Discovery of novel and potent CDK8 inhibitors for the treatment of acute myeloid leukaemia

Figures & data

Figure 1. Chemical structures of some CDK8 inhibitors.

Figure 2. Binding mode of compound C43 with active site of CDK8 (PDB: 5IDN). CDK8 is shown in gray ribbons with selected residues coloured green. Hydrogen bonds are drawn as yellow dashed lines, and pi-pi stacking is drawn as magenta dashed lines. Compound 43 is shown with blue stick. The illustration was generated using PyMOL.

Scheme 1. Synthesis of compounds 1-11^a

^aReagents and conditions: A. K₂CO₃, Pd(dppf)Cl₂, 1,4-dioxne, H₂O, 85 °C, 14 h.

Scheme 2. Synthesis of compounds 12-40^a

^aReagents and conditions: A. K₂CO₃, Pd(dppf)Cl₂, 1,4-dioxne, H₂O, 85 °C, 14 h; B. NaOH, THF, H₂O, 35 °C, 6 h; C. DMF, NIS, 85 °C, 12 h; D. NaOH, CH₃CH₂OH, H₂O, 75 °C, 2 h.

Table 1. The evaluation of the activity of compounds 1-10 on CDK8.

Compounds	R^1	Inhibition rate @ 200 nM^a (%)^a	CDK8 IC50 (nM)^b
1		63.0 ± 4.5	105.9 ± 4.6
2		35.2 ± 2.8	NT^c
3		50.8 ± 2.3	176.5 ± 7.2
4		45.6 ± 4.2	NT
5		55.7 ± 3.8	186.3 ± 4.5
6		75.8 ± 5.9	64.5 ± 3.8
7		59.1 ± 2.1	212.3 ± 6.1
8		51.7 ± 5.4	255.3 ± 4.5
9		60.4 ± 4.3	189.6 ± 4.9
10		38.3 ± 2.2	NT
SEL-120-34A	/	82.5 ± 5.8	37.2 ± 2.5
C43	/	73.2 ± 4.3	52.6 ± 3.5

^a CDK8 inhibition rate at 200 nM was determined by CDK8 enzyme activity assay. Values from two experiments and results were shown as means ± SD.

^b IC₅₀ values were determined by CDK8 enzyme activity assay. Values from three dependent experiments and results were shown as means ± SD. ^cNT: Not Test.

Table 2. The evaluation of the activity of compounds 11-40 on CDK8.

Compounds	R^2	Inhibition rate @ 200 nM (%)^a	CDK8 IC50 (nM)^b
11	Cl	51.8 ± 4.3	NT^c
12		80.9 ± 3.5	39.2 ± 6.3
13		70.4 ± 3.6	65.7 ± 3.1
14		52.5 ± 2.8	185.7 ± 4.6
15		49.1 ± 3.4	NT
16		29.5 ± 2.3	NT
17		46.5 ± 1.8	NT
18		32.5 ± 4.9	NT
19		57.4 ± 1.4	157.7 ± 4.3
20		58.7 ± 3.3	160.5 ± 3.1
21		23.6 ± 2.6	NT
22		36.2 ± 2.7	NT
23		51.6 ± 2.6	192.6 ± 6.6
24		24.6 ± 2.4	NT
25		61.98 ± 4.6	135.7 ± 5.3
26		33.1 ± 4.2	NT
27		15.9 ± 5.2	NT
28		16.9 ± 2.2	NT
29		23.1 ± 2.5	NT
30		65.5 ± 3.8	86.7 ± 6.5
31		30.5 ± 2.3	NT
32		43.97 ± 3.8	NT
33		34.6 ± 4.1	NT
34		23.4 ± 3.3	NT
35		55.8 ± 4.6	NT
36		61.6 ± 5.4	127.9 ± 3.8
37		28.3 ± 3.1	NT
38		26.42 ± 1.6	NT
39		22.3 ± 3.7	NT
40		46.3 ± 1.9	NT
SEL-120-34A	/	82.5 ± 5.8	37.2 ± 2.5
C43	/	73.2 ± 4.3	52.6 ± 3.5

^a CDK8 inhibition rate at 200 nM was determined by CDK8 enzyme activity assay. Values from two experiments and results were shown as means ± SD.

^b IC₅₀ values were determined by CDK8 enzyme activity assay. Values from three dependent experiments and results were shown as means ± SD. ^cNT = Not Test.

Figure 3. Molecular docking of target compounds. (A) The docking model of compound 12 with CDK8 (B) Superposition of spatial structures of compound C43 and 12 within active site of CDK8 (PDB: 5IDN). CDK8 is shown in gray ribbons with selected residues coloured green. Hydrogen bonds are drawn as yellow dashed lines, and pi-pi stacking is drawn as magenta dashed lines. Compound 12 is shown with yellow stick and compound C43 is shown with blue stick. The illustration was generated using PyMOL.

Table 3. Antiproliferative activity and preliminary safety of selected compounds.

Compounds		12	13	30	SEL120-34A
CDK8 IC50 (nM) ^a	/	39.2 ± 6.3	65.7 ± 3.1	86.7 ± 6.5	37.2 ± 2.5
GC50 (μM) ^b	molm-13	0.02 ± 0.01	0.16 ± 0.02	>50	0.02 ± 0.01
	MV4-11	0.03 ± 0.01	0.36 ± 0.12	>50	0.007 ± 0.001
	MGC-803	11.72 ± 1.93	9.19 ± 2.42	>50	16.26 ± 1.86
	MDA-MB-231	>50	31.47 ± 1.27	>50	9.60 ± 0.81
	A375	1.00 ± 0.23	0.95 ± 0.52	>50	5.90 ± 1.43
	A549	15.49 ± 2.78	21.75 ± 1.66	>50	12.06 ± 0.37
	HCT-116	3.66 ± 1.94	2.48 ± 0.99	>50	11.3 ± 1.9
	SW480	6.05 ± 2.26	5.45 ± 2.82	>50	17.7 ± 2.1
	HT-29	8.26 ± 0.65	9.31 ± 1.67	>50	33.8 ± 1.4
	GES-1	25.70 ± 4.97	24.15 ± 3.62	>100	55.3 ± 3.2

^a IC₅₀ values were determined by CDK8 enzyme activity assay. Values from three dependent experiments and results were shown as means ± SD.

^b GI₅₀ values were determined by CCK-8 assay. Values from three dependent experiments and results were shown as means ± SD.

Table 4. CDKs Selectivity of Compound 12.

Compound	CDK2 (nM)	CDK6 (nM)	CDK7 (nM)	CDK9 (nM)	CDK8 (nM)
12	>1000	351.9 ± 36.5	301.5 ± 27.8	473.6 ± 25.3	39.2 ± 6.3

Figure 4. Compound 12 enhanced the thermal stability of intracellular CDK8 protein. (A-B)The CDK8 protein intensity at different temperature.

Figure 5. Compound 12 and biotin bind to CDK8 protein competitively. (A) Compound 12 binds to CDK8 protein in HCT-116 cells. (B)Compound 12 binds to CDK8 protein in HEK293T cells.

Figure 6. Compound 12 inhibited phosphorylation of STAT1 S727 and STAT5 S726. (A) Compound 12 suppressed the phosphorylation of STAT1 S727 in a dose-dependent manner. HCT-116 cells were treated with compound for 12 h. (B) Compound 12 suppressed the phosphorylation of STAT5 S726 in a dose dependent manner. HL-60 cells were treated with or without compound for 12 h. The samples were analysed by Western blot.

Figure 7. (A-B) The concentrations versus time curve of compound 12.

Table 5. In vivo PK properties of compound 12.

Dose/routes	t1/2 (h)	Tmax (h)	MRT (h)	Cmax (μg/L)	AUC 0-∞ (μg/L × h)	CL(L/h/kg)	F(%)
10 mg/kg (po)	0.95	0.88	2.24	821.82	1611.67	6.21	38.80
2 mg/kg (iv)	1.12	0.33	1.64	647.79	829.75	2.45	/

Table 6. Caco-2 permeability determination.

Compound	Papp (10 ^−6 cm/s)		Efflux ratio	Recovery (%)
	A→B	B→A		A→B	B→A
12	27.0	35.1	1.3	107.0	108.1
Nadolol	0.2	0.5	–	99.0	110.9
Propranolol	40.2	34.1	–	99.9	107.0
Digoxin	0.9	5.6	6.2	92.5	99.2

Table 7. CYP inhibition activity.

Compound	IC50 (μM)
	1A2	2C9	2C19	2D6	3A4/5 (M)
12	>50	>50	>50	>50	>50
Positive control	0.209	0.569	9.30	0.0356	0.0118

Figure 8. Compound 12 had low toxicity in vivo. The HE staining of tissues after mice were treated with 1000 mg/kg compound 12. The scan bar: 100 μM.