Modification of N-(6-(2-methoxy-3-(4-fluorophenylsulfonamido)pyridin-5-yl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl)acetamide as PI3Ks inhibitor by replacement of the acetamide group with alkylurea

Article abstract of DOI:10.1016/j.bmc.2015.07.017

N-(6-(2-Methoxy-3-(4-fluorophenylsulfonamido)pyridin-5-yl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl)acetamide exhibits remarkable anticancer effects and toxicity when orally administrated. In present study, alkylurea moiety replaced the acetamide group in the c

Full text of DOI:10.1016/j.bmc.2015.07.017

Bioorganic & Medicinal Chemistry xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Modification of N-(6-(2-methoxy-3-(4-fluorophenylsulfonamido)
pyridin-5-yl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl)acetamide as PI3Ks
inhibitor by replacement of the acetamide group with alkylurea
a
a
b
a
a
c
Xiao-Meng Wang , Shuai Mao , Lei Cao , Xiao-Xiao Xie , Min-Hang Xin , Jia-Fang Lian ,
b
a,
⇑
Yong-Xiao Cao , San-Qi Zhang
a
Department of Medicinal Chemistry, School of Pharmacy, Xi’an Jiaotong University, Xi’an, Shaanxi 710061, PR China
Department of Pharmacology, School of Basic Medical Sciences, Xi’an Jiaotong University, Xi’an, Shaanxi 710061, PR China
Medical Technology Department, Bethune Medical NCO School of PLA, Shijiazhuang, Hebei 050081, PR China
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
N-(6-(2-Methoxy-3-(4-fluorophenylsulfonamido)pyridin-5-yl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl)ac-
etamide exhibits remarkable anticancer effects and toxicity when orally administrated. In present study,
alkylurea moiety replaced the acetamide group in the compound and a series of 1-alkyl-3-(6-(2-meth-
oxy-3-sulfonylaminopyridin-5-yl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl)urea derivatives were synthesized.
The antiproliferative activities of the synthesized compounds in vitro were evaluated against four human
cancer cell lines. Several compounds with potent antiproliferative activities were tested for their acute
oral toxicity and their inhibitory activity against PI3Ks and mTOR. The results indicate that the compound
attached a alkylurea or 2-(dialkylamino)ethylurea moiety at the 2-position of [1,2,4]triazolo[1,5-a]pyri-
dine can retain the antiproliferative activity and the inhibitory activity against PI3Ks and mTOR. In addi-
tion, their acute oral toxicity reduced dramatically. Moreover, the results also indicate that compound 1e
can efficaciously inhibit tumor growth in a mice S180 model. These findings suggest that title compounds
can serve as potent PI3K inhibitors and effective anticancer agents with low toxicity.
Received 10 May 2015
Revised 9 July 2015
Accepted 10 July 2015
Available online xxxx
Keywords:
[
1,2,4]Triazolo[1,5-a]pyridine
Urea
PI3K inhibitors
Anticancer agent
Acute toxicity
Ó 2015 Elsevier Ltd. All rights reserved.
1
. Introduction
human African trypanosomiasis (HAT).4 PI3K
innate immune responses such as immune cell migration.
CZC19945 and CZC24832, derived from 6-aryl-[1,2,4]triazolo[1,5-
a]pyridine, were identified as PI3K inhibitors with good in vivo
c plays a key role in
5,6
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. There are hydrogen bond acceptors and
hydrogen bond donor in the structure of 2-acetylmino-[1,2,4]tria-
zolo[1,5-a]pyridine. Thus, the structure is considered as an ideal
fragment or a scaffold in drug design. The derivatives of [1,2,4]tri-
azolo[1,5-a]pyridine exhibit a broad spectrum of biological activi-
ties such as anticancer, anti-inflammatory, antimicrobial and
antidiabetic activity. CEP-33779, possessing the scaffold of 1,2,4-
triazolo[1,5-a]pyridine, was discovered as a novel, selective, and
c
PK profile and efficacy in vitro and in vivo models of inflamma-
7
tion. Further, CZC24832 was proved to be efficacious in regulate
H
interleukin-17-producing T helper cell (T 17) differentiation and
may be of use for the treatment of autoimmune and inflammatory
8
disorders. Recently, a series of 2-ureido-[1,2,4]triazolo[1,5-a]pyr-
idine derivatives were synthesized and two compounds exhibited
excellent PI3Kc/d potency with high selectivity over the other iso-
9
forms and the general kinome. In addition, multisubstituted
[1,2,4]triazolo[1,5-a]pyridines were discovered as inhibitors of b-
1,6-glucan synthesis with potent antifungal activity.10 2-Ureido-
5,7-disubstituted-[1,2,4]triazolo[1,5-a]pyridine was reported as
good antibacterial agent with potent Gram-positive antibacterial
orally bioavailable JAK2 inhibitor and can be used for cancer ther-
apy or rheumatoid arthritis treatment.1
–3
2-Acylamino-6-aryl-
[
1,2,4]triazolo[1,5-a]pyridines were identified as inhibitors of the
1
1
leishmania cdc 2-related protein kinase CRK3 and may be used
to treat tropical parasitic diseases such as leishmaniasis and
activity. Early, 6-substituted [1,2,4]triazolo[1,5-a] pyridine was
reported as a potent DPP-4 inhibitor and may be used to treat type
1
2
2
diabetes.
Both PI3K (a family of lipid kinase) and mTOR (mammalian tar-
get of rapamycin) have been found to play key regulatory roles in
⇑
Corresponding author.
E-mail address: sqzhang@xjtu.edu.cn (S.-Q. Zhang).
http://dx.doi.org/10.1016/j.bmc.2015.07.017
968-0896/Ó 2015 Elsevier Ltd. All rights reserved.
0
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2
X.-M. Wang et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
many cellular processes, including cell growth, proliferation, differ-
entiation, motility and survival.13 Accumulation evidence supports
the notion that The PI3K/AKT/mTOR signal transduction pathway
2. Chemistry
The synthetic route of compounds 1a–1q is outlined in
Scheme 1. 6-Bromo-[1,2,4]triazolo[1,5-a]pyridine-2-ylamine was
reacted successively with CDI (N,N’-carbonyldiimidazole) and
amine to yield intermediates 2a–2l. In the case of preparing 2l, gly-
cine ethyl ester was used as an amine. The ester group in interme-
diate 2l was hydrolyzed to afford the carboxylic acid 2m, which
was subsequently condensed with pyrrolidine, piperidine or mor-
pholine to produce intermediates 2n–2p. The phenylsulfonamides
3a–3d were prepared from 2-amino-5-bromopyridine according to
is dysregulated expression in many cancers, contributing to cellu-
lar transformation and tumor growth.1 Therefore, PI3K
4
a
and
mTOR, as key nodes of the PI3K/AKT/mTOR pathway, have been
1
5,16
identified as promising kinase targets for cancer therapy.
Recently, several PI3K inhibitors and PI3K/mTOR dual inhibitors
17,18
have been in clinical development.
Among the reported
PI3K/mTOR dual inhibitors, N-(5-(quinilin-6-yl)-pyridin-3-
yl)phenylsulfonamide derivatives are a class of PI3K/mTOR dual
inhibitors with potent anticancer activity in vitro and in vivo.
Two ring nitrogen atoms in pyridine and quinoline are the main
components of pharmacophore. GlaxoSmithKline discovered
2
6
the synthetic route reported in our previous work. Catalyzed by
PdCl (dppf), intermediates 3a–3d were reacted with bis(pinaco-
2
lato)diboron to produce the corresponding arylboronic ester.
1
9
GSK2126458 (Fig. 1) as a potent, orally bioavailable PI3K
a
and
Without further isolation and purification, the produced aryl-
boronic ester was reacted with intermediates 2a–2k or 2n–2p to
afford compounds 1a–1q by Suzuki coupling. The preparation of
arylboronic esters and Suzuki coupling were completed in one
pot. All the structures of compounds 1a–1q were characterized
mTOR dual inhibitor. Amgen Inc. designed, synthesized and evalu-
ated several classes of N-(2,5-disubstituted-pyridin-3-yl)phenyl-
sulfonamides, and discovered that N-(2-chloro-5-(4-morpholino
quinilin-6-yl)pyridin-3-yl)-4-fluorophenylsulfonamide,20
N-(2-
chloro-5-(2-acetylaminobenzo[d]thiazol-6-yl)pyridin-3-yl)-4-fluo-
1
13
by H NMR, C NMR and HRMS.
2
1
rophenylsulfonamide (compound A, Fig. 1) and N-(2-chloro-5-(2-
acetylamonoimidazo[1,2-b]pyridazin-6-yl)pyridin-3-yl)-4-fluoro
3
3
. Results and discussion
2
2
phenylsulfonamide are excellent PI3K/mTOR dual inhibitors and
anticancer agents. According to the X-ray cocrystal structure of
.1. Antiproliferative activities in vitro
We first evaluated the antiproliferative activities of the synthe-
PI3Kc
with GSK2126458,19 we proposed that the structure of an
amide group may take the place of the water molecule bridge.
Thereupon, we synthesized a series of 2-substituted-3-phenylsul-
fonylamino-5-(quinazolin-6-yl or quinolin-6-yl)benzamides and
discovered that the designed compounds are novel PI3K inhibitors
sized compounds against human colon carcinoma cell line (HCT-
16, PI3CA, mutant: H1047R), human breast adenocarcinoma car-
cinoma cell line (MCF-7, PI3CA, mutant: E545K), glioma cell line
1
2
3
and anticancer agents. Thereafter, we combined the benzamide
moiety with 2-aminobenzothiazole to discover novel anticancer
(
(
U87 MG, PTEN null) and lung adenocarcinoma epithelial cell line
A549, KRAS mutant) by applying the MTT (3-(4,5-dimethylthia-
2
4
agents. Recently, it has been reported that PI3K/mTOR dual inhi-
zol-2-yl)-2,5-diphenyltetrazolium bromide) colorimetric assay.
The PI3K and mTOR dual inhibitor BEZ235 was used as the positive
control. The results are summarized in Table 1.
2
5
bitor VS-5584 can preferentially targets cancer stem cells. This
discovery may potentially bring a breakthrough to the treatment
of cancer. Thus, developing new PI3K/mTOR dual inhibitors is still
needed.
As the data shown in Table 1, the designed compounds displayed
significant antiproliferative activities against four human cancer cell
lines, some of which were comparable to that of BEZ235. Moreover,
most of compounds showed more potent activities against HCT-116
than the other cells. Firstly, compounds with a small alkyl urea moi-
ety (1a, 1b and 1c) displayed potent antiproliferative activities
against four cancer cell lines and compound 1c exhibited 3-fold
activity against HCT-116 than that of BEZ235. These results indicate
that alkylureido is a suitable substituent at the 2-position of 1,2,4-
triazolo[1,5-a]pyridine core. Secondly, compounds 1d–1i, produced
by the replacement of acetylamino in compound B with a 2-(dialky-
lamino)ethylureido, retained the potency against HCT-116 and
MCF-7. However, their activity against U87 MG and A549 dropped.
Among compounds 1d–1i, compounds 1e and 1f exhibited the most
potent antiproliferative activities against the four cancer cell lines,
which is comparable to BEZ235. These results suggest that 2-(di-
alkylamino)ethylureido moiety is tolerated at the 2-position of
In an attempt to develop novel anticancer agents, we combined
2
-acetylamino-[1,2,4]triazolo[1,5-a]pyridine with N-pyridin-3-
ylphenylsulfonamide to synthesize a series of [1,2,4]triazolo[1,5-
a]pyridinylpyridines and the anticancer effects of the synthesized
compounds were evaluated in vitro and in vivo. Therefore we dis-
covered that compound B (Fig. 1) displayed potent antiprolifera-
2
6
tive activities in vitro and remarkable anticancer effects in vivo.
However, the body weight of mice dropped below 90% of the start-
ing body weight over the course of oral administration at 5 mg/kg,
which indicated that compound
B displayed some toxicity.
Therefore, the toxicity of compound B caught our attention.
To reduce the toxicity and retain the anticancer effect of com-
pound B, we intend to replace the 2-acetylamide in compound B
with chain-extended urea to search for the novel anticancer agents
with low toxicity (Fig. 2). In this paper, we reported our studies
on the synthesis, biological activities evaluation of a series of
1
,2,4-triazolo[1,5-a]pyridine core. Replacing the 4-fluorophenyl in
1
-alkyl-3-(6-(2-methoxy-3-sulfonylaminopyridin-5-yl)-[1,2,4]tria-
compound 1e with 4-chlorophenyl, 4-methylphenyl or 2,4-difluo-
rophenyl gave compounds 1j–1l, which displayed the similar
zolo[1,5-a]pyridin-2-yl)urea derivatives.
F
S
F
NC
F
F
O
O
O
N
HN
N
S
O
S
O
O
HN
HN
O
N
MeO
N
N
Cl
MeO
N
N
N
N
S
N
N
N
N
NHAc
NHAc
N
GSK2126458
BEZ235
A
B
Figure 1. The structures of PI3K and mTOR dual inhibitors.
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X.-M. Wang et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
3
F
F
O
O
S
S
HN
HN
O
O
H
3
CO
3
H CO
R
O
O
N
N
N
N
CH
3
N
N
NH
N
N
NH
NH
B
Designed compounds
Figure 2. The design strategy of title compounds.
O
5
3
Br
Br
Br
N
N
R1
N
N
N
a
N
b
NH2
NH
a-2l
7
N
1
2
O
O
CO
2
Et
O
CO H
2
R1
Br
N
N
NH
Br
N
N
NH
c
N
N
N
N
NH
NH
NH
2l
2m
2n-2p
2f: R1
NHCH
CH
N
N
O
2k: R1
N
2
2
2
2
2
a: R1
=
NHMe
=
2
2
=
=
=
=
=
b: R1
2g: R1
2h: R1
2i: R1
2j: R1
2
l: R1
2
NHCH COOEt
=
=
NHCH
2
CH
2
CH
3
=
=
=
=
NHCH
2
CH
2
NMe
H
N
c: R1
2n: R1
2o: R1
2p: R1
NHCH CH N
2
2
NHCH CON
2
2
d: R1
NHCH CH NMe
2 2 2
=
=
NHCH
2
CH
2
N
NHCH CON
e: R1
NHCH
2
CH
2
NEt
2
NMe2
O
2
NHCH CON
R2
O
R2
O
S
O
S
HN
HN
O
d
H CO
3
H
3
CO
O
R1
N
N
N
N
Br
NH
N
3
a-3d
1a-1q
1a: R1
1b: R1
=
=
=
R2 = 4-F
1j: R1
R2 = 4-Cl
a: R2 = 4-Cl
=
=
NHCH
NHCH CH NEt
2
CH
NEt
3
NHMe
NHCH CH CH
3
2
2
2
R2 = 4-F
R2 = 4-F
1k: R1
R2 = 4-Me
b: R2 = 4-Me
c: R2 = 2,4-diF
d: R2 = 4-F
2
2
3
3
3
2
2
H
1
1
c: R1
N
1
R2 = 2,4-diF
1l: R =
NHCH CH NEt
2 2 2
1
R2 = 4-F
R2 = 4-F
R2 = 4-F
d: R =
NHCH
NHCH
NHCH CH N
2
CH
2
NMe
2
1m: R1
NHCH CON
R2 = 4-F
R2 = 4-F
R2 = 4-F
R2 = 4-F
R2 = 4-F
=
2
1
e: R1
=
2
CH
2
NEt
2
1
1
1
1
n: R1
o: R1
p: R1
q: R1
=
=
=
=
2
NHCH CON
1
f: R1
g: R1
h: R1
i: R1
=
O
2
2
NHCH CON
O
2
1
1
1
=
=
=
NHCH CH N
2
2
NMe R2 = 4-F
NMe
N
2
R2 = 4-F
R2 = 4-F
2 2
NHCH CH N
NHCH
2
CH N
2
1
Scheme 1. Reagents and conditions: (a) (i) N,N’-carbonyldiimidazole (CDI), NaH, DMF, 60 °C; (ii) R H, DMF, 60 °C, 6 h, 68.0–93.7%; (b) 2 mol/L NaOH, MeOH/H
0.2%; (c) morpholine or piperidine or pyrrolidine, EDCI, HOBt, DMF, rt, 8 h, 85.2–91.0%; (d) (i) bis(pinacolato)diboron, PdCl
or 2n–2p, PdCl (dppf), Na CO , DME/EtOH/H O (7:3:2), reflux, 2 h, 41.5–73.7%.
2
O (1: 1), rt, 4 h,
2
(dppf), KOAc, 1,4-dioxane, reflux, 2 h; (ii) 2a–2k
9
2
2
3
2
potency with compound 1e. These results are consistent with the
observation in our previous work.26 Thirdly, the replacement of
acetylamino in compound B with an aminocarbonylmethylureido
moiety gave compounds 1m–1o, which displayed a drop in cell-
based activity, especially against U87 MG and A549 cells. Fourthly,
To probe the 1,1-dialkyl substituted urea moiety on activity, com-
pounds 1p and 1q were synthesized. The antiproliferative activity
of 1p and 1q displayed proximately a 5-fold loss in potency, which
further verified that the two hydrogen atoms in the urea is
indispensable. The results reveal that 2-methoxy-3-(4-fluo-
rophenylsulfoamino)pyridine moiety linked at the 6-position of
1,2,4-triazolo[1,5-a]pyridine is beneficial for antiproliferative
activity in vitro.
From the above results, we can conclude that the alkyl urea or
the 2-(dialkylamino)ethyl urea linked to the 2-position and 2-
methoxy-3-(4-fluorophenylsulfoamino)pyridine moiety attached
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4
X.-M. Wang et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
Table 1
Table 3
Antiproliferative activities of compounds 1 ( ꢀx ꢀ s, n = 3)
The acute oral toxicity of tested compounds
Compds
IC50
(l
M)
U87 MG
F
HCT-116
MCF-7
A549
O
S
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
a
b
c
d
e
f
g
h
i
j
k
l
m
n
o
p
q
0.18 ± 0.02
0.15 ± 0.04
0.09 ± 0.03
0.22 ± 0.01
0.18 ± 0.07
0.13 ± 0.02
0.26 ± 0.04
0.23 ± 0.01
0.15 ± 0.02
0.12 ± 0.03
0.17 ± 0.01
0.14 ± 0.02
0.14 ± 0.01
0.13 ± 0.02
0.49 ± 0.07
0.76 ± 0.06
1.29 ± 0.31
0.29 ± 0.03
0.25 ± 0.02
0.36 ± 0.08
0.30 ± 0.06
0.56 ± 0.03
0.37 ± 0.04
0.21 ± 0.02
0.50 ± 0.09
0.38 ± 0.13
0.20 ± 0.05
0.68 ± 0.05
0.41 ± 0.05
0.74 ± 0.13
2.13 ± 0.04
0.57 ± 0.06
0.63 ± 0.03
1.56 ± 0.45
1.66 ± 0.44
0.26 ± 0.08
0.64 ± 0.13
0.42 ± 0.11
0.54 ± 0.12
0.98 ± 0.03
0.72 ± 0.10
0.56 ± 0.05
0.76 ± 0.07
1.12 ± 0.14
1.08 ± 0.18
0.76 ± 0.03
0.67 ± 0.13
1.06 ± 0.08
1.00 ± 0.20
0.74 ± 0.03
3.26 ± 0.34
1.78 ± 0.17
1.69 ± 0.24
0.77 ± 0.04
0.23 ± 0.03
0.22 ± 0.02
0.21 ± 0.02
0.80 ± 0.08
0.76 ± 0.06
0.42 ± 0.02
1.05 ± 0.10
0.96 ± 0.16
0.61 ± 0.10
0.68 ± 0.12
0.73 ± 0.04
0.98 ± 0.16
2.19 ± 0.12
2.06 ± 0.31
2.57 ± 0.34
1.19 ± 0.19
3.57 ± 0.23
0.28 ± 0.10
HN
O
3
H CO
O
N
N
N
R
N
NH
Compds
R
LD50 (mg/kg)
95% confidence
interval (mg/kg)
B
CH
3
28
25–33
1
1
1
b
2
NHCH CH
2
CH
3
156
133–175
H
N
c
118
513
101–133
400–530
e
NHCH CH NEt
2 2 2
BEZ235
toxicity of compound 1e, produced by the replacement of 2-acety-
lamino moiety in compound B with a 2-(diethylamino)ethylurea
moiety, reduced dramatically. In summary, we have identified
the new PI3K inhibitor with low toxicity by optimizing compound
B.
at the 6-position of 1,2,4-triazolo[1,5-a]pyridine core are beneficial
for the antiproliferative activities of title compounds.
3
.2. PI3K and mTOR enzymatic activity
Next, to elucidate the mechanism of antiproliferative activities
3.4. Anticancer effect in vivo
of title compounds, compounds 1c and 1e were selected to evalu-
ate their inhibitory activity against PI3Ks and mTOR by performing
Lastly, we evaluated whether compound 1e could inhibit tumor
growth in established mice homograft model. A study using mice
bearing sarcoma S-180 was performed. Compound 1e was dosed
orally at 5 mg/kg, 10 mg/kg or 30 mg/kg once a day for 8 days.
Considering that it is difficult to measure the volumes of S-180
tumor, tumor weights were used as evaluating indicators. The
tumor weights and daily body weights were depicted in Fig. 3.
The inhibitory ratios of compound 1e at 5 mg/kg, 10 mg/kg and
30 mg/kg were 37.3%, 49.0% and 53.6%, respectively, which showed
a dose-dependent effect. In addition, the body weight of all groups
increased during the treatment. These results suggest that com-
pound 1e is an effective anticancer agent with low toxicity.
2
7,28
an ATP depletion assay.
BEZ235 was used as the positive con-
trol. the results of the PI3Ks and mTOR enzymatic activity are sum-
marized in Table 2.
Compounds 1c and 1e displayed remarkable potency against
PI3K and mTOR, especially against PI3K. Compared with BEZ235,
compounds 1c and 1e displayed potent activities against PI3K
and weaker activities against mTOR. The inhibitory activities of
compounds 1c and 1e against PI3Ka were 7 nM and 14 nM, which
are approximately 8-fold and 3-fold more potent than that of
BEZ235, respectively. PIK3CA is one of the most frequently muta-
tions in human cancer. Considering PIK3CA mutations in both
HCT-116 and MCF-7, the excellent potency of compounds 1c and
1
e on PI3K
a
further explained the significant antiproliferative
3.5. Docking study
activities of compounds 1c and 1e against HCT-116 and MCF-7.
These data suggest that compounds 1c and 1e are potent PI3K
inhibitors.
To further explain the potent activities of compounds 1c and 1e,
we performed a docking analysis utilizing the C-DOCKER program
within Discovery Studio 2.5 software package. Docking simulations
2
1
3
.3. Acute oral toxicity in vivo
were carried out on human PI3K
pounds 1c and 1e and the results were depicted in Fig. 4. From
the docking results of compounds 1c and 1e with PI3K , we
c (PDB code 3QK0) with com-
Thirdly, we picked out compounds B, 1b, 1c and 1e to explore
c
their acute oral toxicities in mice by employing an ‘up-and-down
observed that: (1) 2-substituted ureido[1,2,4]triazolo[1,5-a]pyri-
dine formed hydrogen bonds with Val882 and Val885; (2) the
nitrogen atom of pyridine formed hydrogen bonds directly with
Tyr867 and Asp864 or interacted with Asp841 via an ordered water
molecule; the oxygen atom at 2-position and 3-sulfonamido of
pyridine simultaneously formed hydrogen bonds with Lys833. In
addition, the fluorine atom in phenyl of compound 1c formed a
hydrogen bond with Asn951, which may be related to the antipro-
liferative activity of 1c and 1e.
2
9,30
procedure’ (UDP).
Mice were treated orally the solution of
tested drugs and the LD50s (median lethal doses) of tested com-
pounds are listed in Table 3. The LD50 values of compounds 1b,
1
c and 1e were about 5-fold, 4-fold and 18-fold higher than that
of compound B. These data reveal that the acute oral toxicity is clo-
sely related to the structure of R. To our surprise, the acute oral
Table 2
Inhibitory activity of 1c and 1e against PI3K and mTOR (n = 2)
4
. Conclusions
Compds
IC50 (nM)
In present study, a series of 1-alkyl-3-(6-(5,6-disubstituted-
PI3K
a
PI3Kb
PI3K
c
PI3Kd
mTOR
pyridin-3-yl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl)urea
were synthesized and characterized. Their antiproliferative activi-
ties in vitro were evaluated via MTT assay against four cancer cell
derivatives
1
1
c
e
7
14
54
47
41
399
14
22
95
16
12
164
275
123
89
BEZ235
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X.-M. Wang et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
5
Figure 3. (A) The anticancer effect of compound 1e in establishing mice S180 homograft model. (B) The change of tested mice body weights. Mice bearing subcutaneous
cancers were treated orally vehicle, compound 1e (5 mg/kg, 10 mg/kg or 20 mg/kg) once daily for 8 days. **P <0.01.
Figure 4. The comparison of docking mode between 1c and 1e. (A) Docking mode of 1c with PI3K
c and (B) Docking mode of 1e with PI3Kc. Selected residues Ala885, Val882,
Tyr867, Asp841, Lys833, Asp964 and Asn951 are shown. Green dashed lines indicate hydrogen bond.
lines including HCT-116, MCF-7, U87 MG and A549. The SAR of the
title compounds was discussed. Compounds 1c and 1e were tested
for their inhibitory activity against PI3Ks and mTOR. Meanwhile,
the acute oral toxicity of four compounds was investigated by oral
administration. The results indicate that the compound with a
alkyl urea or a 2-(dialkylamino)ethylurea moiety at the 2-position
of [1,2,4]triazolo[1,5-a]pyridine moiety can retain the antiprolifer-
ative activity and the inhibitory activity against PI3K and mTOR. In
addition, their acute oral toxicity reduced dramatically. Moreover,
compound 1e can effectively inhibit tumor growth in a mice S180
homograft model. These findings suggest that designed com-
pounds can serve as potent PI3K inhibitors and anticancer agents
with low toxicity.
quadrupole time-of-flight (QTOF) mass spectrometer (Maxis Q-
TOF, Bruker Inc.).
5.1.1. General procedure for synthesis of compounds 2a–2l
To the suspension of sodium hydride (60%, 0.23 g, 5.66 mmol)
in dried dimethyl formamide (15 mL) was added 6-bromo (or 7-
bromo)-[1,2,4]triazolo[1,5-a]pyridin-2-amine (0.60 g, 2.82 mmol),
0
stirred for 10 min at room temperature, added N,N -carbonyldimi-
dazole (CDI, 1.37 g, 8.46 mmol), stirred at 60 °C until the starting
material consumed, then added amine (9.87 mmol) and stirred at
60 °C for 6 h. The volatile was removed under reduced pressure
to give a white pale yellow residue. Water (30 mL) was added to
the residue. The mixture was stirred. The precipitate was
collected by filtration, dried to afford the expected product as a
white solid.
5
5
. Experimental
5
.1.1.1. 1-(6-Bromo-[1,2,4]triazolo[1,5-a]pyridin-2-yl)-3-methy-
.1. Chemistry
1
lurea (2a).
White solid; yield: 79.6%; mp: 237–239 °C;
): d 10.05 (s, 1H, NH), 9.22 (d, J = 1.2 Hz, 1H, Ar-
= 1.6 Hz, J = 9.6 Hz,
H, Ar-H), 7.64 (d, J = 9.6 Hz, 1H, Ar-H), 2.78 (m, 6H, CH
ꢁ 3),
H
NMR (DMSO-d
H), 8.04 (d, J = 4.4 Hz, 1H, NH), 7.80 (dd, J
6
Unless specified otherwise, all the starting materials, reagents
and solvents were commercially available. All the reactions were
monitored by thin-layer chromatography on silica gel plates (GF-
1
2
1
1
2
+
3
.50 (d, J = 4.8 Hz, 3H, CH ). MS m/z (ESI): 270.0 [M+H] .
2
54) and visualized with UV light. All the melting points were
determined on a Beijing micro melting-point apparatus and ther-
mometer was uncorrected. NMR spectra were recorded on a 400
Bruker NMR spectrometer with tetramethylsilane (TMS) as an
internal reference. All chemical shifts are reported in parts per mil-
lion (ppm). High-resolution exact mass measurements were per-
5
.1.1.2. 1-(6-Bromo-[1,2,4]triazolo[1,5-a]pyridin-2-yl)-3-propy-
1
lurea (2b).
NMR (CDCl
White solid; yield: 91.4%; mp: 188–190 °C;
): d 9.67 (s, 1H, NH), 8.80 (s, 1H, Ar-H), 8.16 (m, 1H,
), 1.70 (m, 2H, CH ),
H
3
NH), 7.63 (m, 2H, Ar-H), 3.42 (m, 2H, CH
.03 (m, 3H, CH ). MS m/z (ESI): 298.1 [M+H] .
2
2
+
1
3
formed using electrospray ionization (positive mode) on
a
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6
X.-M. Wang et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
5
.1.1.3. 1-(6-Bromo-[1,2,4]triazolo[1,5-a]pyridin-2-yl)-3-cyclo-
White solid; yield: 89.0%; mp: 204–206 °C;
): d 9.88 (s, 1H, NH), 8.80 (s, 1H, Ar-H), 8.25 (m,
H, NH), 7.71 (d, J = 9.2 Hz, 1H, Ar-H), 7.61 (dd, J = 1.6 Hz,
= 9.6 Hz, 1H, Ar-H), 2.84 (m, 1H, CH), 0.87 (m, 2H, CH ), 0.70
8,72 (m, 1H, NH), 7.63 (d, J = 1.2 Hz, 2H, Ar-H), 4.28 (m, 2H,
OCH ), 4.02 (s, 2H, CH ), 1.34 (m, 3H, CH ). MS m/z (ESI): 342.0
[M+H] .
propylurea (2c).
2
2
3
1
+
H NMR (CDCl
3
1
1
J
2
2
5.1.2. 2-(3-(6-Bromo-[1,2,4]triazolo[1,5-a]pyridin-2-yl)ureido)-
acetic acid (2m)
+
(
2
m, 2H, CH ). MS m/z (ESI): 296.0 [M+H] .
To the solution of compound 2l (0.68 g, 2 mmol) in methanol
(10 mL) was added NaOH (2 mol/L) in water (10 mL) and the mix-
ture was stirred for 4 h at room temperature. The solvent was
evaporated under reduced pressure to afford a white residue. The
residue was dissolved in water (20 mL) and the solution was
adjusted to pH 4–5 with 6 mol/L HCl whereupon a white precipi-
tate formed. The precipitate was collected by filtration under
5
.1.1.4.
1-(6-Bromo-[1,2,4]triazolo[1,5-a]pyridin-2-yl)-3-(2-
(
dimethylamino)ethyl)urea (2d).
White solid; yield: 77.7%;
): d 9.56 (s, 1H, NH), 8.76 (s, 1H,
1
mp: 195–197 °C; H NMR (CDCl
Ar-H), 8.31 (s, 1H, NH), 7.61 (m, 2H, Ar-H), 3.55 (m, 2H, CH
3
2
),
2
.59 (m, 2H, CH ), 2.36 (s, 6H, CH
2
3
ꢁ 2). MS m/z (ESI): 327.0
+
[
M+H] .
reduced pressure, washed with water and dried to afford com-
1
5
.1.1.5.
1-(6-Bromo-[1,2,4]triazolo[1,5-a]pyridin-2-yl)-3-(2-
pound 2m as a white solid. Yield: 90.2%; mp: 233–235 °C;
H
(
diethylamino)ethyl)urea (2e).
White solid; yield: 68.0%;
): d 9.51 (s, 1H, NH), 8.75 (s, 1H,
NMR (DMSO-d ): d 9.59 (s, 1H, NH), 8.79 (s, 1H, Ar-H), 8.72 (m,
6
1
mp: 148–150 °C; H NMR (CDCl
3
1H, NH), 7.63 (d, J = 1.2 Hz, 2 H, Ar-H), 4.28 (m, 2H, OCH ), 4.02
2
+
Ar-H), 8.40 (s, 1H, NH), 7.60 (m, 2H, Ar-H), 3.51 (m, 2H, CH
.71 (m, 2H, CH ), 2.64 (m, 4H, CH ꢁ 2).
ꢁ 2), 1.11 (m, 6H, CH
MS m/z (ESI): 355.1 [M+H] .
2
),
(s, 2H, CH ), 1.34 (m, 3H, CH ). MS m/z (ESI): 314.0 [M+H] .
2
3
2
2
2
3
+
5
.1.3. General procedure for synthesis of compounds 2n–2p
A mixture of compound 2m (0.63 g, 2 mmol), alkyl amine
5
.1.1.6.
1-(6-Bromo-[1,2,4]triazolo[1,5-a]pyridin-2-yl)-3-(2-
(
8 mmol), N-hydroxybenzotrizole (HOBt, 0.68 g, 5 mmol), 1-
ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 0.76 g,
mmol) and DMF (8 mL) was stirred at room temperature for
h and the solvent was removed under reduced pressure to give
a yellow residue. The residue was suspended into water (20 mL)
whereupon a precipitate formed. The precipitate was collected by
suction, washed with water and dried to afford the compound as
a white solid.
morpholinoethyl)urea (2f).
White solid; yield: 92.3%; mp:
): d 9.51 (s, 1H, NH), 8.78 (s, 1H, Ar-
1
2
18–220 °C; H NMR (CDCl
3
4
8
H), 8.49 (s, 1H, NH), 7.63 (m, 2H, Ar-H), 3.81 (m, 4H, CH
2
ꢁ 2),
3
.58 (m, 2H, CH ), 2.63 (m, 6H, CH
2
2
ꢁ 3). MS m/z (ESI): 369.0
+
[
M+H] .
5
.1.1.7. 1-(6-Bromo-[1,2,4]triazolo[1,5-a]pyridin-2-yl)-3-(2-(4-
methylpiperazin-1-yl)ethyl)urea (2g).
White solid; yield:
): d 9.60 (s, 1H, NH), 8.80
1
8
1.0%; mp: 182–184 °C; H NMR (CDCl
3
5
2
.1.3.1. 1-(6-Bromo-[1,2,4]triazolo[1,5-a]pyridin-2-yl)-3-(2-oxo-
(
s, 1H, Ar-H), 8.46 (s, 1H, NH), 7.63 (m, 2H, Ar-H), 2.60 (m, 10H,
-(pyrrolidin-1-yl)ethyl)urea (2n).
White solid; yield: 91.0%;
+
CH
2
ꢁ 5), 2.36 (s, 3H, CH
3
). MS m/z (ESI): 382.1 [M+H] .
1
3
mp: 248–250 °C; H NMR (CDCl ): d 9.68 (s, 1H, NH), 9.02 (s, 1H,
NH), 8.82 (s, 1H, Ar-H), 7.69 (d, J = 9.2 Hz, 1H, Ar-H), 7.61 (d,
J = 9.2 Hz, 1H, Ar-H), 4.22 (d, J = 3.6 Hz, 2H, CH ), 3.53 (m, 4H,
CH ꢁ 2). MS m/z (ESI): 367.0 [M+H] .
ꢁ 2), 1.98 (m, 4H, CH
5
.1.1.8. 1-(6-Bromo-[1,2,4]triazolo[1,5-a]pyridin-2-yl)-3-(2-
2
(
pyrrolidin-1-yl)ethyl)urea (2h).
White solid; yield: 79.2%;
+
1
2
2
mp: 172–174 °C; H NMR (CDCl
3
): d 9.51 (s, 1H, NH), 8.75 (s, 1H,
Ar-H), 8.39 (s, 1H, NH), 7.61 (m, 2H, Ar-H), 3.61 (m, 2H, CH
.80 (m, 2H, CH ), 2.68 (s, 4H, CH ꢁ 2). MS
ꢁ 2), 1.86 (s, 4H, CH
m/z (ESI): 353.1 [M+H] .
2
),
5
.1.3.2. 1-(6-Bromo-[1,2,4]triazolo[1,5-a]pyridin-2-yl)-3-(2-oxo-
2
2
2
2
+
2-(piperidin-1-yl)ethyl)urea (2o).
mp: 247–249 °C; H NMR (CDCl
White solid; yield: 85.2%;
1
3
): d 9.56 (s, 1H, NH), 9.04 (s, 1H,
NH), 8.82 (s, 1H, Ar-H), 7.69 (d, J = 9.2 Hz, 1H, Ar-H), 7.61 (dd,
= 1.2 Hz, J = 9.2 Hz, 1H, Ar-H), 4.29 (d, J = 3.6 Hz, 2H, CH ), 3.66
(m, 2H, CH ), 3.43 (m, 2H, CH ), 1.66 (m, 6H, CH
ꢁ 3). MS m/z
5
.1.1.9.
1-(6-Bromo-[1,2,4]triazolo[1,5-a]pyridin-2-yl)-3-(2-
J
1
2
2
(
piperidin-1-yl)ethyl)urea (2i).
White solid; yield: 71.1%;
): d 10.03 (s, 1H, NH), 9.19
1
2
2
2
mp: 180–181 °C; H NMR (DMSO-d
6
+
(
ESI): 381.1 [M+H] .
(
d, J = 1.2 Hz, 1H, Ar-H), 8.38 (m, 1H, NH), 7.80 (dd, J
= 9.2 Hz, 1H, Ar-H), 7.57 (d, J = 9.2 Hz, 1H, Ar-H), 3.30 (m, 2H,
CH ), 2.41 (m, 6H, CH
1
= 1.6 Hz,
J
2
5
.1.3.3.
1-(6-Bromo-[1,2,4]triazolo[1,5-a]pyridin-2-yl)-3-(2-
2
2
ꢁ 3), 1.50 (m, 6H, CH ꢁ 3). MS m/z (ESI):
2
+
(morpholino-2-oxoethyl)urea (2p).
White solid; yield:
): d 10.16 (s, 1H, NH),
3
67.1 [M+H] .
1
8
9
8.2%; mp: >250 °C; H NMR (DMSO-d
6
.23 (s, 1H, Ar-H), 8.54 (m, 1H, NH), 7.80 (dd, J
= 9.2 Hz, 1H, Ar-H), 7.63 (d, J = 9.2 Hz, 1H, Ar-H), 4.14 (d,
J = 4.8 Hz, 2H, CH ), 3.52 (m, 8H, CH
ꢁ 4). MS m/z (ESI): 383.0
1
= 1.6 Hz,
5
.1.1.10.
3-(6-Bromo-[1,2,4]triazolo[1,5-a]pyridin-2-yl)-1,1-
J
2
dimethylurea (2j).
White solid; yield: 75.7%; mp: 154–
1
2
2
1
56 °C; H NMR (CDCl
= 2.0 Hz, J
J = 9.2 Hz, 1H, Ar-H), 3.09 (s, 6H, CH
3
): d 8.67 (d, J = 1.2 Hz, 1H, Ar-H), 7.57 (dd,
+
[
M+H] .
J
1
2
= 9.2 Hz, 1H, Ar-H), 7.52 (s, 1H, NH), 7.45 (d,
ꢁ 2). MS m/z (ESI): 284.0
3
+
[
M+H] .
5.1.4. General procedure for synthesis of compounds 1a–1q
The mixture of compounds 3a–3d (0.4 mmol), bis(pinaco-
5
.1.1.11. N-(6-Bromo-[1,2,4]triazolo[1,5-a]pyridin-2-yl)pyrro-
lato)diboron (0.11 g, 0.44 mmol), PdCl
potassium acetate (0.12 g, 1.2 mmol) and 1,4-dioxane (10 mL)
was refluxed for 2 h under N atmosphere. The solvent was evap-
2
(dppf) (0.02 g, 0.03 mmol),
lidine-1-carboxamide (2k).
White solid; yield: 72.8%; mp:
1
2
31–233 °C; H NMR (CDCl
3
): d 8.67 (d, J = 1.2 Hz, 1H, Ar-H), 7.56
2
(
dd, J = 2.0 Hz, J
1
2
= 9.2 Hz, 1H, Ar-H), 7.44 (d, J = 9.6 Hz, 1H, Ar-
orated off under reduced pressure to afford a dark brown residue.
H), 7.26 (s, 1H, NH), 3.53 (s, 4H, CH
MS m/z (ESI): 310.0 [M+H] .
2
ꢁ 2), 2.00 (s, 4H, CH
2
ꢁ 2).
To the residue was added compounds 2a–2k or 2n–2p
+
(0.32 mmol), PdCl
0.13 g, 1.2 mmol), 1,2-dimethoxyethane (10.5 mL), ethanol
(4.5 mL) water (3 mL) and the mixture was refluxed for 2 h under
atmosphere. The volatile was removed under reduced pressure
to afford a dark brown residue and the residue was purified by
2
(dppf) (0.02 g, 0.03 mmol), sodium carbonate
(
5
.1.1.12. Ethyl 2-(3-(6-bromo-[1,2,4]triazolo[1,5-a]pyridin-2-
yl)ureido)acetate (2l).
White solid; yield: 93.7%; mp: 186–
): d 9.59 (s, 1H, NH), 8.79 (s, 1H, Ar-H),
N
2
1
1
88 °C; H NMR (CDCl
3
Please cite this article in press as: Wang, X.-M.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.07.017

X.-M. Wang et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
7
silica
gel
column
chromatography
using
chloro-
228–230 °C; 1H NMR (DMSO-d
6
): d 10.03 (s, 2H, NH), 9.06 (s, 1H,
form/methanol = 15: 1 as the eluent to produce the title compound
as a white solid.
Ar-H), 8.43 (s, 1H, NH), 8.27 (s, 1H, Ar-H), 7.88 (m, 2H, Ar-H),
7.80 (m, 2H, Ar-H), 7.79 (d, J = 9.2 Hz, 1H, Ar-H), 7.38 (m, 2H, Ar-
H), 3.66 (s, 3H, OCH
1.03 (m, 6H, CH
(C), 163.1 (C), 160.0 (CH), 157.3 (C), 151.4 (C), 144.8 (C), 139.8
(C), 135.4 (C), 133.0 (CH), 132.8 (CH), 132.7 (CH), 128.5 (CH),
126.3 (CH), 123.7 (C), 119.3 (CH), 119.2 (CH), 117.2 (CH), 56.5
3
), 3.32 (m, 2H, CH
ꢁ 2). C NMR (DMSO-d
2
), 2.63 (m, 6H, CH
2
ꢁ 3),
1
3
5
.1.4.1. 1-(6-(2-Methoxy-3-(4-fluorophenylsulfonylamino) pyr-
3
6
): d 168.2 (C), 166.6
idin-5-yl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl)-3-methylurea
(
(
1a).
White solid; yield: 43.2%; mp: 237–239 °C; 1H NMR
6
DMSO-d ): d 10.11 (s, 1H, NH), 10.05 (s, 1H, NH), 9.16 (s, 1H, Ar-
H), 8.39 (s, 1H, Ar-H), 8.15 (d, J = 3.6 Hz, 1H, NH), 8.00 (s, 1H, Ar-
H), 7.92 (d, J = 9.2 Hz, 1H, Ar-H), 7.80 (m, 2H, Ar-H), 7.75 (d,
(CH
3
), 53.8 (CH
2
), 50.0 (CH
2
ꢁ 2), 38.5 (CH
2
), 12.4 (CH
S [M+H] : 557.2095; found:
3
ꢁ 2). ESI-
+
HRMS m/z: calcd for C25
H
30FN
8
O
4
J = 9.2 Hz, 1H, Ar-H), 7.41 (m, 2H, Ar-H), 3.63 (s, 3H, OCH
3
), 2.80
557.2089.
13
(
(
(
d, J = 4.0 Hz, 3H, CH ). C NMR (DMSO-d ): d 168.2 (C), 166.6
C), 163.3 (C), 160.0 (CH), 157.4 (C), 157.3 (C), 151.4 (C), 144.8
C), 139.8 (C), 135.4 (CH), 132.9 (CH), 132.8 (CH), 128.6 (CH),
3
6
5.1.4.6. 1-(6-(2-Methoxy-3-(4-fluorophenylsulfonylamino) pyr-
idine-5-yl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl)-3-(2-morpholi-
noethyl)urea (1f).
NMR (DMSO-d
1H, NH), 8.38 (s, 1H, Ar-H), 7.85 (m, 5H, Ar-H), 7.41 (m, 2H, Ar-H),
1
1
26.1 (CH), 123.6 (C), 119.3 (CH), 119.2 (CH), 117.1 (CH), 56.6
White solid; yield: 70.4%; mp: >250 °C; H
+
(
CH
3
), 29.4 (CH
3
). ESI-HRMS m/z: calcd for C20
7
H19FN O
4
S [M+H] :
6
): d 10.06 (bm, 2H, NH), 9.12 (s, 1H, Ar-H), 8.40 (s,
4
72.1203; found: 472.1198.
3
CH
.64 (s, 7H, OCH
3
, CH
2
ꢁ 2), 3.36 (s, 2H, CH
2
), 2.47 (d, J = 3.6 Hz, 6H,
): d 168.2 (C), 166.6 (C), 163.4 (C),
1
3
5
.1.4.2. 1-(6-(2-Methoxy-3-(4-fluorophenylsulfonylamino)pyr-
2
ꢁ 3). C NMR (DMSO-d
6
idin-5-yl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl)-3-propylurea
160.0 (CH), 157.0 (C), 151.4 (C), 144.7 (C), 139.8 (C), 135.3 (C),
(
1b).
White solid; yield: 44.7%; mp: 221–223 °C; 1H NMR
): d 10.11 (s, 1H, NH), 10.00 (s, 1H, NH), 9.17 (s, 1H, Ar-
132.9 (CH ꢁ 2), 132.8 (CH), 128.6 (CH), 126.2 (CH), 123.7 (C),
(
DMSO-d
6
119.3 (CH), 119.2 (CH), 117.2 (CH), 69.5 (CH
2
ꢁ 2), 60.3 (CH
2
),
H), 8.40 (d, J = 2.4 Hz, 1H, Ar-H), 8.30 (m, 1H, NH), 8.00 (d,
J = 2.4 Hz, 1H, Ar-H), 7.92 (dd, J = 9.2 Hz, J = 1.6 Hz, 1H, Ar-H),
.81 (m, 2H, Ar-H), 7.76 (d, J = 9.2 Hz, 1H, Ar-H), 7.41 (m, 2H, Ar-
56.6 (CH
3
), 56.2 (CH
2
ꢁ 2), 39.5 (CH
2
). ESI-HRMS m/z: calcd for
+
1
2
25 8 5
C H28FN O S [M+H] : 571.1887; found: 571.1882.
7
1
3
H), 3.63 (s, 3H, OCH
NMR (DMSO-d ): d 168.2 (C), 166.6 (C), 163.4 (C), 160.0 (CH),
56.8 (C), 151.4 (C), 144.9 (C), 139.7 (C), 135.6 (CH), 132.9 (CH),
32.8 (CH), 128.6 (CH), 128.6 (C), 126.1 (CH), 123.5 (C), 119.3
3
), 3.20 (m, 2H, CH
2
), 1.53 (m, 2H, CH
2
).
C
5.1.4.7. 1-(6-(2-Methoxy-3-(4-fluorophenylsulfonylamino) pyr-
idine-5-yl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl)-3-(2-(4-methyl-
6
1
1
pipe razin-1-yl)ethyl)urea (1g).
mp: 237–239 °C; H NMR (DMSO-d
White solid; yield: 62.5%;
): d 10.02 (s, 2H, NH), 9.09
1
6
(
1
5
CH), 119.1 (CH), 117.2 (CH), 56.6 (CH
3
), 44.2 (CH
2
), 26.0 (CH
2
),
(s, 1H, Ar-H), 8.48 (m, 1H, NH), 8.32 (d, J = 2.0 Hz, 1H, Ar-H), 7.95
(d, J = 2.4 Hz, 1H, Ar-H), 7.91 (dd, J = 9.2 Hz, J = 1.6 Hz, 1H, Ar-
H), 7.80 (m, 2H, Ar-H), 7.71 (d, J = 9.2 Hz, 1H, Ar-H), 7.39 (m, 2H,
Ar-H), 3.64 (s, 3H, OCH ), 3.35 (m, 2H, CH ), 2.48 (m, 10H,
CH ). ESI-HRMS m/z: calcd for
ꢁ 5), 2.25 (s, 3H, NCH
S [M+H] : 584.2204; found: 584.2198.
+
3
4.5 (CH ). ESI-HRMS m/z: calcd for
C
22
H23FN
7
O
4
S
[M+H] :
1
2
00.1516; found: 500.1511.
3
2
5
.1.4.3. 1-Cyclopropyl-3-(6-(2-methoxy-3-(4-fluorophenylsul-
2
3
+
fonylamino)pyridine-5-yl)-[1,2,4]triazolo[1,5-a]pyridin-2-
yl)urea (1c).
NMR (DMSO-d
26 9 4
C H31FN O
White solid; yield: 49.2%; mp: 236–238 °C; 1H
): d 10.11 (s, 1H, NH), 10.04 (s, 1H, NH), 9.17 (s,
6
5.1.4.8. 1-(6-(2-Methoxy-3-(4-fluorophenylsulfonylamino) pyr-
1
H, Ar-H), 8.39 (d, J = 2.0 Hz, 1H, Ar-H), 8.34 (s, 1H, NH), 8.00 (d,
J = 2.4 Hz, 1H, Ar-H), 7.92 (dd, J = 9.2 Hz, J = 1.6 Hz, 1H, Ar-H),
.80 (m, 2H, Ar-H), 7.75 (d, J = 9.2 Hz, 1H, Ar-H), 7.41 (m, 2H, Ar-
idine-5-yl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl)-3-(2-(pyrrolidin-
1
2
1-yl)ethyl)urea (1h).
White solid; yield: 45.8%; mp: 225–
1
7
227 °C; H NMR (DMSO-d
6
): d 10.00 (m, 2H, NH), 9.06 (s, 1H, Ar-
H), 3.63 (s, 3H, OCH
3
), 2.66 (m, 1H, CH), 0.72 (m, 2H, CH
2
), 0.52
H), 8.40 (m, 1H, NH), 8.25 (d, J = 2.0 Hz, 1H, Ar-H), 7.90 (d,
J = 2.4 Hz, 1H, Ar-H), 7.86 (dd, J = 9.2 Hz, J = 1.6 Hz, 1H, Ar-H),
7.80 (m, 2H, Ar-H), 7.71 (d, J = 9.2 Hz, 1H, Ar-H), 7.37 (m, 2H, Ar-
H), 3.66 (s, 3H, OCH ), 3.39 (m, 2H, CH ), 2.73 (m, 2H, CH ), 2.68
(s, 4H, CH ): d
+
13
(
(
(
m, 2H, CH
2
). MS m/z (ESI): [M+H] . C NMR (DMSO-d
6
): d 168.2
1
2
C), 166.6 (C), 163.2 (C), 160.0 (CH), 157.6 (C), 151.4 (C), 144.9
C), 139.7 (C), 135.6 (CH), 132.9 (CH), 132.8 (CH), 128.7 (CH),
3
2
2
1
3
1
28.5 (C), 126.1 (CH), 123.4 (C), 119.3 (CH), 119.1 (CH), 117.2
2
ꢁ 2), 1.76 (s, 4H, CH
2
ꢁ 2). C NMR (DMSO-d
6
(
CH), 56.5 (CH ), 25.6 (CH), 9.5 (CH
3
2
ꢁ 2). ESI-HRMS m/z: calcd
168.0 (C), 166.3 (C), 163.2 (C), 160.0 (CH), 156.9 (C), 151.4 (C),
143.0 (C), 140.7 (C), 133.8 (C), 132.9 (CH), 132.7 (CH), 132.6 (CH),
+
for C22
7
H20FN NaO
4
S [M+Na] : 520.1179; found: 520.1174.
1
28.4 (C), 128.4 (CH), 126.5 (CH), 119.1 (CH), 119.0 (CH), 117.1
(CH), 57.9 (CH ), 56.7 (CH ), 41.2 (CH ), 26.2
ꢁ 2), 56.4 (CH
(CH [M+H] :
ꢁ 2). ESI-HRMS m/z: calcd for
555.1938; found: 555.1933.
5
.1.4.4. 1-(2-(Dimethylamino)ethyl)-3-(6-(2-methoxy-3-(4-fluo-
rophenylsulfonyl amino)pyridine-5-yl)-[1,2,4]triazolo[1,5-
a]pyridin-2-yl)urea (1d). White solid; yield: 73.7%; mp:
): d 9.98 (bm, 2H, NH), 9.09 (s,
2
2
3
2
+
2
25 8 4
C H28FN O S
1
2
1
7
41–243 °C; H NMR (DMSO-d
6
H, Ar-H), 8.29 (d, J = 6.4 Hz, 2H, NH, Ar-H), 7.92 (s, 1H, Ar-H),
.87 (d, J = 8.8 Hz, 1H, Ar-H), 7.80 (m, 2H, Ar-H), 7.73 (d,
5.1.4.9. 1-(6-(2-Methoxy-3-(4-fluorophenylsulfonylamino) pyr-
idine-5-yl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl)-3-(2-(piperidin-1-
J = 9.2 Hz, 1H, Ar-H), 7.38 (m, 2H, Ar-H), 3.66 (s, 3H, OCH
3
), 3.37
), 2.31 (s, 6H,
6
): d 168.0 (C), 166.3 (C), 163.2 (C),
yl)ethyl)urea (1i).
White solid; yield: 63.2%; mp: 219–
221 °C; H NMR (DMSO-d ): d 10.05 (s, 1H, NH), 9.95 (m, 1H,
NH), 9.09 (s, 1H, Ar-H), 8.49 (s, 1H, NH), 8.32 (d, J = 2.0 Hz, 1H,
Ar-H), 7.95 (d, J = 2.0 Hz, 1H, Ar-H), 7.90 (dd, = 9.2 Hz,
= 1.6 Hz, 1H, Ar-H), 7.81 (m, 2H, Ar-H), 7.79 (d, J = 9.2 Hz, 1H,
Ar-H), 7.39 (m, 2H, Ar-H), 3.65 (s, 3H, OCH ), 3.36 (d, J = 5.6 Hz,
2H, CH ), 2.55 (m, 6H, CH ꢁ 2), 1.44 (d,
ꢁ 3), 1.58 (m, 4H, CH
J = 3.6 Hz, 2H, CH ). C NMR (DMSO-d ): d 168.1 (C), 166.4 (C),
63.3 (C), 160.0 (CH), 156.9 (C), 151.4 (C), 143.9 (C), 140.3 (C),
134.5 (C), 132.9 (CH), 132.8 (CH), 132.7 (CH), 128.5 (CH), 126.4
1
(
CH
1
1
d, J = 5.2 Hz, 2H, CH
2
), 2.54 (d, J = 8.8 Hz, 2H, CH
2
6
1
3
3
ꢁ 2). C NMR (DMSO-d
60.0 (CH), 156.9 (C), 151.4 (C), 144.9 (C), 140.6 (C), 135.4 (C),
32.9 (CH), 132.8 (CH), 132.7 (CH), 128.5 (CH), 126.5 (CH), 123.4
J
1
J
2
(
C), 119.1 (CH), 119.0 (CH), 117.2 (CH), 61.3 (CH
2
), 56.5 (CH
3
),
3
4
7.9 (CH
3
ꢁ 2), 40.2 (CH
2
). ESI-HRMS m/z: calcd for C23
H26FN
8
O
4
S
2
2
2
+
13
[
M+H] : 529.1776; found: 529.1782.
2
6
1
5
.1.4.5.
1-(2-(Diethylamino)ethyl)-(6-(2-methoxy-3-(4-fluo-
amino)pyridine-5-yl)-[1,2,4]triazolo[1,5-
a]pyridin-2-yl)urea (1e). White solid; yield: 49.0%; mp:
rophenylsulfonyl
(CH), 124.7 (C), 119.2 (CH), 119.1 (CH), 117.0 (CH), 60.3 (CH
56.8 (CH ), 39.6 (CH ), 28.4 (CH ꢁ 2), 26.8 (CH
ꢁ 2), 56.5 (CH
2
),
).
2
3
2
2
2
Please cite this article in press as: Wang, X.-M.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.07.017

8
X.-M. Wang et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
+
ESI-HRMS m/z: calcd for C26
8
H30FN O
4
S [M+H] : 569.2095; found:
(CH), 117.2 (CH), 56.6 (CH
3
), 47.9 (CH
ꢁ 2). ESI-HRMS m/z: calcd for C25
91.1550; found: 591.1545.
2
ꢁ 2), 45.6 (CH
2
), 26.8
NaO S [M+Na] :
+
5
69.2089.
(CH
2
H
25FN
8
5
5
5
.1.4.10. 1-(2-(Diethylamino)ethyl)-3-(6-(2-Methoxy-3-(4-chl-
amino)pyridine-5-yl)-[1,2,4]triazolo[1,5-
a]pyridin-2-yl)urea (1j). White solid; yield: 59.5%; mp:
): d 10.05 (s, 2H, NH), 9.05 (s, 1H,
Ar-H), 8.44 (m, 1H, NH), 8.25 (d, J = 2.0 Hz, 1H, Ar-H), 7.90 (d,
J = 2.4 Hz, 1H, Ar-H), 7.87 (dd, J = 9.2 Hz, J = 2.0 Hz, 1H, Ar-H),
.74 (d, J = 8.4 Hz, 2H, Ar-H), 7.70 (d, J = 6.8 Hz, 1H, Ar-H), 7.61
d, J = 8.4 Hz, 2H, Ar-H) 3.66 (s, 3H, OCH ), 3.34 (s, 2H, CH ), 2.66
ꢁ 2). C NMR (DMSO-d ): d
63.3 (C), 160.0 (CH), 157.0 (C), 156.9 (C), 151.4 (C), 143.5 (C),
42.8 (C), 140.1 (C), 133.5 (C), 132.9 (CH), 132.0 (CH ꢁ 2), 131.6
orophenylsulfonyl
5
.1.4.14.
1-(6-(2-Methoxy-3-(4-fluorophenylsulfonylamino)-
1
pyridine-5-yl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl)-3-(2-oxo-2-
2
00–202 °C; H NMR (DMSO-d
6
(
piperidin-1-yl)urea (1n).
White solid; yield: 67.8%; mp:
1
>250 °C; H NMR (DMSO-d
6
): d 10.14 (s, 1H, NH), 10.10 (s, 1H,
1
2
NH), 9.15 (s, 1H, Ar-H), 8.65 (s, 1H, NH), 8.40 (d, J = 2.0 Hz, 1H,
Ar-H), 8.00 (d, J = 2.4 Hz, 1H, Ar-H), 7.92 (dd, = 9.2 Hz,
= 1.2 Hz, 1H, Ar-H), 7.82 (m, 2H, Ar-H), 7.74 (d, J = 9.2 Hz, 1H,
Ar-H), 7.42 (m, 2H, Ar-H), 4.13 (d, J = 5.6 Hz, 2H, CH ), 3.63 (s,
H, OCH ), 3.46 (m, 2H, CH ),3.36 (s, 2H, CH ), 1.57 (m, 4H,
CH ). C NMR (DMSO-d ): d
ꢁ 2), 1.46 (d, J = 3.6 Hz, 2H, CH
69.6 (C), 168.2 (C), 166.6 (C), 163.3 (C), 160.0 (CH), 156.7 (C),
151.5 (C), 144.9 (C), 139.7 (C), 135.4 (C), 132.9 (CH), 132.9 (CH),
32.9 (CH), 128.6 (C), 126.1 (CH), 123.5 (CH), 119.3 (CH), 119.2
CH), 117.2 (CH), 56.5 (CH ), 48.0 (CH ), 45.4 (CH ), 44.8 (CH ),
), 27.1 (CH ). ESI-HRMS m/z: calcd for
S [M+Na] : 605.1707; found: 605.1701.
7
J
1
(
(
1
1
3
2
13
J
2
m, 6H, CH
2
ꢁ 3), 1.05 (s, 6H, CH
3
6
2
3
3
2
2
1
3
2
2
6
(
CH ꢁ 2), 128.4 (CH), 128.3 (C), 126.6 (CH), 117.0 (CH), 56.4
CH ), 54.7 (CH ), 49.7 (CH ), 14.4 (CH ꢁ 2). ESI-
ꢁ 2), 40.4 (CH
30ClN S [M+H] : 573.1799; found:
1
(
3
2
2
2
3
+
HRMS m/z: calcd for C25
H
8 4
O
1
(
5
73.1794.
3
2
2
2
2
8.9 (CH
2
), 28.3 (CH
NaO
2
2
5
.1.4.11. 1-(2-(Diethylamino)ethyl)-3-(6-(2-methoxy-3-(4-me-
amino)pyridine-5-yl)-[1,2,4]triazolo[1,5-
+
C
26
H27FN
8
5
thylphenylsulfonyl
a]pyridin-2-yl)urea (1k).
White solid; yield: 68.0%; mp:
1
5.1.4.15.
1-(6-(2-Methoxy-3-(4-fluorophenylsulfonylamino)-
1
88–190 °C; H NMR (DMSO-d
6
): d 10.02 (s, 2H, NH), 9.05 (s, 1H,
pyridine-5-yl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl)-3-(2-(mor-
pholino-2-oxoethyl)urea (1o).
mp: >250 °C; H NMR (DMSO-d
Ar-H), 8.43 (m, 1H, NH), 8.29 (d, J = 2.0 Hz, 1H, Ar-H), 7.90 (d,
J = 2.4 Hz, 1H, Ar-H), 7,86 (dd, J = 9.2 Hz, J = 1.2 Hz, 1H, Ar-H),
.70 (d, J = 9.2 Hz, 1H, Ar-H), 7.64 (d, J = 8.0 Hz, 2H, Ar-H), 7.34
d, J = 8.0 Hz, 2H, Ar-H) 3.66 (s, 3H, OCH ), 3.31 (d, J = 5.6 Hz, 2H,
CH ), 2.60 (m, 6H, CH
ꢁ 3), 1.02 (s, 6H, CH
DMSO-d ): d 163.3 (C), 160.0 (CH), 156.9 (C), 151.4 (C), 146.0
C), 143.4 (C), 140.8 (C), 133.4 (C), 132.8 (CH), 132.4 (CH ꢁ 2),
White solid; yield: 64.2%;
): d 10.16 (s, 1H, NH), 10.10 (s,
1
2
1
6
7
1
H, NH), 9.15 (s, 1H, Ar-H), 8.65 (s, 1H, NH), 8.40 (d, J = 2.0 Hz,
1H, Ar-H), 8.00 (d, J = 2.4 Hz, 1H, Ar-H), 7.92 (dd, J = 9.2 Hz,
= 1.2 Hz, 1H, Ar-H), 7.82 (m, 2H, Ar-H), 7.75 (d, J = 9.2 Hz, 1H,
Ar-H), 7.41 (m, 2H, Ar-H), 4.16 (d, J = 2.0 Hz, 2H, CH ), 3.64 (s,
H, OCH ),3.60 (m, 4H, CH
DMSO-d ): d 170.4 (C), 168.2 (C), 166.5 (C), 163.2 (C), 160.0
CH), 156.8 (C), 151.5 (C), 144.8 (C), 139.7 (C), 135.2 (C), 132.9
(
3
13
1
2
2
3
ꢁ 2). C NMR
J
2
(
(
6
2
13
3
(
(
3
2
ꢁ 2), 3.47 (m, 4H, CH ꢁ 2). C NMR
2
1
29.8 (CH ꢁ 2), 128.4 (CH), 128.4 (C), 126.4 (CH), 125.0 (C), 117.0
6
(
(
[
CH), 56.5 (CH
CH
M+H] : 553.2345; found: 553.2341.
3
), 54.8 (CH
2
), 49.7 (CH
2
ꢁ 2), 40.7 (CH
2
), 24.1
3
), 14.8 (CH
3
33 8 4
ꢁ 2). ESI-HRMS m/z: calcd for C26H N O S
+
(CH), 132.9 (CH), 132.8 (CH), 128.5 (CH), 126.2 (C), 123.5 (CH),
19.3 (CH), 119.1 (CH), 117.2 (CH), 69.1 (CH ), 69.0 (CH ), 56.5
CH ), 47.5 (CH ), 44.9 (CH ), 44.7 (CH ). ESI-HRMS m/z: calcd for
NaO S [M+Na] : 607.1499; found: 607.1494.
1
(
C
2
2
3
2
2
2
5
.1.4.12. 1-(2-(Diethylamino)ethyl)-3-(6-(2-methoxy-3-(2,4-di-
fluorophenyl sulfonylamino)pyridine-5-yl)-[1,2,4]triazolo[1,5-
a]pyridin-2-yl)-[1,2,4]triazolo [1,5-a]pyridin-3-yl)urea
+
25
H25FN
8
6
1
5.1.4.16. 1,1-Dimethyl-3-(6-(2-methoxy-3-(4-fluorophenylsul-
fonylamino)pyridine-5-yl)-[1,2,4]triazolo[1,5-a]pyridin-2-
yl)urea (1p).
NMR (DMSO-d
(
1l).
White solid; yield: 41.5%; mp: 211–213 °C; H NMR
(
DMSO-d ): d 10.08 (s, 2H, NH), 9.02 (s, 1H, Ar-H), 8.44 (s, 1H,
6
White solid; yield: 53.7%; mp: 153–155 °C; 1H
6
): d 10.10 (s, 1H, NH), 9.40 (s, 1H, Ar-H), 9.16 (s,
NH), 8.20 (s, 1H, Ar-H), 7.85 (d, J = 7.6 Hz, 2H, Ar-H), 7.77 (m, 1H,
Ar-H), 7.69 (d, J = 9.2 Hz, 1H, Ar-H), 7.47 (m, 1H, Ar-H), 7.16 (m,
1
H, Ar-H), 8.40 (s, 1H, NH), 7.99 (s, 1H, Ar-H), 7.84 (m, 3H, Ar-
1
(
1
H, Ar-H), 3.68 (s, 3H, OCH
m, 6H, CH
ꢁ 3), 1.07 (s, 6H, CH
67.4 (C, JC-F = 251 Hz), 163.2 (C), 162.2 (C, JC–F = 246 Hz), 160.7
3
), 3.38 (d, J = 5.6 Hz, 2H, CH
2
), 2.75
13
H), 7.72 (d, J = 8.8 Hz, 1H, Ar-H), 7.42 (d, J = 8.4 Hz, 2H, Ar-H),
2
3
ꢁ 2). C NMR (DMSO-d ): d
6
1
3
3
1
1
.63 (s, 3H, OCH
3
), 2.94 (s, 6H, CH
3
ꢁ 2). C NMR (DMSO-d
6
): d
68.2 (C), 166.5 (C), 163.8 (C), 160.0 (CH), 157.6 (C), 151.8 (C),
44.7 (C), 139.8 (C), 135.3 (CH), 132.9 (CH), 132.8 (CH), 132.2
(
(
CH), 157.0 (C), 151.3 (C), 141.9 (C), 134.7 (CH), 133.7 (C), 132.8
CH), 130.1 (C), 128.3 (CH), 128.1 (CH), 126.8 (CH), 125.0 (C),
(
CH), 128.6 (CH), 125.7 (CH), 123.7 (C), 119.3 (CH), 119.1 (CH),
1
17.0 (CH), 114.4 (CH), 108.6 (CH), 56.3 (CH
3
), 54.5 (CH
ꢁ 2). ESI-HRMS m/z: calcd for
39 2 8 4
F N O S [M+H] : 575.2001; found: 575.1995.
2
), 49.8
1
17.5 (CH), 56.5 (CH
3
), 39.5 (CH
3
ꢁ 2). ESI-HRMS m/z: calcd for
(
CH
2
ꢁ 2), 40.1 (CH
2 3
), 14.1 (CH
+
+
C
21
H20FN
7
NaO S [M+Na] : 508.1179; found: 508.1174.
4
C
25
H
5
.1.4.13. 1-(6-(2-Methoxy-3-(4-fluorophenylsulfonylamino)py-
5.1.4.17. 1,1-Dimethyl-N-(6-(5-(4-fluorophenylsulfonamido)-6-
ridine-5-yl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl)-3-(2-oxo-2-
methoxypyridin -3-yl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl)pyrro-
(
pyrrolidin-1-yl)ethyl)urea (1m).
White solid; yield: 61.4%;
lidine-1-carboxamide (1q).
155–156 °C; H NMR (DMSO-d ): d 10.10 (s, 1H, NH), 9.26 (s, 1H,
Ar-H), 9.16 (s, 1H, Ar-H), 8.40 (m, 1H, NH), 7.99 (s, 1H, Ar-H),
White solid; yield: 48.3%; mp:
1
1
mp: >250 °C; H NMR (DMSO-d
6
): d 10.14 (s, 1H, NH), 10.10 (s,
6
1
1
H, NH), 9.14 (s, 1H, Ar-H), 8.64 (s, 1H, NH), 8.40 (d, J = 2.4 Hz,
H, Ar-H), 8.00 (d, J = 2.4 Hz, 1H, Ar-H), 7.92 (dd, J = 9.2 Hz,
= 1.6 Hz, 1H, Ar-H), 7.82 (m, 2H, Ar-H), 7.74 (d, J = 9.2 Hz, 1H,
Ar-H), 7.42 (m, 2H, Ar-H), 4.06 (d, J = 4.4 Hz, 2H, CH ), 3.64 (s,
H, OCH ), 3.44 (m, 2H, CH ), 3.34 (d, J = 6.8 Hz, 2H, CH ), 1.91
m, 4H, CH ꢁ 2). C NMR (DMSO-d ): d
ꢁ 2), 1.79 (m, 4H, CH
1
7.80 (m, 3H, Ar-H), 7.72 (d, J = 8.8 Hz, 1H, Ar-H), 7.41 (m, 2H, Ar-
H), 3.63 (s, 3H, OCH ), 3.40 (s, 4H, CH ꢁ 2), 1.85 (s, 4H, CH ꢁ 2).
J
2
3
2
2
1
3
2
6
C NMR (DMSO-d ): d 168.2 (C), 166.6 (C), 163.8 (C), 160.0 (CH),
3
(
1
1
1
3
2
2
155.6 (C), 151.7 (C), 144.8 (C), 139.8 (C), 135.4 (CH), 132.9 (CH),
132.8 (CH), 132.2 (CH), 128.6 (CH), 125.7 (CH), 123.6 (C), 119.3
(CH), 119.1 (CH), 117.5 (CH), 56.5 (CH ), 49.0 (CH ꢁ 2), 28.1
13
2
2
6
69.7 (C), 168.2 (C), 166.6 (C), 163.2 (C), 160.0 (CH), 156.8 (C),
51.5 (C), 144.8 (C), 139.7 (C), 135.2 (C), 132.9 (CH), 132.9 (CH),
32.9 (CH), 128.5 (C), 126.2 (CH), 123.5 (CH), 119.3 (CH), 119.2
3
2
+
(CH ꢁ 2). ESI-HRMS m/z: calcd for C
2
23 22 7 4
H FN NaO S [M+Na] :
534.1336; found: 534.1330.
Please cite this article in press as: Wang, X.-M.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.07.017

X.-M. Wang et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
9
5
5
.2. Biology
factor sigma of all these compounds was 0.12. Dose progression
was stopped when one of the three stopping rules of the
AOT425StatPgm program was satisfied. The LD50 values and
95% confidence intervals were calculated using the
AOT425StatPgm program.
3
0
.2.1. Cell culture
The four human tumor cell lines including HCT-116, MCF-7,
U87 MG and A549 were grown as a monolayer respectively.
HCT-116, MCF-7 and U87 MG were maintained in DMEM medium
supplemented with 10% heat inactivated fetal bovine serum (FBS).
A549 was maintained in RPMI-1640 medium, with 10% heat inac-
tivated calf serum. All cancer cells were incubated in a humidified
5.2.5. In vivo antitumor effect in established mice S180
homograft models
Mice (20.3 ± 1.6 g) were purchased from Experiment Animal
Center of Xi’an Jiaotong University Health Science Center and fed
in the same place. The experimental protocol was approved by
Ethic Committee of Xian Jiaotong University.
2
atmosphere containing 5% CO at 37 °C.
5
.2.2. MTT assay
The in vitro antiproliferative activities of compounds were
determined by MTT assay. 1500–4000 cells per well were seeded
into 96-well plates in 200 L medium and incubated for 24 h. A
6
3 ꢁ 10 S180 cells were injected subcutaneously into the flank
of the mice. All tumor-bearing mice were randomly divided into
four groups, with 8 mice in each group. The next day, compound
l
series of concentrations of synthesized compounds and BEZ235
were added to the wells with DMSO as vehicle control. The mixture
was incubated at 37 °C, with a final concentration of 1% DMSO.
After 72 h of incubation, 20 lL of MTT solution (5 mg/mL in PBS)
was added to each well and incubated at 37 °C for 4 h. The super-
2
1e was dissolved in NMP/PEG400/H O (1:6:3) and dosed orally at
5 mg/kg, 10 mg/kg or 20 mg/kg once a day for 8 days, respectively.
In the solvent group, the same volume of solvent was administered
orally. Body weights were recorded per day. The mice were anes-
thetized and sacrificed on Day 9. The weights of the body and
the neoplasm were measured and inhibitory ratios of tumor
weight were calculated.
natant of each well was removed and the formed blue formazan
crystals were dissolved in 200
lL of DMSO. The optical density at
4
90 nm wavelength was determined by Varioskan Flash
Multimode Reader (Thermo scientific). Three separate experiments
with triplicate data were performed to obtain mean cell viability.
5.3. Molecule docking
The IC50 value, that is, the concentration (
l
M) of a compound
The protein–ligand complex crystal structure21 of compound A
was able to cause 50% cell death with respect to the control culture,
were calculated by means of GraphPad Prism 5 Software.
with PI3K
modes of 1c and 1e with PI3K
c
was chosen as the template to elucidate the docking
. Protein structure was downloaded
c
from Protein Data Bank (PDB code 3QK0) and the C-DOCKER proto-
5
.2.3. PI3Ks and mTOR kinase assay
col within Discovery Studio 2.5 was used for molecular docking.
Inhibitory PI3Ks enzymatic activities of compounds T1c, T1e
and BEZ235 were evaluated according to the reported method.
Compounds T1c, T1e and BEZ235 were dissolved in DMSO and
diluted to a series of concentrations. Different concentrations of
compounds were added to the enzyme reaction buffer containing
The PI3Kc enzyme was prepared by added hydrogen atoms and
2
6,27
then defined as a receptor. The site sphere was selected on the
basis of the ligand binding location of compound A, which was
replaced by compound 1c or 1e. After end of molecular docking,
10 docking poses were scored and selected based on calculated
C-DOCKER energy.
4
0 mM Tris–HCl, pH 7.4, 10 mM MgCl
DTT, 2.5 M ATP, PI3K (p110 /p85 , p110b/p85
or p110d/p85 ) or mTOR and L- -phosphatidylinositol. The final
reaction volume was 50 L. After incubation for 40 min at 30 °C,
2
, 0.1 mg/mL BSA, 1 mM
l
a
a
a
, p110 /PIK3R5
c
a
a
5.4. Statistical analysis
l
the reaction was terminated by addition of stop solution. The
amount of ADP was then detected via luciferase assay. After incu-
bation for 5 min, the luminescence signal was determined by the
multimode reader (MD-SpectraMax M5). The signal intensity is
proportional to the PI3Ks or mTOR activity.
The data in Table 1 are reported as mean ± standard deviation
(SD) for at least three experiments. Statistical differences were
analyzed according to one way ANOVA test wherein the differences
were considered to be significant at P<0.05. All statistics were cal-
culated using a statistical program GraphPad Prism 5.
The percentage of inhibition was calculated based on the fol-
lowing equation
Acknowledgment
Â
Ã
%
inhibition ¼ 1 ꢂ ðLucompound ꢂ LuminÞ=ðLumax ꢂ LuminÞ ꢁ 100%;
Financial support from National Natural Science Foundation of
China (Grant Nos. 21072156 and 81272448) is gratefully
acknowledged.
where Lucompound is the signal at a given compound concentra-
tion, Lumax is the signal of PI3Ks or mTOR without compound and
Lumin is the signal of background in the absence of enzyme and
compound. The IC50 values were calculated according to the fit of
the dose–response curves by using GraphPad Prism 5 Software.
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