Hybrids of MEK inhibitor and NO donor as multitarget antitumor drugs

Article abstract of DOI:10.1016/j.ejmech.2020.112271

A series of hybrids of MEK inhibitor and nitric oxide donor have been designed and synthesized. Compound 18h [4-(3-((3-(2-fluoro-3-((N-methylsulfamoyl)amino)benzyl)-4-methyl-2-oxo-2H-chromen-7-yl)oxy) propoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide]

Full text of DOI:10.1016/j.ejmech.2020.112271

Journal Pre-proof
Hybrids of MEK inhibitor and NO donor as multitarget antitumor drugs
Chao Wang, Dandan Xi, Han Wang, Yan Niu, Lei Liang, Fengrong Xu, Yihong Peng,
Ping Xu
PII:
S0223-5234(20)30238-5
DOI:
https://doi.org/10.1016/j.ejmech.2020.112271
EJMECH 112271
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 7 February 2020
Revised Date: 3 March 2020
Accepted Date: 23 March 2020
Please cite this article as: C. Wang, D. Xi, H. Wang, Y. Niu, L. Liang, F. Xu, Y. Peng, P. Xu, Hybrids of
MEK inhibitor and NO donor as multitarget antitumor drugs, European Journal of Medicinal Chemistry
(2020), doi: https://doi.org/10.1016/j.ejmech.2020.112271.
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Hybrids of MEK inhibitor and NO donor as multitarget antitumor drugs
Chao Wang, a,1 Dandan Xi, b,1 Han Wang, b Yan Niu, b Lei Liang, b Fengrong Xu, b Yihong Peng, c and Ping Xu*b
a National Pharmaceutical Teaching Laboratory Center, School of Pharmaceutical Sciences, Peking University Health
Science Center, 38 Xueyuan Road, Beijing 100191, China
b Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Peking University Health Science Center, 38
Xueyuan Road, Beijing 100191, China
c Department of Microbiology, School of Basic Medical Sciences, Peking University Health Science Center, 38 Xueyuan
Road, Beijing 100191, China
*Corresponding author:
Email: pingxu@bjmu.edu.cn
1These authors contributed equally to this work.
Abstract
A series of hybrids of MEK inhibitor and nitric oxide donor have been designed and synthesized.
Compound 18h [4-(3-((3-(2-fluoro-3-((N-methylsulfamoyl)amino)benzyl)-4-methyl-2-oxo-2H-chromen-7-
yl)oxy) propoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide] was proven to be more potent than the
clinical compound RO5126766 in MDA-MB-231 cells. Compound 18h can significantly reduce the levels
of pMEK and pERK, induce cell apoptosis in MDA-MB-231 cells, and release NO in cells efficiently,
suggesting that these hybrids, while displaying the properties of both MEK inhibitors and NO donors have
a mechanism of action different from that of MEK inhibitors and NO donors. Thus, we are able to report a
series of multitarget hybrids with better antitumor potency than a known MEK inhibitor and NO donor.
Keywords: Hybrid; MEK; NO donor; multitarget; antitumor.
Introduction
The RAS/RAF/MEK/ERK system is a key signaling pathway involved in the regulation of cell
proliferation, differentiation and apoptosis. Aberrant activation of this pathway contributes to various
cancers and other diseases. The mitogen-activated protein kinase (MEK) is one of the key kinases in this
pathway and the extracellular signal-regulated kinase (ERK) appears to be the main substrate of MEK 1.
Allosteric MEK inhibitors have demonstrated bona fide antitumor efficacy and low toxicity in clinical
trials. Two MEK inhibitors, trametinib (1) and cobimetinib (2) have received FDA approval for the
treatment of metastatic melanoma 2. The preliminary work showed that substituted 3-benzylcoumarins
such as 3-benzyl-4-methyl-2-oxo-2H-chromen-7-yl dimethylcarbamate (4) and 3-benzyl-1,3-benzoxazine-
2,4-dione analogues are potent allosteric MEK inhibitors 3-6. The clinical compound RO5126766 (3),
which is in Phase I clinical trial, is a result of further optimization of the substituted 3-benzyl coumarins 2.
MEK inhibitors have the following characteristics: (1) they bind to the allosteric binding site of the protein
without competing against ATP or ERK, (2) they cause several conformational changes of MEK and lock
the unphosphorylated MEK in a catalytically inactive state, and (3) there is no homologous sequence
between the inhibitor binding site of MEK and other protein kinases 7. These properties made MEK a
highly selective and very promising target for drug discovery.

HN
HO
O
N
O
O
N
O
N
N
O
O
O
F
F
N
H
N
H
O
O
HN
N
N
N
S
N
N
H
H
F
I
F
I
F
O
RO5126766, Phase I
Trametinib, Approved
1
Cobimetinib, Approved
2
3
NO2
O
N
R2
R1
4
N
O
O
O
3
O
N
N
N
N
5
O
O
2
N
O
1
O2N
COOC2H5
JS-K
6
5
4
Fig. 1 The structures of MEK inhibitors and NO donors
In recent years, combination therapy has gained momentum over monotherapies in oncology due to a
decreased likelihood of drug resistance. The combination of MEK inhibitors with other drugs such as
BRAF inhibitors or PI3K/mTOR inhibitors has provided a new treatment choice for various cancers 8. It
has been reported that the combination of an MEK inhibitor and a nitric oxide (NO) donor can
synergistically inhibit proliferation and invasion of cancer cells 9. Hybrids of MEK inhibitor and NO donor
already showed potent antitumor effects in many cancer cell lines10-11. Nitric oxide is a signaling molecule
in many physiological and pathological processes. It has been reported that high levels of NO show very
good anti-proliferation activity in various tumor cells 12. There are several classes of NO donors, including
organic nitrates, diazeniumdiolates, metal-NO complexes, furoxans, S-nitrosothiols and sydnonimines 12.
Of these, furoxans (5) are an important class of NO donors and can release NO when thiol-containing
molecules or proteins attack either C3 or C4 of furoxans 13.
Both MEK inhibitors and NO donors have anti-proliferative activities in cells and we have
designed and synthesized hybrids of a MEK inhibitory motif containing the coumarin core and a
NO donor motif possessing phenylsulfonyl-substituted furoxan as multitarget antitumor drugs (Fig.
2) which would be expected to have synergistic antitumor effects and to be more potent than the
individual known drugs (1, 2 or 3). Their anti-proliferative effects have been evaluated in several
cancer cells and their effects on the RAS/RAF/MEK/ERK pathway. Their pro-apoptotic effects
and NO-releasing effects were also evaluated in an effort to gain a better understanding of the
mechanism of action of these hybrid compounds.
Fig. 2 Strategy for the discovery of hybrids containing a MEK inhibitor and a NO donor.

2. Results and discussion
2.1 Docking
The crystal structure of human MEK1 kinase in a complex with RO5126766 and MgAMP-PNP
(3WIG) was used as the docking MEK protein. Fig. 3 shows the binding mode of 4-(2-((3-(2-
fluoro-3-((N-methylsulfamoyl)amino)benzyl)-4-methyl-2-oxo-2H-chromen-7-yl)oxy)ethoxy)-3-
(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (18a) and the MEK protein (PDB ID: 3WIG) using the
docking program GOLD. A linker was added from position 7 of the coumarin, as this site is on the
exterior of the protein and should have no detrimental effect on the ligand-protein binding.
Fig. 3 (a) Binding mode of 18a to the MEK protein (3WIG). (b) Superposition of the docked
conformation of 18a (pink) and RO5126766 (purple) in the protein 3WIG crystal structure.
2.2 Chemistry
A series of hybrids of the MEK inhibitor and the NO donor with different substituents at the 3-
benzyl group of the coumarin was synthesized using the synthetic route outlined in Scheme 1. The
synthesis of the intermediate phenylsulfonyl-substituted furoxan (10) in three steps from
benzenethiol (
chloroacetic acid to afford 2-(phenylthiol) acetic acid (
to form compound which was converted to 10 by treatment with fuming HNO3. Compounds 16a
16c were prepared according to the synthetic route showed below in Scheme 1. Commercially
available 1-Methyl- 2-fluoro-3-nitrobenzene (11) was allowed to react with -bromosuccinimide
7
) was reported previously10, 14. Briefly, benzenethiol (
), which was then oxidized by 30% H2O2
7) was reacted with
8
9
-
N
(NBS), to give 1-(bromomethyl)-2-fluoro-3-nitrobenzene (12). Compound 12 was reacted with
ethyl acetoacetate affording the alkylation product ethyl 2-(2-fluoro-3-nitrobenzyl)-3-oxobutanoate
(
13). The coumarin (14) was synthesized by reacting compound 13 with resorcinol in a Pechmann
reaction15. Compound 15, which was obtained by reaction of coumarin 14 and halo alcohol (2-
chloroethanol, 3-chloro-1-propanol or 4-chloro-1-butanol), was converted to 16a 16c by reduction
-
of the nitro group. Chlorosulfonylisocyanate was reacted with 2-bromoethanol followed by
reaction of the in situ generated halogenosulfonyloxazolidinone with the appropriate primary

amine, in the presence of Et3N to yield substituted oxazolidin-2-one 19a-19g15. Compound 17a-
17i, which were made by reaction of 16a-16c with 19a-19g 16, was allowed to react with
compound 10 affording the final product (18a-18i) using 8-diazabicyclo[5.4.0]undec-7-ene (DBU)
as a catalyst in CH2Cl2.
Scheme 1 Synthetic routes of target compounds.

SH
7
O
O
O O
S
PhO2S
SO2Ph
a
c
b
S
HO
HO
N
N
O
O
10
9
8
O
O
HO
O
O
f
e
d
Br
NO2
NO2
NO2
NO2
F
F
O
F
F
12
13
11
14
O
n
O
O
O
n
O
O
HO
HO
g
h
NO2
NH2
F
F
16a n=1
16b n=2
16c n=3
15a n=1
15b n=2
15c n=3
O
N
N
O
n
O
O
O
n
O
O
HO
O
i
j
O
10
19
R
R
PhO2S
F
F
18a: R=NHSO2NHCH3
18b: R=NHSO2NHCH2CF3
n=1
n=1
n=1
n=1
n=1
n=1
n=1
n=2
n=3
17a: R=NHSO2NHCH3
n=1
n=1
n=1
n=1
n=1
n=1
n=1
n=2
n=3
17b: R=NHSO2NHCH2CF3
17c: R=NHSO2NH(CH2)2CN
17d: R=NHSO2NHCH(CH3)2
17e: R=NHSO2NHCH2CH3
17f: R=NHSO2NHOCH3
17g: R=NHSO2NHCH2OCH3
17h: R=NHSO2NHCH3
18c: R=NHSO2NH(CH2)2CN
18d: R=NHSO2NHCH(CH3)2
18e: R=NHSO2NHCH2CH3
18f: R=NHSO2NHOCH3
18g: R=NHSO2NHCH2OCH3
18h: R=NHSO2NHCH3
18i: R=NHSO2NHCH3
17i: R=NHSO2NHCH3
O
19a: R'=SO2NHCH3
19e: R'=SO2NHCH2CH3
19f: R'=SO2NHOCH3
19g: R'=SO2NH(CH2)2OCH3
Cl
O
O
k
R'
19b: R'=SO2NHCH2CF3
19c: R'=SO2NH(CH2)2CN
19d: R'=SO2NHCH(CH3)2
C
S
O
N
N
O
Reagents and conditions: (a) (i) NaOH (aq.), ClCH2COOH, rt, 3 h; (ii) 6 mol/L HCl; (b) 30% H2O2, AcOH,
rt, 3 h; (c) fuming HNO3,100 oC, 4 h. (d) NBS, benzoyl peroxide, CCl4, reflux, 13 h; (e) NaH (1.0 equiv),
ethyl acetoacetate (1.0 equiv), THF, 0 oC, 12 h (2 steps); (f) resorcinol (1.0 equiv), conc. H2SO4, 0 oC, 12h;
(g) 2-chloroethanol or 3-chloro-1-propanol or 4-chloro-1-butanol, K2CO3, acetone or DMF, reflux; (h) 10%
Pd/C, H2, MeOH, rt, 12 h. (i) pyridine or Et3N, 12 h; (j) CH2Cl2, 1,8-Diazabicyclo[5.4.0]undec-7-ene
(DBU), -15 oC, 3 h; (k) 2-bromoethanol, amine, Et3N, CH2Cl2.
2.3 Inhibition effects on cell proliferation.
We tested inhibition effects of compounds 17a-17g on the cell growth in HCT116, A549, MDA-
MB-231, A375 and HL60 cell lines (Table 1). The mutant KRAS was found in HCT116 and A549
cells, mutant KRAS and BRAF in MDA-MB-231 cells, mutant BRAF in A375 cells and NRAS

variants in HL60 cells 17-18. In three cell lines, HCT116, A549 and MDA-MB-231, we found that
the IC50 values of compounds 18a-18g are better than the intermediates (17a-17g). In the A375 and
HL60 cell lines the final products with a NO donor showed similar or reduced potency than the
intermediates, suggesting that these NO-coupled MEK inhibitors have better effects in several but
not all the cell lines. The HCT116, A549 and MDA-MB-231 cell lines are more sensitive than the
A375 and HL60 cell lines to the compounds tested.
Table 1 Inhibition of cell viability by compounds in different cell lines (μM).a
R
MDA-MB-231
HCT116
A549
A375
HL60
(KRASG13D /BRAFG464V
)
(KRASG13D
)
(KRASG12S
)
（BRAFV600E
）
（NRASK61L
）
18a
18b
NHSO2NHCH3
NHSO2NHCH2CF3
NHSO2NH(CH2)2CN
NHSO2NHCH(CH3)2
NHSO2CH2CH3
0.093±0.05
0.13±0.09
0.27±0.16
0.86±0.59
0.18±0.11
0.31±0.23
0.38±0.24
0.018±0.011
0.082±0.06
2.90±1.65
0.17±0.17
2.19±1.33
7.72±3.90
5.49±2.55
2.38±1.45
2.48±1.33
ND
0.67±0.31
0.61±0.16
0.76±0.28
1.23±0.82
0.72±0.18
1.01±0.13
0.71±0.23
1.10±0.57
2.51±1.31
2.61±1.31
2.99±0.70
2.57±1.24
3.75±1.89
2.27±0.97
0.46±0.27
0.06±0.05
0.69±0.08
1.24±0.06
>5
0.33±0.05
0.5±0.09
0.53±0.27
0.27±0.11
0.64±0.19
0.55±0.53
0.29±0.10
5.64±1.13
0.31±0.25
18c
0.66±0.21
0.97±0.22
0.54±0.18
0.87±0.31
0.54±0.15
18d
18e
18f
NHSO2NHOCH3
18g
NHSO2NHCH2OCH3
Trametinib
PD0325901
JS-K
RO5126766
17a
0.019±0.012
0.09±0.053
0.50±0.18
0.053±0.017
3.47±2
0.0024±0.00076
0.003±0.0002
0.00077±0.00055 0.0013±0.0003
1.99±0.37
0.017±0.009
0.15±0.03
0.93±0.14
0.65±0.17
0.15±0.08
0.2±0.06
ND
0.53±0.02
0.006±0.001
0.10±0.05
0.78±0.23
0.39±0.04
0.14±0.06
0.15±0.03
ND
NHSO2NHCH3
NHSO2NHCH2CF3
NHSO2NH(CH2)2CN
NHSO2NHCH(CH3)2
NHSO2CH2CH3
17b
10.33±5.26
15.40±3.79
5.09±2.61
5.79±2.08
ND
>5
17c
>5
17d
>5
17e
>5
17f
NHSO2NHOCH3
ND
17g
NHSO2NHCH2OCH3
2.99±1.45
6.50±1.85
>5
0.36±0.05
0.25±0
a Data are representative of 2-3 independent experiments. ND: not determined. MDA-MB-231: human breast cancer cell line.
HCT116: human colorectal carcinoma cell line. A549: human non-small cell lung cancer cell line. A375: human
malignant melanoma cell line. HL60: human promyelocytic leukemia cell line.
Among these 7 substituents at the meta-position of the 3-benzyl group, 18a, which has the smallest
R substituent, is the most potent and is as potent as RO5126766 in A549 cells (1.1 μM vs. 1.24
μM) and more potent (0.093 μM) than the NO donor prodrug JS-K (6) (2.9 μM) and MEK
inhibitor RO5126766 (0.17 μM) in MDA-MB-231 cells. Compared to 18b-18g with larger
substituents, the potency of 18a indicates that the protein pocket in R substituent area is not very
big and small substituents are more favoured. Its intermediate (17a) is also more potent than the
other MEK inhibitor intermediates 17b-17g, suggesting that better inhibition effect of the MEK
inhibitor motif is associated with better cell proliferation inhibition effect of the hybrid.
2.4 Compound 18a reduces pMEK and pERK protein levels

We selected the most potent compound (18a) for western blot assays in the A549 cell line at the 2
h time point and compared it with the NO donor JS-K and the known MEK inhibitor RO5126766
.
The results showed that 18a impacts the MAPK pathway immediately. At the 2 h time point, 18a
at concentrations of 10 μM and 1 μM, efficiently reduced the levels of pMEK and pERK proteins
respectively, although somewhat less efficiently than RO5126766 (Fig.4).
Fig. 4 Western blot results of 18a in A549 cells at the 2 h time point with glyceraldehyde 3-phosphate
dehydrogenase (GAPDH) used as the loading control.
The effects of the best compound 18a were further examined in MDA-MB-231 cells through the
dose-dependent and the time-dependent assays. Fig. 5a shows that 18a can efficiently reduce pMEK
and pERK levels at 1, 3 and 10 μM after treatment for 24 h. Fig. 5b shows that the level of pERK and
pMEK is obviously reduced by 18a at a concentration of 10 μM at 2 h post-treatment. Almost
complete depletion of these proteins was observed within 4 h.
Fig. 5 (a)24 h dose-dependent western blot results of 18a with 1 μM JS-K and RO5126766 as negative
and positive control, respectively. (b) time-dependent western blot results of 18a with 10 μM JS-K and
RO5126766 in MDA-MB-231 cells.
2.5 Optimization of the linker
Focusing on the substituent group of 18a we further optimized the linker connecting the NO donor
moiety and the MEK inhibitor. Two compounds, 18h and 18i, with a longer linker (n=2-3) were
made and compared with 18a (n=1). The results in Table 2 show that 18h with three carbons in the

linker has similar or better activity than 18a, with IC50 values of 0.64 vs. 0.67 μM in HCT116, 1.35
vs. 1.10 μM in A549, and 0.034 vs. 0.093 μM in MDA-MB-231 cells. Compound 18i, with the
longest linker, displayed lower potency suggesting that longer linkers are not beneficial. It was
concluded that a linker with 2-3 carbons is optimal for the activity.
Table 2 IC50 (μM) of compounds on cell viability against different cell lines.a
n
MDA-MB-231
0.17 ± 0.17
0.093 ± 0.05
0.034 ± 0.007
0.20 ± 0.068
2.19 ± 1.33
1.90 ± 1.00
3.20 ± 1.54
2.90±1.65
HCT116
0.053 ± 0.017
0.67 ± 0.31
0.64 ± 0.30
2.78 ± 1.08
3.47 ± 2
A549
1.24 ± 0.06
1.10 ± 0.57
1.35 ± 0.94
3.49 ± 3.08
>5
Vero
>50
HL7702
17.27 ± 1.62
2.78 ± 0.49
5.62 ± 0.82
1.93 ± 0.12
ND
RO5126766
18a
1
2
3
1
2
3
4.34 ± 0.32
21.07 ± 1.32
2.98 ± 0.14
ND
18h
18i
17a
17h
5.34 ± 3.10
7.35 ± 2.15
0.50±0.18
>5
ND
ND
17i
>5
ND
ND
JS-K
0.69±0.08
1.16 ± 1.62
1.99 ± 0.22
determined.
aData
normal African green monkey kidney cell line. HL7702: human normal liver cell line.
are
representative
of
2-3
independent
experiments.
ND:
not
Vero:
We next determined the activity of 18a, 18h-i on normal cell lines including Vero cell line and
HL7702 cell line. All the three compounds showed their activities at micromolar level. 18h showed
the lowest toxicity with IC50 values of 21.07 and 5.62 μM in these two cell lines, which possess
lower toxicity than NO donor JS-K (1.99 μM). Therefore, 18h may have a good therapeutic
window in MDA-MB-231 breast cancer therapy in the future.
2.6 The reduction effect of 18h on the levels of pERK and pMEK
We performed western blot assays in a dose-dependent and time-dependent manner of compound
18h, the compound with the best cell growth inhibitory effect in MDA-MB-231 cells. Figure 6a
shows that at the 2 h time point, 18h at 1 μM and 10 μM concentrations can reduce the pMEK and
pERK levels as efficiently as RO5126766. Figure 6b shows that a majority of pERK or pMEK is
reduced by 18h at 1 μM in 1 h or less.

Fig. 6 (a)The effects of RO5126766, JS-K or 18h on the levels of pERK and pMEK in MDA-MB-231
cells for 2 hours at different concentration and (b) at different time points.
2.7 Compound 18a and 18h induces apoptosis in MDA-MB-231 cells
Apoptosis assays in MDA-MB-231 cells showed that 18a and 18h, each at 10 μM and 1 μM can
obviously induce cell apoptosis after treatment for 24 h. The NO donor JS-K at 10 μM can have
similiar effects on cell apoptosis compared to 18a and 18h (Figure 7). In comparison, RO5126766
has no obvious apoptotic effect on the cells, suggesting that hybrid and the MEK inhibitor may
have different mechanisms of action.
Fig. 7 18a and 18h can induce cell apoptosis in MDA-MB-231 cells after 24 h treatment. MDA-MB-231
cells were treated with DMSO, RO5126766, JS-K, 18a or 18h at the concentrations indicated for 24 h for
flow cytometry analysis. Data are representative of 2 independent experiments. PI: propidium iodide.
2.8 NO release evaluation
NO release was evaluated in HCT116 cells with RO5126766 and JS-K as negative and positive
controls, respectively. The final compounds (18a-i) containing a NO donor can induce release of
NO after 2 h treatment compared to the intermediates (17a-i). Compound 18h can induce release of
the highest amount of NO, even higher than JS-K (Fig.8a). Fig.8b shows the intracellular NO
levels after 2 h treatment with 18a or 18h or the positive NO donor JS-K in HCT116 cells using
the fluorescent indicator 4-Amino-5-methylamino-2', 7'-difluorofluorescein diacetate (DAF-FM-
DA). These data suggest that our synthetic compounds (18a-i) can significantly induce NO release
in cells.

Fig. 8 Evaluation of NO release. (a) The NO-releasing effect in HCT116 cells after 2 h treatment with the
compounds at 100 μM. The statistical significance was determined using 1way ANOVA. P value style
GP:0.1234 (ns). 0.0332 (*). 0.0021 (**). 0.0002 (***). <0.0001 (****). (b) Intracellular NO levels in
HCT116 cells changed 2 h treatment with the compounds as detected using the commercial fluorescent
indicator DAF-FM DA. Experiments were repeated twice.
3. Conclusion
We have designed and synthesized a series of hybrids (18a-i) containing an MEK inhibitor motif
and a NO donor motif. The structure-activity relationship indicated that small substituents at R
position of the protein are more favoured. The linker connecting the NO donor moiety and the
MEK inhibitor with 2-3 carbons is optimal for the activity. Better inhibition effect of the MEK
inhibitor motif is associated with better cell proliferation inhibition effect of the hybrid. The release
of NO can increase the anti-proliferation effect of MEK inhibitor.
Compound 18h with 3 carbon linker induced five-fold more potent inhibition of cell proliferation
than the MEK inhibitor RO5126766 in MDA-MB-231 cells and showed low toxicity to normal
cell lines tested, suggesting a broad therapeutic window. MDA-MB-231 cell line has been proved
to be more sensitive to compound 18h than other cell lines tested in this paper. Further research
will be done to determine whether the compound is only specific to MDA-MB-231 cell line or also
effective in other breast cancer cell lines.
Western blot analysis results in MDA-MB-231 and A549 cells demonstrated that compound 18h
efficiently reduces the levels of pMEK and pERK in a dose-dependent and time-dependent
manner. Most of pERK and pMEK can be reduced by 18h at 1 μM in 1 h, and 18h is as efficient as
the clinical candidate RO5126766 in MDA-MB-231 cells while the NO donors do not affect this
pathway. In contrast to RO5126766, 18a and 18h can also induce cell apoptosis in MDA-MB-231
cells. Qualitative and quantitative analysis of NO levels show that 18a and 18h can efficiently
induce NO release in cells, suggesting that these hybrids have mechanism of action different from
that in MEK inhibitors and NO donors since they produce the effects of both MEK inhibitors and
NO donors. Thus, we are reporting a series of multitarget antitumor hybrids that have the

synergistic effects of both MEK inhibitors and NO donors. Further optimization of 18h to improve
its potency and PK/PD properties for in vivo studies is in progress.

4. Experimental section
4.1 Chemistry
4.1.1 General conditions
All reactions were carried out in oven-dried glassware, and monitored by thin layer chromatography (TLC).
All reagents were reagent grade and purchased from commercial sources unless otherwise indicated. NMR
spectra were recorded with a 400 MHz spectrometer for 1H NMR, 100 MHz for 13C NMR. Chemical shifts
δ are given in ppm relative to the residual proton signals of the deuterated solvent DMSO in 1H and 13C
NMR spectra. Multiplicities are reported as singlet (s), doublet (d), doublet of doublets (dd), triplet (t),
quartet (q) or multiplet (m). High resolution mass spectra were recorded on an AB SCIEX FTMS ESI
spectrometer. For column chromatography, silica gel (200-300 mesh) was used as the stationary phase.
4.1.2 Synthetic procedures
3-(2-Fluoro-3-nitrobenzyl)-7-hydroxy-4-methyl-2H-chromen-2-one (14).
The synthesis of the intermediate 14 from 2-fluoro-1-methyl-3-nitro-benzene has been reported
previously15.
General procedure for compounds 15a-15c.
The corresponding halo alcohol (2-chloroethanol, 3-chloro-1-propanol, 4-chloro-1-butanol) (2 mmol) and
K2CO3 (3 mmol) were added to a stirred solution of 14 (1 mmol) in DMF (5 mL) at 70 °C . The mixture
was refluxed for about 10 h and then poured into H2O (50 mL). After filtration, the residue was washed
with H2O (3 × 10 mL) and dried with an infrared lamp, yielding 15a-15c as a white solid.
3-(2-Fluoro-3-nitrobenzyl)-7-(2-hydroxyethoxy)-4-methyl-2H-chromen-2-one (15a)
The title compound was obtained from 14 and 2-chloro-1-ethanol as a white solid (60% yield, mp 163-
1
165 °C). H NMR (400 MHz, [D6]DMSO): δ 2.49 (s, 3H, CH3), 3.78 (dd, J = 9.9, 5.2 Hz, 2H, CH2OH),
4.07 (s, 2H, CH2), 4.14 (t, J = 4.8 Hz, 2H, OCH2), 4.96 (t, J = 5.5 Hz, 1H, OH), 7.03 (dd, J = 4.6, 2.3 Hz,
2H, ArH), 7.35 (t, J = 8.0 Hz, 1H, ArH), 7.61 (t, J = 6.6 Hz, 1H, ArH), 7.80 (d, J = 9.6 Hz, 1H, ArH), 8.02
(t, J = 7.0 Hz, 1H, ArH); 13C NMR (100 MHz, [D6]DMSO): δ 15.62, 26.26, 59.83, 70.79, 101.47, 113.09,
113.83, 118.85, 124.55, 125.09 (d, JC-F = 4.6 Hz, 1C), 127.31, 129.65 (d, JC-F = 14.7 Hz, 1C), 136.31 (d, JC-
F = 5.3 Hz, 1C), 137.76 (d, JC-F = 8.1 Hz, 1C), 150.25, 153.19 (d, JC-F = 259.4 Hz, 1C), 154.00, 161.28,
161.90; HRMS: exact mass calcd for C19H16FNO6 [M+H]+ 374.1040; found 374.1042.
3-(2-Fluoro-3-nitrobenzyl)-7-(3-hydroxypropoxy)-4-methyl-2H-chromen-2-one (15b)
The title compound was obtained from 14 and 3-chloro-1-propanol as a white solid (50% yield, mp 117-
1
118 °C). H NMR (400 MHz, [D6]DMSO): δ 1.92 (t, J = 6.3 Hz, 2H, CH2 ), 2.48 (s, 3H, CH3), 3.59 (q, J =
6.0 Hz, 2H, CH2OH), 4.07 (s, 2H, CH2), 4.18 (t, J = 6.3 Hz, 2H, OCH2), 4.64 (t, J = 5.1 Hz, 1H, OH), 7.01
(d, J = 3.6 Hz, 2H, ArH), 7.35 (t, J = 8.0 Hz, 1H, ArH), 7.61 (t, J = 6.9 Hz, 1H, ArH), 7.84 – 7.74 (m, 1H,
ArH), 8.01 (t, J = 7.6 Hz, 1H, ArH); 13C NMR (100 MHz, [D6]DMSO): δ 15.60, 26.22 (d, JC-F = 3.6 Hz,
1C), 32.31, 57.57, 65.87, 101.40, 113.01, 113.78, 118.82, 124.53, 125.08 (d, JC-F = 4.6 Hz, 1C), 127.29,
129.64 (d, JC-F = 14.6 Hz, 1C), 136.30 (d, JC-F = 5.2 Hz, 1C), 137.75 (d, JC-F = 8.3 Hz, 1C), 150.22, 153.18
(d, JC-F = 259.5 Hz, 1C), 154.00, 161.27, 161.85; HRMS: exact mass calcd for C20H18FNO6 [M+H]+
388.1196; found 388.1187.

3-(2-Fluoro-3-nitrobenzyl)-7-(4-hydroxybutoxy)-4-methyl-2H-chromen-2-one (15c)
The title compound was obtained from 8 and 4-chloro-1-butanol as a white solid (20% yield, mp 93-94 °C).
1H NMR (400 MHz, [D6]DMSO): δ 1.66 – 1.50 (m, 2H, CH2), 1.88 – 1.73 (m, 2H, CH2), 2.49 (s, 3H, CH3),
3.49 (dd, J = 11.7, 6.3 Hz, 2H, CH2O), 4.07 (s, 2H, CH2), 4.12 (t, J = 6.5 Hz, 2H, OCH2), 4.52 (t, J = 5.1
Hz, 1H, OH), 7.01 (d, J = 7.5 Hz, 2H, ArH), 7.35 (t, J = 8.0 Hz, 1H, ArH), 7.61 (t, J = 6.6 Hz, 1H, ArH),
7.80 (d, J = 9.5 Hz, 1H, ArH), 8.02 (t, J = 7.0 Hz, 1H, ArH); 13C NMR (100 MHz, CDCl3): δ 15.40, 25.52,
26.41, 29.19, 62.45, 68.32, 101.09, 112.93, 113.72, 119.14, 124.01 (d, J = 4.8 Hz, 1C), 124.27, 125.96,
129.23 (d, J = 14.8 Hz, 1C), 136.57 (d, J = 5.4 Hz,1C), 149.10, 153.75 (t, J = 131.4 Hz, 1C), 154.02,
161.70, 161.97,; HRMS: exact mass calcd for C21H20FNO6 [M+H]+ 402.1353; found 402.1355.
3-(3-Amino-2-fluorobenzyl)-7-(2-hydroxyethoxy)-4-methyl-2H-chromen-2-one (16a-16c)
A catalytic amount of Pd/C (0.12 mmol) was added to a stirred solution of 15a-15c (1.2 mmol) in MeOH
(20 mL). The mixture was stirred under a hydrogen atmosphere (0.4 MPa) for 12 h at room temperature
then the catalyst was removed by filtration, and the filtrate was concentrated under reduced pressure. The
fluoroaniline intermediates (16a-16c) were obtained without further purification.
General procedure for compounds 17a-17i.
Compound 19a-19g (2.66 mmol) and Et3N (3.99 mmol) were added to a solution of 16a-16c (1.33 mmol)
in anhydrous MeCN (15 mL) at room temperature. The reaction mixture was heated to 75 oC for 5 -12 h
and then the mixture was cooled to room temperature and diluted with EtOAc. The mixture was washed
with 1 mol/L aqueous HCl, saturated aqueous NaHCO3 and brine. The organic layer was dried over
Na2SO4, filtered and evaporated. The residue was purified by silica gel chromatography (CH2Cl2/CH3OH)
to give compounds 17a-17i.
3-(2-Fluoro-3-(N-methylsulfamoylamino)benzyl)-4-methyl-7-(2-hydroxyethoxy)-2H-chromen-2-one
(17a)
1
The title compound was obtained from 16 reacting with 19a as a white solid (65% yield, mp 85-86 °C). H
NMR (400 MHz, [D6]DMSO): δ 2.40 (s, 3H, CH3), 2.53 (d, J = 5.0 Hz, 3H, NHCH3), 3.75 (dd, J = 9.8, 5.1
Hz, 2H, CH2OH), 3.94 (s, 2H, CH2), 4.10 (t, J = 4.8 Hz, 2H, OCH2), 4.94 (t, J = 5.5 Hz, 1H, OH), 6.85 (t, J
= 6.8 Hz, 1H, ArH), 7.05 – 6.93 (m, 3H, ArH), 7.34 – 7.15 (m, 2H, ArH), 7.74 (d, J = 9.4 Hz, 1H, NH),
9.37 (s, 1H, NH); 13C NMR (100 MHz, [D6]DMSO): δ 15.52, 25.99, 28.78, 59.83, 70.75, 101.44, 113.01,
113.85, 119.65, 122.69, 124.35 (d, JC-F = 4.1 Hz, 1C), 125.23, 126.30 (d, JC-F = 13.4 Hz, 1C), 126.70 (d, JC-
F = 14.4 Hz, 1C), 127.12, 149.65, 153.08 (d, JC-F = 240.0 Hz, 1C), 153.90, 161.42, 161.77; HRMS: exact
mass calcd for C20H21FN2O6S [M+H]+ 437.1183 ; found 437.1173.
3-(2-Fluoro-3-(N-(2,2,2-trifluoroethyl)sulfamoyl-amino)benzy)-l-4-methyl-7-(2-hydroxyethoxy)-2H-
chromen-2-one (17b)
The title compound was obtained from 16 reacting with 19b as a white solid (52% yield, mp 101-102 °C).
1H NMR (400 MHz, [D6]DMSO): δ 2.40 (s, 3H, CH3), 3.73 – 3.64 (m, 2H, CH2CF3), 3.75 (t, J = 4.8 Hz,
2H, CH2OH), 3.94 (s, 2H, CH2), 4.10 (t, J = 4.8 Hz, 2H, OCH2), 6.91 (t, J = 6.7 Hz, 1H, ArH), 7.06 – 6.94
(m, 3H, ArH), 7.29 (dd, J = 7.7, 6.7 Hz, 1H, ArH), 7.81 – 7.68 (m, 1H, ArH), 8.35 (t, J = 6.8 Hz, 1H, NH),
9.60 (s, 1H, NH); 13C NMR (100 MHz, [D6]DMSO): δ 15.52, 26.03, 43.78 (d, JC-F = 34.3 Hz, 1C), 59.84

(2C), 70.77, 101.45, 113.03, 113.87, 119.63, 123.37 (d, JC-F = 8.0 Hz, 1C), 124.44 (d, JC-F = 4.2 Hz, 1C),
125.60 (d, JC-F = 13.4 Hz, 1C), 126.11 (d, JC-F = 15.4 Hz, 1C), 126.85 (d, JC-F = 14.4 Hz, 1C), 127.14,
149.67, 153.54 (d, JC-F = 244.78 Hz, 1C), 153.92, 161.43, 161.79; HRMS: exact mass calcd for
C21H18FNO6 [M+H]+ 505.1056; found 505.1055.
3-(2-Fluoro-3-(N-(2-cyanoethyl)sulfamoylamino)benzyl)-4-methyl-7-(2-hydroxyethoxy)-2H-
chromen-2-one (17c)
1
The title compound was obtained from 16 and 19c as a white solid (49% yield, mp 65-66 °C). H NMR
(400 MHz, [D6]DMSO): δ 2.44 (s, 3H, CH3), 2.70 (dd, J = 19.5, 12.9 Hz, 2H, CH2CN), 3.28 – 3.13 (m, 2H,
NHCH2), 3.78 (d, J = 4.6 Hz, 1H), 3.97 (s, 2H, CH2), 4.13 (t, J = 4.8 Hz, 1H), 4.42 (s, 2H, OCH2), 5.78 (s,
1H, OH), 6.93 (d, J = 7.0 Hz, 1H, ArH), 7.09 – 6.97 (m, 3H, ArH), 7.31 (d, J = 7.6 Hz, 1H, ArH), 7.87 –
7.67 (m, 2H, ArH, NH), 9.53 (s, 1H, NH); 13C NMR (100 MHz, [D6]DMSO): δ 15.55, 18.24, 55.37, 59.93,
66.46, 68.22, 70.83, 101.36, 112.98 (d, JC-F = 9.5 Hz, 1C), 114.09 (d, JC-F = 44.6 Hz, 1C) , 119.80 (d, JC-F
33.9 Hz, 1C), 123.26, 124.43, 125.77 (d, JC-F = 8.5 Hz, 1C), 125.94, 126.86 (d, JC-F = 5.6 Hz, 1C), 127.22
=
(d, JC-F = 16.3 Hz, 1C), 149.64 (d, JC-F = 6.1 Hz, 1C), 153.41 (d, JC-F = 244.09 Hz, 1C), 160.89, 161.39 (d,
C-F = 8.0 Hz, 1C), 161.78; HRMS: exact mass calcd for C22H22FN3O6S [M+H]+ 476.1292; found 476.1286
J
.
3-(2-Fluoro-3-(N-isopropylsulfamoylamino)benzyl)-4-methyl-7-(2-hydroxyethoxy)-2H-chromen-2-
one (17d)
1
The title compound was obtained from 16 and 19d as a white solid (48% yield, mp 178-179 °C). H NMR
(400 MHz, [D6]DMSO): δ 0.99 (d, J = 6.3 Hz, 6H, CH3CH3), 2.39 (s, 3H, CH3), 3.39 (dd, J = 12.9, 6.1 Hz,
1H, CH), 3.75 (t, J = 3.5 Hz, 2H, CH2OH), 3.94 (s, 2H, CH2), 4.10 (s, 2H, OCH2), 4.93 (s, 1H, OH), 6.85
(d, J = 6.6 Hz, 1H, ArH), 7.00 (q, J = 7.6 Hz, 3H, ArH), 7.30 (d, J = 7.7 Hz, 2H, ArH), 7.74 (d, J = 8.9 Hz,
1H, NH), 9.33 (s, 1H, NH); 13C NMR (100 MHz, [D6]DMSO): δ 15.53, 23.53, 25.96, 45.38, 59.84, 70.76,
101.43, 113.01, 113.87, 119.78, 121.63, 124.26, 124.76, 126.52, 126.70 (d, JC-F = 7.1 Hz, 1C), 126.86,
127.10, 149.51, 152.44 (d, JC-F = 243.3 Hz, 1C), 153.90, 161.40, 161.76; HRMS: exact mass calcd for
C22H25FN2O6S [M+H]+ 465.1496 ; found 465.1485.
3-(2-Fluoro-3-(N-ethylsulfamoylamino)benzyl)4-methyl-7-(2-hydroxyethoxy)-2H-chromen-2-one
(17e)
1
The title compound was obtained from 16 and 19e as a white solid (58% yield, mp 87-88 °C). H NMR
(400 MHz, [D6]DMSO): δ 1.02 (t, J = 7.2 Hz, 3H, CH2CH3), 2.43 (s, 3H, CH3), 3.05 – 2.84 (m, 2H,
CH2CH3), 3.78 (dd, J = 9.9, 5.2 Hz, 2H, CH2OH), 3.97 (s, 2H, CH2), 4.13 (t, J = 4.8 Hz, 2H, OCH2), 4.96
(t, J = 5.5 Hz, 1H, OH), 6.88 (t, J = 6.8 Hz, 1H, ArH), 7.11 – 6.94 (m, 3H, ArH), 7.33 (dd, J = 11.2, 5.8 Hz,
2H, ArH), 7.77 (d, J = 9.5 Hz, 1H, NH), 9.34 (s, 1H, NH); 13C NMR (100 MHz, [D6]DMSO): δ 15.04,
15.53, 37.58, 59.84, 70.76, 101.44, 113.44 (d, JC-F = 85.3 Hz, 1C), 119.73, 122.47, 124.31 (d, JC-F = 4.1 Hz,
1C), 125.14, 126.41, 126.57 (d, JC-F = 5.3 Hz, 1C), 126.74, 127.12, 149.59, 151.73, 153.90, 154.17, 161.42,
161.77; HRMS: exact mass calcd for C21H23FN2O6S [M+H]+ 451.1339; found 451.1332.
3-(2-Fluoro-3-(N-methoxylsulfamoylamino)benzyl)-4-methyl-7-(2-hydroxyethoxy)-2H-chromen-2-
one (17f)

1
The title compound was obtained from 16 and 19f as a white solid (45% yield, mp > 300 °C). H NMR
(400 MHz, [D6]DMSO): δ 2.09 (s, 1H, NHO), 2.40 (s, 3H, CH3), 3.62 (s, 3H, OCH3), 3.74 (d, J = 4.6 Hz,
2H, CH2OH), 3.93 (s, 2H, CH2), 4.10 (t, J = 4.7 Hz, 2H, OCH2), 4.94 (s, 1H, OH), 6.89 (s, 1H, ArH), 7.10
– 6.93 (m, 3H, ArH), 7.27 (t, J = 7.3 Hz, 1H, ArH), 7.76 (d, J = 8.8 Hz, 1H, ArH), 9.95 (s, 1H, NH);
HRMS: exact mass calcd for C20H18FNO6 [M+H]+ 453.1132; found 453.1138.
3-(2-Fluoro-3-(N-methoxyethylsulfamoylamino)benzyl)-4-methyl-7-(2-hydroxyethoxy)-2H-chromen-
2-one (17g)
1
The title compound was obtained from 16 and 19g as a white solid (56% yield, mp 56-57 °C). H NMR
(400 MHz, [D6]DMSO): δ 2.43 (s, 3H, CH3), 3.08 (d, J = 5.9 Hz, 2H, CH2O), 3.19 (s, 3H, OCH3), 3.35 (dd,
J = 8.1, 3.9 Hz, 2H, NHCH2), 3.78 (dd, J = 9.9, 5.1 Hz, 2H, OCH2), 3.96 (s, 2H, CH2), 4.13 (t, J = 4.8 Hz,
2H, OCH2), 4.95 (t, J = 5.5 Hz, 1H, OH), 6.88 (t, J = 6.9 Hz, 1H, ArH), 7.08 – 6.96 (m, 3H, ArH), 7.34 (t,
J = 7.4 Hz, 1H, ArH), 7.50 (t, J = 5.8 Hz, 1H, ArH), 7.77 (d, J = 9.6 Hz, 1H, NH), 9.38 (s, 1H, NH); 13C
NMR (100 MHz, [D6]DMSO): δ 15.51, 26.02, 26.79, 42.19, 58.30, 59.83, 70.80, 101.42, 113.00, 113.85,
119.69, 122.35, 124.32 (d, JC-F = 4.0 Hz, 1C), 125.15, 126.39 (d, JC-F = 13.3 Hz, 1C), 126.64 (d, JC-F = 14.2
Hz, 1C), 127.11, 149.58, 152.89 (d, JC-F = 243.7 Hz, 1C), 153.89, 161.40, 161.76; HRMS: exact mass
calcd for C22H25FN2O7S [M+H]+ 481.1445; found 481.1439.
3-(2-Fluoro-3-(N-methylsulfamoylamino)benzyl)-4-methyl-7-(3-hydroxyethoxy)-2H-chromen-2-one
(17h)
1
The title compound was obtained from 16b and 19a as a white solid (60% yield, mp 59-60 °C). H NMR
(400 MHz, [D6]DMSO): δ 1.97 – 1.87 (m, 2H, CH2), 2.43 (s, 3H, CH3), 2.56 (s, 3H, NHCH3), 3.59 (q, J =
5.8 Hz, 2H, CH2O), 3.96 (s, 2H, CH2), 4.18 (t, J = 6.3 Hz, 2H, OCH2), 4.61 (t, J = 4.9 Hz, 1H, OH), 6.88 (t,
J = 7.1 Hz, 1H, ArH), 7.02 (dd, J = 17.1, 7.3 Hz, 3H, ArH), 7.22 (d, J = 5.0 Hz, 1H, ArH), 7.30 (t, J = 8.0
Hz, 1H, ArH), 7.78 (d, J = 9.6 Hz, 1H, NH), 9.36 (s, 1H, NH); 13C NMR (100 MHz, [D6]DMSO): δ 15.53,
26.83, 28.77, 32.32, 57.57, 65.85, 101.40, 112.96, 113.82, 119.63, 122.68, 124.35 (d, JC-F = 4.2 Hz, 1C),
125.21 (d, JC-F = 3.0 Hz, 1C), 126.30 (d, JC-F = 13.1 Hz, 1C), 126.70 (d, JC-F = 14.6 Hz, 1C), 127.15,
149.67, 153.07 (d, JC-F = 245.5 Hz, 1C), 153.93, 161.43, 161.74; HRMS: exact mass calcd for
C21H23FN2O6S [M+H]+ 451.1339; found 451.1336.
3-(2-Fluoro-3-(N-methylsulfamoylamino)benzyl)-4-methyl-7-(4-hydroxyethoxy)-2H-chromen-2-one
(17i)
The title compound was obtained from 16c and 19a as a white solid (40% yield, mp 57-58 °C). 1H NMR
(400 MHz, CDCl3): δ 1.81 – 1.71 (m, 2H, CH2), 1.97 – 1.83 (m, 2H, CH2), 2.40 (s, 3H, CH3), 2.75 (d, J =
5.2 Hz, 3H, NHCH3), 3.64 (t, J = 7.9 Hz, 2H, CH2O), 3.74 (t, J = 6.0 Hz, 2H, CH2), 4.46 (t, J = 7.9 Hz, 2H,
OCH2), 4.71 (d, J = 5.0 Hz, 1H, OH), 6.75 (s, 1H, ArH), 6.81 (s, 1H, ArH), 6.87 (d, J = 8.6 Hz, 1H, ArH),
7.04 – 6.89 (m, 2H, ArH), 7.38 (t, J = 7.5 Hz, 1H, ArH), 7.53 (d, J = 8.9 Hz, 1H, NH); 13C NMR (100
MHz, CDCl3): δ 15.28, 25.54, 29.21, 40.59, 62.43, 64.96, 68.33, 101.14, 112.79, 113.84, 119.57, 119.92,
124.48 (d, JC-F = 4.3 Hz, 1C), 125.44 (d, JC-F = 3.9 Hz, 1C),125.77, 126.40 (d, JC-F = 14.3 Hz, 1C), 148.70,
151.47 (d, JC-F = 242.5 Hz, 1C), 154.01, 160.29, 161.57, 162.07; HRMS: exact mass calcd for
C22H25FN2O6S [M+H]+ 465.1496; found 465.1483.

General Procedure for the preparation of 18a-18i.
Compound 10 (1 mmol) was added to a stirred solution of compound 17a-17i (1.2 mmol) in the presence
of 8-diazabicyclo[5.4.0]undec-7-ene (DBU) (3 mmol) in CH2Cl2. The reaction mixture was stirred at room
temperature for 2-5 h and then washed with H2O (3 × 10 mL). The organic layer was dried with anhydrous
Na2SO4, and the solvent was removed in vacuo. The residue was purified by silica gel chromatography to
give compounds 18a-18i.
4-(2-((3-(2-Fluoro-3-((N-methylsulfamoyl)amino)benzyl)-4-methyl-2-oxo-2H-chromen-7-
yl)oxy)ethoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (18a)
1
The title compound was obtained from 10 and 17a as a white solid (80% yield, mp 167-168 °C). H NMR
(400 MHz, [D6]DMSO): δ 2.43 (s, 3H, CH3), 3.96 (s, 2H, CH2O), 4.51 (s, 2H, CH2), 4.77 (s, 2H, OCH2),
6.86 (d, J = 6.8 Hz, 1H, ArH), 7.14 – 6.96 (m, 3H, ArH), 7.33 – 7.14 (m, 2H, ArH), 7.67 (t, J = 7.7 Hz, 2H,
ArH), 7.83 (dd, J = 14.8, 8.0 Hz, 2H, ArH), 7.98 (d, J = 7.5 Hz, 2H, ArH, NH), 9.38 (s, 1H, NH); 13C NMR
(100 MHz, [D6]DMSO): δ 15.58, 25.99, 28.78, 31.15, 66.57, 70.18, 101.74, 111.00, 113.10, 114.35,
120.03, 122.69, 124.36 (d, JC-F = 4.4 Hz, 1C), 125.22, 126.33 (d, JC-F = 13.5 Hz, 1C), 126.67 (d, JC-F = 14.5
Hz, 1C), 127.32, 128.73, 130.41 (2C), 136.57, 137.63, 149.62, 151.87, 153.89, 154.31, 160.31 (d, JC-F
214.77 Hz, 1C), 161.00; HRMS: exact mass calcd for C28H25FN4O10S2 [M+Na]+ 683.0894; found
683.0874.
=
4-(2-((3-(2-Fluoro-3-((N-(2,2,2-trifluoroethyl)sulfamoyl)amino)benzyl)-4-methyl-2-oxo-2H-chromen-
7-yl)oxy)ethoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (18b)
1
The title compound was obtained from 10 and 17b as a white solid (78% yield, mp 188-190 °C). H NMR
(400 MHz, [D6]DMSO): δ 2.43 (s, 3H, CH3), 3.71 (dd, J = 9.4, 6.9 Hz, 2H, CH2CF3), 3.96 (s, 2H, CH2O),
4.57 – 4.43 (m, 2H, CH2), 4.78 (dd, J = 4.0, 1.6 Hz, 2H, OCH2), 6.91 (t, J = 6.7 Hz, 1H, ArH), 7.12 – 6.96
(m, 3H, ArH), 7.30 (t, J = 7.3 Hz, 1H, ArH), 7.66 (dd, J = 10.8, 5.0 Hz, 2H, ArH), 7.88 – 7.75 (m, 2H,
ArH), 8.04 – 7.93 (m, 2H, ArH), 8.38 (t, J = 6.8 Hz, 1H, NH), 9.63 (s, 1H, NH); 13C NMR (100 MHz,
[D6]DMSO): δ 15.57, 26.02, 43.76 (d, JC-F = 33.6 Hz, 1C), 66.56 (2C), 70.17, 101.72, 111.00, 113.09,
114.34, 119.98, 123.37 (d, JC-F = 9.5 Hz, 1C), 124.45 (d, JC-F = 4.2 Hz, 1C), 125.62 (d, JC-F = 13.3 Hz, 1C),
126.00, 126.81 (d, JC-F = 14.2 Hz, 1C), 127.30, 128.74, 130.40 (2C), 136.56, 137.62, 149.63, 153.53 (d, JC-
F = 244.12 Hz, 1C), 153.90, 159.24 (2C), 161.00, 161.39; HRMS: exact mass calcd for C29H24F4N4O10S2
[M+Na]+ 751.0768; found 751.0742.
4-(2-((3-(3-((N-(2-Cyanoethyl)sulfamoyl)amino)-2-fluorobenzyl)-4-methyl-2-oxo-2H-chromen-7-
yl)oxy)ethoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (18c)
1
The title compound was obtained from 10 and 17c as a white solid (81% yield, mp 161-162 °C). H NMR
(400 MHz, [D6]DMSO): δ 2.43 (s, 3H, CH3), 2.66 (t, J = 6.5 Hz, 2H, CH2CN), 3.16 (q, J = 6.4 Hz, 2H,
NHCH2), 3.96 (s, 2H, CH2O), 4.56 – 4.43 (m, 2H, CH2), 4.82 – 4.70 (m, 2H, OCH2), 6.89 (t, J = 6.7 Hz,
1H, ArH), 7.13 – 6.95 (m, 3H, ArH), 7.29 (t, J = 7.2 Hz, 1H, ArH), 7.67 (dd, J = 8.3, 7.6 Hz, 2H, ArH),
7.88 – 7.73 (m, 2H, ArH), 7.98 (dd, J = 8.5, 1.1 Hz, 2H, ArH, NH), 9.53 (s, 1H, NH); 13C NMR (100 MHz,
[D6]DMSO): δ 15.50, 18.24, 25.89, 38.81, 66.54, 69.97, 101.69, 111.01, 113.10, 114.36, 119.33, 119.99,
123.26, 124.43, 125.72, 125.89 (d, JC-F = 13.5 Hz, 1C), 126.78 (d, JC-F = 14.5 Hz, 1C), 127.32, 128.74 (2C),

130.41 (2C), 136.57, 137.63, 149.63, 153.41 (d, JC-F = 245.4 Hz, 1C), 153.90, 159.24, 161.00, 161.38;
HRMS: exact mass calcd for C30H26FN5O10S2 [M+H]+ 722.1003; found 722.1002.
4-(2-((3-(2-Fluoro-3-((N-isopropylsulfamoyl)amino)benzyl)-4-methyl-2-oxo-2H-chromen-7-
yl)oxy)ethoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (18d)
1
The title compound was obtained from 10 and 17d as a white solid (85% yield, mp 104-105 °C). H NMR
(400 MHz, [D6]DMSO): δ 0.99 (d, J = 6.3 Hz, 6H, CH3CH3), 2.39 (s, 3H, CH3), 2.66 (t, J = 6.5 Hz, 2H,
CH2CN), 3.16 (q, J = 6.4 Hz, 2H, NHCH2), 3.96 (s, 2H, CH2O), 4.56 – 4.43 (m, 2H, CH2), 4.82 – 4.70 (m,
2H, OCH2), 6.89 (t, J = 6.7 Hz, 1H, ArH), 7.13 – 6.95 (m, 3H, ArH), 7.29 (t, J = 7.2 Hz, 1H, ArH), 7.67
(dd, J = 8.3, 7.6 Hz, 2H, ArH), 7.88 – 7.73 (m, 2H, ArH), 7.98 (dd, J = 8.5, 1.1 Hz, 2H, ArH, NH), 9.53 (s,
1H, NH); 13C NMR (100 MHz, [D6]DMSO): δ 15.51, 24.59 (2C), 24.09, 45.27, 66.35, 70.08, 101.71,
110.99, 113.07, 114.35 (2C), 120.15, 121.63, 124.26, 124.75, 126.54 (d, JC-F = 14.5 Hz, 1C), 126.81 (d, JC-
F = 13.0 Hz, 1C), 128.73, 130.40, 136.56 (2C), 137.63 (2C), 149.46, 152.44 (d, JC-F = 244.9 Hz, 1C),
153.66, 159.23, 160.97, 161.34; HRMS: exact mass calcd for C30H29FN4O10S [M+Na]+ 711.1207; found
711.1180.
4-(2-((3-(3-((N-Ethylsulfamoyl)amino)-2-fluorobenzyl)-4-methyl-2-oxo-2H-chromen-7-
yl)oxy)ethoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (18e)
1
The title compound was prepared from 10 and 17e as a white solid (82% yield, mp 95-96 °C). H NMR
(400 MHz, [D6]DMSO): δ 0.99 (t, J = 7.2 Hz, 3H, CH2CH3), 2.42 (s, 3H, CH3), 2.93 (dd, J = 7.2, 5.7 Hz,
2H, CH2CH3), 3.95 (s, 2H, CH2OH), 4.61 – 4.38 (m, 2H, CH2), 4.82 – 4.69 (m, 2H, OCH2), 6.86 (t, J = 6.6
Hz, 1H, ArH), 7.04 (ddd, J = 15.7, 15.2, 5.2 Hz, 3H, ArH), 7.31 (dt, J = 12.2, 6.7 Hz, 2H, ArH), 7.75 –
7.55 (m, 2H, ArH), 7.86 – 7.75 (m, 2H, ArH), 7.98 (dd, J = 8.5, 1.1 Hz, 2H, ArH, NH), 9.35 (s, 1H, NH);
13C NMR (100 MHz, [D6]DMSO): δ 15.58, 26.02, 43.51, 43.83, 66.55, 70.16, 101.69, 112.05 (d, JC-F
=
211.7 Hz, 1C), 114.33, 119.96, 123.35 (d, JC-F = 11.1 Hz, 1C), 124.43, 125.60 (d, JC-F = 13.3 Hz, 1C),
126.06 (d, JC-F = 22.5 Hz, 1C), 126.8 (d, JC-F = 14.4 Hz, 1C), 127.32, 128.74, 130.41 (2C), 136.58, 137.59,
149.66, 152.28, 153.88, 154.73, 159.23, 160.99, 161.39; HRMS: exact mass calcd for
C29H27FN4O10S2[M+K]+ 713.0790; found 713.0405.
4-(2-((3-(2-Fluoro-3-((N-methoxysulfamoyl)amino)benzyl)-4-methyl-2-oxo-2H-chromen-7-
yl)oxy)ethoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (18f)
1
The title compound was obtained from 10 and 17f as a white solid (78% yield, mp 127-128 °C). H NMR
(400 MHz, [D6]DMSO): δ 2.45 (s, 3H, CH3), 3.65 (s, 3H, OCH3), 3.98 (s, 2H, CH2OH), 4.59 – 4.46 (m,
2H, CH2), 4.86 – 4.72 (m, 2H, OCH2), 6.96 (d, J = 6.5 Hz, 1H, ArH), 7.08 (dt, J = 15.6, 5.5 Hz, 3H, ArH),
7 7.30 (t, J = 7.1 Hz, 1H, ArH), 7.69 (t, J = 7.9 Hz, 2H, ArH), 7.90 – 7.74 (m, 2H, ArH), 8.00 (dd, J = 8.4,
1.1 Hz, 2H, ArH), 9.97 (s, 1H, NH), 10.12 (s, 1H, NH); 13C NMR (100 MHz, [D6]DMSO): δ 15.55, 25.86,
64.06, 66.47, 69.96, 101.73, 110.99, 113.10 (2C), 114.34, 119.93, 124.43 (d, JC-F = 9.9 Hz, 1C), 125.11 (d,
JC-F = 13.4 Hz, 1C), 126.49, 126.81 (d, JC-F = 14.5 Hz, 1C), 127.32, 128.73 (2C), 130.40 (2C), 136.56,
137.62, 149.67, 154.05 (d, JC-F = 247.2 Hz, 1C), 153.89, 159.23, 161.00, 161.36; HRMS: exact mass calcd
for C28H25FN4O11S2 [M+Na]+ 699.0843; found 699.0840.

4-(2-((3-(2-Fluoro-3-((N-(2-methoxyethyl)sulfamoyl)amino)benzyl)-4-methyl-2-oxo-2H-chromen-7-
yl)oxy)ethoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (18g)
1
The title compound was obtained from 10 and 17g as a white solid (80% yield, mp 72-73 °C). H NMR
(400 MHz, [D6]DMSO): δ 2.43 (s, 3H, CH3), 3.05 (q, J = 6.0 Hz, 2H, CH2O), 3.16 (s, 3H, OCH3), 3.32 (dd,
J = 10.4, 4.3 Hz, 2H, NHCH2), 3.96 (s, 2H, CH2OH), 4.56 – 4.38 (m, 2H, CH2), 4.86 – 4.70 (m, 2H,
OCH2), 6.86 (t, J = 7.2 Hz, 1H, ArH), 7.14 – 6.94 (m, 3H, ArH), 7.31 (t, J = 7.2 Hz, 1H, ArH), 7.49 (t, J =
5.9 Hz, 1H, ArH), 7.75 – 7.58 (m, 2H, ArH), 7.84 (dt, J = 16.4, 5.0 Hz, 2H, ArH), 8.02 – 7.88 (m, 2H, ArH,
NH), 9.38 (s, 1H, NH); 13C NMR (100 MHz, [D6]DMSO): δ 15.55, 25.74, 42.26, 58.30, 66.35, 70.07,
70.81, 101.73, 111.00, 113.09, 114.36, 120.07, 122.36, 124.35, 125.13, 126.35, 126.50 (d, JC-F = 4.7 Hz,
1C), 126.67, 127.31, 128.73, 130.41, 136.57, 137.63, 149.55, 151.68, 152.90 (d, JC-F = 243.5 Hz, 1C),
153.89, 159.24, 160.99, 161.36; HRMS: exact mass calcd for C30H29FN4O11S2 [M+Na]+ 727.1156; found
727.1144.
4-(3-(3-(2-Fluoro-3-((N-methylsulfamoyl)amino)benzyl)-4-methyl-2-oxo-2H-chromen-7-yl)propoxy)-
3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (18h)
1
The title compound was obtained from 10 and 17h as a white solid (80% yield, mp 81-82 °C). H NMR
(400 MHz, [D6]DMSO): δ 2.33 – 2.18 (m, 2H, CH2), 2.41 (s, 3H, CH3), 3.95 (s, 2H, CH2O), 4.22 (s, 2H,
ArCH2), 4.58 (t, J = 6.0 Hz, 2H, OCH2), 6.86 (d, J = 6.6 Hz, 1H, ArH), 7.08 – 6.94 (m, 3H, ArH), 7.25 (dd,
J = 21.1, 6.4 Hz, 2H, ArH), 7.70 (t, J = 7.9 Hz, 2H, ArH), 7.77 (d, J = 8.8 Hz, 1H, ArH), 7.86 (t, J = 7.5
Hz, 1H, ArH), 8.01 (dd, J = 8.4, 1.1 Hz, 2H, ArH, NH), 9.38 (s, 1H, NH); 13C NMR (100 MHz,
[D6]DMSO): δ 15.57, 25.97, 26.82, 28.16, 28.78, 64.99, 68.70, 101.51, 111.00, 112.94, 114.08, 119.83,
122.67, 124.37 (d, JC-F = 3.6 Hz, 1C), 125.20 (d, JC-F = 3.3 Hz, 1C), 126.33 (d, JC-F = 13.3 Hz, 1C), 126.69
(d, JC-F = 14.4 Hz, 1C), 127.25, 128.81, 130.45 (2C), 136.58, 137.64, 149.67, 151.85, 153.91, 159.31,
160.33 (d, JC-F = 205.7 Hz, 1C), 161.41; HRMS: exact mass calcd for C29H27FN4O10S2 [M+Na]+ 697.1050;
found 697.1068.
4-(4-((3-(2-Fluoro-3-((N-methylsulfamoyl)amino)benzyl)-4-methyl-2-oxo-2H-chromen-7-
yl)oxy)butoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (18i)
1
The title compound was obtained from 10 and 17i as a white solid (85% yield, mp 75-76 °C). H NMR
(400 MHz, [D6]DMSO): δ 2.04 – 1.77 (m, 4H, CH2CH2), 2.43 (s, 3H, CH3), 3.97 (s, 2H, CH2O), 4.21 (t, J
= 6.1 Hz, 2H, ArCH2), 4.51 (t, J = 5.9 Hz, 2H, OCH2), 6.87 (t, J = 6.7 Hz, 1H, ArH), 7.02 (ddd, J = 8.8,
5.8, 2.5 Hz, 3H, ArH), 7.33 – 7.17 (m, 2H, ArH), 7.83 – 7.70 (m, 3H, ArH), 7.91 (t, J = 7.5 Hz, 1H, ArH),
8.03 (dd, J = 8.4, 1.1 Hz, 2H, ArH, NH), 9.38 (s, 1H, NH); 13C NMR (100 MHz, [D6]DMSO): δ 15.54,
25.16, 25.22, 25.89, 28.78, 68.20, 71.65, 101.45, 113.05, 113.91, 119.68, 122.68, 124.36 (d, JC-F = 4.1 Hz,
1C), 125.18 (d, JC-F = 3.1 Hz, 1C), 126.31 (d, JC-F = 13.4 Hz, 1C), 126.70 (d, JC-F = 14.4 Hz, 1C), 127.15,
128.75, 129.61 , 130.47 (2C), 136.59, 137.65, 149.68, 153.08 (d, JC-F = 245.4 Hz, 1C), 153.95, 159.33,
160.42, 161.56; HRMS: exact mass calcd for C30H29FN4O10S2[M+Na]+ 711.1201; found 711.1154.
General Procedure for the Preparation of 19a-19g.
The synthesis of the intermediate 19a-19g was reported previously17
.
4.2 Biology

4.2.1 General
Medium. HCT116 cells were cultured in McCoy’s 5A medium; A549, MDA-MB-231, HL60 and
HL7702 cells were cultured in RPMI-1640 medium. A375 and Vero cells were grown in DMEM
medium. All of the cells were supplemented with 10% fetal bovine serum and cultured at 37 °C in
a humidified 5% CO2 incubator.
Antibodies. Rabbit mAbs for MEK (#9126), pMEK (#9154P), ERK (#9102L), pERK (#9101S)
and GAPDH (#3683S HRP conjugate) were obtained from Cell Signaling Technology. The
secondary antibody conjugated with HRP (A16172) was from Invitrogen.
Reagents. CellTiter Glo assay reagent was purchased from Promega. Annexin V/Dead cell
apoptosis kit (#1825833) was from Invitrogen. RIPA lysis buffer (#89900), protease and
phosphatase inhibitor cocktail (#78442) were obtained from Thermo Scientific.
4.2.2 Anti-proliferation assay
The activity was determined using CellTiter-Glo assay. Cells were plated in 384 well plates,
compounds were added and cells were incubated at 37ꢀ°C for 96 h in a humidified incubator
containing 5% CO2. CellTiter-Glo Reagent was added directly to the cells and OD values were
read in a microplate reader. The IC50 values were calculated using GraphPad Prism.
4.2.3 Western blot assay
Cells were lysed using RIPA lysis buffer supplemented with protease and phosphatase inhibitors
on ice for 20 min, and the supernatant was collected after centrifugation. The protein
concentrations were determined using Bio-Rad protein assay kit. Equal amounts of protein were
loaded into the wells of 4-20% SDS-PAGE gels (Invitrogen) and electrophoresed. Then the
proteins were transferred from the gels to PVDF membranes. After blocking in 5% milk for 1 h at
room temperature, membranes were incubated with indicated primary antibodies at 4 °C overnight,
washed with TBST and incubated with HRP conjugated secondary antibody for 1 h at room
temperature. Membranes were washed three times with TBST. The images were obtained using
Clarity™ Western ECL Substrate from Bio-Rad.
4.2.4 Apoptosis analysis
Apoptosis analysis was performed using an Annexin V/Dead cell apoptosis kit according to the
manufacturer’s instruction.
4.2.5 NO release evaluation
Total NO production was estimated by measurement of the accumulation of nitrite and nitrate
using the Griess reagent in the Total Nitric Oxide Assay Kit (Beyotime, China). Nitrate was
measured after enzymatic conversion to nitrite by nitrate reductase. Briefly, the lysis buffer of
every sample was added in duplicate wells in a 96-well plate at room temperature. The mixture
was incubated with 5 μl of nicotinamide adenine dinucleotide phosphate (NADPH), 10 μl of flavin
adenine dinucleotide (FAD) and 5 μl of nitrate reductase for 30 min at 37 °C. Then, 10 μl of lactate
dehydrogenase (LDH) buffer and 10 μl of LDH were added in the above mixture for another 30
min at 37 °C. Finally, 50 μl of Griess reagent I and 50 μl of Griess reagent II were added into the

wells before incubation for 10 min. Optical density at 540 nm was measured with a FlexStation 3
multi-plate reader. Concentrations were calculated using a standard curve (80, 60, 40
2 μM sodium nitrite).
, 20, 10, 5 and
4.2.6 Intracellular NO measurement
A NO-specific fluorescent probe, DAF-FM DA, purchased from Beyotime Institute of
Biotechnology (China), was diluted to a final concentration of 5 μM with kit diluents. To incubate
the probe and cells sufficiently, HCT116 cells (1 × 107) were pretreated with 5 μM DAF-FM DA at
37 °C for 20 min. The cells were washed 3 times with PBS to remove the remaining DAF-FM DA
that did not enter into the cells. Compounds were incubated at 37 °C for 2 h before NO
determination using a laser confocal scanning microscope (Olympus, Japan) with excitation at 495
nm and emission at 515 nm.
Conflicts of interest
There are no conflicts to declare.
Acknowledgments
This work was supported by the National Natural Science Foundation of China (Grant No.
21807006 and 81872731), Peking University Medicine Seed Fund for Interdisciplinary Research
and the Fundamental Research Funds for the Central Universities. We thank Professor Shaomeng
Wang of University of Michigan for his great support in biology evaluation, Ester Fernandez-Salas
of University of Michigan and Xiaozuo Gao of Royal Melbourne Institute of Technology
University for revising the paper.
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Highlights
A series of hybrids of MEK inhibitor and NO donor were designed and synthesized.
Compound 18h shows better cell proliferation inhibition effects than RO5126766 in MDA-MB-231 cells.
Compound 18h significantly reduces levels of pMEK and pERK in MDA-MB-231 cells.
Compound 18h can induce apoptosis in the MDA-MB-231 cell line.
Compound 18h can release NO in cells.

Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: