Design and synthesis of novel pyrazolopyrimidine-based derivatives as reversible BTK inhibitors with potent antiproliferative activity in mantle cell lymphoma

Development of Bruton’s tyrosine kinase (BTK) inhibitors is of great value and significance in the treatment of B-cell malignancies and autoimmune diseases. Herein, a novel class of pyrazolopyrimidine-based BTK inhibitors were designed and evaluated in mantle cell lymphoma (MCL) cell lines. We demonstrated that target compounds had made great progress in improvement of antiproliferative activity compared to lead compound. Compounds 13c, 13g, 13h, 13l, 13n and 13o demonstrated effectively antiproliferative activity in MCL cells lines with single-digit micromolar potency. Furthermore, compound 13l specifically disturbed mitochondrial membrane potential and increased reactive oxygen species level in Z138 cells in a dose-dependent manner. 13l induced cell apoptosis through the caspase 3- mediated apoptotic pathway in Z138 cells. Overall, this study provides valuable lead compounds for developing antitumor agents. Graphical abstract Graphical abstract


Introduction
Mantle cell lymphoma (MCL) is a rare and incurable B-cell lymphoma defined by the (11;14)(q13;q32) translocation and resultant overexpression of cyclin D 1 [1]. It appears that the incidence of MCL in Asian countries is lower than that in western countries [2]. MCL is most commonly treated with combination chemo-immunotherapy at diagnosis because of the poor prognosis [3]. Long-term followup on chemo-immunotherapy studies has shown durable remissions in some patients; however, long-term toxicities remain a serious concern with chemotherapy. Developing novel treatment strategies is important for patients with MCL. In recent years, the therapeutic options based on MCL pathogenesis have made a breakthrough, especially in blocking B cell receptor (BCR) signaling pathway [4]. In therapy, long-term follow-up on chemo-immunotherapy studies have demonstrated durable remissions in some patients; however, long-term toxicities, especially from second cancers, are a serious concern with chemotherapy.
Bruton's tyrosine kinase (BTK), a member of Tec family kinase, is expressed in B-lineage cells and myeloid cells, and is a key component of BCR signaling pathway, which regulates the survival, activation, proliferation, differentiation and maturation of B cells [5,6]. In BCR signaling pathway, BTK is phosphorylated and activated by SYK and LYN kinases. Activated BTK leads to activation of multiple signaling networks including RAS/RAF/MEK/ERK, PI3K/ AKT/mTOR, and nuclear factor kappa B (NF-κB) pathways, which regulate the activation, survival, and proliferation of B cells [7]. It has been reported that BTK is overexpressed and aberrantly active in the pathogenesis of various B cell hematological malignancies, including MCL, chronic lymphocytic leukemia (CLL), and waldenstrom's macroglobulinemia (WM) [7,8]. BTK is considered as a promising target for the treatment of B-cell malignancies.
During recent decade, various BTK inhibitors have been developed. Based on the binding modes with the BTK catalytic domains, reported inhibitors can be classified into covalent and non-covalent inhibitors. Most covalent inhibitors have Michael acceptor groups, forming a covalent bond with the cysteine 481 (Cys481) residue, whereas noncovalent BTK inhibitors interact with BTK via hydrogen bonds and hydrophobic interactions [8]. Covalent BTK inhibitors, ibrutinib (2013), acalabrutinib (2017), zanubrutinib (2019), tirabrutinib (2020), and orelabrutinib (2020) have been approved to treat B-cell malignancies in the US, Japan and China [9]. Despite the remarkable potency and efficacy of covalent BTK inhibitors against B-cell malignancies in the clinics, a subset of patients loses the initial treatment responses or relapse after the therapy. The mutation of Cys481 to serine (C481S) commonly occurs in patients receiving BTK inhibitor treatment, resulting in disruption of covalent binding and representing the main cause of acquired resistance for covalent BTK inhibitors [10,11].
The ability of these non-covalent BTK inhibitors in overcoming the acquired resistance associated with covalent BTK inhibitors has been proved in multiple clinical studies. Fenebrutinib (GDC-0853, 6), a highly selective reversible BTK inhibitor, is undergoing a phase 1 trial (NCT01991184) which includes relapsed/refractory NHL or CLL patients bearing BTK C481S mutation. ARQ 531 (MK-1026, 7), and pirtobrutinib (LOXO-305, 8) are in phase 2 (NCT04728893) and phase 3 (NCT04662255) for treating relapsed/refractory B cell malignancies, respectively. Compared to covalent BTK inhibitors, the development of non-covalent BTK inhibitors has been relatively limited. No revisable BTK inhibitors have been approved for clinical applications as of now. In addition, the noncovalent binding with the target decreases the toxicity and potential risks associated with covalent inhibitors, such as allergic reactions resulting from haptenization of serum proteins by the Michael acceptors [5,12]. Therefore, development of reversible BTK inhibitors has important clinical value and significance (Fig. 1).

Fig. 1 Structures of representative BTK inhibitors
In previous studies, a reversible BTK inhibitor 9 exhibited moderate inhibitory activity against BTK (IC 50 = 0.23 μM) and low antiproliferative activity in MCL cells [13]. In this work, we performed further structural modifications for compound 9 to improve its antiproliferative activity. As the pyrazolopyrimidine scaffold and 4-phenoxyphenyl group are common pharmacophores for BTK inhibitors, further structural optimization focused on the linker and terminal groups. Based on these, we retained the backbone and hydrophobic groups of lead compound 9, extended the linker and introduced diverse substituents (single, multiple, electronwithdrawing, or electron-donating) on the terminal benzene ring for additional interactions with the amino acid residues around the solvent region. In this way, a series of 3-(4-phenoxyphenyl)-1H-pyrazolo [3,4-d]pyrimidin-4amine-based derivatives are designed (Fig. 2).

Biological activity
In vitro BTK kinase activity The newly synthesized compounds were evaluated for their activity against BTK via a kinase profiler radiometric protein kinase assay. Compounds with BTK residual activity < 20% at 10 μM were further chosen to test the IC 50 values. As shown in Table 1, introduction of nitro or halogen group at R 1 is more beneficial to improve BTK inhibitory activity than trifluoromethyl group (13a vs 13b, 13c and 13d). Extending the length of linker did not increase the BTK inhibitory activities (13e-13g). Electron-withdrawing group (13j-13l) on the terminal phenyl group showed more potent inhibitory activity against BTK than electron-donating group (13i), especially compound 13j, the best candidate from the designed series, demonstrated comparable BTK inhibition potency to the lead 9 (IC 50 values = 0.35 and 0.23 μM, respectively). Compounds 13l and 13o contained nitro group and dicyano group also displayed favorable BTK kinase activity with IC 50 of 1.10 and 1.98 μM, respectively.

Antiproliferative activity in MCL cell lines
In order to further study the structure-activity relationship, we preformed the antiproliferation assay in MCL cell lines (Mino, Jeko-1, Z138 and Maver-1). As listed in Table 2, the antiproliferative activities of most compounds 13 were significantly improved compared with lead compound 9. Most of the tested compounds showed comparable antiproliferative activity in MCL cell lines compared to positive control ibrutinib. Similar to our findings in BTK activity assay, extension of linker did not enhance their antiproliferative activities (13g vs 13e and 13f). Compounds 13j-13l contained electron-withdrawing group on the terminal phenyl group also exhibited more potent growth inhibition activity than that of compound 13i with electrondonating group. The introduction of electron-donating group (13u and 13v) was not conducive to biological activity. The nitro group occupying the R 2 position indicated better antiproliferative activity than the R 1 position (13l vs 13b). Expect for 13q, compounds 13m-13q with multiple substituents at R 1 and R 2 positions showed potent antiproliferative activity against MCL cells. Meaningfully, 13c, 13g, 13h, 13l, 13n and 13o demonstrated improved antiproliferative activity with IC 50 values at the single-digit micromolar levels. BTK-independent mechanisms might be involved for the compounds with potent antiproliferative activities while limited BTK inhibition.

Cell apoptosis assay
Compound 13l with excellent antiproliferative activity was selected for further studies on its apoptosis inductive effect on Z138 and Jeko-1 cells. The results were shown in Fig. 3A and B. After 24 h of treatment, dose-dependent apoptosis effects were observed in Z138 and Jeko-1 cells.
After the treatment at 20 μM, 42.8 and 36.6% of apoptotic cells were detected in Z138 and Jeko-1 cells, respectively. The cleavage level of caspase-3 in Z138 cells after 13l treatment were also measured. Impressively, as shown in Fig. 3C, 13l induced the cleavage of caspase-3 in a dose-dependent manner, indicating that 13l induced cell

Effect on mitochondrial membrane potential
Mitochondrial membrane potential (ΔΨm) loss is an early event in apoptosis. We evaluated the effect of 13l on ΔΨm in MCL via a JC-1 fluorescence probe, which selectively enters mitochondria and changes own color from red to green as a sign for ΔΨm decreases. As shown in Fig. 4A, the red fluorescence intensity gradually decreased and green fluorescence gradually increased as the concentrations of 13l increased, indicating a gradually attenuation of ΔΨm level in Z138 cells.

Regulation of reactive oxygen species levels
Reactive oxygen species (ROS) accumulation has been reported to cause cellular apoptosis by the mitochondriadependent pathway [14]. It has been reported that BTK has a role in negatively regulating ROS levels, and ibrutinib can enhance ROS production in CLL [15,16]. Then we used the DCFH-DA probe to evaluate the effect of 13l on ROS level in Z138 cells. As shown in Fig. 4B, the green fluorescence intensity gradually increased as the concentrations of 13l increased. 13l enhanced ROS production in Z138 cells in a dose-dependent manner, which may be an important component in the potency of the antiproliferative activity.

Induction autophagy in MCL cells
Autophagy is considered a mechanism of cell death induced by death programs. Muqbil et al. reported that BTK inhibitors treatment caused autophagic cell death of Hodgkin's lymphoma cells [17]. Therefore, we investigated the induction of autophagy by 13l in Z138 cells. As shown in Fig. 3C, the western blotting results showed that expression of LC3B, a critical component in regulating the autophagy, was significantly increased in a dose-dependent manner after 13l treatment of Z138 cells.

Conclusion
In this study, we outlined the design, synthesis and biological evaluation of novel pyrazolopyrimidine-based derivatives as reversible BTK inhibitors for treating MCL. Compared to lead compound 9, most target compounds had made great progress in antiproliferative activity. Compounds 13c, 13g, 13h, 13l, 13n and 13o displayed favorable antiproliferative activity in MCL cells lines with singlemicromolar IC 50 values. Especially, compound 13l specifically disturbed mitochondrial membrane potential and induced cell apoptosis through the caspase 3-mediated apoptotic pathway in Z138 cells. Furthermore, the upregulated ROS levels and autophagy induction may be the possible mechanisms for its potent antiproliferative activity. In summary, this study provides valuable lead candidates for developing anti-lymphoma agents.

Experimental
All chemical solvents, catalysts, and starting materials were purchased from commercial suppliers and used without further purification. Reaction progress was examined by SGF254 plates under UV (254 nm). Compounds were purified using silica gel chromatography (60 Å, 200-300 mesh). Melting points of target compounds were determined by a Büchi capillary melting point apparatus without correction. 1 H NMR and 13 C NMR spectra of the compounds were recorded using the Bruker Avance DRX-400 Spectrometer at 400 and 100 MHz, respectively. Chemical shifts (δ) are given in ppm relative to tetramethylsilane (TMS, 0.0 ppm) in CDCl 3 or DMSO-d 6 ; J values are given in Hz. HRMS was recorded using an Thermo Scientific Q Exactive.

General synthesis of target compounds 13
Various substituted amines 10a-10v (2 mmol) and NaHCO 3 (7.4 mmol) were added to 1/1 ethyl acetate/water mixture (5 mL). Subsequently, bromoacetyl bromide (4.6 mmol) was added dropwise to the mixture under ice bath conditions. After dropping, the mixture was stirred at room temperature for 1 h. Progress of the reaction was detected by TCL. Upon completion, the organic layer was separated and the water layer was extracted. The combined organic layer phases were dried and concentrated to obtain 11a-11v. Then compounds 11 (0.6 mmol) was added to a mixture of compound 12 (0.5 mmol) and K 2 CO 3 (0.75 mmol) in N, Ndimethylformamide (5 mL). The mixture was stirred at room temperature for 5 h. Upon completion, the reaction solution was quenched by pouring it into water (50 mL). The suspension was filtered, and the cake was purified by ethyl acetate recrystallization or column chromatography (methanol/dichloromethane, 1/200-1/50) to obtain target compounds 13.

Biological assay
In vitro BTK inhibition activity assay The in vitro BTK inhibition effects of target compounds were tested via a KinaseProfiler radiometric protein kinase assay following the previously described protocol [18].

Cell antiproliferation assay
MCL cell lines Mino, Jeko-1, Z138 and Maver-1 were purchased from the American Type Culture Collection (ATCC). The antiproliferative activity of target compounds were tested using the CellTiter-Glo luminescent cell viability assay according to the previously published protocol [19].

Cell apoptosis assay
Apoptosis was quantified by Annexin V/Propidium Iodide (PI)-binding assay following the previously described protocol [20]. In short, Z138 Cells were plated in 6-well plates with 13l at concentrations of 10 or 20 μM and incubated for 24 h. After washing with cold phosphate buffered saline (PBS) and resuspension in 100 μL binding buffer, 2 μL annexin V-FITC and 5 μL PI were added to the treated cells. The samples were subsequently gently vortexed and incubated for 15 min in the dark at room temperature. Then 200 μL binding buffer was added to the samples. Samples were analyzed by flow cytometry on a FACScan flow cytometer. Results were analyzed by the Flowjo software.

Mitochondrial membrane potential
In total, 2 × 10 5 Z138 cells per well were plated in 6-well plates and treated with compound 13l at concentrations of 2.5, 10, and 20 μM for 24 h. The cells were collected, washed with PBS three times, followed by adding JC-1 binding solution, and then incubated at 37°C for 20 min in the dark, and washed twice with PBS. Then, the effects of compound 13l on mitochondrial membrane potential were taken under a confocal laser scanning microscope (Leica TCS SP8).

ROS assay
The regulatory effect of 13l on ROS level in Z138 cells was determined through DCFH-DA probe according to the protocol published earlier [21].

Western blotting
Z138 cells were cultured with specified concentrations of 12l for 72 h. Then the apoptosis marker and autophagy protein levels of treated cells were detected by western blotting according to the previously reported experimental procedures [21].