Design, synthesis and biological evaluation of 2,4-pyrimidinediamine derivatives as ALK and HDACs dual inhibitors for the treatment of ALK addicted cancer

Abstract Simultaneous inhibition of histone deacetylases (HDACs) and anaplastic lymphoma kinase (ALK) could enhance therapeutic activity against ALK addicted cancer cells. Herein, a new series of 2,4-pyrimidinediamine derivatives as ALK and HDACs dual inhibitors were designed, synthesised and evaluated. Compound 12a which possessed good inhibitory potency against ALKwt and HDAC1, exhibited stronger antiproliferative activity than Ceritinib on ALK positive cancer cell lines though inducing cell apoptosis and cell cycle arrest in vitro and in vivo. In addition, the mechanism is further verified by the down-regulation of p-ALK protein, and up-regulation of Acetylated histone 3 (Ac-H3) protein in cancer cells. These results suggested that 12a would be a potential candidate for the ALK addicted cancer treatment.


Introduction
Anaplastic lymphoma kinase (ALK) is a tyrosine kinase that belongs to the insulin receptor (IR) superfamily 1,2 . ALK alterations are often involved in the development of several types of cancers including non-small cell lung cancer (NSCLC), anaplastic large cell lymphoma (ALCL) and neuroblastoma 3,4 . For example, the echinoderm microtubule-associated protein-like 4 (EML4-ALK) fusion, as a common oncogenic gene fusion detected in NSCLC, promotes the dimerisation and phosphorylation of ALK protein, which finally leads to NSCLC occurrence 5 . As a consequence, small molecular ALK inhibitors such as Crizotinib 6 and Ceritinib 7 ( Figure 1) were approved for the treatment of ALK-driven NSCLC via blocking ALK and its downstream signal transduction pathways. However, the effective application of ALK inhibitors is often limited by the drug resistance that emerges following the prolonged treatment in the clinic 8 .
Histone deacetylases (HDACs) are a class of scavenging enzyme that catalysed the removal of acetyl from lysine residues, leading to chromatin condensation and transcriptional suppression in cells 9,10 . However, aberrant activation of HDACs often contributes to the development and progression of many cancers 11 . Hence, inhibition of HDACs can reverse the genetic aberrations of epigenetic states associated with malignancy 9,12 . Currently, four small molecule HDACs inhibitors (HDACIs) (Figure 1), Vorinostat (SAHA) 13 , Belinostat (PXD-101) 14 , Romidepsin (FK228) 15 and Panobinostat (LBH589) 16 targeting HDACs to suppress the growth of cancers were approved by FDA and Entinostat (MS-275) 17 (Figure 1) was being evaluated in the third stages of clinical trials.
As AXL-dependent epithelial-to-mesenchymal transition (EMT) regulated by HDACs has been proved to be associated with the emergence of drug resistance to ALK inhibitors, simultaneous inhibition of HDACs could reduce the levels of H3K27ac related to AXL to decrease its gene expression, thus improving the efficacy of ALK inhibitors 18 . Increasing evidences have indicated that ALK inhibitors in combination with HDACIs could synergistically induce the anti-proliferative effects on ALK inhibitor resistant cells or xenografts and are more efficient in ALK positive NSCLC patients [19][20][21][22] . In addition to the combinational therapy methods, we conceived that ALK and HDACs dual inhibitors that can concurrently inhibit both targets would be an alternative and attractive therapeutic strategy for ALK addicted cancer.
In our previous work, we have discovered a series of ALK and HDACs novel dual inhibitors via fused pharmacophore approach 23 . Among them, the optimal compound 10f featuring a flexible sidechain exhibited good potency on ALK-positive cancer cell lines. However, compound 10f displayed poor antitumor activities in vivo. It was speculated that the hydroxamic acid group might account for the low permeability or aqueous solubility of compound 10f. Therefore, in this work, a more effective ALK/HDACs dual inhibitor compound 12a, referring to the structure of Ceritinib and Entinostat, was identified and evaluated ( Figure 2). To our delight, 12a showed a remarkable antitumor efficacy against different cancer cell lines in vitro and in vivo. These results indicated that 12a would be a promising anti-NSCLC candidate which deserves further research.

Chemistry
The desired compounds 6a-l and 11a-j were prepared according to the synthetic route depicted in Schemes 1 and 2. 2,5-dichloro-N-(2-(isopropylsulfonyl)phenyl)pyrimidin-4-amine (1) was purchased and used as the starting material. With regard to procedures in Scheme 1, m/p-phenylenediamines (2a-b) were firstly reacted with compound 1 to obtain intermediates 3a-b, respectively. Next, methyl alkanoate derivatives were activated by 1-ethyl-3-(3-dimethyllaminopropyl) carbodiimide hydrochloride (EDCI)/1-hydroxybenzotriazole (HOBT), and subsequently condensed with intermediates 3a-b to produce the key ester intermediates 4a-l in the presence of N, N-Diisopropylethylamine (DIPEA). Then 4a-l were hydrolysed by NaOH to afford carboxylic acid intermediates 5a-l, which were finally condensed with o-phenylenediamine to give the target products 6a-l under the same condensed reaction condition. In Scheme 2, compounds 11a-j were synthesised by following similar procedures from compound 1 and p/m-aminobenzoic acid (7a-b).

Biological evaluation
Anti-proliferative activities of target compounds against cancer cell lines The in vitro antiproliferative effects of the target compounds on A549 (lung cancer cells), MDA-MB-231 (breast carcinoma cells), HepG2 (hepatocellular carcinoma cells), and SK-N-BE(2) (neuroblastoma cells) were detected via CCK-8 assay for 72 h, Ceritinib and Entinostat were selected as two positive controls. As shown in Table 1, most of the synthetic compounds displayed stronger inhibitory activity than the positive controls on selected cancer cell line, especially compound 12a, which inhibited the growth of cells with IC 50 values ranging from 0.0003 to 0.01 lM, demonstrating that the HDACs inhibitory effects of these dual inhibitors may contribute to their antiproliferative capacities. Notably, SK-N-BE(2) cell line harbouring ALK wt mutation seems to be the most sensitive cancer cell line than others, indicating that our compounds may be more effective on ALK positive cancer cell lines. To further investigate the effect of linker on the antitumor activity, compounds 6a-f with different linker length (n ¼ 2-6) were synthesised. However, it was found that increasing the linker length may be unfavourable for enhancing the anti-proliferative activity. Moreover, introducing electron-withdrawing or electron-donating groups such as F, Cl, CH 3 groups on the phenyl ring (12b-h) also lead to decreased inhibition activity. Interestingly, the meta-substituted compounds (6g-l) showed good antitumor activity as well as their para-substituted analog (6a-f), indicated that the substituted position may had little effect on inhibition capacities. Finally, in comparison with 6a-l, compounds 11a-j were made as reversed amide and they exhibited parallel potency compared to compound 6a-l.
Considering the excellent antitumor efficacy, compound 12a was further selected to evaluate its potency on ALK-dependent H2228 lung cancer cells, and 12a showed an IC 50 value of 11 nM against the proliferation of H2228, which was almost 10-fold potent than that of Ceritinib and Entinostat (Table 2). In addition, the binding affinity of compound 12a to ALK and HDAC1 were investigated in kinase assay. As depicted in Table 2, although less efficient than Ceritinib and Entinostat, compound 12a showed good inhibitory potency against ALK wt and HDAC1, with IC 50 values of 9.5 and 1450 nM, respectively ( Table 2), indicating that compound 12a was a dual ALK/HDACs inhibitor.

Compound 12a represses cell invasion and migration in vitro
Next, we detected the effect of compound 12a on the migration and invasion ability of A549, H2228 and SK-N-BE(2) cells by cell scratch assay and Transwell method. As shown in Figure 3(A-C), the results showed that the migration capability of all cancer cells was significantly suppressed by 12a after 24 h treatment, compared with the Ceritinib group and Entinostat group. In Transwell assay, 12a also reduced the metastasis ability in A549 and SK-N-BE(2) cells at 4.0 lM or 0.4 lM concentration ( Figure 3D, E). These results clearly displayed that compound 12a can prevent the cell migration and invasion in a dose-dependent manner.

Compound 12a arrests cell cycle and induces cell apoptosis in vitro
Since we had demonstrated that compound 12a is effective to inhibit proliferation of cells in vitro, the effect of compound 12a on the cell cycle was then investigated via flow cytometry to further explore the mechanism. The results of flow cytometry were illustrated in Figure 4(A, B). After A549 cells were treated with 12a at different concentrations (1.0, 2.0, 4.0 lM) for 24 h, it can be observed that 12a could slightly arrest cell cycle at S phase. When H2228 cells were treated in the same manner, the percentage of cell population in G1 phase was increased from 38.95% (control) to 60.92% (1.0 lM), indicating that 12a could arrest the cell cycle at G1 phase in H2228 cells.
Furthermore, AO/EB and Hoechst 33258 staining assays were utilised to evaluate whether compound 12a could induce apoptosis of cells. The AO/EB staining results were illustrated in Figure  5(A, C), it can be seen that the cells in the control group showed well-distributed green fluorescence. On the contrary, after 24 h treatment of compound 12a at 4.0 lM concentration, the number of orange fluorescent cells increased and the cell morphology gradually changed in A549 and H2228 cells, indicating that the number of late apoptotic cells increased. Similarly, the results of  Hoechst 33258 staining further verified that compound 12a could induce cell death. As illustrated in Figure 5(B, D), the uneven blue fluorescence was emerged and enhanced in a dose-dependent manner in compound treatment groups compared with the control groups. Flow cytometry was further conducted to investigate whether the anti-proliferation effect of 12a was associated with cellular apoptosis in A549 and H2228 cells. As shown in Figure  5(E), when treated with compound 12a, the percentage of apoptotic cells increased in a dose-dependent manner, from 10.9% (control) to 46.7% (1.0 lM), 64.2% (2.0 lM), and 66.98% (4.0 lM). Likewise, the apoptosis rate of cells treated with compound 12a also increased significantly in H2228 cells, much greater than that of control ( Figure 5E). From these results, we conclude that 12a was able to cause apoptotic effects at 1ow concentration in cancer cell lines.
Compound 12a blocks ALK and HDAC signalling pathways Furthermore, the changes of protein expression related to apoptosis were also investigated by western blot assays in A549 and H2228 cells, the results were illustrated in Figure 6(A, B). Not surprisingly, compound 12a could dose-dependently up-regulated the expression of pro-apoptotic protein Bax accompanied with a decreased expression of anti-apoptotic protein Bcl-2, which were correlated with previous outcomes.
Meanwhile, to clarify the mechanism, the relative proteins involved in the ALK-or HDAC-mediated signalling pathway were also tested. It was found that the intracellular levels of p-ALK was significantly suppressed in 12a treatment groups (4.0 lM), compared to that of control group, while the expression of ALK was not changed. On the other hand, after exposure to compound 12a for 24 h, the expression levels of Acetylated histone 3 (Ac-H3) protein increased in  a dose-dependent manner in drug treatment group ( Figure 6C, D). Taken together, these results displayed that compound 12a could block the ALK-and HDAC-mediated signalling pathways simultaneously as a dual inhibitor.

Compound 12a inhibits the growth of SK-N-BE(2) cells in vivo
On the basis of the favourable in vitro anticancer activity, compound 12a was further selected to evaluate its preliminary antitumor efficacy in the SK-N-BE(2) xenografts in vivo 24 . When tumours had reached an average volume of 100 mm 3 , mice were random divided into four groups and intraperitoneally administered with saline, 25 or 100 mg/kg compound 12a and 50 mg/kg Ceritinib every two days for 16 consecutive days, respectively. The results in Figure 7(A, C) showed that compound 12a could significantly suppress the tumour growth. Compared with 50 mg/kg Ceritinib, compound 12a treatment group at a dose of 25 or 100 mg/kg results in 37.2% and 64.7% TGI (tumour growth inhibition), respectively. In addition, no obvious weight loss was observed in all compound treatment groups ( Figure 7B). Moreover, the H&E staining results showed no obvious pathological changes were observed in lung, heart and kidney, but vacuolisation in liver were observed in Ceritinib group and high-dose group, indicating that high dose of 12a may have toxic effects on liver ( Figure 7E).

Molecular docking studies
Docking studies of compound 12a with ALK and HDAC2 were performed ( Figure 8A-C). Similar to Ceritinib, the pyrimidine nitrogen atom in compound 12a establish strong hydrogen bonds at the hinge area with Met1199. In addition, the phenylamine group was found to be projected towards solvent region which didn't form key interactions with ALK ( Figure 8A, B). On the other hand, from the overlap model of 12a, the original ligand and Entinostat in HDAC2, we could see that the NH 2 group and carbonyl group in compound 12a can coordinate with Zn 2þ to form a stable six-ring in ZBG pocket, as the original ligand and Entinostat did, which might explain the good inhibitory activity of 12a against HDAC2 ( Figure 8C). Interestingly, it can be observed that the GAP group in compound 12a and Entinostat adopts a totally opposite orientation, respectively, which might barely affect the HDAC2 inhibitory potency.

Conclusions
In summary, a novel and potent dual ALK and HDACs inhibitor 12a was discovered. Compound 12a exhibited good inhibitory activities against ALK wt or HDAC1 enzymes and synergistically inhibited proliferation of ALK-driven cancer cells via inducing cell apoptosis and cell cycle arrest. Western blot assays were further conducted and confirmed that compound 12a can concurrently inhibits ALK and HDACs signal pathways as an ALK/HDACs dual inhibitor. More importantly, compound 12a possessed desirable in vivo antitumor potency in a SK-N-BE(2) xenograft model. These results suggested compound 12a was expected to be a good candidate for the ALK-positive cancer treatment.

Chemistry
All materials used in this study were obtained from Tansoole, Macklin and Adamas without further purification after purchasing. The conventional 1 H NMR and 13 C HNMR spectra were measured in CDCl 3 /DMSO-d 6 by a Bruker spectrometer with tetramethylsilane (TMS) as internal standard. Coupling constants (J) and chemical shifts (d) are noted in Hz and ppm separately. The melting point of compounds was detected with microscopic melting point apparatus. High-resolution mass spectra (HRMS) were recorded  with Aglient 6470 Triple Quad LC-MS apparatus. Column chromatography was conducted on silica gel (200-300 mesh).
General procedure for the synthesis of intermediates 3a-b To a solution of p-phenylenediamine (1.25 g, 11.6 mmol, 2a) in 50 mL isopropanol, was added 2,5-dichloro-N-(2-(isopropylsulfonyl)phenyl)pyrimidin-4-amine (3.48 g, 10.0 mmol, 1) and 37% HCl solution (0.8 mL). The mixture was heated and stirred at 90 C for 11 h. After reaction completion, the solvent was evaporated in vacuo and extracted with 100 mL EtOAc twice, then the organic layer was sequentially washed with saturated sodium chloride aqueous solution twice, dried over anhydrous Na 2 SO 4 and filtered to give desired compound 3a which can be directly used without further purification. 3b was synthesised in the manner of 3a.
General procedure for the synthesis of intermediates 4a-l Methyl 3-((4-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)phenyl)amino)-3-oxopropanoate (4a                  General procedure for the synthesis of intermediates 5a-l Sodium hydroxide (0.10 g, 2.50 mmol) was dissolved in methanol/ water (80%, 10 mL) mixture and heated to 70 C, compound 4a (0.40 g, 0.77 mmol) was slowly added to the solution and stirred under reflux for 6 h. When the reaction completed, the solvent was evaporated in vacuo, and water (40 mL) was added, after stirring for 0.5 h at room temperature. The reaction mixture was filtered, dried in an oven to give intermediate 5a which can be directly used. 5b-l were synthesised in the manner of 5a.

Enzyme inhibitory activity assay
The enzyme inhibitory activity assay was conducted with a fluorescent assay kit as we previously reported 23 .

Migration assay
For Transwell method, the tumour cells were cultured in serumfree DMEM/RPMI-1640 medium for 24 h, then seeded into the upper chamber at a density of 3 Â 10 5 cells (100 lL/well), and DMEM/RPMI-1640 medium (750 lL/well) with 10% FBS was added to the lower chamber of 24-well plate. The cells of upper chamber were treated with the drug containing medium or DMSO control (100 lL/well) for 24 h at 37 C. Finally, the migrated cells were fixed with 4% formaldehyde, dyed with crystal violet, and washed by PBS. The selected area was photographed by inverted microscope (Nikon, Japan) and determined using ImageJ software.
For wound healing method, A549, SK-N-BE(2) and H2228 cells were seeded in 6-well flat-bottomed plates and incubated overnight. Cells were scratched with a pipette tip to generate a cleanwound area in the cell layer, following washed with PBS and treated with compound 12a, positive control (Ceritinib, Entinostat), then replenished with serium-free DMEM/RPMI-1640 medium. The cells were photographed by inverted microscope at 0 h, 24 h or 72 h. Quantification of the area of scratch were measured by ImageJ software.

Flow cytometric analysis
Flow cytometric (FCM) was performed to detect cell cycle and apoptosis. Briefly, cancer cells (5 Â 10 5 cells/well) were grown in 6-well plate overnight and then incubated with compound 12a, positive control (Ceritinib, Entinostat) for 24 h, then washed with PBS. In terms of cell cycle detection, resuspended with ice-cold ethanol (70%) for 24 h and stained with propidium iodine (PI) and Annexin V-FITC for 30 min. Finally, the samples were detected by FCM (CytoFLEX, Beckman Coulter, US). For apoptosis analysis, the cells were gathered with cold PBS, and then stained with PI and RNase A. Subsequently, the treated samples were tested with FCM.

Western blot analysis
The cancer cells were seeded and treated with compound 12a, positive control (Ceritinib, Entinostat) for 24 h, cells in each well were washed and decomposed in lysis buffer. Proteins were detached by SDS-PAGE and transferred to PVDF membrance. After being blocked by 5% BSA, washed and incubated with primary antibodies (1:1000 diluted) (Changzhou affinity Biosciences, China) at 4 C, followed by incubated with horseradish peroxidase (HRP) conjugated secondary antibodies (1:5000 diluted) (Shanghai Beyotime institute of biotechnology, China). Finally, the enhanced chemiluminescence reagent was conducted to detect the bands. Immunoblotting was analysed by densitometry using the software ImageJ.

Hoechst 33258 and AO/EB staining analysis
The tumour cells were stained with AO/EB or Hoechst 33258 to preliminarily identify apoptotic morphological changes. In short, H2228 or A549 cells were seeded into 6-well plates and treated with compound 12a, positive control (Ceritinib, Entinostat) for 24 h. For AO/EB staining assay, after washing with PBS, cells were gathered and dyed with AO/EB for 15 min. Then cells were photographed using an inverted fluorescence microscope. For Hoechst 33258 staining assay, the cells were fixed with 4% polyformaldehyde for 10 min and stained with Hoechst 33258 solution after washing cells with PBS for twice. Finally, an anti-fluorescence quencher was added in each well for observation by an inverted fluorescence microscope.

SK-N-BE(2) xenograft model assay
All animal studies were conducted according to the guidelines of the Animal Experimental Ethics Committee of Chongqing Medical University (ID. SCXK2018-0003). Female BALB/C nude mice aged 4 weeks, were utilised to establish the SK-N-BE(2) xenograft model for determining the antitumor effect of compound 12a in vivo. To put it briefly, a 100 lL suspension of 5 Â 10 6 SK-N-BE(2) cells was injected subcutaneously into the one flank region of nude mouse (Chengdu Yaokang Bio-Technology Co., LTD, Chengdu, China). Once the size of the tumours reached approximately 100-150 mm 3 , the mice were randomly divided into four groups and intraperitoneally (i.p.) administrated with 0.9% NaCl, compound 12a (25 and 100 mg/kg) or Ceritinib (100 mg/kg) once every two days for 16 days. The tumour volume was calculated with the formula (length Â width 2 )/2. The tumour volume and body weight of each mouse were determined using calliper every 2 days. On day 16, the mice were sacrificed, xenograft tumour were dissected and the weight was measured in each group. The organs were further examined by H&E staining to observe drug toxicity.

Molecular docking
The Molecular docking studies were conducted with AMDOCK software as previously reported 25 . (PDB Code: 4MKC) and (PDB Code: 5IWG) were used and obtained from the Protein Data Bank.

Statistical analysis
The data were analysed using GraphPad Prism 6 software and expressed as mean ± SD of three independent experiments. The statistical analyses were performed using t-test and the statistical differences were measured using a one-way ANOVA with p < 0.05, which was considered as statistically significant.