Hybrids of phenylsulfonylfuroxan and coumarin as potent antitumor agents

Article abstract of DOI:10.1021/jm500613m

Sixteen furoxan-based nitric oxide (NO) releasing coumarin derivatives (6a-c, 8a-g, 10a, 13a,b, 15, and 17a,b) were designed, synthesized, and evaluated against the A549, HeLa, A2780, A2780/CDDP, and HUVEC cell lines. Most derivatives displayed potent antiproliferation activities. Among them, 8b exhibited the strongest antiproliferation activity on the four sensitive cell lines mentioned above and three drug resistant tumor cell lines A2780/CDDP, MDA-MB-231/Gem, and SKOV3/CDDP with IC₅₀ values from 14 to 53 nM and from 62 to 140 nM, respectively. Furthermore, 8b inhibited the growth of A2780 in vivo and displayed lower toxicity on nontumorigenesis T29, showing good selectivity against malignant cells in vitro. Preliminary pharmacological studies showed that 8b induces apoptosis, arrests the cell cycle at the G2/M phase in the A2780 cell line, and disrupts the phosphorylation of MEK1 and ERK1. Overall, the NO-releasing capacity and the inhibition of ERK/MAPK pathway signaling may explain the potent antineoplastic activity of these compounds.

Full text of DOI:10.1021/jm500613m

Article
pubs.acs.org/jmc
Hybrids of Phenylsulfonylfuroxan and Coumarin as Potent
Antitumor Agents
Ming-Ming Liu,† Xiao-Yu Chen, Yao-Qing Huang, Pan Feng, Ya-Lan Guo, Gong Yang,*
,‡,∥
†,∥
†
†
†
,‡,§
,
†
and Ying Chen*
†
Department of Medicinal Chemistry, School of Pharmacy, Fudan University, Shanghai, 201203, China
‡
Cancer Institute, Fudan University Shanghai Cancer Center, and Department of Oncology, Shanghai Medical College, Fudan
University, Shanghai, 200032, China
§
Central Laboratory, The Fifth People’s Hospital of Shanghai, Fudan University, Shanghai, 200240, China
S Supporting Information
*
ABSTRACT: Sixteen furoxan-based nitric oxide (NO) releasing coumarin derivatives (6a−c, 8a−g, 10a, 13a,b, 15, and 17a,b)
were designed, synthesized, and evaluated against the A549, HeLa, A2780, A2780/CDDP, and HUVEC cell lines. Most
derivatives displayed potent antiproliferation activities. Among them, 8b exhibited the strongest antiproliferation activity on the
four sensitive cell lines mentioned above and three drug resistant tumor cell lines A2780/CDDP, MDA-MB-231/Gem, and
SKOV3/CDDP with IC values from 14 to 53 nM and from 62 to 140 nM, respectively. Furthermore, 8b inhibited the growth
5
0
of A2780 in vivo and displayed lower toxicity on nontumorigenesis T29, showing good selectivity against malignant cells in vitro.
Preliminary pharmacological studies showed that 8b induces apoptosis, arrests the cell cycle at the G2/M phase in the A2780 cell
line, and disrupts the phosphorylation of MEK1 and ERK1. Overall, the NO-releasing capacity and the inhibition of ERK/MAPK
pathway signaling may explain the potent antineoplastic activity of these compounds.
1
1
INTRODUCTION
hydroxycoumarin and 3-benzyl-6-chloro-7-(dimethylamino-
■
12
formyloxy)coumarin (G8935, Figure 2), showed antiprolifer-
ation of cancer cells via MEK1 targeting.
In our prior research, a series of S- and O-substituted 7-
mercaptocoumarin analogs were designed, synthesized, and
evaluated in vitro against four human tumor cell lines (KB
The Ras/Raf/MEK/ERK pathway is one of the most important
signal transduction pathways in human cancer, and plays an
important role in cell proliferation and cytokine production.
13
1
−3
Oncogenic mutations to Ras or Raf contribute to the
constitutive activation of this pathway and have been observed
in lung, colon, pancreas, kidney, and ovary primary human
(
nasopharyngeal), KB-vin (vincristine-resistant subline), A549
(lung), and DU145 (prostate)). The 7-(6-chloropyridin-2-
4
tumor samples. MEK is a pivotal node in the ERK/MAPK
13
ylthio)-4-methycoumarin (1, Figure 2), which increased
cellular apoptosis in a concentration-dependent manner and
induced A549 cell cycle arrest at the G2/M phase, exhibited
potent cytotoxic activity. The result prompted us to further
modify the structures for the desirable compounds with the
possible pharmacological mechanism(s) such as MEK1 target.
Additionally, nitric oxide (NO) plays pivotal roles in diverse
physiological and pathophysiological processes. Generally,
high levels of NO generated from NO donors can induce
apoptosis, inhibit metastasis of tumor cells, and sensitize tumor
cells to chemotherapy, radiation, and immunotherapy in vitro
pathway, and extracellular regulated kinase (ERK) is the only
known downstream target of MEK. All these characters make5
MEK a promising molecular target for tumor therapy.
6
Trametinib is the first MEK inhibitor and was approved for
7
the treatment of melanoma by FDA in 2013. Selumetinib and
8
MEK162 are diarylamine derivatives and MEK inhibitors; they
have been at phase II and phase III clinical trials, respectively
Figure 1).
Recently, many coumarin derivatives were widely reported to
have antitumor activity through binding to different targets and
diverse pharmacological mechanisms. For example, 3,8-
dibromo-7-hydroxycoumarin (DBC, Figure 2) inhibited CK2
function. The 7,8-dihydroxy-4-methylcoumarin (DHMC,
Figure 2) induced apoptosis of human lung adenocarcinoma
cells by a ROS-independent mitochondrial pathway through
partial inhibition of ERK/MAPK signaling. Some other
coumarin derivatives, such as 3-(benzo[d]thiazol-2-yl)-7-
14
(
15−17
9
and in vivo.
Furoxans are an important class of NO donors
that can produce high levels of NO in vitro and inhibit the
growth of tumors in vivo. The mechanisms of NO release by
10
18
Received: April 19, 2014
©
XXXX American Chemical Society
A
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Figure 1. Approved or in clinical trial MEK1 inhibitors.
Figure 2. Some bioactive coumarin derivatives.
Figure 3. Compounds 20−22, their corresponding furoxan hybrids 23−25, and compound 19.
furoxans were complex. Generally, it is hypothesized that
thiolate attacked the position 3 or 4 followed by ring opening
to give a nitroso derivative. The broken derivative then released
of cancer cells and can be an effective and promising strategy
26
for cancer treatment.
To enhance the pharmacological activity of compound 1, the
furoxan based NO donor was introduced at 7-position of the
coumarin core to design and synthesize 16 coumarin/furoxan
hybrids. At the same time, their antitumor activities were
screened in vitro and in vivo. Furthermore, MEK1 inhibitory
activities, apoptosis inductive activities, and NO-releasing ability
were also biologically evaluated to uncover the mechanisms of
action underlying them.
−
the nitroxyl anion NO , which was further oxidized to
1
9−21
NO.
The NO release can be modulated by varying the
22
nature of the substituents. Zhang and Tian’s group reported
hybrids from furoxan based NO donor and different active
compounds like 20 (glycyrrhetinic acid, GA), 21 (farnesylth-
23
2
4
iosalicylic acid, FTS), and 22 (N-(3-((5-chloro-2-((2-
methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)pyrimidin-4-
25
yl)oxy)phenyl)acrylamide, WZ4002). Hybrids like 23 (GA/
2
3
24
RESULTS AND DISCUSSION
furoxan hybrid), 24 (FTS/furoxan hybrid), and 25
(
■
2
5
WZ4002/furoxan hybrid) displayed potent antitumor
Chemistry. The synthesis of target compounds is illustrated
in Scheme 1. 3,4-Diphenylsulfonyl-1,2,5-oxadiazole 2-oxide (4)
was synthesized in a three-step sequence according to literature
activity both in vivo and in vitro (Figure 3). Besides, it has
also been reported that combined treatment of NO donor and
MEK inhibitor synergistically inhibits proliferation and invasion
27
procedures. Commercial available reagents 5a−e and 14 were
B
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a
Scheme 1. Synthetic Routes of Target Compounds
a
Reagents and conditions: (a) ClCH COOH, NaOH (aq), 140 °C, 2 h; (b) 30% H O , AcOH, room temperature, 3.5 h; (c) fuming HNO , 90 °C,
2
2
2
3
4
h; (d) CH Cl , DBU, −15 °C, 3 h, 95%; (e) corresponding halo alcohol, K CO , acetone or DMF, reflux; (f) ClCH COOH, K CO , DMF, reflux;
2
2
2
3
2
2
3
(
g) DMAP, EDC, 0 °C to room temperature; (h) CH I, K CO , DMF, reflux; (i) corresponding halo alcohol, K CO , acetone or DMF, reflux; (j) 2-
3 2 3 2 3
methoxyethanol, CH Cl , DBU, room temperature, 3 h, 46%.
2
2
treated with various halo alcohols to provide the intermediates
of 4 with various hydroxyl linkers coupling compounds 5a−c,
7a−g, 9a, 12a,b, 14, 16a,b, and 2-methoxyethanol provided the
16 target compounds 6a−c, 8a−g, 10a, 13a,b, 15, 17a,b, and
the NO donor moiety compound 18 using 8-
diazabicyclo[5.4.0]undec-7-ene (DBU) as a catalyst in CH Cl .
Activities of Antiproliferation in Vitro. Sixteen newly
synthesized coumarin/furoxan hybrids (6a−c, 8a−g, 10a,
7
9
a−g and 16a,b. Methylation of compound 7g with CH I gave
3
a. Compounds 5a,b were reacted with 2-chloroactic acid to
form 11a,b, which then condensed with ethanediol in the
presence of 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide
hydrochloride (EDC) and 4-(dimethylamino)pyridine
2
2
(DMAP) to gain intermediates 12a,b. Finally, condensation
C
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3a,b, 15, and 17a,b) were screened for antiproliferation in
Article
1
cancer cell lines MDA-MB-231/gem and SKOV3/CDDP as
well as their sensitive parent cell lines MDA-MB-231 and
SKOV3.
A549, HeLa, A2780, and A2780/CDDP with 1, 4, and a NO
28
releasing prodrug 19 (JS-K, Figure 3) as references. Because it
is a first-line drug in the clinic for patients with ovarian cancer,
cisplatin was also chosen as the positive control. As shown in
Table 1, most target compounds showed considerable
As shown in Table 2, compound 8b had an activity against
both of these sensitive and drug resistant cancer cell lines with
0.15, 0.14, 0.083, and 0.14 μM IC50 values, respectively. The
results indicated that 8b might be a broad-spectrum inhibitor
for both sensitive and drug resistant cancer cell lines. Moreover,
angiogenesis is necessary for tumor growth and metastasis,
which made it a promising target for cancer treatment. Human
umbilical vein endothelial cells (HUVECs) are a normal cell
useful for assessing antiangiogenic potential. The HUVEC line
was also screened in company with the previous four cancer cell
lines. Eight compounds showed potent activity with IC values
Table 1. Antiproliferation Activities for 6a−c, 8a−g, 10a,
1
3a,b, and 17a,b
a
IC50 (μM)
b
c
d
e
f
compd
A549
HeLa
A2780
A2780/CDDP
HUVEC
g
cisplatin
25.23
3.25
2.35
8.86
5.55
11.68
0.26
0.024
0.12
0.28
0.65
1.00
1.44
1.02
>20
2.07
1.48
1.45
4.55
>20
>20
0.11
1.06
1.35
0.88
0.11
2.99
9.35
0.17
0.014
0.036
0.35
0.66
0.046
2.45
0.95
>20
>50
1.00
0.89
3.44
3.15
>20
0.20
0.062
0.14
0.36
0.99
0.33
5.45
2.38
>20
7.36
2.99
2.88
0.89
1.05
ND
1
4
5.89
4.33
6.75
5.43
>20
50
less than 1 μM. The 8f and 17a demonstrated activity against
HUVEC with EC50 of 0.097 and 1.03 μM, respectively. Again,
compound 8b exhibited the highest potency with an IC value
6
6
6
8
8
8
8
8
8
8
1
1
1
1
1
1
1
a
b
50
c
of 0.034 μM. The results implied that these new compounds
might also be active on angiogenesis targets like vascular
endothelial growth factor receptor (VEGFR) and stimulated us
to explore their pharmacological mechanism in the future.2
a
0.37
0.034
0.22
0.68
0.25
0.097
3.04
0.89
>20
b
0.053
0.024
0.26
0.32
0.25
1.99
0.51
>20
c
8,29
d
Considering that 19 exerted selective antitumor activity,
e
the selectivity of 8b on tumor and nontumorigenic cells also
was studied via the inhibition of compound 8b testing on the
proliferation of nontumorigenic T29 cells (immortalized but
nontumorigenic ovarian epithelial cells) in vitro. As Table 3 and
f
g
0a
3a
3b
5
6.92
3.29
5.45
0.40
1.26
4.31
>20
3.65
0.13
3.33
0.29
0.63
>20
Table 3. Antiproliferation Activities for 8b, 7-Hydroxy-4-
methylcoumarin, and 18
3.35
1.03
0.35
5.39
7a
7b
9
0.83
0.19
3.56
a
IC50
b
c
compd
T29
A2780
14 nM
a
d
The antiproliferation activities of individual compound to tumor cells
were determined by the MTT assay. The data are the mean of
8b
>2 μM
>2 μM
>2 μM
7-hydroxy-4-methylcoumarin
>2 μM
>2 μM
b
triplicate determinations. A549 is a human lung cancer cell line.
18
c
d
HeLa is a human cervical carcinoma cell line. A2780 is a human
a
The antiproliferation activities of individual compound to tumor cells
were determined by the MTT assay. The data are the mean from at
least three independent experiments. T29 cells are immortalized but
nontumorigenic ovarian epithelial cells. A2780 is ovarian cancer cell
e
ovarian cancer cell line. A2780/CDDP is a cisplatin-resistant human
ovarian cancer cell line. HUVEC are human umbilical vein endothelial
cells. Not determined.
f
b
g
c
d
line. Data presented here were the first batch that was agreed with the
second batch.
anticancer activities and had much more potency than that of
the NO donor moiety 4, hit 1, cisplatin, and 19. Among them,
compounds 8a−f and 17b exhibited activities in the nanomolar
range. In particular, 4-(2-(4-methyl-2-oxo-2H-chromen-7-
yloxy)ethanoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide
Figure 4 illustrate, the IC value of compound 8b on T29 cells
50
(IC50 > 2 μM) was at least 140-fold more than that of
compound 8b on A2780 cells (IC50 = 14 nM), indicating that
8b has selective inhibition on the proliferation of A2780 cell in
vitro. Notably, antiproliferation activity of 8b (IC50 = 14 nM)
against A2780 cells was much more potent than that of the
coumarin core moiety 7-hydroxy-4-methylcoumarin (IC50 > 2
μM) and the NO donor moiety compound 18 (IC > 2 μM),
(
8b) was the most potent compound. It not only inhibited
the proliferation of A549, HeLa, and A2780 with IC values at
5
0
2
4, 53, and 14 nM, respectively, but also had a very strong
cytotoxicity for drug resistance in A2780/CDDP with IC50
value of 62 nM. This result encouraged us to further measure
its antiproliferative activity on the other two drug resistant
50
Table 2. Antiproliferation Activities for 8b against Drug Resistant Cell Lines
a
IC50 (μM)
b
c
d
e
compd
MDA-MB-231
0.15
MDA-MB-231/Gem
SKOV3
SKOV3/CDDP
8
b
0.14
0.083
5.95
0.14
cisplatin
>100
gemcitabine
0.025
>10
a
The antiproliferation activities of individual compound to tumor cells were determined by the MTT assay. The data are the mean from at least
b
c
three independent experiments. MDA-MB-231 is a human breast cancer cell. MDA-MB-231/Gem is a gemcitabine resistant breast cancer cell.
d
e
SKOV3 is a human ovarian carcinoma cell line. SKOV3/CDDP is a cisplatin resistant human ovarian cancer cell line.
D
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Figure 4. Antiproliferation activities of compound 8b against A2780
tumor cells and nontumor T29 cells. T29 cells are immortalized but
nontumorigenic ovarian epithelial cells.
Figure 5. Capillary-like tube formation assay. (A) Tube formation for
the vehicle control. HUVECs formed robust tube structures when they
were only treated with vehicle (DMSO). (B) Tubule formation was
significantly suppressed with 10 nM 8b treatment. (C) 100 nM 8b
completely inhibits the formation of tube structures. (D) Compound
which suggested that the combination of the NO donor moiety
with coumarin core might lead to a synergetic antitumor effect.
Structure−Activity Relationships (SARs). According to
the activities mentioned above, the preliminary SAR of target
compounds can be inferred. First, the bioisosteric modification
at the 7-position with O, S, and N showed that oxygen atom
substitution was more favorable for antiproliferation activity.
Second, the replacement of the coumarin scaffold with 4H-
chromen-4-one core decreased the antiproliferation activity.
Most importantly, for both the coumarin scaffold and its analog
1
9 was used as a positive control.
cells, and its activity was diminished by pretreatment with a NO
scavenger in a dose-dependent manner (Figure 6B and Figure
6C). The results above agreed with the study that 8b might
have selective inhibition on the proliferation of A2780 cell in
vitro. This indicated that the potent antiproliferation activities
and selectivity may be partly attributed to the release of nitric
oxide.
Next we determined the intracellular NO level produced by
compound 8b and the reference compound 19 using
fluorescent indicator DAF-FM DA. As shown in Figure 6D,
when compared with the DMSO treated group, exposure of
A2780 cells to 8b for 1 h led to an increase in DAF-FM
fluorescence in a dose-dependent manner. And the fluorescence
intensity induced by 8b was higher than that induced by 19,
which may indicate the better NO-releasing activity of 8b
compared with that of 19.
4
H-chromen-4-one scaffold, the length of the spacers
connecting the NO donors was crucial to preserve the strong
anticancer activity. For example, the coumarin/furoxan
derivative 8b with a C2 spacer was much more potent than
those with no (6b) or longer spacers (8d, 8e), respectively.
Similarly, the 4H-chromen-4-one/furoxan derivative, however,
with a C3 spacer (17b) would be better than those with C2
(
17a) or no linkers (15).
Compound 8b Inhibited Angiogenesis in Vitro. To
further evaluate the antiangiogenesis activity of compound 8b,
we performed capillary-like tube formation assay in vitro.
Endothelial cells could form capillary-like structures in Matrigel,
which are useful indicators of angiogenesis potential.
Compound 19 was employed as a positive control. As shown
in Figure 5, HUVEC cells formed a complete network structure
overnight with serum stimulation. After compound 8b
treatment, the tubule formation was significantly suppressed
even in a nonantiproliferative concentration (10 nM). At the
concentration of 100 nM, 8b completely disrupted the
formation of tube structures.
Compound 8b Inhibited Colony Formation and
3
1
Blocked the Cell Cycle. We tested the colony formation
ability of cells after treatment with compound 8b. As shown in
Figure 7 (parts A and B), the exposure of A2780 and MDA-
MB-231/Gem cell lines to nanomolar concentrations of
compound 8b resulted in a significant inhibition of colony
formation. The 8b completely abolished growth of A2780 cell
line at doses above 20 nM. Moreover, because chemical
antitumor agents can inhibit cell proliferation through
induction of cell cycle arrest, DNA-based cell cycle analysis
was performed by flow cytometry to understand how the cell
growth was inhibited by compound 8b. As illustrated in Figure
7C, the treatment of 8b caused blockage of the cell cycle in the
G2/M phase. Compared with the control cells treated with
DMSO, when A2780 cells were treated with increasing
concentrations of 8b (20, 40, and 80 nM), the mean percentage
of cells in the G2/M phase increased from 10.5% to 64.9%, and
the percentages of cells in S and G0/G1 phase decreased
concomitantly (Figure 7D).
NO Releasing Assessment. With the ability to produce
1
8,30
high levels of NO in vitro,
furoxans are an important class
of NO donors. As shown in Figure 6A, the intracellular NO
production capability of these furoxan/coumarin hybrids (6a−
c, 8a−g, 10a, 13a,b, 15, and 17a,b) and furoxan moiety 4, 18
was determined and presented as that of nitrite in the cell
lysates using a Griess assay, with 19 and compound 1 as
positive and negative controls, respectively. As expected,
compound 1 without furoxan group was hardly detected with
nitrite in A2780 cells, whereas the compounds bearing the
furoxan moiety could produce various levels of nitrite
intracellularly with the releasing percentage range from 21 to
9
7 μM, in which the most active compound 8b released the
Compound 8b Induced Cellular Apoptosis. Consider-
32
highest concentration of nitrite (97 μM) and higher than the
positive control 19 (48 μM). Most importantly, compound 8b
released more NO in A2780 tumor cancer cells than in T29
ing that NO induces cellular apoptosis, our coumarin/furoxan
hybrids with the ability to release NO might also have a similar
mechanism of inducing apoptosis against tumor cells. To
E
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Figure 6. (A) Variable levels of NO produced by some coumarin/furoxan hybrids in A2780 cells. Results are indicated as the mean ± SD (standard
error) of three independent experiments. (B) Effects of hemoglobin on the antiproliferative effect of 8b. A2780 and A2780/CDDP cells were
pretreated with the indicated concentrations of hemoglobin (0, 1.25, 2.5, 5, 10, or 20 μM) for 1 h and treated with 500 nM 8b for 24 h. The results
are expressed as the percentage of cell growth inhibition relative to control cells. Data are the mean value ± SD obtained from three determinations.
(
C) 8b releases a higher level of NO in A2780 cells than in T29 cells, P < 0.05. Results are indicated as the mean ± SD (standard error) of three
independent experiments. (D) Intracellular NO level produced by compound 8b or the reference compound 19 using fluorescent indicator DAF-FM
DA.
3
3
determine the number and stage of apoptotic cells, the annexin-
V/PI double staining assay was applied to quantitate 8b treated
A2780 cells using flow cytometry. As depicted in Figure 8A and
Figure 8B, the total proportion of annexin V+/PI− (the right
lower quadrant, representing early apoptotic) and annexin V
apoptosis. These results suggested that 8b triggers cellular
apoptosis through regulation of apoptosis-associated proteins.
Compound 8b induced the expression and the phosphor-
ylation levels of p53, which subsequently promoted Bax and
reduced Bcl-2 expression. The increase of Bax/Bcl-2 ratio may
be responsible for the concomitant execution phase of
apoptosis which included disruption of mitochondrial mem-
brane potential and finally cleavage and inactivation of key
cellular proteins such as PARP-1.
+/PI+ (the right upper quadrant representing late apoptotic
and necrotic) cells increased from 2.5% to 29.5% after they
were exposed to 20, 40, and 80 nM 8b for 24 h.
We next investigated the signaling pathway involved in
compound 8b induced apoptosis. Western blot analysis showed
that 8b significantly up-regulated the expression of proapop-
totic protein Bax but down-regulated the expression of the
antiapoptotic proteins Bcl-2 in a dose dependent manner
Effects of Compound 8b on MEK/ERK Signaling and
Kinase Inhibitory Activity. Our newly synthesized coumar-
in/furoxan hybrids also shared the coumarin scaffold. It
promoted us to study the effect of these compounds on the
ERK/MAPK pathway signaling, and 8b was selected to explore
the effect on the protein expression of MEK1, P-MEK1, ERK1,
and P-ERK1. As described in Figure 9, the phosphorylation of
MEK1 (P-MEK1) and ERK1 (P-ERK1) was decreased after
treatment with 8b. The result means that 8b not only could
prevent the inactive MEK1 being phosphorylated by upstream
(
Figure 8C). Meanwhile, 8b could also up-regulate the
expression and the phosphorylation levels of p53, an important
tumor suppressor. Moreover, compared with control cells,
exposure to 8b significantly induced the cleavage of poly ADP-
ribose polymerase 1 (PARP-1), a marker of cells undergoing
F
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Figure 7. Compound 8b inhibited colony formation and blocked cell cycle. (A) Treatment of ovarian cancer cell line A2780 and breast cancer drug
resistant subline MDA-MB-231/gem with the compound 8b resulted in a does-dependent inhibition of colony formation. (B) The profiles show the
number of colonies. One colony was defined to be an aggregate of >50 cells. Results represent the mean ± SD from three independent experiments:
(
∗) p < 0.05, (∗∗) p < 0.01 compared to control. (C) 8b dose-dependent induced cell cycle arrest in G2/M phase of A2780 cells. (D) The profiles
showed the proportions (%) in each phase of A2780 cells treated with 8b and diluent (DMSO). The experiments were repeated three times, and a
representative experiment is shown: (∗) p < 0.05, (∗∗) p < 0.01 compared to control.
kinase B-RAF triggering but also inhibited the active MEK1
activating the downstream protein ERK1.
The in vivo antitumor activity of 8b was further evaluated in
a human ovarian cancer xenograft mouse model established by
subcutaneous inoculation of the A2780 cells in the female
BALB/C nude mice. After establishment of solid tumor, the
mice were randomized and treated with 15 or 30 mg/kg 8b or
vehicle once every 3 days, respectively. As shown in Figure 10A,
treatment of 15 mg/kg 8b significantly inhibited the tumor
growth and produced a 55.8% reduction by the end of the
observation period (15th day) versus vehicle only. Treatment
with 30 mg/kg 8b enhanced the inhibitory effect on the tumor
growth with an inhibition rate of 65.5%. When the observation
time ended, the mice were sacrificed and the tumors were
excised and weighed. The results (Figure 10B) again showed a
statistically significant reduction in tumor weight in both doses
of 8b-treated mice. Moreover, there was no significant weight
loss in animals treated with 8b compared with those in the
vehicle control group (Figure 10C).
On the basis of the Western blot assay, we subsequently
examined the inhibitory effects of 1, 6a−c, 8a−g, 10a, 13a,b,
1
5, and 17a,b on MEK1. As depicted in Table 4, most of these
NO release compounds had weak inhibitory ratios of less than
0% except compound 8c with 53.4% inhibitory effect for
5
MEK1 at a concentration of 5 μM. Merging the MEK
inhibitory activity with the antitumor activity showed an
interesting result that the antitumor action of compounds (6a−
c, 8g, 13a,b, and 15) with very poor MEK inhibitory activity
(
<
inhibitory ratio of <15%) generally was not very potent (IC50
1 μM) even if they could release high levels of NO. On the
contrary, compounds 8a−f exhibited stronger antiproliferation
activity that might be attributed to their higher MEK inhibitory
activity together with high NO release capability. This
phenomenon was in accordance with the report that NO
donor combined with MEK inhibitor exerted synergistically
2
6
anticancer effect.
CONCLUSIONS
In Vivo Antitumor Activity of Compound 8b. To
evaluate the safety of compound 8b, groups of female KM mice
were injected intraperitoneally with a single dose of 8b at 500,
■
In summary, a series of hybrids from phenylsulfonylfuroxan and
coumarin derivatives (6a−c, 8a−g, 10a, 13a,b, 14, and 17a,b)
were synthesized and biologically evaluated. Most of them
could produce high levels of NO and show strong
antiproliferation activities for A549, HeLa, A2780, and
A2780/CDDP cell lines. The antiproliferation of these hybrids
300, 275, 250, 225, 200 mg/kg or vehicle control. As illustrated
in Table 5, treatment with 8b less than 200 mg/kg caused no
abnormality throughout the observation period similar to
control. The LD50 value calculated was 263.6 mg/kg.
G
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Figure 8. Apoptosis induced by compound 8b. (A) Apoptotic cells were detected with annexin V/PI double staining after incubation with various
concentrations of compound 8b or diluent (DMSO) for 24 h. (B) The profiles showed the apoptotic proportion (%) of A2780 cells. The
experiments were performed three times: (∗) p < 0.05, (∗∗) p < 0.01 compared to control. (C) Western blotting analysis showed that 8b
significantly up-regulated the expression of proapoptotic protein Bax but down-regulated the expression of the antiapoptotic proteins Bcl-2 in a dose
dependent manner. Meanwhile, 8b could also up-regulate the expression and the phosphorylation levels of p53 and induced the cleavage of PARP.
and three drug resistant tumor cell lines (A2780/CDDP, MDA-
MB-231/Gem, and SKOV3/CDDP) with nearly comparative
IC50 values. Interestingly, 8b had a distinguishing antiprolifer-
ative selectivity between nontumorigenic T29 cell and A2780
tumor cell line. In addition, compound 8b also inhibited human
xenograft growth in the A2780 ovarian tumor-bearing nude
mice in vivo. In our investigation of pharmacological
mechanism, most of compounds were weakly active against
MEK1, but the introduction of the NO group resulted in
considerable antiproliferation activity against several cancer cell
lines. In particular, 8b showed cellular apoptosis, cell cycle
arrest at the G2/M phase, and inhibition of MEK1 and ERK1
Figure 9. Effects of 8b on the ERK/MAPK signaling pathway.
phosphorylation in A2780 cells. The preliminary SAR showed
that the two to three C length of linker between the NO moiety
against HUVEC combined with the result that 8b disrupted the
and coumarin core was crucial to increase the anticancer
formation of tube structures indicated that these compounds
activity. A detailed SAR study is ongoing. In conclusion, the
might have antiangiogenesis effect and pushed us to explore
NO-releasing capacity and the inhibition of ERK/MAPK
active targets such as VEGFR in the next study. By far, the most
pathway signaling, which exerts synergistic multitarget actions,
may explain the potent antineoplastic activity of these
compounds.
potent compound 8b displayed a wide-spectrum anticancer
activity against eight cancer cell lines including the five sensitive
cell lines (A549, HeLa, A2780, MDA-MB-231, and SKOV3)
a
Table 4. In Vitro MEK1 Inhibitory Activities of Target Compounds
b
compd
1
6a
6b
6c
8a
8b
8c
8d
8e
8f
8g
10a
13a
13b
15
17a
17b
MEK1 (%)
1.6
8.4
10
11.3
22.7
35.2
53.4
31.6
43
48.1
5.7
47.6
16.4
14.4
10.9
29.8
20.4
a
b
The data are the mean from two independent experiments. The concentrations of tested compounds were 5 μM.
H
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a
Table 5. Acute Toxicity of 8b in Mice
no. of dead mice
dose (mg/kg)
no. of mice
1 h
4 h
1 day
2 days
3 days
4 days
5−14 days
total death
survivial (%) on day 14
5
3
2
2
2
2
00
00
75
50
25
00
10
10
10
10
10
10
10
0
0
0
0
0
0
0
1
1
0
0
0
0
0
3
2
2
1
0
0
0
2
2
2
2
1
0
0
0
1
1
0
0
0
0
1
0
0
0
0
0
0
2
1
1
1
0
0
0
9
7
6
4
1
0
0
10
30
40
60
90
100
100
vehicle
a
LD50 = 263.6 mg/kg.
EXPERIMENTAL SECTION
■
Chemistry. Melting points were measured on a SGW X-4
microscopy melting point apparatus without correction. H and
1
13
C
NMR spectral data were recorded with a Varian 400 MHz
spectrometer at 303 K using TMS as an internal standard. Mass
spectra were recorded on Agilent Technologies 1260 infinity LC/MS
instrument, and HRMS spectra were recorded on an Agilent
Technologies LC/MSD TOF instrument. Analytical and preparative
TLC was performed on silica gel HSGF/UV 254. The chromatograms
were conducted on silica gel (100−200 mesh) and visualized under
UV light at 254 and 365 nm. Tested compounds were analyzed by
reverse-phase HPLC (ODS-C18 column (5 μm, 4.6 mm × 150 mm or
3
.5 μm, 4.6 mm × 150 mm) using a Waters 1525 binary HPLC pump
system equipped with a Waters 2489 UV/visible detector. The mobile
phase was 70% CH CN/30% H O (v/v) and with a total flow rate of 1
3
2
mL/min. The purity was determined by monitoring at 254 nm. The
purities of the synthesized compounds were confirmed to be ≥95% by
this HPLC analysis.
3,4-Diphenylsulfonyl-1,2,5-oxadiazole 2-Oxide (4). The syn-
thesis of the intermediate 4 from benzenethiol in a three-step sequence
was reported previously in ref 27.
2-Ethyl-7-hydroxy-4H-chromen-4-one (14). The synthesis of
the intermediate 14 from resorcinol was reported previously in ref 34.
General Procedure for the Preparation of 7a−g. To a stirred
solution of 5a−e (1 mmol) in DMF (5 mL) at room temperature were
added corresponding halo alcohol (2 mmol) and K CO (3 mmol).
2
3
The mixture was refluxed for about 2−10 h and then poured into
water (50 mL). After filtration, the residue was washed with water (3 ×
1
7
0 mL) and dried with infrared lamp, yielding a yellow or white solid
a−g (40−96%).
7-(2-Hydroxyethoxy)-2H-chromen-2-one (7a). The title com-
pound was obtained starting from 5a and 2-chloroethanol (white solid,
8
8% yield, mp 85−86 °C). Analytical data for 7a: ESI-MS m/z 207.1
+
1
[
M + H] ; H NMR (400 MHz, CDCl ) δ 7.64 (d, 1H, −CHCH−,
3
J = 9.5 Hz), 7.38 (d, 1H, ArH, J = 8.6 Hz), 6.87 (dd, 1H, ArH, J = 8.6,
2
.3 Hz), 6.82 (d, 1H, ArH, J = 2.0 Hz), 6.26 (d, 1H, −CHCH−, J =
9
.5 Hz), 4.19−4.11 (m, 2H, −OCH −), 4.01 (m, 2H, −CH O−), 2.15
2
2
(
s, 1H,OH).
7-(2-Hydroxyethoxy)-4-methyl-2H-chromen-2-one (7b). The
title compound was obtained starting from 5b and 2-chloroethanol
(
white solid, 90% yield, mp 145−147 °C). Analytical data for 7b: ESI-
+
1
MS m/z 221.1 [M + H] ; H NMR (400 MHz, CDCl ) δ 7.50 (d, 1H,
3
ArH, J = 8.8 Hz), 6.89 (d, 1H, ArH, J = 8.8 Hz), 6.82 (s, 1H, ArH),
6
.14 (s, 1H, −CH), 4.14 (d, 2H, −CH O−, J = 3.0 Hz), 4.02 (s, 2H,
2
Figure 10. Inhibition of human xenograft growth in vivo by compound
b. (A) A2780 human ovarian tumor-bearing nude mice were
−CH O−), 2.40 (s, 3H, −CH ), 2.20 (s, 1H, OH).
2
3
8
7-(2-Hydroxyethylthio)-4-methyl-2H-chromen-2-one (7c).
The title compound was obtained starting from 5d and 2-
chloroethanol (yellow solid, 89% yield, mp 117−118 °C). Analytical
administered 15 or 30 mg/kg 8b or the vehicle, as controls (n = 6).
Injections were given intraperitoneally once every 3 days. The figures
show the average measured tumor volumes (A), the weight of the
excised tumor at the end of the observation period (B), and the body
weights of the mice recorded at the end of the observation period (C).
Data are expressed as the mean ± SD (n = 6): (∗) p < 0.05 and (∗∗) p
+
1
data for 7c: ESI-MS m/z 237.1 [M + H] ; H NMR (400 MHz,
CDCl ) δ 7.48 (d, 1H, ArH, J = 8.2 Hz), 7.26−7.18 (m, 2H, ArH),
3
6.23 (d, 1H, CH−, J = 1.1 Hz), 3.86 (q, 2H, −OCH −, J = 5.6 Hz),
2
3.22 (t, 2H, −CH S−, J = 6.0 Hz), 2.41 (d, 3H, −CH , J = 1.1 Hz),
2
3
<
0.01 vs control.
2.07 (s, 1H, OH).
7
-(3-Hydroxypropoxy)-4-methyl-2H-chromen-2-one (7d).
The title compound was obtained starting from 5b and 3-
I
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chloropropan-1-ol (white solid, 96% yield, mp 77−80 °C). Analytical
1H, ArH), 6.28 (d, 1H, −CHCH−, J = 9.5 Hz), 4.81 (s, 2H,
+
1
data for 7d: ESI-MS m/z 235.1 [M + H] ; H NMR (400 MHz,
−COCH O−).
2
CDCl ) δ 7.48 (dd, 1H, ArH, J = 8.7, 2.6 Hz), 6.90−6.79 (m, 2H,
2-(4-Methyl-2-oxo-2H-chromen-7-yloxy)acetic Acid (11b).
The title compound was obtained starting from 5b and 2-chloroacetic
acid (white solid, 85% yield, mp 201−203 °C). Analytical data for 11b:
3
ArH), 6.12 (d, 1H, CH−, J = 1.0 Hz), 4.17 (t, −CH O−, J = 6.0
2
Hz), 3.87 (d, 2H, −CH −, J = 2.7 Hz), 2.39 (d, 3H, −CH , J = 1.1
2
3
+
1
Hz), 2.08 (p, −CH −, J = 6.0 Hz).
ESI-MS m/z 235.1 [M + H] ; H NMR (400 MHz, DMSO-d
(d, 1H, ArH, J = 9.0 Hz), 7.01−6.91 (m, 2H, ArH), 6.21 (s, 1H,
−CH), 4.81 (s, 2H, −COCH O−), 2.38 (s, 3H, −CH ).
General Synthetic Procedure for 12a,b. A mixture of 11a,b (2
mmol), EDC (2.1 mmol), and DMAP (0.6 mmol) in CH Cl (5 mL)
6
) δ 7.67
2
7-(4-Hydroxybutoxy)-4-methyl-2H-chromen-2-one (7e). The
title compound was obtained starting from 5b and 4-chlorobutan-1-ol
white solid, 72% yield, mp 127−130 °C). Analytical data for 7e: ESI-
2
3
(
+
1
MS m/z 249.1 [M + H] ; H NMR (400 MHz, CDCl ) δ 7.49 (dd,
2
2
3
1
−
H, ArH, J = 8.8, 3.0 Hz), 6.90−6.72 (m, 2H, ArH), 6.13 (s, 1H,
was stirred at 0 °C for 0.5 h. After addition of glycol (4 mL) to the
solution, the mixture was further stirred at room temperature for 24 h.
The solution was washed with water (3 × 20 mL). After removal of
the solvent in vacuo, the crude product was purified by column
CH), 4.53 (t, 2H, −OCH −, J = 6.12 Hz), 4.13 (t, 2H, −CH O−,
2
2
J = 5.92 Hz), 2.39 (d, J = 1.2 Hz, 3H), 2.08 (m, 4H, −CH CH −).
2
2
7
-(2-Hydroxyethoxy)-4,8-dimethyl-2H-chromen-2-one (7f).
The title compound was obtained starting from 5c and 2-
chloroethanol (white solid, 84% yield, mp 116−118 °C). Analytical
data for 7f: ESI-MS m/z 235.1 [M + H] ; H NMR (400 MHz,
chromatography (eluent, CH
51%).
Cl /MeOH 50:1) to give 12a,b (40−
2 2
+
1
2-Hydroxyethyl 2-(2-Oxo-2H-chromen-7-yloxy)acetate
12a). The title compound was obtained starting from 11a and glycol
(
CDCl ) δ 7.42 (d, 1H, ArH, J = 8.8 Hz), 7.26 (s, 1H, ArH), 6.85 (d,
3
(
white solid, 40% yield, mp 85−87 °C). Analytical data for 12a: ESI-
1
H, ArH, J = 8.8 Hz), 6.15 (d, 1H, −CH, J = 1.0 Hz), 4.23−4.15
+ 1
MS m/z 265.1 [M + H] ; H NMR (400 MHz, CDCl ) δ 7.66 (d, 1H,
(
m, 2H, −CH O), 4.08−3.99 (m, 2H, −CH O), 2.40 (s, 3H, CH ),
3
2
2
3
−
CHCH−, J = 9.5 Hz), 7.42 (d, 1H, ArH, J = 8.6 Hz, 1H), 6.91
2
.34 (s, 3H, CH ), 1.98 (t, 1H, OH, J = 6.3 Hz).
3
(
dd, 1H, ArH, J = 8.6, 2.5 Hz), 6.81 (d, 1H, ArH, J = 2.4 Hz), 6.29 (d,
7-(2-Hydroxyethylamino)-4-methyl-2H-chromen-2-one (7g).
1
2
Hz).
H, −CHCH−, J = 9.5 Hz), 4.77 (s, 2H, −COCH O−), 4.38 (dd,
The title compound was obtained starting from 5e and 2-
bromoethanol (white solid, 40% yield, mp 145−147 °C). Analytical
data for 7g: ESI-MS m/z 220.1 [M + H] ; H NMR (400 MHz,
DMSO-d ) δ 7.40 (d, 1H, ArH, J = 8.7 Hz), 6.61 (dd, 1H, ArH, J =
8
2
H, −CH O−, J = 5.4, 3.9 Hz), 3.90 (dd, 2H, −CH O−, J = 5.4, 3.9
2
2
+
1
2
-Hydroxyethyl 2-(4-Methyl-2-oxo-2H-chromen-7-yloxy)-
6
acetate (12b). The title compound was obtained starting from 11b
and glycol (white solid, 51% yield, mp 111−112 °C). Analytical data
.7, 1.7 Hz), 6.41 (d, 1H, ArH, J = 1.7 Hz), 5.89 (s, 1H, −CH), 3.54
(
3
t, 2H, −OCH −, J = 5.8 Hz), 3.19−3.10 (m, 2H, −CH N−), 2.28 (s,
2
2
+ 1
for 12b: ESI-MS m/z 279.1 [M + H] ; H NMR (400 MHz, CDCl ) δ
3
H,-CH ).
3
7
6
.52 (d, 1H, ArH, J = 8.8 Hz), 6.92 (dd, 1H, ArH, J = 8.8, 2.6 Hz),
.79 (d, 1H, ArH, J = 2.5 Hz), 6.16 (d, 1H, −CH, J = 1.1 Hz), 4.75
General Procedure for the Preparation of 16a,b. The
procedure was identical to that used in the preparation of 7a−g
75−93%).
-(2-Hydroxyethoxy)-2-ethyl-4H-chromen-4-one (16a). The
title compound was obtained starting from 14 and 2-bromoethanol
(
2
s, 2H, −COCH O−), 4.37 (t, 2H, −OCH −, J = 4.6 Hz), 3.89 (t,
2
2
(
H, −CH O−, J = 4.6 Hz), 2.40 (d, 3H, −CH , J = 1.1 Hz).
2
3
7
General Procedure for the Target Compounds 6a−c, 8a−g,
1
0a, 13a,b, 15, and 17a,b as Well as the NO Donor Moiety
(
white crystal, 93% yield, mp 97−98 °C). Analytical data for 16a: ESI-
Compound 18. Compound 4 (1 mmol) was added to a stirred
solution of compound alcohol selected from the group 5a−c, 7a−g,
+
1
MS m/z 235.1 [M + H] ; H NMR (400 MHz, CDCl ) δ 8.07 (d, 1H,
3
ArH, J = 8.9 Hz), 6.95 (dd, 1H, ArH, J = 8.9, 2.4 Hz), 6.84 (d, 1H,
9
a, 12a,b, 14, 16a,b, or 2-methoxyethanol (1.2 mmol) in the presence
ArH, J = 2.4 Hz), 6.12 (s, 1H, −CH), 4.21−4.13 (m, 2H, −CH −),
2
of 8-diazabicyclo[5.4.0]undec-7-ene (DBU) (3 mmol) in CH Cl . The
2
2
4
1
.07−3.98 (m, 2H, −CH −), 2.68−2.58 (m, 2H, −CH −), 2.26 (t,
2
2
reaction mixture was stirred at room temperature for 2−5 h and then
washed with water (3 × 10 mL). The organic layer was dried with
anhydrous Na SO , and the solvent was removed in vacuo to give an
H, −OH, J = 6.2 Hz), 1.30 (t, 3H, CH , J = 7.5 Hz).
3
7
-(3-Hydroxypropoxy)-2-ethyl-4H-chromen-4-one (16b).
2
4
The title compound was obtained starting from 14 and 3-
chloropropan-1-ol (white solid, 75% yield, mp 71−73 °C). Analytical
data for 16b: ESI-MS m/z 249.1 [M + H] ; H NMR (400 MHz,
oil. The oil was added methanol to precipitate. After filtration, the filter
residue was purified by recrystallizing using ethyl acetate or ethanol to
give the respective title products listed below.
+
1
DMSO-d ) δ 7.86 (d, 1H, ArH, J = 8.8 Hz), 7.07 (s, 1H, ArH), 6.99
6
4
-(2H-Chromen-2-one-7-yloxy)-3-(phenylsulfonyl)-1,2,5-ox-
(
d, 1H, ArH, J = 8.9 Hz), 6.11 (s, 1H, −COCH), 4.59 (t, 1H, OH, J
adiazole 2-Oxide (6a). The title compound was obtained starting
from 5a and 4 (white solid, 84% yield, mp 177−179 °C). Analytical
=
=
−
4.7 Hz), 4.15 (t, 2H, −OCH −, J = 6.0 Hz), 3.54 (q, 2H, −CH −, J
2
2
5.4 Hz), 2.62 (q, 2H, −CH O−, J = 7.4 Hz), 2.05−1.67 (m, 2H,
+
+ 1
2
data for 6a: ESI-MS m/z 387.0 [M + H] , 409.0 [M + Na] ; H NMR
CH CH ), 1.20 (dd, 3H, −CH CH , J = 8.1, 6.9 Hz).
2
3
2
3
(400 MHz, CDCl ) δ 6.16 (d, 1H, −HCCH−, J = 9.56 Hz), 7.28
3
General Synthetic Procedure for 9a. A mixture of 7g (100 mg,
(dd, 1H, ArH, J = 8.54, 2.40 Hz), 7.32 (d, 1H, ArH, J = 2.12 Hz), 7.58
0
.46 mmol) and CH I (133 mg, 0.92 mmol) in DMF (3 mL) was
3
(d, 1H, ArH, J = 8.48 Hz), 7.67 (t, 2H, ArH, J = 7.56 Hz), 7.72 (d, 1H,
refluxed for 2 h. Then the reaction mixture was poured into ice−water
−
CHCH−, J = 9.60 Hz), 7.81 (t, 1H, ArH, J = 7.56 Hz) 8.09 (d,
(
30 mL) and filtrated. The filtrate was washed with water (3 × 5 mL)
13
2
1
1
H, ArH, J = 7.44 Hz); C NMR (101 MHz, CDCl ) δ 159.79,
3
and dried under infrared lamp to give 7-(N-(2-hydroxyethyl)-N-
methylamino)-4-methyl-2H-chromen-2-one (9a), which was used in
the next reaction without further purification. ESI-MS m/z 234.1 [M +
57.37, 154.80, 154.57, 147.47, 137.55, 136.02, 129.84, 129.29, 128.64+,
17.23, 116.71, 116.09, 110.59, 108.46; ESI-HRMS (m/z) [M + H]
calcd for C H N O S 387.0287, obsd 387.0292, ppm error 1.3.
17
10
2
7
+
H] (95 mg, 88%).
4
-(4-Methyl-2H-chromen-2-one-7-yloxy)-3-(phenylsulfonyl)-
General Procedure for the Preparation of 11a,b. At room
1,2,5-oxadiazole 2-Oxide (6b). The title compound was obtained
starting from 5b and 4 (white solid, 90% yield, mp 201−203 °C).
temperature, 2-chloroacetic acid (2 mmol) and K CO (3 mmol) were
2
3
+
+
added into a stirred solution of 5a−e (1 mmol) in DMF (5 mL). After
reflux for 1 h, water (20 mL) was added to dissolve the mixture and
further acidified by 2 N aq HCl to reprecipit the solid. After filtration,
the residue was washed with water (3 × 10 mL) and dried with
infrared lamp to gain white solid 11a,b (69−85%).
Analytical data for 6b: ESI-MS m/z 401.0 [M + H] , 423.0 [M + Na] ;
1
H NMR (400 MHz, CDCl ) δ 2.46 (s, 3H, −CH C), 6.32 (s, 1H,
3
3
−CH CCHCOO), 7.31 (m, 2H, ArH), 7.68 (m, 3H, ArH), 7.81 (t,
3
13
1H, ArH, J = 6.4 Hz), 8.10 (d, 2H, ArH, J = 7.48 Hz); C NMR (101
MHz, CDCl ) δ 159.98, 157.44, 154.44, 154.30, 151.59, 137.55,
3
2
-(2-Oxo-2H-chromen-7-yloxy)acetic Acid (11a). The title
compound was obtained starting from 5a and 2-chloroacetic acid
white solid, 69% yield, mp 198−200 °C). Analytical data for 11a: ESI-
136.01, 129.84, 128.64, 126.16, 118.48, 115.80, 115.05, 110.60, 108.50,
+
18.77. ESI-HRMS (m/z) [M + H] calcd for C H N O S 401.0443,
18
12
2
7
(
obsd 401.0446, ppm error 0.7.
4-(4,8-Dimethyl-2H-chromen-2-one-7-yloxy)-3-(phenylsul-
fonyl)-1,2,5-oxadiazole 2-Oxide (6c). The title compound was
+
1
MS m/z 221.0 [M + H] ; H NMR (400 MHz, DMSO-d ) δ 8.01−
6
7
.88 (m, 2H, ArH), 7.62 (d, 1H, −CHCH−, J = 9.3 Hz), 6.93 (m,
J
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Journal of Medicinal Chemistry
Article
obtained starting from 5c and 4 (white solid, 80% yield, mp 243−245
Hz), 4.53 (t, 2H, −OCH −, J = 6.12 Hz), 6.14 (s, 1H, −CH C
2
3
+
°
C). Analytical data for 6c: ESI-MS m/z 415.0 [M + H] , 437.0 [M +
CH−COO−), 6.82 (d, 1H, ArH, J = 2.20 Hz), 6.87 (dd, 1H, ArH, J =
8.76, 2.36 Hz),7.51 (d, 1H, ArH, J = 8.80 Hz), 7.61 (t, 3H, ArH, J =
8.00 Hz), 7.76 (t, 1H, ArH, J = 7.32 Hz), 8.05 (d, 2H, ArH, J = 8.16
+
1
Na] ; H NMR (400 MHz, CDCl ) δ 2.39 (s, 3H, CCH ), 2.46 (s,
3
3
3
H, ArCH ), 6.33 (s, 1H, −CH CCH−COO−), 7.27 (m, 1H,
3
3
ArH), 7.54 (d, 1H, ArH, J = 8.76 Hz), 7.68 (t, 2H, ArH, J = 7.68 Hz),
.83 (t, 3H, ArH, J = 7.48 Hz), 8.14 (d, 2H, ArH, J = 7.40 Hz); 13
NMR (101 MHz, CDCl ) δ 160.22, 157.88, 152.78, 152.63,
Hz); 13C NMR (101 MHz, CDCl ) δ 161.73,161.27, 158.89, 155.19,
3
7
C
152.55, 137.91, 135.62, 129.62, 128.48, 125.59, 113.65, 112.44, 111.98,
110.43, 101.39, 71.08, 67.64, 25.41, 25.34, 18.68; ESI-HRMS (m/z)
3
+
1
1
52.01,137.73, 135.99, 129.88, 128.57,122.80, 118.76, 118.51, 115.84,
14.86, 110.48, 18.89, 9.12; ESI-HRMS (m/z) [M + H] calcd for
[M + H] calcd for C H N O S 473.1019, obsd 473.1021, ppm error
22
20
2
8
+
0.4.
C H N O S 415.0600, obsd 415.0600, ppm error 0.
4-(2-(4,8-Dimethyl-2-oxo-2H-chromen-7-yloxy)ethanoxy)-3-
(phenylsulfonyl)-1,2,5-oxadiazole 2-Oxide (8f). The title com-
pound was obtained starting from 7f and 4 (yellow solid, 84% yield,
1
9
14
2
7
4
-(2-(2-Oxo-2H-chromen-7-yloxy)ethanoxy)-3-(phenylsul-
fonyl)-1,2,5-oxadiazole 2-Oxide (8a). The title compound was
+
obtained starting from 7a and 4 (white solid, 84% yield, mp 139−141
mp 181−185 °C). Analytical data for 8f: ESI-MS m/z 459.1 [M + H] ,
+
+ 1
°
C). Analytical data for 8a: ESI-MS m/z 431.0 [M +H] , 453.0 [M +
481.0 [M + Na] ; H NMR (400 MHz, CDCl ) δ 7.98 (d, 2H, ArH, J
3
+
1
Na] ; H NMR (400 MHz, CDCl ) δ 8.02 (d, 2H, ArH, J = 7.4 Hz),
= 7.6 Hz), 7.69 (t, 1H, ArH, J = 7.5 Hz), 7.50 (t, 2H, ArH, J = 7.9 Hz),
7.44 (d, 1H, ArH, J = 8.8 Hz), 6.89 (d, 1H, ArH, J = 8.8 Hz), 6.16 (s,
3
7.73 (t, 1H, ArH, J = 7.5 Hz), 7.67 (d, 1H, −CHCH−, J = 9.5 Hz),
7.56 (t, 2H, ArH, J = 7.9 Hz), 7.44 (d, 1H, ArH, J = 8.6 Hz), 6.90 (dd,
1H, ArH, J = 8.6, 2.3 Hz), 6.85 (d, 1H, ArH, J = 2.1 Hz), 6.30 (d, 1H,
1H, −CH), 4.84-.79 (m, 2H, −OCH −), 4.51−4.44 (m, 2H,
2
13
−OCH −), 2.40 (s, 3H, −CH ), 2.26 (s, 3H, −CH ). C NMR (101
MHz, CDCl ) δ 161.32, 158.76, 158.69, 152.68, 152.61, 137.91,
2
3
3
−
CHCH−, J = 9.5 Hz), 4.86−4.73 (m, 2H, −CH O−), 4.47−4.37
2
3
13
(
m, 2H, −CH O−). C NMR (101 MHz, CDCl ) δ 161.32, 158.76,
135.68, 129.62, 128.36, 122.64, 114.69, 114.46, 112.37, 107.74, 69.30,
2
3
+
1
58.79, 152.68, 152.61, 137.91, 135.68, 129.62, 128.36, 122.64, 114.69,
66.03, 18.77, 8.29. ESI-HRMS (m/z) [M + H] calcd for
1
14.46, 112.37, 110.35, 107.74, 69.30, 66.03, 18.77, 8.29; ESI-HRMS
C H N O S 459.0862, obsd 459.0864, ppm error 0.4.
21
18
2
8
+
(m/z) [M + H] calcd for C H N O S 431.0549, obsd 431.0557,
4-(2-(4-Methyl-2-oxo-2H-chromen-7-azyl)ethanoxy)-3-(phe-
nylsulfonyl)-1,2,5-oxadiazole 2-Oxide (8g). The title compound
was obtained starting from 7g and 4 (yellow solid, 80% yield, mp
19
14
2
8
ppm error 1.8.
4
-(2-(4-Methyl-2-oxo-2H-chromen-7-yloxy)ethanoxy)-3-
+
(
phenylsulfonyl)-1,2,5-oxadiazole 2-Oxide (8b). The title com-
193−195 °C). Analytical data for 8g: ESI-MS m/z 444.0 [M + H] ,
+
1
pound was obtained starting from 7b and 4 (white solid, 93% yield,
mp 162−165 °C). Analytical data for 8b: ESI-MS m/z 445.0 [M +
466.0 [M + Na] ; H NMR (400 MHz, DMSO-d ) δ 2.31 (s, 3H, 
6
CCH ), 3.60 (m, 2H, −CH NH−), 4.53 (t, 2H, −OCH −, J = 5.04
3
2
2
+
+ 1
H] , 467.0 [M + Na] ; H NMR (400 MHz, CDCl ) δ 2.43 (s, 3H,
Hz), 5.93 (s, 1H, −CH CCH−COO−), 6.54 (d, 1H, ArH, J = 1.52
3
3
CCH ), 4.45 (t, 2H, −CH O−, J = 4.12 Hz), 4.81 (t, 2H, −OCH −, J
Hz), 6.67 (d, 1H, ArH, J = 8.72 Hz), 6.87 (t, 1H, ArH, J = 5.64 Hz),
7.47 (d, 1H, ArH, J = 8.76 Hz), 7.66 (t, 2H, ArH, J = 7.72 Hz), 7.85 (t,
1H, ArH, J = 7.36 Hz), 7.96 (d, 2H, ArH, J = 7.88 Hz); 13C NMR (101
3
2
2
=
=
7
4.20 Hz), 6.18 (s, 1H, −CH CCH−COO−), 6.85 (d, 1H, ArH, J
3
2.32 Hz), 6.92 (dd, 1H, ArH, J = 8.80, 2.36 Hz), 7.57 (m, 3H, ArH),
.73 (t, 1H, ArH, J = 7.57 Hz), 8.03 (d, 2H, ArH, J = 7.72 Hz); 13
C
MHz, DMSO-d ) δ 160.62, 158.93, 155.59, 153.66, 151.93, 137.08,
6
NMR (101 MHz, CDCl ) δ 161.05, 160.97, 158.65, 155.09, 152.42,
136.06, 129.88, 128.30, 126.04, 110.54, 110.31, 109.12, 107.80, 96.53,
69.68, 40.93, 18.00; ESI-HRMS (m/z) [M + H]+ calcd for
C H N O S 444.0865, obsd 444.0867, ppm error 0.4.
3
1
37.89, 135.68, 129.63, 128.51, 125.87, 114.27, 112.46, 112.20, 110.33,
+
1
01.81, 69.03, 65.63, 18.71; ESI-HRMS (m/z) [M + H] calcd for
20
17
3
7
C H N O S 445.0706, obsd 445.0712, ppm error 1.3.
4-(2-(Methyl(4-methyl-2-oxo-2H-chromen-7-yl)amino)-
2
0
16
2
8
4
-(2-(4-Methyl-2-oxo-2H-chromen-7-ylthio)ethanoxy)-3-
ethoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-Oxide (10a).
The title compound was obtained starting from 9a and 4 (yellow
solid, 40% yield, mp 181−185 °C). Analytical data for 10a: ESI-MS m/
(
phenylsulfonyl)-1,2,5-oxadiazole 2-Oxide (8c). The title com-
pound was obtained starting from 7c and 4 (white solid, 83% yield, mp
+
+
+ 1
1
4
62−165 °C). Analytical data for 8c: ESI-MS m/z 461.0 [M + H] ,
z 458.0 [M + H] , 480.0 [M + Na] ; H NMR (400 MHz, CDCl ) δ
3
+
1
83.0 [M + Na] ; H NMR (400 MHz, CDCl ) δ 2.42 (s, 3H, 
2.36 (s, 3H, CCH ), 3.19 (s, 3H, −NCH ), 3.95 (t, 2H, −CH N−, J
3
3
3
2
CCH ), 3.49 (t, 2H, −CH S−, J = 6.76 Hz), 4.63 (t, 2H, −OCH −, J
=
= 5.32 Hz), 4.63 (t, 2H, −OCH −, J = 5.28 Hz), 6.02 (s, 1H,
3
2
2
2
6.76 Hz), 6.25 (s, 1H, −CH CCH−COO−), 7.28 (m, 2H, ArH),
−CH CCH−COO−), 6.59 (d, 1H, ArH, J = 2.52 Hz), 6.76 (dd,
3
3
7
1
.53 (d, 1H, ArH, J = 8.8 Hz), 7.63 (t, 2H, ArH, J = 7.68 Hz), 7.77 (t,
1H, ArH, J = 8.92, 2.56 Hz), 7.46 (d, 1H, ArH, J = 8.92 Hz), 7.57 (t,
2H, ArH, J = 7.88 Hz), 7.74 (t, 1H, ArH, J = 7.48 Hz), 7.95 (d, 2H,
H, ArH, J = 7.44 Hz), 8.05 (d, 2H, ArH, J = 8.24 Hz); 13C NMR (101
MHz, CDCl ) δ 160.33, 158.46, 153.77, 152.00, 140.25, 137.79,
ArH, J = 7.40 Hz); 13C NMR (101 MHz, CDCl ) δ 206.05, 161.81,
3
3
1
6
35.72, 129.68, 128.54, 125.12, 123.81, 118.13, 115.50, 114.50, 110.31,
8.91, 30.74, 18.71; ESI-HRMS (m/z) [M + H] calcd for
158.72, 155.64, 152.79, 151.27, 137.71, 135.72, 129.64, 128.41, 125.72,
+
110.32, 109.90, 108.92, 98.48, 68.51, 50.64, 39.34, 18.51; ESI-HRMS
+
C H N O S 461.0477, obsd 461.0489, ppm error 2.6.
(m/z) [M + H] calcd for C H N O S 458.1022, obsd 458.1033,
2
0
16
2
7
2
21 19
3
7
4
-(3-(4-Methyl-2-oxo-2H-chromen-7-yloxy)propanoxy)-3-
ppm error 2.4.
(
phenylsulfonyl)-1,2,5-oxadiazole 2-Oxide (8d). The title com-
4-(2-(2-(2-Oxo-2H-chromen-7-yloxy)acetyl)oxyethoxy)-3-
(phenylsulfonyl)-1,2,5-oxadiazole 2-Oxide (13a). The title
compound was obtained starting from 12a and 4 (white solid, 50%
yield, mp 174−177 °C). Analytical data for 13a: ESI-MS m/z 488.9
pound was obtained starting from 7d and 4 (white solid, 79% yield,
mp 116−118 °C). Analytical data for 8d: ESI-MS m/z 459.0 [M +
+
+ 1
H] , 480.9 [M + Na] ; H NMR (400 MHz, CDCl ) δ 2.40 (m, 5H,
3
+
+ 1

CCH and −CH −), 4.23 (t, 2H, −CH O−, J = 5.84 Hz), 4.65 (t,
[M + H] , 510.9 [M + Na] ; H NMR (400 MHz, CDCl ) δ 4.68 (s,
3
2
2
3
2
6
H, −OCH −, J = 6.00 Hz), 6.16 (s, 1H, −CH CCH−COO−),
4H, −OCH CH O−), 4.80 (s, 2H, −COCH O−), 6.28 (d, 1H,
2
3
2
2
2
.84 (d, 1H, ArH, J = 1.84 Hz), 6.89 (dd, 1H, ArH, J = 8.76, 2.28
−HCCH−, J = 9.44 Hz), 6.83 (s, 1H, ArH), 6.90 (d, 1H, ArH, J =
8.64 Hz),7.41 (d, 1H, ArH, J = 8.52 Hz),7.63 (m, 3H, ArH and
−HCCH−), 7.76 (t, 1H, ArH, J = 7.28 Hz), 8.07 (d, 2H, ArH, J =
Hz),7.52 (d, 1H, ArH, J = 8.80 Hz),7.58 (t, 2H, ArH, J = 7.60 Hz),
.74 (t, 1H, ArH, J = 7.40 Hz), 8.02 (d, 2H, ArH, J = 8.32 Hz); 13
NMR (101 MHz, CDCl ) δ161.46, 161.17, 158.78, 155.16, 152.49,
7
C
7.60 Hz); 13C NMR (101 MHz, CDCl ) δ 167.85,160.82, 160.52,
3
3
1
37.86, 135.67, 129.63, 128.44, 125.67, 113.84, 112.39, 112.15, 110.41,
158.49, 155.56, 143.14, 137.74, 135.70, 129.65, 129.08, 128.61, 113.88,
1
09.72, 101.46, 67.78, 63.99, 28.24, 18.68; ESI-HRMS (m/z) [M +
113.45, 112.49, 101.91, 68.41, 65.11, 61.91, 29.67; ESI-HRMS (m/z)
+
+
H] calcd for C H N O S 459.0862, obsd 459.0864, ppm error 0.4.
[M + H] calcd for C H N O S 489.0604, obsd 489.0613, ppm
21
18
2
8
21 16
2
10
4
-(4-(4-Methyl-2-oxo-2H-chromen-7-yloxy)butanoxy)-3-
phenylsulfonyl)-1,2,5-oxadiazole 2-Oxide (8e). The title com-
pound was obtained starting from 7e and 4 (white solid, 70% yield, mp
error 1.8.
(
4-(2-(2-(4-Methyl-2-oxo-2H-chromen-7-yloxy)acetyl)-
oxyethoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-Oxide (13b).
The title compound was obtained starting from 12b and 4 (white
solid, 65% yield, mp 113−115 °C). Analytical data for 13b: ESI-MS
+
1
4
53−155 °C). Analytical data for 8e: ESI-MS m/z 473.3 [M + H] ,
95.0 [M + Na] ; H NMR (400 MHz, CDCl ) δ 2.08 (m, 4H,
CH CH −), 2.40 (s, 3H, CCH ), 4.13 (t, 2H, −CH O−, J = 5.92
+ 1
3
+
+ 1
−
m/z 502.9 [M + H] , 525.0 [M + Na] ; H NMR (400 MHz, CDCl )
2
2
3
2
3
K
dx.doi.org/10.1021/jm500613m | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
δ 2.39 (s, 3H, CCH ), 4.67 (s, 4H, −OCH CH O−), 4.80 (s, 2H,
150 μL of DMSO per well was added to dissolve the formazan crystals
newly formed. Absorbance of each well was determined by a
microplate reader (Synergy H4, Bio-Tek) at a 570 nm wavelength.
The inhibition rates of proliferation were calculated with the following
equation:
3
2
2
−
COCH O−), 6.16 (s, 1H, −CH CCH−COO−), 6.82 (d, 1H,
2
3
ArH, J = 2.32 Hz), 6.92 (dd, 1H, ArH, J = 8.88, 2.08 Hz), 7.53 (d, 1H,
ArH, J = 8.80 Hz), 7.62 (t, 3H, ArH, J = 7.69 Hz), 7.76 (t, 1H, ArH, J
=
7.44 Hz), 8.06 (d, 2H, ArH, J = 8.16 Hz); 13C NMR (101 MHz,
CDCl ) δ 167.91, 160.94, 160.35, 158.50, 154.96, 152.35, 137.74,
3
1
6
35.70, 129.66, 128.60, 125.91, 114.55, 112.58, 112.15, 110.37, 101.90,
8.44, 65.09, 61.93, 18.67; ESI-HRMS (m/z) [M + H] calcd for
ODDMSO − ODcompd
+
inhibition ratio (%) =
× 100
ODDMSO − ODblank
C H N O S 503.0760, obsd 503.0762, ppm error 0.4.
2
2
18
2
10
4
-(2-Ethyl-4H-chromen-4-one-7-yloxy)-3-(phenylsulfonyl)-
The concentrations of the compounds that inhibited cell growth by
0% (IC ) were calculated using GraphPad Prism, version 6.0. For
the NO scavenge experiment, cells were pretreated with the indicated
concentrations of hemoglobin (Hb) (0, 1.25, 2.5, 5, 10, or 20 μM) for
1 h and treated with 500 nM 8b for 24 h. Then the viability of the cells
was determined by MTT reagent as described above.
1
,2,5-oxadiazole 2-Oxide (15). The title compound was obtained
5
50
starting from 14 and 4 (white solid, 89% yield, mp 159−161 °C).
Analytical data for 15: ESI-MS m/z 415.0 [M + H] , 437.0 [M + Na] ;
H NMR (400 MHz, CDCl ) δ 1.32 (t, 3H, CH CH , J = 7.56 Hz),
+
+
1
3
2
3
2
7
7
.47 (q, 2H, −CH CH , J = 7.56 Hz), 6.21 (s, 1H, −OCCH−),
2
3
.32 (dd, 1H, ArH, J = 8.80, 2.36 Hz), 7.46 (d, 1H, ArH, J = 2.32 Hz),
.66 (t, 2H, ArH, J = 8.28 Hz), 7.81 (t, 1H, ArH, J = 7.56 Hz), 8.08
Capillary-like Tube Formation Assay. Capillary-like tube
formation assay was performed by following the procedure published
36
(
dd, 2H, ArH, J = 1.28, 8.48 Hz), 8.27 (d, 1H, ArH, J = 8.80 Hz); 13
C
previously. Briefly, 70 μL per well Matrigel (Corning, NY) was
NMR (101 MHz, CDCl ) δ 177.22, 171.33, 157.12, 156.97, 155.89,
added to 96-well plates and incubated at 37 °C for 30 min to allow
gelation to occur. HUVECs were added to the top of the gel at a
density of 30 000 cells/well in the presence of tested compound or
vehicle control (DMSO). Cells were incubated at 37 °C with 5% CO2
overnight, and pictures were captured with a CCD Sensicam camera
mounted on a Olympus inverted microscope.
3
1
1
37.55, 135.99, 129.83, 128.60, 128.01, 121.93, 116.62, 110.61, 109.20,
08.60, 27.41, 10.85; ESI-HRMS (m/z) [M + H]+ calcd for
C H N O S 415.0600, obsd 415.0604, ppm error 0.9.
1
9
4
14
2
7
-(2-(2-Ethyl-4H-chromen-4-one-7-yloxy)ethanoxy)-3-(phe-
nylsulfonyl)-1,2,5-oxadiazole 2-Oxide (17a). The title compound
was obtained starting from 16a and 4 (white solid, 81% yield, mp
67−169 °C). Analytical data for 17a: ESI-MS m/z 458.9 [M + H] ,
80.9 [M + Na] ; H NMR (400 MHz, CDCl ) δ 1.31 (t, 3H,
Nitrite Measurement in Vitro. The levels of NO released by
tested compounds in the cells are presented as that of nitrite and were
determined by the Griess reagent (Beyotime, China), according to the
+
1
4
−
(
6
1
1
+
1
3
2
5
7
CH CH , J = 7.52 Hz), 2.65 (q, 2H, −CH CH , J = 7.56 Hz), 4.49
2
3
2
3
literature with some modifications. Briefly, cells (1 × 10 per 10 cm
dish) were treated with a 100 μM concentration of each compound for
150 min. Subsequently, the cells were harvested and lysed with 100 μL
of RIPA lysis buffer (Beyotime, China) for 30 min on ice. The cell
lysates were mixed with Griess for 30 min in a dark place, followed by
measurement by a microplate reader (Synergy H4, Bio-Tek) at 540
nm wavelength. The cells treated with diluent were used to determine
the background levels of nitrite production, while sodium nitrite at
different concentrations was measured to generate a standard curve.
Measurement of Intracellular NO. Intracellular NO was
measured with 3-amino,4-aminomethyl-2′,7′-difluorescein, diacetate
t, 2H, −CH O−, J = 4.44 Hz), 4.82 (t, 2H, −OCH −, J = 4.28 Hz),
2
2
.14 (s, 1H, −CCH−), 6.90 (d, 1H, ArH, J = 2.36 Hz), 6.97 (dd,
H, ArH, J = 8.82, 2.24 Hz), 7.53 (t, 2H, ArH, J = 7.76 Hz), 7.71 (t,
H, ArH, J = 7.52 Hz), 8.00 (d, 2H, ArH, J = 7.56 Hz), 8.14 (d, 1H,
13
ArH, J = 8.84 Hz); C NMR (101 MHz, CDCl ) δ 177.80, 170.52,
62.22, 158.63, 158.01, 137.87, 135.66, 129.62, 128.46, 127.31, 118.14,
14.12, 110.31, 108.81, 101.09, 69.04, 65.60, 27.37, 10.91; ESI-HRMS
m/z) [M + H] calcd for C H N O S 459.0862, obsd 459.0859,
3
1
1
(
+
21
18
2
8
ppm error 0.6.
4
-(3-(2-Ethyl-4H-chromen-4-one-7-yloxy)propanoxy)-3-
(
phenylsulfonyl)-1,2,5-oxadiazole 2-Oxide (17b). The title
(
DAF-FM DA, purchased from beyotime,China), as previously
37
compound was obtained starting from 16b and 4 (yellow solid, 74%
yield, mp 153−155 °C). Analytical data for 17b: ESI-MS m/z 473.0
M + H] , 495.0 [M + Na] ; H NMR (400 MHz, CDCl ) δ 1.28 (t,
described. Briefly, A2780 cells were pretreated with 5 μM DAF-
FM DA at 37 °C for 20 min. And then the cells were washed with PBS
three times and incubated with tested compound or vehicle control
(DMSO) for 2 h. Finally, the cells were observed under a fluorescence
microscope (Olympus, Japan) and pictures were captured with a CCD
Sensicam camera.
+
+ 1
[
3
−
2
3
H, −CH CH , J = 7.52 Hz), 2.40 (m, 2H, −CCH C−), 2.62 (q, 2H,
2
3
2
CH CH , J = 7.60 Hz), 4.25 (t, 2H, −CH O−, J = 5.84 Hz), 4.64 (t,
2
3
2
H, −OCH −, J = 6.00 Hz), 6.11 (s, 1H, −CCH−), 6.86 (d, 1H,
2
3
8
3
ArH, J = 2.32 Hz), 6.93 (dd, 1H, ArH, J = 8.86, 2.32 Hz), 7.51 (t, 2H,
ArH, J = 8.16 Hz), 7.69 (t, 1H, ArH, J = 7.56 Hz), 7.96 (d, 2H, ArH, J
Colony Formation Assay. 1×10 cells per well were seeded in
six-well plates at a single cell density. At 48 h later, the cells were
treated with DMSO or different concentrations of compound 8b for
48 h. Then the medium was replaced with fresh medium to allow cell
growth for at least 1 week. The cells were fixed with methyl alcohol for
15 min and stained by gentian violet staining for 30 min. The colonies
consisting of more than 50 cells were counted.
=
7.32 Hz), 8.09 (d, 1H, ArH, J = 8.84 Hz); 13C NMR (101 MHz,
CDCl ) δ 177.87, 170.41, 162.73, 158.77, 158.10, 137.81, 135.64,
29.60, 128.41, 127.11, 117.77, 114.20, 110.40, 108.74, 100.79, 67.66,
3.96, 28.23, 27.35, 10.92; ESI-HRMS (m/z) [M + H] calcd for
3
1
6
+
C H N O S 473.1019, obsd 473.1011, ppm error 1.7.
2
2
4
20
2
8
-(2-Methoxyethanoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole
Cell Cycle Analysis. Cell cycle status was detected by flow
39
2
-Oxide (18). The title compound was obtained starting from 2-
cytometry according to a previously published method and were
analyzed by Multicycle AV (for Windows, version 320) software.
Briefly, cells were first treated with DMSO or different concentrations
of compound 8b for 24 h and then harvested, washed twice with 1×
PBS, and resuspended in 200 μL of 1× PBS. The cells were fixed in 4
mL of ice-cold 75% ethanol at −20 °C overnight and stained with 500
μL of propidium iodide (50 μg/mL, Sigma) containing 0.1% RNase (1
mg/mL, Sigma) for 15 min in dark conditions at room temperature.
The cells were then analyzed by flow cytometry (Cytomics FC 500
MPL, Beckman Coulter). The results were indicated as mean values
from three independent determinations.
methoxyethanol and 4 (white solid, 46% yield, mp 104−106 °C). ESI-
MS m/z 301.0 [M + H] , 323.0 [M + Na] ; H NMR (400 MHz,
CDCl ) δ 8.08 (d, 2H, ArH, J = 7.8 Hz), 7.75 (d, 1H, ArH, J = 7.4
+
+ 1
3
Hz), 7.62 (t, 2H, ArH, J = 7.8 Hz), 4.61−4.53 (m, 2H, −CH −, 2H),
2
13
3
.85−3.77 (m, 2H, −CH −), 3.46 (s, 3H, −CH ); C NMR (101
2
3
MHz, DMSO-d ) δ 159.26, 138.33, 135.89, 129.91, 128.91, 110.77,
6
7
0.76, 70.00, 59.59.
In Vitro Antiproliferative Assay. The in vitro antiproliferation of
the chemical compounds was measured by the MTT reagent, as
described in the literature. Briefly, 5 × 10 cells in 100 μL of medium
per well were plated in 96-well plates. After incubated for 24 h, the
cells were treated with different concentration of tested compound or
DMSO (as negative control) for 48 h. Then the medium with
compound or DMSO was replaced with 200 μL of fresh medium
containing 10% MTT (5 mg/mL in PBS) in each well and incubated
at 37 °C for 4 h. Last, the MTT-containing medium was discarded and
35
3
Cell Apoptosis Analysis. Cell apoptosis was detected by flow
4
0
cytometry according to a previously published method. Briefly, cells
were incubated with DMSO or different concentrations of compound
8b for 24 h. The cells were harvested, washed twice with cold 1× PBS,
5
and resuspended in 200 μL of binding buffer at a density of 1 × 10
cells/mL. The cells were then stained with 5 μL of annexin-V and PI
L
dx.doi.org/10.1021/jm500613m | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
for 15 min in dark conditions at room temperature and subjected to
analysis by flow cytometry (Cytomics FC 500 MPL, Beckman
Coulter). The early apoptosis was evaluated based on the percentage
of cells with annexin V+/PI−, while the late apoptosis was that of
annexin V+/PI+. The results were indicated as mean values from three
independent determinations.
Western Blot Analysis. A2780 cells were treated with DMSO or
different concentrations of compound 8b for 24 h. Cells were
harvested, washed with cold 1× PBS, and lysed with RIPA lysis buffer
Author Contributions
M.-M.L. and X.-Y.C. are co-first-authors and contributed
equally to this work.
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
∥
Notes
The authors declare no competing financial interest.
(
1
Beyotime, China) for 30 min on ice, then centrifuged at 12000g for
5 min at 4 °C. The total protein concentration was determined by
ACKNOWLEDGMENTS
■
BCA protein assay kit (Beyotime, China). Equal amounts (30 μg per
load) of protein samples were subjected to SDS−PAGE electro-
phoresis and transferred onto polyvinylidene fluoride (PVDF)
membranes (Millipore) which were then blocked in 10% nonfat
milk (BD Biosciences) and reacted with primary antibodies. The
antibodies against Bcl-2, Bax, p53, MEK 1/2, phosphor-MEK1/
This study was supported by grants from the Shanghai Natural
Science Foundation (Grant 14ZR1401900 for Y.C.), National
Natural Science Foundation of China (Grant 81071839 for M.-
M.L. and Grants 91129721 and 81372797 for G.Y.), China
Postdoctoral Science Foundation (Grant 2013M531126 for M.-
M.L.), Shanghai Pujiang Program (Grant 11PJ1402200) from
the Shanghai Municipal Government of China (for G.Y.), and
the Doctoral Fund of Ministry of Education of China (Grant
2
(Ser217/221), ERK1/2, phosphor-ERK1/2(Thr202/Tyr204) were
purchased from Santa Cruz Biotechnology, and phosphor-p53,
cleaved-PARP, and β-actin were purchased from Cell Signaling
Technology. The secondary antibodies conjugated with horseradish
peroxidase (HRP) were from Cell Signaling Technology. The protein
bands were developed by the chemiluminescent reagents (Millipore).
Acute Toxicity. Female KM mice were purchased from Depart-
ment of Laboratory Animal Science, Fudan University (Shanghai,
China), and housed individually in a specific pathogen free facility.
Groups of mice (n = 10 per group) were injected intraperitoneally
once with different concentrations of 8b (500, 300, 275, 250, 225, 200
mg/kg) or vehicle control, respectively. The mouse death was
monitored daily and recorded up to 14 days after injection. The
animal experimental protocols were approved by the Animal Ethics
Committee of School of Pharmacy, Fudan University.
2
0120071110079 for G.Y). We thank LetPub (www.letpub.
com) for its linguistic assistance during the preparation of this
manuscript.
ABBREVIATIONS USED
■
NO, nitric oxide; MEK, mitogen-activated protein kinase
kinase; ERK, extracellular regulated protein kinase; MAPK,
mitogen-activated protein kinase; VEGFR, vascular endothelial
growth factor receptor; EDC, 1-(3-dimethylaminopropyl)-3-
ethylcarbodiimide hydrochloride; DMAP, 4-(dimethylamino)-
pyridine; SAR, structure−activity relationship; DBU, 8-
diazabicyclo[5.4.0]undec-7-ene; FDA, Food and Drug Admin-
istration; JS-K, 4-(2,4-dinitrophenoxy)-NNO-azoxy]piperazine-
1-carboxylic acid ethyl ester
In Vivo Tumor Growth Inhibition Assessment. Female BALB/
c nude mice at 4−5 weeks of age were purchased from Shanghai Slac
Laboratory Animal Co. Ltd. (Shanghai, China) and housed
individually in a specific pathogen free facility. The mice were
6
inoculated subcutaneously with A2780 cells (2 × 10 suspended in 0.2
mL of PBS for each mouse). After reaching an average tumor volume
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