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

1 Jiangsu Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, PR China

2 Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, PR China

 

 

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.

 

 

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 (WM).5,6 Recently, the second generation Btk inhibitor acalabrutinib (Calquence®) 7 (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 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 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.

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.

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.

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.

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.

 

 

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 pyridine in compound 16b could bind to the residue Lys779 of the affinity pocket 39 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 NX-5948, 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