Design, synthesis, and evaluation of benzofuran derivatives as novel anti-pancreatic carcinoma agents via interfering the hypoxia environment by targeting HIF-1α pathway

Article abstract of DOI:10.1016/j.ejmech.2017.05.042

Pancreatic ductal adenocarcinoma (PDAC) is one of the most common type of pancreatic cancer, and has still been the medicinal mystery. New drugs and treatment strategies are urgently needed. In this study, 32 benzofuran derivatives are designed, synthesized and evaluated as potential agents against the pancreatic cancer. Among them, compound 9o with the best physicochemical and pharmacokinetic properties exhibited excellent cytotoxicity against many tumor cell lines. In vivo study showed that compound 9o dramatically suppressed the tumor growth of nude mice. Furthermore, compound 9o could affect the hypoxia environment through Hif-1α/VEGF pathway, resulting in the anti-angiogenic activity. These studies indicated that compound 9o was a promising candidate for the treatment of PDAC, deserving further studies.

Full text of DOI:10.1016/j.ejmech.2017.05.042

Accepted Manuscript
Design, synthesis, and evaluation of benzofuran derivatives as novel anti-pancreatic
carcinoma agents via interfering the hypoxia environment by targeting HIF-1α
pathway
Xiao-li Xu, Ying-rui Yang, Xiao-fei Mo, Jin-lian Wei, Xiao-jin Zhang, Qi-dong You
PII:
S0223-5234(17)30403-8
10.1016/j.ejmech.2017.05.042
EJMECH 9472
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 16 March 2017
Revised Date: 16 May 2017
Accepted Date: 19 May 2017
Please cite this article as: X.-l. Xu, Y.-r. Yang, X.-f. Mo, J.-l. Wei, X.-j. Zhang, Q.-d. You, Design,
synthesis, and evaluation of benzofuran derivatives as novel anti-pancreatic carcinoma agents via
interfering the hypoxia environment by targeting HIF-1α pathway, European Journal of Medicinal
Chemistry (2017), doi: 10.1016/j.ejmech.2017.05.042.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Design, synthesis, and evaluation of benzofuran derivatives as
novel anti-pancreatic carcinoma agents via interfering the hypoxia
environment by targeting HIF-1α pathway
Xiao-li Xu a,b, Ying-rui Yang a,b, Xiao-fei Moa,b, Jin-lian Weia,b, Xiao-jin Zhang a,c, Qi-dong
You a,b*
a
State Key Laboratory of Natural Medicines, and Jiangsu Key Laboratory of Drug Design and Optimization, China
Pharmaceutical University, Nanjing 210009, China
b Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, China
c Department of Organic Chemistry, School of Science, China Pharmaceutical University, Nanjing, 210009, China
* Corresponding author. e-mail: youqd@163.com (Q. You)
.
Abstract
Pancreatic ductal adenocarcinoma (PDAC) is one of the most common type of pancreatic
cancer，and has still been the medicinal mystery. New drugs and treatment strategies are
urgently needed. In this study, 32 benzofuran derivatives are designed, synthesized and
evaluated as potential agents against the pancreatic cancer. Among them, compound 9o with
the best physicochemical and pharmacokinetic properties exhibited excellent cytotoxicity
against many tumor cell lines. In vivo study showed that compound 9o dramatically suppressed
the tumor growth of nude mice. Furthermore, compound 9o could affect the hypoxia
environment through Hif-1α/VEGF pathway, resulting in the anti-angiogenic activity. These
studies indicated that compound 9o was a promising candidate for the treatment of PDAC,
deserving further studies.
Key words Pancreatic cancer, Benzofuran derivatives, Anti-tumour activity,
Structure-activity relationships, Apoptosis, HIF-1α pathway, Anti-angiogenesis
1. Introduction
Pancreatic ductal adenocarcinoma (PDAC), as the most common form of pancreatic
cancer (PC), is a kind of digestive-tract malignant tumor with highly invasive and metastatic
features and has become the fourth most common cause of the lethal of malignancies in many
countries[1, 2]. At the present, the 5-year survival rate of PDAC is lower than 5%[3], and it is
estimated that PC will surpass the breast and colorectal cancer to become the second disease
that cause the mortality within a decade in developed countries.[4] However, the management
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of PDAC is still frustrating that mostly due to the poor detection on the early stages of
pancreatic cancer and most patients with PC were only diagnosed at advanced-stage. Besides
survival of PC patients over 5 years is only 15-20% [5], unclear actionable therapeutic targets,
lack of effective drugs and inefficient therapeutic strategies also make PC become a great
health burden for patients and medical researchers [6]. Thus, it is critical to identify novel and
effective therapeutic drugs or strategies against pancreatic adenocarcinoma.
Administration of gemcitabine has been used as the standard therapy for patients with
metastatic pancreatic cancer in the past decades [5, 7]. The investigational drugs are ongoing
early clinical trials in metastatic pancreatic cancer including the cytotoxic agents such as
irinotecan [8], PARP inhibitors such as iniparib [9, 10], Janus kinase inhibitors such as
ruxolitinib [11], agents targeting Kras pathway including Mek or PI3K inhibitors such as
trametinib and everolimus [12-16] and some monoclonal antibodies for immunotherpay and so
on [17] (Fig.1).
Fig.1 Drugs targeting pancreatic cancer cells and title compounds.
Cytotoxic agents with new formulation has been proved to be one of the effective drugs,
holding the promise for targeting pancreatic cancer cells [18, 19]. Recently, both nab-paclitaxel
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plus gemcitabine and FOLFIRINOX (folinic acid, 5-fluorouracil [5‑FU], irinotecan, and
oxaliplatin) have performed better in the treatment of patients with advanced-stage metastatic
pancreatic cancer [20-22]. Classical cytotoxic drugs with novel formulations such as MM-398
with nanoliposomal formulation of irinotecan has a longer duration of action and could
aggregate at the tumor tissue [23]. DNA alkylating agent such as evofosfamide (knows as
TH-320, hypoxia-activated prodrug of a nitrogen mustard derivative) is proved to increase the
potency of gemcitabine alone for the advanced-stage pancreatic cancer (6.0 versus 3.6 months;
P = 0.008) in the randomized trial [24](Fig.1). As a consequence, development of small
molecules that could induce death of pancreatic cells is still urgent for the therapy of pancreatic
cancer.
Although the clear mechanisms of PDAC remained vague and largely unknown, recent
research achievements have shorten the distance between patients to PDAC and made its
pathological mechanism more clear. It has been found that constitutively activation of
oncogenes such as Kras and/or mutations or the loss of tumor suppressor genes (including p53,
p16 and smad4) would trigger the PDAC tumors[25]. A profound role of TP53 is related in
cancer progression including pancreatic cancer [26, 27]. Therefore, molecules or chemotherapy
against p53-independent pancreatic cancer cells could be a better therapeutic approach for
pancreatic cancer. Additionally, the hypoxic tumor microenvironment and hypoxia-induced
factors are critical drivers for metastasis and progression of PDAC [28]. Hypoxia appears in
the microenvironment of many solid tumors, including pancreatic cancer cells with high level
of HIF-1α and VEGF, angiopoietin-1 and so on [29, 30]. Therefore, targeting HIF-1α pathway
of PDAC is an interesting attempt to improve the microenvironment for combating the
resistance to chemotherapy and radiation.
We sought to develop inhibitors that can be used to improve the activity and drug-like
ability against the pancreatic cancer. In our previous studies, we have described that
benzofuran derivatives suppressed p53-independent malignant cancer cells through inhibition
of HIF-1 pathway [31]. Preliminary SAR found that 4-halogen phenylsulfonyl and N
containing alkyl chains contributed significantly to the antiproliferative activities [31]. In
continuation with previous work, we intended to design and synthesize some novel series of
benzofuran derivatives with new scaffolds and to evaluate their cell-based activities,
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biochemical and physicochemical properties as well as in vivo activity against pancreatic
cancer. Compared to previous compounds, new compound with heteroatom-containing group
exhibited better physicochemical and pharmacokinetic properties. This work led to the
identification of 9o as the respective compound with the best cytotoxic activities against tumor
cells. Moreover, the anti-tumor activity is related to anti-angiogenic activities against HIF-1α
pathway. These inhibitors have potential to become drug candidates for the treatment of
pancreatic cancer.
Scheme 1. Reagents and conditions: (a) Ac2O, DCM, r.t., 6 h; (b) AcCl, AlCl3, DCM, reflux,
12 h; (c) BrCH2COOEt, K2CO3, KI, acetone, reflux, 6 h; (d) EtONa, DMF, microwave
o
irradiation (350W), 145 C, 7 min; (e) 3 M HCl, EtOH, reflux, 6 h; (f) pyridine,
4-bromobenzenesulfonyl chloride, DCM, 6 h; (g) p-bromobenzyl chloride, K2CO3, KI,
acetone, reflux, 2 h; (h) NaOH, EtOH, reflux, 30 min; then diluted HCl; (i) EDCI, HOBt,
DIPEA, corresponding amines, r.t., 12 h; (j) CF3COOH, DCM, r.t., 12 h; (k) EDCI, HOBt,
DIPEA, corresponding amines, r.t., 12 h.
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Scheme 2. Reagents and conditions: (a) HSCH2COOC2H5, K2CO3, C2H5OH, r.t., reflux,
overnight; (b) ethyl 2-oxobutanoate, C2H5OH, reflux, 30 min; (c) PPA, dixoane, reflux, 48 h,
(d) ethyl 2-oxoacetate, dioxane, 50 oC, then CAN, reflux, 6 h; (e) H2, Pd/C, THF, r.t., overnight;
(f) 4-bromobenzene-1-sulfonyl chloride, pyridine, TEA, DCM, r.t., overnight; (g)
1-bromo-4-(bromomethyl)benzene, K2CO3, KI, acetone, 4 h; (h) NaOH aqueous, C2H5OH,
reflux, 3 h; (i) corresponding amines, EDCI, HOBT, DCM, r.t, overnight.
2. Results and Discussion
2.1 Chemistry
In previous study, it was found that a series of benzofuran derivatives as new chemotype
exhibited potential inhibitory against p53-independent malignant tumor cells.[31] In this study,
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in order to discover new chemical entity and improve the drug-like properties, a new series
derivatives based on the main scaffold of benofuran were designed and synthesized as outlined
in Scheme 1. In general, introduction of heteroatom-containing group is a good method to
improve the physicochemical property for compounds. We changed the substitutes on the ring
of benzofuran with heteroatom-containing group, including straight flexible side chains, and
heterocyclic side chains.
The derivatives 9a-x were designed and synthesized by utilizing the synthetic route
shown in Scheme 1. First, acylation of p-methoxyaniline produced the amino-protected
intermediate 2. Friedel-crafts acylation and simultaneous demethylation of 2 afforded 3.
Intermediate 4 was prepared using bromoacetic acid ethyl ester and potassium carbonate in a
polar aprotic solvent (acetone). Cyclization of 4 with sodium ethoxide under microwave
irradiation gave intermediate 5 and then compound 6 was obtained by deacylation of 5.
Subsequently, sulfonation of 6 with 4-bromobenzenesulfonyl chloride in the presence of
pyridine produced 7, and then refluxed with p-bromobenzyl in presence of potassium carbonate
and potassium iodide in acetone and followed by hydrolysis reaction to obtain the key
intermediate 8. Substitution of the carboxyl with corresponding amines produced the target
compounds 9a-9x.
At the same time, we attempted to identify the core that could server a main scaffold to
extend the SAR. So the second series of derivatives where the benzofunan ring replaced with
three kinds of benzoheterocyclic core were prepared and described in Scheme 2. Cyclization of
1-(2-chloro-5-nitrophenyl)ethan-1-one and 2-mercaptoacetate ethyl produced 10 with
benzothiophene ring. The Fischer indole synthesis reaction afforded the derivatives with indole
ring. Condensation between p-nitrophenyl hydrazine and ethyl 2-oxobutanoate in refluxing
alcohol gave 16 and then cyclized with dixoane in the presence of PPA. Cyclization of
2-amino-5-nitrophenol with ethyl 2-oxoacetate in the presence of CAN afforded 23. Reduction
of 10, 17 and 23 using Pd/C catalytic hydrogenation gave 11, 18 and 24, and then followed by
sulfonylation using 4-bromobenzene-1-sulfonyl chloride to produce 12, 19 and 25, respectively.
Bromobenzyl group was then introduced in the presence of TEA to give 13, 20 and 26, which
were hydrolyzed to give the key intermediates 14, 21, 27. Condensation of the carboxylic acid
intermediates and corresponding hydrophilic amines produced the target compounds 15a-c,
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22a-c, 28a-c.
2.2 Biological Evaluation
2.2.1 Evaluation of Antiproliferative Activity
In order to evaluate the antiproliferative activity of the synthesized compounds 9a-9x,
15a-c, 22a-c, 28a-c, we chose a panel of pancreatic cancer cells including pancreatic
(PANC-1, BxPC-3) and some other cell lines with high malignancy and prognosis such as
colon (HTC116, p53 -/-), breast (MCF-7), lung (A549), and triple negative breast cancer cells
(MDA-MB-231).
Anti-proliferative
activity
was
measured
by
using
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Doxorubicin
was used as positive control and the results were shown in table 1-2. In general, compounds
with a long side chain such as 9a-9i performed remarkable anti-proliferative activity with IC50
values between 1-10 µM, whereas the length of the side chains had little influence on the
activity. However, the activity of compounds 9d, 9f-9h were much lower than 9a and 9b,
suggesting that physicochemical property of the compounds might in part explain this
behavior. Heterocyclic substituted compounds such as 9i-9o kept the anti-proliferative activity,
and heterocyclic aromatic substituted compounds such as 9p, 9u-9x, showed moderate or poor
activity, indicating that flexible side chains were more suitable for the cytotoxic activity.
SAR analysis was also performed with three scaffolds made on the benzofunan ring.
Exploration of these heterocyles at this position was expected to be a promising approach to
increase the potency. However, replacement by benzothiophene, indole ring and benzoxazole
resulted in a slightly decrease against three cancer cell lines.
Based on the results of Table 2, replacement of benzofunan with benzothiophene, indole
ring and benzoxazole, as in 15a-c, 22a-c, and 28a-c, resulted in slightly lower activity than
those of benzofunan compounds, as in 9b-9c and 9o. Compounds with methylpiperazine
substitution (9o) were highly potent, regardless of whether the scaffold of benzoheterocycle
were benzothiophene (15c), indole ring (22c) and benzoxazole (28c). In summary, although
compounds with benzoheterocycle kept the cytotoxic activity, to some extent, the core of
benzofunan provided superior potency. Moreover, among the series of benzofunan derivatives,
9c and 9o exhibited an outstanding activity against PANC-1, BxPC3, HCT116, HCT116
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(p53-/-), MCF7, A549, MDA-MB-231 in vitro, with IC50 value of 1.07 ± 0.09, 0.65 ± 0.01,
1.81 ± 0.18, 1.61 ± 0.03, 2.39 ± 0.33, 2.68 ± 0.30, 1.90 ± 0.05, and 1.52 ± 0.12, 1.08 ± 0.07,
2.39 ± 0.08, 1.66 ± 0.05, 2.84 ± 0.35, 2.98 ± 0.73, 3.73 ± 0.10, respectively, which were
chosen as representative for the further biological evaluation.
Table 1. Cell proliferation inhibition results of compound 9a-9x.
Anti-proliferation activities IC50 (µM)a
HCT116
(p53-/-)
MDA-MB-
Compd
9a
R
PANC-1
BxPC3
HCT116
MCF7
A549
231
2.49±0.17 5.13±0.12 2.44 ±0.02 2.37±0.48 4.37±0.38 3.08±0.36
5.94±0.16 4.79±0.78 3.64±1.07 1.20±0.13 3.66±0.15 5.40±0.13
1.52±0.12 1.08±0.07 2.39±0.08 1.66±0.05 2.84±0.35 2.98±0.73
7.96±0.75
9b
8.24±0.54
3.73±0.10
9c
9d
6.25±0.18
28.3±4.6
10.4±0.6
3.11±0.68 10.9±0.19 5.50±0.23
9.44±0.45
9e
9f
9g
9h
9i
2.30±0.76 2.02±0.20 3.07±0.26 3.73±0.27 3.71±0.51 4.31±0.91
6.68±0.05
28.6±1.7
>40
24.6±0.6
34.8±6.6
>40
24.2±0.8
>40
11.2±0.1
7.05±1.03
39.7±4.8
21.3±3.4
>40
>40
>40
>40
12.2±0.26 20.4±1.26
>40
>40
>40
>40
29.3±1.19 27.56±1.3
27.8±1.1
11.5±0.2
36.5±2.09 30.5±2.09
16.5±0.69
9.04±0.71
>40
9j
9k
9l
8.38±0.91
>40
19.6±1.2
>40
3.00±0.58 3.30±0.10 9.72±0.33 8.68±0.22
8.55±0.26 5.47±0.18 >40 22.3±0.3
22.1±1.16 26.4±1.43 15.4±0.76 19.1±0.86
23.8±1.27
29.1±2.5
25.1±1.56
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9m
9n
>40
>40
7.88±1.24
21.9±0.8
28.3±1.2
>40
>40
>40
26.22±1.4 16.65±1.4 23.12±1.1
32.78±2.2 33.69±1.7
19.4±0.7
9o
9p
9q
9r
9s
1.07±0.09 0.65±0.01 1.81±0.18 1.61±0.03 2.39±0.33
>40 >40 >40 >40 >40
2.68±0.3
>40
1.90±0.05
>40
2.78±0.31 2.36±0.15 2.67±0.10 2.29±0.35 5.44±0.84 5.29±0.40
6.94±1.43 3.71±0.24 2.23±0.06 2.84±0.33 3.36±0.18 5.17±0.44
32.12±1.6 27.2±1.28 10.5±0.79 13.28±1.1 17.2±0.69 21.37±1.4
8.17±0.15
9.26±0.22
23.5±1.19
7.50±0.33
>40
9t
5.04±0.86
>40
2.6±0.56
>40
3.81±1.12 2.59±0.42 5.66±0.17 5.03±0.07
9u
9v
9x
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
5.59±1.07
19.2±2.3
>40
>40
doxorub
icin
0.41±0.04
0.56±0.06 1.01±0.09 1.23±0.11 0.67±0.08
0.89±0.1
0.49±0.04
a Values shown are the mean ± SD (n = 3).
Table 2. SAR exploration of the R position and the core.
Anti-proliferation activities IC50 (µM)a
Compd
X
S
Y
R
PANC-1
BxPC3
HCT116
C-Me
7.89 ± 0.52
9.0 ± 0.76
9.8 ± 0.86
15a
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S
S
C-Me
C-Me
C-Me
C-Me
C-Me
C-Me
N
7.65 ± 0.53 7.88 ± 0.81 10.9 ± 1.03
15b
15c
22a
22b
22c
28a
28b
28c
9b
5.2 ± 0.78
11.2 ± 0.92
10.5 ± 0.9
3.67 ± 0.18
7.1 ± 0.66
6.98 ± 0.4
10.1 ± 0.87
16.6 ± 1.23
N
N
N
O
O
O
O
O
O
17.4 ± 1.81 15.8 ± 1.72
7.5 ± 0.63
14.5 ± 0.92
13.6 ± 1.09 13.7 ± 0.93 14.3 ± 1.09
12.6 ± 1.10
5.91 ± 0.5
16.3 ± 1.2
14.3 ± 0.78
N
10.16 ± 1.4 7.32 ± 0.66
N
5.94 ± 0.16 4.79 ± 0.78 3.64 ± 1.07
1.52 ± 0.12 1.08 ± 0.07 2.39 ± 0.08
1.07 ± 0.09 0.65 ± 0.01 1.81 ± 0.18
C-Me
C-Me
9c
9o
0.41
0.04
±
Doxorub
icin
0.56 ± 0.06 1.01 ± 0.09
a Values shown are the mean ± SD (n = 3).
2.2.2 Physicochemical Properties and in vivo Pharmacokinetics Study
Parallel to the cytotoxicity evaluation, two representative compounds with better cytotoxic
activity were selected for the evaluation of their potential drug-like properties. Solubility and
permeability as two well-established markers of physicochemical properties were determined
by PION and a standard parallel artificial membrane permeability assay (PAMPA). 9c and 9o
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as the representative of the branched chain substituents exhibited similar properties. As the
results shown in Fig.2A, compound 9o with the moiety of methyl piperazine exhibited the
better favorable Log D7.4 (3.149) and membrane permeability ( 6.54
10-6 cm/s) as well as the
×
aqueous solubility of 30.2 µg/mL. Compound 9o with the best cytotoxicity exhibited balanced
physicochemical properties. Thus, compound 9o was chosen for further in vivo
pharmacokinetic studies in healthy rats. In general, oral and intraperitoneal administration
showed considerable bioavailability, as shown in the Fig. 2B. For intravenous dosing,
compound 9o exhibited acceptable half-life (7.68 h) and clearance rate (1.34 L/h/kg),
suggesting elimination was slow and widely distributed. After the treatment of 9o for 36 h by
intraperitoneal and intravenous administration, the concentration persisted in plasma was more
than 10 µg/L (Fig. 2C), illustrating 9o have possibility to keep higher concentration and longer
retention in the tumor. Taken together, compound 9o have a good pharmacokinetics data
deserving the further study.
Fig.2 (A) Intrinsic solubility, Log D7.4 and permeability were determined. Ketoprofen (4.5
×
10
×
6 cm/s) and propranolol (112.8
×
10-6 cm/s) are internal standards in permeability
determinations. (B) Pharmacokinetic analysis in healthy rats. (C) Plasma concentration vs time
profile of compound 9o in rat.
2.2.3 Compound 9o Induces Apoptosis of Pancreatic Cancer Cell
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To further evaluate the cytotoxicity of compound 9o, we detected whether 9o could induce
the cell death via apoptosis. PANC-1 cells were treated with compound 9o for 24 h and the
apoptotic cells were calculated by double staining of Annexin-V and PI. The results showed
that apoptosis occurred in a dose-dependent manner, after the treatment of compound at 0.1, 1,
2 µM for 24 h, 9%, 45%, 51% apoptotic cells were induced, respectively (Fig.3A, B). In
addition, 9o also dramatically decreased colony formation of PANC-1 in a dose-dependent
manner, suggesting the growth of PANC-1 cells were inhibited due to the apoptosis induced by
9o. Moreover, compound 9o led to the higher level of pro-apoptotic protein including cleaved
Caspase-3, 9 and PARP, and decreased the anti-apoptotic protein such as Caspase-3, 9 and
PARP, compared with control group (Fig.3D, E). Together, compound 9o led to the apoptosis
of PANC-1 cell via a mitochondrial apoptosis pathway.
Fig.3 (A) Treatment of compound 9o for 24 h induced the apoptosis of PANC-1 cell in a
dose-dependent manner. (B) The rate of late apoptotic cell and total apoptosis were quantified
by using flow cytometry. (C) Clonogenicity assay of PANC-1 in response to compound 9o for
two weeks. (D) The expression of apoptotic related proteins in PANC-1 cells treated with
compound 9o for 24 h. GAPDH is used as the control for protein loading. (E) Densitometry
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analysis of the apoptotic related protein levels were shown as normalized (to GAPDH) ratios.
2.2.4 In vivo Anti-tumor Activity
Results of in vivo antitumor evaluation of compounds 9o against mice bearing PANC-1
xenograft tumors were shown in Fig.4A. Doxorubicin was used as a standard agent. Compound
9o was administered intraperitoneally after the tumor were established every two days for 21
days. As shown in Fig. 4C, twice-daily treatment with 15, 30, and 60 mg/kg 9o caused
significant tumor inhibition of the tumor weight by 39.0%, 66.4%, and 75.8%, respectively,
compared with doxorubicin group (71.9%). Also, compound 9o displayed considerable
decrease in tumor volume. The 60 mg/kg dose showed equivalent tumor growth inhibition
compared to the reference doxorubicin (10 mg/kg) (Fig. 4B). Meanwhile, all mice had normal
weights and quality of life during the test. These results suggested that compound 9o could
effectively suppress the tumor development in vivo.
Fig.4 (A) Antitumor efficacy of compound 9o in PANC-1 human pancreatic cancer xenografts.
The PANC-1 exnograft-bearing mice were intraperitoneally injected with 15, 30, 60 mg/kg 9o
for 21 days. (B) Tumor volumes were calculated by measuring the tumor diameters. (C)
Tumors were weighted when the mice were sacrificed after 21 days. **p < 0.01, ***p < 0.001;
Student’s t-test (n = 6).
2.2.5 Compound 9o Shows Anti-Angiogenesis Activity on the PANC-1 Cell Under
Hypoxia
It has been reported that benzofuran derivatives displayed an inhibitory against malignant
cancer cells through inhibition of HIF-1 pathway [31]. So we continued the mechanism studies
of compound 9o with anti-tumor activity based on the pathway of HIF-1α and angiogenesis.
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Treatment of compound 9o inhibited hypoxia-activated HIF-1α-driven expression of luciferase
in a concentration dependent manner by a cell based luciferase reporter assay (Fig.5A). Using
qRT-PCR assay, we identified that exposure of PANC-1 cells to hypoxia for 24 h with the
treatment of compound 9o resulted in the inhibition of VEGF transcriptional activity, which is
a typical target of HIF-1α transcription (Fig.5B). Compound 9o suppressed hypoxia-induced
migration of Human umbilical vein endothelial cells (HUVECs) with the medium from
PANC-1 cells in a time-dependent manner at 1 µM compared to the control group (Fig.5C).
Tube-formation assay displayed that compound 9o could impaired the tube-forming ability of
HUVECs, suggesting that 9o significantly suppressed the invasion activity of HUVECs
(Fig.5D). Then we sought to examine whether HIF-1α was involved in 9o-induced inhibition of
in vivo tumor angiogenesis. CD31 is normally found on endothelial cells, so monitoring CD31
in histological tissue sections is used primarily to evaluate the degree of tumor angiogenesis
[32]. Immunohistochemical study for HIF-1α, CD31 and VEGF in PANC-1 tumors showed
that treatment of 9o caused an inhibitory of tumor angiogenesis with low expression level of
CD31 and VEGF. However, the expression of HIF-1α was not influenced much (Fig.5E).
Altogether, these evidences suggested that compound 9o showed an inhibitory on the function
of HIF-1α transcription, resulting in an anti-angiogenesis activity which in part contributing the
growth inhibitory on pancreatic tumor in vivo.
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Fig.5 (A) Effects of Compound 9o on HIF-1α–driven luciferase expression in HEK293 cells
under hypoxic versus normoxic conditions (48 h). (B) Compound 9o inhibited the ability of
HUVECs on the formation of HUVECs after the treatment for 6 h. (C) Treatment of 9o
inhibited the motility of HUVECs for 6 h in the wound-healing assay. (D) The transcription of
VEGF was inhibited by Compound 9o in a dose-dependent way. (E) The expression of CD31,
HIF-1α, VEGF in tumor microenvironment were examined by immunochemistry using
corresponding antibodies.
3. Conclusion
In conclusion, a series of benzofuran derivatives as anti-tumor agents were designed,
synthesized and evaluated for their druggability and biological activity. All compounds were
assayed by MTT assay against many cancer cell lines. The preliminary SAR analysis suggested
that introduction of hydrophilic heteroatom-containing groups on the ring exhibited prominent
antiproliferative activity, and replacement of the main scaffold basically kept the activity,
especially benzofuran performed better. Finally, Compound 9o with N-methylpiperazine group
showed the most potent cytotoxicity as well as the balanced physicochemical property,
illustrating the feasibility of inhibitor with heteroatom-containing groups. The flow-cytometry
15

ACCEPTED MANUSCRIPT
revealed that treatment of 9o induced the apoptosis of PANC-1 cells. WB analysis further
confirmed that significant apoptotic proteins appeared after the treatment of 9o, indicating the
caspase-dependent way was triggered. In addition, it was found that 9o inhibited the function
of HIF-1α as transcription factor for VEGF. The wound and tube formation assay of HUVECs
and histopathological examination provided insights into the evidence that the hypoxia
environment of tumor was interfered by 9o, laying the foundation for targeting the pancreatic
cancer by interfering the hypoxic microenvironment through HIF-1/VEGF pathway. Overall,
9o as the representative compound indicated that the series of benzofuran derivatives could be
further studied for the therapeutic treatment of pancreatic cancer.
4. Experimental section
4.1 Chemistry
4.1.1 General materials and methods
All material and reagents were purchased from commercial sources and, unless otherwise
stated, were used without further purification. Concentration of solutions from reactions and
extractions in a rotary evaporator (Büchi Rotavapor) below 45°C at reduced pressure.
Reactions were monitored by thin-layer chromatography (TLC) using Merck Kieselgel 60
F254 plates and visualized under UV light at 254 nm. Flash column chromatography was
performed on silica gel (60e120 mesh size).Melting points were measured with a Melt-Temp II
apparatus and were uncorrected. IR spectra were recorded by using a Nicolet iS10 Avatar
1
13
FT-IR spectrometer with KBr film. The H NMR and C NMR spectra were recorded on a
Bruker AV-300 instrument. Chemical shift (d) are in ppm, referenced to tetramethylsilane
(TMS). Multiplicities were abbreviated as s (singlet), d (doublet), t (triplet) or m (multiplet),
broad (br) or some combination of them. Coupling constants (J) are recorded in hertz and
rounded to 0.5 Hz. High resolution mass spectra (HRMS) were recorded on a Water Q-Tof
micro mass spectrometer. The purity (≥ 95%) of the compounds is verified by HPLC.
4.1.2 N-(4-methoxyphenyl)acetamide (2)
p-Methoxyaniline (12.3 g, 0.1 mol) was dissolved in 120 mL of CH2Cl2. Acetic anhydride
(10.2 g, 9.44 mL, 0.1 mol) was added dropwise to this solution in an ice/water bath for 6 h at
room temperature. The reaction mixture was quenched and filtered by petroleum ether. The
16

ACCEPTED MANUSCRIPT
product is white power (14.4 g, 87.3% yield). m.p. 127-129 °C.
4.1.3 N-(3-acetyl-4-hydroxyphenyl)acetamide (3)
Intermediate 2 (4.2 g, 0.025 mol) was dissolved in 120 mL of CH2Cl2. AlCl3 (13.3 g, 0.1 mol)
and acetylchloride (7.9 g, 7.4 mL, 0.1 mol) were added slowly to this solution in an ice/water
bath. The reaction mixture was then heated to reflux for 12 h. Then the reaction were quenched
by ice water, and the supernatant was abandon. Then flake ice and 1 N HCl/ice water were
added to the mixture for 30 mins with stir. The reaction mixture was concentrated under
reduced pressure and the residue was purified via column chromatography to give the
compound 3 as yellow-green solid (4.13 g, 84.1% yield). m.p. 162-164 oC. 1H NMR (300 MHz,
DMSO-d6) δ 11.55 (s, 1H), 9.93 (s, 1H), 8.06 (d, J = 2.58 Hz, 1H), 7.65 (dd, J = 2.62, 8.90 Hz,
1H), 6.91 (d, J = 8.90 Hz, 1H), 2.58 (s, 3H), 2.01 (s, 3H). HRMS(ESI): calcd for C10H12NO3
[M+H]+ 194.0812, found 194.0815.
4.1.4 Ethyl 2-(4-acetamido-2-acetylphenoxy)acetate (4)
Compound 3 (3.8 g, 19.7 mmol) was dissolved in 120 mL of acetone. Ethyl bromoacetate (4.35
mL，39.3 mmol), K2CO3 (5.44 g, 39.3 mmol) and KI (0.635 g, 3.93 mmol) were added to this
solution. Heat the reaction mixture to reflux for 6 h. The reaction mixture was concentrated
under reduced pressure and the residue was purified via column chromatography to give the
1
compound 4 as white solid (5.0 g, 91.2% yield). m.p. 165-166 °C . H NMR (300 MHz,
DMSO-d6) δ 9.95 (s, 1H), 7.80 – 7.66 (m, 2H), 7.05 (d, J = 8.95 Hz, 1H), 4.89 (s, 2H), 4.17 (q,
J = 7.10 Hz, 2H), 2.60 (s, 3H), 2.00 (s, 3H), 1.21 (t, J = 7.11 Hz, 4H). HRMS(ESI): calcd. for
C14H18NO5 [M+H]+ 280.1179, found 280.1175.
4.1.5 Ethyl 5-acetamido-3-methylbenzofuran-2-carboxylate (5)
Add compound 4 (2.0 g, 7.16 mmol) and K2CO3 (1.4 g, 10.74 mmol) to microwave tube with
10 ml DMF. The reaction was carried out at 145 oC for 7 min under microwave irridiation. The
reaction mixture was concentrated under reduced pressure and the residue was purified via
column chromatography to give the compound 5 as white solid (1.1 g, 58.8% yield). m.p.
159-161 °C. 1H NMR (300 MHz, DMSO-d6) δ 10.10 (s, 1H), 8.11 (d, J = 1.57 Hz, 1H), 7.65 –
7.46 (m, 2H), 4.34 (q, J = 7.09 Hz, 2H), 2.50 (s, 3H), 2.06 (s, 3H), 1.33 (t, J = 7.10 Hz, 3H).
17

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HRMS(ESI): calcd. for C14H16NO4 [M+H]+ 262.1074; found 262.1072.
4.1.6 Ethyl 5-amino-3-methylbenzofuran-2-carboxylate (6)
Compound 5 (1.2 g, 4.59 mmol) was dissolved in the mixture solution of 3 M HCl and ethanol.
Heat the reaction to reflux with the protection of N2 for 6 h. The reaction mixture was
concentrated under reduced pressure and the residue was purified via column chromatography
to give the compound 6 as yellow solid (0.74 g, 63.0 % yield). m.p. 218-220 °C. 1H NMR (300
MHz, DMSO-d6) δ 10.56 (brs, 2H), 7.84 – 7.71 (m, 2H), 7.56 – 7.44 (m, 1H), 4.35 (q, J = 7.09
Hz, 2H), 2.53 (s, 3H), 1.34 (t, J = 7.10 Hz, 3H). HRMS(ESI): calcd for C12H14NO3 [M+H]+
220.0968, found 220.0964.
4.1.7 Ethyl 5-(4-bromophenylsulfonamido)-3-methylbenzofuran-2-carboxylate (7)
Compound 6 (1.1 g, 4.30 mmol), p-bromobenzenesulfonyl chloride (1.36 g, 6.45 mmol)
pyridine (1.7 g, 1.74 ml, 21.5 mmol) were added to the anhydrous dichloromethane. The
reaction mixture was stirred for 12 h at room temperature, and monitored by TLC, and washed
by diluted hydrochloric acid and saturated brines. The reaction were dried over anhydrous
Na2SO4, concentrated under reduced pressure and the residue was purified via column
chromatography to give the compound 7 as yellow solid (1.54 g, 81.7% yield). m.p. 167-169
1
oC. H NMR (300 MHz, DMSO-d6): δ 10.39 (s, 1H), 7.80 – 7.69 (m, 2H, Ar-H), 7.68 – 7.52
(m, 3H), 7.47 – 7.39 (m, 1H), 7.17 (dd, J = 2.23, 8.89 Hz, 1H), 4.33 (q, J = 7.10 Hz, 2H, ), 2.46
13
(s, 3H), 1.32 (t, J = 7.10 Hz, 3H). C NMR (75 MHz, CDCl3): δ 159.30, 150.95, 141.21,
138.44, 133.03, 132.31, 128.87, 128.68, 126.75, 124.91, 122.74, 113.65, 112.70, 60.84, 14.08,
8.98; HRMS(ESI): calcd. for C18H17BrNO5S [M+H]+ 438.0005; found 437.9998;. IR ( KBr,
cm-1):3415, 3237, 2923, 2847, 2310, 1714, 1581, 1467, 1456, 1401, 1337, 1308,
1159,1136,1087,1009,821,748,539; HPLC (75% methanol in water), tR = 4.5 min, 96.8%.
4.1.8
Ethyl-5-(4-bromo-N-(4-bromobenzyl)phenylsulfonamido)-3-methylbenzofuran-2-carboxylate
(8)
To a stirring solution of compound 7 (282 mg, 0.64 mmol) in acetone (10 mL) was added
bromobenzyl bromide (241 mg, 0.97 mmol), K2CO3 (266 mg, 1.9 mmol) and KI (100 mg, 0.06
mmol). The reaction mixture was heated to reflux for 2 h, and then filtered. The filtration was
concentrated under reduced pressure. The residue was added to a mixture of NaOH/EtOH
(v:v=1:1, 20 mL). Heat it to reflux and regulate pH to 4-5 with diluted HCl, the solid appeared
18

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was filtered and purified via column chromatography to give compound 8 (1.5 g, 84.6% yield).
m.p. 191-193 oC.1H NMR (300 MHz, DMSO-d6): δ 7.82 (d, J = 8.57 Hz, 2H), 7.60 – 7.38 (m,
6H), 7.22 (d, J = 8.37 Hz, 2H), 7.13 (dd, J = 2.09, 8.85 Hz, 1H), 4.81 (s, 2H), 2.40 (s, 3H). 13C
NMR (75 MHz, DMSO-d6): δ 160.72, 152.32, 142.39, 136.51, 135.44, 133.72, 132.43, 131.28,
130.40, 129.50, 128.98, 128.37, 127.34, 124.26, 121.77, 120.65, 112.25, 53.23, 8.98. HRMS
+
(ESI): calcd for C23H18Br2NO5S [M+H] 577.9267; found 577.9268; IR (KBr, cm-1):3376,
3111, 3095, 1718, 1604, 1573, 1469, 1336, 1163, 1148, 1016, 1007, 879, 818, 762, 654; HPLC
(0.1% TFA in methanol) : tR = 3.4 min, 94.9%.
4.1.9 Tert-butyl
(2-(5-((4-bromo-N-(4-bromobenzyl)phenyl)sulfonamido)-3-methylbenzofuran-2-carboxamido)
ethyl)(methyl)carbamate (8a)
To a stirring solution of compound 8 (140 mg, 0.24 mmol) in DCM (10 mL) was added EDCI
( 91 mg, 0.47 mmol), HOBt (64 mg, 0.47 mmol) and N,N-Dimethylethylenediamine (42 mg,
0.47 mmol) with a drop of DIPEA. The reaction mixture was stirred for 12 h at room
temperature, then washed with diluted HCl and saturated brines. The organic layer were
combined and dried over anhydrous Na2SO4, concentrated in vacuo, and then purified by flash
chromatography to give compound 8a as solid. (87 mg, 52.3% yield).
4.1.10 5-(4-bromo-N-(4-bromobenzyl)phenylsulfonamido)-3-methyl-N-(2
(methylamino)ethyl)benzofuran-2-carboxamide (9a)
To a stirring solution of compound 8a (93 mg, 0.13 mmol) in DCM (10 mL) was added 1 mL
of trifluoroacetic (1 mL). Then heated the reaction to reflux for 12 h. The reaction mixture was
washed with saturated NaHCO3 and extracted with CH2Cl2, dried over anhydrous Na2SO4,
concentrated in vacuo, and then purified by flash chromatography to give 9a as yellow solid
(58 mg, 72.2% yield). m.p. 80-83 oC. 1H NMR (300 MHz, DMSO-d6) δ 8.40 (s, 1H), 7.84 (d, J
= 8.08 Hz, 2H), 7.55 (d, J = 8.22 Hz, 2H), 7.51 – 7.36 (m, 4H), 7.23 (d, J = 8.09 Hz, 2H), 7.10
(d, J = 8.77 Hz, 1H), 4.82 (s, 2H), 2.61 (t, J = 6.31 Hz, 2H), 2.38 (s, 3H), 2.27 (s, 3H). 13C
NMR (75 MHz, DMSO-d6) δ 158.97, 151.39, 135.48, 133.63, 132.40, 131.26, 130.40, 129.47,
127.33, 120.64, 120.34, 111.85, 53.33, 50.45, 38.05, 35.72, 8.48. HRMS (ESI): calcd for
C26H26Br2N3O4S [M + H]+ 634.0005, found 634.0011. IR (KBr, cm-1): 3414, 2920, 2849, 1654,
1612, 1573, 1522, 1487, 1467, 1388, 1351, 1280, 1164, 1089, 1068, 1009, 857, 821, 747, 734,
649; HPLC: (0.1% Et3N and 90% methanol in water), tR =5.6 min, 95.3 %.
General procedure for preparation of 9b-9h, 9k-9x
To a stirring solution of compound 8 (200 mg, 0.35 mmol) in DCM (10 mL) was added EDCI
19

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(91 mg, 0.47 mmol), HOBt (64 mg, 0.47 mmol) and the corresponding diamine derivatives
(0.35 mmol) with a drop of DIPEA. The reaction mixture was stirred for 12 h at room
temperature, then washed with diluted HCl and saturated brines. The organic layer were
combined and dried over anhydrous Na2SO4, concentrated in vacuo, and then purified by flash
chromatography to give product 9b-9h, 9k-9v, respectively.
4.1.11
5-(4-bromo-N-(4-bromobenzyl)phenylsulfonamido)-N-(2-(dimethylamino)ethyl)-3-methylbenzo
furan-2-carboxamide (9b)
(87 mg, 63.3% yield). m.p. 89-91 oC. 1H NMR (300 MHz, CDCl3) δ 7.63 (d, J = 8.22 Hz, 2H),
7.49 (d, J = 8.31 Hz, 2H), 7.40 – 7.25 (m, 3H), 7.20 – 7.05 (m, 4H), 6.89 (d, J = 8.62 Hz, 1H),
4.71 (s, 2H), 3.52 (q, J = 5.37 Hz, 2H), 2.56 – 2.50 (m, 2H), 2.49 (s, 3H), 2.29 (s, 6H). 13C
NMR (75 MHz, DMSO) δ 159.35, 151.88, 144.65, 136.98, 135.97, 134.15, 132.90, 131.76,
130.89, 129.97, 128.26, 127.83, 121.72, 121.13, 120.94, 112.37, 58.39, 53.80, 45.60, 36.90,
8.98.HRMS (ESI): calcd for C27H28Br2N3O4S [M + H]+ 648.0162, found 648.0157. IR (KBr,
cm-1): 3413, 2942, 2856, 2820, 2772, 2359, 2342, 1649, 1600, 1575, 1522, 1466, 1388, 1352,
1263, 1164, 1068, 1009, 855, 819, 649, 603, 550. HPLC:( 0.1% Et3N and 90% methanol in
water), tR = 6.0 min, 94.8 %
4.1.12
5-(4-bromo-N-(4-bromobenzyl)phenylsulfonamido)-N-(2-(diethylamino)ethyl)-3-methylbenzofu
ran-2-carboxamide (9c)
o
1
(108 mg, 57.7% yield). m.p. 105-107 C. H NMR (300 MHz, CDCl3) δ 7.69- 7.48 (m, 4H),
7.41 -7.28 (m, 3H), 7.22 – 7.06 (m, 4H), 6.90 (d, J = 8.88 Hz, 1H), 4.72 (s, 2H), 3.56 – 3.43 (m,
2H), 2.70 – 2.54 (m, 6H), 2.51 (s, 3H), 1.06 (t, J = 7.07 Hz, 6H). HRMS (ESI): calcd for
C29H32Br2N3O4S [M + H]+ 676.0475, found 676.0462. IR (KBr, cm-1): 3413, 2967, 2809, 2361,
1647, 1617, 1575, 1521, 1466, 1389, 1351, 1280, 1164, 1069, 1010, 819, 647, 604, 551.
HPLC:( 0.1% Et3N and 90% methanol in water), tR =7.8min, 96.0 %.
4.1.13
5-((4-bromo-N-(4-bromobenzyl)phenyl)sulfonamido)-N-(2-(diisopropylamino)ethyl)-3-methylb
enzofuran-2-carboxamide (9d)
o
1
(94 mg, 61.9% yield). m.p.129-131 C. H NMR (300 MHz, CDCl3) δ 7.71 - 7.46 (m, 4H),
7.37 (d, J = 8.45 Hz, 3H), 7.21- 7.06 (m, 3H), 6.89 (dd, J = 8.79, 2.18 Hz, 1H), 4.72 (s, 2H),
20

ACCEPTED MANUSCRIPT
3.50 – 3.34 (m, 2H), 3.13 – 3.02 (m, 2H), 2.69 (t, J = 5.89 Hz, 2H), 2.51 (s, 3H), 1.06 (d, J =
6.55 Hz, 12H). HRMS (ESI): calcd for C31H36Br2N3O4S [M + H]+ 704.0788, found 704.0790.
IR (KBr, cm-1):75, 2963, 2922, 2849, 1661, 1608, 1573, 1505, 1486, 1467, 1386, 1352, 1284,
1171, 1122, 1087, 1068, 1054, 1008, 930, 871, 836, 750, 703, 651. HPLC :( 0.1% Et3N and 90%
methanol in water), tR =12.7 min, 97.3 %.
4.1.14
5-(4-bromo-N-(4-bromobenzyl)phenylsulfonamido)-N-(3-(dimethylamino)propyl)-3-methylbenz
ofuran-2-carboxamide (9e)
o
1
(134 mg, 67.1% yield). m.p.125-127 C. H NMR (300 MHz, CDCl3) δ 8.15 (t, J = 5.05 Hz,
1H), 7.69 -– 7.45 (m, 4H), 7.42-7.32 (m, 2H), 7.20-7.06 (m, 3H), 6.89 (dd, J = 8.74, 2.18 Hz,
1H), 4.72 (s, 2H), 3.54 (q, J = 5.86 Hz, 2H), 2.50 (s, 3H), 2.49-2.43 (m, 2H), 2.30 (s, 6H),
1.85-1.70 (m, 3H). HRMS (ESI): calcd for C28H30Br2N3O4S [M + H]+ 662.0318, found
662.0327. IR (KBr, cm-1): 3415, 2942, 2858, 2817, 2770, 1655, 1611, 1572, 1522, 1466, 1405,
1388, 1352, 1263, 1164, 1089, 1068, 1009, 856, 822, 749, 648, 603. HPLC:( 0.1% Et3N and 90%
methanol in water), tR =6.4 min, 95.4 %.
4.1.15 3-(dimethylamino)propyl 5-((4-bromo-N-(4-bromobenzyl)phenyl)sulfonamido)-3-
methylbenzofuran-2-carboxylate (9f)
(112 mg, 62.4% yield). m.p.167-169 oC. 1H NMR (300 MHz, DMSO-d6) δ 7.83 (d, J = 8.37 Hz,
2H), 7.62 – 7.47 (m, 4H), 7.43 (d, J = 8.08 Hz, 2H), 7.27 – 7.07 (m, 3H), 4.82 (s, 2H), 4.31 (t,
J = 6.45 Hz, 2H), 2.42 (s, 3H), 2.31 (t, J = 7.02 Hz, 2H), 2.12 (s, 6H), 1.88 – 1.77 (m, 2H). 13C
NMR (75 MHz, DMSO-d6) δ 159.27, 152.46, 141.42,136.49, 135.40, 133.89, 132.43, 131.27,
130.40, 129.48, 128.77, 128.72, 127.36, 125.04, 121.90, 120.66, 112.35, 63.27, 55.43, 53.21,
45.07, 26.21, 8.98. HRMS (ESI): calcd for C28H29Br2N2O5S [M + H]+ 663.0158, found
663.0167. IR(KBr, cm-1)：3416, 3101, 2924, 2855, 2818, 2762, 2367, 2334, 1723, 1575, 1464,
1397, 1349, 1315, 1284, 1163, 1144, 1087, 1068, 1011, 861, 751, 649, 602. HPLC:( 0.1% Et3N
and 90% methanol in water), tR =7.9 min, 96.3 %.
4.1.16
N-(2-acetamidoethyl)-5-(4-bromo-N-(4-bromobenzyl)phenylsulfonamido)-3-methylbenzofuran-
2-carboxamide (9g)
o
1
(98 mg, 68.1% yield). m.p. 187-190 C. H NMR (300 MHz, CDCl3) δ 7.63 (d, J = 8.03 Hz,
2H), 7.49 (d, J = 8.14 Hz, 2H), 7.39 – 7.20 (m, 4H), 7.09 (d, J = 8.12 Hz, 2H), 6.88 (d, J = 8.04
21

ACCEPTED MANUSCRIPT
Hz, 1H), 6.30 (s, 1H), 4.70 (s, 2H), 3.84 – 3.27 (m, 4H), 2.49 (s, 3H), 1.98 (s, 3H). 13C NMR
(75 MHz, CDCl3) δ 170.72, 160.37, 151.86, 143.19, 136.80, 134.00, 133.28, 131.80, 131.19,
130.41, 129.86, 129.78, 128.72, 127.57, 126.99, 122.04, 121.71, 121.47, 111.76, 54.39, 39.71,
38.98, 22.70, 8.37. HRMS (ESI): calcd for C27H26Br2N3O5S [M + H]+ 661.9954, found
661.9946. IR (KBr, cm-1):3413, 3318, 2365, 2342, 1646, 1617, 1535, 1487, 1466, 1389, 1346,
1284, 1157, 1089, 1069, 1010, 857, 600, 543. HPLC: (0.1% Et3N and 90% methanol in water),
tR =4.9min, 94.9 %.
4.1.17
5-((4-bromo-N-(4-bromobenzyl)phenyl)sulfonamido)-3-methyl-N-(2-(methylsulfonyl)ethyl)benz
ofuran-2-carboxamide (9h)
o
1
(116 mg, yield, 49.1%). mp:182-184 C. H NMR (300 MHz, CDCl3) δ 7.70 – 7.45 (m, 4H),
7.41 – 7.30 (m, 3H), 7.23 (d, J = 8.23 Hz, 1H), 7.10 (d, J = 8.50 Hz, 2H), 6.89 (dd, J = 8.83,
2.16 Hz, 1H), 4.72 (s, 2H), 4.00 (q, J = 6.14 Hz, 2H), 3.42 – 3.31 (m, 2H), 3.00 (s, 3H), 2.51 (s,
3H). HRMS (ESI): calcd for C26H24Br2N2NaO6S [M + Na]+ 704.9335, found 704.9340. IR
(KBr, cm-1):3412, 2920, 2849, 2363, 2342, 1654, 1615, 1573, 1521, 1487, 1466, 1388, 1352,
1294, 1163, 1131, 1089, 1068, 1009, 858, 749, 735, 603. HPLC: (0.1% Et3N and 90%
methanol in water), tR =4.6 min, 95.9 %.
4.1.18
5-((4-bromo-N-(4-bromobenzyl)phenyl)sulfonamido)-N-(2,3-dihydroxypropyl)-3-methylbenzofu
ran-2-carboxamide (9i)
Compound 8 (300 mg, 0.52 mmol) was dissolved in 7 mL of SOCl2 and refluxed for 1 h. Then
o
add the anhydrous acetone (10 mL) to the mixture for cooling to -10 C. The pre-cooling
mixture of 3-amino-1,2-propanediol (188 mg, 1.55 mmol) and water (5 mL H2O) was added to
o
the reaction for 30 min at -10 C. Acetone was removed by reduced pressure distillation. The
reaction mixture was extracted with EA/water, and the organic extracts were combined,
purified by flash chromatography to afford white solid (100 mg, 24.4% yield). mp:117-120 oC.
1H NMR (300 MHz, CDCl3) δ 7.70 – 7.60 (m, 2H), 7.55 – 7.46 (m, 2H), 7.41 – 7.32 (m, 2H),
7.20 (d, J = 1.84 Hz, 1H), 7.10 (d, J = 8.48 Hz, 2H), 6.93 (dd, J = 8.79, 2.20 Hz, 1H), 4.72 (s,
2H), 3.69 – 3.53 (m, 4H), 2.51 (s, 3H). HRMS (ESI): calcd for C26H25Br2N2O6S [M + H]+
650.9795, found 650.979791. IR (KBr, cm-1): 3414, 3088, 2921, 2360, 2342, 1653, 1609, 1573,
1527, 1487, 1466, 1388, 1351, 1279, 1195, 1163, 1110, 1089, 1069, 1009, 917, 857, 822, 748,
649, 602. HPLC: (0.1% Et3N and 90% methanol in water), tR =4.6 min, 95.0 %.
22

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4.1.19 Tert-butyl
4-(5-((4-bromo-N-(4-bromobenzyl)phenyl)sulfonamido)-3-methylbenzofuran-2-carbonyl)piper
azine-1-carboxylate (8b)
The compound was prepared by a procedure identical to the preparation of 8a.
4.1.20
4-bromo-N-(4-bromobenzyl)-N-(3-methyl-2-(piperazine-1-carbonyl)benzofuran-5-yl)benzenesu
lfonamide (9j)
The compound 9j was prepared by a procedure identical to the preparation of 9a (143 mg,
o
64.0 % yield over three steps). m.p. 95-97 C. 1H NMR (300 MHz, DMSO-d6) δ 7.84 (d, J =
8.70 Hz, 2H), 7.61 – 7.43 (m, 4H), 7.43 – 7.35 (m, 2H), 7.23 (d, J = 8.48 Hz, 2H), 7.04 (dd, J =
8.84, 2.14 Hz, 1H), 4.81 (s, 2H), 3.48 – 3.40 (m, 4H), 2.77 – 2.61 (m, 4H), 2.19 (s, 3H).HRMS
(ESI): calcd for C27H26Br2N3O4S [M + H]+ 646.0005, found 646.0006. IR (KBr, cm-1)：3415,
3086, 2917, 2850, 2359, 2338, 1629, 1573, 1458, 1388, 1350, 1267, 1164, 1109, 1089, 1068,
1009, 920, 856, 820, 797, 735, 703, 642, 600. HPLC: (0.1% Et3N and 90% methanol in water),
tR =5.0 min, 94.9 %.
4.1.21
4-bromo-N-(4-bromobenzyl)-N-(3-methyl-2-(4-methylpiperazine-1-carbonyl)benzofuran-5-yl)b
enzenesulfonamide (9k)
The compound 9k was prepared by a procedure identical to the preparation of 9a (97 mg, 42.5%
o
1
yield). m.p.119-121 C. H NMR (300 MHz, CDCl3) δ 7.70 – 7.46 (m, 4H), 7.36 (d, J = 7.21
Hz, 2H), 7.29 – 7.23 (m, 1H), 7.20 – 7.05 (m, 3H), 6.86 (d, J = 8.57 Hz, 1H), 4.71 (s, 2H), 3.97
– 3.48 (m, 4H), 2.48 (s, 4H), 2.34 (s, 3H), 2.31 (s, 3H). HRMS (ESI): calcd for
C28H28Br2N3O4S [M + H]+ 660.0162, found 660.017. IR (KBr, cm-1): 3415, 2928, 2789, 2360,
2341, 1636, 1574, 1463, 1350, 1295, 1262, 1163, 1088, 1069, 1007, 924, 881, 860, 824, 757,
738, 639. HPLC: ( 0.1% Et3N and 90% methanol in water), tR =5.7 min, 96.0 %.
4.1.22
4-bromo-N-(4-bromobenzyl)-N-(3-methyl-2-(morpholine-4-carbonyl)benzofuran-5-yl)benzenes
ulfonamide (9l)
The compound 9l was prepared by a procedure identical to the preparation of 9a (158 mg, 70.6%
23

ACCEPTED MANUSCRIPT
yield). m.p. 92-94 oC. 1H NMR (300 MHz, CDCl3) δ 7.69 – 7.46 (m, 4H), 7.41 – 7.28 (m, 3H),
7.20 – 7.03 (m, 3H), 6.87 (dd, J = 8.78, 2.17 Hz, 1H), 4.72 (s, 2H), 4.03 – 3.54 (m, 8H), 2.34 (s,
3H).HRMS (ESI): calcd for C27H25Br2N2O5S [M + H]+ 646.9845, found 646.9836. IR (KBr,
cm-1)： 3420, 3087, 2961, 2919, 2853, 2359, 2338, 1635, 1573, 1487, 1456, 1436, 1388, 1352,
1277, 1256, 1164, 1115, 1089, 1068, 1009, 855, 822, 752, 736, 643, 601. HPLC: (0.1% Et3N
and 90% methanol in water), tR =5.6 min, 95.1 %.
4.1.23
4-bromo-N-(4-bromobenzyl)-N-(3-methyl-2-(3-oxopiperazine-1-carbonyl)benzofuran-5-yl)benz
enesulfonamide (9m)
o
1
(107 mg, 46.9% yield). m.p. 207-210 C. H NMR (300 MHz, CDCl3) δ 7.71 – 7.45 (m, 4H),
7.36 (d, J = 6.92 Hz, 2H), 7.20 (s, 1H), 7.10 (d, J = 7.12 Hz, 2H), 6.96 – 6.68 (m, 2H), 4.71 (s,
2H), 4.42 (s, 2H), 3.95 (brs, 2H), 3.52 (brs, 2H), 2.37 (s, 3H). HRMS (ESI): calcd for
C27H24Br2N3O5S [M + H]+ 659.9798, found 659.9786. IR (KBr, cm-1)：3414, 2922, 2363, 2342,
1679, 1662, 1628, 1573, 1487, 1458, 1388, 1352, 1261, 1226, 1193, 1163, 1110, 1089, 1068,
1009, 857, 822, 752, 737, 640, 551. HPLC: (0.1% Et3N and 90% methanol in water), tR =4.6
min, 94.6 %.
4.1.24
4-bromo-N-(4-bromobenzyl)-N-(3-methyl-2-(3-methylpiperidine-1-carbonyl)benzofuran-5-yl)b
enzenesulfonamide (9n)
o
1
(151 mg, 66.2% yield). m.p. 160-161 C. H NMR (300 MHz, CDCl3) δ 7.70 – 7.46 (m, 4H),
7.42 – 7.28 (m, 3H), 7.19 – 7.06 (m, 3H), 6.84 (dd, J = 8.75, 2.17 Hz, 1H), 4.72 (s, 2H), 4.54 –
4.37 (m, 1H), 4.13 – 3.62 (m, 1H), 3.27 – 2.48 (m, 2H), 2.28 (s, 3H), 1.96 – 1.51 (m,5H), 1.38
– 1.28 (m, 2H). HRMS (ESI): calcd for C29H29Br2N2O4S [M + H]+ 659.0209, found 659.0202.
IR (KBr, cm-1): 3415, 2955, 2930, 2851, 1625, 1589, 1488, 1469, 1440, 1388, 1352, 1277,
1165, 1121, 1087, 1069, 1015, 1007, 920, 877, 823, 754, 604, 551, 472. HPLC:( 0.1% Et3N
and 90% methanol in water), tR =8.7 min ,94.3 %.
4.1.25
5-(4-bromo-N-(4-bromobenzyl)phenylsulfonamido)-3-methyl-N-(1-methylpiperidin-4-yl)benzof
uran-2-carboxamide (9o)
o
1
(118 mg, 50.6% yield). m.p. 195-197 C. H NMR (300 MHz, CDCl3) δ 7.68 – 7.45 (m, 4H),
7.41 – 7.29 (m, 3H), 7.18 (d, J = 2.06 Hz, 1H), 7.10 (d, J = 8.36 Hz, 2H), 6.91 (dd, J = 8.77,
2.17 Hz, 1H), 6.43 (d, J = 8.09 Hz, 1H), 4.72 (s, 2H), 4.07 – 3.84 (m, 1H), 2.92 – 2.76 (m, 2H),
2.50 (s, 3H), 2.32 (s, 3H), 2.16 (t, J = 11.50 Hz, 2H), 2.08 – 1.95 (m, 2H), 1.70 – 1.51 (m, 2H).
24

ACCEPTED MANUSCRIPT
13C NMR (75 MHz, CDCl3) δ 159.23, 152.19, 143.63, 137.11, 134.49, 133.71, 132.28, 131.63,
130.35, 130.25, 129.19, 128.07, 127.46, 122.46, 122.11, 121.89, 112.21, 54.85, 54.17, 45.10,
44.85, 30.91, 8.86.HRMS (ESI): calcd for C29H30Br2N3O4S [M + H]+ 674.0318, found
674.0329. IR (KBr, cm-1)：3338, 2925, 2852, 2778, 1643, 1615, 1575, 1525, 1487, 1466, 1389,
1377, 1344, 1344, 1285, 1200, 1157, 1110, 1089, 1069, 1011, 857, 843, 735, 649, 605. HPLC:
( 0.1% Et3N and 90% methanol in water), tR =6.6 min, 96.9 %.
4.1.26
5-(4-bromo-N-(4-bromobenzyl)phenylsulfonamido)-3-methyl-N-(5-methylpyridin-2-yl)benzofur
an-2-carboxamide (9p)
o
(157mg, 67.9% yield). m.p. 148-150 C. 1H NMR (300 MHz, DMSO-d6) δ 10.08 (s, 1H), 8.20
(s, 1H), 8.02 (d, J = 8.02 Hz, 1H), 7.84 (d, J = 6.66 Hz, 2H), 7.67 – 7.38 (m, 7H), 7.23 (d, J =
6.77 Hz, 2H), 7.14 (d, J = 8.86 Hz, 1H), 4.82 (s, 2H), 2.44 (s, 3H), 2.25 (s, 3H). 13C NMR (75
MHz, DMSO-d6) δ 157.53, 151.64, 148.69, 147.92, 143.33, 138.61, 136.48, 135.40, 133.82,
132.44, 131.28, 130.42, 129.48, 129.34, 128.25, 127.37, 122.62, 121.50, 120.67, 113.87,
112.27, 53.29, 17.26, 8.67; HRMS (ESI): calcd for C29H24Br2N3O4S [M + H]+ 667.9849, found
667.9839. IR (KBr, cm-1)：3417, 2918, 2360, 2342, 1670, 1515, 1466, 1384, 1356, 1302, 1166,
1147, 1085, 1069, 1007, 846, 753, 736, 701, 653. HPLC: (0.1% Et3N and 90% methanol in
water), tR =9.7 min, 95.5 %.
4.1.27
5-(4-bromo-N-(4-bromobenzyl)phenylsulfonamido)-3-methyl-N-(2-(pyrrolidin-1-yl)ethyl)benzo
furan-2-carboxamide (9q)
o
1
(122 mg, 52.3% yield). m.p. 152-154 C. H NMR (300 MHz, CDCl3) δ 7.69 – 7.47 (m, 4H),
7.41 – 7.29 (m, 3H), 7.19 (d, J = 2.04 Hz, 1H), 7.14 – 7.04 (m, 2H), 6.89 (dd, J = 8.78, 2.20 Hz,
1H), 4.72 (s, 2H), 3.67 – 3.59 (m, 2H), 2.87 – 2.79 (m, 2H), 2.78 – 2.61 (m, 4H), 2.50 (s, 3H),
2.01 – 1.70 (m, 4H). HRMS (ESI): calcd for C29H30Br2N3O4S [M + H]+ 674.0318, found
674.0311. IR (KBr, cm-1): 3423, 2965, 2796, 2367, 2342, 1664, 1609, 1574, 1516, 1462, 1342,
1280, 1157, 1094, 1068, 858, 751, 652, 547 HPLC: ( 0.1% Et3N and 90% methanol in water),
tR =7.1 min, 97.4 %.
4.1.28
5-((4-bromo-N-(4-bromobenzyl)phenyl)sulfonamido)-3-methyl-N-(2-(piperidin-1-yl)ethyl)benz
ofuran-2-carboxamide (9r)
o
1
(129 mg, 54.2% yield). m.p. 135-138 C. H NMR (300 MHz, CDCl3) δ 7.68 – 7.46 (m, 5H),
25

ACCEPTED MANUSCRIPT
7.41 – 7.29 (m, 3H), 7.18 (d, J = 2.03 Hz, 1H), 7.11 (d, J = 8.46 Hz, 2H), 6.90 (dd, J = 8.77,
2.18 Hz, 1H), 4.72 (s, 2H), 3.59 – 3.50 (m, 2H), 2.64 – 2.53 (m, 2H), 2.51 (s, 3H), 2.49 – 2.45
(m, 2H), 1.66 – 1.56 (m, 6H), 1.53 – 1.44 (m, 2H). HRMS (ESI): calcd for C30H32Br2N3O4S [M
+ H]+ 688.0475, found 688.0464. IR (KBr, cm-1): 3422, 2922, 2851, 2360, 2341, 1664, 1608,
1575, 1533, 1488, 1464, 1388, 1342, 1263, 1163, 1121, 1090, 1070, 1009, 849, 744, 647, 602.
HPLC:( 0.1% Et3N and 90% methanol in water), tR =8.4 min ,95.2 %.
4.1.29
5-(4-bromo-N-(4-bromobenzyl)phenylsulfonamido)-3-methyl-N-(2-morpholinoethyl)benzofuran
-2-carboxamide (9s)
o
1
(144 mg, 60.3% yield). m.p. 163-165 C. H NMR (300 MHz, CDCl3) δ 7.70 – 7.46 (m, 4H),
7.41 – 7.29 (m, 3H), 7.19 (d, J = 1.90 Hz, 1H), 7.11 (d, J = 8.44 Hz, 3H), 6.91 (dd, J = 8.81,
2.14 Hz, 1H), 4.72 (s, 2H), 3.81 – 3.71 (m, 4H), 3.61 – 3.52 (m, 2H), 2.62 (t, J = 6.00 Hz, 2H),
2.57 – 2.44 (m, 7H).HRMS (ESI): calcd for C29H30Br2N3O5S [M + H]+ 690.0267, found
690.0262. IR (KBr, cm-1)：3364, 2923, 2853, 2809, 2355, 1652, 1612, 1573, 1528, 1486, 1467,
1389, 1342, 1265, 1158, 1115, 1089, 1068, 1010, 861, 821, 651, 548. HPLC: (0.1% Et3N and
90% methanol in water), tR =5.8 min, 96.7 %.
4.1.30
5-(4-bromo-N-(4-bromobenzyl)phenylsulfonamido)-3-methyl-N-(2-(4-methylpiperazin-1-yl)eth
yl)benzofuran-2-carboxamide (9t)
o
1
(99 mg, 40.7% yield). m.p.142-145 C. H NMR (300 MHz, CDCl3) δ 7.66 – 7.47 (m, 4H),
7.36 (d, J = 8.40 Hz, 2H), 7.22 – 7.03 (m, 4H), 6.91 (dd, J = 8.74, 2.14 Hz, 1H), 4.72 (s, 2H),
3.73 (q, J = 7.02 Hz, 2H), 3.60 – 3.49 (m, 2H), 2.68 – 2.55 (m, 8H), 2.50 (s, 3H), 2.37 (s, 3H),
1.28 – 1.20 (m, 4H). HRMS (ESI): calcd for C30H33Br2N4O4S [M + H]+ 703.0548, found
703.0592. IR (KBr, cm-1)：3414, 2933, 2791, 2342, 1659, 1609, 1573, 1515, 1465, 1344, 1261,
1162, 1087, 1069, 1010, 918, 877, 820, 743, 703, 647. HPLC: (0.1% Et3N and 90% methanol
in water), tR =5.9 min, 95.7 %.
4.1.31
5-(4-bromo-N-(4-bromobenzyl)phenylsulfonamido)-3-methyl-N-(2-(phenylamino)ethyl)benzofu
ran-2-carboxamide (9u)
(78 mg, 32.4% yield). m.p. 178-180 oC. 1H NMR (300 MHz, DMSO-d6) δ 8.63 (t, J = 5.90 Hz,
1H), 7.88 – 7.79 (m, 2H), 7.59 – 7.50 (m, 2H), 7.50 – 7.37 (m, 4H), 7.22 (d, J = 8.45 Hz, 2H),
26

ACCEPTED MANUSCRIPT
7.15 – 7.00 (m, 3H), 6.59 (d, J = 7.64 Hz, 2H), 6.51 (t, J = 7.25 Hz, 1H), 5.71 (t, J = 5.73 Hz,
1H), 4.82 (s, 2H), 3.45 – 3.35 (m, 2H), 3.23 – 3.12 (m, 2H), 2.41 (s, 3H). 13C NMR (75 MHz,
DMSO-d6) δ 159.19, 151.42, 148.53, 144.15, 136.49, 135.49, 133.68, 132.42, 131.28, 130.41,
129.50, 128.90, 127.86,127.34, 121.26, 120.65, 120.62, 115.62, 111.82, 53.29, 42.21, 37.80,
8.53. HRMS (ESI): calcd for C31H28Br2N3O4S [M + H]+ 696.0162, found 696.0157. IR (KBr,
cm-1)：3380, 1647, 1603, 1574, 1515, 1486, 1466, 1430, 1350, 1271, 1159, 1089, 1068, 1010,
854, 748, 650, 602. HPLC: (0.1% Et3N and 90% methanol in water), tR =7.0 min, 95.3 %.
4.1.32
5-(4-bromo-N-(4-bromobenzyl)phenylsulfonamido)-3-methyl-N-(2-(pyridin-2-yl)ethyl)benzofur
an-2-carboxamide (9v)
o
1
(113 mg, 47.9% yield). m.p. 172-174 C. H NMR (300 MHz, CDCl3) δ 8.59 (d, J = 4.37 Hz,
1H), 7.70 – 7.47 (m, 6H), 7.36 (d, J = 8.34 Hz, 2H), 7.25 – 7.04 (m, 5H), 6.89 (dd, J = 8.72,
2.06 Hz, 1H), 4.71 (s, 2H), 3.88 (q, J = 5.89 Hz, 2H), 3.12 (t, J = 6.29 Hz, 2H), 2.50 (s, 3H).
13C NMR (75 MHz, DMSO) δ 159.18, 151.41, 148.52, 144.14, 136.48, 135.48, 133.67, 132.41,
131.27, 130.40, 129.49, 128.89, 127.86, 127.34, 121.26, 120.64, 120.62, 115.62, 111.82, 42.21,
37.79, 8.53. HRMS (ESI): calcd for C30H26Br2N3O4S [M + H]+ 682.0005, found 682.002. IR
(KBr, cm-1)：3412, 2920, 1662, 1609, 1588, 1575, 1533, 1486, 1465, 1389, 1339, 1279, 1263,
1159, 1120, 1089, 1071, 1010, 918, 883, 853, 763, 703, 648, 601, 545. HPLC:( 0.1% Et3N and
90% methanol in water), tR =6.0 min, 94.5 %.
4.1.33
N-(3-(1H-imidazol-1-yl)propyl)-5-(4-bromo-N-(4-bromobenzyl)phenylsulfonamido)-3-methylbe
nzofuran-2-carboxamide (9w)
o
1
(126 mg, 53.2% yield). m.p. 97-100 C. H NMR (300 MHz, DMSO-d6) δ 8.69 – 8.62 (m,
1H), 7.83 (d, J = 8.37 Hz, 2H), 7.56 – 7.39 (m, 6H), 7.22 (d, J = 8.29 Hz, 3H), 7.19 – 7.05 (m,
2H), 6.89 (s, 1H), 4.82 (s, 2H), 3.98 (t, J = 6.89 Hz, 2H), 3.26 – 3.17 (m, 2H), 2.40 (s, 3H),
1.97 – 1.90 (m, 2H). HRMS (ESI): calcd for C29H27Br2N4O4S [M + H]+ 685.0014, found
685.012. IR (KBr, cm-1)：3388, 3088, 2925, 1655, 1608, 1573, 1510, 1487, 1467, 1388, 1351,
1280, 1163, 1109, 1088, 1068, 1009, 918, 857, 703, 649, 602. HPLC: (0.1% Et3N and 90%
methanol in water), tR =5.0 min, 95.8 %.
4.1.34
5-((4-bromo-N-(4-bromobenzyl)phenyl)sulfonamido)-3-methyl-N-(4-sulfamoylphenethyl)benzof
27

ACCEPTED MANUSCRIPT
uran-2-carboxamide(9x)
The compound was prepared by a procedure identical to the preparation of 9i. (181mg, 45.6%
o
1
yield). m.p. 168-170 C. H NMR (300 MHz, CDCl3) δ 7.88 (d, J = 8.36 Hz, 2H), 7.70 – 7.60
(m, 2H), 7.55 – 7.46 (m, 2H), 7.45 – 7.32 (m, 4H), 7.24 – 7.16 (m, 1H), 7.10 (d, J = 8.37 Hz,
2H), 6.90 (dd, J = 8.77, 2.16 Hz, 1H), 6.65 (t, J = 6.06 Hz, 1H), 4.86 (s, 2H), 4.71 (s, 2H), 3.73
(q, J = 6.81 Hz, 2H), 3.03 (t, J = 7.02 Hz, 2H), 2.51 (s, 3H); HRMS (ESI): calcd for
C31H28Br2N3O6S2 [M + H]+ 759.9781, found 759.9777. IR (KBr, cm-1)：3373, 3088, 2923, 1653,
1609, 1573, 1525, 1487, 1467, 1341, 1280, 1161, 1089, 1068, 1009, 858, 749, 649, 602, 549.
HPLC: (0.1% Et3N and 90% methanol in water), tR =4.5 min, 95.0 %.
4.1.35 ethyl 3-methyl-5-nitrobenzo[b]thiophene-2-carboxylate (10)
Ethyl 2-mercaptoacetate (8.21 mL, 7.52 mmol) was added into a solution of
1-(2-chloro-5-nitrophenyl)ethanone (1.0 g, 5.01 mmol) in dry ethanol (25 mL), followed by
addition of K2CO3 (830.93 mg, 6.01 mmol) with stirring overnight at room temperature. The
reaction was monitored by TLC (PE:EA=10:1).Then the mixture solution was poured into 100
mL water with stirring, and the precipitate was filtered and dried to afford a solid of green. (1.3
o
g, 99% yield). m.p. 164.0-166.1 C. 1H NMR (300 MHz, CDCl3) δ 8.76 (d, J = 1.95 Hz 1H),
8.32 (dd, J = 8.92, 2.16 Hz, 1H), 7.96 (d, J = 8.90, 1H), 4.44 (q, J = 7.15 Hz, 2H), 2.86 (s, 3H),
1.45 (t, J = 7.13 Hz, 3H). HRMS(ESI): calcd, for C12H12NO4S [M+H]+ 266.0485, found
266.0482.
4.1.36 ethyl 2-(2-(4-nitrophenyl)hydrazono)butanoate (16)
A mixture of (4-nitrophenyl)hydrazine (1.0 g, 6.53 mmol) and ethyl 2-oxobutanoate (2.0 g,
13.06 mmol) in ethanol (50 mL) was refluxed 30 min with stirring. Then the solution was
concentrated in vacuo and purified by silica gel column chromatography (PE:EA=10:1) to give
solid. (346 mg, 20% yield). m.p. 212.5-215.6 oC. 1H NMR (300 MHz, CDCl3) δ 12.36 (s, 1H),
8.19 (d, J = 9.12 Hz 2H), 7.21 (d, J = 9.90 Hz 2H), 4.32 (q, J = 7.11 Hz, 2H), 2.38 – 2.15 (m,
5H), 1.39 (t, J = 7.13 Hz, 3H). HRMS(ESI): calcd, for C12H12NO4S [M+H]+ 265.0409, found
266.0482.
4.1.37 ethyl 3-methyl-5-nitro-1H-indole-2-carboxylate (17)
A solution of 16 (300 mg, 1.13 mmol) in dioxane (20 mL) was added polyphosphoric acid (1 g)
and refluxed 48 h with vigorously stirring. Then the solution was poured into water (100 mL)
and the precipitate was filtered and dried to give solid (157 mg, 57% yield). m.p. 237.1-240.4
oC. 1H NMR (300 MHz, DMSO-d6) δ 9.15 (s, 1H), 8.68 (d, J = 2.22 Hz, 1H), 8.47 (dd, J = 9.13,
2.67 Hz, 1H), 8.00 (d, J = 9.14 Hz, 1H), 4.55 (q, J = 7.05 Hz, 2H), 2.67 (s, 3H), 1.46 (t, J =
28