Synthesis and identification of GZD856 as an orally bioavailable Bcr-Abl^T315I inhibitor overcoming acquired imatinib resistance

Figures & data

Figure 1. (A) Chemical structures of FDA-approved Bcr-Abl inhibitors; (B) design of GZD856 as new Bcr-Abl inhibitor by scaffold hopping based on ponatinib.

Scheme 1. Synthetic route of compound GZD856. Reagents and conditions: (a) i. Pd(dppf)₂Cl₂, CuI, Et₃N, DMF, 80 °C; ii. K₂CO₃, MeOH, rt, 90%; (b) Pd(PPh₃) Cl₂, CuI, DIPEA, DMF, 80%; (c) t-BuOK, THF, −20 °C, 75%.

Table 1. Inhibitory activities of GZD856 against Bcr-Abl^WT and Bcr-Abl^T315I mutant.

	Kinase inhibition (IC50 nM)
Compounds	Bcr-Abl^WT	Bcr-Abl^T315I
GZD856	19.9	15.4
Imatinib	98.2	5155
Nilotinib	43.5	702.4
Ponatinib*	8.6	40

* Reported data19.

Figure 2. The predicted binding mode of GZD856 with Bcr-Abl^T315I. Hydrogen bonds are indicated by yellow hatched lines to key amino acids.

Table 2. GZD856 selectively and potently inhibited the proliferation of Bcr-Abl positive leukemia cells.

	IC50 values (nM)
Cell lines	GZD856	Imatinib	Nilotinib	Ponatinib	Taxol
K562	2.2	189	6.5	0.5	5.1
Ba/F3^WT	0.64	500	22	0.16	ND
Ba/F3^T315I	10.8	10 160	1461	6.5	ND
K562R (Q252H)	67.0	6050	350	ND	7.0
MOLT-4	499.4	48 500	20 400	7.8	2.7
U937	2001.0	16 000	9520	1.0	2.3

ND: not detected.

Figure 3. GZD856 inhibits Bcr-Abl signaling in K562 and Ba/F3 stable cell lines expressing Bcr-Abl^WT and Bcr-Abl^T315I. Cells were treated with GZD856 at the indicated concentrations for 4.0 h, and whole cell lysates were then subjected to Western blot analyses. The results represent three independent experiments.

Figure 4. GZD856 potently suppressed tumor growth in K562 xenograft model of human CML. Mice bearing K562 xenografts were dosed orally once a day with GZD856 at 10 mg/kg dosages for 16 consecutive days. The data are representative of three independent experiments.

Figure 5. GZD856 suppressed tumor growth in xenograft models of Ba/F3 cells expressing Bcr-Abl^T315I. Mice bearing xenograft Ba/F3 Bcr-Abl^T315I cells were orally dosed with GZD856 and imatinib at the indicated doses. Tumor sizes were monitored every 2 days (n = 10 for each group; error bar represents SE).

Huang WS, Metcalf CA, Sundaramoorthi R, et al. Discovery of 3-[2-(imidazo[1,2-b]pyridazin-3-yl)ethynyl]-4-methyl-N-{4-[(4-methyl-piperazin-1-yl)methyl]-3-(trifluoromethyl)phenyl}benzamide (AP24534), a potent, orally active pan-inhibitor of breakpoint cluster region-abelson (BCR-ABL) kinase including the T315I gatekeeper mutant. J Med Chem 2010;53:4701–19.

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