Design, synthesis and evaluation of some 1,6-disubstituted-1H-benzo[d]imidazoles derivatives targeted PI3K as anticancer agents

Article abstract of DOI:10.1016/j.bioorg.2019.103283

Phosphatidylinositol 3-kinase (PI3K) pathway regulates various cellular processes, such as proliferation, growth, autophagy and apoptosis. Class I PI3K is frequently mutated and overexpressed in a lot of human cancers and PI3K was considered as a target for therapeutic treatment of cancer. In this study, we designed and synthesized a series of 1,6-disubstituted-1H-benzo[d]imidazoles derivatives and evaluated their anticancer activity and the compound 8i was identified as a lead compound. Compound 8i with the most potent antiproliferative activity was selected for further biological mechanism. The PI3K kinase assay have shown potent efficiency against four subtypes of PI3K with an IC₅₀ of 0.5–1.9 nM. Molecular docking showed a possible formation of H-bonding with essential amino acid residues. Meanwhile, western blot assay indicated that 8i inhibited cell proliferation via suppression of PI3K kinase activity and subsequently blocked PI3K/Akt pathway activation in HCT116 cells. In addition, 8i could inhibit the migration and invasion ability of HCT116 cells and could induce apoptosis of HCT116 cells.

Full text of DOI:10.1016/j.bioorg.2019.103283

Journal Pre-proofs
Design, synthesis and evaluation of some 1,6-disubstituted-1H-benzo[d]imida-
zoles derivatives targeted PI3K as anticancer agents
Huai-Wei Ding, Lu Yu, Meng-xuan Bai, Xiao-Chun Qin, Man-tong Song, Qing-
Chun Zhao
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S0045-2068(19)30813-2
DOI:
https://doi.org/10.1016/j.bioorg.2019.103283
YBIOO 103283
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Bioorganic Chemistry
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Please cite this article as: H-W. Ding, L. Yu, M-x. Bai, X-C. Qin, M-t. Song, Q-C. Zhao, Design, synthesis and
evaluation of some 1,6-disubstituted-1H-benzo[d]imidazoles derivatives targeted PI3K as anticancer agents,
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Design, synthesis and evaluation of some 1,6-disubstituted-1H-benzo[d]imidazoles
derivatives targeted PI3K as anticancer agents
Huai-Wei Ding a, Lu Yu b, Meng-xuan Bai a, Xiao-Chun Qin b, Man-tong Song c,*, Qing-Chun Zhao b,*
a Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education, Shenyang Pharmaceutical
University, Shenyang 110016, China
b School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, China
c School of Public Health, Shenyang Medical College, Shenyang 110034, China
* Corresponding authors.
Email address: zhaoqingchun1967@163.com, songmtong@163.com.

Abstract：
Phosphatidylinositol 3-kinase (PI3K) pathway regulates various cellular processes, such as proliferation,
growth, autophagy and apoptosis. Class I PI3K is frequently mutated and overexpressed in a lot of human
cancers and PI3K was considered as a target for therapeutic treatment of cancer. In this study, we designed
and synthesized a series of 1,6-disubstituted-1H-benzo[d]imidazoles derivatives and evaluated their
anticancer activity and the compound 8i was identifified as a lead compound. Compound 8i with the most
potent antiproliferative activity was selected for further biological mechanism. The PI3K kinase assay have
shown potent effificiency against four subtypes of PI3K with an IC50 of 0.5 nM to 1.9 nM. Molecular
docking showed a possible formation of H-bonding with essential amino acid residues. Meanwhile, western
blot assay indicated that 8i inhibited cell proliferation via suppression of PI3K kinase activity and
subsequently blocked PI3K/Akt pathway activation in HCT116 cells. In addition, 8i could inhibit the
migration and invasion ability of HCT116 cells and could induce apoptosis of HCT116 cells.
Keywords: benzo[d]imidazole, antitumor, PI3K

1. Introduction
As a member of lipid kinases, the phosphatidylinositol 3-kinase (PI3K) are divided into four different
classes: Class I, Class II, Class III and Class IV. The class I PI3K, the most commonly studied, can be
further divided into PI3Kα, PI3Kβ, PI3Kδ and PI3Kγ [1-3]. Much evidence indicate that the PI3K pathway
plays a key role in various cellular processes, such as proliferation, growth, cell cycle and apoptosis [4-5].
Up to now, lots of PI3K inhibitors had been developed and evaluated in preclinical studies and early clinical
trials [6]. Among them, there were four PI3K inhibitors as antineoplastic drugs approved by the FDA (Fig.1),
Idelalisib approved in 2014 [7], Copanlisib approved in 2017 [8], Duvelisib approved in 2018 [9] and
Alpelisib approved in 2019 [10].
F
O
Cl
O
N
N
N
N
HN
N
HN
N
N
N
N
N
Idelalisib
Duvelisib
NH
NH
O
O
N
O
F
F
F
NH2
O
N
O
H2N
N
N
N
O
N
N
HN
S
NH
N
N
Alpelisib
Copanlisib
Fig.1. Four PI3K inhibitors approved by the FDA.
B
O
N
O
O
O
S
N
NH
O
N
O
4
3
R2
S
N
O
O
O
O
2
N
S
N
NH
O
O
NH
H
N
R1
N
H
1
N
O
GSK2292767
7
4
6
N
1
N
N
3
N
2
2
5
N
3
N
H
1
4
O
O
S
NH
Target compounds
Transition structure
O
O
2
4
N
3
N
N
1
HS-173
Fig.2. The design strategy based on GSK2292767 and HS-173.
The design strategy for the target compounds in this work was inspired by GSK2292767 [11] and
HS-173 [12-14] (Fig.2) which are PI3K inhibitors. From the structure of these two compounds, it was
found that they had similar molecular characteristics as effective PI3K inhibitors. They contain
six-membered rings and five-membered heterocycles, and pyridyl group at position-5. The difference is

that GSK2292767 has a substituent at position-4 and HS-173 has a substituent at position-3. According
to the mentioned above literature of compound HS-173 and our previous work [15], it suggested that the
substituent group should be at position-3 with better activity compare to at position-4. From the
perspective of economics of synthesis, it was found that benzoimidazole group could effectively
combine the GSK2292767 and HS-173. Not only that, the more active groups of the group R2 are
methyl and 2,4 difluorophenyl base on the conclusion of structure-activity relationships (SARs) in
literatures [16-19] . For this reason, our design strategies are concentrated on these two R2 substituent
groups and focusing on the effect of different R1 on the activity. Interestingly, due to the IUPAC naming
rules, the substituent at position-3 of GSK2292767 becomes the position-1 of our target compounds at
last by the replacing of pyrazole ring with an imidazole ring base on GSK2292767. Finally, we designed
a series of derivatives with benzoimidazoles as the mother nucleus and had different substituents at 1
and 6 positions.
As a result, with the goal of developing some new PI3K inhibitors which might serve as potential
drugs for the treatment of cancer, we synthesized a series of compounds containing hydrophilic group at
position-1 in benzo[d]imidazole. They were prepared and the anti-tumor effects were investigated in
vitro. The final results indicate that these series of compounds are a class of pan-PI3K inhibitors and
neither like the GSK2292767 as a selective PI3Kγ inhibitor nor like HS-173 as a PI3Kα inhibitor. We
speculate that the change in the position of the substituent perhaps led to this result. The second perhaps
reason is that although HS-173 is reported to be an inhibitor of PI3Kα subtype [13], but the difference in
selectivity between four subtypes is not big enough.
2. Results and discussion
2.1. Chemistry
The synthetic routes are showed in Scheme 1. Intermediate 1 was reacted with three amines to yield
2a-c, which were then be reduced by iron powder in glacial acetic acid and directly added the
trimethylorthoformate to afford 3a-c. 3a-c were reacted with different halogenated hydrocarbon to abtain
4a-o, which were then coupled with 7a-b via Suzuki reaction to afford the target compounds 8a-s.

4a: R2
4b: R2
4c: R2
4d: R2
4e: R2
4f: R2
=
=
=
=
=
=
OH
OH
O
N
Br
F
R2
N
O
O
N
N
O
NO2
1
Br
N
N
a
O
4a-f 67~88%
R1
R1
Br
NH
Br
N
b
c
4g: R2
=
R
R
R
R
R
R
R
R
R
4 = H
OH
OH
N
NO2
4 = H
R4
O
4h: R2
4i: R2
4j: R2
4k: R2
4l: R2
4m: R2
4n: R2
4o: R2
=
=
=
=
=
=
=
=
2a-c 85~95%
3a-c 68~75%
R3
4 = H
NH
N
NH
N
OH
R5
S
O
3a: R1
3b: R1
3c: R1
=
=
=
2a: R1
=
=
=
O
O
O
R2
N
4 = H
O
NH
OH
OH
OH
OH
N
2b: R1
2c: R1
Br
N
4 = H
N
O
N
4 = H
N
N
N
N
4g-o 52~92%
O
O
O
4 = OCH3
4 = OCH3
4 = OCH3
OH
OH
f
8a-h 42~61%
N
R5
S
R4
O
O
O
O
NH
R3
R5
S
O
R5
O
O
O
O
S
NH2
O
O
NH
NH
N
N
N
O
e
d
N
N
O
N
Br
B
Br
8i-s 41~68%
5
O
7a-b 74~78%
6a-b 85~89%
6a: R5
6b: R5
=
=
7a: R5
7b: R5
=
=
F
F
F
F
Scheme 1. (a) NH2-R1, DMF, DIPEA, 25 ºC, 12 h; (b) i) Fe/CH3COOH, 90 ºC, 6 h; 2) TrimethylOrthoformate, 90 ºC, 6 h;
(c) Cl-R2, K2CO3, DMF, 60 ºC, 12 h; (d) ClSO2-R5, pyridine, rt, 24 h; (e) bis(pinacolato)diborane, Pd(dppf)2Cl2, AcOK,
DMF, 100 ºC, 8 h; (f) Bis(triphenylphosphine)palladium (II) dichloride, Cs2CO3, DMF/H2O, 95 ºC, 8 h.
2.2. Antiproliferative assays in vitro
All the synthesized compounds were evaluated for their cytotoxicities in vitro towards the T47D,
MCF-7 and HCT116 cell lines. The results were summarized in Table 1. As shown in Table 1, the
cytotoxicities results showed that most of the compounds exhibited potent antiproliferative effects. As the
representative, the SARs was discussed with HCT116 cell line. From the overall results, it showed that the
activity of hydrophilic groups at position-3 was better than that of hydrophobic groups, including 8b vs.8e,
8d vs. 8f and 8i vs. 8o. That's why most of the compounds we designed contain alcohol hydroxyl groups.
The activity of compounds containing phenyl ring group at position-3 is slightly better than that of
compounds containing pyrazoly at the same position, it could be reflected in 8b vs. 8i, 8d vs. 8l and 8h vs.
8o. From the activity of 8i, 8l and 8n, it was possible to draw such a conclusion that the further the
hydrophilic group was from the parent nucleus, the lower the activity would be. It also could find that all of
compounds with 2,4-difluoro-phenyl group substituent on R5 position displayed much better
antiproliferative activities than those substituted by methyl on the same position, including 8a vs.8b, 8c vs.
8d, 8k vs. 8l and 8r vs. 8s. Introducing methoxy group into benzene ring at R3 position could not improve
the activity, but lead to the decrease of the activity, such as, 8j, 8m and 8q. After the analysis, we

hypothesize that introducing methoxy group into the benzene ring will increase hydrophobicity and it is
inconsistent with the principle that hydrophilic groups are needed in the ribose pocket.
Table 1 Antiproliferative activities of target compounds against three cancer cell lines (IC50 Valuesa in μM).
R5
S
R5
S
R4
O
O
O
O
O
O
O
R2
N
NH
NH
R3
N
N
N
N
N
N
N
8a-h
8i-s
Cells
Comp.
8a
T47D
MCF-7
HCT116
R2
R3
R4
R5
CH3
>20
9.43±0.14
0.57±0.09
3.92±0.12
0.13±0.03
-
-
-
-
-
-
-
-
-
-
OH
OH
F
0.27±0.05
>20
8b
8c
8d
8e
8f
F
OH
CH3
16.81±1.12 12.74±0.76
-
F
F
F
F
F
F
F
OH
0.29±0.04
3.26±0.24
1.14±0.32
3.58±0.16
0.67±0.08
0.36±0.03
0.62±0.10
3.99±0.19
0.45±0.02
0.75±0.08
0.89±0.10
3.92±0.22
0.54±0.06
0.68±0.06
0.66±0.09
4.80±0.18
0.76±0.14
1.06±0.12
0.29±0.04
0.31±0.02
0.76±0.02
1.44±0.12
0.59±0.03
1.02±0.12
0.96±0.12
4.80±0.34
0.41±0.02
0.48±0.02
0.27±0.06
0.84±0.10
0.59±0.08
1.01±0.11
0.41±0.06
0.14±0.02
0.73±0.08
0.86±0.09
0.85±0.06
2.42±0.14
0.74±0.08
3.51±0.25
0.14±0.03
0.57±0.04
-
F
F
F
F
F
F
F
O
-
O
O
-
O
8g
8h
8i
-
N
N
-
-
O
H
OH
8j
-
OCH3
H
OH
OH
CH3
8k
8l
-
F
F
F
F
F
F
OH
OH
-
H
F
F
F
F
F
F
8m
8n
8o
8p
8q
-
OCH3
H
-
OH
O
-
H
O
N
-
H
O
O
N
-
OCH3

O
O
CH3
11.22±0.86
0.29±0.02
0.62±0.12
4.82±0.32
0.41±0.03
0.26±0.04
3.17±0.28
0.34±0.03
0.54±0.13
8r
8s
-
-
H
H
N
N
F
F
HS-1
73
a IC50 values are the mean of triplicate measurements.
2.3 8i inhibits PI3K and blocks the PI3K-Akt pathway
In order to elucidate the mechanism of antiproliferative activities of these active compounds, we
selected the representative compounds 8i and 8l for the further PI3K enzymatic activity assays. HS-173 were
selected as the positive drugs. As shown in Table 2, kinase inhibition activity assay indicated that the
activity of 8i and 8l has reached the picomolar level. It found that the kinase activities of 8i and 8l were
consistent with cellular activities. Two compounds above showed a higher inhibitory activity against PI3Kα
when compared to that of other class I PI3Ks (PI3Kβ, PI3Kγ and PI3Kδ).
Docking simulations were conducted to predict the binding model of 8i in the binding site of PI3K. The
structure of PI3K kinase was obtained from protein data bank (PDB entry 3L08, a PI3Kγ isoform [16]). As
shown in Fig.3 (A) or (C), the nitrogen of benzo[d]imidazole group formed a hydrogen bond with the side
chain of Val882 in the hinge binder region of PI3K. Besides, the oxygen of methoxy group formed an
additional hydrogen bond interaction with Lys833. Moreover, the nitrogen of pyridyl group formed a
hydrogen bond with the conserved water molecule. In addition to these, the hydrogen of hydroxyl group
formed a hydrogen bond interaction with Thr887. As shown in Fig.3 (B) and (D), the folded form of
compound 8i in the protein pocket was very similar to that of Omipalisib which was the original ligand in
3L08. Apart from the interaction of hydrogen bonds, the aromatic rings of 8i also formed Pi-Pi interactions
with multiple amino acid residues.
The docking analysis indicated that compound 8i could fit into the binding site of PI3K kinase, which
also indicated that this compound may be a potent PI3K inhibitor.
Table 2 Enzyme activities of 8i and 8l and HS-173 in Class I PI3K (IC50 Values in nM)
PI3Kα
0.50
0.82
1.1
PI3Kβ
1.9
PI3Kγ
1.8
PI3Kδ
0.74
1.3
8i
8l
5.5
2.9
HS-173
59.2
104
87

A
B
D
C
F
F
F
F
Fig.3. Docking mode of 8i with protein crystal structure of PI3K. (A) Key interactions of compound 8i in the active site of
PI3K (PDB: 3L08). (B) The binding pose of 8i and Omipalisib in the active site of PI3K. Omipalisib was highlighted with
yellow. (C) Two-dimensional graph of compound 8i in the active site of PI3K. (D) Two-dimensional graph of Omipalisib in
the active site of PI3K.
To further determine whether the PI3K signaling were affected by compound 8i, Western blot assay
was used to evaluate the effects of 8i on the PI3K related protein levels including Akt and phospho-Akt
(p-Akt, S473) in HCT116 cells. As shown in Fig.4, compared with control group, 8i experimental group can
reduce the protein level of p-Akt in a concentration-dependent manner, indicating that compound 8i can
reduce the activity of p-Akt, and the expression of total protein Akt does not show a change trend.
According to the results of Western blot, we can conclude that compound 8i may inhibit the activity of Akt
protein by inhibiting the activation of Akt phosphorylation, thus inhibiting the proliferation of HCT116

cells.
Fig.4. The inhibition effects of compound 8i (0.3 μM, 1 μM and 3 μM) on the expression of p-Akt, Akt in HCT116 cells are
depicted. β-Actin was used as internal control.
2.4. Colony formation assay of 8i on HCT116 cells
In order to further investigate the antiproliferation of 8i on HCT116 cells, the colony formation assay
was used to measure the cell viability. As shown in Fig.5, 8i inhibited the formation of HCT116 cell clones
in a concentration-dependent manner, and the high concentration group (3.0 μM) could almost completely
inhibit the production of cell clones. The results indicated that HCT116 cells treated with 8i would lose the
ability of cell proliferation.
Fig.5. The colony formation assay of compound 8i. Treatment with compound 8i, representative photographs of colony
formation are shown. Data are presented as the mean for three independent experiments. ** P < 0.01, *** P < 0.001
compared with control.
2.5. Colonic carcinoma cell invasion and migration ability by compound 8i
The PI3K/Akt signaling pathway plays an important role in cancer cell migration and invasion. To
determine the effects of 8i on colonic carcinoma cell migration and invasion ability, we performed wound
healing and invasion assays. For the Transwell invasion assay, 8i significantly inhibited HCT116 cell
metastasis and invasiveness in a dose-dependent manner, after treatment with 8i for 48 h (Fig.6. A and B).
For wound healing assay, the statistical results (Fig.6. C, D and E) showed that the inhibition ability
increased in a concentration-dependent manner.

Fig.6. Transwell invasion and wound healing assays of compound 8i. (A) Treatment with compound 8i, representative
photographs of invasion are shown. (B) Quantification of the percentages of cells at different concentration. (C)
Photographs of wound healing assay. (D) Quantification of the percentages of transfection group (24h). (E) Quantification
of the percentages of transfection group (48h). Data are presented as the mean for three independent experiments. ** P <
0.01, *** P < 0.001 compared with control.
2.6. Apoptosis induced by compound 8i
Annexin V-PI double staining assay was used to examine the effect of 8i on cells apoptosis. As shown
in Fig.7 (A and B), compared with the control group, the proportion of apoptotic cells in compound 8i
treatment group increased significantly and showed a concentration-dependent manner. In summary, the
data showed that compound 8i could induce apoptosis of HCT116 cells. In order to further evaluate
8i-induced apoptosis in HCT116 cells, cells were stained with Hoechst33342 and analyzed by fluorescence
microscope. As shown in Fig.7 (C), treated with different concentrations of 8i, the brighter fluorescence was
seen. Compared with control which had normal nuclear morphology after Hoechst33342 staining, 8i
treatment group marked with nuclear fragmentation and condensation of chromatin.

Fig.7. 8i induced apoptosis in HCT116 cells. (A) AV-PI staining show early and late apoptosis of HCT116 cells induced by
compound 8i. (B) Quantification of early and late apoptosis. (C) The nuclei morphology changes of HCT116 cells treated
with 8i visualized by Hoechst 33342. Data are presented as the mean for three independent experiments. *** P < 0.001
compared with control.
2.7. ADME evaluation
Table 3 ADME evaluation of 8i
Ligand
8i
Mol.wt.
(g/mol)
552.55
No. HBA
9
No. HBD
2
TPSA(A2)
123.95
cLogp
4.10
Lipinski’s rule
Yes; 1 violation: MW>500
a HBA: Hydrogen Bond Acceptor, HBD: Hydrogen Bond Donor.
Drug likeliness properties of the compound 8i was calculated by the help of SwissADME online
software. The results are presented in Table 3. According to Lipinski’s rule of five, total number of HBD
should not be more than 5, and total number of HBA should not be greater than 10. Number of HBD and
HBA of 8i are determined 2 and 9 respectively. Topological polar surface area (TPSA, >140 A2 is linked
with low blood-brain barrier (BBB) penetration, and poor membrane permeability [20].) is a very useful
parameter for prediction of transport of drug molecule and the TPSA of 8i was 123.95 A2 . Estimation of
lipophilicity is determined by clogP, which is the log of octanol/water partition coefficient. The clogP value

should not be greater than 5. Predicted by software, 8i has a favourable clogP value (4.10) and it
demonstrates that could have good membrane permeability. Unfortunately, 8i has a molecular weight of 552
which violates the Lipinski’s rule of five. However, many anticancer drugs approved by FDA also have a
molecular weight greater than 500, such as Quizartinib (MW: 560) and Neratinib (MW: 557), so this may
not particularly affect the compound 8i as a good potential candidate.
3. Conclusion
In summary, some 1,6-disubstituted-1H-benzo[d]imidazoles derivatives were designed and synthesized
and the antiproliferative activities against three cancer cell lines, including T47D, HCT116 and MCF-7.
Compound 8i with the most potent antiproliferative activity was selected for further biological evaluation.
The PI3K kinase assay and western blot assay indicated that 8i inhibited cell proliferation via blocking the
PI3K/Akt pathway in HCT116 cells. In addition, the colony formation assay and apoptosis assay suggested
that 8i could inhibit the migration and invasion ability of HCT116 cells. ADME screening demonstrated that
8i possessed drug-like properties to become biologically active molecules. According to these results,
compound 8i could be as a potential PI3K inhibitor and could be considered as a potential candidate for
anticancer drug development.
4. Experimental section
4.1. Chemistry and chemical methods
The reagents and solvents were commercially available without further purification. 13C NMR spectra
and 1H NMR spectra were tested on 400 and 600 Bruker NMR spectrometer with tetramethylsilane (TMS)
as an internal standard and the chemical shifts are reported in ppm (δ) and coupling constants (J) are in hertz
(Hz). The melting points were determined on a Beijing micromelting-point apparatus and thermometer was
uncorrected. High-resolution exact mass measurements were performed using electrospray ionization
(positive mode) on a quadrupole time-of-flight (QTOF) mass spectrometer (microTOF-Q, Bruker Inc.).
4.1.1. N-(5-bromo-2-nitrophenyl)-1H-pyrazol-4-amine (2a)
To a solution of 4-bromo-2-fluoro-1-nitrobenzene (1, 2.19 g, 10 mmol) in DMF (50 ml) and
DIPEA (10ml) at 25 ºC was added 1H-pyrazol-4-amine (1.25 g, 15 mmol). Then the mixture was
stirred at room temperature 12 h. DMF and DIPEA were removed at reduced pressure and add water
(100 ml), and the residue was purified through a column chromatography on silica with
chloroform/methanol (V:V 20:1) as a red solid. (2.68 g, 95.0% yield). mp 94-96 ºC.1H NMR (400
MHz, DMSO-d6) δ 13.03 (s, 1H, NH), 9.19 (s, 1H, NH), 8.03 (d, J = 9.1 Hz, 1H, Ar-H), 7.96 - 7.58
(m, 2H, Ar-H), 7.06 (d, J = 2.0 Hz, 1H, Ar-H), 6.93 (dd, J = 9.1, 2.0 Hz, 1H, Ar-H). 13C NMR (100
MHz, DMSO-d6) δ 145.63, 136.48, 131.50, 130.93, 128.51, 125.80, 120.00, 119.94, 118.08.
ESI-MS: m/z 281.1 [M-H]+.

Compounds 2b-c was synthesized according to the procedure described in 2a.
4.1.2. 4-((5-bromo-2-nitrophenyl)amino)phenol (2b)
94.4% yield. mp 155-157 ºC. 1H NMR (400 MHz, DMSO-d6) δ 9.66 (s, 1H, OH), 9.42 (s, 1H, NH),
8.03 (d, J = 9.3 Hz, 1H, Ar-H), 7.14 (d, J = 8.6 Hz, 2H, Ar-H), 6.93 (s, 1H, Ar-H), 6.90 (d, J = 2.0 Hz, 1H,
Ar-H), 6.86 (d, J = 8.7 Hz, 2H, Ar-H). 13C NMR (100 MHz, DMSO-d6) δ 156.57, 145.43, 131.49, 130.62,
129.48, 128.61, 128.18 (2C), 119.81, 118.19, 116.75 (2C). ESI-MS: m/z 309.0 [M+H]+.
4.1.3. 4-((5-bromo-2-nitrophenyl)amino)-2-methoxyphenol (2c)
85.6% yield. mp 138-141 ºC. 1H NMR (400 MHz, DMSO-d6) δ 9.42 (s, 1H, OH), 9.22 (s, 1H, NH),
8.03 (d, J = 9.1 Hz, 1H, Ar-H), 7.01 (d, J = 2.0 Hz, 1H, Ar-H), 6.94 (s, 1H, Ar-H), 6.91 (d, J = 2.0 Hz, 1H,
Ar-H), 6.86 (d, J = 8.3 Hz, 1H, Ar-H), 6.76 (dd, J = 8.3, 2.2 Hz, 1H, Ar-H), 3.77 (s, 3H, CH3). 13C NMR
(100 MHz, DMSO-d6) δ 148.78, 145.78, 145.32, 131.50, 130.63, 129.76, 128.54, 119.91, 118.96, 118.43,
116.31, 111.35, 56.21. ESI-MS: m/z 338.9 [M+H]+.
4.1.4. 6-bromo-1-(1H-pyrazol-4-yl)-1H-benzo[d]imidazole (3a)
A solution of the 2a (2.82 g, 10 mmol), iron powder (2.8 g, 50 mmol) in CH3CH2COOH (100 ml) was
stirred at 90 ºC for 6 h. Without purified and directly added the TrimethylOrthoformate (10ml), then the
solution stirred at 90 ºC for another 6 h. CH3CH2COOH was removed under reduced pressure and add water
(100 ml), whereby a white solid precipitate formed. The precipitate was washed with water (20 ml), dried to
give compound 3a as a white solid (1.97 g, 75.2% yield). mp 179-182 ºC.1H NMR (400 MHz, DMSO-d6) δ
13.36 (s, 1H, NH), 8.50 (s, 1H, Ar-H), 8.22 (d, J = 165.2 Hz, 2H, Ar-H), 7.74 (d, J = 1.6 Hz, 1H, Ar-H),
7.71 (d, J = 8.6 Hz, 1H, Ar-H), 7.44 (dd, J = 8.6, 1.7 Hz, 1H, Ar-H). 13C NMR (100 MHz, DMSO-d6) δ
145.19, 142.81, 135.37, 133.90, 125.76, 123.85, 121.92, 118.49, 116.29, 113.90. ESI-MS: m/z 260.5
[M+H]+.
Compounds 3b-c were synthesized according to the procedure described in 3a.
4.1.5. 4-(6-bromo-1H-benzo[d]imidazol-1-yl)phenol (3b)
72.3% yield. mp 153-155 ºC.1H NMR (400 MHz, DMSO-d6) δ 9.93 (s, 1H, OH), 8.48 (s, 1H, Ar-H),
7.72 (d, J = 8.5 Hz, 1H, Ar-H), 7.62 (s, 1H, Ar-H), 7.48 – 7.44 (m, 2H, Ar-H), 7.42 (s, 1H, Ar-H), 6.99 (d, J
= 8.5 Hz, 2H, Ar-H). 13C NMR (100 MHz, DMSO-d6) δ 157.86, 145.00, 143.05, 135.44, 127.15, 126.22
(2C), 125.62, 122.02, 116.84 (2C), 116.07, 113.67. ESI-MS: m/z 289.0 [M+H]+.
4.1.6. 4-(6-bromo-1H-benzo[d]imidazol-1-yl)-2-methoxyphenol (3c)
68.8% yield. mp 202-204 ºC. 1H NMR (400 MHz, DMSO-d6) δ 9.50 (s, 1H, OH), 8.50 (s, 1H, Ar-H),
7.72 (d, J = 8.6 Hz, 1H, Ar-H), 7.68 (d, J = 1.7 Hz, 1H, Ar-H), 7.43 (dd, J = 8.6, 1.8 Hz, 1H, Ar-H), 7.21 (d,
J = 2.3 Hz, 1H, Ar-H), 7.04 (dd, J = 8.3, 2.4 Hz, 1H, Ar-H), 6.98 (d, J = 8.4 Hz, 1H, Ar-H), 3.86 (s, 3H,
CH3). 13C NMR (100 MHz, DMSO-d6) δ 148.83, 147.12, 145.11, 143.01, 135.48, 127.29, 125.64, 121.98,

117.18, 116.29, 116.09, 113.84, 109.66, 56.42.. ESI-MS: m/z 318.7 [M+H]+
4.1.7. 2-(4-(6-bromo-1H-benzo[d]imidazol-1-yl)-1H-pyrazol-1-yl)ethan-1-ol (4a)
A solution of the 3a (0.26 g, 1 mmol) in DMF (20 ml) to add K2CO3 (0.28 g, 2 mmol) at 25 ºC and this
solution was stirred at 25 ºC for 10 minutes. Then added into 1H-pyrazol-4-amine (1.25 g, 15 mmol) and the
solution was stirred at 60 ºC for 12 h. DMF was removed under reduced pressure and the residue was
purified through a column chromatography on silica with chloroform/methanol (V:V 10:1) as a white oil
(0.19 g, 60.4% yield).1H NMR (400 MHz, DMSO-d6) δ 8.56 (s, 1H, Ar-H), 8.45 (s, 1H, Ar-H), 8.02 (s, 1H,
Ar-H), 7.83 – 7.81 (m, 1H, Ar-H), 7.77 (d, J = 8.6 Hz, 1H, Ar-H), 7.51 – 7.47 (m, 1H, Ar-H), 5.06 (s, 1H,
OH), 4.29 (t, J = 5.5 Hz, 2H, CH2), 3.88 (t, J = 5.5 Hz, 2H, CH2). 13C NMR (100 MHz, DMSO-d6) δ 145.04,
142.76, 136.02, 135.31, 128.43, 125.42, 120.50, 116.77, 115.12, 113.23, 60.35, 55.29. ESI-MS: m/z 307.1
[M+H]+.
Compounds 4b-o were synthesized according to the procedure described in 4a.
4.1.8. 3-(4-(6-bromo-1H-benzo[d]imidazol-1-yl)-1H-pyrazol-1-yl)propan-1-ol (4b)
81.2% yield. Oil.1H NMR (400 MHz, DMSO-d6) δ 8.51 (s, 1H, Ar-H), 8.44 (s, 1H, Ar-H), 7.98 – 7.95
(m, 1H, Ar-H), 7.77 (d, J = 1.6 Hz, 1H, Ar-H), 7.71 (d, J = 8.6 Hz, 1H, Ar-H), 7.44 (dd, J = 8.6, 1.8 Hz, 1H,
Ar-H), 4.73 (s, 1H, OH), 4.26 (t, J = 7.1 Hz, 2H, CH2), 3.46 (t, J = 6.1 Hz, 2H, CH2), 2.01 (p, J = 6.4 Hz, 2H,
CH2). 13C NMR (100 MHz, DMSO-d6) δ 144.96, 142.77, 135.15, 133.43, 125.82, 125.14, 121.95, 118.39,
116.35, 113.89, 58.15, 49.72, 33.40. ESI-MS: m/z 321.1[M+H]+.
4.1.9. methyl 2-(4-(6-bromo-1H-benzo[d]imidazol-1-yl)-1H-pyrazol-1-yl)acetate (4c)
88.6% yield. mp 96-98ºC.1H NMR (400 MHz, DMSO-d6) δ 8.56 (s, 1H, Ar-H), 8.48 (s, 1H, Ar-H),
8.05 (s, 1H, Ar-H), 7.74 (s, 1H, Ar-H), 7.72 (d, J = 5.7 Hz, 1H, Ar-H), 7.45 (dd, J = 8.6, 1.8 Hz, 1H, Ar-H),
5.21 (s, 2H, CH2), 3.74 (s, 3H, CH3). 13C NMR (100 MHz, DMSO-d6) δ 168.89, 162.76, 144.87, 142.81,
135.05, 134.30, 126.39, 125.89, 122.06, 119.10, 116.39, 113.74, 53.51, 52.84. ESI-MS: m/z 334.9 [M+H]+.
4.1.10. methyl 3-(4-(6-bromo-1H-benzo[d]imidazol-1-yl)-1H-pyrazol-1-yl)propanoate (4d)
83.4% yield. mp 80-83 ºC. 1H NMR (400 MHz, DMSO-d6) δ 8.51 (s, 1H, Ar-H), 8.45 (s, 1H, Ar-H),
7.98 (s, 1H, Ar-H), 7.76 (d, J = 1.7 Hz, 1H, Ar-H), 7.71 (d, J = 8.6 Hz, 1H, Ar-H), 7.44 (dd, J = 8.6, 1.8 Hz,
1H, Ar-H), 4.46 (t, J = 6.7 Hz, 2H), 3.64 (s, 3H, CH3), 2.99 (t, J = 6.7 Hz, 2H, CH2). 13C NMR (100 MHz,
DMSO-d6) δ 171.50, 144.90, 142.76, 135.13, 133.77, 125.85, 125.38, 121.97, 118.50, 116.37, 113.86, 52.08,
48.06, 34.30. ESI-MS: m/z 348.7 [M+H]+.
4.1.11. 2-(4-(6-bromo-1H-benzo[d]imidazol-1-yl)-1H-pyrazol-1-yl)-N,N-dimethylethan-1-amine (4e)
67% yield. Oil. 1H NMR (400 MHz, DMSO-d6) δ 8.52 (s, 1H, Ar-H), 8.45 (s, 1H, Ar-H), 7.96 (s, 1H,
Ar-H), 7.75 (d, J = 1.7 Hz, 1H, Ar-H), 7.71 (d, J = 8.6 Hz, 1H, Ar-H), 7.44 (dd, J = 8.6, 1.8 Hz, 1H, Ar-H),
4.29 (t, J = 6.5 Hz, 2H, CH2), 2.74 (t, J = 6.5 Hz, 2H, CH2), 2.21 (s, 6H, CH3). 13C NMR (100 MHz,

DMSO-d6) δ 144.91, 142.79, 135.12, 133.32, 125.82, 125.26, 121.98, 118.42, 116.33, 113.84, 58.79, 50.51,
45.53 (2C). ESI-MS: m/z 333.7 [M+H]+.
4.1.12. 4-(2-(4-(6-bromo-1H-benzo[d]imidazol-1-yl)-1H-pyrazol-1-yl)ethyl)morpholine (4f)
71.4% yield. Oil.1H NMR (400 MHz, DMSO-d6) δ 8.52 (s, 1H, Ar-H), 8.45 (s, 1H, Ar-H), 7.95 (s, 1H,
Ar-H), 7.74 (d, J = 1.7 Hz, 1H, Ar-H), 7.72 (d, J = 8.6 Hz, 1H, Ar-H), 7.44 (dd, J = 8.6, 1.8 Hz, 1H, Ar-H),
4.32 (t, J = 6.4 Hz, 2H, CH2), 3.59 – 3.56 (m, 4H, CH2), 2.77 (t, J = 6.5 Hz, 2H, CH2), 2.45 (s, 4H, CH2). 13C
NMR (100 MHz, DMSO-d6) δ 144.92, 142.78, 135.21, 133.49, 125.81, 125.55, 122.00, 118.33, 116.32,
113.79, 66.70 (2C), 58.00, 53.56 (2C), 49.75. ESI-MS: m/z 375.8 [M+H]+.
4.1.13. 2-(4-(6-bromo-1H-benzo[d]imidazol-1-yl)phenoxy)ethan-1-ol (4g)
69% yield. Oil. 1H NMR (400 MHz, DMSO-d6) δ 8.52 (s, 1H, Ar-H), 7.73 (d, J = 8.6 Hz, 1H, Ar-H),
7.65 (d, J = 1.4 Hz, 1H, Ar-H), 7.59 (d, J = 8.7 Hz, 2H, Ar-H), 7.44 (dd, J = 8.5, 1.4 Hz, 1H, Ar-H), 7.18 (d,
J = 8.8 Hz, 2H, Ar-H), 4.95 (t, J = 4.6 Hz, 1H, OH), 4.09 (t, J = 4.9 Hz, 2H, CH2), 3.80 – 3.74 (m, 2H, CH2).
13C NMR (100 MHz, DMSO-d6) δ 158.88, 144.99, 143.10, 135.31, 128.58, 126.18, 126.12 (2C), 125.73,
122.07, 116.19 (2C), 113.68, 70.47, 59.98. ESI-MS: m/z 333.0 [M+H]+.
4.1.14. 3-(4-(6-bromo-1H-benzo[d]imidazol-1-yl)phenoxy)propan-1-ol (4h)
85.7% yield. Oil. 1H NMR (400 MHz, DMSO-d6) δ 8.51 (s, 1H, Ar-H), 7.73 (d, J = 8.6 Hz, 1H, Ar-H),
7.65 (d, J = 1.7 Hz, 1H, Ar-H), 7.58 (d, J = 8.9 Hz, 2H, Ar-H), 7.44 (dd, J = 8.6, 1.8 Hz, 1H, Ar-H), 7.17 (d,
J = 8.9 Hz, 2H, Ar-H), 4.61 (t, J = 5.1 Hz, 1H, OH), 4.14 (t, J = 6.3 Hz, 2H, CH2), 3.60 (q, J = 6.0 Hz, 2H,
CH2), 1.91 (t, J = 6.3 Hz, 2H, CH2). 13C NMR (100 MHz, DMSO-d6) δ 158.86, 144.97, 143.10, 135.31,
128.52, 126.12 (2C), 125.72, 122.06, 116.16, 116.12 (2C), 113.68, 65.52, 57.70, 32.50. ESI-MS: m/z 347.0
[M+H]+.
4.1.15. 4-(4-(6-bromo-1H-benzo[d]imidazol-1-yl)phenoxy)butan-1-ol (4i)
52.2% yield. Oil. 1H NMR (400 MHz, DMSO-d6) δ 8.51 (s, 1H, Ar-H), 7.73 (d, J = 8.6 Hz, 1H, Ar-H),
7.65 (d, J = 1.7 Hz, 1H, Ar-H), 7.58 (d, J = 8.9 Hz, 2H, Ar-H), 7.44 (dd, J = 8.6, 1.8 Hz, 1H, Ar-H), 7.17 (d,
J = 8.9 Hz, 2H, Ar-H), 4.61 (t, J = 5.1 Hz, 1H, OH), 4.14 (t, J = 6.3 Hz, 2H, CH2), 3.60 (q, J = 6.0 Hz, 2H,
CH2), 1.91 (t, J = 6.3 Hz, 2H, CH2). 13C NMR (100 MHz, DMSO-d6) δ 158.86, 144.97, 143.10, 135.31,
128.52, 126.12 (2C), 125.72, 122.06, 116.16, 116.12 (2C), 113.68, 65.52, 57.70, 32.50. ESI-MS: m/z 347.0
[M+H]+.
4.1.16. methyl 2-(4-(6-bromo-1H-benzo[d]imidazol-1-yl)phenoxy)acetate (4j)
91.2% yield. mp 167-168 ºC. 1H NMR (400 MHz, DMSO-d6) δ 8.53 (s, 1H, Ar-H), 7.74 (d, J = 8.6 Hz,
1H, Ar-H), 7.66 (d, J = 1.6 Hz, 1H, Ar-H), 7.61 (d, J = 8.9 Hz, 2H, Ar-H), 7.44 (dd, J = 8.6, 1.8 Hz, 1H,
Ar-H), 7.18 (d, J = 8.9 Hz, 2H, Ar-H), 4.93 (s, 2H, CH2), 3.74 (s, 3H, CH3). 13C NMR (100 MHz, DMSO-d6)
δ 169.54, 157.70, 144.98, 143.12, 135.21, 129.31, 126.08 (2C), 125.77, 122.08, 116.32 (2C), 116.20, 113.69,

65.30, 52.37. ESI-MS: m/z 361.0 [M+H]+.
4.1.17. 4-(2-(4-(6-bromo-1H-benzo[d]imidazol-1-yl)phenoxy)ethyl)morpholine (4k)
88.7% yield. mp 115-118 ºC. 1H NMR (400 MHz, DMSO-d6) δ 8.52 (s, 1H, Ar-H), 7.73 (d, J = 8.6 Hz,
1H, Ar-H), 7.66 – 7.63 (m, 1H, Ar-H), 7.58 (d, J = 8.8 Hz, 2H, Ar-H), 7.44 (dd, J = 8.5, 1.7 Hz, 1H, Ar-H),
7.18 (d, J = 8.8 Hz, 2H, Ar-H), 4.19 (t, J = 5.1 Hz, 2H, CH2), 3.60 (s, 4H, CH2), 2.81 – 2.71 (m, 2H, CH2),
2.56 – 2.50 (m, 4H, CH2). 13C NMR (100 MHz, DMSO-d6) δ 158.58, 144.98, 143.11, 135.31, 128.70,
126.21, 126.12 (2C), 125.73, 122.07, 116.23 (2C), 116.17, 113.68, 66.58 (2C), 66.16, 57.34, 54.02 (2C).
ESI-MS: m/z 402.0 [M+H]+.
4.1.18. 4-(3-(4-(6-bromo-1H-benzo[d]imidazol-1-yl)phenoxy)propyl)morpholine (4l)
92.1% yield. mp 132-133 ºC. 1H NMR (400 MHz, DMSO-d6) δ 8.51 (s, 1H, Ar-H), 7.73 (d, J = 8.6 Hz,
1H, Ar-H), 7.64 (d, J = 1.6 Hz, 1H, Ar-H), 7.58 (d, J = 8.9 Hz, 2H, Ar-H), 7.44 (dd, J = 8.6, 1.8 Hz, 1H,
Ar-H), 7.16 (d, J = 8.9 Hz, 2H, Ar-H), 4.10 (t, J = 6.3 Hz, 2H, CH2), 3.60 – 3.57 (m, 4H, CH2), 2.45 (t, J =
7.0 Hz, 2H, CH2), 2.39 (s, 4H, CH2), 1.92 (p, J = 6.7 Hz, 2H, CH2). 13C NMR (100 MHz, DMSO-d6) δ
158.78, 144.97, 143.11, 135.30, 128.57, 126.12 (2C), 125.73, 122.07, 116.15 (2C), 113.68, 113.63, 66.70
(2C), 66.67, 55.25, 53.84 (2C), 26.27. ESI-MS: m/z 416.0 [M+H]+.
4.1.19. 2-(4-(6-bromo-1H-benzo[d]imidazol-1-yl)-2-methoxyphenoxy)ethan-1-ol(4m)
55.4% yield. Oil. 1H NMR (400 MHz, DMSO-d6) δ 7.84 (s, 1H, Ar-H), 7.44 (s, 1H, Ar-H), 7.26 (s, 1H,
Ar-H), 7.18 (d, J = 8.6 Hz, 1H, Ar-H), 7.14 (s, 1H, Ar-H), 4.93 (t, J = 5.4 Hz, 1H, OH), 4.07 (t, J = 5.0 Hz,
2H, CH2), 3.85 (s, 3H, CH3), 3.77 (q, J = 5.2 Hz, 2H, CH2). 13C NMR (100 MHz, DMSO-d6) δ 150.26,
150.07, 148.47, 136.26, 133.96, 129.14, 125.61, 122.41, 116.68, 116.25, 114.23, 114.05, 109.34, 70.97,
60.00, 56.33. ESI-MS: m/z 363.0 [M+H]+.
4.1.20. 3-(4-(6-bromo-1H-benzo[d]imidazol-1-yl)-2-methoxyphenoxy)propan-1-ol (4n)
73% yield. Oil. 1H NMR (400 MHz, DMSO-d6) δ 8.53 (s, 1H, Ar-H), 7.73 (d, J = 8.6 Hz, 1H, Ar-H),
7.71 – 7.70 (m, 1H, Ar-H), 7.44 (dd, J = 8.6, 1.8 Hz, 1H, Ar-H), 7.28 – 7.25 (m, 1H, Ar-H), 7.17 (s, 1H,
Ar-H), 7.16 (s, 1H, Ar-H), 4.59 (t, J = 4.9 Hz, 1H, CH2), 4.18 – 4.13 (m, 2H, CH2), 4.11 (d, J = 6.3 Hz, 2H,
CH2), 3.85 (s, 3H, OCH3), 1.93 (dp, J = 12.6, 6.3 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) δ 150.24,
148.46, 145.06, 143.09, 135.35, 128.69, 125.73, 122.02, 116.71, 113.87, 113.80, 109.34, 66.11, 57.81, 56.40,
32.58. ESI-MS: m/z 376.9 [M+H]+.
4.1.21. methyl 4-((6-bromoquinazolin-4-yl)amino)-2-methoxybenzoate (4o)
68.5% yield. Oil. 1H NMR (400 MHz, DMSO-d6) δ 8.53 (s, 1H, Ar-H), 7.73 (d, J = 8.6 Hz, 1H, Ar-H),
7.70 (d, J = 1.4 Hz, 1H, Ar-H), 7.44 (dd, J = 8.6, 1.5 Hz, 1H, Ar-H), 7.26 (d, J = 2.0 Hz, 1H, Ar-H), 7.20 (d,
J = 8.5 Hz, 1H, Ar-H), 7.16 (dd, J = 8.5, 2.1 Hz, 1H, Ar-H), 4.17 (t, J = 5.8 Hz, 2H, CH2), 3.85 (s, 3H,
OCH3), 3.62 – 3.57 (m, 4H, CH2), 2.74 (t, J = 5.4 Hz, 2H, CH2), 2.51 (s, 4H, CH2). 13C NMR (100 MHz,

DMSO-d6) δ 150.31, 148.26, 145.06, 143.09, 135.33, 128.95, 125.75, 122.03, 116.70, 116.18, 114.27,
113.86, 109.44, 67.03, 66.66, 57.44, 56.46, 54.13. ESI-MS: m/z 431.9 [M+H]+.
4.1.22. N-(5-bromo-2-methoxypyridin-3-yl)methanesulfonamide (6a)
4.1.23. N-(5-bromo-2-methoxypyridin-3-yl)-2,4-difluorobenzenesulfonamide (6b)
4.1.24. N-(2-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-3-yl)methanesulfonamide (7a)
4.1.25.
2,4-difluoro-N-(2-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-3-yl)benzenesulfonamide
(7b)
Compounds 6a, 6b, 7a and 7b were synthesized according to the procedure described in our our
previous article [15].
4.1.26. N-(5-(4-((2,4-difluorophenyl)amino)quinazolin-6-yl)-2-methoxypyridin-3-yl)methanesulfonamide
(8a)
A solution of the 4a (0.153 g, 0.5 mmol), 7a (0.164 g, 0.5 mmol), Cs2CO3 (0.33 g, 0.56 mm
ol) and Bis(triphenylphosphine)palladium(II) Dichloride (0.018 g, 0.025 mmol) in DMF (10 ml) und
er an atmosphere of N2 was stirred at 95 ºC for 4 h. DMF was removed under reduced pressure a
nd the residue was purified through a column chromatography on silica with chloroform/methanol (
1
V:V 20:1) as a white solid (0.13g, 57.0% yield). mp 109-111 ºC. H NMR (600 MHz, DMSO-d6)
δ 10.16 (s, 1H, NH), 8.50 (s, 1H, Ar-H), 8.45 (s, 1H, Ar-H), 8.33 (d, J = 2.0 Hz, 1H, Ar-H), 8.0
3 (s, 1H, Ar-H), 7.89 (d, J = 2.1 Hz, 1H, Ar-H), 7.83 (d, J = 8.4 Hz, 1H, Ar-H), 7.75 (s, 1H, A
r-H), 7.53 (d, J = 8.4 Hz, 1H, Ar-H), 5.03 (s, 1H, OH), 4.25 (t, J = 5.5 Hz, 2H, CH2), 3.96 (s,
3H, OCH3), 3.82 (t, J = 5.5 Hz, 2H, CH2), 3.05 (s, 3H, CH3). 13C NMR (150 MHz, DMSO-d6) δ
156.59, 144.65, 143.28, 140.59, 134.54, 133.39, 133.01, 131.27, 130.88, 125.41, 122.74, 122.01, 120.7
0, 118.88, 108.99, 60.37, 55.25, 54.09, 41.06. HRMS (ESI+) m/z calcd for C19H21N6O4S [M+H]+, 4
29.1356; found, 429.1340.
Compounds 8b-8i were synthesized according to the procedure described in 8a.
4.1.27.
2,4-difluoro-N-(5-(1-(1-(2-hydroxyethyl)-1H-pyrazol-4-yl)-1H-benzo[d]imidazol-6-yl)-2-methoxypyridin-3-y
l)benzenesulfonamide (8b)
58.6% yield, mp 85-88 ºC. 1H NMR (600 MHz, DMSO-d6) δ 10.28 (s, 1H, NH), 8.49 (s, 1H, Ar-H),
8.44 (s, 1H, Ar-H), 8.31 (s, 1H, Ar-H), 8.03 (s, 1H, Ar-H), 7.87 (s, 1H, Ar-H), 7.82 (d, J = 8.3 Hz, 1H,
Ar-H), 7.74 (d, J = 8.2 Hz, 1H, Ar-H), 7.70 (s, 1H, Ar-H), 7.56 – 7.51 (m, 1H, Ar-H), 7.50 – 7.48 (m, 1H,
Ar-H), 7.17 (t, J = 9.4 Hz, 1H, Ar-H), 5.01 (s, 1H, OH), 4.26 (t, J = 5.6 Hz, 2H, CH2), 3.85 – 3.82 (m, 2H,
CH2), 3.66 (s, 3H, OCH3). 13C NMR (150 MHz, DMSO-d6) δ 167.41, 166.95, 160.80 – 158.77 (m), 157.64,

143.09, 135.01, 133.46, 133.31, 132.41, 132.29 (d, J = 10.0 Hz), 132.25, 132.15, 130.80, 129.11, 125.38 (d,
J = 28.0 Hz), 123.77, 122.70, 121.87, 120.80, 119.87, 118.85, 112.25 (d, J = 23.3 Hz), 111.11, 109.00,
106.24 (t, J = 22.2 Hz), 60.39, 55.26, 53.76. HRMS (ESI+) m/z calcd for C24H21F2N6O4S [M+H]+, 527.1308;
found, 527.1307.
4.1.28.
N-(5-(1-(1-(3-hydroxypropyl)-1H-pyrazol-4-yl)-1H-benzo[d]imidazol-6-yl)-2-methoxypyridin-3-yl)methanes
ulfonamide (8c)
42.8% yield, mp 170-172 ºC. 1H NMR (600 MHz, DMSO-d6) δ 9.80 (s, 1H, NH), 8.51 (s, 1H, Ar-H),
8.49 (s, 1H, Ar-H), 8.36 – 8.32 (m, 1H, Ar-H), 8.03 (s, 1H, Ar-H), 7.90 (d, J = 2.0 Hz, 1H, Ar-H), 7.84 (d, J
= 8.3 Hz, 1H, Ar-H), 7.75 (s, 1H, Ar-H), 7.54 (d, J = 8.2 Hz, 1H, Ar-H), 4.68 (s, 1H, OH), 4.28 (t, J = 7.0
Hz, 2H, CH2), 3.96 (s, 3H, OCH3), 3.47 (t, J = 6.0 Hz, 2H, CH2), 3.05 (s, 3H, CH3), 2.01 (p, J = 6.4 Hz, 2H,
CH2). 13C NMR (150 MHz, DMSO-d6) δ 156.59, 144.67, 143.30, 140.78, 134.51, 133.27, 132.99, 131.41,
130.88, 124.89, 122.55, 122.01, 120.71, 118.92, 108.99, 58.15, 54.11, 49.72, 41.10, 33.45. HRMS (ESI+)
m/z calcd for C20H23N6O4S [M+H]+, 541.1464; found, 541.1469.
4.1.29.
2,4-difluoro-N-(5-(1-(1-(3-hydroxypropyl)-1H-pyrazol-4-yl)-1H-benzo[d]imidazol-6-yl)-2-methoxypyridin-
3-yl)benzenesulfonamide (8d)
52.7% yield, mp 98-101 ºC. 1H NMR (600 MHz, DMSO-d6) δ 10.32 (s, 1H, NH), 8.53 (s, 1H, Ar-H),
8.50 (s, 1H, Ar-H), 8.41 (d, J = 2.3 Hz, 1H, Ar-H), 8.05 (s, 1H, Ar-H), 7.94 (d, J = 2.3 Hz, 1H, Ar-H), 7.84
(d, J = 8.4 Hz, 1H, Ar-H), 7.77 (d, J = 2.7 Hz, 1H, Ar-H), 7.74 (d, J = 9.8 Hz, 1H, Ar-H), 7.61 – 7.56 (m, 1H,
Ar-H), 7.52 (dd, J = 8.4, 1.4 Hz, 1H, Ar-H), 7.20 (td, J = 8.5, 2.1 Hz, 1H, Ar-H), 4.68 (s, 1H, OH), 4.28 (t, J
= 7.1 Hz, 2H, CH2), 3.64 (s, 3H, CH3), 3.47 (t, J = 5.9 Hz, 2H, CH2), 2.03 (q, J = 6.6 Hz, 2H, CH2). 13C
NMR (150 MHz, DMSO-d6) δ 165.50 (dd, J = 254.2, 11.7 Hz), 162.75, 159.83 (dd, J = 257.7, 13.1 Hz),
157.65, 144.77, 143.37, 143.14, 135.08, 133.31, 132.42, 132.27 (d, J = 10.4 Hz), 130.80, 125.57 (dd, J =
14.1, 3.2 Hz), 124.91, 121.88, 120.78, 119.85, 118.88, 112.24 (d, J = 23.0 Hz), 109.00, 106.24 (t, J = 25.9
Hz), 58.16, 53.76, 49.73, 33.46. HRMS (ESI+) m/z calcd for C25H23F2N6O4S [M+H]+, 541.1464; found,
541.1469.
4.1.30. methyl
2-(4-(6-(5-((2,4-difluorophenyl)sulfonamido)-6-methoxypyridin-3-yl)-1H-benzo[d]imidazol-1-yl)-1H-pyrazo
l-1-yl)acetate (8e)
53.4% yield, mp 103-105 ºC. 1H NMR (600 MHz, DMSO-d6) δ 10.34 (s, 1H, NH), 8.55 (s, 1H, Ar-H),
8.52 (s, 1H, Ar-H), 8.28 (s, 1H, Ar-H), 8.11 (s, 1H, Ar-H), 7.85 (s, 1H, Ar-H), 7.83 (d, J = 8.4 Hz, 1H,
Ar-H), 7.75 (q, J = 8.5 Hz, 1H, Ar-H), 7.67 (s, 1H, Ar-H), 7.51 (d, J = 9.1 Hz, 1H, Ar-H), 7.49 (d, J = 9.5

Hz, 1H, Ar-H), 7.19 – 7.13 (m, 2H, Ar-H), 5.22 (s, 2H, CH2), 3.73 (s, 3H, OCH3), 3.66 (s, 3H, OCH3). 13C
NMR (150 MHz, DMSO-d6) δ 168.90, 166.15 – 164.09 (m), 162.56, 159.77 (dd, J = 257.1, 13.4 Hz), 157.56,
144.60, 143.30, 141.26 (d, J = 5.7 Hz), 134.44, 134.22 (2C), 132.93, 132.20 (d, J = 10.7 Hz), 130.66, 126.53,
126.18, 121.94, 120.82, 119.58, 112.02 (d, J = 21.1 Hz), 108.73, 106.08 (t, J = 25.9 Hz), 53.65, 53.53, 52.83.
HRMS (ESI+) m/z calcd for C25H21F2N6O5S [M+H]+, 555.1257; found, 555.1255.
4.1.31.methyl
3-(4-(6-(5-((2,4-difluorophenyl)sulfonamido)-6-methoxypyridin-3-yl)-1H-benzo[d]imidazol-1-yl)-1H-pyrazo
l-1-yl)propanoate (8f)
61.9% yield, mp 110-112 ºC. 1H NMR (600 MHz, DMSO-d6) δ 10.32 (s, 1H, NH), 8.52 (s, 1H, Ar-H),
8.51 (s, 1H, Ar-H), 8.41 (d, J = 2.2 Hz, 1H, Ar-H), 8.05 (s, 1H, Ar-H), 7.93 (d, J = 2.2 Hz, 1H, Ar-H), 7.84
(d, J = 8.4 Hz, 1H, Ar-H), 7.76 (d, J = 8.5 Hz, 1H, Ar-H), 7.74 (d, J = 5.2 Hz, 1H, Ar-H), 7.61 – 7.56 (m, 1H,
Ar-H), 7.52 (dd, J = 8.4, 1.4 Hz, 1H, Ar-H), 7.20 (td, J = 8.5, 2.2 Hz, 1H, Ar-H), 4.48 (t, J = 6.7 Hz, 2H,
CH2), 3.64 (s, 3H, OCH3), 3.63 (s, 3H, OCH3), 3.01 (t, J = 6.7 Hz, 2H, CH2). 13C NMR (150 MHz,
DMSO-d6) δ 171.52, 166.44 – 164.50 (m), 162.75, 159.83 (dd, J = 257.8, 13.3 Hz), 157.68, 144.69, 143.37,
143.07, 135.07, 134.49, 133.66, 132.45, 132.31, 132.24, 130.77, 125.18, 121.89, 120.81, 118.96, 112.23 (d,
J = 22.0 Hz), 108.95, 106.23 (t, J = 26.1 Hz), 53.75, 52.09, 48.04, 34.32. HRMS (ESI+) m/z calcd for
C26H23F2N6O5S [M+H]+, 569. 1413; found, 569.1392.
4.1.32.
N-(5-(1-(1-(2-(dimethylamino)ethyl)-1H-pyrazol-4-yl)-1H-benzo[d]imidazol-6-yl)-2-methoxypyridin-3-yl)-2,
4-difluorobenzenesulfonamide (8g)
42.6% yield, mp 80-82 ºC. 1H NMR (600 MHz, DMSO-d6) δ 11.49 (s, 1H, NH), 8.53 (s, 2H, Ar-H),
8.37 (d, J = 2.1 Hz, 1H, Ar-H), 8.06 (s, 1H, Ar-H), 7.91 (d, J = 2.3 Hz, 1H, Ar-H), 7.84 (d, J = 8.4 Hz, 1H,
Ar-H), 7.75 – 7.73 (m, 1H, Ar-H), 7.72 (d, J = 7.4 Hz, 1H, Ar-H), 7.57 – 7.54 (m, 1H, Ar-H), 7.52 (dd, J =
8.4, 1.4 Hz, 1H, Ar-H), 7.20 (dt, J = 8.4, 4.3 Hz, 1H, Ar-H), 4.38 (d, J = 6.2 Hz, 2H, CH2), 3.65 (s, 3H,
OCH3), 2.93 (d, J = 7.1 Hz, 2H, CH2), 1.91 (s, 6H, NCH3). 13C NMR (100 MHz, DMSO-d6) δ 165.33 (dd, J
= 253.9, 11.7 Hz), 159.81 (dd, J = 257.3, 13.4 Hz), 157.55, 142.05, 133.89, 133.47, 132.67, 132.25 (d, J =
10.7 Hz), 130.73, 125.83, 125.37, 125.25, 121.93, 121.85, 121.16, 120.77, 118.98, 118.52, 116.35, 113.84,
112.22, 111.98, 108.85, 106.11 (t, J = 26.1 Hz), 58.35, 53.69, 50.03, 45.08 (2C). HRMS (ESI+) m/z calcd for
C25H23F2N6O4S [M+H]+, 554.1780; found, 554.1780.
4.1.33.
2,4-difluoro-N-(2-methoxy-5-(1-(1-(2-morpholinoethyl)-1H-pyrazol-4-yl)-1H-benzo[d]imidazol-6-yl)pyridin
-3-yl)benzenesulfonamide (8h)

53.2% yield, mp 66-68 ºC. 1H NMR (600 MHz, DMSO-d6) δ 10.32 (s, 1H, NH), 8.53 (s, 1H, Ar-H),
8.51 (s, 1H, Ar-H), 8.39 (d, J = 2.1 Hz, 1H, Ar-H), 8.04 (s, 1H, Ar-H), 7.93 (d, J = 2.1 Hz, 1H, Ar-H), 7.84
(d, J = 8.4 Hz, 1H, Ar-H), 7.77 – 7.73 (m, 1H, Ar-H), 7.72 (s, 1H, Ar-H), 7.60 – 7.56 (m, 1H, Ar-H), 7.54 –
7.50 (m, 1H, Ar-H), 7.22 – 7.18 (m, 1H, Ar-H), 4.35 (t, J = 6.3 Hz, 2H), 3.64 (s, 3H, OCH3), 3.56 (s, 4H,
CH2), 2.88 – 2.78 (m, 2H, CH2), 2.51 (s, 4H, CH2). 13C NMR (150 MHz, DMSO-d6) δ 165.49 (dd, J = 253.8,
11.7 Hz), 159.83 (dd, J = 257.4, 13.3 Hz), 157.65, 144.74, 143.39, 143.07, 135.03, 134.56, 133.41, 132.45,
132.27 (d, J = 11.1 Hz), 130.81, 125.57 (d, J = 13.2 Hz), 125.24, 121.90, 120.82, 119.90, 118.90, 112.25 (d,
J = 21.7 Hz), 108.95, 106.23 (t, J = 25.8 Hz), 66.52 (2C), 57.93, 53.76, 53.50 (2C), 40.89. HRMS (ESI+) m/z
calcd for C28H28F2N7O4S [M+H]+, 596.1886; found, 596.1880.
4.1.34.
2,4-difluoro-N-(5-(1-(4-(2-hydroxyethoxy)phenyl)-1H-benzo[d]imidazol-6-yl)-2-methoxypyridin-3-yl)benzen
esulfonamide
(8i)
53.9% yield, mp 104-106 ºC. 1H NMR (600 MHz, DMSO-d6) δ 10.30 (s, 1H, NH), 8.53 (s, 1H, Ar-H),
8.35 (d, J = 2.4 Hz, 1H, Ar-H), 7.88 (d, J = 2.3 Hz, 1H, Ar-H), 7.85 (d, J = 8.4 Hz, 1H, Ar-H), 7.74 (q, J =
8.2, 7.8 Hz, 1H, Ar-H), 7.66 (d, J = 2.4 Hz, 1H, Ar-H), 7.66 – 7.64 (m, 2H, Ar-H), 7.60 – 7.55 (m, 1H,
Ar-H), 7.52 (dd, J = 8.4, 1.7 Hz, 1H, Ar-H), 7.20 (d, J = 8.8 Hz, 2H, Ar-H), 7.17 (d, J = 10.1 Hz, 1H, Ar-H),
4.95 (t, J = 5.5 Hz, 1H, OH), 4.10 (t, J = 4.9 Hz, 2H, CH2), 3.77 (q, J = 4.9 Hz, 2H, CH2), 3.63 (s, 3H,
OCH3). 13C NMR (100 MHz, DMSO-d6) δ 165.50 (dd, J = 253.9, 11.9 Hz), 159.81 (dd, J = 257.0, 13.4 Hz),
158.75, 157.58, 144.83, 143.74, 142.92, 134.76, 132.33, 132.20, 130.88, 129.03, 126.05 (2C), 125.89,
125.60 (d, J = 13.8 Hz), 121.84, 120.87, 119.95, 116.20 (2C), 112.25 (dd, J = 22.0, 3.0 Hz), 108.89, 106.23
(t, J = 26.3 Hz), 70.47, 60.02, 53.77. HRMS (ESI+) m/z calcd for C27H23F2N4O5S [M+H]+, 553.1352; found,
553.1349.
4.1.35.
2,4-difluoro-N-(5-(1-(4-(2-hydroxyethoxy)-3-methoxyphenyl)-1H-benzo[d]imidazol-6-yl)-2-methoxypyridin-
3-yl)benzenesulfonamide (8j)
42.7% yield, mp 58-60 ºC. 1H NMR (600 MHz, DMSO-d6) δ 10.30 (s, 1H, NH), 8.53 (d, J = 13.5 Hz,
1H, Ar-H), 8.26 (d, J = 12.2 Hz, 1H, Ar-H), 7.84 (s, 1H, Ar-H), 7.83 (s, 1H, Ar-H), 7.75 – 7.71 (m, 1H,
Ar-H), 7.67 (d, J = 12.3 Hz, 1H, Ar-H), 7.52 (s, 1H, Ar-H), 7.48 (s, 1H, Ar-H), 7.32 (d, J = 11.3 Hz, 1H,
Ar-H), 7.23 (d, J = 8.5 Hz, 1H, Ar-H), 7.20 (d, J = 8.2 Hz, 1H, Ar-H), 7.14 – 7.10 (m, 1H, Ar-H), 4.95 –
4.91 (m, 1H, OH), 4.09 – 4.06 (m, 2H, CH2), 3.87 (s, 3H, OCH3), 3.78 – 3.75 (m, 2H, CH2), 3.66 (s, 3H,
OCH3). 13C NMR (150 MHz, DMSO-d6) δ 165.21 (dd, J = 252.2, 11.1 Hz), 159.76 (dd, J = 257.2, 13.5 Hz),
157.47, 150.22, 148.25, 144.83, 143.66, 134.69, 132.21, 132.14, 130.75, 129.25, 126.17 (dd, J = 23.6, 7.1

Hz), 121.77, 120.81, 116.50, 114.03, 112.04 (d, J = 22.3 Hz), 109.15, 108.95, 106.12 (t, J = 26.1 Hz), 70.94,
63.24, 56.30, 53.69. HRMS (ESI+) m/z calcd for C28H25F2N4O6S [M+H]+, 583.1457; found, 541.1469.
4.1.36.
N-(5-(1-(4-(3-hydroxypropoxy)phenyl)-1H-benzo[d]imidazol-6-yl)-2-methoxypyridin-3-yl)methanesulfonami
de (8k)
47.6% yield, mp 77-79 ºC. 1H NMR (600 MHz, DMSO-d6) δ 9.35 (s, 1H, NH), 8.51 (s, 1H, Ar-H),
8.33 (d, J = 2.2 Hz, 1H, Ar-H), 7.88 (d, J = 2.1 Hz, 1H, Ar-H), 7.85 (d, J = 8.4 Hz, 1H, Ar-H), 7.66 (s, 1H,
Ar-H), 7.64 (d, J = 8.8 Hz, 2H, Ar-H), 7.56 – 7.53 (m, 1H, Ar-H), 7.17 (d, J = 8.8 Hz, 2H, Ar-H), 4.62 (t, J =
4.8 Hz, 1H, OH), 4.13 (t, J = 6.3 Hz, 2H, CH2), 3.96 (s, 3H, OCH3), 3.59 (q, J = 5.5 Hz, 2H, CH2), 3.07 (s,
3H, CH3), 1.91 (p, J = 6.2 Hz, 2H, CH2). 13C NMR (150 MHz, DMSO-d6) δ 158.66, 156.56, 144.76, 143.67,
141.41, 134.64, 132.70, 132.02, 130.98, 128.96, 125.99 (2C), 121.98, 121.62, 120.81, 116.08 (2C), 108.94,
65.46, 57.69, 54.19, 41.19, 32.51. HRMS (ESI+) m/z calcd for C23H25N4O5S [M+H]+, 469.1540; found,
469.1534.
4.1.37.
2,4-difluoro-N-(5-(1-(4-(3-hydroxypropoxy)phenyl)-1H-benzo[d]imidazol-6-yl)-2-methoxypyridin-3-yl)benz
enesulfonamide (8l)
51.2.0% yield, mp 72-74 ºC. 1H NMR (600 MHz, DMSO-d6) δ 10.30 (s, 1H, NH), 8.53 (s, 1H, Ar-H),
8.35 (d, J = 2.2 Hz, 1H, Ar-H), 7.88 (d, J = 2.1 Hz, 1H, Ar-H), 7.86 (d, J = 8.4 Hz, 1H, Ar-H), 7.77 – 7.73
(m, 1H, Ar-H), 7.66 (s, 1H, Ar-H), 7.66 – 7.61 (m, 2H, Ar-H), 7.56 (d, J = 8.8 Hz, 1H, Ar-H), 7.54 – 7.51
(m, 1H, Ar-H), 7.19 (d, J = 8.8 Hz, 2H, Ar-H), 7.16 (d, J = 2.3 Hz, 1H, Ar-H), 4.62 (t, J = 5.0 Hz, 1H, OH),
4.15 (d, J = 6.3 Hz, 2H, CH2), 3.64 (s, 3H, OCH3), 3.60 (q, J = 6.6, 5.7 Hz, 2H, CH2), 1.92 (p, J = 6.2 Hz, 2H,
CH2). 13C NMR (150 MHz, DMSO-d6) δ 165.48 (dd, J = 253.9, 11.6 Hz), 160.79 – 160.54 (m), 158.96 (d, J
= 13.7 Hz), 158.70, 157.59, 144.82, 143.73, 142.94, 134.83, 134.71, 132.31, 132.22, 130.87, 128.94, 126.05
(2C), 125.58 (dd, J = 14.3, 2.9 Hz), 121.84, 120.86, 119.92, 116.09 (2C), 112.24 (d, J = 22.2 Hz), 108.89,
106.23 (t, J = 26.2 Hz), 65.49, 57.71, 53.76, 32.53. HRMS (ESI+) m/z calcd for C28H25F2N4O5S [M+H]+,
567.1508; found, 567.1502.
4.1.38.
2,4-difluoro-N-(5-(1-(4-(3-hydroxypropoxy)-3-methoxyphenyl)-1H-benzo[d]imidazol-6-yl)-2-methoxypyridi
n-3-yl)benzenesulfonamide (8m)
52.6% yield, mp 69-71 ºC. 1H NMR (600 MHz, DMSO-d6) δ 10.30 (s, 1H, NH), 8.56 (s, 1H, Ar-H),
8.39 – 8.32 (m, 1H, Ar-H), 7.90 (s, 1H, Ar-H), 7.86 (d, J = 8.3 Hz, 1H, Ar-H), 7.78 – 7.73 (m, 1H, Ar-H),
7.71 (s, 1H, Ar-H), 7.57 (d, J = 9.4 Hz, 1H, Ar-H), 7.54 (d, J = 8.8 Hz, 1H, Ar-H), 7.36 – 7.31 (m, 1H,
Ar-H), 7.28 – 7.23 (m, 1H, Ar-H), 7.20 (d, J = 10.8 Hz, 1H, Ar-H), 7.17 (d, J = 9.5 Hz, 1H, Ar-H), 4.59 (s,

1H, OH), 4.15 – 4.12 (m, 2H, CH2), 3.87 (s, 3H, OCH3), 3.65 (s, 3H, OCH3), 3.60 (t, J = 5.5 Hz, 2H, CH2),
3.49 – 3.43 (m, 2H, CH2). 13C NMR (150 MHz, DMSO-d6) δ 165.50 (dd, J = 254.1, 11.7 Hz), 160.81 –
158.67 (m), 157.50, 150.21, 148.28, 144.84, 143.59, 142.81, 134.68, 134.56, 132.29, 132.21, 130.79, 129.06,
125.66 – 125.15 (m), 121.85, 120.83, 119.95, 116.59, 113.82, 112.24 (d, J = 21.8 Hz), 109.13, 109.01,
106.19 (t, J = 26.3 Hz), 66.09, 57.85, 56.37, 53.80, 32.53. HRMS (ESI+) m/z calcd for C29H27F2N4O6S
[M+H]+, 597.1614; found, 597.1610.
4.1.39.
2,4-difluoro-N-(5-(1-(4-(4-hydroxybutoxy)phenyl)-1H-benzo[d]imidazol-6-yl)-2-methoxypyridin-3-yl)benzen
esulfonamide (8n)
47.2% yield, mp 101-103 ºC. 1H NMR (600 MHz, DMSO-d6) δ 10.31 (s, 1H, NH), 8.57 (s, 1H, Ar-H),
8.35 (d, J = 2.3 Hz, 1H, Ar-H), 7.88 (d, J = 2.3 Hz, 1H, Ar-H), 7.86 (d, J = 8.4 Hz, 1H, Ar-H), 7.77 – 7.72
(m, 1H, Ar-H), 7.67 (s, 1H, Ar-H), 7.65 (s, 2H, Ar-H), 7.59 – 7.55 (m, 1H, Ar-H), 7.54 (dd, J = 8.4, 1.5 Hz,
1H, Ar-H), 7.19 (d, J = 2.9 Hz, 1H, Ar-H), 7.17 (d, J = 4.3 Hz, 2H, Ar-H), 4.49 (s, 1H, OH), 4.09 (t, J = 6.5
Hz, 2H, CH2), 3.64 (s, 3H, OCH3), 3.48 (t, J = 6.4 Hz, 2H, CH2), 1.80 (p, J = 6.6 Hz, 2H, CH2), 1.61 (p, J =
6.5 Hz, 2H, CH2). 13C NMR (150 MHz, DMSO-d6) δ 165.48 (dd, J = 253.9, 11.5 Hz), 159.81 (dd, J = 257.8,
13.1 Hz), 158.73, 157.61, 144.80, 143.41, 142.97, 134.85, 132.40, 132.29, 132.22, 130.83, 128.86, 126.08,
125.53, 121.95, 120.74, 119.91, 116.10, 112.24 (d, J = 22.3 Hz), 108.97, 106.23 (t, J = 26.0 Hz), 68.34,
60.84, 53.76, 29.43, 25.88. HRMS (ESI+) m/z calcd for C29H27F2N4O5S [M+H]+, 581.1665; found,
581.1679.
4.1.40. methyl
2-(4-(6-(5-((2,4-difluorophenyl)sulfonamido)-6-methoxypyridin-3-yl)-1H-benzo[d]imidazol-1-yl)phenoxy)ac
etate (8o)
68.6% yield, mp 78-80 ºC. 1H NMR (600 MHz, DMSO-d6) δ 10.30 (s, 1H, NH), 8.55 (s, 1H, Ar-H),
8.35 (d, J = 2.3 Hz, 1H, Ar-H), 7.88 (d, J = 2.3 Hz, 1H, Ar-H), 7.86 (d, J = 8.4 Hz, 1H, Ar-H), 7.77 – 7.72
(m, 1H, Ar-H), 7.70 – 7.67 (m, 2H, Ar-H), 7.66 – 7.64 (m, 1H, Ar-H), 7.59 – 7.55 (m, 1H, Ar-H), 7.53 (dd,
J = 8.4, 1.6 Hz, 1H, Ar-H), 7.20 (d, J = 8.9 Hz, 2H, Ar-H), 7.17 (dd, J = 8.5, 2.2 Hz, 1H, Ar-H), 4.94 (s, 2H,
CH2), 3.74 (s, 3H, OCH3), 3.64 (s, 3H, OCH3). 13C NMR (150 MHz, DMSO-d6) δ 169.57, 166.44 – 164.49
(m), 159.81 (dd, J = 257.4, 13.4 Hz), 157.61, 157.53, 144.83, 143.73, 142.99, 134.89, 134.59, 132.36,
132.26 (d, J = 11.0 Hz), 130.86, 129.74, 125.98 (2C), 125.66 – 125.48 (m), 121.90, 120.88, 119.89, 116.29
(2C), 112.25 (d, J = 23.8 Hz), 108.93, 106.24 (t, J = 26.1 Hz), 65.29, 53.76, 52.35. HRMS (ESI+) m/z calcd
for C28H23F2N4O6S [M+H]+, 581.1301; found, 581.1302.

4.1.41.
2,4-difluoro-N-(2-methoxy-5-(1-(4-(2-morpholinoethoxy)phenyl)-1H-benzo[d]imidazol-6-yl)pyridin-3-yl)be
nzenesulfonamide (8p)
47.6% yield, mp 68-70 ºC.1H NMR (600 MHz, DMSO-d6) δ 10.32 (s, 1H, NH), 8.52 (d, J = 4.4 Hz, 1H,
Ar-H), 8.38 – 8.30 (m, 1H, Ar-H), 7.89 – 7.86 (m, 1H, Ar-H), 7.75 – 7.72 (m, 1H, Ar-H), 7.66 (d, J = 8.8 Hz,
2H, Ar-H), 7.64 (s, 1H, Ar-H), 7.59 (d, J = 8.6 Hz, 1H, Ar-H), 7.56 (d, J = 9.2 Hz, 1H, Ar-H), 7.53 (d, J =
8.3 Hz, 1H, Ar-H), 7.23 – 7.19 (m, 2H, Ar-H), 7.17 (d, J = 7.1 Hz, 1H, Ar-H), 4.24 – 4.19 (m, 2H, CH2),
3.64 (s, 3H, OCH3), 3.61 (s, 4H, CH2), 2.79 – 2.75 (m, 2H, CH2), 2.53 (s, 4H, CH2). 13C NMR (150 MHz,
DMSO-d6) δ 165.46 (dd, J = 254.1, 11.1 Hz), 162.74, 161.07 – 158.74 (m), 158.56, 158.42, 157.56, 144.94,
144.79, 143.72, 142.77, 134.70, 134.62, 132.36, 132.29, 132.22, 130.86, 129.12, 126.09, 126.03 (2C),
116.20 (2C), 112.21 (d, J = 21.0 Hz), 108.87, 106.21 (t, J = 26.3 Hz), 66.54 (2C), 66.11, 57.34, 54.00 (2C),
53.75. HRMS (ESI+) m/z calcd for C31H29F2N5NaO5S [M+Na]+, 644.1750; found, [M+H]+, 644.1749.
4.1.42.
2,4-difluoro-N-(2-methoxy-5-(1-(3-methoxy-4-(2-morpholinoethoxy)phenyl)-1H-benzo[d]imidazol-6-yl)pyri
din-3-yl)benzenesulfonamide (8q)
41.8% yield, mp 92-94 ºC. 1H NMR (600 MHz, DMSO-d6) δ 10.30 (s, 1H, NH), 8.55 (s, 1H, Ar-H),
8.34 (s, 1H, Ar-H), 7.89 (s, 1H, Ar-H), 7.86 (d, J = 8.3 Hz, 1H, Ar-H), 7.74 (q, J = 8.1 Hz, 1H, Ar-H), 7.70
(s, 1H, Ar-H), 7.57 (d, J = 8.8 Hz, 1H, Ar-H), 7.53 (d, J = 8.7 Hz, 1H, Ar-H), 7.33 (s, 1H, Ar-H), 7.24 (d, J
= 8.1 Hz, 1H, Ar-H), 7.22 (d, J = 8.5 Hz, 1H, Ar-H), 7.17 (t, J = 7.6 Hz, 1H, Ar-H), 4.19 (t, J = 5.4 Hz, 2H,
CH2), 3.87 (s, 3H, OCH3), 3.65 (s, 3H, OCH3), 3.60 (s, 4H, CH2), 2.77 (t, J = 4.9 Hz, 2H, CH2), 2.54 (s, 4H,
CH2). 13C NMR (150 MHz, DMSO-d6) δ 165.45 (dd, J = 253.8, 11.6 Hz), 159.80 (dd, J = 257.5, 13.3 Hz),
157.52, 150.29, 148.01, 144.88, 143.73, 142.72, 134.68, 134.62, 132.31, 132.20, 130.82, 129.41, 125.61 (d,
J = 12.0 Hz), 121.80, 120.85, 120.13, 116.51, 114.29, 112.22 (d, J = 23.8 Hz), 109.23, 109.05, 106.23 (t, J =
25.9 Hz), 66.91, 66.56, 57.40, 56.43, 54.07, 53.77. HRMS (ESI+) m/z calcd for C32H32F2N5O6S [M+H]+,
652.2036; found, 652.2035.
4.1.43.
N-(2-methoxy-5-(1-(4-(3-morpholinopropoxy)phenyl)-1H-benzo[d]imidazol-6-yl)pyridin-3-yl)methanesulfon
amide (8r)
45.8% yield, mp 94-96 ºC. 1H NMR (600 MHz, DMSO-d6) δ 9.42 (s, 1H, NH), 8.52 (s, 1H, Ar-H),
8.32 (d, J = 2.0 Hz, 1H, Ar-H), 7.88 (d, J = 2.0 Hz, 1H, Ar-H), 7.86 (d, J = 8.4 Hz, 1H, Ar-H), 7.66 (d, J =
5.6 Hz, 2H, Ar-H), 7.64 (s, 1H, Ar-H), 7.55 (d, J = 8.3 Hz, 1H, Ar-H), 7.17 (d, J = 8.8 Hz, 2H, Ar-H), 4.11
(t, J = 6.2 Hz, 2H, CH2), 3.96 (s, 3H, OCH3), 3.58 (s, 4H, CH2), 3.07 (s, 3H, CH3), 2.45 (t, J = 7.1 Hz, 2H,
CH2), 2.43 – 2.33 (m, 4H, CH2), 1.94 – 1.90 (m, 2H, CH2). 13C NMR (150 MHz, DMSO-d6) δ 158.59,

156.55, 144.75, 143.68, 141.27, 134.64, 132.74, 131.88, 130.97, 129.01, 125.99 (2C), 121.98, 121.79,
120.82, 116.11 (2C), 108.93, 66.67 (2C), 55.26, 54.17, 53.85 (2C), 41.17, 26.30. HRMS (ESI+) m/z calcd for
C27H32N5O5S [M+H]+, 538.2199; found, 538.2099.
4.1.43.
2,4-difluoro-N-(2-methoxy-5-(1-(4-(3-morpholinopropoxy)phenyl)-1H-benzo[d]imidazol-6-yl)pyridin-3-yl)b
enzenesulfonamide (8s)
42.5% yield, mp 79-81 ºC. 1H NMR (600 MHz, DMSO-d6) δ 10.30 (s, 1H, NH), 8.52 (s, 1H, Ar-H),
8.33 (d, J = 2.3 Hz, 1H, Ar-H), 7.87 – 7.84 (m, 2H, Ar-H), 7.76 – 7.72 (m, 1H, Ar-H), 7.66 (s, 1H, Ar-H),
7.65 (s, 1H, Ar-H), 7.63 (s, 1H, Ar-H), 7.58 – 7.54 (m, 1H, Ar-H), 7.52 (dd, J = 8.4, 1.4 Hz, 1H, Ar-H), 7.18
(d, J = 8.9 Hz, 2H, Ar-H), 7.16 (dd, J = 8.5, 2.2 Hz, 1H, Ar-H), 4.12 (t, J = 6.3 Hz, 2H, CH2), 3.64 (s, 3H,
OCH3), 3.61 – 3.59 (m, 4H, CH2), 2.51 – 2.50 (m, 4H, CH2), 2.45 (s, 2H, CH2), 1.95 (q, J = 6.7 Hz, 2H,
CH2). 13C NMR (150 MHz, DMSO-d6) δ 172.47, 165.43 (dd, J = 254.0, 11.6 Hz), 162.75, 159.80 (dd, J =
257.6, 13.3 Hz), 158.58, 157.57, 144.84, 143.72, 142.67, 134.58, 132.36, 130.84, 129.02, 126.11, 126.06
(2C), 125.87 – 125.51 (m), 121.83, 120.87, 120.24, 116.11 (2C), 112.20 (d, J = 22.1 Hz), 108.87, 106.22 (t,
J = 26.2 Hz), 66.59, 66.43 (2C), 55.15, 53.66 (2C), 26.07. HRMS (ESI+) m/z calcd for C32H32F2N5O5S
[M+H]+, 636.2087; found, 636.2114.
4.2. Biological assay methods
4.2.1. Cell culture
T47D, HCT116 and MCF-7 cells were purchased from the American Type Culture Collection
(Manassas, VA, USA). Cells were grown in DMEM (T47D, MCF-7) or RPMI1640 (HCT116)
supplemented with 10% FBS and antibiotics-antimycotics (PSF; 100 units/mL penicillin G sodium, 100
μg/ml streptomycin and 250 ng/ml amphotericin B) in a humidified incubator containing 5% CO2 at 37 °C.
4.2.2. Antiproliferative activity
The activity was evaluated using the sulforhodamine B (SRB) cellular protein-staining method with
minor modifications. Briefly, cells were treated with various concentrations of compounds in 96-well plates
and incubated at 37 ºC in a humidified atmosphere with 5% CO2 for 72 h. After treatment, the cells were
fixed with 10% TCA solution, and cell viability was determined with SRB assay. The percentage of
cell-growth inhibition was calculated using the formulae below. The IC50 values were calculated using a
non-linear regression analysis (percent growth versus concentration). Percent growth inhibition = 100 - 100
× (ODsample - ODDay0) / (ODneg control - ODDay0).
4.2.3. PI3K enzymatic activity assay
The PI3K enzymatic activity were tested at Shanghai ChemPartner Co., Ltd.
4.2.4. Colony formation assay

HCT116 cells were seeded in a 6-well plates with a density of 3×103 cells/well and then were cultured
with different concentrations (0, 1.0, 2.5, 5.0 μM) of 8i about 2 weeks. Cells were fixed by ethanol and
stained with crystal violet.
4.2.5. Transwell and wound healing assays
For the invasion assay, dilute matrigel with DMEM (1:6) and then placed 60 μl in the upper chamber of
the 24-well plate, which was incubated at 37 ºC in a humidified atmosphere with 5% CO2 for 12-24 h.
HCT116 cells were resuspended in DMEM without serum (2×104/200 μl ), added 100 μl cells and 100 μl 8i
to each upper chamber, added 700 μl DMEM with 10% FBS to each lower chamber, and incubated at 37 ºC
in a humidified atmosphere with 5% CO2 for 48 h. Cells that crossed to the underside of the membrane were
fixed in 70% ethanol, infiltrated with 100% methanol, then stained with 0.1% crystal violet, and observed
and taked pictures under microscope. For wound healing assay, 2 ml HCT116 cells with a density of
5×105/well were placed in 6-well plate and incubated at 37 ºC in a humidified atmosphere with 5% CO2 for
12-24 h and grown to confluency. Then we used 200 μl tip to scratch cell monolayers to create a cell-free
zone. Washed each well 3 times with PBS buffer and added 2 ml 8i to each well, and then incubated at 37 ºC
in a humidified atmosphere with 5% CO2 for 48 h. Photographed at 0 h, 24 h, 48 h under the microscope to
observe cell migration.
4.2.6. Annexin V-FITC and propidium iodide (PI) double staining assay
Apoptotic cells were quantified with an annexin-V-fluorescein isothiocyanate (FITC)/propidium iodide
(PI) apoptosis detection kit. HCT116 cells were seeded in a 6-well plate (3×105 cells/well) and treated with
8i mentioned above. At the end of treatments, the cells were washed by PBS and harvested by trypsin
without EDTA. Annexin-V/PI double staining was performed according to the manufacturers' instructions.
Stained cells were analyzed with a flow cytometer. Annexin-V-/PI- was used to represent viable cells,
Annexin-V+/PI- was used to represent early apoptotic cells, Annexin-V+/PI+ was used to represent late
apoptotic cells.
4.2.7. Hoechst33342 staining assay
Hoechst33342 staining assay was performed according to manufacturers' instructions. HCT116 cells
were seeded in a 6-well plate (3×105 cells/well) and treated with 8i mentioned above. At the end of
treatments, the cells were washed by PBS 2 times, and then we added 1 ml Hoechst 33342 (c = 10 μg/ml) to
each well, which was incubated at 37 ºC in a humidified atmosphere with 5% CO2 for 15 min. The cells
were washed by PBS 2 times and we added 1 ml PBS to each well. Photographed under the microscope to
observe cell morphology and apoptosis.
4.2.8. Western blot analysis
Cells were seeded into 100mm dishes and allowed to adhere overnight prior to treatment. After

treatment, cells were collected and lysed in RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1%
NP-40, 0.5% sodium deoxycholate, 0.1% SDS), and centrifuged at 13,000 r for 15 min in the 4 ℃ centrifuge.
Supernatants were collected, and the total protein concentration was quantified with a bicinchoninic acid
(BCA) assay kit. The protein concentration was determined, equal amounts of proteins (30 μg) were loaded
onto SDS-PAGE gels, and separated proteins were transferred to PVDF membranes. After blocking with 5%
BSA at room temperature for 2 h, the membranes were incubated with primary antibodies against AKT and
p-AKT, a monoclonal antibody against β-actin was used as a protein loading control. The membranes were
washed three times with TBST buffer for 30 min, 10 min at a time, then incubated with HRP-conjugated
secondary antibody for 2 h. After washing with the TBST buffer again, membranes were scanned with the
Odyssey Infrared Imaging System.
4.2.9. Molecular docking studies
The specific operation steps are shown in our previous work [15].
Acknowledgments
Financial supported by Liaoning Natural Fund Guidance Plan (Grant No. 2019-ZD-0446 and Grant No.
201602701) is gratefully acknowledged.
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O
N
O
O
O
S
N
NH
F
O
N
O
HO
4
3
N
O
O
O
F
2
N
S
N
O
O
O
O
NH
N
H
1
S
N
N
NH
O
GSK2292767
4
3
N
N
N
2
N
H
1
O
O
8i
S
NH
PI3Kα 0.50 nM
PI3Kβ 1.9 nM
PI3Kγ 1.8 nM
PI3Kδ 0.74 nM
O
O
2
4
N
H
N
3
N
N
1
N
HS-173
Synthesis of 1,6-disubstituted-1H-benzo[d]imidazoles derivatives.
Cytotoxicity evaluation.
Evaluation of PI3K inhibition.
Study on anti-tumor mechanisms.