield: 48%; Mp: 225.0-228.0 °C; H-NMR (DMSO-d6): δ 11.12 (s, 1H, NH),
10.06 (s, 1H, NH), 9.10 (s, 1H, Ar-H), 7.93 (m, 2H, Ar-H), 7.81 (m, 3H, 2Ar-H,
CONH), 7.73 (s, 1H, Ar-H), 7.62 (m, 2H, CONH, Ar-H), 7.44 (t, J = 8.8 Hz, 2H,
Ar-H), 3.47 (s, 3H, OCH3), 2.07 (s, 1H, CH), 0.85 (d, J = 6.0 Hz, 4H, 2CH2).
ESI-HRMS m/z: calc’d for C24H22FN6O5S [M+H]+: 525.1356; found 525.1353.
4.1.11.17.
5-(2-Amino-[1,2,4]triazolo[1,5-a]pyridin-7-yl)-3-(4-fluorophenyl
sulfonylamino)-2-methoxybenzamide (3a)
1
Yield: 38%; Mp: 210.0-213.0 °C; H NMR (DMSO-d6): δ 10.08 (s, 1H, NH),
8.60 (d, J = 6.8 Hz, 1H, Ar-H), 7.92 (m, 2H, Ar-H), 7.85 (s, 1H, CONH), 7.70 (s, 1H,
Ar-H), 7.62 (s, 2H, CONH2, Ar-H), 7.46 (m, 3H, Ar-H), 7.07 (d, J = 6.8 Hz, 1H, Ar-H),
6.10 (s, 2H, NH2). ESI-HRMS m/z: calc’d for C22H20FN6O5S [M+H]+: 499.1200;
found 499.1201.
20

4.1.11.18.
5-(2-Acetamido-[1,2,4]triazolo[1,5-a]pyridin-7-yl)-3-(4-fluorophenyl
sulfonylamino)-2-methoxybenzamide (3b)
1
Yield: 42%; Mp: 225.0-227.0 °C; H NMR (DMSO-d6): δ 10.84 (s, 1H, NH)
10.10 (s, 1H, NH), 8.91 (d, J = 6.8 Hz, 1H, Ar-H), 7.92 (m, 2H, Ar-H), 7.86 (s, 1H,
CONH), 7.83 (s, 1H, Ar-H), 7.78 (s, 1H, Ar-H), 7.62 (s, 1H, Ar-H), 7.63 (s, 1H,
CONH), 7.44 (t, J = 8.8 Hz, 2H, Ar-H), 7.34 (d, J = 6.8 Hz, 1H, Ar-H), 3.50 (s, 3H,
OCH3), 2.17 (s, 3H, CH3). ESI-HRMS m/z: calc’d for C22H20FN6O5S [M+H]+:
499.1200; found 499.1205.
4.1.11.19.
5-(2-(Cyclopropanecarboxamido)-[1,2,4]triazolo[1,5-a]pyridin-7-yl)-3-
(4-fluorophenylsulfonylamino)-2-methoxybenzamide (3c)
1
Yield: 42%; Mp: 233.0-235.0 °C; H NMR (DMSO-d6): δ 11.12 (s, 1H, NH)
10.10 (s, 1H, NH), 8.90 (d, J = 6.8 Hz, 1H, Ar-H), 7.92 (m, 2H, Ar-H), 7.86 (s, 1H,
CONH), 7.83 (s, 1H, Ar-H), 7.77 (s, 1H, Ar-H), 7.68 (s, 1H, Ar-H), 7.63 (s, 1H,
CONH), 7.44 (t, J = 8.8 Hz, 2H, Ar-H), 7.34 (d, J = 7.2 Hz, 1H, Ar-H), 3.50 (s, 3H,
OCH3), 2.06 (s, 1H, CH), 0.84 (d, J = 6.0 Hz, 4H, CH2). ESI-HRMS m/z: calc’d for
C24H22FN6O5S [M+H]+: 525.1356; found 525.1354.
4.2. Biological assay methods
4.2.1. Cell culture
The human cell lines HCT-116, A549, U-87 MG or MCF-7 were maintained as a
monolayer culture in DMEM, supplemented with 10% FBS in a humidified
atmosphere (5% CO2) at 37 °C.
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,
5-dimethyl-2-thiazolyl]-2, 5-diphenyl-2H- tetrazolium bromide (MTT) was purchased
21

from Sigma (St. Louis, MO, USA). Cellular chemosensitivity was determined by
using a modified MTT method assay in vitro. In brief, HCT-116, A549, U-87 MG or
MCF-7 cells in 200 µl culture medium were seeded into 96-well microplates at
3000-5000 cells per well respectively and cultured in DMEM 10% FBS, 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 µM of tested
compounds simultaneously and incubated for 48 h and then 20 µl of MTT solution (5
mg/ml in medium) was added to each well and incubated for 4 h. The formed blue
formazan crystals were pelleted to the bottom of the well by centrifugation, separated
from the supernatant, and dissolved in 200 µl of DMSO. The optical density at 490
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 (µM) of a compound was able to cause 50%
cell death with respect to the control culture, was calculated according to the
inhibition ratios.
4.2.3. Western blot assay
The suppressive activity of AKT and p-AKT473 in HCT-116 cells was determined
by western blot. HCT-116 cells were seeded into six-well plates at 1×106 cells per
well respectively and cultured in DMEM 10% FBS, incubated at 37 °C for 16 h prior
to drug exposure. Cells were treated with compounds 1g, A and DMSO and incubated
at 37 °C for 1 h. Cells were washed twice with ice-cold PBS and scraped into ice-cold
cell lysis buffer. Then the cell lysates were clarified by centrifugation at 12000 rpm
for 20 min at 4 °C and the supernatant was collected. Protein content was measured
by BCA protein Assay Kit and the proteins were separated on SDS-PAGE and then
transferred onto nitrocellulose membranes. The membranes were incubated with
antibodies against AKT (Cell Signaling Technology), p-Ser473 AKT (Cell Signaling
Technology) and β-Acin (Santa Cruz), washed by TBST and then incubated with
mouse and rabbit horseradish peroxidase-conjugated secondary antibodies. The
22

protein-antibody complexs were detected by chemiluminescence with an
GeneGnome5 system (Syngene, UK). Protein bands were quantified using ImageJ
software (National Institutes of Health, Bethesda, MD, USA).
4.2.4. Anticancer effects in established nude mouse HCT-116 xenograft model in
vivo
Mice (BALB/C, SPF grade, male, 18-20 g) were purchased from Shanghai
Slakey Laboratory Animal Co., LTD and fed at Shanghai Institute of Pharmaceutical
Industry. The experimental protocol was approved by Ethic Committee of Shanghai
Institute of Pharmaceutical Industry.
HCT-116 cells at 3 × 106 were injected subcutaneously into the flank. Once the
tumor xenografts reached 100 mm3, all tumor-bearing nude mice were randomly
divided into four groups, with 8 mice in control group and 5 mice in each drug group.
In the control group, the same volume of solvent was administered orally. BEZ235
and 1g were dissolved in NMP/PEG300 (1: 9). BEZ235 was dosed orally at 20 mg/kg
to positive group. 1g was dosed orally at 10 mg/kg and 30 mg/kg for the low and high
dosage groups once a day for 14 days, respectively. Tumor volumes and body weights
were recorded at intervals of 3 or 4 days. Tumor volume was calculated as length ×
width × width/2 and is reported in mm3. Results are expressed as the mean standard
error. The mice were anesthetized and sacrificed on day 26. The weights of the body,
the neoplasm, and the volumes of neoplasm, were measured and inhibitory ratios for
tumor volume and weight was calculated.
Molecular modeling
The protein-ligand complex crystal structure of compound A bound to PI3Kγ
was chosen as the template to compare the docking mode compound A bound to
PI3Kγ and 1g 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.
23

Compound A was removed and compound 1g 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. 21072156 and 81272448) is gratefully acknowledged.
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26

Figure and scheme Legends
Figure 1. The structures of some PI3Kα and mTOR dual inhibitors
Figure 2. Discovery of novel PI3K inhibitor based on bioisostere
Figure 3. Proposed novel structures
Figure 4. Effect of 1g and BEZ235 on AKT in HCT-116 cells. (A) Effect of
compound 1g and BEZ235 on the AKT and p-AKT473. (B) The quantified effect of
compounds 1g and BEZ235 on the AKT and p-AKT473. Results are x s for three
independent experiments each performed in duplicate. **P<0.01 vs DMSO group.
Figure 5. In vivo efficacy of 1g in HCT-116 xenograft tumor model on a once daily
dosing schedule. (A) the change of tumor volume; (B) the change of mouse body
weight. ***, p < 0.001 compared with the vehicle control group. (mpk: mg/kg)
Figure 6. Docking mode of compound 1g with PI3Kγ. Selected residues Val882,
Tyr867, Lys833, Asp964 and Asp841 are shown. Green dashed lines indicate
hydrogen bond.
Scheme 1. Reagents and conditions: (a) Ac2O, 85% HCO2H, 60°C, 2 h; then, rt, 4a,
ether, overnight; or 4a or 4b, Ac2O, DMAP, CH2Cl2, rt, overnight; or 4a or 4b,
cPrCOCl, TEA, CH2Cl2, reflux, 3 h; (b) 5a, BrCH2CO2Et, NaH, DMF, rt, 4 h; (c) 4a
or 4b, H3PO4, NaNO2, -10°C, 1 h, then CuBr, 40% HBr, rt, 1 h, 40°C, 2h; (d) 7a or 7b,
cyclopropylamine, 1,4-dioxane, 45°C, 4 h; (e) 9a-9c, SnCl2·2H2O, EtOAc, reflux, 4 h;
(f) RSO2Cl, Py., THF, rt, overnight; (g) 10a - 10f, bis(pinacolato)diboron, AcOK,
PdCl2(dppf), 1,4-dioxane, reflux under N2, 3-5 h; (h) 5b-5e or 6 or 8a-8b, PdCl2(dppf),
K2CO3, 1,4-dioxane/water (5:1), reflux under N2, 1-5 h.
Scheme 2. Reagents and conditions: (a) (i) 11 or 13, Ac2O, DMAP, CH2Cl2, rt,
27

overnight; or (ii) 11 or 13, cPrCOCl, Et3N, CH2Cl2, reflux, 3 h; (b) 10,
bis(pinacolato)diboron, AcOK, PdCl2(dppf), 1,4-dioxane, reflux under N2, 3-5 h; (h)
12a-12b or 14a-14b, PdCl2(dppf), K2CO3, 1,4-dioxane/water, reflux under N2, 1-3 h.
Table 1 Antiproliferative activities of synthesized compounds against HCT-116,
MCF-7, A549 and U-87 MG ( x s , n = 3)
28

Table 1 Antiproliferative activities of synthesized compounds against HCT-116,
MCF-7, A549 and U-87 MG (
, n = 3)
x
s
O
O
O
R1
R1
R1
S
S
S
HN
HN
HN
O
O
O
R2
R2
O
R2
O
H2N
H2N
X
H2N
N
N
S
N
N
N
H
N
H
N
H
N
R3
R3
N
R3
O
N
1
2
3
IC50(µM)
Compds
R1
R2
R3
X
HCT-116
MCF-7
nt
A549
nt
U-87 MG
4-FPh
4-FPh
4-FPh
cPr
H
cPrCO
cPrCO
cPrCO
cPrCO
cPr
CH
CH
CH
CH
CH
>50
nt
1a
1b
1c
Cl
11.40 2.35 >50
>50
17.93 2.86
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
2.01 0.95
1.58 0.24
6.34 2.22
46.30 8.65
nt
2.87 0.40
14.50 1.88 nt
8.22 0.08
1d
1e
4-FPh
4-FPh
4-FPh
4-FPh
4-FPh
4-FPh
cPr
>50
nt
nt
nt
CH2CO2Et CH
>50
nt
nt
1f
Ac
CH
N
1.95 0.66
4.25 1.06
2.50 0.89
0.50 0.24
1.70 1.50
4.75 1.92
1g
1h
1i
Ac
12.54 3.60 4.31 1.90
22.10 3.60 8.05 1.91
21.70 5.35
cPrCO
cPr
N
20.47 3.82
N
35.72 8.26 33.13 9.20 30.25 7.38
41.68 9.15
1j
Ac
36.33 4.16 nt
27.33 2.08 nt
31.67 5.69 nt
25.40 3.61 nt
nt
nt
2a
2b
2c
4-MePh OMe
Ac
nt
nt
4-ClPh
4-FPh
4-FPh
4-FPh
4-FPh
4-FPh
4-FPh
OMe
OMe
Cl
Ac
nt
nt
Ac
nt
nt
2d
2e
Ac
>50
43.50 5.07 nt
>50 nt
32.63 8.30 nt
nt
nt
nt
OMe
OMe
OMe
OMe
cPrCO
H
nt
nt
2f
nt
nt
3a
3b
3c
Ac
nt
nt
cPrCO
>50
nt
nt
nt
0.51 0.10
0.32 0.39
0.52 0.41
0.55 0.23
0.20 0.08
0.10 0.06
2.79 1.06
2.34 0.36
A
BEZ235
nt: not tested
29