Discovery of novel anaplastic lymphoma kinase (ALK) and histone deacetylase (HDAC) dual inhibitors exhibiting antiproliferative activity against non-small cell lung cancer

Figures & data

Figure 1. FDA-approved ALK and HDAC inhibitors and reported 2,4-pyrimidinediamine derivative ALK/HDAC dual inhibitor.

Figure 2. Design of benzimidazole dual ALK/HDAC inhibitors. The dashed boxes in the lead compounds indicated the preserved pharmacophore structure.

Scheme 1. Synthesis of compounds 3a, 3b, 3d, 3e. Reagents and conditions: (a) NH₂XCO₂Me or NH₂XCO₂Et, DIPEA, ACN, 80 °C, 12 h, 89–98%; (b) (i) NaOH, H₂O, MeOH, rt, 12 h; iPrNH₂, EDCI, HOBt, TEA, DMF, rt, 20 h, 88%; (c) ethyl acrylate, Herrmann’s palladacycle, [(tBu)₃PH]BF₄, Cy₂NMe, DMF, MW, 30 min, 58–69%; (d) Fe, NH₄Cl, H₂O, EtOH, 80 °C, 6 h; (e) (i) benzoyl isothiocyanate, THF, 0 °C, 15 min; (ii) EDCI, DIPEA, 60 °C, 2 h, then rt, 16 h, 36–75% (for step d and e); (f) (i) CNBr, EtOH, 25 °C, 16 h; (ii) RCOCl, TEA, DCM, 0 °C to rt, 2.5 h, 47% (for step d and f); (g) (i) LiOH, H₂O, MeOH, rt, 20 h; (ii) NH₂OTHP, EDCI, HOBt, TEA, DMF, rt, 20 h; (h) TFA, MeOH, rt, 6 h, 36–74% (for step g and h).

Scheme 2. Synthesis of compound 6. Reagents and conditions: (a) (i) Fe, NH₄Cl, H₂O, EtOH, 80 °C, 6 h; (ii) benzoyl isothiocyanate, THF, 0 °C, 15 min; EDCI, DIPEA, 60 °C, 2 h, then rt, 16 h, 33%.

Scheme 3. Synthesis of compound 3c. Reagents and conditions: (a) methyl cis-1,4-aminocyclohexanecarboxylate hydrochloride, DIPEA, I, 80 °C, 12 h, 99%; (b) (i) NaOH, H₂O, MeOH, rt, 12 h; (ii) iPrNH₂, EDCI, HOBt, TEA, DMF, rt, 20 h, 41%; (c) ethyl acrylate, Herrmann’s palladacycle, [(tBu)₃PH]BF₄, Cy₂NMe, DMF, MW, 30 min, 68%; (d) Fe, NH₄Cl, H₂O, EtOH, 80 °C, 6 h; (e) (i) benzoyl isothiocyanate, THF, 0 °C, 15 min; (ii) EDCI, DIPEA, 60 °C, 2 h, then rt, 16 h, 63% (for step d and e); (f) (i) LiOH, H₂O, MeOH, rt, 20 h; (ii) NH₂OTHP, EDCI, HOBt, TEA, DMF, rt, 20 h; (g) TFA, MeOH, rt, 6 h, 75% (for step f and g).

Scheme 4. Synthesis of compound 3f. Reagents and conditions: (a) NaN₃, CuI, N,N-dimethylethylenediamine, NaCO₃, DMSO, 110 °C, 5 h; (b) monomethyl suberate, EDCI, HOBt, TEA, DMF, rt, 20 h, 58% (for step a and b); (c) (i) Fe, NH₄Cl, H₂O, EtOH, 80 °C, 6 h; (ii) benzoyl isothiocyanate, THF, 0 °C, 15 min; (iii) EDCI, DIPEA, 60 °C, 2 h, then rt, 16 h, 65%; (d) (i) LiOH, H₂O, MeOH, rt, 20 h; (ii) NH₂OTHP, EDCI, HOBt, TEA, DMF, rt, 20 h; (e) TFA, DCM, 0 °C to rt, 12 h, 71% (for step d and e).

Table 1. Structure-activity relationships for test compounds (3a–3f and 6).

Compound	R^1	R^2	R^3	IC50 (μM)
				ALK^a	HDAC1^b	HDAC6^b	HDAC8^b	HDAC11^b
6			5-Br	(18.8%)^c	ND^d	ND^d	ND^d	ND^d
3a				5.75	6.18	0.81	2.95	9.35
3b				0.016	10.80	1.03	1.69	16.60
3c				0.0052	>30	2.34	2.87	>30
3d				3.73	>30	2.47	7.77	>30
3e				>10	0.84	0.21	1.78	1.42
3f				0.015	2.29	0.10	2.52	1.72
Crizotinib				0.026^e	ND^d	ND^d	ND^d	ND^d
Ceritinib				<0.001^e	ND^d	ND^d	ND^d	ND^d
1				0.001 ^f	ND^d	ND^d	ND^d	ND^d
Staurosporine				0.0013	ND^d	ND^d	ND^d	ND^d
Trichostatin A				ND^d	0.019	0.005	0.65	0.05

^a Data from Reaction Biology Corporation. Compounds were tested in duplicate in ten-dose IC₅₀ mode with three-fold serial dilutions starting at 10 μM. Control compound (staurosporine) was tested in ten-dose IC₅₀ mode with three-fold serial dilutions starting at 20 μM. Reactions were carried out in 20 μM ATP. Average values are shown.

^b Data from Reaction Biology Corporation. Compound and control (trichostatin A) were tested once in ten-dose IC₅₀ mode with three-fold serial dilutions starting at 30 µM.

^c Relative inhibition of enzymatic activity with vehicle as 100%. Compounds were tested in duplicate in single dose at a concentration of 10 μM. Average value is shown.

^d ND: not determined.

^e Data from reference.³⁴

^f Data from reference.³⁵

Table 2. Antiproliferative activity of compound 3b and 3c in cancer cell lines.

compound	A549	HepG2	MCF7	U87MG	H2228
3b	0.33 ± 0.03	0.59 ± 0.09	0.55 ± 0.01	0.62 ± 0.10	0.44 ± 0.06
3c	7.26 ± 2.66	ND^b	0.98 ± 0.03	11.12 ± 0.81	0.21 ± 0.07
3f	9.45 ± 2.56	ND^b	3.97 ± 0.28	19.78 ± 1.45	ND^b
crizotinib	0.025 ± 0.001	0.027 ± 0.001	0.095 ± 0.024	0.048 ± 0.008	0.13 ± 0.05
Pracinostat (2)	ND^b	ND^b	ND^b	ND^b	0.69 ± 0.05
SAHA	3.42 ± 0.51	3.38 ± 0.56	4.17 ± 0.43	2.09 ± 0.72	3.10 ± 0.57

^a Cancer cell lines were treated with compounds for 48 h and antiproliferative activity was tested by SRB assay.

^b ND: not detected.

Figure 3. In vitro enzymatic activity of 3b against ALK mutants (IC₅₀ in nM). No inhibitor control as 100% enzyme activity. The assay was tested in duplicated, and the results were indicated separately as black squares and hollow inverted triangles.

Table 3. In Vitro Enzymatic Activity of 3b Against Various Receptor Tyrosine Kinases (IC₅₀ in μM).^a

Compound	EGFR	VEGFR2	c-Met	IGF1R	c-Src	JAK2
1	–	–	–	0.10^Table Footnoteb	0.42^Table Footnoteb	1.07^b
3b	>10	>10	>10	4.84	3.38	>10
Staurosporine	0.16	0.021	0.18	0.041	0.0025	0.0004

^a Data from Reaction Biology Corporation. Compounds were tested in duplicate in ten-dose IC₅₀ mode with three-fold serial dilutions starting at 10 μM. Control compound (staurosporine) was tested in ten-dose IC₅₀ mode with three-fold serial dilutions starting at 20 μM. Reactions were carried out at 20 μM ATP. Average values are shown.

^b Data from reference.³⁵

Table 4. In Vitro enzymatic activity of 3b against various ALK Mutants (IC₅₀ in nM).^a

Compound	ALK Mutants (IC50 in nM)^a
	C1156Y	L1196M	F1174L	G1202R	G1269A
3b	9.7	4.9	59	51	50
Crizotinib	ND^d	16^b	32^c	12^b	166^c
Staurosporine	2.9	1.8	2.9	5.6	5.9

^a Data from Reaction Biology Corporation. Compounds were tested in duplicate in ten-dose IC₅₀ mode with three-fold serial dilutions starting at 10 μM. Control compound (staurosporine) was tested in ten-dose IC₅₀ mode with three-fold serial dilutions starting at 20 μM. Reactions were carried out at 20 μM ATP. Average values are shown.

^b Data from reference³⁴.

^c Data from reference³⁸.

^d ND: not determined.

Figure 4. Effect of test compounds on H2228 cells. Human NSCLC H2228 cell line was treated with 3b (2 µM), 3c (4 µM), or positive controls, SAHA as pan-HDAC inhibitor, pracinostat as class I and IIa HDAC inhibitor, and crizotinib as ALK inhibitor, with DMSO (as negative control) for 6 h. (A) Western blots of phosphorylated tyrosine residue 1640 on ALK as well as acetylated (Ac) α-tubulin and histone H3 with their corresponding total proteins for normalisation. GAPDH was used as a loading control. (B) Quantification of western blotting results using Image J. Error bars indicate ± S.D.E. *p-Value ≤ 0.05, **p-value ≤ 0.01, ***p-value ≤ 0.001.

Figure 5. The predicted binding mode for 3b in the binding site of ALK^wt (PDB ID: 4MKC). (A) The yellow lines indicated hydrogen bonds and the estimated lengths of bonds was shown in purple number. (B) The purple arrows indicated hydrogen bonds.

Figure 6. The predicted binding mode for 3b in the binding site of HDAC6 (PDB ID: 5EDU). (A) The yellow lines indicated hydrogen bonds and the cyan lines indicated pi-pi interactions. (B) The purple arrows indicated hydrogen bonds, the green arrows indicated π–π interactions, and the gray line indicated metal interaction.

Figure 7. Tumour volume and body weight following treatment of human cancer A549 xenografts in mice with 3b. Indicated doses were administered daily up to day 21 (dashed line). Data are mean ± SEM.

Table 5. Inhibition of CYP450 Enzymes and hERG by 3b (IC₅₀ in μM).^a

Compound	CYP1A2	CYP2C9	CYP2C19	CYP2D6	CYP3A4	hERG
3b	12.70	2.65	7.86	>10	9.37	>10
Furafylline	0.59
Ketoconazole	0.59^b	1.92	1.30	4.83	0.056
E-4031						0.0069

^a Data from Reaction Biology Corporation.

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