Synthesis and biological evaluation of novel 2,3-dihydrochromeno[3,4-d]imidazol-4(1H)-one derivatives as potent anticancer cell proliferation and migration agents

Article abstract of DOI:10.1016/j.ejmech.2016.01.035

In this study, a series of novel molecules containing chromeno [3,4-d] imidazol-4(1H)-one was synthesized and their biological activities were evaluated. Among them, compound 35 showed a dramatic anticancer activity against HCT116 and MCF-7, and the flow cytometry assays demonstrated that it could arrest G0/G1 cell-cycle and induce apoptosis of SW620 cells in a dose-dependent manner. Besides, it also blocked MCF-7 cancer cell migration. Moreover, it inhibited tumor growth in HCT116 subcutaneously implanted xenografted mice. Taken together, compound 35 may be a promising candidate for anti-cancer agent as well as metastatic one.

Full text of DOI:10.1016/j.ejmech.2016.01.035

Accepted Manuscript
Synthesis and biological evaluation of novel 2,3-dihydrochromeno[3,4-
d]imidazol-4(1H)-one derivatives as potent anticancer cell proliferation and migration
agents
Xuan Han, Jiang Luo, Feng Wu, XueYan Hou, Guoyi Yan, Meng Zhou, Mengqi
Zhang, Chunlan Pu, Rui Li
PII:
S0223-5234(16)30035-6
10.1016/j.ejmech.2016.01.035
EJMECH 8325
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 4 June 2015
Revised Date: 18 January 2016
Accepted Date: 19 January 2016
Please cite this article as: X. Han, J. Luo, F. Wu, X. Hou, G. Yan, M. Zhou, M. Zhang, C. Pu, R. Li,
Synthesis and biological evaluation of novel 2,3-dihydrochromeno[3,4-d]imidazol-4(1H)-one derivatives
as potent anticancer cell proliferation and migration agents, European Journal of Medicinal Chemistry
(2016), doi: 10.1016/j.ejmech.2016.01.035.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
1
2
Synthesis and biological evaluation of novel
2,3-dihydrochromeno[3,4-d]imidazol-4(1H)-one derivatives as
potent anticancer cell proliferation and migration agents
3
4
a, 1
a, 1
a
a
a
b
a
a
5
6
Xuan Han , Jiang Luo , Feng Wu , XueYan Hou , Guoyi Yan , Meng Zhou , Mengqi Zhang , Chunlan Pu ,
a,
Rui Li
*
a
7
8
National Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, and
Collaborative Innovation Center for Biotherapy, Chengdu, 610041 Sichuan, P. R. China
b
9
0
1
Engineering Research Center for the Development and Application of Ethnic Medicine and TCM (Ministry of
1
1
Educa-tion), School of Pharmacy, Guiyang Medical University, 9 Beijing Road, Guiyang, Guizhou, 550004, P. R.
China.
1
1
1
1
1
1
1
1
2
2
2
3
4
5
6
7
8
9
0
1
ABSTRACT: In this study, a series of novel molecules containing chromeno [3,4-d]
imidazol-4(1H)-one was synthesized and their biological activities were evaluated. Among them,
compound 35 showed a dramatic anticancer activity against HCT116 and MCF-7, and the flow
cytometry assays demonstrated that it could arrest G0/G1 cell-cycle and induce apoptosis of
SW620 cells in a dose-dependent manner. Besides, it also blocked MCF-7 cancer cell migration.
Moreover, it inhibited tumor growth in HCT116 subcutaneously implanted xenografted mice.
Taken together, compound 35 may be a promising candidate for anti-cancer agent as well as
metastatic one.
2
2
2
3
Keywords: chromeno [3,4-d] imidazol-4(1H)-one; anti-cancer agents, anti-metastasis
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
1. Introduction
Cancer is becoming a leading cause of death in developing and developed countries. An
estimated 14.1 million new cancer cases occurred in 2012 worldwide and Eastern Asia accounted
for 29.4% of it [1]. There were 8.2 million cancer deaths in 2012 all over the world, and cancer
has also been the first reason of death in China under the age of 65 [1, 2].
A pharmacophore hybrid approach for exploration of novel and highly active compounds is an
effective and commonly used direction in modern medicinal chemistry. Hybridization of two
different bioactive molecules with complementary pharmacophoric functions often showed
synergistic effects [3, 4]. For instance, pyrazole [5] and stilbene [6] conjugated with coumarin
derivatives possess good antiproliferative activities. Given the important role of this fusion
strategy, previously, we also synthesized a series of triazol and coumarin fused derivatives which
showed efficient anticancer activities [7].
The coumarin and its derivatives are widely used for antitumor agents [8-11]. The structural
modification and structure-activity relationship analysis of coumarins on anticancer effect have
1
/ 21

ACCEPTED MANUSCRIPT
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
6
6
6
6
6
6
6
6
6
7
7
7
7
7
7
7
7
7
7
8
8
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
been fueled by various academics and industries [12-14]. Another prevalent scaffold, imidazole
template, is a privileged structure fragment in current medicinal chemistry [15-21]. In particular,
benzimidazole and imidazoquinoline derivatives are well known for their pharmaceutical
properties, which have been extended to widespread inhibitors of proton pump [22], hypertension,
virus [23] and cancer [24-26]. (Fig. 1.) Because there are few reports on the biological research on
the coumarin and imidazole fused skeleton, in this study, a series of compounds possessing a
chromeno [3,4-d] imidazol-4(1H)-one core was synthesized and their structure-activity
relationships (SAR) were studied.
2. Results and discussion
2.1 Structure and activity relationship
At the beginning of our research, the coumarin analogues were substituted by the simple phenyl
on the N-1 position and aryl fragments on C-6 position. As displayed in Table 1, phenyl groups
including pyridin-3-yl, 3-NH -C H , 2-Me-C H , 4-OH-C H and 3-NHSO -(4-F-C H )-C H on
2
6
4
6
4
6
4
2
6
4
6
4
the C-6 position showed no bioactivity. When the pyridin-4-yl and quinolin-3-yl substituted the
C-6 position of coumarin, the inhibitory activities of compounds 15 and 16 against three tested
cancer cell lines were improved.
Furthermore, we turned our attention to the phenyl ring at the N-1 position. Compounds 17 - 25
with 4-CH CN in the phenyl ring displayed slight improvement in inhibitory activities against
2
U87-MG, PC-3 and HCT116 cancer cell lines. Specifically, when 3-NH -C H was introduced to
2
6
4
the C-6 position, compound 19 showed moderate activity against PC-3 cell line with IC value of
50
36.51 ꢀM. Furthermore, compound 21 that containing a 4-OH-C H , exhibited some bioactivity
6
4
with value of 29.37 ꢀM. Besides, pyridine-3-yl (22) or quinolin-3-yl (25) at C-6 position showed
better activities with IC50 values of 29.37 ꢀM and 28.4 ꢀM against HCT116 cells, indicating that
the nitrogen atom on the heterocyclic substitute is crucial to the bioactivity, which may provide a
hydrogen acceptor site.
To further investigate the SAR of N-1 group, with pyridin-4-yl, pyridine-3-yl or quinolin-3-yl
retained at C6-position, the phenyl was replaced by 4-OMe-C H and 2-Me-C H . However, all
6
4
6
4
the compounds (compounds 26 to 31) showed no bioactivity, suggesting that the cyano of
4-CH CN-C H group is a very crucial moiety which may provide a hydrogen acceptor site. When
2
6
4
N4-C(CH ) -C H group was introduced to the N-1 position, all compounds showed an improved
3
2
6
4
inhibitory activities. Compound 32 with pyridin-3-yl at C-6 position showed antitumor activity
against U87-MG cells with IC value of 25.62 ꢀM and 33 inhibited U87-MG and PC-3 with IC
50
50
values of 28.55 ꢀM and 29.01 ꢀM, respectively. What’s more, 34 with quinolin-3-yl displayed
higher bioactivity against the U87-MG, PC-3 and HCT116 cell lines with IC50 values of 33.42,
19.76, 20.40 ꢀM.
Out of our expectation, when a methyl group was attempted to introduce to N-3 position,
tert-butoxy was accidentally inserted into C-2 of imidazole ring. To our delight, compound 35
possesses attractive antitumor activity against HCT116 with IC50 value of 1.40 ꢀM, 15 fold
stronger than that of 34. (Table 2) It can be concluded that the good inhibitory activity does not
simply reside in the quinolin-3-yl at C-6 position, but also the methyl and tert-butoxy at the ring of
imidazole.
2
/ 21

ACCEPTED MANUSCRIPT
8
8
8
8
8
8
8
9
9
9
9
9
9
9
9
9
9
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
2.2 Biological evaluation
2.2.1 Compound 35 inhibited many kinds of cancer cells with low toxicity to normal LO2 cells
In order to investigate whether 35 has widely anti-cancer activities, the proliferation inhibition
on colon carcinoma SW420 cell, CT26 cell and HCT116 cell, human breast carcinoma MCF-7 cell
and lung carcinoma A549 cell were tested. As a result, compound 35 displayed attractive
antitumor activities against SW620, CT26, HCT116, MCF-7, and A549 with IC50 values of 3.55,
3.50, 1.40, 1.70 and 9.50 ꢀM, respectively (Fig. 2A). Meanwhile, it also expressed relatively low
cytotoxicity to LO2 normal cell with the IC value of 37.0 ꢀM.
50
2.2.2 Compound 35 inhibited the formation of colony in HCT116
As indicated in Fig. 2B, after the treatment of 35, the size of the colonies was apparently
smaller than that of the non-treated group. When the concentration of 35 was increased to 2.50 ꢀM,
nearly no colony formation was observed.
2.2.3 Compound 35 induced G0/G1 phase arrest and apoptosis
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
00
01
02
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
The effects of 35 on SW620 cell cycle distribution were shown in Fig. 3A. Under the treatment
of 35, SW620 cells were arrested significantly at the G0/G1 phase compared to the untreated
group, with the percentage of G0/G1 increased from 38.47% (control) to 38.78% (0.3125 ꢀM),
40.30% (0.625 ꢀM), 42.97% (1.25 ꢀM), 47.49% (2.50 ꢀM) and 60.04% (5.0 ꢀM), respectively.
Meanwhile, the number of cells in S and G2/M phase decreased. Furthermore, flow cytometry
analysis using Annexin V-FITC and propidium iodide (PI) staining showed that 35 treatment at
0.3125, 0.625, 1.25, 2.50 and 5.0 ꢀM increased the percentage of apoptosis cells from 2.83% to
15.71%, 25.19%, 26.22% and 42.90%, respectively. From the above experiments, it can be
concluded that 35 could induce G0/G1 arrest and apoptosis of SW620 cells in a dose-dependent
manner.
2.2.4 Compound 35 blocked MCF-7 cancer cell migration
To further test the effect of 35 on the cancer cell migration potential, wound healing assay was
conducted on MCF-7 cells. As shown in Fig. 4, after treatment with 35 at 5.0 ꢀM for 24 h, the
breast cancer cell migration was suppressed obviously.
2.2.5 Compound 35 inhibited tumor growth in HCT116 mouse mammary tumor model
To assess the antitumor effect of 35 in vivo, nude mice were administered 35 by subcutaneous
injection at the dose of 40 mg/kg and 80 mg/kg. As shown in Fig. 5A, 35 Amarkedly suppressed
tumor growth in a dose-dependent manner with the inhibition of tumor progression at 52.96% in
the 80 mg/kg group. In addition, 35 treatment was well tolerated and did not cause significant
body weight loss (Fig. 5B).
3. Conclusion
In this research, a series of chromeno [3,4-d] imidazol-4(1H)-one derivatives was synthesized
and their antitumor activities in vitro against U87-MG cells, PC-3 cells and HCT116 cells were
3
/ 21

ACCEPTED MANUSCRIPT
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
tested. Among these compounds, 35 exhibits excellent broad spectrum anticancer activities in vitro
with IC values of 1.40 ꢀM, 3.55 ꢀM, 1.70 ꢀM, 3.50 ꢀM and 9.50 ꢀM against HCT116, SW620,
5
0
MCF-7, CT26 and A549 respectively. In addition, structure activity relationship research reveals
that 4-C(CH ) -CN-C H at N-1 position of coumarin core is the best optimal for bioactivity; a
3
2
6
4
methyl group at N-3 position and tert-butoxy inserted into C-2 of imidazole are indispensable for
the improvement of potency; the quinolin-3-yl at C-6 position of coumarin core can make a
positive contribution to the activity. Flow cytometry experiment demonstrates that compound 35
can arrest the cell cycle of SW620 at G0/G1 and induce apoptosis in a concentration-dependent
manner. Further in vivo anticancer experiments on subcutaneous HCT116 tumor model in nude
mice show that 35 can inhibit tumor growth by 52.96% at 80 mg/kg/48h for 20 days. Altogether,
our results provide a novel scaffold with better potency as antitumor agents. All the results
encourage us to continue the development and the further validation of mechanism at the
molecular level.
4. Experimental section
4.1 General methods for chemistry
All the reagents and solvents were purchased from commercial vendors and dried by standard
1
13
methods in advance and distilled before used. H and C NMR spectra were recorded on with a
Bruker AC-E400 spectrometer (operating at 400 MHz), with chemical shift in parts per million
(ppm, δ) downfield from TMS as an internal standard. Mass spectrometry (MS) data were
obtained using a BrukerAmjzon spectrometer. Compound purity was determined by a Waters 1525
series HPLC system with a confirming purity of ≥ 95% for all of the final biologically tested
compounds. Flash column chromatography was completed by silica gel.
3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide (MTT), DMSO, DMEMꢀwere
from Sigma. The Annexin V-FITC Apoptosis Detection Kit was purchased from KeyGEN
BioTECH.
4.2 General procedure for the synthesis of 1 – 5
4.2.1 Synthesis of 1
The mixture of 4-bromophenol (116 mmol) and acetic anhydride (464 mmol) in pyridine (60
mL) was stirred at 100 °C for 3.5 h. Then the reaction mixture was cooled to room temperature
and quenched with water. The reaction was acidized with hydrochloric acid and extracted with
ethyl acetate (40 mL×3). The combined organic layer was washed by saturated sodium chloride
solution for three times, dried over anhydrous Na SO and concentrated under reduced pressure.
2
4
The residue was purified by silica gel chromatography. 1: yield 100%.
4.2.2 Synthesis of 2
A suspension of 1 (46 mmol) and aluminum chloride (69 mmol) was stirred at 150 °C. After
3.5 h the mixture was cooled to room temperature and the diluted hydrochloric acid was added to
it slowly for quenching aluminum chloride until all solid disappeared. Then the solution was
extracted with ethyl acetate (20 mL×3), and the combined organic layer was washed by saturated
4
/ 21

ACCEPTED MANUSCRIPT
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
00
01
02
03
04
05
06
07
08
09
10
11
12
13
14
sodium chloride solution for three times, dried over anhydrous Na SO and concentrated under
2
4
1
reduced pressure. The decolorized solid was recrystallized with n-hexane. 2: yield 72%. H NMR
(400 MHz, CDCl ): δ 7.38 (d, J = 8.0 Hz, 1H), 7.32 (s, 1H), 7.23 (d, J = 7.9 Hz, 1H), 7.06 (d, J =
3
8.2 Hz, 1H), 2.28 (s, 3H) ppm.
4.2.3 Synthesis of 3
2 (74 mmol) and diethyl carbonate (111 mmol) in toluene (160 mL) were added dropwise to
sodium hydride (369 mmol) in toluene (50 mL) in ice-water bath. The mixture was stirred at ice
bath for 30 min and at 100 °C for 4 h. The sodium hydride was quenched with water, acidized
with hydrochloric acid and then the suspension was filtered separately, and the solid collected
from this filtration was washed with toluene and water for three times. The solid dried at room
1
temperature with phosphorus pentoxide. 3: yield 71%. H NMR (400 MHz, CDCl ): δ 12.17 (s,
3
1H), 7.83 (d, J = 2.45, 1H), 7.55 (dd, J = 2.4 Hz, 8.8 Hz, 1H), 6.90 (d, J = 9.2 Hz, 1H), 2.63 (s, 3H)
ppm.
4.2.4 Synthesis of 4
6-bromo-4-hydroxy-2H-chromen-2-one (0.15 molꢁwas dissolved in dichloromethane (300 mL)
and then stirred at room temperature. The solution was acidified with hydrochloric acid and the
yellow solid was filtered off and subsequently washed with water and acetic acid. 4: yield 94%.
-
1
ESI-MS: Calcd for C H BrNO [M-H] : 463.98, Found 284.93, 286.93, found 283.9, 285.9. H
9
4
5
NMR (400 MHz, d6-DMSO): δ 7.93 (d, J = 2.4 Hz, 1H), 7.68 (dd, J = 2.4 Hz, 8.8 Hz, 1H), 7.84
(d, J = 8.8 Hz, 1H) ppm.
4.2.5 Synthesis of 5
To a suspension of 6-bromo-4-hydroxy-3-nitro-2H-chromen-2-one (10 mmol) in phosphorus
oxychloride (15 mL) was added triethylamine slowly. The mixture was stirred and refluxed for 1 h.
The phosphorus oxychloride was quenched with water carefully, then the suspension was filtered
and the decolorized solid was recrystallized with ethyl acetate and petroleum ether. 5: yield 87%.
1
H NMR (400 MHz, d6-DMSO): δ 7.92 (d, J = 2.4 Hz, 1H), 7.67 (dd, J = 2.6 Hz, 9.0 Hz, 1H),
7.18 (d, J = 8.8 Hz, 1H) ppm.
4.3 General procedure for the synthesis of 6a - 6e
A mixture of corresponding phenylamine (20 mmol) and 5 (20 mmol) was added into DMF (50
mL). The reaction mixture was stirred at room temperature for 5 h. Then, it was diluted with water
and the separated solid was filtered off, washed with water, ethanol and ethyl acetate.
4.3.1 6-bromo-3-nitro-4-(phenylamino)-2H-chromen-2-one (6a)
-
1
Yield: 95%. ESI-MS: Calcd for C H BrN O [M-H] : 358.97, 360.98, Found 358.9, 360.9. H
15
9
2
4
NMR (400 MHz, DMSO) δ 10.36 (s, 1H), 8.35 (d, J = 8.7 Hz, 1H), 7.84 (d, J = 1.7 Hz, 1H), 7.74
(dd, J = 8.7, 1.7 Hz, 1H), 7.37 (t, J = 7.7 Hz, 2H), 7.25 (t, J = 7.4 Hz, 1H), 7.20 (d, J = 7.7 Hz, 2H)
ppm.
4.3.2 2-(4-((6-bromo-3-nitro-2-oxo-2H-chromen-4-yl)amino)phenyl)acetonitrile (6b)
5
/ 21

ACCEPTED MANUSCRIPT
+
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
Yield: 93%. ESI-MS: Calcd for C H BrN O [M+Na] : 421.99, 423.99, Found 422.0, 423.7.
17
10
3
4
1
H NMR (400 MHz, DMSO): δ 10.37 (s, 1H), 8.74 (s, 1H), 7.96 (d, J = 7.2 Hz, 1H), 7.47 (d, J =
8.8 Hz, 1H), 7.35 (d, J = 8.4 Hz, 2H), 7.23 (d, J = 8.4 Hz, 2H), 4.08 (s, 2H) ppm.
4.3.3 6-bromo-4-((4-methoxyphenyl)amino)-3-nitro-2H-chromen-2-one (6c)
-
1
Yield: 95%. ESI-MS: Calcd for C H BrN O [M-H] : 388.99, 390.98, Found 389.0, 391.1. H
16
11
2
5
NMR (400 MHz, DMSO): δ 10.25 (s, 1H), 8.33 (d, J = 8.8 Hz, 1H), 7.82 (d, J = 1.8 Hz, 1H), 7.73
(dd, J = 8.7, 1.8 Hz, 1H), 7.15 (d, J = 8.9 Hz, 2H), 6.92 (d, J = 8.9 Hz, 2H), 3.77 (s, 3H) ppm.
4.3.4 6-bromo-3-nitro-4-(o-tolylamino)-2H-chromen-2-one (6d)
+
Yield: 97%. ESI-MS: Calcd for C H BrN O [M+Na] : 396.99, 398.99, Found 397.2, 399.2.
16
11
2
4
1
H NMR (400 MHz, CDCl ) δ 11.43 (s, 1H), 7.63 (d, J = 8.8 Hz, 1H), 7.42 (m, 2H), 7.32 (t, J =
3
6.7 Hz, 1H), 7.20 (m, 2H), 7.14 (d, J = 7.8 Hz, 1H), 2.36 (s, 3H) ppm.
4.3.5 2-(4-((6-bromo-3-nitro-2-oxo-2H-chromen-4-yl)amino)phenyl)-2-methylpropanenitrile (6e)
-
1
Yield: 95%. ESI-MS: Calcd for C H BrN O [M-H] : 426.02, 429.01, Found 426.1, 428.1. H
19
14
3
4
NMR (400 MHz, DMSO) δ 10.37 (s, 1H), 8.33 (d, J = 8.9 Hz, 1H), 7.85 (d, J = 1.8 Hz, 1H), 7.75
(dd, J = 8.7, 1.7 Hz, 1H), 7.51 (d, J = 8.6 Hz, 2H), 7.25 (d, J = 8.5 Hz, 2H), 1.69 (s, 6H) ppm.
4.4 General procedure for the synthesis of 7a - 7e
A 250 mL 3-necked round-bottom flask was charged with a solution of iron (166.2 mmol) and
NH Cl (50 mmol) in 3:1 ethanol: water (70 mL), heated to 100 °C, charged with 6 (6 g, 16.62
4
mmol) in several batches and stirred for 5 h. The reaction mixture was cooled, and added into a
solution of Na CO . The separated solid was filtered off and extracted with ethyl acetate. The
2
3
combined organic layers were dried over sodium sulfate, filtered and concentrated in vacuo.
4.4.1 3-amino-6-bromo-4-(phenylamino)-2H-chromen-2-one (7a)
+
1
Yield: 59%. ESI-MS: Calcd for C H BrN O [M+H] : 331.00, 333.00, Found 330.6, 332.7. H
15
11
2
2
NMR (400 MHz, CDCl ): δ 7.57 (d, J = 1.6 Hz, 1H), 7.41 (dd, J = 2.0, 8.8 Hz, 1H), 7.29 (m, 2H),
3
7.22 (d, J = 8.4 Hz, 1H), 6.94 (t, J = 14.4, 7.2 Hz, 1H), 7.21 (d, J = 8.0 Hz, 2H), 5.50 (s, 1H), 4.13
(br, 2H) ppm.
4.4.2 2-(4-((3-amino-6-bromo-2-oxo-2H-chromen-4-yl)amino)phenyl)acetonitrile (7b)
+
Yield: 63 %. ESI-MS: Calcd for C H BrN O [M+Na] : 392.01, 394.01, Found 389.9, 391.9.
17
12
3
2
1
H NMR (400 MHz, CDCl ): δ 7.53 (d, J = 2.4 Hz, 1H), 7.43 (dd, J = 8.8, 2.4 Hz, 1H), 7.22 (m,
3
4H), 6.71 (d, J = 8.4 Hz, 1H), 5.44 (s, 1H), 4.13 (br, 2H), 3.75 (s, 2H) ppm.
4.4.3 3-amino-6-bromo-4-((4-methoxyphenyl)amino)-2H-chromen-2-one (7c)
+
1
Yield: 54%. ESI-MS: Calcd for C H BrN O [M+H] : 361.01, 363.01, Found 360.6, 363.7. H
16
13
2
3
NMR (400 MHz, CDCl ): δ 7.55 (s, 1H), 7.42 (d, J = 8.8 Hz, 1H), 7.23 (d, J = 8.6 Hz, 1H), 6.86
3
(d, J = 8.1 Hz, 2H), 6.73 (d, J = 8.2 Hz, 2H), 5.41 (s, 1H), 3.79 (s, 3H), 1.26 (s, 2H) ppm.
4.4.4 3-amino-6-bromo-4-(o-tolylamino)-2H-chromen-2-one (7d)
6
/ 21

ACCEPTED MANUSCRIPT
+
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
3
3
3
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
00
01
02
Yield: 52%. ESI-MS: Calcd for C16H13BrN2O2 [M+ K] : 383.02, 385.01, Found 383.2, 384.5.
1
H NMR (400 MHz, DMSO) δ 7.95 (s, 1H), 7.42 (dd, J = 8.7, 1.6 Hz, 1H), 7.32 (d, J = 7.7 Hz,
2H), 7.17 (d, J = 7.3 Hz, 1H), 6.95 (t, J = 7.5 Hz, 1H), 6.89 (s, 1H), 6.78 (t, J = 7.3 Hz, 1H), 6.31
(d, J = 7.9 Hz, 1H), 5.27 (s, 1H), 2.37 (s, 3H) ppm.
4.4.5 2-(4-((3-amino-6-bromo-2-oxo-2H-chromen-4-yl)amino)phenyl)-2-methylpropanenitrile (7e)
+
Yield: 61%. ESI-MS: Calcd for C H BrN O [M+ Na] : 420.04.02, 422.04, Found 420.2,
19
16
3
2
1
6
422.5. H NMR (400 MHz, d -DMSO): δ7.96 (s, 1H), 7.55 (d, J = 2.0 Hz, 1H), 7.45 (dd, J = 1.8
Hz, 8.6 Hz, 1H), 7.35 (s, 1H), 7.32 (d, J = 9.2 Hz, 2H), 6.67 (d, J = 8.8 Hz, 2H), 5.24 (s, 2H), 1.63
(s, 6H) ppm.
4.5 General procedure for the synthesis of 8a - 8e
Compound 7 (9 mmol) and 1, 1'-carbonyldiimidazole (45 mmol) in acetic acid (50 mL) were
stirred for 12 h at 120 °C. The reaction mixture was cooled and concentrated in vacuo to remove
most of the acetic acid. The residual solution was added into water. The precipitate was filtered
and extracted with ethyl acetate and then it was purified by silica gel chromatography.
4.5.1 8-bromo-1-phenyl-1,3-dihydrochromeno[3,4-d]imidazole-2,4-dione (8a)
+
1
Yield: 74%. ESI-MS: Calcd for C H BrN O [M+ H] : 356.98, 358.98, Found 355.1, 357.1. H
16
9
2
3
6
NMR (400 MHz, d -DMSO): δ 12.12 (s, 1H), 7.68 (d, J = 3.2 Hz, 3H), 7.61 (m, 3H), 7.46 (d, J =
8.8 Hz, 1H), 6.62 (d, J = 2.0 Hz, 1H) ppm.
4.5.2 2-(4-(8-bromo-2,4-dioxo-2,3-dihydrochromeno[3,4-d]imidazol-1(4H)-yl)phenyl)acetonitrile
(8b)
+
1
Yield: 70%. ESI-MS: Calcd for C H BrN O [M+ H] : 395.99, 397.99 Found 393.9, 395.9. H
18
10
3
3
6
NMR (400 MHz, d -DMSO): δ7.76 (s, 4H), 7.65 (m, 1H), 7.50 (d, J = 8.8 Hz, 1H), 6.62 (d, J =
2.0 Hz, 1H), 4.30 (s, 2H) ppm
4.5.3 8-bromo-1-(4-methoxyphenyl)-1,3-dihydrochromeno[3,4-d]imidazole-2,4-dione (8c)
+
1
Yield: 45%. ESI-MS: Calcd for C H BrN O [M+ H] : 386.99, 388.99 Found 385.0, 387.0. H
17
11
2
4
NMR (400 MHz, d6-DMSO): δ7.62 (m, 3H), 7.47 (d, J = 8.9 Hz, 1H), 7.29 (d, J = 8.7 Hz, 2H),
+
6.67 (d, J = 2.0 Hz, 1H), 3.92 (s, 3H) ppm. ESI-MS: 385, 387 [M+H] .
4.5.4 8-bromo-1-(o-tolyl)-1,3-dihydrochromeno[3,4-d]imidazole-2,4-dione (8d)
+
1
Yield: 62%. ESI-MS: Calcd for C H BrN O [M+ H] : 370.00, 371.99 Found 369.1, 371.1. H
17
11
2
3
6
NMR (400 MHz, d -DMSO): δ 7.65 (m, 5H), 7.49 (d, J = 8.8 Hz, 1H), 6.45 (d, J = 1.4 Hz, 1H),
1.98 (s, 3H) ppm.
4.5.5
2-(4-(8-bromo-2,4-dioxo-2,3-dihydrochromeno[3,4-d]imidazol-1(4H)-yl)phenyl)-2-methylpropane
nitrile (8e)
+
Yield: 56%. ESI-MS: Calcd for C H BrN O [M+ Na] : 446.02, 448.02 Found 444.3, 446.3.
20
14
3
3
1
6
H NMR (400 MHz, d -DMSO): δ7.93 (d, J = 8.8 Hz, 2H), 7.81 (d, J = 8.4 Hz, 2H), 7.63 (dd, J =
2.0 Hz, 8.8 Hz, 1H), 7.49 (d, J = 8.8 Hz, 1H), 6.41 (d, J = 2.4 Hz, 1H), 1.83 (s, 6H) ppm.
7
/ 21

ACCEPTED MANUSCRIPT
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
4.6 General procedure for the synthesis of 9-35
A mixture of 8 (0.1 mmol), boric acid (0.11 mmol), PdCl (dppf) CH Cl (0.05 mmol) and
2
2
2
K CO (0.3 mmol) in dioxane/water (3/1 mL) was bubbled with nitrogen for 10 minutes, and
2
3
heated in a sealed tube at 50 °C for 5 h. After cooling the reaction mixture was partitioned with
water. The aqueous layer was further extracted with ethyl acetate. The organic extract was washed
with brine, dried over sodium sulfate, and it was purified by silica gel chromatography.
4.6.1 1,8-diphenyl-1,3-dihydrochromeno[3,4-d]imidazole-2,4-dione (9)
+
1
Yield: 44%. ESI-MS: Calcd for C H N O [M+ H] : 355.10 Found 353.2. H NMR (400 MHz,
22
14
2
3
CDCl ) δ 7.71 (s, 3H), 7.61 (d, J = 8.6 Hz, 1H), 7.49 (d, J = 8.4 Hz, 3H), 7.32 (m, 3H), 7.19 (d, J
3
= 7.2 Hz, 2H), 6.84 (s, 1H) ppm.
4.6.2 1-phenyl-8-(pyridin-3-yl)-1,3-dihydrochromeno[3,4-d]imidazole-2,4-dione (10)
+
1
Yield: 43%. ESI-MS: Calcd for C H N O [M+ Na] : 378.10 Found 376.0. H NMR (400
21
13
3
3
MHz, CDCl ) δ 8.54 (d, J = 3.7 Hz, 1H), 8.41 (d, J = 1.8 Hz, 1H), 7.73 (dt, J = 4.2 Hz, 5.4 Hz, 3H,
3
3H), 7.60 (dd, J = 8.6, 2.1 Hz, 1H), 7.55 (m, 2H), 7.49 (dd, J = 7.7, 1.7 Hz, 2H), 7.29 (dd, J = 8.0,
4.9 Hz, 1H), 6.82 (d, J = 2.0 Hz, 1H) ppm.
4.6.3 8-(3-aminophenyl)-1-phenyl-1,3-dihydrochromeno[3,4-d]imidazole-2,4-dione (11)
+
1
Yield: 57%. ESI-MS: Calcd for C H N O [M+ H] : 370.11 Found 368.3. H NMR (400 MHz,
22
15
3
3
DMSO) δ 7.76 (d, J = 5.7 Hz, 5H), 7.62 (d, J = 8.6 Hz, 1H), 7.54 (d, J = 8.6 Hz, 1H), 6.97 (t, J =
7.7 Hz, 1H), 6.72 (s, 1H), 6.50 (d, J = 8.0 Hz, 1H), 6.47 (s, 1H), 6.28 (d, J = 7.5 Hz, 1H), 5.11 (s,
2H) ppm.
4.6.4 1-phenyl-8-(o-tolyl)-1,3-dihydrochromeno[3,4-d]imidazole-2,4-dione (12)
+
1
Yield: 55%. ESI-MS: Calcd for C H N O [M+ Na] : 391.12 Found 389.0. H NMR (400
23
16
2
3
MHz, CDCl ) δ 7.62 (s, 3H), 7.49 (d, J = 8.6 Hz, 1H), 7.43 (s, 2H), 7.35 (d, J = 8.6 Hz, 1H), 7.18
3
(m, 3H), 7.00 (d, J = 7.2 Hz, 1H), 6.66 (s, 1H), 2.38 (s, 3H) ppm.
4.6.5 8-(4-hydroxyphenyl)-1-phenyl-1,3-dihydrochromeno[3,4-d]imidazole-2,4-dione (13)
-
1
Yield: 52%. ESI-MS: Calcd for C H N O [M- H] : 369.10 Found 367.1. H NMR (400 MHz,
22
14
2
4
DMSO) δ 9.59 (s, 1H), 7.75 (d, J = 3.1 Hz, 5H), 7.65 (dd, J = 8.7, 2.2 Hz, 1H), 7.50 (d, J = 8.7 Hz,
1H), 7.01 (d, J = 8.6 Hz, 2H), 6.71 (d, J = 8.6 Hz, 2H), 6.65 (d, J = 2.0 Hz, 1H) ppm.
4.6.6
N-(3-(2,4-dioxo-1-phenyl-1,2,3,4-tetrahydrochromeno[3,4-d]imidazol-8-yl)phenyl)-4-fluorobenzen
esulfonamide (14)
-
1
Yield: 56%. ESI-MS: Calcd for C H FN O S [M- H] : 526.10 Found 524.3. H NMR (400
28
18
3
5
MHz, DMSO) δ 10.40 (s, 1H), 7.81 (m, 2H), 7.74 (s, 5H), 7.58 (s, 2H), 7.40 (t, J = 8.3 Hz, 2H),
7.22 (t, J = 7.8 Hz, 1H), 7.10 (s, 1H), 7.00 (d, J = 8.0 Hz, 1H), 6.80 (d, J = 7.7 Hz, 1H), 6.71 (s,
1H) ppm.
8
/ 21

ACCEPTED MANUSCRIPT
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
4.6.7 1-phenyl-8-(pyridin-4-yl)-1,3-dihydrochromeno[3,4-d]imidazole-2,4-dione (15)
+
1
Yield: 57%. ESI-MS: Calcd for C H N O [M+ H] : 356.10 Found 354.0. H NMR (400 MHz,
21
13
3
3
CDCl ) δ 8.57 (d, J = 4.8 Hz, 2H), 7.74 (m, 3H), 7.66 (dd, J = 8.7, 1.9 Hz, 1H), 7.52 (m, 3H), 7.09
3
(d, J = 5.4 Hz, 2H), 6.87 (s, 1H) ppm.
4.6.8 1-phenyl-8-(quinolin-3-yl)-1,3-dihydrochromeno[3,4-d]imidazole-2,4-dione (16)
+
1
Yield: 40%. ESI-MS: Calcd for C H N O [M+ Na] : 428.11 Found 426.1. H NMR (400
25
15
3
3
MHz, CDCl ) δ 8.72 (d, J = 2.2 Hz, 1H), 8.11 (d, J = 8.4 Hz, 1H), 8.00 (d, J = 1.9 Hz, 1H), 7.75
3
(m, 6H), 7.59 (t, J = 8.0 Hz, 2H), 7.52 (m, 2H), 6.94 (d, J = 2.0 Hz, 1H) ppm.
4.6.9
2-(4-(2,4-dioxo-8-(o-tolyl)-2,3-dihydrochromeno[3,4-d]imidazol-1(4H)-yl)phenyl)acetonitrile (17)
-
1
Yield: 52%. ESI-MS: Calcd for C H N O [M- H] : 406.13 Found 404.2. H NMR (400 MHz,
25
17
3
3
CDCl ) δ 7.63 (d, J = 8.1 Hz, 2H), 7.49 (dd, J = 8.2, 6.0 Hz, 3H), 7.37 (dd, J = 8.6, 2.0 Hz, 1H),
3
7.19 (m, 3H), 7.00 (d, J = 7.1 Hz, 1H), 6.65 (d, J = 1.9 Hz, 1H), 3.88 (s, 2H), 2.37 (s, 3H) ppm.
4.6.10
N-(3-(1-(4-(cyanomethyl)phenyl)-2,4-dioxo-1,2,3,4-tetrahydrochromeno[3,4-d]imidazol-8-yl)phen
yl)-4-fluorobenzenesulfonamide (18)
-
1
Yield: 43%. ESI-MS: Calcd for C H FN O S [M- H] : 565.11 Found 563.2. H NMR (400
30
19
4
5
MHz, DMSO) δ 10.43 (s, 1H), 7.78 (m, 6H), 7.61 (s, 2H), 7.39 (t, J = 8.8 Hz, 2H), 7.25 (t, J = 7.9
Hz, 1H), 7.16 (s, 1H), 6.94 (d, J = 8.1 Hz, 1H), 6.79 (d, J = 7.9 Hz, 1H), 6.74 (s, 1H), 4.25 (s, 2H)
ppm.
4.6.11
2-(4-(8-(3-aminophenyl)-2,4-dioxo-2,3-dihydrochromeno[3,4-d]imidazol-1(4H)-yl)phenyl)acetonit
rile (19)
+
1
Yield: 55%. ESI-MS: Calcd for C H N O [M+ H] : 409.12 Found 407.3. H NMR (400 MHz,
24
16
4
3
CDCl ) δ 7.72 (d, J = 7.9 Hz, 2H), 7.56 (dd, J = 15.8, 8.9 Hz, 3H), 7.46 (d, J = 8.6 Hz, 1H), 7.15 (t,
3
J = 7.8 Hz, 1H), 6.76 (s, 1H), 6.63 (d, J = 8.5 Hz, 1H), 6.57 (d, J = 7.5 Hz, 1H), 6.50 (s, 1H), 3.96
(s, 2H), 3.79 (s, 2H) ppm.
4.6.12
2-(4-(2,4-dioxo-8-phenyl-2,3-dihydrochromeno[3,4-d]imidazol-1(4H)-yl)phenyl)acetonitrile (20)
-
1
Yield: 55%. ESI-MS: Calcd for C H N O [M- H] : 392.11 Found 390.2. H NMR (400 MHz,
24
15
3
3
CDCl ) δ 7.72 (d, J = 8.2 Hz, 2H), 7.62 (dd, J = 8.7, 2.1 Hz, 1H), 7.56 (d, J = 8.2 Hz, 2H), 7.50 (d,
3
J = 8.6 Hz, 1H), 7.39 (t, J = 7.4 Hz, 2H), 7.32 (t, J = 7.3 Hz, 1H), 7.20 (d, J = 7.2 Hz, 2H), 6.79 (d,
J = 2.0 Hz, 1H), 3.95 (s, 2H) ppm.
4.6.13
2-(4-(8-(4-hydroxyphenyl)-2,4-dioxo-2,3-dihydrochromeno[3,4-d]imidazol-1(4H)-yl)phenyl)aceto
nitrile (21)
-
1
Yield: 55%. ESI-MS: Calcd for C H N O [M-H] : 308.11 Found 306.2. H NMR (400 MHz,
24
15
3
4
6
d -DMSO): δ9.64 (s, 1H), 7.79 (m, 4H), 7.70 (dd, J = 2.0 Hz, 8.8 Hz, 1H), 7.52 (d, J = 8.8 Hz,
9
/ 21

ACCEPTED MANUSCRIPT
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
91
92
93
94
95
96
97
98
99
00
01
02
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
1H), 7.05 (d, J = 8.8 Hz, 2H), 6.79 (d, J = 8.4 Hz, 2H), 6.67 (d, J = 2.0 Hz, 1H), 4.30 (s, 2H) ppm.
4.6.14
2-(4-(2,4-dioxo-8-(pyridin-3-yl)-2,3-dihydrochromeno[3,4-d]imidazol-1(4H)-yl)phenyl)acetonitril
e (22)
+
1
Yield: 51%. ESI-MS: Calcd for C H N O [M+H] : 395.11 Found 393.1. H NMR (400 MHz,
23
14
4
3
CDCl ): δ 8.55 (s, 1H), 8.45 (s, 1H), 7.23 (d, J = 8.4 Hz, 2H), 7.59 (d, J = 8.4 Hz, 3H), 7.51 (t, J =
3
9.6 Hz, 2H), 7.35 (m, 1H), 6.78 (d, J = 1.6 Hz, 1H), 3.99 (s, 2H) ppm.
4.6.15
2-(4-(8-(4-(methylsulfonyl)phenyl)-2,4-dioxo-2,3-dihydrochromeno[3,4-d]imidazol-1(4H)-yl)phen
yl)acetonitrile (23)
+
1
Yield: 49%. ESI-MS: Calcd for C H N O S [M+H] : 472.09 Found 470.2. H NMR (400
25
17
3
5
MHz, CDCl ) δ 7.96 (d, J = 8.2 Hz, 2H), 7.73 (d, J = 7.7 Hz, 2H), 7.64 (d, J = 7.3 Hz, 1H), 7.56
3
(m, 3H), 7.37 (d, J = 8.2 Hz, 2H), 6.80 (s, 1H), 3.96 (s, 2H), 3.08 (s, 3H) ppm.
4.6.16
2-(4-(2,4-dioxo-8-(pyridin-4-yl)-2,3-dihydrochromeno[3,4-d]imidazol-1(4H)-yl)phenyl)acetonitril
e (24)
+
1
Yield: 58%. ESI-MS: Calcd for C H N O [M+H] : 395.11 Found 393.0. H NMR (400 MHz,
23
14
4
3
CDCl ) δ 8.62 (s, 2H), 7.74 (d, J = 8.2 Hz, 2H), 7.67 (dd, J = 8.6, 2.1 Hz, 1H), 7.56 (d, J = 7.6 Hz,
3
3H), 7.11 (d, J = 2.8 Hz, 2H), 6.84 (d, J = 2.0 Hz, 1H), 3.97 (s, 2H) ppm.
4.6.17
2-(4-(2,4-dioxo-8-(quinolin-3-yl)-2,3-dihydrochromeno[3,4-d]imidazol-1(4H)-yl)phenyl)acetonitri
le (25)
+
1
Yield: 52%. ESI-MS: Calcd for C H N O [M+H] : 445.12 Found 443.3. H NMR (400 MHz,
27
16
4
3
CDCl ): δ 8.72 (s, 1H), 8.10 (d, J = 8.4 Hz, 1H), 8.01 (s, 1H), 7.84 (d, J = 8.0 Hz, 1H), 7.75 (m,
3
4H), 7.60 (m, 4H), 7.27 (s, 1H), 6.92 (s, 1H), 3.99 (s, 2H) ppm.
4.6.18 1-(4-methoxyphenyl)-8-(pyridin-3-yl)-1,3-dihydrochromeno[3,4-d]imidazole-2,4-dione (26)
+
1
Yield: 47%. ESI-MS: Calcd for C H N O [M+H] : 386.11 Found 384.0. H NMR (400 MHz,
22
15
3
4
CDCl ) δ 8.56 (d, J = 4.2 Hz, 1H), 8.50 (s, 1H), 7.56 (m, 3H), 7.39 (d, J = 8.6 Hz, 2H), 7.30 (m,
3
1H), 7.18 (d, J = 8.6 Hz, 2H), 6.92 (s, 1H), 3.96 (d, J = 7.8 Hz, 3H) ppm.
4.6.19 1-(4-methoxyphenyl)-8-(pyridin-4-yl)-1,3-dihydrochromeno[3,4-d]imidazole-2,4-dione (27)
+
1
Yield: 43%. ESI-MS: Calcd for C H N O [M+H] : 386.11 Found 384.0. H NMR (400 MHz,
22
15
3
4
CDCl ) δ 8.59 (s, 2H), 7.66 (d, J = 8.2 Hz, 1H), 7.54 (d, J = 8.6 Hz, 1H), 7.40 (d, J = 8.4 Hz, 2H),
3
7.19 (m, 4H), 6.94 (s, 1H), 3.97 (s, 3H) ppm.
4.6.20
1-(4-methoxyphenyl)-8-(quinolin-3-yl)-1,3-dihydrochromeno[3,4-d]imidazole-2,4-dione
(28)
+
1
Yield: 53%. ESI-MS: Calcd for C H N O [M+H] : 436.12 Found 334.1. H NMR (400 MHz,
26
17
3
4
CDCl ) δ 8.85 (s, 2H), 7.79 (m, 4H), 7.60 (d, J = 8.1 Hz, 1H), 7.42 (d, J = 7.9 Hz, 3H), 7.22 (d, J
3
1
0 / 21

ACCEPTED MANUSCRIPT
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
= 7.0 Hz, 2H), 7.03 (s, 1H), 3.97 (s, 3H) ppm.
4.6.21 8-(quinolin-3-yl)-1-(o-tolyl)-1,3-dihydrochromeno[3,4-d]imidazole-2,4-dione (29)
+
1
Yield: 55%. ESI-MS: Calcd for C H N O [M+H] : 420.13 Found 418.2. H NMR (400 MHz,
26
17
3
3
CDCl ) δ 8.65 (s, 1H), 8.05 (d, J = 8.2 Hz, 1H), 7.92 (s, 1H), 7.73 (d, J = 8.0 Hz, 1H), 7.66 (m,
3
3H), 7.59 (d, J = 7.3 Hz, 1H), 7.49 (m, 3H), 7.36 (d, J = 7.6 Hz, 1H), 6.76 (s, 1H), 2.01 (s, 3H)
ppm.
4.6.22 8-(pyridin-3-yl)-1-(o-tolyl)-1,3-dihydrochromeno[3,4-d]imidazole-2,4-dione (30)
+
1
Yield: 51%. ESI-MS: Calcd for C H N O [M+H] : 370.11 Found 368.3. H NMR (400 MHz,
22
15
3
3
CDCl ) δ 8.48 (s, 1H), 8.34 (s, 1H), 7.51 (m, 6H), 7.33 (d, J = 7.7 Hz, 1H), 7.22 (s, 1H), 6.65 (d, J
3
= 1.4 Hz, 1H), 1.98 (d, J = 5.8 Hz, 3H) ppm.
4.6.23 8-(pyridin-4-yl)-1-(o-tolyl)-1,3-dihydrochromeno[3,4-d]imidazole-2,4-dione (31)
+
1
Yield: 46%. ESI-MS: Calcd for C H N O [M+H] : 370.11 Found 368.3. H NMR (400 MHz,
22
15
3
3
CDCl ) δ 8.57 (s, 2H), 7.66 (dd, J = 11.3, 4.3 Hz, 2H), 7.56 (dd, J = 12.8, 7.7 Hz, 3H), 7.42 (d, J =
3
7.6 Hz, 1H), 7.08 (d, J = 4.2 Hz, 2H), 6.77 (d, J = 1.8 Hz, 1H), 2.06 (s, 3H) ppm.
4.6.24
2-(4-(2,4-dioxo-8-(pyridin-3-yl)-2,3-dihydrochromeno[3,4-d]imidazol-1(4H)-yl)phenyl)-2-methylp
ropanenitrile (32)
+
1
Yield: 60%. ESI-MS: Calcd for C H N O [M+Na] : 445.14 Found 443.1. H NMR (400
25
18
4
3
MHz, CDCl ) δ 8.54 (s, 1H), 8.43 (s, 1H), 7.85 (d, J = 8.1 Hz, 2H), 7.55 (m, 4H), 7.28 (s, 2H),
3
6.69 (s, 1H), 1.87 (s, 6H) ppm.
4.6.25
2-(4-(2,4-dioxo-8-(pyridin-4-yl)-2,3-dihydrochromeno[3,4-d]imidazol-1(4H)-yl)phenyl)-2-methylp
ropanenitrile (33)
+
1
Yield: 56%. ESI-MS: Calcd for C H N O [M+Na] : 445.14 Found 443.1. H NMR (400
25
18
4
3
MHz, CDCl ) δ 8.56 (d, J = 4.8 Hz, 2H), 7.87 (d, J = 8.2 Hz, 2H), 7.67 (m, 2H), 7.56 (t, J = 8.5
3
Hz, 3H), 7.08 (d, J = 4.5 Hz, 2H), 6.78 (s, 1H), 1.88 (s, 6H) ppm.
4.6.26
2-(4-(2,4-dioxo-8-(quinolin-3-yl)-2,3-dihydrochromeno[3,4-d]imidazol-1(4H)-yl)phenyl)-2-methyl
propanenitrile (34)
+
1
Yield: 48%. ESI-MS: Calcd for C H N O [M+Na] : 495.15 Found 493.2. H NMR (400
29
20
4
3
6
MHz, d -DMSO): δ 8.71 (d, J = 2.0 Hz, 1H) , 8.28 (s, 1H), 7.98 (m, 5H), 7.86 (d, J = 8.4 Hz, 2H),
7.78 (t, J = 7.8 Hz, 1H), 7.71 (d, J = 8.4 Hz, 1H), 7.65 (t, J = 7.4 Hz, 1H), 6.82 (d, J = 1.6 Hz, 1H),
2.65 (s, 6H) ppm.
4.7 Procedure for the synthesis of 35a
A mixture of compound 8e (1 mmol), CH I (1 mmol), and potassium tert-butanolate (3 mmol)
3
in DMF was stirred at 0 °C for 20 minutes. The reaction mixture was partitioned with water. Then
the aqueous layer was further extracted with ethyl acetate. The organic extract was washed with
brine, dried over sodium sulfate, and it was purified by silica gel chromatography.
1
1 / 21

ACCEPTED MANUSCRIPT
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
00
01
02
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
20
21
22
2-(4-(8-bromo-2-(tert-butoxy)-2,3-dimethyl-4-oxo-2,3-dihydrochromeno[3,4-d]imidazol-1(4H)-yl)
phenyl)-2-methylpropanenitrile (35a)
+
1
Yield: 7%. ESI-MS: Calcd for C H BrN O [M+H] : 510.13, 512.13 Found 510.2, 512.2 H
26
28
3
3
NMR (400 MHz, DMSO): δ 7.55 (d, J = 8.3 Hz, 2H), 7.44 (dd, J = 8.8, 2.3 Hz, 1H), 7.37 (d, J =
2.3 Hz, 1H), 7.29 (d, J = 8.2 Hz, 2H), 6.84 (d, J = 8.9 Hz, 1H), 3.49 (s, 3H), 2.18 (s, 3H), 1.66 (s,
+
6H), 1.23 (s, 9H) ppm. ESI-MS: 510.2, 512.2 [M+H] .
4.7.1
2-(4-(2-(tert-butoxy)-2,3-dimethyl-4-oxo-8-(quinolin-3-yl)-2,3-dihydrochromeno[3,4-d]imidazol-1
(4H)-yl)phenyl)-2-methylpropanenitrile (35)
+
1
Yield: 55%. ESI-MS: Calcd for C H N O [M+H] : 559.26 Found 559.3. H NMR (400 MHz,
35
34
4
3
CDCl ): δ 9.03 (s, 1H), 8.16 (s, 1H), 8.11 (d, J = 8.4 Hz, 1H), 7.84 (d, J = 8.1 Hz, 1H), 7.71 (t, J =
3
7.6 Hz, 1H), 7.64 (d, J = 8.5 Hz, 1H), 7.57 (t, J = 7.5 Hz, 1H), 7.52 (s, 1H), 7.46 (d, J = 8.2 Hz,
2H), 7.16 (d, J = 8.1 Hz, 2H), 6.90 (d, J = 8.6 Hz, 1H), 3.64 (s, 3H), 2.36 (s, 3H), 1.68 (s, 6H),
13
1.36 (s, 9H) ppm. C NMR (400 MHz, DMSO): δ 161.71, 157.12, 149.03, 146.46, 144.82, 131.86,
131.62, 131.21, 130.16, 129.23, 128.86, 128.61, 128.33, 128.17, 127.90, 127.67, 127.01, 125.93,
124.27, 119.72, 111.69, 78.85, 55.40, 36.45, 28.05, 27.60, 13.64 ppm. Mp = 205- 207 °C.
4.8. Biological evaluation
4.8.1 Cell Culture and reagents preparation
Mouse colorectal carcinoma CT26 cell, human colorectal carcinoma HCT116 and SW620 cells,
human breast carcinoma MCF-7 cell, lung carcinoma A549 cell and human hepatocytes LO2 cell
were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). Cells
were propagated in DMEM or RPMI 1350 media containing 10% fetal bovine serum (FBS; Gibco,
Auckland, N. Z.) and 1% antibiotics (penicillin and streptomycin) in 5% CO at 37 °C.
2
4.8.2 MTT Assay
The cell viability was performed by MTT assay. Briefly, cells were seeded in 96-well plates
(2000 - 3000 cells/plate). After 24h incubation, the cells were treated with various concentrations
of compounds. After treatment for 72 h, a volume of 20 ꢀL of MTT solution (5 mg/mL) was added
to each well and incubated for additional 2 - 4 h incubation at 37 °C. Then the medium was
discarded, and the formazan salt was dissolved with 150 mL DMSO for 15 - 20 minutes. The
absorbance of each well was measured at 570 nm using a Spectra MAX M5 microplate
spectrophotometer (Molecular Devices, CA, USA), and the median inhibitory concentration (IC )
50
values were calculated. Three replicate wells were used for each analysis. The results were
obtained from three separate experiments.
4.8.3 Colony Formation Assay
To test the survival of cells treated with compound 35, HCT116 cells (500 cells/well) were
plated in a 6-well plate and incubated overnight at 37 °C, followed by various concentrations of 35
treatment (0 - 5.0 ꢀM) for 15 days with fresh medium. Finally, the cells were washed with cold
phosphate-buffered saline (PBS) for twice. Colonies were fixed with 4% paraformaldehyde and
stained with a 0.5% crystal violet solution for 15 minutes, and the colonies (>50 cells) were
1
2 / 21

ACCEPTED MANUSCRIPT
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
counted under microscope. The data are expressed from three independent experiments. The
number of colonies in treated cultures was expressed as a percentage of the control cultures.
4.8.4 Flow Cytometry
The cell cycle distribution of SW620 cells after 24 h exposure to various concentrations of 35
was monitored by flow cytometric analysis using a FACScan flow cytometer (Becton Dickinson
5
USA). Briefly, SW620 cells (2×10 cells/well) were seeded in 6-well plates and incubated
overnight at 37 °C. After 24 h treatment, the cells were harvested and washed with cold PBS twice,
and fixed with 75% alcohol at 4 °C. 24h later, cells were stained with propidium iodide (PI) and
analyzed by flow cytometry. Then data were analyzed with FlowJo software.
4.8.5 Apoptotic Assay
To further confirm the apoptosis inducing effect of compound 35, Annexin V-FITC apoptosis
5
detection kit was used. HCT116 cells (2×10 cells/well) were seeded in 6-well plates and incubated
overnight at 37 °C. After 24 h exposure to various concentrations of 35, cells were harvested and
washed with cold PBS twice. After centrifugation, cells were stained with Annexin V-FITC and PI,
and then analyzed with FCM (Becton- Dickinson, USA). Then data were analyzed with FlowJo
software.
4.8.6 Wound-healing migration assay
The inhibition of tumor cell migration by compound 35 was determined by wound-healing
migration assay. MCF-7 cells were seeded onto 6-well plates, when grew to 80 - 90% confluence
and then the “wound” were scraped by a 0.1 mL sterile pipette tips. Fresh medium containing 10%
fetal bovine serum and different concentrations of 35 were added. Cells were photographed after 0,
12, 24 h incubation at 37 °C, respectively.
4.8.7 Mice and tumor model
All animal experiments were approved and conducted by the Institutional Animal Care and
Treatment Committee of Sichuan University in China. 100 mL tumor cell (HCT116) suspension
7
containing 1×10 cells were injected subcutaneously into the right flank of the seven-week-old
female BALB/c athymic nude mice. After one week, when average tumor volume reached
3
approximately 100 mm , mice were separated into three groups randomly (8 mice per group), and
received intraperitoneally injection (i.p.) of 35 at dose of 40 mg/kg, 80 mg/kg or vehicle,
respectively once two days for 24 days. Tumor growth and body weight were measured every two
days during the treatment. The tumor volumes were calculated according to the formula as follow:
3
2
volume (mm ) = (L × W ) ×0.5, where L is the length and W is the width. Growth inhibition was
calculated from the start of treatment by comparison of the mean change in tumor volume for
vehicle and treated group as before.
Acknowledgements
The authors are grateful for the support from the National Science Foundation of China (81472780)
and the National Key Program of China (2012ZX09103101-022).
1
3 / 21

ACCEPTED MANUSCRIPT
5
5
5
5
67
68
69
70
Supporting Information
Characterization data of compounds.
1
4 / 21

ACCEPTED MANUSCRIPT
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
00
01
02
03
04
05
06
07
08
09
10
11
12
13
14
Reference
[1] L.A. Torre, F. Bray, R.L. Siegel, J. Ferlay, J. Lortet-Tieulent, A. Jemal, Global cancer statistics,
2012, CA: a cancer journal for clinicians, 65 (2015) 87-108.
[2] P. Guo, Z.L. Huang, P. Yu, K. Li, Trends in cancer mortality in China: an update, Annals of
oncology : official journal of the European Society for Medical Oncology / ESMO, 23 (2012)
2755-2762.
[3] B. Meunier, Hybrid molecules with a dual mode of action: dream or reality?, Accounts of chemical
research, 41 (2008) 69-77.
[4] D. Havrylyuk, B. Zimenkovsky, O. Vasylenko, A. Gzella, R. Lesyk, Synthesis of new
4-thiazolidinone-, pyrazoline-, and isatin-based conjugates with promising antitumor activity, J Med
Chem, 55 (2012) 8630-8641.
[5] S. Lee, K. Sivakumar, W.S. Shin, F. Xie, Q. Wang, Synthesis and anti-angiogenesis activity of
coumarin derivatives, Bioorganic & medicinal chemistry letters, 16 (2006) 4596-4599.
[6] X.H. Liu, H.F. Liu, J. Chen, Y. Yang, B.A. Song, L.S. Bai, J.X. Liu, H.L. Zhu, X.B. Qi, Synthesis
and molecular docking study of novel coumarin derivatives containing 4,5-dihydropyrazole moiety as
potential antitumor agents, Bioorganic & medicinal chemistry letters, 20 (2010) 5705-5708.
[7] W. Zhang, Z. Li, M. Zhou, F. Wu, X. Hou, H. Luo, H. Liu, X. Han, G. Yan, Z. Ding, R. Li,
Synthesis and biological evaluation of 4-(1,2,3-triazol-1-yl)coumarin derivatives as potential antitumor
agents, Bioorganic & medicinal chemistry letters, 24 (2014) 799-807.
[8] M.A. Musa, J.S. Cooperwood, M.O.F. Khan, A Review of Coumarin Derivatives in
Pharmacotherapy of Breast Cancer, Current medicinal chemistry, 15 (2008) 2664-2679.
[9] S.S. Bhattacharyya, S. Paul, S.K. Mandal, A. Banerjee, N. Boujedaini, A.R. Khuda-Bukhsh, A
synthetic coumarin (4-Methyl-7 hydroxy coumarin) has anti-cancer potentials against DMBA-induced
skin cancer in mice, European Journal of Pharmacology, 614 (2009) 128-136.
[10] M.A. Musa, J.S. Cooperwood, M.O. Khan, A review of coumarin derivatives in pharmacotherapy
of breast cancer, Current medicinal chemistry, 15 (2008) 2664-2679.
[11] A. Lacy, R. O'Kennedy, Studies on coumarins and coumarin-related compounds to determine their
therapeutic role in the treatment of cancer, Current pharmaceutical design, 10 (2004) 3797-3811.
[12] M. Baba, Y.R. Jin, A. Mizuno, H. Suzuki, Y. Okada, N. Takasuka, H. Tokuda, H. Nishino, T.
Okuyama, Studies on cancer chemoprevention by traditional folk medicines XXIV. Inhibitory effect of
a
coumarin derivative, 7-isopentenyloxycoumarin, against tumor-promotion, Biological
&
Pharmaceutical Bulletin, 25 (2002) 244-246.
[13] N.E.B. Saidu, S. Valente, E. Bana, G. Kirsch, D. Bagrel, M. Montenarh, Coumarin polysulfides
inhibit cell growth and induce apoptosis in HCT116 colon cancer cells, Bioorganic & Medicinal
Chemistry, 20 (2012) 1584-1593.
[14] M.A. Musa, V.L.D. Badisa, L.M. Latinwo, T.A. Patterson, M.A. Owens, Coumarin-based
Benzopyranone Derivatives Induced Apoptosis in Human Lung (A549) Cancer Cells, Anticancer
Research, 32 (2012) 4271-4276.
[15] S.M. Maira, F. Stauffer, J. Brueggen, P. Furet, C. Schnell, C. Fritsch, S. Brachmann, P. Chene, A.
De Pover, K. Schoemaker, D. Fabbro, D. Gabriel, M. Simonen, L. Murphy, P. Finan, W. Sellers, C.
Garcia-Echeverria, Identification and characterization of NVP-BEZ235, a new orally available dual
phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor with potent in vivo antitumor
activity, Molecular cancer therapeutics, 7 (2008) 1851-1863.
[16] M.A. Dawson, R.K. Prinjha, A. Dittmann, G. Giotopoulos, M. Bantscheff, W.I. Chan, S.C. Robson,
1
5 / 21

ACCEPTED MANUSCRIPT
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
C.W. Chung, C. Hopf, M.M. Savitski, C. Huthmacher, E. Gudgin, D. Lugo, S. Beinke, T.D. Chapman,
E.J. Roberts, P.E. Soden, K.R. Auger, O. Mirguet, K. Doehner, R. Delwel, A.K. Burnett, P. Jeffrey, G.
Drewes, K. Lee, B.J. Huntly, T. Kouzarides, Inhibition of BET recruitment to chromatin as an effective
treatment for MLL-fusion leukaemia, Nature, 478 (2011) 529-533.
[17] C.A. Bernhart, P.M. Perreaut, B.P. Ferrari, Y.A. Muneaux, J.L. Assens, J. Clement, F. Haudricourt,
C.F. Muneaux, J.E. Taillades, M.A. Vignal, et al., A new series of imidazolones: highly specific and
potent nonpeptide AT1 angiotensin II receptor antagonists, J Med Chem, 36 (1993) 3371-3380.
[18] S.N. Mokale, D. Lokwani, D.B. Shinde, Synthesis, biological activity and docking study of
imidazol-5-one as novel non-nucleoside HIV-1 reverse transcriptase inhibitors, Bioorg Med Chem, 20
(2012) 3119-3127.
[19] A.A. Adjei, R.B. Cohen, W. Franklin, C. Morris, D. Wilson, J.R. Molina, L.J. Hanson, L. Gore, L.
Chow, S. Leong, L. Maloney, G. Gordon, H. Simmons, A. Marlow, K. Litwiler, S. Brown, G. Poch, K.
Kane, J. Haney, S.G. Eckhardt, Phase I pharmacokinetic and pharmacodynamic study of the oral,
small-molecule mitogen-activated protein kinase kinase 1/2 inhibitor AZD6244 (ARRY-142886) in
patients with advanced cancers, Journal of clinical oncology : official journal of the American Society
of Clinical Oncology, 26 (2008) 2139-2146.
[20] D.A. Altomare, L.L. Zhang, J. Deng, A. Di Cristofano, A.J. Klein-Szanto, R. Kumar, J.R. Testa,
GSK690693 Delays Tumor Onset and Progression in Genetically Defined Mouse Models Expressing
Activated Akt, Clinical Cancer Research, 16 (2010) 486-496.
[21] El-Nassan, H. B., Recent progress in the identification of BRAF inhibitors as anti-cancer agents.
European journal of medicinal chemistry, 72(2014) 170-205.
[22] M. Classen, H.G. Dammann, W. Domschke, K.J. Hengels, W. Huttemann, W. Londong, M. Rehner,
B. Simon, L. Witzel, J. Berger, Short-Duration Treatment of Duodenal-Ulcer with Omeprazole and
Ranitidine - Results of a Multi-Centre Trial in Germany, Deut Med Wochenschr, 110 (1985) 210-215.
[23] Y.F. Li, G.F. Wang, P.L. He, W.G. Huang, F.H. Zhu, H.Y. Gao, W. Tang, Y. Luo, C.L. Feng, L.P. Shi,
Y.D. Ren, W. Lu, J.P. Zuo, Synthesis and anti-hepatitis B virus activity of novel benzimidazole
derivatives, Journal of Medicinal Chemistry, 49 (2006) 4790-4794.
[24] K.Y. Chang, S.Y. Tsai, C.M. Wu, C.J. Yen, B.F. Chuang, J.Y. Chang, Novel Phosphoinositide
3-Kinase/mTOR Dual Inhibitor, NVP-BGT226, Displays Potent Growth-Inhibitory Activity against
Human Head and Neck Cancer Cells In Vitro and In Vivo, Clinical Cancer Research, 17 (2011)
7116-7126.
[25] M.A. Dawson, R. Prinjha, A. Dittman, G. Giotopoulos, M. Bantscheff, W.I. Chan, S. Robson, C.W.
Chung, C. Hopf, M. Savitski, K. Dohner, A.K. Burnett, R. Delwel, H. Carola, E. Gudgin, D. Lugo, S.
Beinke, P. Soden, K.R. Auger, O. Mirguet, P. Jeffrey, G. Drewes, K. Lee, T. Kouzarides, B.J.P. Huntly,
Inhibition of BET Recruitment to Chromatin As An Effective Treatment for MLL-Fusion Leukaemia,
Blood, 118 (2011) 27-28.
[26] C.H. Huang, D. Mandelker, O. Schmidt-Kittler, Y. Samuels, V.E. Velculescu, K.W. Kinzler, B.
Vogelstein, S.B. Gabelli, L.M. Amzel, The structure of a human p110 alpha/p85 alpha complex
elucidates the effects of oncogenic PI3K alpha mutations, Science, 318 (2007) 1744-1748.
6
6
55
56
1
6 / 21

ACCEPTED MANUSCRIPT
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
Fig. 1. A few of benzimidazole and imidazoquinoline derivatives with antitumor activities.
Fig. 2. The effect of compound 35 on cancer cells viability (A) Proliferation of HCT116, MCF-7,
SW620, CT26, A549 and LO2 cells treated with various concentrations (0 - 20 ꢀM) of 35 and
Cisplatin for 72 h, respectively. Cell viability was detected by MTT assay. The data are expressed
as the means ± SD from three independent experiments. (B) and (C) The colony clusters were
detected after a 20-day 35 treatment. Quantification is shown in the right panels. Images shown
are representatives of three independent experiments. (**p < 0.01).
Fig. 3. Compound 35 induced G0/G1 cell-cycle arrest and apoptosis of SW620 cells. (A) SW620
cells were treated with various concentrations of 35 (0, 0.3125, 0.625, 1.25, 5.0 ꢀM) for 24 h. (B)
SW620 cells were exposed to 35 at indicated doses for 24 h, and the level of apoptosis was
evaluated using the Annexin V/PI dual-labeling technique, and analyzed by flow cytometry. Data
shown are representative of three independent experiments.
6
6
6
6
6
6
6
72
73
74
75
76
77
78
Fig. 4. Compound 35 inhibited breast cancer cell MCF-7 migration in vitro.
Fig. 5. Effects of compound 35 on the growth of xenografts in nude mice in vivo. (A) HCT116
tumor-bearing nude mice were treated as described with vehicle, 35 at 40 and 80 mg/kg, the mean
tumor volumes ± SD of five mice per every group. (B) After the tumor cell inoculation, the body
weight of the 35 treatment and vehicle groups were counted, and there were no significant
difference among the groups.
6
79
1
7 / 21

ACCEPTED MANUSCRIPT
6
6
6
6
80
81
82
83
Tables
Table 1. In Vitro inhibition of tumor cell growth of compounds 9 - 34.
6
6
84
85
a
IC (ꢀM)
5
0
1
2
Compd.
R
R
U87-MG
>40
PC-3
>40
>40
>40
ꢂ-
HCT116
9
C
C
C
C
C
6
6
6
6
6
H
H
H
H
H
5
5
5
5
5
C
6
H
5
ꢂ-
ꢂ-
ꢂ-
ꢂ-
ꢂ-
1
0
pyridin-3-yl
>40
11
C3-NH
2
-C
6
H
4
>40
1
2
3
2-Me-C
6
H
4
>40
1
4-OH-C
6
H
4
>40
ꢂ-
1
4
C
6
H
5
>40
>40
-
>40
15
16
17
C
C
6
6
H
H
5
5
pyridin-4-yl
38.74 ± 1.64
35.6 ± 2.87
>40
-
quinolin-3-yl
34.05 ± 3.43
>40
31.5 ± 4.89
>40
4
4
4
-CH CN-C6
2
2-Me-C H
6 4
H4
-CH CN-C6
2
1
8
9
>40
>40
>40
>40
-
H4
-CH CN-C6
2
1
3-NH
2
-C
6
H
4
36.51 ± 3.32
H4
1
8 / 21

ACCEPTED MANUSCRIPT
4
4
4
4
4
4
-CH CN-C6
2
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
C
6
H
5
31.42 ± 5.46
29.59 ± 4.30
>40
35.31 ± 2.56
30.59 ± 3.21
>40
>40
H4
-CH CN-C6
2
4-OH-C
H
30.13 ± 4.44
6
4
H4
-CH CN-C6
2
pyridin-3-yl
29.37 ± 5.56
H4
-CH CN-C6 4-(methylsulfo
2
39.72 ± 0.22
36.42 ± 1.45
34.9 ± 2.40
>40
29.61 ± 2.32
37.06 ± 3.50
30.25 ± 3.60
>40
-
H4
nyl)phenyl
-CH CN-C6
2
pyridin-4-yl
37.2 ± 2.10
H4
-CH CN-C6
2
quinolin-3-yl
pyridin-3-yl
pyridin-4-yl
quinolin-3-yl
quinolin-3-yl
pyridin-3-yl
pyridin-4-yl
pyridin-3-yl
pyridin-4-yl
quinolin-3-yl
-
28.40 ± 2.60
H4
4-OMe-C
4-OMe-C
4-OMe-C
H
H
H
>40
6
6
6
4
4
4
>40
>40
>40
>40
>40
>40
2-Me-C
2-Me-C
2-Me-C
H
H
H
>40
>40
>40
6
6
6
4
4
4
>40
>40
>40
>40
>40
>40
4
4
4
-C(CH ) -C
3 2
6
6
6
25.62 ± 3.80
28.55 ± 2.30
33.42 ± 2.50
>20
38.20 ± 2.10
29.01 ± 4.60
19.76 ± 1.60
>20
-
H4
-C(CH ) -C
3
2
-
H4
-C(CH ) -C
3
2
20.40 ± 3.60
12.3
H4
Cisplatin
-
a
6
6
6
6
86
87
88
89
Inhibitory activity was assayed by exposure for 72 h to substances and expressed as
concentration required to inhibit tumor cell proliferation by 50% (IC ). SD, standard deviation (n
50
= 3).
1
9 / 21

ACCEPTED MANUSCRIPT
6
6
90
91
Table 2. In vitro Inhibition of tumor cell growth of compound 35.
6
6
92
93
a
IC (ꢀM)
5
0
2
Compd.
R
U87-MG
>31.0 ± 4.50
>20
PC-3
HCT116
1.40 ± 0.50
12.3
3
5
quinolin-3-yl
-
18.0 ± 2.40
>20
Cisplatin
a
6
6
6
94
95
96
Inhibitory activity was assayed by exposure for 72 h to substances and expressed as
concentration required to inhibit tumor cell proliferation by 50% (IC ). SD, standard deviation (n
50
= 3).
6
6
97
98
Schemes
6
7
99
00
7
7
7
7
01
02
03
04
Scheme 1. Reagents and conditions: (i) acetic anhydride, pyridine, 100 °C, 3.5 h, 92%; (ii) AlCl3,
150 °C, 3.5 h, 72%; (iii) diethyl carbonate, NaH, Toluene, 100 °C, 4 h, 71%; (iv) HNO , DCM,
3
0 °C, 2 h, 94%; (v) POCl , Et N, reflux, 1 h, 87%.
3
3
2
0 / 21

ACCEPTED MANUSCRIPT
7
05
7
7
7
7
7
06
07
08
09
10
Scheme 2. Reagents and conditions: (i) DMF, r.t., 2h, 93-97%; (ii) Fe, ethanol, 75%, NH Cl, r.t.,
4
4-5 h, 52-63 %; (iii) CDI, CH COOH, 2 h, 100 °C, 45-74%; (iv) K CO , PdCl (dppf) CH Cl ,
3
2
3
2
2
2
1,4-dioxane : H O=3:1, 65 °C, 4-5 h, 40-60%.
2
7
7
11
12
7
7
7
7
13
14
15
16
Scheme 3. Reagents and conditions: (i) CH I, Potassium tert-butanolate, DCM, 0 °C, 1 h, 7%; (ii)
3
K CO , [PdCl (dppf)] CH Cl , 1,4-dioxane : H O=3 : 1, 65 °C, 4-5 h, 55%.
2
3
2
2
2
2
2
1 / 21

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
We have found a novel scaffold with better potency as antitumor agents.
Target compound 35 could arrest G0/G1 cell-cycle and induce apoptosis of SW620 cells in a
dose-dependent manner.
3
3
5 blocked MCF-7 cancer cell migration with low toxicity to normal LO2 cells.
5 inhibited tumor growth by 52.96% at 80 mg/kg/48h for 20 days.