Optimization of novel benzofuro[3,2-b]pyridin-2(1H)-one derivatives as dual inhibitors of BTK and PI3Kδ

Article abstract of DOI:10.1016/j.ejmech.2018.12.055

BTK and PI3Kδ play crucial roles in the progression of leukemia, and studies confirmed that the dual inhibition against BTK and PI3Kδ could provide superior anticancer agents to single targeted therapies. Herein, a new series of novel benzofuro[3,2-b]pyridin-2(1H)-one derivatives were optimized based on a BTK/PI3Kδ inhibitor 2 designed by our group. Biological studies clarified that compound 6f exhibited the most potent inhibitory activity (BTK: IC₅₀ = 74 nM; PI3Kδ: IC₅₀ = 170 nM) and better selectivity than 2. Moreover, 6f significantly inhibited the proliferation of Raji and Ramos cells with IC₅₀ values of 2.1 μM and 2.65 μM respectively by blocking BTK and PI3K signaling pathways. In brief, 6f possessed of the potency for further optimization as an anti-leukemic drug by inhibiting BTK and PI3Kδ kinase.

Full text of DOI:10.1016/j.ejmech.2018.12.055

Accepted Manuscript
Optimization of novel benzofuro[3,2-b]pyridin-2(1H)-one derivatives as dual inhibitors
of BTK and PI3Kδ
Linyi Liu, Xinyu Li, Yu Cheng, Lianjian Wang, Huizhu Yang, Jiurong Li, Siying He,
shuangjie Wu, Qianqian Yin, Hua Xiang
PII:
S0223-5234(18)31101-2
DOI:
https://doi.org/10.1016/j.ejmech.2018.12.055
EJMECH 10989
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 28 September 2018
Revised Date: 8 December 2018
Accepted Date: 22 December 2018
Please cite this article as: L. Liu, X. Li, Y. Cheng, L. Wang, H. Yang, J. Li, S. He, shuangjie Wu, Q.
Yin, H. Xiang, Optimization of novel benzofuro[3,2-b]pyridin-2(1H)-one derivatives as dual inhibitors
of BTK and PI3Kδ, European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/
j.ejmech.2018.12.055.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Optimization of novel benzofuro[3,2-b]pyridin-2(1H)-one derivatives as dual
inhibitors of BTK and PI3Kδ
Linyi Liu1,2,3; Xinyu Li1,2; Yu Cheng1,2; Lianjian Wang1,2; Huizhu Yang1,2; Jiurong Li1,2; Siying
He1,2; shuangjie Wu2; Qianqian Yin3, **; Hua Xiang1,2, *,
1Jiangsu Key Laboratory of Drug Design and Optimization, China Pharmaceutical
University, 24 Tongjiaxiang, Nanjing 210009, China
2Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University,
24 Tongjiaxiang, Nanjing 210009, China
3Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University,
Pudong, Shanghai, 201210, China
*Corresponding author. Department of Medicinal Chemistry, School of Pharmacy, China
Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, PR China. Tel.: +86 025 83271096; Fax:
+86 025 83271096. E-mail address: xianghua@cpu.edu.cn (H. Xiang).
**Corresponding author. E-mail address: yinqq@shanghaitech.edu.cn.
Abstract
BTK and PI3Kδ play crucial roles in the progression of leukemia, and studies confirmed
that the dual inhibition against BTK and PI3Kδ could provide superior anticancer agents to
single targeted therapies. Herein, a new series of novel benzofuro[3,2-b]pyridin-2(1H)-one
derivatives were optimized based on a BTK/PI3Kδ inhibitor 2 designed by our group.
Biological studies clarified that compound 6f exhibited the most potent inhibitory activity
(BTK: IC50 = 74 nM; PI3Kδ: IC50 = 170 nM) and better selectivity than 2. Moreover, 6f
significantly inhibited the proliferation of Raji and Ramos cells with IC50 values of 2.1 µM
and 2.65 µM respectively by blocking BTK and PI3K signaling pathways. In brief, 6f
possessed of the potency for further optimization as an anti-leukemic drug by inhibiting BTK
and PI3Kδ kinase.
Keywords: Dual inhibitor; Oncology; B-cell malignancies; BTK; PI3Kδ.
1. Introduction
B cell receptor (BCR), a transmembrane receptor located on the cell surface of B
lymphocytes, is essential for normal B-cell development and adaptive immunity [1,2].
However, aberrantly activated BCR signaling supports the survival and growth of malignant
B cells [3]. Inhibition of BCR signaling has been used clinically to treat B-cell malignancies.
These kinases such as LYN, SYK, BTK and PI3K in BCR pathway have become potential
targets to develop kinase inhibitors for the treatment of B cell malignancies [4]. Among them,
BTK and PI3K are gaining increasing attention as effective targets to develop therapeutic
agents in clinic for the treatment of leukemia and lymphoma [5]. Ibrutinib as the first BTK
inhibitor approved by FDA in 2013, exhibited significant clinical benefit in treating leukemia
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and lymphoma, including chronic lymphocytic leukemia (CLL), mantle cell lymphoma
(MCL), and Waldenström’s macroglobulinemia (WM) [6]. Acalabrutinib, the second
generation of BTK inhibitor, has been approved by FDA in 2017 for the treatment of MCL [7].
Meanwhile, PI3Kδ inhibitor idelalisib, the first approved PI3K inhibitor, has been applied to
treat CLL and follicular lymphoma (FL) [8]. Recently, copanlisib, another PI3K inhibitor
against PI3Kα and δ has been approved by FDA for the treatment of FL [9].
Unfortunately, acquired mutations has occurred frequently with single target drugs in
disease progression and patients with drug resistance have a poor survival [11,12]. Nowadays,
this drawback has been deemed to be overcome by multiple target drugs. On one hand,
multiple target drugs could increase therapeutic effectiveness and keep cancer cells from
developing resistance. On the other hand, they could also avoid the risks involved in
multicomponent drugs or drug cocktails, such as poor patient compliance, unpredictable
pharmacokinetic or pharmacodynamics profiles and drug−drug interactions [12,13]. In view
of the cross-linking of BTK and PI3Kδ [14-16], the dual inhibition of BTK and PI3K is an
attractive strategy to achieve more durable patient responses as well as preventing or delaying
resistance [17,18]. Recently, Brahmam et al. reported a series of pyrazolopyrimidine
derivatives as novel dual inhibitors of BTK and PI3Kδ. MDVN1003 [19] (1, Figure 1) which
exhibited better oral bioavailability could reduce tumor growth in a B cell lymphoma
xenograft model, and was more effective compared with either ibrutinib or idelalisib,
although this compound showed relatively poor target selectivity [20]. Our group previously
has reported a series of novel benzofuro[3,2-b]pyridine-2(1H)-one derivatives as BTK/PI3Kδ
kinases inhibitors [21]. Further biological studies showed that compound 2 (Figure 1)
exhibited better activities against BTK and PI3Kδ. Moreover, compound 2 significantly
inhibited the growth of Raji cells and HL60 cells in vitro.
Figure 1. Synthesized BTK/PI3Kδ inhibitors.
According to the docking model of 2 with BTK and PI3Kδ (Figure S1) [21], we found
that 2 occupied the ATP pocket of BTK and PI3Kδ, and its skeleton benzofuro[3,2-b]pyridin-
2(1H)-one was extended towards the hinge region. Moreover, this binding mode exhibited
two key hydrogen bonds: between the furan oxygen atom and MET477 of BTK as well as
between the pyridine N and LYS799 of PI3Kδ. Nevertheless, the deficiency of interactions
resulted in unsatisfactory activity compared with the lead compounds BTK inhibitor QL47 (3)
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[22] and PI3K/mTOR inhibitor BEZ235 (4) [23] which both interact to BTK or PI3K with
three different binding sites, respectively (Figure 2). These studies clarified compound 2 as a
lead compound needs further optimization. Thus, we further designed and synthesized new
benzofuro[3,2-b]pyridine-2(1H)-one derivatives to explore the structure-activity relationship
and improve the biological activity based on the docking model of 2 (Figure 2).
Figure 2. Optimization of 2 based on docking studies. Blue mark represents
hydrogen bond receptor sites which form hydrogen bonds with residues of BTK
or PI3K, while red mark represents covalent sites.
2. Results and discussion
2.1 Chemistry
The general reactions used for the synthesis of the novel targeted derivatives are outlined
in Schemes 1~ 4. The starting material S5, 5-bromobenzofuran-3(2H)-one, was synthesized
via several common reaction from methyl salicylate [20], and S17,
5-bromofuro[2,3-b]pyridin- 3(2H)-one, was synthesized from 2-hydroxynicotinic acid
(Supplement information, Scheme 5). The furnished furan-3(2H)-one S5 was subsequently
condensated with p-nitroaniline, or m-nitroaniline affording intermediate S6a or S6b in a
quantitative yield. Then, the key intermediate S9a or S9b was obtained through the acetylation,
Vilsmeier-Haack cyclization and reduction reaction of S6 continually with corresponding reagents
as showed in Scheme 1. Further, the intermediate S9a was reacted with chloroacetyl chloride,
closely following dimethylamine to provide the dimethylamine acetamide intermediate S12 which
was coupled with arylboronic acid to afford the final targeted benzofuro[3,2-b]pyridine-2(1H)-one
derivatives 5a~5d (Scheme 2). Meanwhile, 5e was obtained from 5d under the catalysis of
trifluoroacetic acid. On the other hand, S9a was reacted subsequently with acryloyl chloride, and
2-fluoropyridine-5-boronic acid to provide acryloyl derivative 5f.
In addition, the synthesis of final meta-substitutional derivatives 6a~6g were performed as
depicted in Scheme 3 when the amino was placed in meta-position. Amino derivative S14 was
obtained through the Suzuki coupling reaction of S9b with 2-methoxy-5-pyridineboronic acid.
Then S14 was reacted with chloroacetyl chloride, following appropriate aniline to provide
alkylamino derivatives 6a~6d. When acryloyl chloride was introduced into the compound S9-B
firstly, the obtained intermediate S16 was reacted with arylboronic acid to afford the target
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molecules 6e~6g.
Scheme 1. Reagents and reaction conditions: a) p-nitroaniline, or m-nitroaniline, p-TSA, reflux,
100%; b) AcCl, NaH, 0 °C, 71%; c) POCl3, DMF, 0 °C to 90 °C, 35%; d) Fe/NH4Cl, reflux, 80%.
Scheme 2. Reaction conditions and reagents: a) Chloroacetyl chloride, K2CO3, DMF, 0 °C, 80%;
b) Dimethylamine, K2CO3, KI, DMF, 60 °C, 70%; c) ArB(OH)2, K2CO3, Pd(PPh3)2Cl2, dioxane,
100 °C, 60%~80%; d) Acryloyl chloride, K2CO3, DMF, 0 °C, 80%; e) 2-fluoropyridine-5-boronic
acid, K2CO3, Pd(PPh3)2Cl2, dioxane, 100 °C, 60%~80%; f) Trifluoroacetic acid, DCM, 60%.
Scheme 3. Reaction conditions and reagents: a) 2-methoxy-5-pyridineboronic acid, K2CO3,
Pd(PPh3)2Cl2, dioxane, 100 °C, 80%; b) Chloroacetyl chloride, K2CO3, DMF, 0 °C, 75%; c)
Appropriate aniline, K2CO3, KI, DMF, 60 °C, 60~90%; d) Acryloyl chloride, K2CO3, DMF, 0 °C,
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80%; e) ArB(OH)2, K2CO3, Pd(PPh3)2Cl2, dioxane, 100 °C, 60%~80%.
Finally, the synthesis of final furo[2,3-b:4,5-b']dipyridin-2(1H)-one derivatives 7a~7d were
prepared using the synthetic route showed in Scheme 4. The starting material S17 was allowed to
react with p-nitroaniline and acetylchloride to give acetamide intermediate S19 which in turn was
reacted with Vilsmeier-Haack reagent and Fe/NH4Cl to afford the furo[2,3-b:4,5-b']
dipyridin-2(1H)-one intermediate S21. Next, several reactions were performed to give 7a~7d
derivatives through the coupling reaction and acylation reaction. At the end, all of the synthesized
compounds have been confirmed by NMR and mass spectrometry.
Scheme 4. Reaction conditions and reagents: a) p-nitroaniline, p-TSA, reflux, 100%; b) AcCl,
NaH, 0 °C, 71%; c) POCl3, DMF, 0 °C to 90 °C, 35%, d) Fe/NH4Cl, reflux, 80%; e)
2-methoxy-5-pyridineboronic acid, K2CO3, Pd(PPh3)2Cl2, dioxane, 100 °C, 80%; f) Chloroacetyl
chloride, K2CO3, DMF, 0 °C, 70%; g) appropriate aniline, K2CO3, KI, DMF, 60 °C, 60~90%; h)
Acyl chloride, K2CO3, DMF, 0 °C, 70%; i) 3-pyridylboronic acid, K2CO3, Pd(PPh3)2Cl2, dioxane,
100 °C, 80%.
2.2 Biological activity
2.2.1 Anticancer activity and BTK, PI3Kδ assays
All these compounds were evaluated for their activity against BTK and PI3Kδ enzymes using
ADP-Glo™ Kinase Assay. For comparison, BTK inhibitor ibrutinib and PI3K inhibitor BEZ235
were also tested as reference compounds. As shown in Table 1, these compounds effectively
inhibited BTK with different levels at the concentrations of 200 nM, and part of them also
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inhibited PI3Kδ. In particular, compound 6f exhibited excellent inhibition with IC50 values of 74
nM against BTK and 170 nM against PI3Kδ, respectively. SAR analysis revealed that pyridyl
substituent was significantly beneficial to anti-BTK activity, 5b and 6f possessed of better
inhibitory activity, with IC50 values of 50 nM and 74 nM, respectively. Replacement of pyridyl
substituent in 5b with other heterocyclic groups or analogues yielded 5a or 5c~e, which were less
potent than 5b in inhibition of BTK. When attaching methoxy group to C-2 of pyridyl substituent
in 6f, the generated compound 6e achieved an IC50 value of 0.08 nM in inhibition of BTK and
exhibited similar inhibitory activity compared to 6f. While introducing fluorine in pyridyl group,
the inhibitory activities against BTK of compound 6g declined significantly, indicating that the
pyridyl group could achieve good potent BTK inhibitory activity while the stronger electron
withdrawing group F was inappropriate to be installed on the C-2’ position of pyridine.
Furthermore, replacement of acrylamide group in 6e with alkylamine generated 6a~6d, which
result in the decrease of inhibitory potency. This demonstrated locating the acrylamide group at
meta-position of phenyl ring was beneficial for efficient inhibition against BTK. Actually, the
acrylamide group was also beneficial to anti-PI3Kδ activity, these acryloyl analogues 5f, 6e, 6f, 6g
and 7d exhibited different PI3Kδ inhibitory activity. However, alkyl amino substituents, such as
dimethylamino (5b), morpholine (6c) and piperidine (7c), were unfavorable to anti-PI3Kδ activity.
The introduction of N at C-6 position (derivatives 7a-~7d) did not improve the inhibitory activity
of these analogs towards either BTK or PI3Kδ as depicted in Table 1, indicating the N atom was
inappropriate to be installed here.
Based on the encouraging enzymes inhibitory activities of the newly synthesized analogs,
the anticancer activity in vitro of these analogs was assessed using two typical B cell
lymphoblastic leukemic cell lines Raji (Burkitt's lymphoma cell) and Ramos (Burkitt's
lymphoma cell). These cell lines were chosen because they both express BTK and PI3K. The
results were summarized in Table 1. The tested compounds showed variable anticancer
activities against these two cell lines. Compound 6f which showed the most dual potency
against BTK and PI3Kδ exhibited slightly stronger inhibitory activity against Raji (IC50 = 2.1
µM) and Ramos (IC50 = 2.65 µM) cells. Furo[2,3-b:4,5-b']dipyridin-2(1H)-one derivatives
7a~7c showed minimal activity in Raji cells, indicating that the introduction of N at C-6
position adversely affected antiproliferative activity. These data taken together showed that
this new series of furo[3,2-b]pyridine derivatives inhibited B-cell lymphoblastic leukemic
cells by serving as potent dual inhibitors of BTK and PI3K
effective agent with anti-lymphoma cell activity.
δ, and compound 6f was an
Table 1. Effect of compounds on BTK, PI3Kδ and the cell viability of B cells: Raji and Ramos
cell lines.
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Anticancer
BTK
PI3Kδ
Inh%/200
activity IC50/µM
Entry
Ar
R
X
Inh%/200 nM IC50/µM
IC50/µM
Raji
18.8
7.1
Ramos
nM
5a
5b
5c
5d
5e
5f
C
C
C
C
C
C
C
C
C
C
C
C
C
N
N
N
N
47.2±2.9
63.4±1.3
51.7±5.4
49.2±2.1
33.1±4.8
32.1±4.1
51.4±0.7
55±0.1
-
NI
-
-
0.050
NI
NI
NI
NI
-
3.43
0.150
-
1.8
-
-
-
7.0
-
-
-
-
2.4
-
31.7±3.1
NI
1.27
7.3
1.65
6a
6b
6c
6d
6e
6f
0.153
0.119
-
-
18.0
7.2
-
NI
-
-
37.6±0.7
19±0.1
NI
-
>40
20.8
2.4
-
-
-
NI
-
67.3±2.6
86.8±1.3
40.2±4.1
58.8±1.9
32±4.2
0.08
0.074
0.55
0.150
-
11.3±3.0
58.1±1.4
34.0±2.9
NI
-
0.17
0.92
-
1.3
2.65
1.86
-
2.1
N
6g
7a
7b
7c
7d
2.2
>40
33.7
>40
7.4
NI
-
-
55±2.0
0.125
-
NI
-
-
38.7±5.5
34.2±3.9
1.0
1.33
ibrutinib
BEZ235
-
-
0.0007
-
-
-
-
14.5
0.22
5.8
0.022
0.0068
NI: no inhibition
2.2.2 Effect of 6f on Raji cell viability
As shown in Table 1, compound 6f exhibited the most potent biological activity, thus we
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further investigated the effect of 6f on cell viability of Raji cells. As depicted in Figure 3, ibrutinib
didn’t significantly inhibit the growth of Raji cells at the concentration of 5 µM before 24h, but
induced substantial suppression of cell viability after 24h and reached the maximum inhibition
after 48h. In addition, PI3K inhibitor BEZ235 exhibited a rapid and robust anti-proliferative effect
on Raji cells at 5 µM, superior to that of ibrutinib. Importantly, 6f displayed superior potency to
that of ibrutinib or BEZ235 with more rapid suppressive effect. This result indicated that 6f could
significantly suppress the growth of Raji cells in time-dependent manner.
Figure 3. Effect of compounds on the temporal dependence of Raji cells viability
2.2.3 Selectivity of 6f on PI3K isoforms and mTOR
Based on its impressive dual BTK/PI3Kδ kinase inhibitory activity and anti-proliferative
effects, compound 6f was selected for further study. It’s known that BEZ235 is a pan inhibitor of
PI3K and mTOR (the key kinase downstream of PI3K), so the selectivity of compound 6f was
evaluated against PI3K isoforms including PI3Kα, PI3Kβ, PI3Kγ as well as mTOR. The results in
Table 2 showed that 6f was less potent than BEZ235 against PI3K. Nevertheless, 6f displayed
more potent inhibition and higher selectivity to PI3Kδ compared with compound 2. Moreover, 6f
didn’t inhibit mTOR but compound 2 has IC50 value of 228 nM. These data demonstrated that
shifting the acryloyl group in compound 2 to meta-position was a reasonable optimization.
Table 2. Selectivity of 6f to PI3K isoforms and mTOR
IC50 /nM
PI3Kα
855
254
4
PI3Kβ
2821
838
PI3Kγ
2183
684
5
mTOR
>10000
228
Entry
PI3K
170
275
7
δ
6f
2
BEZ235*
75
6
* Data from reference [21].
2.2.4 Effect of 6f on BTK and PI3K mediated signaling pathway
In addition, the effect of 6f on BTK and PI3K mediated signaling pathways in Ramos cells
was further studied (Figure 4). The phosphorylation of BTKTyr223 and its downstream signaling
factor PLCγ-2 was significantly up-regulated under anti-IgM stimulation, but were inhibited
substantially in concentration-dependent way following the treatment of 6f or ibrutinib (Figure 4a).
In addition, 6f also blocked PI3K pathway (Figure 4b). The phosphorylation of Ser473 of Akt and
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Ser2481 of mTOR was inhibited with the 6f treatment, although the inhibitory activity is weaker
than BEZ235. All the result clarified that 6f could effectively block the BCR signaling by through
inhibiting BTK and PI3K signaling pathway, thereby suppressing Raji and Ramos cell
proliferation.
Figure 4 Effect of 6f on BTK and PI3K mediated signaling pathway in the Ramos
cell line
2.2.5 Effect of 6f on cell apoptosis and cycle
In order to explore the anti-proliferative mechanism of 6f on B-cell leukemia cells, the effect
on apoptosis and on the cell cycle distribution of 6f on Ramos cells was detected using flow
cytometry analysis. As shown in Figure 5, BEZ235 treatment significantly induced Ramos cell
apoptosis (Figure 5d) while ibrutinib treatment only leaded to slight apoptosis after 48h incubation
(Figure 5b). Furthermore, 6f induced signally apoptosis of Ramos cells (positive Annexin-V% was
89.7%) with the low dose of 5 µM (Figure 5c). Moreover, 6f could induce the growth arrest of
Ramos cells at the G0/G1 phase compared with vehicle treatment. The G0/G1 phase arrest was also
found with the treatment of ibrutinib or BEZ235.
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Figure 5. Compound 6f induced Ramos cell apoptosis in vitro. The cells were
incubated with compound 6f (5 µM) for 48h, and the cells were stained with
annexin V/FITC, followed by flow cytometry analysis.
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Figure 6. Effect of 6f (5 µM) on Ramos cell cycle arrest detected by flow
cytometry assay.
2.3 ADME properties study
Next, the preADMET server was used to predict ADME (Table 3). The calculated results
show that the compound 6f has good predicted solubility and better extracorporeal colon
cancer cell permeability (caco-2) than that of BEZ235 and QL47, while human intestinal
absorption is comparable and all values are greater than 97% [24]. All the data suggested that
6f may have good predicted oral absorption and utilization. In addition, 6f also showed good
plasma protein binding rate (PPB, > 90%), but low BBB value (< 0.1), indicating that the
compound has a long half-life and is less likely to be toxic to CNS. MDCK is an index to
investigate the renal efflux of drugs, and the general value greater than 25 indicates better
efflux [25,26]. From the predicted results, the three compounds showed no obvious efflux
effect. In addition, preADMET provides information related to CYP450 metabolism.
According to the results, 6f is not a metabolic substrate of CYP_2D6 and CYP_3A4, which
has better metabolic stability compared with BEZ235 and QL47. These data indicated that 6f
has better ADME properties than BEZ235 and QL47.
Table 3. Predicted ADME properties of BEZ235, QL47 and 6f
Compounds
SN
ADME properties
BEZ235
6.41
QL47
6f
Water solubility in buffer system (SK
atomic types, mg/L)
1
2
1.49
13.39
in vitro Caco-2 cell permeability
(nm/sec)
27.82
38.77
42.64
Human intestinal absorption (HIA, %)
in vitro plasma protein binding (%)
98.03
93.83
97.76
98.22
97.08
94.47
3
4
in vivo blood-brain barrier penetration
(C.brain/C.blood)
5
6
0.14
0.04
0.05
0.08
0.01
0.39
in vitro MDCK cell permeability
(nm/sec)
CYP_2D6_substrate
CYP_3A4_inhibition
CYP_3A4_substrate
Non
Non
Non
Non
Non
7
8
9
Inhibitor
Substrate
Inhibitor
Substrate
2.4 Docking study
The biological studies above highlighted the utility of the newly synthesized
benzofuro[3,2-b]pyridine-2(1H)-one derivatives as anti-leukemia agents. Thus, a molecular
docking study was further performed in an attempt to gain some structural insights into their
potential binding patterns and possible interactions with both BTK and PI3K
Accordingly, the most active derivative 6f was docked inside the active sites of both BTK and
PI3K kinases. As shown in Figure 7a, it could be found that the furan oxygen atom of 6f
δ kinases.
δ
formed hydrogen bond with Met477 of BTK. Meanwhile, the acrylamide group formed
covalent bonds with cysteine residue C481. Obviously, the covalent bond could significantly
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increase the affinity of 6f to BTK compared to 2. In contrast to BTK, the inhibitory activity of
6f against PI3Kδ was owed to the interaction of the pyridine with LYS799 and the lactam
with SER831 (Figure 7b). Compared with 2, the added interaction of lactam with SER831
improved the inhibitory activity of 6f against PI3Kδ.
Figure 7. The docking model of 6f with BTK (a, PDB: 3PIY) and PI3Kδ (b, 5O83)
3. Conclusion
In this study, a novel series of benzofuro[3,2-b]pyridin-2(1H)-one derivatives based on
previous studies were synthesized and screened for their anticancer potential against two
cancer cell lines at cellular level and two kinases at biochemical level. Among the newly
synthesized analogs, compound 6f exhibited the best dual BTK/PI3Kδ kinase inhibitory
activity along with impressive anti-proliferative effects in Raji and Ramos leukemia cell lines.
Additional studies identified 6f significantly blocked the BCR/BTK pathway and
PI3K/Akt/mTOR pathway. Moreover, 6f also significantly arrested the cell cycle distribution
and induced cell apoptosis. And the preADME server predicted the ADME properties of the
obtained compound 6f. The docking studies were performed to predict the possible binding
patterns of the potent compound 6f into the ATP-active sites of BTK and PI3Kδ kinases.
Overall, we obtained a more potent compound 6f based on the optimization of its derivative 2.
The docking simulation study, along with the in vitro assay results identified a promising duel
BTK/ PI3Kδ inhibitor 6f for the further development in the treatment of B-cell lymphoblastic
leukemia.
4. Experimental protocols
4.1. Chemistry
Chemical reagents and solvents were obtained from commercial sources. Solvents were dried
by standard methods when necessary. Reactions were monitored by thin-layer chromatography
(TLC) using precoated silica gel plates (silica gel GF/UV 254), and spots were visualized under
UV light (254 nm). Melting points (uncorrected) were determined on a Mel-TEMP II melting
point apparatus and are uncorrected. 1H-NMR and 13C-NMR spectra were recorded with a Bruker
Avance 300 MHz spectrometer at 300 MHz and 75 MHz, respectively in DMSO-d6 or CDCl3. MS
spectra or high-resolution mass spectra (HRMS) were recorded on an Agilent 1946A-MSD (ESI)
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Mass Spectrum or Agilent 6230 Series Accurate-Mass Time-Of-Flight (TOF) LC/MS. Chemical
shifts were reported on the d scale and J values were given in Hz.
4.1.1 General procedures for the synthesis
4.1.1.1 General procedures A: Reduction of nitroarenes.
A suspension of nitroarene S8 or S20 in ethanol was heated at reflux. To this mixture was
added iron (10 equiv) followed by a solution of NH4Cl (10 equiv, 2.5 N) in H2O. The resulting
suspension was heated at reflux for 2 h. The hot mixture was then filtered through a Celite pad,
and the filtrate was evaporated under vacuum. The residue was dissolved in EtOAc and washed
with H2O, and the aqueous phase was further extracted with ethyl acetate (2×20 mL). The organic
extracts were combined, dried over Na2SO4, filtered, and evaporated under vacuum to obtain
compounds S9 or S21.
4.1.1.2 General procedures B: N-acetylation.
To a solution of arylamine in dimethylformamide at 0 °C was subsequently added K2CO3 (2
equiv), then dropwise added acyl chloride (1.2 equiv). The solution was stirred for 1 h at room
temperature and quenched with H2O (150 mL). The aqueous phase was further extracted with
ethyl acetate (2×20 mL). The organic extracts were combined, dried over Na2SO4, filtered and
concentrated, the residue was subjected to column purification (CH2Cl2/ MeOH) to furnish the
desired compounds.
4.1.1.3 General procedures C: halide displacement by amine
To a solution of chloracetyl compounds in dimethylformamide was subsequently added
K2CO3 (2 equiv) and KI (catalytic amount), then added the amine (5 equiv). The solution was
stirred for 2 h at r.t. and quenched with H2O (150 mL). The aqueous phase was further extracted
with ethyl acetate (2×20 mL). The organic extracts were combined, dried over Na2SO4, filtered
and concentrated, the residue was subjected to column purification (CH2Cl2/ MeOH) to furnish the
desired compounds.
4.1.1.4 General procedures D: Suzuki coupling
To a solution of bromoaryl compounds in 1,4-dioxane at room temperature was subsequently
added PdCl2(Ph3P)2 (0.1 equiv), K2CO3(3 equiv), and boronic acids or pinacol boronate esters and
a few drops of water. After degassing, the resulting mixture was heated to 80 °C for 4-12 h before
cooling to room temperature and filtering through Celite. Upon removal of the solvents, the
residue was subjected to column purification (CH2Cl2/ MeOH) to furnish the desired compounds.
4.1.2 The synthesis of series 5a~5f compounds
4.1.2.1 5-bromo-N-(4-nitrophenyl)benzofuran-3-amine (S6a)
To a solution of compound S5 (1.3 g, 6.1 mmol) in toluene was subsequently paranitroaniline
13

ACCEPTED MANUSCRIPT
(0.9 g, 6.l mmol). The resulting suspension was heated at reflux for 2 h with and concentrated,
1
then the resulting yellow precipitate was recovered by filtration. (yield: 100%, 2.03 g). H-NMR
(300MHz, d6-DMSO): δ ppm 9.19 (s, 1H), 8.33 (s, 1H), 8.10 (d, J = 9.2 Hz, 2H), 7.89 (d, J =
1.95Hz,1H), 7.58 (d, J = 8.7 Hz, 1H), 7.52 (dd, J = 1.9, 8.7 Hz, 1H), 7.04 (d, J = 9.2 Hz, 2H).
4.1.2.2 N-(5-bromobenzofuran-3-yl)-N-(4-nitrophenyl)acetamide (S7a)
To a solution of compound S6a (0.4 g, 1.2 mmol) in dimethylformamide was subsequently
added 60% sodium hydride (86 mg, 2.16 mmol) in batches at ice bath. Until there is no bubble, the
acetylchloride (86 mg, 2.16 mmol) was dropped slowly into the reaction, then stirred 0.5h
continually. The reaction mixture was poured into water and was further extracted with ethyl
acetate. The organic extracts were combined, dried over Na2SO4, filtered, and evaporated under
vacuum and was subjected to column purification (CH2Cl2/ MeOH) to furnish the desired
compound S7 (yield: 71%, 0.32 g), MS (ESI, m/z): 374 [M+H]+.
4.1.2.3 8-bromo-1-(4-nitrophenyl)benzofuro[3,2-b]pyridin-2(1H)-one (S8a)
To dimethylformamide (2.8 ml, 36.6 mmol) was added phosphoryl chloride (3.4 ml, 36.6
mmol) slowly at ice bath. After finished, the reaction mixture stirred 0.5h continually. Then the S7
(6.85 g, 18.3 mmol) in dimethylformamide was added into the reaction solution, which was
subsequently heated at 90 °C for 2h. The reaction mixture was cooled and poured into water,
further extracted with ethyl acetate. The organic extracts were combined, dried over Na2SO4,
filtered, and evaporated under vacuum and was subjected to column purification (CH2Cl2/MeOH)
to furnish the desired compound S8 (yield: 35.5%, 2.5 g). 1H-NMR (300MHz, d6-DMSO): δ ppm
8.54 (d, J = 8.94 Hz, 2H), 8.22 (d, J = 9.87 Hz, 1H), 7.92 (d, J = 8.94 Hz, 2H), 7.76 (d, J = 8.91
Hz, 1H), 7.63 (dd, J = 1.74, 8.91 Hz, 1H), 6.72 (d, J = 9.87 Hz, 1H), 6.3 (d, J = 1.74 Hz, 1H).
4.1.2.4 8-bromo-1- (4-aminophenyl)-benzofuro [3,2-b]pyridin-2(1H)-one (S9a）
The preparation of S9a was from S8a according to the general procedure A, yellow solid
1
(yield: 80%). H-NMR (300 MHz, d6-DMSO): δ ppm 8.09 (d, J = 9.8 Hz, 1H), 7.69 (d, J = 8.9
Hz, 1H), 7.58 (dd, J = 8.9, 2.0 Hz, 1H), 7.08 (d, J = 8.6 Hz, 2H), 6.78 (d, J = 8.6 Hz, 2H), 6.61 (d,
J = 9.8 Hz, 1H), 6.39 (d, J = 1.9 Hz, 1H), 5.58 (s, 2H). MS (ESI, m/z), [M+Na]+: 377.
4.3.5
N-(4-(8-bromo-2-oxobenzofuro[3,2-b]pyridin-1(2H)-yl)phenyl)-2-(dimethylamino)aceta–
mide (S12)
The preparation of S12 was from S9a according to the general procedure A and B, yellow
solid (yield: 70%). 1H-NMR (300 MHz, d6-DMSO): δ ppm 9.41 (s, 1H), 7.93 (d, J = 8.7Hz, 2H),
7.80 (d, J = 9.7 Hz, 1H), 7.47 (dd, J = 2.0, 8.9 Hz, 1H), 7.42 (d, J = 8.7 Hz, 2H), 6.75 (d, J = 9.7
Hz, 1H), 6.52 (d, J = 1.8 Hz, 1H), 3.16 (s, 3H), 2.44 (s, 6H).
4.1.2.5
1-(4-dimethylamino-acetamido-phenyl)-8-(4-indol)-benzofuro[3,2-b]pyridin-2(1H)-one
(5a)
1
General procedure D, yellow solid (yield: 54%). M.p. 146-151 °C; H-NMR (300 MHz,
d6-DMSO): δ ppm 11.21 (s, 1H), 9.93 (s, 1H), 8.12 (d, J = 9.7, 2H), 7.95 (d, J = 8.7 Hz, 2H), 7.78
(d, J = 8.6 Hz, 1H), 7.72 (dd, J = 1.6, 8.3 Hz, 1H), 7.48 (d, J =8.7 Hz, 2H), 7.29 (t, J = 2.7 Hz, 1H),
14

ACCEPTED MANUSCRIPT
7.08 (t, J= 7.4 Hz, 1H), 6.92 (d, J = 6.9 Hz, 1H), 6.63 (s, 1H), 6.62 (d, J = 9.7 Hz, 1H), 6.18 (s,
1H), 3.11 (s, 2H), 2.29 (s, 6H). 13C-NMR (75 MHz, d6-DMSO): δ ppm 168.92, 161.05, 154.33,
139.69, 138.94, 136.27, 136.06, 132.46, 131.61, 129.32, 128.34, 128.14, 125.70, 125.03, 121.19,
120.19, 119.30, 118.76, 118.49, 112.77, 110.87, 99.94, 63.34, 45.31. MS (ESI, m/z): 424 [M-H]-.
4.1.2.6 1-(4- dimethylamino-acetamido-phenyl)-8-(3-pyridinyl)-benzofuro[3,2-b]pyridin-2(1H)-
one (5b)
1
General procedure D, yellow solid (yield: 70%). M.p. 196-200 °C; H-NMR (300 MHz,
d6-DMSO): δ ppm 11.21 (s, 1H), 10.08 (s, 1H), 8.13 (d, J = 10.0 Hz, 1H), 7.93 (d, J = 8.1 Hz, 2H),
7.80 (m, 2H), 7.70 (m, 1H), 7.47 (d, J = 8.1 Hz, 2H), 7.40 (m, 2H), 6.62 (d, J= 9.7 Hz, 1H), 6.25
(s, 1H), 3.15 (s, 2H), 2.32 (s, 6H). 13C-NMR (75 MHz, d6-DMSO): δ ppm 168.67, 161.46, 155.51,
148.98, 147.73, 140.12, 139.69, 138.66, 134.39, 133.14, 132.85, 131.69, 131.59, 129.25, 128.73,
128.59, 127.30, 124.44, 121.18, 119.86, 118.43, 113.85, 63.92, 45.45. HRMS (ESI) m/z calcd for
C26H22N4O3 [M+H]+ 439.1770, found 439.1771.
4.1.2.7
1-(4-dimethylamino-acetamido-phenyl)-8-(4-hydroxyphenyl)-benzofuro[3,2-b]pyridin-
2(1H)–one (5c)
1
General procedure D, yellow solid (yield: 70%). M.p. 276-280 °C; H-NMR (300 MHz,
d6-DMSO): δ ppm 10.11 (s, 1H), 9.58 (s, 1H), 8.11 (d, J = 9.7 Hz, 1H), 7.96 (d, J = 8.2 Hz, 2H),
7.70 (d, J = 8.6 Hz, 1H), 7.64 (d, J = 9.2 Hz, 1H), 7.48 (d, J = 8.3 Hz, 2H), 7.12 (d, J = 8.1 Hz,
2H), 6.73 (d, J = 8.3 Hz, 2H), 6.61 (d, J = 9.8 Hz, 1H), 6.14 (s, 1H), 3.18 (s, 2H), 2.34 (s, 6H).
13C-NMR (75 MHz, d6-DMSO): δ ppm 169.06, 160.89, 157.04, 154.18, 139.60, 138.91, 135.50,
132.65, 130.03, 129.39, 128.64, 128.12, 127.43, 126.09, 120.58, 118.99, 118.79, 116.81, 115.71,
112.70, 63.37, 45.28. MS (ESI, m/z): 454 [M+H]+.
4.1.2.8 1-(4-dimethylamino-acetamido-phenyl)-8-(4-N-t-butyloxycarboryl-piperidyl)-benzofuro
[3,2-b]pyridin-2(1H)-one (5d)
1
General procedure D, yellow solid (yield: 74%). M.p. 155-160 °C; H-NMR (300 MHz,
d6-DMSO): δ ppm 10.12 (s, 1H), 8.08 (d, J = 9.8 Hz, 1H), 7.96 (t, J = 11.2 Hz, 2H), 7.61 (d, J =
8.8 Hz, 1H), 7.53 (dd, J = 8.9, 1.6 Hz, 1H), 7.44 (d, J = 8.7 Hz, 2H), 6.59 (d, J = 9.8 Hz, 1H), 5.95
(s, 1H), 5.89 (s, 1H), 3.88 (s, 2H), 3.17 (s, 2H), 2.34 (s, 6H), 2.08 (s, 2H), 1.41 (s, 9H). 13C-NMR
(75 MHz, d6-DMSO): δ ppm 168.97, 160.82, 154.40, 153.78, 139.52, 138.83, 134.97, 133.12,
132.63, 129.33, 128.59, 128.06, 124.67, 120.81, 118.77, 118.49, 115.57, 112.33, 78.84, 63.31,
54.85, 45.25, 28.04, 26.31. MS (ESI, m/z): 543[M-H]-.
4.1.2.9
1-(4-dimethylamino-acetamido-phenyl)-8-(4-piperidyl)-benzofuro[3,2-b]pyridin-2(1H)-
one (5e)
Compound 5c was solved in DCM, trifluoroacetic acid was added dropwise in ice bath. Then
15

ACCEPTED MANUSCRIPT
the reaction was stirred at 0 °C for 2h and was monitored by TLC to confirm its completion. The
pH was adjusted to basicity with sodium carbonate, and the reaction mixture was extracted with
ethyl acetate for twice. The organic extracts were combined, dried over Na2SO4, filtered, and
evaporated under vacuum and was subjected to column purification (CH2Cl2/ MeOH) to furnish
1
the desired compound (yield: 60%). M.p. 120-126 °C; H-NMR (300 MHz, d6-DMSO): δ ppm
10.85 (s, 1H), 8.11 (d, J = 9.8 Hz, 1H), 7.90 (d, J = 8.6 Hz, 2H), 7.67 (t, J = 9.2 Hz, 1H), 7.60 (d, J
= 8.8 Hz, 1H), 7.49 (d, J = 8.6 Hz, 2H), 6.61 (d, J = 9.8 Hz, 1H), 6.03 (s, 1H), 5.95 (s, 1H), 3.72 (s,
2H), 3.65 (s, 4H), 3.19 (d, J = 5.4 Hz, 2H), 2.58 (d, J = 24.4 Hz, 6H), 2.34 (s, 1H). 13C-NMR (75
MHz, d6-DMSO): δ ppm 166.57, 160.85, 154.65, 139.09, 139.01, 133.89, 133.08, 132.94, 129.13,
128.73, 128.19, 124.70, 121.30, 118.98, 118.52, 117.10, 115.70, 112.33, 78.84, 63.31, 45.35,
44.39, 41.20, 22.91. HRMS (ESI) m/z calcd for C26H28N4O3 [M+H]+ 445.2240, found 445.2241.
4.1.2.10 1-(4-acryloylamino-phenyl)-8-(3-(6-fluopyridinyl))-benzofuro[3,2-b]pyridin-2(1H)-one
(5f)
1
General procedure D, yellow solid (yield: 73%). M.p. 278-286 °C; H-NMR (300 MHz,
d6-DMSO): δ ppm 10.49 (s, 1H), 8.09 (t, J = 8.6 Hz, 2H), 7.89 (dd, J = 22.8, 8.1 Hz, 3H), 7.74 (dd,
J = 16.2, 8.6 Hz, 2H), 7.47 (dd, J = 17.2, 8.4 Hz, 2H), 7.11 (d, J = 8.2 Hz, 1H), 6.62 (d, J = 9.8 Hz,
1H), 6.57 – 6.42 (m, 1H), 6.34 (d, J = 16.0 Hz, 1H), 6.21 (d, J = 15.8 Hz, 1H), 5.82 (d, J = 9.0 Hz,
1H). 13C-NMR (75 MHz, d6-DMSO): δ ppm 163.45, 160.88, 154.87, 144.89, 144.69, 139.84,
139.17, 133.53, 132.55, 131.57, 131.09, 128.84, 128.13, 127.54, 126.76, 120.49, 120.18, 119.34,
119.11, 117.79, 113.26, 109.96, 109.46. MS (ESI, m/z): 425 [M+H]+.
4.1.3 The synthesis of series 6a~6g compounds
4.1.3.1 1-(3-aminophenyl)-8-bromobenzofuro[3,2-b]pyridin-2(1H)-one (S9b)
The preparation of S9b was synthesized with the similar procedure of S9a, yellow solid
(yield: 75%).1H-NMR (300 MHz, d6-DMSO): δ ppm 8.09 (d, J = 9.8 Hz, 1H), 7.69 (d, J = 8.9
Hz, 1H), 7.58 (dd, J = 8.9, 2.0 Hz, 1H), 7.32 (t, J = 7.9 Hz, 1H), 6.86 (t, J = 7.9 Hz, 1H), 6.75 –
6.54 (m, 2H), 6.43 (s, 1H), 6.39 (d, J = 1.9 Hz, 1H), 5.55 (s, 2H). MS (ESI, m/z), [M+Na]+: 377.
4.1.3.2 8-(6-methoxypyridin-3-yl)-1-(3-aminophenyl)benzofuro[3,2-b]pyridin-2(1H)-one (S14)
1
General procedure D, yellow solid (yield: 81%). M.p. 271-274 °C; H-NMR (300 MHz,
d6-DMSO): δ ppm 8.20 – 8.03 (m, 1H), 7.74 (dd, J = 20.3, 8.8 Hz, 2H), 7.32 (t, J = 7.9 Hz, 1H),
6.86 (t, J = 7.9 Hz, 1H), 6.75 – 6.54 (m, 2H), 6.43 (s, 1H), 5.55 (s, 1H), 3.88 (s, 2H). 13C-NMR
(75 MHz, d6-DMSO): δ ppm 163.44, 161.11, 155.06, 150.94, 144.60, 139.38, 138.81, 137.67,
132.66, 130.56, 129.44, 129.17, 128.33, 126.84, 119.57, 117.95, 114.88, 113.37, 111.14, 53.80.
MS (ESI, m/z): 382 [M-H]-.
4.1.3.3
8-(6-methoxypyridin-3-yl)-1-(3-(1-N-methyl-piperazinyl)-acetamido-phenyl)benzofuro
16

ACCEPTED MANUSCRIPT
[3,2-b]pyridin-2(1H)-one (6a)
The preparation of 6a was obtained for two steps according the general procedure B and C,
yellow solid (yield: 71%). M.p. 225-230 °C; 1H-NMR (300 MHz, d6-DMSO): δ ppm 10.06 (s, 1H),
8.13 (d, J = 9.7 Hz, 1H), 8.04 (s, 1H), 7.95 (s, 1H), 7.88 (d, J = 8.1 Hz, 1H), 7.76 (d, J = 8.6 Hz,
1H), 7.72 – 7.58 (m, 3H), 7.26 (d, J = 7.1 Hz, 1H), 6.81 (d, J = 8.6 Hz, 1H), 6.64 (d, J = 9.7 Hz,
1H), 6.29 (s, 1H), 3.85 (s, 3H), 3.21 – 3.08 (m, 2H), 2.51 (s, 4H), 2.39 (s, 4H), 2.17 (s, 3H).
13C-NMR (75 MHz, d6-DMSO): δ ppm 169.29, 163.44, 161.16, 155.09, 144.67, 140.54, 139.61,
138.24, 137.72, 132.83, 130.62, 129.20, 129.11, 128.73, 127.03, 123.58, 120.62, 119.68, 119.51,
119.43, 117.69, 113.58, 111.15, 62.11, 54.72, 53.76, 52.79, 45.84. MS (ESI, m/z): 524 [M+H]+.
4.1.3.4
8-(6-methoxypyridin-3-yl)-1-(3-dimethylamino-acetamido-phenyl)benzofuro[3,2-b]
pyridin-2(1H)-one (6b)
The preparation of 6c was obtained for two steps according the general procedure B and C,
yellow solid (yield: 86%). M.p. 223-225 °C; 1H-NMR (300 MHz, d6-DMSO): δ ppm 10.09 (s, 1H),
8.16 (d, J = 10.1 Hz, 1H), 8.08 (s, 1H), 7.97 (s, 1H), 7.87 (d, J = 7.6 Hz, 1H), 7.81 (d, J = 9.0 Hz,
1H), 7.71 (d, J = 9.0 Hz, 1H), 7.64 (s, 2H), 7.28 (s, 1H), 6.84 (d, J = 8.3 Hz, 1H), 6.65 (d, J = 9.1
13
Hz, 1H), 6.32 (s, 1H), 3.86 (s, 3H), 3.09 (s, 2H), 2.25 (s, 6H). C-NMR (75 MHz, d6-DMSO): δ
ppm 169.75, 163.40, 161.18, 155.05, 144.64, 140.66, 139.57, 138.17, 137.62, 132.74, 130.55,
129.12, 128.66, 126.91, 123.48, 120.66, 119.50, 117.66, 113.47, 111.05, 63.61, 53.72, 45.64. MS
(ESI, m/z): 469 [M+H]+.
4.1.3.5
8-(6-methoxypyridin-3-yl)-1-(3-(1-morpholinyl)-acetamido-phenyl)benzofuro[3,2-b]
pyridin-2(1H)-one (6c)
The preparation of 6c was obtained for two steps according the general procedure B and C,
yellow solid (yield: 75%). M.p. 265-268 °C; 1H-NMR (300 MHz, d6-DMSO): δ ppm 10.06 (s, 1H),
8.16 (d, J = 9.7 Hz, 1H), 8.04 (s, 1H), 7.95 (s, 1H), 7.88 (d, J = 8.1 Hz, 1H), 7.76 (d, J = 8.6 Hz,
1H), 7.72 – 7.58 (m, 3H), 7.26 (d, J = 7.1 Hz, 1H), 6.81 (d, J = 8.6 Hz, 1H), 6.64 (d, J = 9.7 Hz,
1H), 6.29 (s, 1H), 3.86 (s, 3H), 3.59 (m, 4H), 3.14 (s, 2H), 2.39 (s, 4H). 13C-NMR (75 MHz,
d6-DMSO): δ ppm 169.29, 163.11, 161.14, 155.11, 144.70, 140.51, 139.61, 138.24, 137.73,
132.83, 130.60, 129.21, 128.70, 127.01, 123.60, 120.67, 119.67, 117.69, 113.58, 111.15, 66.46,
62.51, 53.76, 53.58. MS (ESI, m/z): 533 [M+Na]+.
4.1.3.6 8-(6-methoxypyridin-3-yl)-1-(3-amino-acetamido-phenyl)benzofuro[3,2-b]pyridin-2(1H)-
one (6d)
The preparation of 6d was obtained for two steps according the general procedure B and C,
yellow solid (yield: 74%). M.p. 205-207 °C; 1H-NMR (300 MHz, d6-DMSO): δ ppm 8.11 (d, J =
9.8 Hz, 1H), 8.01 (s, 1H), 7.90 (s, 1H), 7.83 (d, J = 8.2 Hz, 1H), 7.74 (d, J = 8.7 Hz, 1H), 7.66 (d,
17

ACCEPTED MANUSCRIPT
J = 7.6 Hz, 2H), 7.61 (dd, J = 6.4, 4.1 Hz, 1H), 7.25 (d, J = 7.7 Hz, 1H), 6.82 (d, J = 8.6 Hz, 1H),
6.61 (t, J = 8.6 Hz, 1H), 6.23 (s, 1H), 3.83 (s, 3H), 3.47 (s, 2H). 13C-NMR (75 MHz, d6-DMSO): δ
ppm 170.01, 162.95, 160.70, 154.58, 144.14, 140.06, 139.13, 137.83, 137.31, 132.32, 130.29,
128.69, 128.59, 128.30, 126.58, 123.02, 119.84, 119.15, 118.89, 118.69, 117.04, 113.15, 110.75,
53.33, 43.93. MS (ESI, m/z): 441 [M+H]+.
4.1.3.7
8-(6-methoxypyridin-3-yl)-1-(3-acrylamido-phenyl)benzofuro[3,2-b]pyridin-2(1H)-one
(6e)
The preparation of 6e was obtained for two steps according the general procedure B and D,
yellow solid (yield: 74%). M.p. 290-295 °C; 1H-NMR (300 MHz, d6-DMSO): δ ppm 3.87 (s, 3H),
5.77 (d, J = 9.6 Hz, 1H), 6.26 (d, J = 16.3 Hz, 1H), 6.34 (s, 1H), 6.45 (dd, J = 10.1, 12.7 Hz, 1H),
6.65 (d, J = 9.5 Hz, 1H), 6.83 (d, J = 8.5 Hz, 1H), 7.31 (d, J = 7.89 Hz, 1H), 7.65 (d, J = 7.9 Hz,
2H), 7.72 (d, J = 8.6 Hz, 1H), 7.81 (m, 2H), 7.97 (s, 1H), 8.09 (s, 1H), 8.15 (d, J=9.51 Hz, 1H),
10.45 (s, 1H). 13C-NMR (75 MHz, d6-DMSO): δ ppm 164.02, 163.58, 161.19, 155.14, 152.07,
144.71, 144.04, 140.87, 139.69, 138.33, 137.81, 132.91, 131.97, 130.75, 128.80, 127.97, 127.11,
123.71, 120.58, 119.73, 119.53, 117.68, 114.63, 113.67, 111.19, 53.82. MS (ESI, m/z): 460
[M+Na]+.
4.1.3.8 1-(3-acryloylamino-phenyl)-8-(3-pyridinyl)-benzofuro[3,2-b]pyridin-2(1H)-one (6f)
The preparation of 6f was obtained for two steps according the general procedure B and D,
yellow solid (yield: 80%). M.p. 294-296 °C; 1H-NMR (300 MHz, d6-DMSO): δ ppm 10.57 (s, 1H),
8.49 (s, 2H), 8.17 (d, J = 9.9 Hz, 1H), 8.01 (s, 1H), 7.81 (d, J = 8.4 Hz, 3H), 7.69 (dd, J = 18.8, 8.1
Hz, 2H), 7.40 (s, 1H), 7.31 (d, J = 7.2 Hz, 1H), 6.67 (d, J = 9.7 Hz, 1H), 6.45 (dd, J = 16.6, 10.5
13
Hz, 1H), 6.37 (s, 1H), 6.25 (d, J = 16.9 Hz, 1H), 5.77 (d, J = 10.6 Hz, 1H). C-NMR (75 MHz,
d6-DMSO): δ ppm 163.51, 160.67, 154.96, 148.47, 147.17, 140.40, 139.24, 137.79, 134.99,
133.91, 132.40, 131.43, 130.31, 128.59, 128.34, 127.54, 126.95, 123.89, 123.22, 120.08, 119.37,
118.98, 117.92, 113.35. MS (ESI, m/z): 406 [M-H]-.
4.1.3.9 1-(3-acryloylamino-phenyl)-8-(3-(6-fluo-pyridinyl))-benzofuro[3,2-b]pyridin-2(1H)-one
(6g)
The preparation of 6g was obtained for two steps according the general procedure B and D,
yellow solid (yield: 76%). M.p. 281-286 °C; 1H-NMR (300 MHz, d6-DMSO): δ ppm 10.45 (s, 1H),
8.09 (d, J = 1.3 Hz 1H), 8.07 (s, 1H), 7.85 (m, 2H), 7.75 (m, 3H), 7.56 (m, 1H), 7.19 (d, J = 6.3
Hz 1H), 7.11 (d, J = 5.9 Hz, 1H), 6.57 (d, J = 1.3 Hz, 1H), 6.28 (m, 1H), 6.22 (s, 1H), 6.13 (),d, J
13
= 16.92, 1H), 5.66 (d, J = 9.84 Hz, 1H). C-NMR (75 MHz, d6-DMSO): δ ppm 163.49, 160.65,
154.96, 144.96, 144.75, 140.17, 139.27, 137.74, 131.40, 131.25, 130.70, 130.31, 128.74, 128.35,
127.59, 126.99, 123.25, 120.15, 119.45, 119.06, 117.95, 113.37, 110.10, 109.61. MS (ESI, m/z):
18

ACCEPTED MANUSCRIPT
425 [M+H]+.
4.1.4 The synthesis of series 7a~7d compounds
4.1.4.1 5-bromo-N-(4-nitrophenyl)furo[2,3-b]pyridin-3-amine (S18)
To a solution of compound S17 (4.3 g, 20.2 mmol) in toluene was subsequently
paranitroaniline (2.8 g, 20.2 mmol). The resulting suspension was heated at reflux for 2 h with and
concentrated, then the resulting yellow precipitate was recovered by filtration. (yield: 92%, 6.2 g).
1H-NMR (300 MHz, d6-DMSO): δ ppm 7.06 (d, J = 9.2 Hz, 2H), 8.12 (d, J=9.2 Hz, 2H), 8.4 (d, J
= 2.2 Hz, 1H), 8.43 (s, 1H), 8.46 (d, J = 2.2 Hz, 1H), 9.29 (s, 1H).
4.1.4.2 N-(5-bromofuro[2,3-b]pyridin-3-yl)-N-(4-nitrophenyl)acetamide (S19)
To a solution of compound S18 (6.24 g, 18.68 mmol) in dimethylformamide was
subsequently added 60% sodium hydride (1.12 g, 28 mmol) in batches at ice bath. Until there is no
bubble, the acetylchloride (2 ml, 28 mmol) was dropped slowly into the reaction, then stirred 0.5h
continually. The reaction mixture was poured into water, filtered and dried on infrared. The solid
was used without further purified.
4.1.4.3 8-bromo-1-(4- nitrophenyl)furo[2,3-b:4,5-b’]dipyridin-2(1H)-one (S20)
To dimethylformamide (2.8 ml, 36.6 mmol) was added phosphoryl chloride (3.4 ml, 36.6
mmol) slowly at ice bath. After finished, the reaction mixture stirred 0.5h continually. Then the
S19 (6.85 g, 18.3 mmol) in dimethylformamide was added into the reaction solution, which was
subsequently heated at 90 °C for 3h. The reaction mixture was cooled and poured into water,
further extracted with ethyl acetate. The organic extracts were combined, dried over Na2SO4,
filtered, and evaporated under vacuum and was subjected to column purification (CH2Cl2/ MeOH)
to furnish the desired compound S20 (yield: 35.5%, 2.5 g). 1H-NMR (300 MHz, d6-DMSO): δ
ppm 6.75 (d, J = 9.8 Hz, 1H), 6.82 (d, J = 2.2 Hz, 1H), 7.86 (d, J = 8.9 Hz, 2H), 8.22 (d, J = 9.8
Hz, 1H), 8.49 (d, J = 8.9 Hz, 2H), 8.51 (d, J = 2.2 Hz, 1H).
4.1.4.4 1-(4-aminophenyl)-8-bromofuro[2,3-b:4,5-b']dipyridin-2(1H)-one (S21)
The preparation of S21 was from S20 according to the general procedure A，yellow solid
(yield: 86%). 1H-NMR (300 MHz, d6-DMSO): δ ppm 8.51 (d, J = 2.1 Hz, 1H), 8.22 (d, J = 9.8 Hz,
1H), 7.08 (d, J = 8.6 Hz, 2H), 6.82 (d, J = 2.1 Hz, 1H), 6.78 (d, J = 8.6 Hz, 2H), 6.75 (d, J = 9.8
Hz, 1H).
4.1.4.5 1-(4-aminophenyl)-8-(6-methoxypyridin-3-yl)furo[2,3-b:4,5-b’]dipyridin-2(1H)-one (S22)
General procedure D, yellow solid (yield: 86%). 1H-NMR (300 MHz, d6-DMSO): δ ppm 3.98
(s, 3H), 5.55 (s, 2H), 6.67 (d, J = 9.7 Hz, 1H), 6.78 (s, 1H), 6.79 (d, J = 9.0 Hz, 2H), 6.91 (d, J =
8.6 Hz, 1H), 7.14 (d, J = 9.0 Hz, 2H), 7.79 (dd, J = 2.5, 8.6 Hz, 1H), 8.13 (d, J = 9.7 Hz, 1H), 8.17
(d, J = 2.2 Hz, 1H), 8.65 (d, J = 2.2 Hz,1H).
4.1.4.6
2-(dimethylamino)-N-(4-(8-(6-methoxypyridin-3-yl)-2-methylenefuro[2,3-b:4,5-b’]
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dipyridin-1(2H)-yl)phenyl)acetamide (7a)
The preparation of 7a was obtained for two steps according the general procedure B and C,
yellow solid (yield: 70%). M.p. 210-215 °C; 1H-NMR (300 MHz, d6-DMSO): δ ppm 9.42 (s, 1H),
8.50 (s, 1H), 8.11 (s, 1H), 7.90 (t, J = 9.6 Hz, 3H), 7.55 (dt, J = 11.2, 5.6 Hz, 1H), 7.45 (t, J = 8.9
Hz, 2H), 6.82 (dd, J = 8.9, 6.8 Hz, 3H), 3.94 (s, 3H), 3.15 (s, 2H), 2.42 (s, 6H). 13C-NMR (75
MHz, d6-DMSO): δ ppm 169.26, 164.06, 161.83, 160.88, 145.55, 145.07, 139.24, 138.63, 137.51,
132.50, 130.38, 128.53, 127.88, 127.23, 126.29, 121.44, 120.63, 112.00, 111.44, 63.65, 53.68,
46.06. HRMS (ESI) m/z calcd for C26H23N5O4 [M+H]+ 470.1828, found 470.1824.
4.1.4.7
N-(4-(8-(6-methoxypyridin-3-yl)-2-oxofuro[2,3-b:4,5-b’]dipyridin-1(2H)-yl)phenyl)-2-
(piperazin-1-yl)acetamide (7b)
The preparation of 7b was obtained for two steps according the general procedure B and C,
1
yellow solid (yield: 40%). M.p. 195-198 °C; H-NMR (300 MHz, d6-DMSO): δ ppm 2.31 (brs,
4H), 2.49 (brs, 4H), 3.23 (s, 2H), 3.85 (s, 3H), 6.57 (s, 1H), 6.79 (d, J = 9.8 Hz, 1H), 6.83 (d, J =
8.6 Hz, 1H), 7.51 (d, J = 8.5 Hz, 2H), 7.68 (dd, J = 2.2, 8.58 Hz, 1H), 7.93 (d, J = 8.6 Hz, 2H),
8.13 (s, 1H), 8.15 (d, J = 9.9 Hz, 1H), 8.63 (d, J = 1.9 Hz, 1H), 10.11 (s, 1H). 13C-NMR (75 MHz,
d6-DMSO): δ ppm 168.68, 163.37, 160.61, 160.23, 154.72, 154.21, 145.11, 144.62, 139.58,
137.81, 137.48, 132.14, 129.23, 128.52, 128.22, 127.63, 126.28, 125.79, 120.84, 120.52, 111.46,
110.92, 107.23, 61.57, 54.21, 53.34, 52.17, 45.43. HRMS (ESI) m/z calcd for C28H26N6O4 [M+H]+
511.2094, found 511.2092.
4.1.4.8
N-(4-(8-(6-methoxypyridin-3-yl)-2-oxofuro[2,3-b:4,5-b’]dipyridin-1(2H)-yl)phenyl)-2-
(piperidin-1-yl)acetamide (7c)
The preparation of 7c was obtained for two steps according the general procedure B and C,
yellow solid (yield: 65%). M.p. 178-182 °C; 1H-NMR (300 MHz, d6-DMSO): δ ppm 9.57 (s, 1H),
8.49 (d, J = 2.1 Hz, 1H), 8.10 (d, J = 2.3 Hz, 1H), 7.89 (d, J = 4.3 Hz, 1H), 7.86 (d, J = 5.4 Hz,
2H), 7.55 (dd, J = 8.6, 2.5 Hz, 1H), 7.46 (d, J = 8.7 Hz, 2H), 6.87 – 6.81 (m, 1H), 6.80 (d, J = 3.9
Hz, 1H), 6.78 (s, 1H), 3.92 (s, 3H), 3.13 (s, 2H), 2.57 (d, J = 4.7 Hz, 4H), 1.83 – 1.56 (m, 4H),
1.51 (d, J = 4.6 Hz, 2H). 13C-NMR (75 MHz, d6-DMSO): δ ppm 169.58, 163.84, 161.10, 160.71,
145.59, 145.09, 140.08, 138.29, 137.94, 137.52, 132.61, 129.69, 129.01, 128.86, 128.71, 128.12,
126.73, 126.25, 121.33, 120.99, 120.80, 111.94, 111.38, 100.50, 63.19, 54.51, 53.81, 25.87, 24.02.
HRMS (ESI) m/z calcd for C29H27N5O4 [M+H]+ 510.2141, found 510.2140.
4.1.4.9 N-(4-(2-oxo-8-(pyridin-3-yl)furo[2,3-b:4,5-b']dipyridin-1(2H)-yl)phenyl)acrylamide (7d)
The preparation of 7d was obtained for two steps according the general procedure B and D,
yellow solid (yield: 65%). M.p. 256-260 °C; 1H-NMR (300 MHz, d6-DMSO): δ ppm 10.52 (s, 1H),
8.74 (s, 1H), 8.58 (s, 2H), 8.20 (d, J = 9.8 Hz, 1H), 7.95 (d, J = 8.6 Hz, 2H), 7.84 (d, J = 7.7 Hz,
20

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1H), 7.56 (d, J = 8.6 Hz, 2H), 7.49 – 7.38 (m, 1H), 6.74 (d, J = 9.7 Hz, 1H), 6.72 (s, 1H), 6.50 (dd,
13
J = 16.9, 9.9 Hz, 1H), 6.33 (d, J = 17.1 Hz, 1H), 5.83 (d, J = 10.2 Hz, 1H). C-NMR (75 MHz,
d6-DMSO): δ ppm 163.44, 160.64, 149.16, 147.37, 145.62, 139.93, 137.94, 134.30, 132.25,
132.16, 131.55, 129.30, 128.68, 128.29, 127.58, 127.05, 124.03, 121.01, 120.43, 111.58. MS (ESI,
m/z): 407 [M-H]-.
4.2 Biological screening
4.2.1. Cell culture
The human cell lines Raji (Burkitt lymphoma cell line), Ramos cells (Burkitt lymphoma cell line
cell line) were obtained from Chinese academy of sciences cell bank. Cancer cell lines were
maintained as a monolayer culture in PRAM1640 or IMDM (Keygentech, CN), supplemented
with 10% FBS (Gibco) in a humidified atmosphere (5% CO2) at 37°C.
4.2.2. Antiproliferative assays
Cellular chemo-sensitivity was determined by using a modified CCK-8 (Dojindo) method assay in
vitro. In brief, Raji and Ramos cells in 200 ml culture medium were seeded into 96-well microplates at
3000-5000 cells per well respectively and cultured in PRAM1640 or IMDM with 10% FBS, incubated
at 37 °C for 12-24 h prior to drug exposure. Cell numbers were titrated to keep control cells growing in
the exponential phase throughout the 48 h incubation period. Cells were treated with several
concentrations of tested compounds simultaneously and incubated for 48 h and then 10 ml of CCK-8
was added to each well and incubated for 4 h. The optical density at 450 nm was determined by
Varioskan Flash Multimode Reader. The IC50 value, that is, the concentration (µM) of a compound was
able to cause 50% cell death with respect to the control culture, was calculated according to the
inhibition ratios. For temporal dependence of Raji cells viability, cells were collected at different time,
then determined the cell viability with CCK-8.
4.2.3 In vitro kinase enzymatic assay
The BTK kinase enzyme assay system (Catalog: V9071, Promega, PI3K-Glo™ Class I
Profiling Kit (Promega) and mTOR Lance Ultra Assay were used in this test. Concentrations
consisting of suitable 200 nM were used for all of the tested compounds and 1nM~5µM were used
for part of the tested compounds. The experiments were performed according to the instructions of
the manufacturer. Measure the luminescence with a plate-reading luminometer or charge-coupled
device (CCD) camera.
4.2.4 Western Blot
Cells was treatment with compounds for 4h, then stimulated with or not anti-human IgM F(ab’)2
(Jackson Immuno Research Laboratories), and collected celles were washed twice with PBS, then
collected and lysed in lysis buffer (100 mM of Tris–Cl, pH 6.8, 4% (m/v) SDS, 20% (v/v) glycerol, 200
mM of β-mercaptoethanol, 1mM of PMSF, 0.1 mM NaF and DTT) for 0.5 h on the ice. Heat sample to
95–100°C for 5 min; then cool on ice for 5 min; for 3 times. Protein concentration in the supernatants
was detected by BCA protein assay (Thermo, Waltham, MA). Then equal amount of protein was
separated with 12% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes
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(Millipore, Bedford, MA) using a semi-dry transfer system (Bio-rad, Hercules, CA). Proteins were
detected using specific antibodies overnight at 4 °C followed by HRP-conjugated secondary antibodies
for 1 h at 37 °C. All of the antibodies were diluted in PBST containing 1% BSA. Enhanced
chemi-luminescent reagents (Beyotime, Jiangsu, China) were used to detect the HRP on the
immunoblots, and the visualized bands were captured by film.
4.2.5 Flow cytometry assay
The Ramos cells (1 to 5 ×105 cells/well) incubated in 6-well plates were treated with solvent
control (DMSO), BEZ235, ibrutinib, or compound 6f in medium containing 5% FBS for 48 h. Then,
collected and fixed with 70% ethanol at 4 °C overnight. After being fixed with 75% ethanol at 4 °C for
24 h, the cells were stained with Annexin V-FITC (5 µL)/propidium iodide (5 µL), and analyzed by
flow cytometry assay (Becman Coulter, USA). For cell cycle analysis, Ramos cells at a density of
approximately 1×106 cells/well were incubated in 6-well plates, treated with different concentrations of
inhibitors for 48 h, collected and fixed with 70% ethanol at 4 °C overnight. After fixation, the cells
were washed with PBS and stained with propidium iodide (PI) for 10 min under subdued light. Stained
cells were analyzed flow cytometry assay (Becman Coulter, USA), and the results were performed
using FCS Express flow cytometry analysis software (ModFit LT 3.1).
4.3 Docking study
The docking study was used with the CDOCKER model of Discovery Studio 3.0. The general step
was step by step according to the course of Discovery Studio software. At last, the docking results were
presented with Discovery Studio visualization.
For the in silico prediction, the PreADMET server application was used. The PreADMET
approach is based on different classes of molecular parameters which are considered for generating
quantitative structure properties.
Acknowledgement
We are grateful to the Program of Jiangsu Key Laboratory of Drug Design and Optimization,
China Pharmaceutical University, postgraduate innovation fund of Huahai medical corporation
and National Fund for Fostering talents of Basic Science for the financial support of this research.
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ò
Simultaneously inhibiting BTK and PI3Kδ benefit of the treatment of leukemia
ò A series of new benzofuro[3,2-b]pyridine-2(1H)-one derivatives were optimized from hit
BTK/PI3Kδ inhibitor
ò
6f exhibited anti-leukemia activity by selectively inhibiting BTK kinase and PI3Kδ kinase