Identification of 1H-pyrazolo[3,4-b]pyridine derivatives as potent ALK-L1196M inhibitors

Abstract Anaplastic lymphoma kinase (ALK) has been recognised as a promising molecular target of targeted therapy for NSCLC. We performed SAR study of pyrazolo[3,4-b]pyridines to override crizotinib resistance caused by ALK-L1196M mutation and identified a novel and potent L1196M inhibitor, 10g. 10g displayed exceptional enzymatic activities (<0.5 nM of IC50) against ALK-L1196M as well as against ALK-wt. In addition, 10g is an extremely potent inhibitor of ROS1 (<0.5 nM of IC50) and displays excellent selectivity over c-Met. Moreover, 10g strongly suppresses proliferation of ALK-L1196M-Ba/F3 and H2228 cells harbouring EML4-ALK via apoptosis and the ALK signalling blockade. The results of molecular docking studies reveal that, in contrast to crizotinib, 10g engages in a favourable interaction with M1196 in the kinase domain of ALK-L1196M and hydrogen bonding with K1150 and E1210. This SAR study has provided a useful insight into the design of novel and potent inhibitors against ALK gatekeeper mutant.

As part of continuing studies aimed at discovering structurally distinct and potent ALK-L1196M inhibitors, we carried out SAR investigation using pyrazolopyridine derivatives bearing 3-fluorophenyl sulfone moiety. The substances utilised in this study were designed based on the reported 3-amino-5-substituted indazolebased ALK inhibitors including entrectinib 11,38 . SAR exploration using novel pyrazolopyridine derivatives led to identification of 10 g as a novel and potent inhibitor against ALK-L1196M (IC 50 < 0.5 nM) as well as against ALK-wt (IC 50 < 0.5 nM). It is worthwhile noting that entrectinib 11,38 inhibits ALK-wt with an IC 50 value of 12 nM.
Reaction progress was monitored by TLC analysis using a UV lamp, ninhydrin, or p-anisaldehyde stain for detection purposes. All solvents were purified by standard techniques. Purification of reaction products was carried out by silica gel column chromatography using Kieselgel 60 Art. 9385 (230 À 400 mesh). The purities of all compounds were shown to be over 95% by using Waters LCMS system (Waters 2998 photodiode array detector, a Waters 3100 mass detector, a Waters SFO system fluidics organiser, a Water 2545 binary gradient module, a Waters reagent manager and a Waters 2767 sample manager) using a SunFireTM C18 column (4.6 mm Â 50 mm, 5 mm particle size): solvent gradient ¼ 60% (or 95%) A at 0 min, 1% A at 5 min. Solvent A ¼ 0.035% TFA in H 2 O; solvent B ¼ 0.035% TFA in MeOH; flow rate 3.0 (or 2.5) mL/ min. 1 H and 13 C NMR spectra were obtained by using a Bruker 400 MHz FT-NMR (400 MHz for 1 H, and 100 MHz for 13 C) spectrometer. Standard abbreviations are used for denoting the signal multiplicities.

5-((3-Fluorophenyl)sulfonyl)-1-trityl-1H-pyrazolo[3,4-b]pyridin-3amine (8)
A suspension of compound 7 (1 g, 1.5 mmol) and hydrazine monohydrate (0.22 mL, 4.5 mmol) in THF (15 mL) and EtOH (15 mL) was stirred at room temperature for 1 h. The mixture was concentrated and the generated residue was subjected to silica gel column chromatography (20-50% EtOAc/hexane) to afford 8 (680 mg, 85%) as a pale yellow solid. 1 To a solution of 8 (500 mg, 0.94 mmol) and DMAP (50 mg, 0.41 mmol) in pyridine (10 mL) was added 4-(1,3-dioxoisoindolin-2-yl)benzoyl chloride (400 mg, 1.4 mmol) dropwise at 0 C under nitrogen atmosphere. The mixture was stirred at room temperature for 1 h, then diluted with water, and extracted with DCM. The organic layer was dried over Na 2 SO 4 , filtered and concentrated. The resulting crude was subjected to silica gel column chromatography (25-80% EtOAc/hexane) to afford 9i (680 mg, 92%) as a yellow solid. 1  was stirred at room temperature for 1 h. The mixture was concentrated and the generated residue was subjected to silica gel column chromatography (50-80% EtOAc/hexane) to afford 11 (150 mg, 88%) as a pale yellow solid. 1  General procedure A for the synthesis of compounds 10a-10h To a solution of 8 (1 equiv) in pyridine (0.1 M) was added various acid chlorides (1.05 equiv) and DMAP (0.6 equiv) at 0 C under nitrogen atmosphere. The mixture was stirred at room temperature for 0.5 h, then diluted with water, and extracted with DCM. The organic layer was dried over Na 2 SO 4 , filtered, and concentrated. The residue was diluted with dry DCM (0.5 M), and added slowly with TFA (2 equiv) at 0 C. The mixture was stirred at room temperature for 0.5 h, diluted with water, and extracted with DCM. The organic phase was washed with brine, dried over Na 2 SO 4 , filtered and concentrated. The resulting crude product was subjected to flash column chromatography on silica gel.
General procedure A was used to transform 8 (50 mg, 0.09 mmol) to the target compound. The resulting residue was subjected to flash column chromatography on silica gel (50-100% THF/hexane) to afford 10a (14.2 mg, 37%) as a yellow solid. 1 General procedure A was used to transform 8 (50 mg, 0.09 mmol) to the target compound. The resulting residue was subjected to flash column chromatography on silica gel (40-90% THF/hexane) to afford 10 b (21.5 mg, 51%) as a white solid. 1

N-(5-((3-Fluorophenyl)sulfonyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)-4-(trifluoromethyl)benzamide (10c)
General procedure A was used to transform 8 (50 mg, 0.09 mmol) to the target compound. The resulting residue was subjected to flash column chromatography on silica gel (40-90% THF/hexane) to afford 10c (18.7 mg, 45%) as a white solid. 1 General procedure A was used to transform 8 (50 mg, 0.09 mmol) to the target compound. The resulting residue was subjected to flash column chromatography on silica gel (30-70% THF/hexane) to afford 10d ( General procedure A was used to transform 8 (50 mg, 0.09 mmol) to the target compound. The resulting residue was subjected to flash column chromatography on silica gel (50-90% THF/hexane) to afford 10e (10.1 mg, 26%) as a yellow solid. 1 General procedure A was used to transform 8 (50 mg, 0.09 mmol) to the target compound. The resulting residue was subjected to flash column chromatography on silica gel (50-90% THF/hexane) to afford 10f (11.5 mg, 27%) as a white solid. 1  N-(5-((3-Fluorophenyl)sulfonyl)-1H-pyrazolo [3,4-b]pyridin-3-yl)-4-(4-methylpiperazin-1-yl)benzamide (10g) General procedure A was used to transform 8 (50 mg, 0.09 mmol) to the target compound. The resulting residue was subjected to flash column chromatography on silica gel (50-100% THF/hexane) to afford 10 g (16 mg, 36%) as a white solid. 1 General procedure A was used to transform 8 (50 mg, 0.09 mmol) to the target compound. The resulting residue was subjected to flash column chromatography on silica gel (50-100% THF/hexane) to afford 10 h (9 mg, 20%) as a white solid. 1 To a solution of 11 (50 mg, 0.076 mmol) in DCM (1 mL) was added TFA (0.1 mL, 0.15 mmol) dropwise. The mixture was stirred at room temperature for 0.5 h, diluted with water, and extracted with DCM. The organic phase was washed with brine, dried over Na 2 SO 4 , filtered and concentrated. The resulting crude product was subjected to flash column chromatography on silica gel (40-80% THF/hexane) to afford 12 (16 mg, 51%) as a pale yellow solid. 1  General procedure B for the synthesis of compounds 13a-13c To a solution of 11 (1 equiv) in pyridine (0.1 M) was added various acid chlorides (1.05 equiv) and DMAP (0.6 equiv) at 0 C under nitrogen atmosphere. The mixture was stirred at room temperature for 0.5 h, then diluted with water, and extracted with DCM. The organic layer was dried over Na 2 SO 4 , filtered, and concentrated. The residue was diluted with dry DCM (0.5 M) and treated slowly with TFA (2 equiv) at 0 C. The mixture was stirred at room temperature for 0.5 h, diluted with water, and extracted with DCM. The organic phase was washed with brine, dried over Na 2 SO 4 , filtered and concentrated. The resulting crude product was subjected to flash column chromatography on silica gel.

Biochemical kinase assay
Biochemical kinase assay was performed by methods previously reported 39 . Kinase reactions of all test compounds except 10g were carried out at 10 mM ATP. Kinase reaction of 10g was performed at three different concentrations of ATP (10/50/100 mM).

Anti-proliferation assay
Cells (ALK wt-TEL, ALK L1196M-TEL and Parental Ba/F3: 1 Â 10 4 , H2228: 5 Â 10 3 ) were plated in 96 well tissue culture plates. Each compound was added to each well at 10 dose points of three-fold serial dilution in DMSO. After treatment with each compound for 72 h, CTG assay solution (Promega, # G7572) was added to each well. Cell proliferation was assessed by measuring the luminescence using a 96 well plate reader (EnVision 2013).

Apoptosis analysis and cell cycle arrest
After 24-48 h following compound treatment, cells were harvested and stained with annexin V (1:500 diluted in annexin V buffer) for 30 min and PI (1:200) solution for 30 min. 1 Â 10 6 cells were analysed using flow cytometric analysis. For the cell cycle arrest analysis, cells were harvested by trypsinisation and fixed with 70% ethanol overnight at À20 C. The next day, cells were harvested and washed with cold DPBS at 500 Â g. The cells were suspended in RNase/PI solution (Cell Signalling Technologies, # 4087).

Permeability assessment for 10g using Caco-2 cells
Stock solutions (10 mM) of reference compounds and 10g were diluted to a concentration of 10 mM with the transport buffer (HBSS þ 1%BSA) and the test compounds were applied to the apical or basolateral side of the cell monolayer. Permeability of the test compounds from A to B direction or B to A direction was assessed in duplicate over a 120 min incubation at 37 C and 5% CO 2 with a relative humidity of 95%. In addition, the efflux ratio of each compound was also determined. Reference compounds and 10g were quantified by LC-MS/MS analysis based on the peak area ratio of analyte/the internal standard.

Molecular docking study
X-ray co-crystal structures of ALK kinase domain complexed with crizotinib (PDB code: 2XP2) and ALK kinase domain in complex with entrectinib (PDB code: 5FTO) were retrieved from Protein Data Bank. Molecular docking studies were carried out by methods previously reported 40 .

Statistics
Statistical analysis was performed using GraphPad Prism (Ver 6.01). All values are expressed as the mean standard deviation.

Results and discussion
Chemistry As shown in Scheme 1, synthesis of the pyrazolo[3,4-b]pyridine derivatives commenced with bromination of commercially available 2-oxo-1,2-dihydropyridine-3-carbonitrile 1 using bromine in AcOH to give bromide 2, which was substituted with 3-fluorobenzenethiol using CuI and K 2 CO 3 to afford 3 in 90% yield. Sulphide 3 was treated with PCl 5 /POCl 3 to give the chloride 4 (93%), which was oxidised using m-CPBA to furnish the sulfone 5. Subjection of 5 to aminopyrazole ring formation conditions using hydrazine followed by protection of the resulting amine group with phthalic anhydride yielded 6 in 72% yield. The pyrazole moiety of 6 was protected with a trityl group to give 7 and phthalimide protecting group of 7 was removed using hydrazine to afford the aminopyrazole 8. Amide coupling reactions of 8 with various benzoyl chlorides were performed in the presence of DMAP to produce the amides 9a-i. The trityl groups in 9a-h and 11 were removed using TFA to yield the target the pyrazolo[3,4-b]pyridine derivatives 10a-h and 12, respectively. The aniline 11 was transformed to the corresponding urea, amide and sulfonamide followed by removal of trityl group to give the target derivatives 13a-c.

Rationale for the design of pyrazolo[3,4-b]pyridines and SAR study
The goal of the investigation described below was to identify unique and potent inhibitors against both ALK-wt and ALK-L1196M. Based on the observation that entrectinib 11 strongly inhibits ALK-L1196M 41 , we selected the indazole platform design strategy because substances in this family have not been widely explored in efforts focusing on the discovery of ALK inhibitors while they have been investigated 11 in our other kinase programs. Additional considerations led us to choose the closely related pyrazolo [3,4-b]pyridine group, which is superior to indazole in terms of cLogP, as the core structure. It should be noted that ALK inhibition properties of this type of substances have not been elucidated previously. It was reported 42 that a fluorine group at the C3 position of the phenyl group in crizotinib contributes to potency and binding efficiency, and that entrectinib contains a related fluorophenyl moiety. These findings prompted us to incorporate a 3fluorophenyl group in the newly designed pyrazolo [3,4-b]pyridine based ALK inhibitors. Moreover, we envisioned that the methylene linker between indazole core and difluorophenyl ring of entrectinib could be optimised to promote additional interaction with ALK. In addition, the results of docking studies suggested that the methylene linker in entrectinib could be replaced by a sulfone group. These considerations led us to design a new family potential ALK inhibitors that contain a common 5-((3-fluorophenyl)sulfonyl)-1H-pyrazolo [3,4-b]pyridine moiety and various head groups that should reside in solvent exposed region. The inhibitory activities of the pyrazolopyridine derivatives against both ALK-wt and ALK-L1196M were assessed by using an in vitro biochemical assay. As shown in Table 1, the kinase-inhibitory activities of the derivatives were highly dependent on the R 1 group. For example, 10a containing a 4-cyano group displayed a reasonable inhibitory activity (IC 50 ¼ 453 nM) on ALK-wt. Introduction of a trifluoromethyl group as R 1 (10b and 10c) resulted in little to no activity against ALK-wt. In contrast, the 4-methoxy containing derivative 10d has an enhanced activity against ALK-wt (IC 50 ¼ 69 nM) and it possesses a high activity (IC 50 ¼ 19 nM) against ALK-L1196M gatekeeper mutation, a value that is 50-fold higher than that (IC 50 ¼ 980 nM) of crizotinib. Moreover, replacement of the 4-methoxy group by a 4-dimethylamino group led to 10e, which was found to exhibit picomolar activity against ALK-L1196M. It is worthwhile to note that 10e is more potent against ALK-L1196M (IC 50 ¼ 0.7 nM) than against ALKwt (IC 50 ¼ 7.3 nM). Picomolar inhibitory activity against ALK-wt was achieved with the 4-morpholino derivative 10f, which is also extremely active against ALK-L1196M (IC 50 ¼ 1.4 nM). The SAR study led us to identify 10g containing a 4-methylpiperazin-1-yl group as the most potent inhibitor against both ALK-wt (IC 50 < 0.5 nM) and ALK-1196M (IC 50 < 0.5 nM).

Antiproliferative activities of selected pyrazolo[3,4-b]pyridines
Based on the results arising from studies of the kinase-inhibitory activities of the pyrazolo [3,4-b]pyridine derivatives against ALK-wt and ALK-L1196M gatekeeper mutant, we selected the most potent inhibitors and measured their antiproliferative activities on Ba/F3 cells transformed with ALK-wt/ALK-L1196M and on H2228 nonsmall cell lung cancer cells harbouring EML4-ALK. Ba/F3 cell lines transformed with ALK-wt and ALK-L1196M mutant were employed to assess the ALK inhibition capability of the derivatives in a cellular context and parental Ba/F3 cells were utilised as controls to  39 39 a Radiometric kinase assay. b 'Inactive' means that kinase activity is inhibited by less than 50% even at 10 lM concentration of compound. c Activity value from the reference 15 .
determine differential cytotoxicities. The antiproliferative activities of the selected pyrazolo [3,4-b]pyridines were further elucidated using the H2228 NSCLC cell line, which is an EML4-ALK positive cancer cell line. As the data in Table 2 show, the overall cellular activities of the selected pyrazolo [3,4-b]pyridines are relatively moderate compared with their enzymatic activities. In particular, it is difficult to understand why 10d is potent against ALK enzyme but inactive on H2228 and ALK-driven Ba/F3 cells. Among the compounds tested, 10g most strongly suppressed proliferation of both H2228 (GI 50 ¼ 0.219 mM) and ALK-driven Ba/F3 cells (GI 50 < 0.205 mM).
In order to understand the discrepancy between enzymatic and cellular activities, we first assessed the cell permeability of 10g using the human colon carcinoma cell line Caco-2. It was found that 10g has moderate permeability and is not a substrate of P-glycoprotein (P-gp) as evidenced by the fact that the efflux ratio of 10g is 1.85 (Table 3). This finding indicates that the cell permeability of 10g is not the reason for the discrepancy. We next measured the kinase-inhibitory activities of 10g against ALKwt at three different ATP concentrations because the IC 50 value derived from biochemical kinase assay depends on both K i and K m , which are defined by ATP concentration 43,44 . As described in Table 4, it was observed that a 10-fold increase in ATP concentration resulted in a 50-fold decrease in IC 50 (IC 50 ¼ 24 nM at 100 mM ATP). It should be noted that the physiological ATP concentration is around 1 mM and the IC 50 value of 10g should be much higher than 24 nM at 1 mM ATP concentration, which may explain the discrepancy.

Kinase profile of 10g
The inhibitory activities of 10g against ROS1, c-Met, IRK (insulin receptor kinase), c-Src, and Lyn were assessed by estimating IC 50 values derived from biochemical kinase assay ( Figure 2). As is expected by considering its structural similarity to entrectinib, 10g inhibits ROS1 (IC 50 < 1 nM) as well as ALK-wt (IC 50 < 0.5 nM). As a matter of fact, 10g is the most extremely potent inhibitor of ROS1 (IC 50 < 1 nM). The activity of 10g against c-Met is low (IC 50 ¼ 3775 nM) and it is over 7000-fold more potent against ALKwt (IC 50 < 0.5 nM) than c-Met. This observation indicates that 10g possesses extremely high selectivity over c-Met. It is notable that crizotinb inhibits c-Met as well as it does ALK with IC 50 values less than 1 nM 23 . Therefore, 10g is superior to crizotinib in terms of its selectivity over c-Met. IRK is much less inhibited (IC 50 ¼ 404 nM) by 10g than is ALK, which could be significantly advantageous in terms of toxicity because the alteration of the insulin receptor kinase activity could causes insulin resistance 45 . It is of interest to 9.215 ± 1.92 inactive 4.708 ± 3.02 5.625 ± 2.34 a GI 50 represents the concentration at which a compound causes half-maximal growth inhibition. GI 50 value for parental, Ba/F3 transformed with ALK and H2228 cell lines were shown as the means ± standard deviation (SD) of three independent experiments. b 'Inactive' means that the proliferation was suppressed by less than 10% even at 50 lM concentration of compound.  The IC 50 values of 10g against ALK-wt were measured depending on ATP concentrations ranging from 10 to 100 lM. a Radiometric biochemical kinase assay.
point out that 10g significantly inhibits both c-Src (IC 50 ¼ 7 nM) and Lyn (IC 50 ¼ 33 nM), a Src family kinase, because it was reported that Lyn 46 regulates activation of EGFR in lung cancer cells and c-Src 47 is a potential therapeutic target in alectinib-resistant patients. Therefore, it is anticipated that the remarkable activities of 10g against both c-Src and Lyn contribute to its potential as lead for lung cancer chemotherapy. We next measured the kinase-inhibitory activities of 10g against other clinically relevant ALK mutants including DFG motif mutant F1174L, aC helix mutant C1156Y, T1151-L1152insT. As shown in Table 5, it was observed that 10g strongly inhibits all of these mutants with single-digit nanomolar IC 50 values.
Inhibition by 10g of ALK signalling in ALK-driven cell lines In order to elucidate whether 10g is capable of decreasing the level of ALK phosphorylation and deactivating downstream signalling molecules in a cellular context, western blot analysis was carried out using H2228 and Ba/F3 cells transformed with ALK-wt/ ALK-L1196M. In agreement with the results of biochemical kinase assays and antiproliferative activity assays, 10g (1 and 10 mM) effectively attenuates ALK autophosphorylation in ALK wt-TEL Ba/ F3, ALK L1196M-TEL Ba/F3 and H2228 cell lines ( Figure 3). Also, we found that the phosphorylation levels of STAT3, ERK and PLCgamma, which are ALK downstream signalling molecules, are moderately suppressed by 10g in a dose-dependent manner. Consistent with the results of biochemical kinase assays and antiproliferative activity assays, 10g is more effective than crizotinib in inhibiting ALK autophosphorylation of ALK L1196M-TEL Ba/F3 cells. These results indicate that 10g effectively inhibits the gatekeeper mutant ALK-L1196M as well as ALK-wt in a cellular context.

Effects of 10g on apoptosis and cell cycle arrest in ALK-driven cell lines
Apoptosis plays a pivotal role in anticancer therapy and several ALK inhibitors are able to induce cancer cell apoptosis. Therefore, we determined whether 10g is capable of inducing apoptosis in Ba/F3 cells transformed with ALK-TEL. Annexin V-FITC and PI staining was performed to estimate the rate of formation of apoptotic cells after 10g treatment in Ba/F3 cells transformed with ALK wt-TEL and ALK L1196M-TEL. FACS analysis showed that treatment with 10g for 24 h increases the number of apoptotic cells in a dose-dependent manner (Figure 4(a,b,d,e)). Furthermore, 10g induces apoptosis of ALK L1196M-TEL Ba/F3 cells more effectively than does crizotinib (Figure 4(d,e)). In order to confirm that 10g induces apoptosis of Ba/F3 cells transformed with ALK wt-TEL and ALK L1196M-TEL, the presence of apoptosis related protein markers was assessed using western blotting. Treatment with 10g results in increases in the levels of cleaved PARP and cleaved caspase-3 ( Figure 4(c,f)). The results demonstrate that the antiproliferative activity of 10g against ALK-driven Ba/F3 cells is associated with its apoptosis induction capability. The effects of 10g on apoptosis and cell cycle arrest in H2228 NSCLC cells harbouring EML4-ALK were also determined. FACS analysis revealed that treatment with 10g for 48 h induces apoptosis in H2228 cells in a dose   dependent fashion (Figure 5(a,b)). Also, treatment with 1 mM 10g for 24 h leads to a significant enhancement of G1-S arrest in H2228 cells (Figure 5(c)), suggesting that 10g inhibits cell proliferation via apoptosis and cell cycle arrest.

Molecular docking studies of 10g with ALK-wt and ALK-L1196M
To better understand the high kinase-inhibitory activity of 10g on ALK, molecular docking studies were carried out using the X-ray co-crystal structures of the kinase domain of ALK complexed with crizotinib (PDB code: 2XP2) and entrectinib (PDB code: 5FTO) using Glide (Figures 6 and 7). Analysis of the results reveals that 10g forms three hydrogen bonds with the backbone carbonyl oxygen of E1197 and backbone NH-carbonyl group of M1199 in the hinge region. It is worthwhile to note that 10g is capable of engaging in a favourable interaction with M1196 in the kinase domain of ALK-L1196M (Figure 6(b)). It is important to note that M1196 sterically clashes with crizotinib and creates unfavourable    interactions with amino group and methyl substituents of crizotinib 28 . Moreover, the sulfone group of 10g participates in H-bonding with K1150 in the kinase domain of ALK-L1196M, which might be the reason for the improved binding affinity of 10g with both wt and L1196M. In contrast to crizotinib, 10g is involved in a Hbonding interaction with E1210 in solvent exposed region, which might also contribute to extremely high potency of 10g on ALK enzyme. In addition, the predicted binding mode of 10g was superimposed to the X-ray binding mode of entrectinib (Figure 7). This superimposition reveals that the sulfone linker in 10g makes a water molecule-mediated hydrogen bond with K1150 and D1270 while the methylene linker of entrectinib is not capable of forming this H-bond.

Conclusions
In current effort, we designed and synthesised novel 3-amino-5substituted pyrozolopyridine derivatives and assessed their kinaseinhibitory activities against ALK-L1196M gatekeeper mutant as well as against ALK-wt, and their antiproliferative activities on Ba/ F3 cells transformed with ALK-wt/ALK-L1196M and on H2228 nonsmall cell lung cancer cells harbouring EML4-ALK. The pyrozolopyridine derivative 10g was found to have exceptional kinaseinhibitory activities against both ALK-L1196M (IC 50 < 0.5 nM) and ALK-wt (IC 50 < 0.5 nM). It was reported that entrectinib 11,38 inhibits ALK-wt with IC 50 value of 12 nM and crizotinib inhibits ALK-L1196M with IC 50 value of 980 nM. Moreover, 10g is extremely potent against ROS1 (IC 50 < 0.5 nM) and it possesses a high selectivity (>7000 fold) over c-Met. Meanwhile, the high activities of 10g on both c-Src (IC 50 ¼ 7 nM) and Lyn (IC 50 ¼ 33 nM) could contribute to its potential as a novel lead for lung cancer treatment. Also, 10g strongly suppresses the proliferation of both H2228 cells and ALK-driven Ba/F3 cells. It should be emphasised that 10g more profoundly (GI 50 ¼ 0.129 mM) blocks proliferation of ALK-L1196M Ba/F3 cells than does crizotinib (GI 50 ¼ 0.726 mM). Moreover, 10g is 27-fold more potent against ALK-L1196M Ba/F3 cells than on parental Ba/F3 cells and, in comparison to crizotinib, it exhibits a much more favourable differential cytotoxicity. The results of western blot analysis reveal that 10g dose-dependently attenuates phosphorylation of ALK downstream signalling molecules (STAT3, ERK and PLC-gamma) as well as ALK autophosphorylation in ALK wt-TEL Ba/F3, ALK L1196M-TEL Ba/F3 and H2228 cell lines. Also, 10g inhibits ALK autophosphorylation on ALK L1196M-TEL Ba/F3 cells more potently than does crizotinib. The results also show that 10g exerts its antiproliferative effect by inducing apoptosis, as evidenced by the fact that it markedly induces apoptotic markers (cleaved PARP and cleaved caspase 3) on H2228 cells as well as on ALK-driven Ba/F3 cells. The results of docking study of 10g on ALK-wt/ALK-L1196M kinase domains demonstrate that 10g engages in three hydrogen bonds with backbone E1197 and M1199 in the hinge region. In contrast to crizotinib, 10g participates in favourable interactions with M1196 in the kinase domain of ALK-L1196M and two additional hydrogen bonds with K1150 and E1210, which likely contributes to its exceptional potency against ALK enzyme. The investigation described above has provided insight into new strategies to design novel and potent ALK-L1196M inhibitors that circumvent crizotinib resistance.