Rational design and synthesis of 2-(1H-indazol-6-yl)-1H-benzo[d]imidazole derivatives as inhibitors targeting FMS-like tyrosine kinase 3 (FLT3) and its mutants

Figures & data

Figure 1. (Left) X-ray crystallography of FLT3 kinase (PDB:4RT7); (Right) The region and amino acid residues in which FLT3 mutations commonly occur; (Blue) Juxtamembrane; (Red) Activation loop of the kinase domain.

Figure 2. FLT3 inhibitors approved for AML treatment.

Figure 3. Docking structures of a quinazoline derivative with an indazole fragment in FLT3 (PDB: 4RT7) and Design of a benzimidazole analog with an indazole moiety from studies on the binding mode of a previous FLT3 inhibitor.

Scheme 1. Synthesis of N-(2-(1H-indazol-6-yl)-1H-benzo[d]imidazol-5-yl)benzamide derivatives. (i) 3,4-dihydro-2H-pyran, Pyridinium p-toluenesulfonate, µW, 50 °C, 5 h; (ii) LiAlH₄ in THF, THF, 0 °C; (iii) Dess-Martin periodinane, MC/THF = 1:1, rt; (iv) 4-Nitrobenzene-1,2-diamine, NH₄Cl, EtOH, reflux; (v) H₂, Pd/C, EtOH; (vi) Benzoic acid, EDC, HOBt, TEA, THF, rt; (vii) 20% TFA, CH₂Cl₂ or 5% HCl in EtOH.

Scheme 2. Synthesis of 1–(2-(1H-indazol-6-yl)-1H-benzo[d]imidazol-5-yl)-3-phenylurea derivatives. (i) (1) 4-Nitrophenyl chloroformate, DIPEA, THF, 0 °C; (2) RNH₂, THF, 50 °C; (ii) 20% TFA, CH₂Cl₂.

Table 1. Enzymatic activities of N-(2-(1H-indazol-6-yl)-1H-benzo[d]imidazol-5-yl)benzamide and 1–(2-(1H-indazol-6-yl)-1H-benzo[d]imidazol-5-yl)-3-phenylurea derivatives

No.	R	FLT3	FLT3 (D835Y)	No.	R	FLT3	FLT3 (D835Y)
		IC50 (µM)				IC50 (µM)
8a		0.181	0.021	8n		0.287	0.113
8b		0.639	0.110	8o		0.841	0.120
8c		0.470	0.108	8p		0.409	0.131
8d		1.03	0.151	8q		0.525	0.174
8e		0.154	0.044	8r		0.0416	0.006
8f		2.51	0.370	8s		0.158	0.025
8g		1.10	0.063	8t		0.285	0.087
8h		0.702	0.082	8u		0.289	0.055
8i		0.676	0.084	8v		0.107	0.009
8j		0.514	0.103	8w		0.29	0.013
8k		0.761	0.191	8x		0.743	0.145
8l		1.17	0.261	8y		0.145	0.042
8m		3.58	1.010	8z		0.066	0.046
Staurosporine						1.82 nM	0.073 nM

Figure 4. Docking structure of compound 8a at the active site of FLT3 (Left) and at the hydrophobic pocket adjacent to the ATP-binding site (Right).

Table 2. Enzymatic inhibitory activities of compound 8r against FLT3 mutants.

Kinase	IC50
FLT3 wild type	41.6 nM
FLT3 (ITD)-NPOS	41.5 nM
FLT3 (ITD)-W51	22.8 nM
FLT3 (D835Y)	5.64 nM
FLT3 (F594_R595 ins R)	37.3 nM
FLT3 (F594_R595 ins REY)	41.6 nM
FLT3 (R595_E596 ins EY)	24.8 nM
FLT3 (Y591_V592 ins VDFREYEYD)	49.5 nM

Figure 5. The profiles of the compound 8r (1 µM) are shown.

Table 3. IC₅₀ for the enzymatic inhibitory activity of compound 8r.

	IC50
	ABL1	c-Kit	CAMKK1	TRKC
8r	3.09 µM	4.57 µM	0.395 µM	1.52 µM

Figure 6. Docking structure of 8r in FLT3 (PDB: 4RT7)²⁶.

Smith CC, Lin KC, Zhang Y, et al. Characterizing and overriding the structural mechanism of the quizartinib-resistant FLT3 “gatekeeper” F691L Mutation with PLX3397. Cancer Discov 2015;5:60.

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