key: cord-0012768-0cvrhah4
authors: Lai, Meng-zhen; Song, Pei-ran; Dou, Dou; Diao, Yan-yan; Tong, Lin-jiang; Zhang, Tao; Xie, Hua; Li, Hong-lin; Ding, Jian
title: Discovery and biological evaluation of N-(3-(7-((2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)-4-methyl-2-oxo-2H-pyrimido[4,5-d][1,3]oxazin-1(4H)-yl)phenyl)acrylamide as potent Bruton’s tyrosine kinase inhibitors
date: 2019-07-17
journal: Acta Pharmacol Sin
DOI: 10.1038/s41401-019-0250-8
sha: 8bee9f4612fa395c36dc6c6a5ba17c9ed06b7879
doc_id: 12768
cord_uid: 0cvrhah4

Bruton’s tyrosine kinase (BTK) is a key component of the B cell receptor (BCR) signaling pathway and plays a crucial role in B cell malignancies and autoimmune disorders; thus, it is an attractive target for the treatment of B cell related diseases. Here, we evaluated the BTK inhibitory activity of a series of pyrimido[4,5-d][1,3]oxazin-2-one derivatives. Combining this evaluation with structure-activity relationship (SAR) analysis, we found that compound 2 exhibited potent BTK kinase inhibitory activity, with an IC(50) of 7 nM. This derivative markedly inhibited BTK activation in TMD8 B cell lymphoma cells and thus inhibited the in vitro growth of the cells. Further studies revealed that compound 2 dose dependently arrested TMD8 cells at G(1) phase, accompanied by decreased levels of Rb, phosphorylated Rb, and cyclin D1. Moreover, following treatment with compound 2, TMD8 cells underwent apoptosis associated with PARP and caspase 3 cleavage. Interestingly, the results of the kinase activity assay on a small panel of 35 kinases showed that the kinase selectivity of compound 2 was superior to that of the first-generation inhibitor ibrutinib, suggesting that compound 2 could be a second-generation inhibitor of BTK. In conclusion, we identified a potent and highly selective BTK inhibitor worthy of further development.

The Bruton's tyrosine kinase (BTK) protein is a nonreceptor tyrosine kinase belonging to the TEC family [1] [2] [3] . BTK is expressed in the cells of all hematopoietic lineages except for T and plasma cells [4, 5] . BTK is a critical downstream molecule of the B cell receptor (BCR) signaling pathway and plays an important role in the development of B cells [6, 7] . Dysregulation of BTK usually causes severe B cell related lymphomas and autoimmune diseases. These characteristics of BTK make it a very attractive treatment target for B cell malignancies [8] [9] [10] .

There are two BTK inhibitors that have received marketing authorization for the treatment of B cell malignancies. The firstgeneration agent ibrutinib (PCI-32765) was approved by the US Food and Drug Administration (FDA) in 2013 to treat mantle cell lymphoma (MCL) and was subsequently approved for various indications, such as chronic lymphocytic leukemia (CLL), Waldenstrom's macroglobulinemia (WM), and marginal zone lymphoma [11] [12] [13] . However, ibrutinib not only irreversibly binds to BTK but also induces off-target inhibition [14] [15] [16] , which might cause bleeding, rash, diarrhea, and atrial fibrillation during clinical treatment [17, 18] . To overcome the abovementioned limitations, the second-generation BTK inhibitor acalabrutinib (ACP-196), with better selectivity than ibrutinib, has been developed and received FDA approval in 2017 [19] [20] [21] [22] .

In our previous work, we identified a series of pyrimido [4,5-d] [1, 3] oxazin-2-one derivatives as potent mutant-selective epidermal growth factor receptor (EGFR) inhibitors [23] (Table 1) . Recently, we unexpectedly found that compound 1 exhibited a moderate IC 50 value of 28.8 nM against EGFR and an excellent IC 50 value of 4.7 nM against BTK. To obtain selective BTK inhibitors, we began our study of pyrimido [4,5-d] [1, 3] oxazin-2-one-derived BTK inhibitors by investigating structure-activity relationships of previously synthesized pyrimido [4,5-d] [1, 3] oxazin-2-one irreversible EGFR inhibitors. Then, from the the kinase activity and selectivity results, we found that compound 2 could be a representative BTK inhibitor for further exploration.

We used covalent docking to evaluate the binding pattern of the compound through the software of Maestro 10.1. We chose the PDB code 5P9L from the Protein Date Bank, and this PDB includes the crystal structure of BTK in complex with a small molecule CC-2922 [24] . As we expected, the compound binds to the ATPbingding pocket of BTK, and the acrylamide of this compound proceeds Michael addition reaction with the thiol of Cys481. The position of inhibitor's core should be constrained to a maximum acceptable RMSD value of 1.0 Å from that of the binding ligand CC-292. The docking mode was defined as predicted pose, which find the precise docking pose through the full protocol. Glide was used to assess the affinity score between the inhibitor and BTK [25] .

Cell culture and reagents The human diffuse large B cell lymphoma (DLBCL) cell line TMD8 was obtained from the American Type Culture Collection (Manassas, VA, USA) and was cultured in RPMI-1640 medium (Gibco, USA) supplemented with 10% FBS (Gibco, USA). The TMD8 cell line was maintained at 37°C in 5% CO 2. Kinase assay An enzyme-linked immunosorbent assay (ELISA) was used to evaluate the kinase inhibitory activity of compound 2, ibrutinib, and ACP-196. The kinases were purchased from Eurofins (Brussels, Belgium). The experiment was performed according to standard ELISA procedures as previously described, and the absorbance was read at 490 nm with a multiwell spectrophotometer [26] .

Cell proliferation assay A Cell Counting Kit-8 (CCK8) assay was used to evaluate cell proliferation. TMD8 cells were seeded in 96-well plates at a density of 10,000 cells/well and incubated for 2 h, followed by exposure to compound 2, ibrutinib, and ACP-196 at various concentrations for another 72 h. Subsequently, 10 μL of CCK8 solution (5 mg/mL) was added to each well for 1-2 h, and the results were assessed by measuring the absorbance at 450 nm using SoftMax Pro Software. The cell proliferation inhibition rate was calculated as follows: ½1 − ðA 450 treated =A 450 control Þ × 100% [27] . The logit method was used to obtain the IC 50 values.

Western blot analysis TMD8 cells were lysed in RIPA lysis buffer as previously described after washing with PBS [28] . Subsequently, TMD8 protein samples were used for Western blot analysis. Primary antibodies against the following proteins were used: actin (#3700), BTK (#8547), p-BTK (#87141), Rb (#9309), p-Rb (#2181), cyclin D1 (#2922), PARP (#9532S), caspase 3 (#9665S), and cleaved caspase 3 (#9664S). Antibodies were obtained from Cell Signaling Technologies (Cambridge, MA, USA).

Cell cycle analysis TMD8 cells were seeded in 12-well plates, incubated for 2 h, and exposed to compound 2, ibrutinib, and ACP-196 at various concentrations for another 48 h. A cell cycle analysis kit (Beyotime Biotechnology, Shanghai, China) was used to assess the cell cycle arrest of TMD8 cells. The cell cycle distribution was analyzed with a Becton-Dickinson FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA), and the data were analyzed with Flow Jo software [29] .

Cell apoptosis analysis TMD8 cells were seeded in 12-well plates and incubated for 2 h and exposed to compound 2, ibrutinib, and ACP-196 at various concentrations for another 72 h. An Annexin V-FITC kit (Vazyme Biotechnology, Nanjing, China) was used to detect the apoptosis of TMD8 cells. The cell apoptosis distribution was analyzed with a Becton-Dickinson FACS Calibur flow cytometer (BD Biosciences, San Jose, CA, USA), and the data were analyzed by Flow Jo software [29] .

Statistical analysis Data are presented as the means ± standard deviations (SDs). The significance of differences between experimental groups was evaluated with a two-tailed Student's t test. A P value of <0.05 was considered statistically significant. Significant differences are indicated as *P < 0.05, **P < 0.01, and ***P < 0.001.

In our previous research, we identified a series of pyrimido[4,5-d] [1, 3] oxazin-2-one derivatives as epidermal growth factor receptor (EGFR) inhibitors [23] , among which compound 1 also exhibited inhibitory activity against BTK. An in vitro enzymatic activity assay was performed, and the results are shown in Table 1 . The left N-methylpiperazine ring extends to the solvent-exposed region, and it is poised in a suitable position to form favorable electrostatic interactions with Glu488 and hydrophobic interactions with Asn484. As expected, the electrophilic acrylamide group is covalently bonded to Cys481. The hydrogen atom at the C-5 position of compound 1 directly points to the side chain of the gatekeeper residue Thr474, but there is still enough space to accommodate larger groups (Fig. 1a) . Therefore, we discuss the effects of different substituents with varying volumes on the disparity in kinase activity between BTK and EGFR. On the one hand, diverse alkyl groups were introduced at the R 2 position, while R 1 was maintained as a hydrogen atom. Guided by this idea, compounds 2-5 were synthesized. On the other hand, R 1 and R 2 were simultaneously substituted with the same alkyl groups of different lengths, and the corresponding synthesized compounds were designated compounds 6-8. The inhibitory potencies of compounds 1-8 against BTK kinase were evaluated using an ELISA-based kinase assay. When we changed only R 2 , the kinase activity against BTK and the selectivity over EGFR showed a clear difference (Table 1 ). In general, the introduction of an alkyl group at the C-5 position led to a decrease in the activities of 1-8 against BTK. In addition, the kinase activity against BTK decreased as the length of the alkyl chain increased. However, compounds 1 and 2 still exhibited relatively high kinase activities against BTK, with IC 50 values of 4.7 nM and 7.0 nM, respectively. Because our purpose was to discover highly selective BTK inhibitors, we considered not only the kinase activity against BTK but also the kinase activity against EGFR for the compounds reported in this study.

To further explore the effect of group size on BTK kinase activity and selectivity, the binding pose of compound 2 was also predicted with Maestro 10.1 (Fig. 1b) . The methyl group of compound 2 lies directly beneath the side chain of the gatekeeper residue Thr474. Due to the limited space in the pocket near the gatekeeper residue, compound 3, with an ethyl group at R 2 , displayed a slight decrease in activity. Given the docking mode, we hypothesized that as the steric hindrance is enhanced, the kinase activity against BTK decreases. The results confirmed our hypothesis (Table 1 ). Compound 4, with a propyl group at R 2 , has an IC 50 of 211.0 nM against BTK, indicating that this compound was 45-fold less potent than compound 1 and 30-fold less potent than compound 2. The addition of the isopropyl group at R 2 in compound 5 resulted in an activity loss of~124-fold relative to compound 1 (IC 50 = 583.9 vs. 4.7 nM) and of 83-fold relative to compound 2 (IC 50 = 583.9 vs. 7.0 nM).

In accordance with the activity trend observed above, compounds 6-8, with two alkyl groups at the C-5 position, showed a greater loss of potency against BTK. In the ELISA-based kinase assay, the IC 50 value of compound 6 indicated a 47-fold activity loss relative to compound 1. Moreover, the addition of the second methyl group resulted in a 32-fold activity loss for compound 6 relative to compound 2 (IC 50 = 221.6 vs. 7.0 nM). It should be noted that compounds 2-6, to which a single alkyl group was introduced at the C-5 position, displayed higher activity against BTK than against EGFR. In contrast, compounds 6-8, bearing two alkyl substituents at the same position, exhibited reversed selectivity between the two enzymes, providing significant clues for further optimization of selective BTK inhibitors.

As mentioned above, compound 2 potently inhibited the kinase activity of BTK, with an IC 50 value of 7.0 nM, was weaker than ibrutinib, with an IC 50 value of 0.6 nM but better than ACP-196, with an IC 50 value of 8.6 nM. In addition, compound 2 exhibited greater than 6-fold higher selectivity for BTK than for EGFR (IC 50 = 7.0 vs. 47.4 nM). Therefore, compound 2 was chosen for further evaluation at the cellular level.

Antiproliferative effects and target inhibition of compound 2 in TMD8 cells We then investigated the antiproliferative activity of compound 2 in TMD8 lymphoma cells, which express high levels of BTK and are widely used to evaluate the antitumor effect of BTK inhibitors [18, [30] [31] [32] . Ibrutinib and ACP-196 were used as the positive control compounds [22, 33, 34] . The results revealed that compound 2 inhibited the proliferation of TMD8 cells with an IC 50 value of 0.028 μM, indicating that compound 2 was slightly less potent than ibrutinib (IC 50 = 0.010 μM) and ACP-196 (IC 50 = 0.014 μM) ( Table 2) . Moreover, the Western blot analysis results revealed that compound 2, ibrutinib and ACP-196 effectively suppressed the phosphorylation of BTK in TMD8 cells, confirming that the target inhibition by these compounds accounts for their antiproliferative activity (Fig. 2) . The percentage of the G 1 phase cells was quantitatively depicted. Statistically significant differences were presented as *P < 0.05, **P < 0.01, compared with the control group. c TMD8 cells were treated with 2, ibrutinib and ACP-196 for 48 h, and the total cell lysates were analyzed by Western blot to evaluate the alterations of G 1 phase related protein Fig. 4 Compound 2 induced cell apoptosis in TMD8 cells. a 2 significantly upregulated the apoptotic level of TMD8 cells equivalent to ibrutinib and ACP-196. b Apoptosis rates were quantitatively depicted. Statistically significant differences were presented as *P < 0.05, **P < 0.01, compared with the control group. c The expression of cleaved PARP and cleaved caspase 3 in TMD8 cells was determined by Western blot analysis

Compound 2 arrested the cell cycle at the G 1 phase and induced apoptosis in TMD8 cells To gain insight into the mechanism underlying the inhibition of cell growth by compound 2, we further evaluated the effects of this compound on the cell cycle and apoptosis in TMD8 cells. After treatment with the indicated concentrations of the compounds for 48 h, compound 2 was found to markedly arrest cell cycle progression at the G 1 phase in a dose-dependent manner (Fig. 3a,  b) . Approximately 74.44% and 82.70% of the cells were arrested in G 1 phase when treated with concentrations of 100 nM and 1000 nM, respectively, equivalent to the results with ibrutinib and ACP-196. In agreement with these results, compound 2 caused a reduction in the levels of cell cycle-related proteins, including the Rb, phosphorylated Rb, and cyclin D1 proteins, consistent with the observation of G 1 arrest (Fig. 3c) . We then tested the effects of compound 2 on the apoptosis of TMD8 cells. Compound 2 triggered concentration-dependent apoptosis of the cells with apoptosis rates of 44.46%, 57.95%, and 69.46% for concentrations of 10 nM, 100 nM, and 1000 nM, respectively; the rate was 12.77% in untreated control cells (Fig. 4a,  b) . We further investigated the mechanism involved in the apoptosis induced by compound 2, and the results showed that the cleavage of the poly (ADP-ribose) polymerase (PARP) and caspase 3 proteins was increased in a dose-dependent manner, indicating that compound 2 induced apoptosis through a caspase-related mechanism (Fig. 4c) .

In conclusion, the results indicated that compound 2 exerted its antitumor effects through arresting the cell cycle at the G 1 phase and inducing apoptosis.

Kinase selectivity of compound 2 We then evaluated the kinase selectivity profile of compound 2 against a panel of 35 kinases available in our lab (Fig. 5) . At a concentration of 1000 nM, compound 2 exhibited greater than 50% inhibition against seven kinases, including kinases sharing a conserved cysteine at the same position (such as EGFR, ErbB2, ErbB4, and ITK). Compound 2 did not appreciably inhibit the other kinases tested in this work. The kinase profile of compound 2 was quite similar to that of the 2nd generation BTK inhibitor ACP-196.

Lymphoma is a malignancy with high morbidity and mortality, and mature B cell lymphoma is the most common type of lymphoma [35] . The BCR is an important B cell antigen receptor that can regulate various B cell functions, including proliferation, survival, differentiation, and apoptosis [36] . The most important component in the BCR signaling pathway is BTK [37] and dysregulation of BTK generally causes severe B cell related lymphomas. Among the different treatment approaches, small molecule inhibitors targeting BTK might be an effective method to cure B cell lymphoma. To date, only two BTK inhibitors, ibrutinib, and ACP-196, have been approved for the treatment of BTKinduced diseases, and it is very important to develop novel BTK inhibitors with increased kinase selectivity and more potent antitumor activity.

In our study, the inhibitory efficacy of a series of pyrimido[4,5-d] [1, 3] oxazin-2-one derivatives against BTK was evaluated using an in vitro enzymatic activity assay. Compound 2 was identified as the most potent compound, with an IC 50 of 7.0 nM against BTK. Moreover, this compound inhibited the activation of BTK in TMD8 lymphoma cells and thus repressed the in vitro growth of the cells. Further research showed that compound 2 induced cell death by triggering apoptosis and arresting the cell cycle at the G 1 phase. Interestingly, the kinase selectivity of compound 2 was superior to that of ibrutinib and equal to that of ACP-196 based on our data from a panel of 35 kinases. These data indicated that compound 2 might have fewer side effects than the first-generation BTK inhibitor ibrutinib. The activity of compound 2 against more kinases should be measured to exploit additional advantages of this compound. Moreover, it will also be meaningful to investigate the synergistic activity of compound 2 in combination with other drugs, such as antibody drugs and other BCR pathway inhibitors. Overall, our results identified compound 2 as a novel BTK inhibitor worthy of further investigation.

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Competing interests: The authors declare no competing interests.