Synthesis and anticancer activity evaluation of a series of [1,2,4] triazolo[1,5-a]pyridinylpyridines in vitro and in vivo

Article abstract of DOI:10.1016/j.ejmech.2013.06.052

A series of [1,2,4]triazolo[1,5-a]pyridinylpyridines were synthesized and characterized. Their anti-proliferative activities in vitro were evaluated by MTT against three human cancer cell lines including HCT-116, U-87 MG and MCF-7 cell lines. The SAR of target compounds was preliminarily discussed. The compounds 1c and 2d with potent antiproliferative activities were tested for their effects on the AKT and p-AKT⁴⁷³. The anticancer effect of 1c was evaluated in mice bearing sarcoma S-180 model. The results suggest that the title compounds are potent anticancer agents.

Full text of DOI:10.1016/j.ejmech.2013.06.052

European Journal of Medicinal Chemistry 67 (2013) 243e251
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and anticancer activity evaluation of a series of [1,2,4]
triazolo[1,5-a]pyridinylpyridines in vitro and in vivo
Xiao-Meng Wang a, Jing Xu b, Yi-Ping Li a, Huan Li a, Cong-Shan Jiang b, Guang-De Yang a,
,
She-Min Lu b, San-Qi Zhang a
*
a Department of Medicinal Chemistry, School of Pharmacy, Health Science Center, Xi’an Jiaotong University, Xi’an 710061, PR China
b Department of Genetics and Molecular Biology, School of Medicine, Health Science Center, Xi’an Jiaotong University, Xi’an 710061, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of [1,2,4]triazolo[1,5-a]pyridinylpyridines were synthesized and characterized. Their anti-
proliferative activities in vitro were evaluated by MTT against three human cancer cell lines including
HCT-116, U-87 MG and MCF-7 cell lines. The SAR of target compounds was preliminarily discussed. The
compounds 1c and 2d with potent antiproliferative activities were tested for their effects on the AKT and
p-AKT473. The anticancer effect of 1c was evaluated in mice bearing sarcoma S-180 model. The results
suggest that the title compounds are potent anticancer agents.
Received 17 May 2013
Received in revised form
15 June 2013
Accepted 19 June 2013
Available online 4 July 2013
Ó 2013 Elsevier Masson SAS. All rights reserved.
Keywords:
[1,2,4]Triazolo[1,5-a]pyridine
Synthesis
Antiproliferative activities
Anticancer effect
1. Introduction
immune cell migration [5,6]. Bell et al. described the identification
and SAR of 6-aryl-[1,2,4]triazolo[1,5-a]pyridines and discovered
The chemical and biological study of heterocyclic compounds
has been an interesting field in medicinal chemistry for a long
time. [1,2,4]triazolo[1,5-a]pyridine consists of a triazole ring fused
with a pyridine ring. 2-Amino-[1,2,4]triazolo[1,5-a]pyridine, with
two hydrogen bond acceptors and a hydrogen bond donor, is
considered as an ideal fragment or a scaffold in drug design. Its
derivatives exhibit a broad spectrum of biological activities such as
anticancer, anti-inflammatory, antimicrobial and antidiabetic ac-
tivity. Dugan et al. reported the structureeactivity relationships
and biological evaluation of a novel series of compounds based on
1,2,4-triazolo[1,5-a]pyridine scaffold and discovered that CEP-
33779 is a novel, selective, and orally bioavailable inhibitor of
JAK2 [1]. Thus CEP-33779 can be used in anticancer therapy [2]
and rheumatoid arthritis treatment [3]. Wyatt et al. identified 2-
acylamino-6-aryl[1,2,4]triazolo[1,5-a]pyridines as inhibitors of
the leishmania cdc 2-related protein kinase CRK3 and the in-
hibitors may be used to cure tropical parasitic diseases such as
leishmaniasis and human African trypanosomiasis (HAT) [4].
that CZC19945 and CZC24832 display potent inhibitory activities
against PI3K with good in vivo PK profile [7]. Bergamini et al.
g
further proved that CZC24832 is efficacious in in vitro and in vivo
model of inflammatory diseases in rodents and humans and it will
be of use for the treatment of autoimmune and inflammatory
disorders [8]. Ellard et al. synthesized a series of 2-ureido-[1,2,4]
triazolo[1,5-a]pyridine and discovered two compounds exhibit
excellent PI3Kg/d potency with high selectivity over the other
isoforms and the general kinome [9]. In addition, Kuroyanagi et al.
found that polysubstituted [1,2,4]triazolo[1,5-a]pyridines is the
specific inhibitor of b-1,6-glucan synthesis with potent antifungal
growth inhibition [10]. East et al. reported that 2-ureido-5,7-
disubstituted-[1,2,4]triazolo[1,5-a]pyridine displays good antibac-
terial activity, particularly against gram-positive organisms [11].
Edmondson et al. reported that 6-substituted [1,2,4]triazolo[1,5-a]
pyridine is a potent DPP-4 inhibitor and may be used to treat type
2 diabetes mellitus [12].
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 various cancer treatments. The toxicity
and resistance of traditional chemotherapeutic drugs makes it urgent
todevelopnewtargetsandnoveldrugs forthecancertherapy. Kinases
are well-known targets for a variety of diseases and disorders [13].
PI3Kg plays a key role in innate immune responses such as
* Corresponding author. Tel./fax: þ86 29 82657040.
E-mail address: sqzhang@mail.xjtu.edu.cn (S.-Q. Zhang).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.06.052

244
X.-M. Wang et al. / European Journal of Medicinal Chemistry 67 (2013) 243e251
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 [14]. In many cancers the PI3K/AKT/mTOR signal
transduction pathway is dysregulated contributing to cellular trans-
formation and tumor growth [15]. Therefore, PI3Ka and mTOR have
become the targets of intense research for anticancer drug discovery
[16,17]. In recent years, a significant progress has been made on the
design, synthesis and evaluation of PI3Ka and mTOR dual inhibitors,
and thereupon the pharmacophore of the dual inhibitors has been
proposed [18]. 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 inpyridine and quinoline
are the main components of pharmacophore. GlaxoSmithKline com-
pany identified GSK2126458, N-(2-methoxy-5-(4-pyridazin-4-yl)
nitropyridine 6b afforded corresponding amines. Without further
purification, the amines were converted to sulfamides 7ae7g [26].
Catalyzed by PdCl2(dppf), sulfamides 7ae7g were reacted with bis(-
pinacolato)diboron to produce arylboronic esters. Without isolation of
arylboronic esters, intermediate 3, or 4 or 5, PdCl2(dppf), water and
sodium carbonate as well were added to the above reaction mixture.
The resulted mixture was refluxed for 1 h to produce the title com-
pounds 1ae1p. In the case of preparation of 1m, the refluxing was
lasted for 5 h to make hydrolysis of ester group. The preparation of
arylboronic esters and Suzuki coupling were completed in one pot.
Formyl group in intermediate 5 was removed in last step.
The synthetic route to obtain the compounds 1q and 1r is shown
in Scheme 2. Ammonolysis of ester group in compound 1p pro-
duced the compounds 1q and 1r.
To expand the structural diversity of the title compounds, 7-
position of 2-acetylamino-[1,2,4]triazolo[1,5-a]pyridine was com-
bined with 3-sulfonamidopyridinyl to yield compound 2. The
acetylation of 7-bromo-[1,2,4]triazolo[1,5-a]pyridine-2-ylamine 8
produced compound 9. Compounds 2ae2d were synthesized by
Suzuki coupling in a same manner as preparation of compounds
1ae1p. The synthetic route for the compounds 2ae2d is shown in
Scheme 3.
quinilin-6-yl)pyridine-3-yl)-2,4-difluorobenzenesulfonamide, as
a
potent, orally bioavailable inhibitor of PI3K and mTOR [19]. Amgen
a
Inc. designed, synthesized and evaluated several classes of N-(2, 5-
disubstituted-pyridine-3-yl)phenylsulfonamides. Thus they discov-
ered that N-(2-chloro-5-(4-morpholinoquinilin-6-yl)pyridine-3-yl)-4-
fluorophenylsulfonamide [20], N-(2-chloro-5-(2-acetylamonobenzo[d]
thiazol-6-yl)pyridine-3-yl)-4-fluorophenyl sulfonamide (compound A,
Fig. 1) [21], N-(2-chloro-5-(2-acetylamonoimidazo[1,2-b]pyridazin-6-
yl)pyridine-3-yl)-4-fluorophenylsulfonamide [22] and AMG 511 [23]
2.2. In vitro antiproliferative assays
are excellent PI3Ka/mTOR dual inhibitors or PI3Ka selective inhibitor,
and orally bioavailable anticancer agents as well.
We evaluated antiproliferative activities of compounds 1ae1r
and 2ae2d against three human cancer cell lines including human
colon carcinoma cell line (HCT-116), glioma cell line (U-87 MG) and
human breast adenocarcinoma cell line (MCF-7) by applying the
MTT colorimetric assay. The PI3K and mTOR dual inhibitors A and
BEZ235 were used as the positive controls. The results of the cell
viability assay of compounds are summarized in Table 1.
First we synthesized a series of compound 1 by changing sub-
stituent R1, R2 and R3. As expected, all compounds exhibited sig-
nificant effects on antiproliferative activities in vitro, especially
In an attempt to develop novel anticancer agents, we try to
combine 2-amino-[1,2,4]triazolo[1,5-a]pyridine with N-pyridin-3-
ylphenylsulfonamide to design and synthesize the analogues of
compound A. Herein, the benzo[d]thiazole moiety was replaced by
[1,2,4]triazolo[1,5-a]pyridine (Fig. 1). Compared with compound A,
the designed compounds 1 and 2 have the same pharmacophore
with compound A. Here, we report the synthesis and anticancer
activity evaluation of a series of [1,2,4]triazolo[1,5-a]pyridinylpyr-
idines in vitro and in vivo.
against HCT-116. This may be related to PI3K
The fact that compounds 1c (IC50 ¼ 0.85
and 1e (IC50 ¼ 0.60
compounds 1a (IC50
a
mutant in HCT-116.
mM against HCT-116)
2. Results and discussion
m
M against HCT-116) were more potent than
¼
9.23 against HCT-116) and 1b
mM
2.1. Chemistry
(IC50 ¼ 2.65
mM against HCT-116) indicated that the compound with
a methoxy at 2-position (R1) of pyridine ring may improve the
antiproliferative activity. Compounds 1c, 1d, 1e and 1f exhibited
similar activities against HCT-116, U-87 MG and MCF-7, which
suggests that the substituent at the phenyl ring of R2 has less effect
The synthetic route to obtain the compounds 1ae1p is outlined
in Scheme 1.
6-Bromo-[1,2,4]triazolo[1,5-a]pyridin-2-ylamine 3 was acylated
to give acetyl amide 4a, cyclopropylcarbonyl amide 4b and form-
amide 4c, respectively [24]. Reaction of 4c with alkyl halides
afforded 5ae5c in the presence of sodium hydride [25]. Reduction
of 5-bromo-2-chloro-3-nitropyridine 6a or 5-bromo-2-methoxy-3-
on the antiproliferative activity. However, 1g (IC50 ¼ 2.22
against HCT-116) was less potent than 1c, 1e, 1d and 1f
(IC50 ¼ 0.60e0.85 M against HCT-116), indicating that aromatic
mM
m
group at position of R2 is more beneficial to the activity than
cyclopropyl group. To further study structureeactivity relationship
(SAR) of compound 1, we replaced acetyl at position of R3 with
different groups. When acetyl at position of R3 was removed, the
activity of compounds 1je1l decreased against all three human cell
lines. Compared with compounds 1a and 1c with an acetyl group at
R3, compounds 1h and 1i with cyclopropylcarbonyl group at R3
were less effect against three human cells. In addition, we replaced
acetyl at R3 with substituted methyl to produce 1m, 1p, 1q and 1r or
with substituted ethyl to produce 1n and 1o. The antiproliferative
activities of compounds 1me1r were not improved. Therefore, the
acetyl at R3 position is effective on antiproliferative activity in vitro.
In our second approach, we changed the pyridinyl from 6-
position of 2-amino-[1,2,4]triazolo[1,5-a]pyridine to 7-position to
produce compound 2. Compared with compounds 1c, 1d and 1e,
compounds 2a, 2b and 2c were less potent. Interestingly, com-
pound 2d (IC50 ¼ 0.82
m
M against HCT-116) with H at R3 is more
M) with acetyl at position R3, which is
Fig. 1. The structures of compound A and target compounds 1 and 2.
potent than 2c (IC50 ¼ 1.32
m

X.-M. Wang et al. / European Journal of Medicinal Chemistry 67 (2013) 243e251
245
Scheme 1. Reagents and conditions: (a) (i) AcCl or cPrCOCl, THF, rt; (ii) 7 N NH3 in MeOH, rt, overnight; (b) MeCOOCHO, Et2O, rt; (c) NaH, RX, DMF; (d) SnCl2$2H2O, conc. HCl, rt; (e)
RSO2Cl, pyridine, rt or reflux; (f) K2CO3, MeOH, rt; (g) bis(pinacolato)diboron, PdCl2(dppf), KOAc, 1,2-dimethoxyethane, reflux; (h) 3 or 4ae4c or 5ae5c, PdCl2(dppf), Na2CO3, EtOH/
H2O 3:2, reflux.
different from compound 1. From above mentioned results we
could reveal a SAR in which methoxy at R1, aryl at R2 and acetyl at R3
are favorable for antiproliferative activity. The IC50s of compounds
1c and 2d are comparable to that of PI3K and mTOR dual inhibitor
BEZ235. Therefore, compounds 1c and 2d were further
investigated.
distinctly exhibited inhibiting effect on tumor growth. Mouse body
weights loss of tested animals was not observed at 1 mg/kg group.
This is likely due to the low dosage. However, the body weight
dropped below 90% of the starting body weight over the course of
the study at 5 mg/kg group, which indicated that compound 1c still
has some toxicity at the dosage.
2.3. Western blot assay
2.5. Docking studies
To determine whether these compounds inhibit the PI3K, we
Docking analysis utilizing the C-DOCKER program within Dis-
covery Studio 2.5 software package was performed to further
explore the binding mode of the target compounds 1a and 1c.
evaluated the suppressive effects of compounds 1c and 2d at 10 mM
and positive compound A on AKT and p-AKT473 in HCT-116 cells
through Western blot. As shown in Fig. 2, the suppressive effects of
compounds 1c and 2d on p-AKT473 were slightly weaker than that
of positive compound A.
Docking simulations were performed on human PI3K
g (PDB code
3QKO) [21] (Fig. 3). The docking result of compound 1a with PI3K
g
(Fig. 3A) indicated that: (a) the 2-acetyl[1,2,4]triazolo[1,5-a]pyri-
dine forms two hydrogen bonds with Val882; (b) the nitrogen of 2-
chloropyridine forms a hydrogen bond with Asp964 and two
hydrogen bonds are formed via an ordered water molecule located
between the 2-chloropyridine ring nitrogen and the Tyr867 and
Asp841 residues; (c) the two oxygens of sulfonylamide form two
hydrogen bonds with Lys833. By contrast, compound 1c formed an
extra hydrogen bond via the oxygen of methoxypyridine (Fig. 3B),
which explains the methoxy is more suitable than chloro in com-
pound 1.
2.4. Anticancer effects in established mouse S180 homograft models
in vivo
We next evaluated whether compounds 1c and 2d could inhibit
tumor growth in established mouse homograft models. Though
favorable activity of compound 2d against human cancer cells, it led
to death of mice at dose of 20 mg/kg. Compound 1c can be tolerated
by mice at dose of 20 mg/kg and then we evaluated the activity of
compound 1c inhibiting tumor growth. A study using mice bearing
sarcoma S-180 was performed. Compound 1c was dosed orally at
1 mg/kg or 5 mg/kg once a day. In this model the volumes of S-180
tumor is difficult to measure, so tumor weights were used as
evaluating indicators which are illustrated in Table 2.
3. Conclusion
A series of 5-(6-[1,2,4]triazolo[1,5-a]pyridinyl)pyridines and 5-
(7-[1,2,4]triazolo[1,5-a]pyridinyl)pyridines were synthesized. Their
antiproliferative activities were evaluated in vitro against human
cancer cell lines including HCT-116, U-87 MG and MCF-7.
The inhibitory ratios at 1 mg/kg and 5 mg/kg were 20.0% and
75.8%, respectively. The results suggested that compound 1c
Scheme 2. Reagents and conditions: (a) NH3 or MeNH2 in MeOH, rt.

246
X.-M. Wang et al. / European Journal of Medicinal Chemistry 67 (2013) 243e251
Scheme 3. Reagents and conditions: (a) (i) AcCl, THF, rt; (ii) 7 N NH3 in MeOH, rt, overnight; (b) bis(pinacolato)diboron, PdCl2(dppf), KOAc, 1,2-dimethoxyethane E, reflux; (c) 8 or 9,
PdCl2(dppf), Na2CO3, EtOH/H2O, 3:2, reflux.
Compounds displayed IC50 around 1
mM. The SAR of these com-
4. Experimental protocols
pounds indicated that methoxy at 2-position, arylsulfonamino at 3-
positon and 2-acetylamino-[1,2,4]triazolo[1,5-a]pyridinyl at 5-
position of pyridine ring can improve the inhibitory activity. The
Western blot assay results of compounds 1c and 2d suggested that
these compounds can inhibit the PI3K/AKT/mTOR pathway. Com-
pound 1c distinctly exhibited inhibiting effect on tumor growth in
mice bearing sarcoma S-180 model. These results suggest the title
compounds are potent anticancer agents.
4.1. Chemistry and chemical methods
3-[4, 5-Dimethyl-2-thiazolyl]-2, 5-diphenyl-2H-tetrazolium bro-
mide (MTT) was purchased from Sigma (St. Louis, MO, USA). 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
Table 1
Antiproliferative activities against HCT-116, U-87 MG and MCF-7 (x ꢀ s, n ¼ 3).
NHSO2R2
NHSO2R2
R1
R1
N
N
N
N
N
N
NHR3
NHR3
N
N
2a-2d
1a-1r
Compds
R1
R2
R3
IC50
(
mM)
HCT-116
U-87 MG
MCF-7
1a
1b
1c
1d
1e
1f
1g
1h
1i
Cl
Cl
4-FPh
4-MePh
4-FPh
4-ClPh
4-MePh
2,4-diFPh
cPr
4-FPh
4-FPh
4-FPh
4-ClPh
4-MePh
4-FPh
4-FPh
4-FPh
4-FPh
4-FPh
4-FPh
4-ClPh
4-MePh
4-FPh
4-FPh
Ac
Ac
Ac
Ac
Ac
Ac
Ac
cPrCO
cPrCO
H
H
H
CH2CO2H
CH2CH2OMe
CH2CH2OEt
CH2CO2Et
CH2CONH2
CH2CONHMe
Ac
9.23 ꢀ 1.00
2.65 ꢀ 0.48
0.85 ꢀ 0.40
0.74 ꢀ 0.43
0.60 ꢀ 0.09
0.74 ꢀ 0.34
2.22 ꢀ 1.23
1.74 ꢀ 0.30
9.98 ꢀ 2.89
2.97 ꢀ 2.19
2.50 ꢀ 1.99
3.40 ꢀ 0.51
8.64 ꢀ 2.88
4.61 ꢀ 2.90
9.35 ꢀ 3.77
4.37 ꢀ 1.93
6.10 ꢀ 1.61
4.91 ꢀ 2.53
1.33 ꢀ 0.49
1.38 ꢀ 0.48
1.32 ꢀ 0.33
0.82 ꢀ 0.06
1.16 ꢀ 0.15
nt
6.95 ꢀ 0.95
3.76 ꢀ 0.37
1.82 ꢀ 0.48
2.09 ꢀ 0.89
1.60 ꢀ 0.27
2.00 ꢀ 0.46
3.73 ꢀ 0.91
4.85 ꢀ 1.33
10.96 ꢀ 4.00
3.95 ꢀ 0.90
3.11 ꢀ 0.72
2.51 ꢀ 0.99
9.81 ꢀ 2.91
6.39 ꢀ 1.02
8.15 ꢀ 1.52
7.94 ꢀ 2.17
5.80 ꢀ 1.89
7.29 ꢀ 2.24
2.12 ꢀ 0.13
2.03 ꢀ 0.47
2.41 ꢀ 0.51
1.77 ꢀ 0.34
1.32 ꢀ 0.41
nt
19.79 ꢀ 2.84
3.25 ꢀ 0.65
0.84 ꢀ 0.30
2.01 ꢀ 0.78
0.81 ꢀ 0.25
0.87 ꢀ 0.21
1.64 ꢀ 0.69
2.59 ꢀ 1.27
>60
1.93 ꢀ 1.13
1.82 ꢀ 1.03
1.55 ꢀ 0.41
12.64 ꢀ 2.94
2.41 ꢀ 0.64
1.35 ꢀ 0.32
4.41 ꢀ 2.23
8.40 ꢀ 3.57
2.54 ꢀ 0.69
1.24 ꢀ 0.30
1.36 ꢀ 0.54
1.73 ꢀ 0.31
0.92 ꢀ 0.22
0.73 ꢀ 0.21
0.23 ꢀ 0.09
OMe
OMe
OMe
OMe
OMe
OMe
Cl
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
1j
1k
1l
1m
1n
1o
1p
1q
1r
2a
2b
2c
Ac
Ac
H
2d
BEZ235
A
nt: not tested.

X.-M. Wang et al. / European Journal of Medicinal Chemistry 67 (2013) 243e251
247
Fig. 2. Effect of compounds A, 1c and 2d on AKT in HCT116 cells. (A) Effect of compounds 1c, 2d and A on the AKT and p-AKT473. (B) The quantified effect of compounds 1c, 2d and A
on the AKT and p-AKT473. Results are x ꢀ s for three independent experiments each performed in duplicate. ***P < 0.001.
UV light. All the melting points were determined on a Beijing
micromelting-point apparatus and thermometer was uncorrected.
1H NMR spectra were recorded on a 400 MHz Bruker NMR spec-
NH), 8.82 (d, J ¼ 5.6 Hz, 1H, AreH), 8.05 (s, 1H, AreH), 7.30 (d,
J ¼ 5.6 Hz, 1H, AreH), 2.14 (s, 3H, CH3). ESIeHRMS m/z: calc’d for
C8H7BrN4NaO [M þ Na]þ: 276.9701; found 276.9699.
trometer with tetramethylsilane (TMS) as an internal reference. 13
C
NMR spectra were recorded on a 100 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 spec-
trometer (Maxis Q-TOF, Bruker Inc.).
4.1.2. N-(6-bromo-[1,2,4]triazolo[1,5-a]pyridin-2-yl)formamide
(4c)
The mixture of acetic anhydride (0.76 ml, 8 mmol) and 88%
formic acid (0.34 ml, 8 mmol) was stirred at 60 ꢁC for 2 h, cooled to
room temperature. To the resulting anhydride 6-bromo-[1,2,4]tri-
azolo[1,5-a]pyridin-2-ylamine 3 (0.43 mg, 2 mmol) and ether
(10 ml) were added. The mixture was stirred at room temperature
overnight, filtered. The solid was washed by ether and dried to
afford a white solid. Yield: 97.5%, mp 217e220 ꢁC. 1H NMR (DMSO-
4.1.1. General procedures for the synthesis of compounds 4a, 4b and 9
To a stirred solution of 6-bromo-[1,2,4]triazolo[1,5-a]pyridin-2-
ylamine 3 (2.00 g, 9.3 mmol) or 7-bromo-[1,2,4]triazolo[1,5-a]
pyridine-2-ylamine 8 (2.00 g, 9.3 mmol) in THF (40 ml) was added
N, N-diisopropylethylamine (4.87 ml, 27.9 mmol), and then acyl
chloride (27.9 mmol) was added. The reaction mixture was stirred
at room temperature until all starting material was consumed. The
solvent was evaporated under vacuum. The solution of ammonia in
methanol (7 N, 20 ml) was added to the residue, and the resulted
mixture was stirred at room temperature overnight. The solvent
was evaporated under vacuum. Ether (20 ml) was added to the
residue, and the solid was collected by filtered, washed with water
(20 ml), acetone (20 ml) and then dried to afford a off-white solid.
4.1.1.1. N-(6-bromo-[1,2,4]triazolo[1,5-a]pyridin-2-yl)acetamide (4a).
Yield 84.6%, mp 222e225 ꢁC. 1H NMR (DMSO-d6):
d 10.87 (s,1H, NH),
9.29 (s, 1H, AreH), 7.78 (d, J ¼ 9.2 Hz, 1H, AreH), 7.78 (d, J ¼ 9.2 Hz,
1H, AreH), 2.13 (s, 3H, CH3). ESIeHRMS m/z: calc’d for C8H7BrN4NaO
[M þ Na]þ: 276.9701; found 276.9699.
4.1.1.2. N-(6-bromo-[1,2,4]triazolo[1,5-a]pyridin-2-yl)cyclo-
propanecarboxamide (4b). Yield 77.1%, mp 216e219 ꢁC. 1H NMR
(DMSO-d6):
d
11.14 (s, 1H, NH), 9.28 (s, 1H, AreH), 7.78 (d, J ¼ 9.6 Hz,
1H, AreH), 7.66 (d, J ¼ 9.2 Hz, 1H, AreH), 2.03 (m, 1H, CH), 0.83 (d,
J ¼ 5.6 Hz, 4H, CH2). 13C NMR (100 MHz, DMSO-d6):
d 171.65, 159.25,
148.68, 133.81, 129.66, 115.90, 106.91, 14.41, 8.34, 8.34. ESIeHRMS m/
z: calc’d for C10H9BrN4NaO [M þ Na]þ: 302.9857; found 302.9857.
4.1.1.3. N-(7-bromo-[1,2,4]triazolo[1,5-a]pyridin-2-yl)acetamide (9).
Yield 62.1%, mp 228e230 ꢁC. 1H NMR (DMSO-d6):
d
10.87 (s, 1H,
Table 2
Inhibitory ratios on cancer weights (x ꢀ s, n ¼ 6).
Groups
Dosage
(mg/kg)
Average
weights (g)
Inhibition rate
in weight (%)
Fig. 3. Docking mode comparison between 1a and 1c. (A) Ribbon show of compound
1a bound to PI3Kg; (B) ribbon show of compound 1c bound to PI3Kg. Compounds 1a
Solvent
1c
1c
10 ml/kg
1.0
5.0
0.95 ꢀ 0.35
0.76 ꢀ 0.25
0.23 ꢀ 0.16
and 1c are shown in yellow backbones. Selected residues Val882, Tyr867, Lys833,
Asp841 and Asp964 are shown based on elements and green dashed lines indicate
hydrogen bonds. (For interpretation of the references to color in this figure legend, the
reader is referred to the web version of this article.)
20.0*
75.8***
*P < 0.05 vs solvent; ***P < 0.001 vs solvent.

248
X.-M. Wang et al. / European Journal of Medicinal Chemistry 67 (2013) 243e251
d6):
d
11.32 (d, J ¼ 10.0 Hz, NH), 9.28 (s, 1H, AreH), 9.13 (d,
OCH3). HRMS m/z: calc’d for C12H10BrFN2NaO3S [M þ Na]þ: 382.9477;
J ¼ 10.0 Hz, CHO), 7.82 (d, J ¼ 9.2 Hz, 1H, AreH), 7.67 (d, J ¼ 9.2 Hz,
found 382.9482.
1H, AreH). 13C NMR (100 MHz, DMSO-d6):
d
163.06, 160.05, 149.33,
134.26, 129.70, 115.77, 107.14. ESIeHRMS m/z: calc’d for
C7H5BrN4NaO [M þ Na]þ: 262.9544; found 262.9543.
4 . 1. 4 . 4 . N - [ 5 - b r o m o - 2 - m e t h y l o x y - 3 - p y r i d i n y l ] - 4 -
methylphenylsulfonamide (7d). Yield 86.0%, mp 126e129 ꢁC. 1H NMR
(CDCl3):
d
7.89 (d, J ¼ 7.2 Hz, 2H, NH, AreH), 7.72 (d, J ¼ 7.6 Hz, 2H,
4.1.3. General procedures for the synthesis of compounds 5ae5c
Compound 4c (0.41 g, 1.7 mmol) was added to the suspension of
sodium hydride (60%, 0.07 g, 1.7 mmol) in DMF (6 ml) and the
mixture was stirred at room temperature for 0.5 h. The alkyl halide
(1.83 mmol) was added dropwise to the reaction mixture and then
the mixture was stirred at room temperature until all starting
material was consumed. Ice-water (20 ml) was poured into the
mixture and the mixture was filtered, washed by water and dried to
afford the white solid [25].
AreH), 7.29 (d, J ¼ 6.8 Hz, 2H, AreH), 6.95 (s, 1H, AreH), 3.85 (s, 3H,
OCH3), 2.42 (s, 3H, CH3). ESIeHRMS m/z: calc’d for C13H13BrN2NaO3S
[M þ Na]þ: 378.9728; found 378.9736.
4 . 1. 4 . 5 . N - [ 5 - b r o m o - 2 - m e t h y l o x y - 3 - p y r i d i n y l ] - 4 -
chlorophenylsulfonamide (7e). Yield 86.3%, mp 154e157 ꢁC. 1H NMR
(CDCl3):
d
7.92 (d, J ¼ 6.8 Hz, 2H, NH, AreH), 7.77 (d, J ¼ 8.0 Hz, 2H,
AreH), 7.48 (d, J ¼ 8.0 Hz, 2H, AreH), 6.95 (s, 1H, AreH), 3.84 (s, 3H,
OCH3). ESIeHRMS m/z: calc’d for C12H10BrClN2NaO3S [M þ Na]þ:
398.9182; found 398.9187.
4.1.3.1. Ethyl
mamido)acetate (5a). Yield 97.3%, mp 140e142 ꢁC. 1H NMR (DMSO-
d6):
9.58 (s, 1H, CHO), 8.62 (s, 1H, AreH), 7.63 (d, J ¼ 8.4 Hz, 1H,
2-(N-(6-bromo-[1,2,4]triazolo[1,5-a]pyridin-2-yl)for-
4 .1. 4 . 6 . N - [ 5 - b r o m o - 2 - m e t h yl o x y - 3 - p y r i d i n yl ] - 2 , 4 -
d
difluorophenylsulfonamide (7f). Yield 88.2%, mp 158e160 ꢁC.1H NMR
AreH), 7.52 (d, J ¼ 9.2 Hz, 1H, AreH), 4.75 (s, 2H, CH2), 4.25 (dd,
(CDCl3):
d
10.48 (s, 1H, NH), 8.13 (s, 1H, AreH), 7.76 (t, J ¼ 11.6 Hz, 2H,
J1 ¼14.4 Hz, J2 ¼ 14.4 Hz, 2H, OCH2), 1.31 (t, J ¼ 6.8 Hz, 3H, CH3). 13
C
AreH), 7.56 (t, J ¼ 9.8 Hz, 1H, AreH), 7.23 (t, J ¼ 8.6 Hz, 1H, AreH).
ESIeHRMS m/z: calc’d for C12H9BrF2N2NaO2S [M þ Na]þ: 400.9383;
found 400.9386.
NMR (100 MHz, DMSO-d6):
d 166.13, 161.47, 161.09, 149.39, 134.89,
129.95, 116.08, 107.75, 61.67, 43.26, 14.45. ESIeHRMS m/z: calc’d for
C
11H11BrN4NaO3 [M þ Na]þ: 348.9912; found 348.9907.
4.1. 4. 7. N-[5-bromo-2-methyloxy-3-pyridinyl]-cyclo-
4.1.3.2. N-(6-bromo-[1,2,4]triazolo[1,5-a]pyridin-2-yl)-N-(2-
propylcarbonylsulfonamide (7g). Yield 95.8%, mp 127e129 ꢁC. 1H
ethoxyethyl)formamide (5b). Yield 66.9%, mp 145e147 ꢁC. 1H NMR
NMR (CDCl3):
d
7.95 (d, J ¼ 12.8 Hz, 2H, NH, AreH), 6.73 (s, 1H, Are
(DMSO-d6):
d
9.52 (s, 1H CHO), 8.63 (s, 1H, AreH), 7.63 (d,
H), 4.02 (s, 3H, OCH3), 2.54 (m, 1H, CH),1.25 (m, 2H, CH2), 1.03 (m,
2H, CH2). ESIeHRMS m/z: calc’d for C9H11BrN2NaO3S [M þ Na]þ:
328.9571; found 328.9573.
J ¼ 9.6 Hz, 1H, AreH), 7.51 (d, J ¼ 9.2 Hz, 1H, AreH), 4.23 (t,
J ¼ 6.2 Hz, 2H, OCH2), 3.74 (t, J ¼ 6.2 Hz, 2H, CH2), 3.54 (dd,
J1 ¼ 14.0 Hz, J2 ¼ 14.0 Hz, 2H, OCH2), 1.56 (t, J ¼ 7.0 Hz, 3H, CH3).
13C NMR (100 MHz, DMSO-d6):
d
161.75, 161.57, 149.36, 134.67,
4.1.5. General procedures for the synthesis of compounds 1ae1p
and 2ae2d
129.89, 116.02, 107.51, 66.47, 65.77, 41.17, 15.47. ESIeHRMS m/z:
calc’d for
335.0118.
C
11H13BrN4NaO2 [M
þ
Na]þ: 335.0120; found
To a round bottomed flask were added compound 7 (0.4 mmol),
bis(pinacolato)diboron (0.13 g, 0.5 mmol), potassium acetate
(0.10 g, 1.0 mmol), PdCl2(dppf) (0.22 g, 0.03 mmol) and 1,2-
dimethoxyethane (10.5 ml) and the mixture was refluxed for 4 h
under N2 atmosphere, cooled to room temperature. Then, com-
pound 3 or 4 or 5 or 8 or 9 (0.3 mmol), sodium carbonate (0.74 g,
0.7 mmol) and EtOH/H2O (3:2, v/v, 7.5 ml) as well were added to the
mixture. The mixture was refluxed under stirring for 1 h under N2
atmosphere. The solvent was evaporated under vacuum and the
residue was purified by chromatography on silica gel
(CHCl3:MeOH ¼ 30:1, v/v) to produce a white or off-white solid.
4.1.3.3. N-(6-bromo-[1,2,4]triazolo[1,5-a]pyridin-2-yl)-N-(2-
methoxyethyl)formamide (5c). Yield 53.5%, mp 121e123 ꢁC. 1H NMR
(DMSO-d6):
d 9.52 (s, 1H, CHO), 8.64 (s, 1H, AreH), 7.63 (d,
J ¼ 9.2 Hz, 1H, AreH), 7.52 (d, J ¼ 9.2 Hz, 1H, AreH), 4.24 (t,
J ¼ 5.8 Hz, 2H, CH2), 3.71 (t, J ¼ 5.8 Hz, 2H, CH2), 3.37 (s, 3H, CH2). 13
C
NMR (100 MHz, DMSO-d6): d 161.78, 161.56, 149.35, 134.66, 129.88,
116.01, 107.51, 68.73, 58.40, 41.02. ESIeHRMS m/z: calc’d for
C
10H11BrN4NaO2 [M þ Na]þ: 320.9963; found 320.9964.
4.1.4. General procedure for the synthesis of N-(5-bromo-3-pyridinyl)-
4-arylsulfonamide (7ae7g)
Compounds 7ae7g were synthesized according to the proce-
dure described in Ref. [26].
4.1.5.1. N-(6-(6-chloro-5-(4-fluorophenylsulfonamido)pyridin-3-yl)-
[1,2,4]triazolo[1,5-a]pyridin-2-yl)acetamide (1a). Yield 65.2%, mp
234e235 ꢁC. 1H NMR (DMSO-d6):
d 10.90 (s, 1H, NH), 10.56 (s, 1H,
NH), 9.39 (s, 1H, AreH), 8.71 (s, 1H, AreH), 8.18 (s, 1H, AreH), 7.97
(d, J ¼ 9.2 Hz, 1H, AreH), 7.85 (m, 3H, AreH), 7.43 (m, 2H, AreH),
4.1.4.1. N-(5-bromo-2-chloro-3-pyridinyl)-4-fluorophenylsulfonamide
2.17 (s, 3H, CH3). 13C NMR (100 MHz, DMSO-d6):
d 166.31, 163.80,
(7a). Yield 82.1%, mp 175e178 ꢁC. 1H NMR (CDCl3):
d
8.22 (s,1H, NH),
159.73, 149.34, 145.89, 145.66, 136.94, 135.17, 132.35, 131.06, 130.35,
130.25, 129.99, 127.38, 121.98, 117.16, 116.94, 115.33, 24.18. ESIe
HRMS m/z: calc’d for C19H14ClFN6NaO3S [M þ Na]þ: 483.0418;
found 483.0409.
8.17 (s, 1H, AreH), 7.85 (m, 2H, AreH), 7.21 (t, J ¼ 8.4 Hz, 2H, AreH),
7.01 (s, 1H, AreH). ESIeHRMS m/z: calc’d for C11H7BrClFN2NaO2S
[M þ Na]þ: 363.9084; found 363.9087.
4.1.4.2. N-(5-bromo-2-chloro-3-pyridinyl)-4-methylphenylsulfonamide
4.1.5.2. N-(6-(6-chloro-5-(4-methylphenylsulfonamido)pyridin-3-
(7b). Yield 74.7%, mp 162e165 ꢁC. 1H NMR (CDCl3):
d
8.17 (d,
yl)-[1,2,4]triazolo[1,5-a pyridin-2-yl)acetamide (1b). Yield 57.4%, mp
J ¼ 5.6 Hz, 2H, NH, AreH), 7.72 (d, J ¼ 7.6 Hz, 2H, AreH), 7.32 (d,
J ¼ 6.8 Hz, 2H, AreH), 7.03 (s, 1H, AreH), 2.44 (s, 3H, CH3). HRMS m/z:
calc’d for C12H10BrClN2NaO2S [M þ Na]þ: 382.9233; found 382.9238.
246e248 ꢁC. 1H NMR (DMSO-d6):
d 10.89 (s, 1H, NH), 10.39 (s, 1H,
NH), 9.34 (s, 1H, AreH), 8.68 (s, 1H, AreH), 8.10 (s, 1H, AreH), 7.91
(d, J ¼ 9.2 Hz, 1H, AreH), 7.82 (d, J ¼ 9.2 Hz, 1H, AreH), 7.65 (d,
J ¼ 8.0 Hz, 2H, AreH), 7.38 (d, J ¼ 7.6 Hz, 2H, AreH), 2.39 (s, 3H,
4.1.4.3. N-[5-bromo-2-methoxy-3-pyridinyl]-4-fluorophenylsulfonamide
CH3), 2.16 (s, 3H, CH3). 13C NMR (100 MHz, DMSO-d6):
d 167.40,
(7c). Yield 85.0%, mp 154e157 ꢁC. 1H NMR (CDCl3):
d
7.92 (s, 1H, AreH),
159.73, 149.32, 145.61, 145.26, 144.15, 137.59, 134.34, 132.20, 131.14,
130.26, 130.26, 129.63, 127.31, 127.25, 127.25, 122.04, 115.37, 24.18,
7.85 (m, 2H, AreH), 7.18 (m, 2H, AreH), 6.92 (s, 1H, AreH), 3.85 (s, 3H,

X.-M. Wang et al. / European Journal of Medicinal Chemistry 67 (2013) 243e251
249
21.49. ESIeHRMS m/z: calc’d for C20H17ClN6NaO3S [M þ Na]þ:
OCH3), 2.07 (m, 1H, CH), 0.85 (s, 4H, CH2). 13C NMR (100 MHz,
DMSO-d6): 171.66, 163.59, 159.31, 157.37, 148.96, 142.34, 137.11,
479.0669; found 479.0668.
d
132.95,130.30,130.20,130.03,126.20,126.03,123.40,120.80,116.73,
4.1.5.3. N-(6-(6-methoxy-5-(4-fluorophenylsulfonamido)pyridin-3-
yl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl)acetamide (1c). Yield 70.4%, mp
116.51, 115.18, 53.92, 14.41, 8.30, 8.30. ESIeHRMS m/z: calc’d for
C
22H19FN6NaO4S [M þ Na]þ: 505.1070; found 505.1071.
239e241 ꢁC. 1H NMR (DMSO-d6):
d 10.84 (s, 1H, NH), 10.08 (s, 1H,
NH), 9.23 (s, 1H, AreH), 8.40 (s, 1H, AreH), 8.00 (s, 1H, AreH), 7.90
(d, J ¼ 9.2 Hz, 1H, AreH), 7.81 (m, 3H, AreH), 7.41 (m, 2H, AreH),
3.64 (s, 3H, OCH3), 2.16 (s, 3H, CH3). 13C NMR (100 MHz, DMSO-d6):
4.1.5.9. N-(6-(6-chloro-5-(4-fluorophenylsulfonamido)pyridin-3-yl)-
[1,2,4]triazolo[1,5-a]pyridin-2-yl)cyclopropanecarboxamide (1i).
Yield 48.9%, mp 236e238 ꢁC. 1H NMR (DMSO-d6):
11.12 (s, 1H, NH),
d
d
166.04, 163.54, 159.45, 157.36, 148.96, 142.33, 137.12, 132.95,
10.56 (s, 1H, NH), 9.37 (s, 1H, AreH), 8.71 (s, 1H, AreH), 8.18 (s, 1H,
AreH), 7.97 (d, J ¼ 9.2 Hz, 1H, AreH), 7.82 (d, J ¼ 8.4 Hz, 3H, AreH),
130.30, 130.21, 130.01, 126.21, 126.02, 123.39, 120.80, 116.73, 116.50,
115.18, 53.92, 24.15. ESIeHRMS m/z: calc’d for C20H17FN6NaO4S
[M þ Na]þ: 479.0914; found 479.0909.
7.43 (m, 2H, AreH), 2.06 (m, 1H, CH), 0.85 (d, J ¼ 5.6 Hz, 4H, CH2). 13
C
NMR (100 MHz, DMSO-d6):
d 171.69, 163.79, 159.59, 149.32, 145.88,
145.64, 136.25, 135.16, 132.35, 131.07, 130.34, 130.25, 129.99, 127.36,
121.99, 117.16, 116.93, 115.32, 14.44, 8.35, 8.35. ESIeHRMS m/z: calc’d
for C21H16ClFN6NaO3S [M þ Na]þ: 509.0575; found 509.0574.
4.1.5.4. N-(6-(6-methoxy-5-(4-chlorophenylsulfonamido)pyridin-3-
yl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl)acetamide (1d). Yield 55.6%,
mp 233e235 ꢁC. 1H NMR (DMSO-d6):
d 10.84 (s, 1H, NH), 10.15 (s,
1H, NH), 9.23 (s, 1H, AreH), 8.41 (s, 1H, AreH), 8.00 (s, 1H, AreH),
7.90 (d, J ¼ 9.2 Hz, 1H, AreH), 7.76 (m, 3H, AreH), 7.65 (d, J ¼ 8.0 Hz,
2H, AreH), 3.64 (s, 3H, OCH3), 2.16 (s, 3H, CH3). 13C NMR (100 MHz,
4.1.5.10. N-(5-(2-amino-[1,2,4]triazolo[1,5-a]pyridin-6-yl)-2-
methoxypyridin-3-yl)-4-fluorophenylsulfonamide (1j). Yield 49.1%,
mp 247e250 ꢁC. 1H NMR (DMSO-d6):
d 10.06 (s, 1H, NH), 8.89 (s, 1H,
DMSO-d6):
d
166.01, 159.46, 157.40, 148.97, 142.39, 139.63, 138.17,
AreH), 8.34 (s,1H, AreH), 7.92 (s,1H, AreH), 7.81 (s, 2H, AreH), 7.68
(d, J ¼ 8.4 Hz, 1H, AreH), 7.42 (m, 3H, AreH), 6.11 (s, 2H, NH2), 3.63
133.05, 130.02, 129.59, 129.59, 129.10,129.10,126.22,126.04,123.38,
120.75, 115.18, 53.89, 24.16. ESIeHRMS m/z: calc’d for
(s, 3H, OCH3). 13C NMR (100 MHz, DMSO-d6):
d 167.00, 163.45,
C
20H17ClN6NaO4S [M þ Na]þ: 495.0618; found 495.0614.
157.09, 150.17, 141.34, 137.44, 132.13, 130.25, 130.16, 128.42, 126.50,
125.01, 121.46, 121.35, 116.66, 116.43, 112.96, 53.82. ESIeHRMS m/z:
calc’d for C18H15FN6NaO3S [M þ Na]þ: 437.0808; found 437.0805.
4.1.5.5. N-(6-(6-methoxy-5-(4-methylphenylsulfonamido)pyridin-3-
yl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl)acetamide (1e). Yield 66.8%,
mp 249e252 ꢁC. 1H NMR (DMSO-d6):
d
10.84 (s,1H, NH), 9.94 (s,1H,
4.1.5.11. N-(5-(2-amino-[1,2,4]triazolo[1,5-a]pyridin-6-yl)-2-
NH), 9.18 (s, 1H, AreH), 8.35 (s, 1H, AreH), 7.93 (s, 1H, AreH), 7.85
(d, J ¼ 9.2 Hz, 1H, AreH), 7.77 (d, J ¼ 9.2 Hz, 1H, AreH), 7.66 (d,
J ¼ 7.6 Hz, 2H, AreH), 7.36 (d, J ¼ 8.0 Hz, 2H, AreH), 3.67 (s, 3H,
OCH3), 2.36 (s, 3H, CH3), 2.16 (s, 3H, CH3). 13C NMR (100 MHz,
methoxypyridin-3-yl)-4-chlorophenylsulfonamide (1k). Yield 57.1%,
mp 210e213 ꢁC. 1H NMR (DMSO-d6):
d 10.13 (s, 1H, NH), 8.84 (s, 1H,
AreH), 8.89 (s, 1H, AreH), 8.35 (s, 1H, AreH), 7.75 (d, J ¼ 7.6 Hz, 2H,
AreH), 7.66 (t, J1 ¼ 8.4 Hz, J2 ¼ 8.0 Hz, 3H, AreH), 7.45 (d, J ¼ 8.8 Hz,
1H, AreH), 6.11 (s, 2H, NH2), 3.62 (s, 3H, OCH3). 13C NMR (100 MHz,
DMSO-d6):
d 165.88, 159.45, 157.02, 148.95, 143.73, 141.66, 137.77,
131.57, 129.97, 129.89, 129.89, 127.23, 127.23, 126.12, 125.88, 123.50,
DMSO-d6):
d 167.02, 157.11, 150.20, 142.02, 139.62, 138.17, 132.82,
121.35, 115.22, 53.04, 24.16, 21.43. ESIeHRMS m/z: calc’d for
C
129.58, 129.58, 129.11, 129.11, 128.42, 126.57, 125.09, 121.19, 120.65,
21H20N6NaO4S [M þ Na]þ: 475.1164; found 475.1162.
112.96, 53.83. ESIeHRMS m/z: calc’d for
[M þ Na]þ: 453.0513; found 453.0513.
C18H15ClN6NaO3S
4.1.5.6. N-(6-(6-methoxy-5-(2,4-difluorophenylsulfonamido)pyridin-
3-yl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl)acetamide (1f). Yield 50.0%,
4.1.5.12. N-(5-(2-amino-[1,2,4]triazolo[1,5-a]pyridin-6-yl)-2-
mp 216e218 ꢁC. 1H NMR (400 MHz, DMSO-d6):
d
10.85 (s, 1H, NH),
methoxypyridin-3-yl)-4-methylphenylsulfonamide (1l). Yield 49.8%,
10.35 (s, 1H, NH), 9.26 (s, 1H, AreH), 8.45 (s, 1H, AreH), 8.03 (s, 1H,
AreH), 7.93 (d, J ¼ 9.2 Hz, 1H, AreH), 7.76 (m, 2H, AreH), 7.59 (m,
1H, AreH), 7.20 (m, 1H, AreH), 3.64 (s, 3H, OCH3), 2.16 (s, 3H, CH3).
mp 235e237 ꢁC. 1H NMR (DMSO-d6):
d 9.92 (s, 1H, NH), 8.84 (s, 1H,
AreH), 8.29 (s, 1H, AreH), 7.86 (s, 1H, AreH), 7.64 (s, 3H, AreH),
7.44 (d, J ¼ 7.6 Hz, 1H, AreH), 7.36 (d, J ¼ 6.0 Hz, 2H, AreH), 6.10 (s,
2H, NH2), 3.65 (s, 3H, OCH3), 2.36 (s, 3H, CH3). 13C NMR (100 MHz,
13C NMR (100 MHz, DMSO-d6):
d 166.84, 164.32, 159.46, 158.64,
158.29, 148.96, 143.12, 135.03, 132.30, 129.99, 126.22, 126.06,
123.25, 120.12, 115.17, 112.38, 112.14, 106.24, 53.89, 24.15. ESIe
HRMS m/z: calc’d for C20H16F2N6NaO4S [M þ Na]þ: 497.0819; found
497.0821.
DMSO-d6): d 167.06, 156.72, 150.23, 143.74, 141.32, 137.73, 131.33,
129.90, 129.90, 128.37, 127.24, 127.24, 126.39, 125.50, 121.32, 121.21,
113.00, 53.89, 21.43. ESIeHRMS m/z: calc’d for C19H18N6NaO3S
[M þ Na]þ: 433.1059; found 433.1058.
4.1.5.7. N-(6-(6-methoxy-5-(4-cyclopropylcarbonylsulfonamido)pyr-
4.1.5.13. 2-(6-5-(4-Fluorophenylsulfonamido)-6-methoxypyridin-3-
idin-3-yl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl)acetamide (1g). Yield
4
yl)-[1,2,4]triazol [1,5-a]pyridin-2-ylamino)acetic acid (1m). Yield
7.0%, mp 240e242 ꢁC.1H NMR (DMSO-d6):
d
10.83 (s, 1H, NH), 9.43 (s,
89.0%, mp 225e228 ꢁC.1H NMR (DMSO-d6):
d 8.99 (s.1H, AreH), 8.33
1H, NH), 9.28 (s, 1H, AreH), 8.42 (s, 1H, AreH), 8.00 (s, 1H, AreH),
7.94 (d, J ¼ 9.2 Hz, 1H, AreH), 7.70 (d, J ¼ 9.2 Hz, 1H, AreH), 3.99 (s,
(d, J ¼ 2.0 Hz, 1H, AreH), 7.93 (d, J ¼ 2.4 Hz, 1H, AreH), 7.81 (m, 2H,
AreH), 7.72 (d, J ¼ 9.2 Hz, 1H, AreH), 7.51 (d, J ¼ 9.2 Hz, 2H, AreH),
7.41 (t, J ¼ 8.8 Hz, 2H, AreH), 6.98 (t, J ¼ 6.4 Hz, 1H, NH), 3.93 (d,
J ¼ 6.8 Hz, 2H, CH2), 3.63 (s, 3H, OCH3). 13C NMR (100 MHz, DMSO-
3H, OCH3), 2.87 (m, 1H, CH), 2.16 (s, 3H, CH3), 0.95 (m, 4H, CH2). 13
C
NMR (100 MHz, DMSO-d6): d 166.82, 159.41, 157.24, 148.94, 141.31,
131.36, 130.14, 126.22, 125.99, 126.64, 122.04, 115.13, 54.32, 30.97,
d6):
d 172.94, 166.89, 163.53, 157.09, 150.16, 141.80, 137.15, 132.54,
34.15, 5.62, 5.62. ESIeHRMS m/z: calc’d for
C
17H18N6NaO4S
130.30, 130.20, 128.77, 126.44, 125.35, 121.50, 120.87, 116.72, 116.49,
113.26, 53.88, 44.63. ESIeHRMS m/z: calc’d for C20H18FN6O5S
[M þ H]þ: 473.1043; found 473.1046.
[M þ Na]þ: 425.1008; found: 425.1009.
4.1.5.8. N-(6-(6-methoxy-5-(4-fluorophenylsulfonamido)pyridin-3-
yl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl)cyclopropanecarboxamide (1h).
4.1.5.14. N-(5-(2-(2-methoxyethylamino)-[1,2,4]triazolo[1,5-a]pyr-
Yield 56.7%, mp 257e260 ꢁC. 1H NMR (DMSO-d6):
d
11.12 (s, 1H,
idin-6-yl)-2-methoxypyridin-3-yl)-4-fluorophenylsulfonamide (1n).
NH), 10.09 (s, 1H, NH), 9.21 (s, 1H, AreH), 8.40 (s, 1H, AreH), 8.00 (s,
1H, AreH), 7.86 (m, 4H, AreH), 7.41 (m, 2H, AreH), 3.64 (s, 3H,
Yield 57.5%, mp 205e207 ꢁC. 1H NMR (DMSO-d6):
d
10.07 (s, 1H,
NH), 8.97 (s. 1H, AreH), 8.35 (s, 1H, AreH), 7.93 (s, 1H, AreH), 7.81

250
X.-M. Wang et al. / European Journal of Medicinal Chemistry 67 (2013) 243e251
(t, J ¼ 6.2 Hz, 2H, AreH), 7.70 (d, J ¼ 9.2 Hz, 1H, AreH), 7.48 (d,
J ¼ 8.8 Hz, 1H, AreH), 7.41 (t, J ¼ 8.4 Hz, 2H, AreH), 6.70 (t,
J ¼ 5.4 Hz, 1H, NH), 3.63 (s, 3H, OCH3), 3.50 (t, J ¼ 5.6 Hz, 2H, OCH2),
3.41 (d, J ¼ 6.0 Hz, CH2), 3.27 (s, 3H, OCH3). 13C NMR (100 MHz,
d 166.05, 163.55, 159.60, 157.92, 150.08, 142.81, 138.65, 137.08,
132.81, 130.30, 130.21, 129.39, 127.25, 120.90, 116.76, 116.53, 112.36,
111.08, 54.04, 24.16. ESIeHRMS m/z: calc’d for C20H17FN6NaO4S
[M þ Na]þ: 479.0914; found 479.0912.
DMSO-d6):
d 166.03, 163.53, 157.06, 141.86, 137.14, 137.11, 132.58,
130.30, 130.20, 128.62, 126.49, 125.27, 121.24, 120.79, 116.71, 116.49,
4.1.5.20. N-(5-(2-amino-[1,2,4]triazolo[1,5-a]pyridin-7-yl)-2-
113.03, 71.23, 58.42, 53.86, 42.52. ESIeHRMS m/z: calc’d for
C
methoxypyridin-3-yl)-4-fluorophenylsulfonamide (2d). Yield 65.5%,
21H21FN6NaO4S [M þ Na]þ: 495.1227; found 495.1231.
mp 232e235 ꢁC. 1H NMR (DMSO-d6):
d 10.12 (s, 1H, NH), 8.61 (d,
J ¼ 6.8 Hz, 1H, AreH), 8.45 (d, J ¼ 2.4 Hz, 1H, AreH), 7.97 (d,
J ¼ 2.4 Hz, 1H, AreH), 7.81 (m, 2H, AreH), 7.60 (d, J ¼ 1.2 Hz, 1H, Are
H), 7.42 (m, 2H, AreH), 7.14 (d, J ¼ 6.8 Hz, 1H, AreH), 3.65 (s, 3H,
4.1.5.15. N-(5-(2-(2-ethoxyethylamino)-[1,2,4]triazolo[1,5-a]pyridin-
6-yl)-2-methoxypyridin-3-yl)-4-fluorophenylsulfonamide (1o).
Yield 58.6%, mp 209e212 ꢁC. 1H NMR (DMSO-d6):
10.08 (s, 1H,
d
OCH3).13C NMR (100 MHz, DMSO-d6):
d 167.16, 163.56, 157.64, 151.25,
NH), 8.99 (s. 1H, AreH), 8.35 (s, 1H, AreH), 7.93 (s, 1H, AreH), 7.81
(m, 2H, AreH), 7.70 (d, J ¼ 9.2 Hz, 1H, AreH), 7.48 (d, J ¼ 8.8 Hz, 1H,
AreH), 7.41 (m, 2H, AreH), 6.68 (t, J ¼ 5.8 Hz, 1H, NH), 3.63 (s, 3H,
OCH3), 3.54 (t, J ¼ 6.0 Hz, 2H, OCH2), 3.47 (dd, J1 ¼ 14.0 Hz,
J2 ¼ 14.4 Hz, 2H, OCH2), 3.40 (d, J ¼ 6.0 Hz, CH2), 1,20 (t, J ¼ 7.0 Hz,
142.42, 137.22, 137.06, 132.46, 130.30, 130.20, 128.15, 127.72, 120.84,
116.77, 116.55, 110.04, 108.99, 55.40. ESIeHRMS m/z: calc’d for
C
18H15FN6NaO3S [M þ Na]þ: 437.0808; found 437.0810 [M þ Na]þ.
4.1.6. General procedures for the synthesis of compounds 1qe1r
The solution of methylamine or ammonia in methanol (2 mmol)
was added to the solution of compound 1p (0.05 g, 0.1 mmol) in
methanol (10 ml). The mixture was stirred at room temperature for
48 h. Then the solvent was evaporated under vacuum. Water
(10 ml) was added to the residue, and the suspension was filtered,
washed by water and dried to get a white solid.
3H, CH3). 13C NMR (100 MHz, DMSO-d6):
d 167.03, 163.54, 157.06,
150.23, 141.89, 137.12, 132.61, 130.25, 130.20, 128.63, 126.50, 125.26,
121.22, 120.76, 116.72, 116.49, 113.02, 69.10, 65.92, 53.86, 42.78,
15.62. ESIeHRMS m/z: calc’d for C22H23FN6NaO4S [M þ Na]þ:
509.1383; found 509.1390.
4.1.5.16. Ethyl 2-(6-(6-methoxy-5-(4-fluorophenylsulfonamido)pyr-
idin-3-yl)-[1,2,4]
triazolo[1,5-a]pyridin-2-ylamino)acetate
(1p).
4.1.6.1. 2-(6-5-(4-Fluorophenylsulfonamido)-6-methoxypyridin-3-yl)-
Yield 66.0%, mp 202e204 ꢁC.1H NMR (DMSO-d6):
d
10.07 (s,1H, NH),
[1,2,4]triazolo[1,5-a]pyridin-2-ylamino)acetamide (1q). Yield 78.5%,
8.99 (s. 1H, AreH), 8.35 (d, J ¼ 1.2 Hz, 1H, AreH), 7.94 (s, 1H, AreH),
7.81 (m, 2H, AreH), 7.73 (d, J ¼ 9.2 Hz, 1H, AreH), 7.52 (d, J ¼ 8.8 Hz,
2H, AreH), 7.41 (m, 2H, AreH), 7.13 (t, J ¼ 6.4 Hz, 1H, NH), 4.11 (dd,
J1 ¼14.0 Hz, J2 ¼ 14.0 Hz, 2H, OCH2), 4.02 (d, J ¼ 6.4 Hz, 2H, CH2), 3.63
(s, 3H, OCH3), 1,20 (t, J ¼ 7.0 Hz, 3H, CH3). 13C NMR (100 MHz, DMSO-
mp 249e251 ꢁC. 1H NMR (DMSO-d6):
d 9.92 (s, 1H, NH), 8.84 (s, 1H,
AreH), 8.29 (s, 1H, AreH), 7.86 (s, 1H, AreH), 7.64 (s, 3H, AreH), 7.44
(d, J ¼ 7.6 Hz, 1H, AreH), 7.36 (d, J ¼ 6.0 Hz, 2H, AreH), 6.10 (s, 2H,
NH2), 3.65 (s, 3H, OCH3), 2.36 (s, 3H, CH3).13C NMR (100 MHz, DMSO-
d6):
d 167.13, 166.05, 163.56, 157.64, 151.23, 142.43, 137.24, 137.03,
d6):
d
168.21, 163.54, 161.52, 157.44, 149.63, 142.36, 137.12, 132.88,
132.46, 130.30, 130.20, 128.16, 127.71, 120.83, 116.78, 116.55, 110.07,
108.98, 54.00, 53.94. ESIeHRMS m/z: calc’d for C20H18FN7NaO4S
[M þ Na]þ: 494.1023; found 494.1030.
131.04, 130.29, 130.20, 126.45, 125.69, 124.19, 120.91, 116.71, 116.49,
115.35, 61.66, 53.94, 43.27, 14.47. ESIeHRMS m/z: calc’d for
C
22H21FN6NaO5S [M þ Na]þ: 523.1176; found 523.1178.
4.1.6.2. 2-(6-5-(4-Fluorophenylsulfonamido)-6-methoxypyridin-3-
4.1.5.17. N-(7-(6-methoxy-5-(4-chlorophenylsulfonamido)pyridin-3-
yl)-[1,2,4]triazolo[1,5-a]pyridin-2-ylamino)-N-methylacetamide (1r).
yl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl)acetamide (2a). Yield 72.5%,
Yield 68.4%, mp 229e231 ꢁC. 1H NMR (DMSO-d6):
d 8.99 (s. 1H, Are
mp 226e229 ꢁC. 1H NMR (DMSO-d6):
d
10.85 (s, 1H, NH), 10.21 (s,
H), 8.33 (s, 1H, AreH), 7.92 (s, 1H, AreH), 7.81 (s, 2H, AreH), 7.72 (d,
J ¼ 8.4 Hz, 1H, AreH), 7.51 (d, J ¼ 8.8 Hz,1H, AreH), 7.41 (m, 2H, Are
H), 6.92 (s, 1H, NH), 3.82 (d, J ¼ 3.6 Hz, 2H, CH2), 3.63 (s, 3H, OCH3),
3.63 (s, 3H, OCH3), 2.59 (d, J ¼ 2.0 Hz, CH3). 13C NMR (100 MHz,
1H, NH), 8.91 (d, J ¼ 6.8 Hz, 1H, AreH), 8.52 (s, 1H, AreH), 8.08 (s,
1H, AreH), 7.96 (s, 1H, AreH), 7.75 (d, J ¼ 8.0 Hz, 2H, AreH), 7.65 (d,
J ¼ 8.0 Hz, 2H, AreH), 7.42 (d, J ¼ 6.8 Hz, 1H, AreH), 3.65 (s, 3H,
OCH3), 2.16 (s, 3H, CH3). 13C NMR (100 MHz, DMSO-d6):
d
168.16,
DMSO-d6): d 170.80, 166.88, 163.52, 157.18, 150.18, 141.87, 137.14,
159.62, 157.95, 150.09, 142.79, 139.64, 138.65, 138.17, 132.863,
129.60, 129.60, 129.39, 129.10, 129.10, 127.27, 120.95, 112.37, 111.09,
132.59, 130.30, 130.20, 128.78, 126.42, 125.35, 121.47, 120.85, 116.72,
116.49, 113.27, 53.87, 46.44, 26.01. ESIeHRMS m/z: calc’d for
54.00, 24.16. ESIeHRMS m/z: calc’d for
C
20H17ClN6NaO4S
C
21H20FN7NaO4S [M þ Na]þ: 508.1179; found 508.1184.
[M þ Na]þ: 495.0618; found 495.0621.
4.2. Biological assay methods
4.1.5.18. N-(7-(6-methoxy-5-(4-methylphenylsulfonamido)pyridin-
3-yl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl)acetamide (2b). Yield 74.3%,
4.2.1. Cell culture
mp 216e218 ꢁC. 1H NMR (DMSO-d6):
d
10.85 (s, 1H, NH), 10.01 (s, 1H,
The human cell lines HCT-116, U-87 MG or MCF-7 were main-
tained as a monolayer culture in DMEM, supplemented with 10%
FBS in a humidified atmosphere (5% CO2) at 37 ꢁC.
NH), 8.91 (d, J ¼ 6.4 Hz,1H, AreH), 8.46 (s, 1H, AreH), 7.99 (s,1H, Are
H), 7.90 (s, 1H, AreH), 7.65 (d, J ¼ 7.6 Hz, 3H, AreH), 7.36 (d,
J ¼ 6.8 Hz, 3H, AreH), 3.68 (s, 3H, OCH3), 2.36 (s, 3H, CH3), 2.16 (s, 3H,
CH3). 13C NMR (100 MHz, DMSO-d6):
d
165.15, 159.62, 157.56, 150.09,
4.2.2. Antiproliferative assays
143.78, 142.15, 138.77, 137.71, 131.36, 129.93, 129.93, 129.43, 127.23,
127.23, 127.10, 121.043, 112.34, 110.99, 54.07, 24.16, 21.44. ESIeHRMS
m/z: calc’d for C21H20N6NaO4S [M þ Na]þ: 475.1164; found 475.1168.
Cellular chemosensitivity was determined by using a modified
MTT method assay in vitro. In brief, HCT-116, U-87 MG or MCF-7
cells in 200 ml culture medium were seeded into 96-well micro-
plates at 3000e5000 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 48 h incubation period. Cells
were treated with final concentrations of 100.0, 10.0, 1.0, 0.1 and
4.1.5.19. N-(7-(6-methoxy-5-(4-fluorophenylsulfonamido)pyridin-3-
yl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl) acetamide (2c). Yield 85.0%,
mp 233e235 ꢁC. 1H NMR (DMSO-d6):
d 10.85 (s, 1H, NH), 10.14 (s,
1H, NH), 8.91 (d, J ¼ 4.4 Hz, 1H, AreH), 8.51 (s, 1H, AreH), 8.05 (s,
1H, AreH), 7.95 (s, 1H, AreH), 7.81 (s, 2H, AreH), 7.41 (s, 3H, AreH),
3.65 (s, 3H, OCH3), 2.16 (s, 3H, CH3). 13C NMR (100 MHz, DMSO-d6):
0.01
mM of tested compounds simultaneously and incubated for
48 h and then 20
ml of MTT solution (5 mg/ml in medium) was

X.-M. Wang et al. / European Journal of Medicinal Chemistry 67 (2013) 243e251
251
added to each well and incubated for 4 h. The formed blue for-
mazan crystals were pelleted to the bottom of the well by centri-
fugation, separated from the supernatant, and dissolved in 200 l of
DMSO. The optical density at 490 nm was determined by Varioskan
Flash Multimode Reader (Thermo scientific). Three separate ex-
periments with triplicate data were performed to obtain mean cell
viability. The IC50 value, that is, the concentration (mM) of a com-
pound was able to cause 50% cell death with respect to the control
culture, was calculated according to the inhibition ratios.
References
[1] B.J. Dugan, D.E. Gingrich, E.F. Mesaros, K.L. Milkiewicz, M.A. Curry, A selective,
orally bioavailable 1,2,4-triazolo[1,5-a]pyridine-based inhibitor of Janus ki-
nase 2 for use in anticancer therapy: discovery of CEP-33779, J. Med. Chem. 55
(2012) 5243e5254.
[2] M.M. Seavey, L.D. Lu, K.L. Stump, N.H. Wallace, W. Hockeimer, T.M. O’Kane,
B. Ruggeri, P. Dobrzanski, Therapeutic efficacy of CEP-33779, a novel selective
JAK2 inhibitor, in a mouse model of colitis-induced colorectal cancer, Mol.
Cancer Ther. 11 (2011) 984e993.
[3] K.L. Stump, L.D. Lu, P. Dobrzanski, C. Serdikoff, D.E. Gingrich, B.J. Dugan,
T.S. Angeles, M.S. Albom, M.A. Ator, B.D. Dorsey, B.A. Ruggeri, M.M. Seavey,
A highly selective, orally active inhibitor of Janus kinase 2, CEP-33779, ablates
disease in two mouse models of rheumatoid arthritis, Arthritis Res. Ther. 13
(2011) R68.
[4] L.A.T. Cleghorn, A. Woodland, I.T. Collie, L.S. Torrie, N. Norcross, T. Luksch,
C. Mpamhanga, R.G. Walker, J.C. Mottram, R. Brenk, A.F. Julie, H.G. Ian,
G.W. Paul, Identification of inhibitors of the leishmania cdc2-related protein
kinase CRK3, ChemMedChem 6 (2011) 2214e2224.
[5] P.T. Hawkins, K.E. Anderson, K. Davidson, L.R. Stephens, Signalling through
class I PI3Ks in mammalian cells, Biochem. Soc. Trans. 34 (2006) 647e662.
[6] T.D. Cushing, D.P. Metz, D.A. Whittington, L.R. McGee, PI3Kd and PI3Kg as targets
for autoimmune and inflammatory diseases, J. Med. Chem. 55 (2012) 8559e8581.
[7] K. Bell, M. Sunose, K. Ellard, A. Cansfield, J. Taylor, SAR studies around a series
of triazolopyridines as potent and selective PI3K
Chem. Lett. 22 (2012) 5257e5263.
[8] G. Bergamini, K. Bell, S. Shimamura, T. Werner, A. Cansfield, A selective in-
hibitor reveals PI3Kg dependence of TH17 cell differentiation, Nat. Chem. Biol.
8 (2012) 576e583.
m
4.2.3. Western blot assay
The suppressive activity of AKTand p-AKT473 in HCT-116 cells was
determined by western blot. HCT-116 cells were seeded into six-well
plates at 1 ꢂ 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 final concentrations of 10 mM of compounds 1c,
2d, 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 12,000 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 SDSePAGE and then transferred onto nitrocellulose
membranes. The membranes were incubated with antibodies
against AKT (Cell Signaling Technology), p-Ser473 AKT (Cell
g inhibitors, Bioorg. Med.
[9] K. Ellard, M. Sunose, K. Bell, N. Ramsden, G. Bergamini, G. Neubauer, Discovery
of novel PI3Kg/d inhibitors as potential agents for inflammation, Bioorg. Med.
Chem. Lett. 22 (2012) 4546e4549.
[10] J. Kuroyanagi, K. Kanai, Y. Sugimoto, T. Fujisawa, C. Morita, Novel antifungal
Signaling Technology) and b-Acin (Santa Cruz), washed by TBST and
then incubated with Mouse and rabbit horseradish peroxidase-
conjugated secondary antibodies. The proteineantibody complexes
were detected by chemiluminescence with a GeneGnome5 system
(Syngene, UK). Protein bands were quantified using ImageJ software
(National Institutes of Health, Bethesda, MD, USA).
agents: triazolopyridines as inhibitors of b-1,6-glucan synthesis, Bioorg. Med.
Chem. 18 (2010) 5845e5854.
[11] S.P. East, C.B. White, O. Barker, S. Barker, J. Bennett, DNA gyrase (GyrB)/
topoisomerase IV (ParE) inhibitors: synthesis and antibacterial activity, Bio-
org. Med. Chem. Lett. 19 (2009) 894e899.
[12] S.D. Edmondson, A. Mastracchio, R.J. Mathvink, J.F. He, B. Harper, (2S,3S)-3-
Amino-4-(3,3-difluoropyrrolidin-1-yl)-N, N-dimethyl-4-oxo-2-(4-[1,2,4]tri-
azolo[1,5-a]-pyridin-6-ylphenyl)butanamide:
dipeptidyl peptidase IV inhibitor for the treatment of type 2 diabetes, J. Med.
Chem. 49 (2006) 3614e3627.
a selective a-amino amide
4.2.4. Anticancer effects in sarcoma S-180 model in vivo
Mice (20.1 ꢀ 2.0 g) were purchased from Experiment Animal
Center of Xi’an Jiaotong University College of Medicine and fed in
the same place. The experimental protocol was approved by Ethic
Committee of Xi’an Jiaotong University.
[13] S.K. Grant, Therapeutic protein kinase inhibitors, Cell. Mol. Life Sci. 66 (2009)
1163e1177.
[14] J.A. Engelman, J. Luo, L.C. Cantley, The evolution of phosphatidylinositol 3-
kinases as regulators of growth and metabolism, Nat. Rev. Genet. 8 (2006)
606e619.
S-180 cells at 3 ꢂ 106 were injected subcutaneously into the
flank of mice. All tumor-bearing mice were randomly divided into
three groups, with 6 mice in each group. In the solvent group, the
same volume of solvent was administered orally. Compound 1c was
dissolved in DMSO/PEG400/5% glucose (volume ratio, 10:60:30)
and dosed orally at 1 mg/kg and 5 mg/kg for the low and high
dosage groups once a day for 10 days, respectively. Body weights
were recorded per day. The mice were anesthetized and sacrificed
on Day 11. The weights of the body and the neoplasm were
measured and inhibitory ratios for tumor weight was calculated.
[15] L. Salmena, A. Carracedo, P.P. Pandolfi, Tenets of PTEN tumor suppression, Cell
133 (2008) 403e414.
[16] P. Liu, H. Cheng, T.M. Roberts, J. Zhao, Targeting the phosphoinositide 3-kinase
pathway in cancer, Nat. Rev. Drug Discov. 8 (2009) 627e644.
[17] Q. Liu, C. Thoreen, J.H. Wang, D. Sabatini, N.S. Gray, mTOR mediated anti-
cancer drug discovery, Drug Discov. Today Ther. Strategy 6 (2009) 47e55.
[18] P. Wu, Y.Z. Hu, Small molecules targeting phosphoinositide 3-kinases, Med.
Chem. Commun. 3 (2012) 1337e1355.
[19] S.D. Knight, N.D. Adams, J.L. Burgess, Discovery of GSK2126458, a highly
potent inhibitor of PI3K and the mammalian target of rapamycin, ACS Med.
Chem. Lett. 1 (2010) 39e43.
[20] N. Nishimura, A. Siegmund, L. Liu, Phospshoinositide 3-kinase (PI3K)/
mammalian target of rapamycin (mTOR) dual inhibitors: discovery and
structureeactivity relationships of a series of quinoline and quinoxaline de-
rivatives, J. Med. Chem. 54 (2011) 4735e4751.
4.3. Molecular modeling
[21] N.D. D’Angelo, T.-S. Kim, K. Andrews, S.K. Booker, Discovery and optimization of
a series of benzothiazole phosphoinositide 3-kinase (PI3K)/mammalian target of
rapamycin (mTOR) dual inhibitors, J. Med. Chem. 54 (2011) 1789e1811.
[22] M.M. Stec, K.L. Andrews, S.K. Booker, S. Caenepeel, Structureeactivity re-
lationships of phosphoinositide 3-kinase (PI3K)/mammalian target of rapa-
mycin (mTOR) dual inhibitors: investigations of various 6,5-heterocycles to
improve metabolic stability, J. Med. Chem. 54 (2011) 5174e5184.
[23] M.H. Norman, K.L. Andrews, Y.Y. Bo, S.K. Booker, S. Caenepeel, V.J. Cee, Se-
lective class I phosphoinositide 3-kinase Inhibitors: optimization of a series of
pyridyltriazines leading to the identification of a clinical candidate, AMG 511,
J. Med. Chem. 55 (2012) 7796e7816.
[24] N. Ramsden, K. Bell, A. Cansfield, J. Taylor, M. Sunose, D. Middlemiss, G.
Neubauer, Amino triazoles as PI3K inhibitors, WO 2009/068482 A1, P. C. T.
Int., 4 June 2009.
[25] A. Pappne Behr, Z. Kapui, P. Aranyi, S. Batori, V. Bartane Bodor, M. Verga, E.
mikus, K. Urban-Szabo, J. Vargane Szeredi, T. Szabo, E. Susan, M. Kovacs,
Amino-alkyl-amide derivatives as CCR3 receptor ligands, WO 2007/034253
A1, P. C. T. Int., 29 Mar 2007.
The proteineligand complex crystal structure of compound A
bound to PI3K
mode between compound 1a bound to PI3K
PI3K . 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 PI3K
g
was chosen as the template to compare the docking
g
and 1c bound to
g
g
enzyme was defined as a receptor and the site sphere was selected
on the basis of the ligand binding location of compound A. Com-
pound A was removed and compounds 1a and 1c were placed. After
end of molecular docking, ten docking poses was scored and
selected based on calculated C-DOCKER energy.
Acknowledgments
[26] N.D. Adams, J.L. Burgess, M.G. Darcy, S.D. Knight, K.A. Newlander, L.H. Ridgers,
S.J. Schmidt, Quinazoline derivatives as PI3 kinase inhibitors, WO 2008/
157191 A2, P. C. T. Int., 24 Dec 2008.
Financial support from The National Natural Science Foundation
of China (Grant No. 21072156) is gratefully acknowledged.