Design and synthesis of benzofuro[3,2-b]pyridin-2(1H)-one derivatives as anti-leukemia agents by inhibiting Btk and PI3Kδ

Article abstract of DOI:10.1016/j.bmc.2018.07.047

Btk inhibitors and PI3Kδ inhibitors play crucial roles in the treatment of leukemia, and studies confirmed that the synergetic inhibition against Btk and PI3Kδ could gain an optimal response. Herein, a series of novel benzofuro[3,2-b]pyridin-2(1H)-one derivatives were designed and synthesized as dual Btk/PI3Kδ kinases inhibitors for the treatment of leukemia. Studies indicated that most compounds could suppress the proliferation of multiple leukemia or lymphoma cells (Raji, HL60 and K562 cells) at low micromolar concentrations in vitro. Further kinase assays identified several compounds could simultaneously inhibit Btk kinase and PI3Kδ kinase. Thereinto, compound 16b exhibited the best inhibitory activity (Btk: IC₅₀ = 139 nM; PI3Kδ: IC₅₀ = 275 nM) and showed some selectivity against PI3Kδ compared to PI3Kβ/γ. Finally, the SAR of target compounds was preliminarily discussed combined with docking results. In brief, 16b possessed of the potency for the further optimization as anti-leukemia drugs by inhibiting simultaneously Btk kinase and PI3Kδ kinase.

Full text of DOI:10.1016/j.bmc.2018.07.047

Accepted Manuscript
Design and synthesis of benzofuro[3,2-b]pyridin-2(1H)-one derivatives as anti-
leukemia agents by inhibiting Btk and PI3Kδ
Linyi Liu, Bingyu Shi, Xinyu Li, Xiangqian Wang, Xiang Lu, Xuerong Cai, Ali
Huang, Guoshun Luo, Qidong You, Hua Xiang
PII:
S0968-0896(18)30638-2
https://doi.org/10.1016/j.bmc.2018.07.047
BMC 14484
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
28 March 2018
18 July 2018
26 July 2018
Please cite this article as: Liu, L., Shi, B., Li, X., Wang, X., Lu, X., Cai, X., Huang, A., Luo, G., You, Q., Xiang,
H., Design and synthesis of benzofuro[3,2-b]pyridin-2(1H)-one derivatives as anti-leukemia agents by inhibiting
Btk and PI3Kδ, Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.07.047
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Design and synthesis of benzofuro[3,2-b]pyridin-2(1H)-one derivatives as
anti-leukemia agents by inhibiting Btk and PI3Kδ
Linyi Liu1,2; Bingyu Shi1,2; Xinyu Li1,2; Xiangqian Wang1,2; Xiang Lu1,2
;
Xuerong Cai1,2; Ali Huang1,2; Guoshun Luo1,2; Qidong You1,2; Hua Xiang1,2,ǂ
1Jiangsu Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, 24 Tongjiaxiang,
Nanjing 210009, PR China
2Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, 24 Tongjiaxiang,
Nanjing 210009, PR China
ǂ 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).
Abstract
Btk inhibitors and PI3Kδ inhibitors play crucial roles in the treatment of leukemia, and studies confirmed that the synergetic
inhibition against Btk and PI3Kδ could gain an optimal response. Herein, a series of novel benzofuro[3,2-b]pyridin-2(1H)-one
derivatives were designed and synthesized as dual Btk/PI3Kδ kinases inhibitors for the treatment of leukemia. Studies indicated
that most compounds could suppress the proliferation of multiple leukemia or lymphoma cells (Raji, HL60 and K562 cells) at
low micromolar concentrations in vitro. Further kinase assays identified several compounds could simultaneously inhibit Btk
kinase and PI3Kδ kinase. Thereinto, compound 16b exhibited the best inhibitory activity (Btk: IC50 =139 nM; PI3Kδ: IC50 = 275
nM) and showed some selectivity against PI3Kδ compared to PI3Kβ/γ. Finally, the SAR of target compounds was preliminarily
discussed combined with docking results. In brief, 16b possessed of the potency for the further optimization as anti-leukemia
drugs by inhibiting simultaneously Btk kinase and PI3Kδ kinase.
Keywords: benzofuro[3,2-b]pyridin-2(1H)-one; Synthesis; leukemia; Btk; PI3K.
1. Introduction
Leukemia is a malignant tumor disease that severely affects the normal function of hematopoietic system. It is developed
due to perturbed cell development process. Any abnormal changes including cytokines and growth factors from stem cells to
blood cells are the pathogenic factors which may lead to the leukemia. At present, allogeneic stem cell transplantation is still the
main curative therapy. Fortunately, after the approval of Bcr-Abl inhibitor imatinib, the targeted therapy rather than
chemotherapy as the safe and effective strategy for the treatment of leukemia has got great development. Nowadays, more than
ten kinase inhibitors by targeting to Bcr-Abl, Bruton’s tyrosine kinase (Btk) and phosphoinositide 3-kinase δ (PI3Kδ) have been
applied in the clinical for the treatment of leukemia.
Btk is a member of the TEC family which include Btk, Tec, Bmx, Itk and Txk, and mostly expressed in lymphoma B cells,
mast cells and macrophages.1,2 The expression and function of Btk has implicated it in the treatment of malignant leukemia.3,4
Ibrutinib (1, Fig. 1) as the first Btk inhibitor, has been shown to have significantly clinical functions in treating leukemias and
lymphomas, including chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), Waldenström’s macroglobulinemia
7
(WM).5,6 Recently, the second generation Btk inhibitor acalabrutinib (Calquence®) (2, Fig. 1) has been approved by FDA for
the treatment of MCL.
PI3Kδ belongs to the class I lipid kinases PI3K family which comprises four isoforms, α, β, γ, and δ that catalyze the
phosphorylation of phosphatidylinositol-4,5-biphosphate (PIP2) to phosphatidylinositol-3,4,5- triphosphate (PIP3).8 It’s known

that PIP3 is a key signaling molecule impacting cellular growth, proliferation, chemotaxis, differentiation, and survival. So PI3K
plays a vital role in cellular activity. Studies found that the expression of the catalytic p110δ and p110γ subunits is mainly
restricted to leukocytes, whereas p110α and p110β are expressed by all cell types. Thus, PI3Kδ (p110δ contained) and PI3Kγ
(p110γ contained) are key enzymes in leukocyte signaling and therefore represent promising targets for the intervention of PI3K
signaling pathways that are involved in inflammatory and auto immune diseases.9 At present, the clinical efficacy of PI3Kδ has
been confirmed, PI3Kδ inhibitors idelalisib (3, Fig.1)10 and copanlisib (4, Fig.1) 11 have been shown to be beneficial for the
treatment of CLL and follicular lymphoma in clinical.
Fig. 1. The approved Btk inhibitors and PI3Kδ inhibitors
The approval of ibrutinib and idelalisib demonstrated the therapeutic benefit of inhibiting Btk and PI3Kδ for the treatment
of CLL and MCL. However, recent studies reported that about 10% of patients discontinued ibrutinib therapy as a result of
disease progression.11 The emergence of resistance-conferring mutations to ibrutinib as a key reason in CLL and MCL
progression had been identified.12-15 Idelalisib has the same problem with the unclear resistant mechanisms at present.16
Nowadays, many studies identified that one kind of kinase resistant mutations may not simultaneously confer resistance to the
other target inhibitors, and multi-target therapy or drug combination are better at controlling complex disease systems over
single-target therapy.17-19 In view of the intracellular function and the cross-linking of Btk and PI3Kδ as well as the significant
efficacy of Btk inhibitors and PI3Kδ inhibitors to leukemia,20,21 the combination of Btk and PI3Kδ is considered to be an
attractive strategy for potentially achieving deeper and durable responses as well as preventing resistance.22-25 In fact, several
studies reported that the dual tyrosine kinases/Phospholipids kinases inhibitors exhibited better potency in cancer treatment.26, 27
This suggested that the idea is feasible. Herein, we reported the design and synthesis of benzofuro[3,2-b]pyridine -2(1H)-one
derivatives as novel anti-leukemia agents by simultaneously inhibiting Btk and PI3Kδ.
2. Design and synthesis of compounds
Dactolisib (BEZ235, 5, Fig. 2) and NVP-BGT226 (6, Fig. 2) which were developed by Novartis are novel selective class I
PI3K/mTOR inhibitors, and both entered clinical trials for a number of cancers including advanced solid tumors and metastatic
breast cancers.28,29 Further studies showed the efficacies of 5 and 6 in a broad set of hematological malignancies, including
aggressive lymphoma, multiple myeloma, and acute leukemia.30,31 QL47 (7, Fig. 2)32 and QL-X-138 (8, Fig. 2) 33 which were
optimized from mTOR inhibitor Torin 2 (9, Fig. 2)34 are alike to 5 and 6 in structure. However, 7 and 8 are selective Btk
inhibitors with good anti-proliferative activity of hematological malignancy cells. Recognizing the structural similarities between
5 or 6 and 7 or 8, we speculated that appropriately designed molecules could maintain reasonable affinity for Btk while also
inhibit PI3K.

Fig. 2. The quinoline compounds possessed of PI3K or Btk inhibiting activity
It benefited of our compounds’ design if we could establish the structure-activity relationship (SAR) of these compounds, so
we looked toward to the docking modes of BEZ235 and QL47 which were previously reported (Fig. 3). BEZ235 could bind to
the hinge region of PI3Kγ through three H-bond interactions (Fig. 3A).28 While QL47 could occupy the ATP binding pocket and
form a covalent bond with Cys481 in Btk32 (Fig. 3B). The interaction sites of BEZ235 in PI3Kγ including the N of
imidazo[4,5-c]quinoline ring, the
N of quinoline ring and the nitrile group respectively correspond to the N of
benzo[h][1,6]naphthyridin ring, the N of pyrazol ring and the carbonyl of amide carbonyl QL47 in Btk (Fig. 3). In fact, QL47
could also inhibit PI3K with IC50 0.69 μM.32 These results powerfully impelled us to take these tricyclic molecules as the lead
compounds to design new molecules. However, the poor microsomal stability of QL47 limited it further study in vivo.32
Interestingly, Cheng et al. illustrated a possible mechanism of the poor microsomal stability with a similar structure. 35 The
aldehyde oxidase could oxidize 10 to 11 at C-2 of naphthyridine, leading to the rapidly cleared (Fig. 3C). Hence, we speculated
that the remove of C-2 of naphthyridine or quinoline could be helpful to the stability, and pyran ring replaced the pyridine as the
H-bond acceptor may be suitable. At the end, we designed and synthesized a series of benzofuro[3,2-b]pyridin- 2(1H)-one
derivatives (Fig. 3D).
Fig. 3. (A) BEZ235 and its proposed interactions with human PI3K-γ.28 (B) QL47 and its proposed
interactions with human Btk.32 (C) The aldehyde oxidase oxidation of 10.35 (D) The designed compounds with
benzofuro[3,2-b]pyridin-2(1H)-one skeleton.
Shown in Scheme 1 was the general synthetic route for preparing the target tricyclic derivatives discussed in this paper. The
intermediate S5 was prepared from the available starting material S1 with 4 steps which included bromization, etherification,
hydrolyzation and cyclization.[36] S5 was reacted with p-nitroaniline to afford compound S6. Treatment of S6 with acetylchloride
yielded compound S7. Then the key intermediate S8 was prepared by Vilsmeier–Haack reaction from S7, which was subjected to
Suzuki coupling conditions with a boronic acid or boronic ester derivative to give compound 12. The reduction of 12 under
Fe/NH4Cl obtained 13 which subsequent reacted with acyl chloride or anhydride to give the target compounds 14a-g. The further

derivatization of 14g could give 15a-h. Another route for several compounds 16a-h was performed from S8 with different
reacting order. Finally, 24 compounds were synthesized and identified by NMR and MS.
Scheme 1. General synthetic route of the benzofuro[3,2-b]pyridin-2(1H)-one derivatives
Reaction condition and reagent: a) Br2, DCM, rt.; b) ethyl bromoacetate, K2CO3, KI, DMF, 60 °C; c) NaOH, EtOH,
80 °C; d) Ac2O, AcOH, NaOAc, 150 °C, then HCl, MeOH, 100 °C; e) p-nitroaniline, p-TSA, reflux; f) AcCl, NaH,
0 °C; g) POCl3, DMF, 0~90 °C; h) boronic acid/boronic ester, K2CO3, Pd(PPh3)Cl2, dioxane, 100 °C; i) Fe/NH4Cl,
reflux; j) chloroacetyl chloride or acyl chloride or anhydride, K2CO3, DMF, 0 °C; or ethyl chloroacetate, K2CO3,
DMF, 60 °C; k) appropriate aniline, K2CO3, KI, DMF, 60 °C.
3. Results and Discussion
It’s sure that Raji cells (Burkitt lymphoma cell line, B cell), HL60 cells (acute myeloid leukemia cell line, promyeloblast)
and K562 cells (chronic myelogenous leukemia, lymphoblast) were obtained from different origin, but Btk and PI3K are also
expressed in these hematopoietic cells, including Raji, HL6037 and K56238 (The expression of Btk in K562 was confirmed, see
supplement data 1.2.6) cell lines. Thus these cells were chosen for the preliminary activity evaluation. We firstly evaluated
antiproliferative activities of several 2-methoxylpyridine derivatives in vitro. For comparison, the Btk inhibitor ibrutinib and
PI3K inhibitor BEZ235 were used as the positive controls. The results were summarized in Table 1. It showed that most
compounds could inhibit the growth of tumor cells with different level. Among these compounds, the antiproliferative activity
tested in cell level revealed that the lipophilic group at the C-4’ position of the benzene ring was unfavorable to suppress the
growth of tumor cell, such as nitro (12) and ethyl aminoacetate (14e). While the introduction of dimethylamine (15a) could
significantly improve the activity compared to acetyl derivative (14a). It’s speculated that the polarity of compounds had a great
influence on the proliferation activity and the dimethylamine group might improve the physical and chemical properties of the
compounds. Subsequently, ADP-Glo™ kinase assay was used for evaluation of Btk inhibitory activities. As shown in Table 1,
these tested compounds could inhibit Btk kinase activity at 200 nM concentration, although its inhibitory rates were still weak
and significantly lower than ibrutinib. Importantly, the active compound 15a against leukemia cells was also very potent against
the Btk kinase.

Table 1. The Anti-proliferative activities and Btk inhibition activities assay of synthesized 2-methoxylpyridine derivatives
Antiproliferative activity
Inh%/Btk
(200 nM)
IC50/Btk
IC50/μM or Inh%*
Entry
R1
(μM)
Raji
NI
HL60
K562
28.8%
17.6%
NO2
NH2
NI
-
-
-
12
13
41.5%
21.5%
32.2±2.6
39
-
47.5%
17
-
-
-
-
-
14a
14b
14c
14d
10%
35%
16.7
35.4%
17.9
15.5
38.3±1.9
41.8±1.7
12.7±1.0
31
16.2
NI
46%
12.7
29.4%
46%
16.2±0.8
0
-
-
14e
14f
39.5
12.8
4
4.1
48.7±0.4
0.237
15a
14.2
4.3
8
1
7.5
5.7
100%
-
0.001
-
Ibrutinib
BEZ235
* Percentage for anti-proliferative activity represent inhibition ratio at 40 μM;
NI: no inhibition at 40 μM.
Considering the relatively good anti-proliferative activity and enzyme inhibitory activity of 15a, we explored a series of
amino-acetamide derivatives. The results were depicted in Table 2. All compounds exhibited significantly effects on
antiproliferative activities against Raji, HL60 and K562 in vitro. And 15c, 15e, 15g and 15h possessed of better inhibitory
activity against Btk than 15a. Especially, 15e with IC50 value of 106 nM, was 2-fold more potent for suppressing the Btk kinase
than 15a. From these tested results shown in Table 2, the diethylamino group derivative (15b), methylamino derivative (15d) and
piperazinyl derivative (15f) significantly lost the capacity for inhibiting Btk compared to other amino-acetamide derivatives. It is
clearly that the diethylamino group possess of bigger bulk than pyrrole, piperidine, and morpholine, so we speculated that the
bulky diethylamino group caused the steric exclusion of the Btk binding pocket, which greatly decreased the binding forces
between the synthesized compounds (15b) and the Btk kinase. And the polar methylamino (15d) or piperazinyl group (15f) was
excluded by the Btk hydrophobic region, which also decreased the activity. Anyway, although the antiproliferative activities were
better, the inhibitory activity of these compounds against Btk still was significantly weaker than ibrutinib and QL47.
Table 2. The Anti-proliferative activities and Btk inhibition activities assay of synthesized amino-acetamide derivatives

Antiproliferative activity
Inh%/Btk
(200 nM)
IC50/Btk
R2
IC50/μM
Entry
(μM)
Raji
15.29
13.2
6.26
8.01
5.84
9.72
11.3
HL60
8.4
4
K562
5.9
10
22.6±7.4
50±0.8
-
15b
15c
15d
15e
15f
0.2
3.2
4.9
5.5
3.2
5.4
3.5
2.6
1.3
4.7
6.5
25±0.7
-
52.3±1.5
12.1±7.2
58.1±7.2
56.8±0.7
0.106
-
0.139
0.183
15g
15h
Thus, in view of the above unsatisfactory results and the important role of the acrylamide in ibrutinib and QL47 or other Btk
inhibitors, and 14c also possessed of some inhibition, we next evaluated the biological activities of acrylamide derivatives which
changed the 2-methoxylpyridine to other heterocyclic aromatic. The results were shown in Table 3. Compared with 14c, these
compounds exhibited better antiproliferative activities except of compounds 16a, 16c and 16f. However, only 16b and 16h
showed better enzyme inhibitory activity than 14c, and still were comparable to 15c, 15e, 15g and 15h. Just like 14a~f, when R3
was the lipophilic group, these derivatives lost the capacity for the suppression of tumor cells. Moreover, R3 containing hydrogen
bond receptor (16b and 16h) but not hydrogen bond donor (16d and 16e) may benefit Btk inhibitory activity.
Table 3. The Anti-proliferative activities and Btk inhibition activities assay of synthesized acrylamide derivatives
Antiproliferative activity
Inh%/Btk
(200 nM)
IC50/Btk
R3
IC50/μM or Inh%*
Entry
(μM)
Raji
NI
HL60
K562
28.3%
11.1
39%
6.9
-
-
-
16a
16b
16c
16d
16e
5.2
25
4.2
28.2
10
51.9±0.5
-
0.136
-
-
-
5.3
3.9
28.3±0.2
23.2±2.8
3.9
5.7

38%
15.7
2.4
22.2%
7.3
NI
10.2
9.6
22.1±0.9
38.3±4.0
47.5±0.5
-
-
16f
16g
16h
12.6
0.217
* Percentage for anti-proliferative activity represent inhibition ratio at 40 μM;
NI: no inhibition at 40 μM.
That’s all, we got a series of benzofuro[3,2-b]pyridin-2(1H)-one derivatives with good antiproliferative effect to leukemia
cells (Raji, HL60 and K562) but moderate or weak enzyme inhibitory activity to Btk, such as 15d, 15f, 16d and 16e. In order to
confirm whether the antiproliferative effect were related to the suppression of PI3K, several active compounds including 14c, 15a,
15c, 15e, 15h, 16b and 16h which possessed of better antiproliferation and Btk inhibitory activity were chosen to further study.
These compounds were performed with ADP-Glo luminescent assay against PI3Kδ. The result was shown in Table 4.
Unfortunately, most compounds didn’t have inhibitory activity to PI3Kδ at 200 nM concentration, only 15c, 16b and 16h
exhibited weak inhibitory activity. Although weaker than positive control BEZ235 which could inhibit 64.4% activity of PI3Kδ
(IC50 = 22 nM), 16b could inhibit PI3Kδ with about 45% inhibitory rate at 200nM (IC50 = 275 nM). Finally, we got a novel
compound 16b which could suppress the leukemia cells’ growth by simultaneously inhibiting Btk and PI3Kδ.
Table 4. The PI3Kδ inhibition activities assay of synthesized derivatives
Inh%/PI3Kδ
Inh%/PI3Kδ
Entry
Entry
(200 nM)
(200 nM)
NI
NI
NI
14c
15a
15c
15e
15f
15g
15h
16b
45.8±9.3
(IC50 = 275 nM)
11.6±2.0
NI
16d
NI
4.2±4
4.8±2.7
16e
NI
16h
64.4±1.3
(IC50 = 22 nM)
NI
BEZ235
NI: no inhibition at 200 nM.
Obviously, it’s remarkable that there are inconsistencies of several compounds (such as 15a, 15d, 15f, 16d and 16e) between
the antiproliferative activity and enzymatic data on Btk or PI3Kδ. And as we known that BEZ235 also inhibits other isoforms of
PI3K and mTOR, so we performed a western blot experiment to confirm whether the antiproliferative effect against the tumor
cells were related to the suppression of PI3K pathway. Thus, several active compounds including 14c, 15a, 15e, 15f, 16b and 16h
were chosen to further study. It’s known that PI3K kinase can recuit Akt to cytomembrane, then PDK-1 and mTORC2 active the
Akt by phosphorylating Thr308 and Ser473 of Akt respectively. So the expression of p-Akt (Ser473) can correspond to the
activity of PI3K pathway. As shown in Fig. 4, all tested compounds could significantly suppress the expression of p-Akt (Ser473)
compared with control at 1μM, and 15a and 15e exhibited equivalent activity to positive control BEZ235. Thus we identified
these new molecules could inhibit the PI3K/Akt/mTOR pathway, and then suppressed the growth of K562, Raji or HL60 cell
lines. The blotting result hinted that these compounds which could not significantly inhibit Btk and PI3Kδ at 200 nM might
suppress the growth of tumor cells by inhibiting other members of PI3K pathway.

Fig. 4. Effect of compounds on AKT phosphorylation in K562 cells. (A). Effect of synthesized compounds on
the Akt and p-Akt473. (B). Quantified effect of synthesized compounds on the Akt and p-Akt473.
In order to identified the effect of these compounds to other PI3K isoforms and mTOR, a kinase profile study of
16b against lipid kinases and mTOR was further performed. As shown in Table 5, 16b could inhibit all PI3K isoforms
and mTOR as speculative result of western blotting experiment. Fortunately, 16b exhibited better activity against
PI3Kδ, PI3Kα and mTOR than PI3Kβ and PI3Kγ. This result identified 16b possessed of selectivity to PI3K, and the
selectivity of 16b was better than that of BEZ235 even though the inhibitory activity of 16b was significantly lower
than BEZ235.
Table 5. The kinase profile study of 16b against lipid kinases and mTOR
IC50 /nM
PI3Kδ
275
7
PI3Kα
254
4
PI3Kβ
838
PI3Kγ
684
5
mTOR
228
6
Entry
16b
BEZ235*
75
* Data from reference [28]
.
4. Docking study
The results from the biological evaluation revealed that the synthesized compounds could inhibit Btk kinase activity and
block the PI3K pathway by inhibiting PI3K isoforms and mTOR. Here, we focused on the discovery of novel dual Btk/PI3Kδ
inhibitors. Therefore, a docking study was performed to analysis the interactive mode of these derivatives and focused targets
(Btk and PI3Kδ). As shown in Fig. 5, both compounds 16b and 15e which exhibited the best Btk inhibitory activity could occupy
the ATP pocket of Btk (PDB:3PIY). And the O of furan formed a hydrogen bond with amino acid residue Met477 of the adenine
pocket (Fig. 5A and 5B). This was consistent with the QL47, and matched with our design. Moreover, the methoxy group of 15e
also formed a hydrogen bond with Lys430 (Fig. 5A), it might be the partial reason that the Btk inhibitory activity of 15e was
better than 16b. Unexpectedly, far from the key residue C481, there was no interaction between the para-substituents of benzene
on N of benzofuro[3,2-b]pyridin-2(1H)-one and Btk, no matter acrylamido or N-piperidinyl-acetamido. Thus, these compounds
could not strongly inhibit the kinase activity of Btk. While it's worth noting that a hydrophobic semi-open area near Cys481
could tolerate finite steric and lipophilic substituents (Fig. 5C). This explained that the inhibitory rate of 15c, 15e, 15g and 15h
were better than other 2-methoxylpyridine derivatives. Next, we docked compound 16b to PI3Kδ (Fig. 6, PDB: 5O83). The N of
39
pyridine in compound 16b could bind to the residue Lys779 of the affinity pocket in PI3Kδ (Fig. 6A). Unfortunately, there is
no interaction between the O of furan and the adenine pocket. From the docking result, we found that the O of furan closed to the
phenol of residue Typ813, but not the residue Val828 which is the core active site residue of the adenine pocket, this maybe lead
to its low activity. Moreover, it’s clear that the narrow cavity of PI3Kδ which could tolerate pyridyl and acrylamide (Fig. 6B),
while 2-methyloxy-pyridyl and N-piperidinyl-acetamido limited 15e embedding to hydrophobic pocket of PI3Kδ, this prevented
15e from binding to the kinase. Anyway, based on the docking results, three further optimizations maybe improve the kinases
inhibitory activity: the replace of 2-methyloxy-pyridyl to 2-chloro-pyridyl or 2-fluoro-pyridyl and a heteroatom as a
hydrogen-bonding acceptor added into the lactam ring of benzofuro[3,2-b]pyridin-2(1H)-one could improve PI3Kδ inhibitory
activity; the acrylamide was transferred to meta-position of benzene could improve Btk inhibitory activity by interacting with

Cys481.
Fig. 5. The docking models of 15e (A) and 16b (B) to Btk and the overlay (C) of 15e (dark blue) and 16b
(brilliant blue) in ATP pocket of Btk kinase domain (PDB: 3PIY).
Fig. 6. Shown are the binding mode of 16b in the active site of p110δ (PDB: 5O83).
5. Conclusion
In summary, a series of benzofuro[3,2-b]pyridin-2(1H)-one derivatives were synthesized for the preliminary study in the
treatment of lymphoma and leukemia. Among these newly obtained compounds, most compounds could suppress the
proliferation of Raji, HL60 and K562 at low micromolar concentrations, and inhibitors 15a, 15c, 15e, 15g, 15h, 16b and 16h
displayed better activity against the Btk kinase with more or less than 50% inhibitory rate at 200nM. Further study identified that
some derivatives also inhibited PI3Kδ, and 16b exhibited better inhibitory activity to PI3Kδ at 200 nM concentration. Moreover,
the results of western blot and kinases assay clarified that these compounds could block the phosphorylation of Akt and 16b also
inhibit other three PI3K isoforms and mTOR. Thus, we speculated that these compounds suppressed the cell growth by
simultaneously inhibiting Btk or PI3K pathway. Even though 16b also inhibited PI3Kα and mTOR with similar value of IC50, this
compound could simultaneously inhibit Btk and PI3Kδ with more or less than 50% inhibitory rate at 200 nM (Btk: IC50 =139 nM;
PI3Kδ: IC50 = 275 nM), Overall, these studies provided a promising compound 16b which could inhibit Btk and PI3Kδ as a lead
compound for the further optimization.
Acknowledgments
This work was supported by grants from the National Natural Science Foundation of China (81373279), the Twelfth
Five-Year Plan Major Project of Candidate Drugs (Ministry of National Science and Technology, 2012ZX09103101048).
Supplementary material
Supplementary material associated with this article can be found, in the online version.
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

Multi-targeted therapy or drugs combination are better at controlling complex disease systems
The therapeutic benefit of inhibiting Btk and PI3Kδ for the treatment of leukemia has been
identified

A series of designed benzofuro[3,2-b]pyridine-2(1H)-one derivatives could simultaneously inhibit
Btk and PI3K kinase activity