Design and biological evaluation of substituted 5,7-dihydro-6H-indolo[2,3-c]quinolin-6-one as novel selective Haspin inhibitors

Figures & data

Figure 1. Structures of Staurosporine and its simplified derivatives, which have entered clinical trials as kinase inhibitors, and Caulersin.

Figure 2. Lamellarin D, chromenoindoles, and envisioned indoloquinolinone chemical series.

Scheme 1. Reagents and conditions: (i) EtOH, conc. H₂SO₄ 10 mol%, reflux, 12 h, 6–9 quant.; (ii) I₂ (1.5 equiv.), KOH (4.0 equiv.), DMF, r.t., 4 h; (iii) NaH (1.5 equiv.), CH₃I (1.2 equiv.), DMF, 0–20 °C.

Scheme 2. Reagents and conditions: (i) PhMgCl 2 M in THF (1.2 equiv.), B(OCH₃)₃ (1.2 equiv.), 30 min. at -78 °C, then aq. HCl 2 M at -10 °C, 10 min; (ii) PdCl₂(dppf) (0.03 equiv.), KOAc (2.0 equiv.), Bis-(pinacolato)diboron (1.5 equiv.), 1,4-dioxane/H₂O 10/1, 80 °C, 14 h.

Table 1. Suzuki-Miyaura reaction using 10–14 and boronylated derivatives 15–24.

Entry	Starting materials	Product^a(Formula/number)	Entry	Starting materials	Product^a(Formula/number)
1	10 + 15		11	11 + 22
2	10 + 17		12	11 + 23
3	10 + 21		13	11 + 24
4	10 + 22		14	12 + 15
5	11 + 15		15	12 + 17
6	11 + 16		16	13 + 17
7	11 + 17		17	13 + 22
8	11 + 18		18	14 + 22
9	11 + 19		19	14 + 19
10	11 + 21		20	14 + 20

^aYields are indicated in the experimental section.

Table 2. “One pot” reduction of nitro group and lactam formation of derivatives 25–44 leading to final compounds 45–64.

Entry	Starting materials	Product^a(Formula/number)	Entry	Starting materials	Product^a (Formula/number)
1	25		11	35
2	26		12	36
3	27		13	37
4	28		14	38
5	29		15	39
6	30		16	40
7	31		17	41
8	32		18	42
9	33		19	43
10	34		20	44

^aYields are indicated in the experimental section.

Table 3. Residual activity on a representative panel of 8 protein kinases at 1 μM.

Entry	Compound	% Residual activity at 1 μM^a
		Haspin	CLK1	DYRK1A	CDK2	CDK5	CDK9	GSK3β	PIM1
1	45	44	67	61	100	100	63	100	86
2	46	6	13	18	80	78	62	86	32
3	47	28	22	33	61	59	20	47	37
4	48	1	4	12	84	87	49	100	63
5	49	2	5	7	73	77	40	39	41
6	50	3	10	5	77	81	51	44	19
7	51	1	5	14	62	70	33	29	36
8	52	2	1	5	58	63	37	22	29
9	53	33	11	36	76	56	56	63	31
10	54	0	11	2	35	59	18	48	9
11	55	0	14	2	38	53	17	20	22
12	56	2	3	7	72	79	37	39	65
13	57	3	2	10	100	100	72	58	37
14	58	59	49	55	62	55	55	51	48
15	59	13	12	21	74	51	41	41	34
18	60	9	31	44	97	100	100	100	100
19	61	0	2	9	57	91	41	71	38
20	62	0	0	2	31	22	6	31	75
21	63	51	19	29	67	68	45	44	42
22	64	39	11	19	100	100	59	81	82

^aResidual kinase activity was determined at 1 μM concentration for each compound. The data mean (n = 2) expressed as percentage of maximum activity of the DMSO control.

Table 4. Measured IC₅₀ values (nM) on Haspin, CLK1 and DYRK1A.

Entry	Compound	Kinase inhibition (IC50)^a (IC50 in nM)			Selectivity index
		Haspin	CLK1	DYRK1A	CLK1/Haspin	DYRK/Haspin
Ref	1a	ND	ND	74	ND	ND
Ref	1b	ND	ND	67	ND	ND
1	46	7	51	68	7	10
2	48	6	17	19	3	3
3	49	1	13	15	13	15
4	50	7	88	196	13	28
5	51	4	10	41	3	10
6	52	16	78	242	5	15
7	54	4	82	97	21	24
8	55	2	33	130	17	65
9	56	14	213	226	15	16
10	57	34	259	280	8	8
11	59	30	94	73	3	2
12	60	19	122	143	6	8
13	61	5	78	104	16	21
14	62	18	124	131	7	7

^aIC₅₀ values were determined on Haspin, CLK1 and DYRK1A when the residual kinase activity was ≤25% on Haspin at a compound concentration of 1 μM in Table 3. They were calculated from a dose-response curve for which each point was measured in duplicate, and reported in nM. Selectivity indexes (SI) were calculated as follows: IC₅₀ DYRK1A or CLK1/IC₅₀ Haspin.

Table 5. Effects on cell viability, EC₅₀ on RPE1 and U-2 OS cell lines^a.

Entry	Compound	RPE1 EC50 in µM	U-2 OS EC50 in µM
1	46	>25	>25
2	48	7.3	7
3	49	>25	>25
4	50	20.8	4.3
5	51	>25	12.6
6	54	>25	>25
7	55	10.7	3.4
8	56	>25	>25
9	61	19.1	8.7

^aCells were incubated with increasing doses of each compound (up to 50 µM) for 48 h. Cell viability was determined by MTS assay in triplicate and EC₅₀ (µM) were calculated from the dose-response curves.

Figure 3. Selectivity profile of compound 55 evaluated on 486 kinases at 1 μM in duplicate (SelectScreen Whole Panel, Life Technologies) and represented on a dendrogram (courtesy of Cell Signalling Technology). Kinases whose activities are inhibited by 80% and above are shown as dots, Haspin is visualised as a green dot, CLKs as purple dots and DYRKs as orange dots.

Figure 4. Superimposition of the ATP site of the three protein kinases, CLK1, DYRK1A and Haspin (residues and ribbons are coloured regarding their localisation in the kinase structure: hinge region (yellow), DFG motif (cyan), G loop (green), Catalytic K (orange), αC-helix (purple) and HRD region (red)), drawn with MOE software.²⁴ Non-polar hydrogen atoms are hidden for clarity. Catalytic lysine and residues in the hinge region forming hydrogen bonds with the ligand are highlighted. CLK1: Leu244 and Lys191, DYRK1A: Leu241 and Lys188, and Haspin: Gly608, Gly609 and Lys 511.

Figure 5. Binding mode representation of 49 (green) and 62 (purple) in ATP site of Haspin. Hydrogen bond interactions are represented in dashed lines.

Figure 6. Effects of compounds on cell viability. Cell viability was assessed on the following human cell lines: U-2 OS (osteosarcoma), HCT116 (colorectal cancer), MDA-MB231 (breast cancer), SH-SY5Y (neuroblastoma) and RPE-1 (retinal fibroblast immortalised with hTERT). Cells were incubated with 25 μM of selected compounds or 10 μM of staurosporine or 0.1% of DMSO for 48 h. Cell viability was evaluated in triplicate via MST assay and results expressed as percentage of DMSO control (mean set at 100%). Results on graphs are mean ± SD.

Figure 7. Effects of compounds on U-2 OS and HCT116 spheroid viability. Cell viability in spheroids from U-2 OS and HCT116 cells was measured after 7 days of treatment with DMSO (0.5%), CHR6494 (CHR), SBS018 or Cpd55 at a single dose of 2.5, 5, and 10 µM, on day 0. Cell viability is expressed in percentage of the DMSO control. n = 3, results are mean ± s.e.m.

Figure 8. Cellular endogenous Haspin inhibition. U-2 OS cells were treated for 16 h with 0.5 µM of each compound or 0.1% of DMSO (CHR: CHR-6494). Haspin activity was monitored by immunofluorescence staining of phosphorylated Histone H3 on threonine 3 (H3T3ph, green); α-Tubulin was visualised in red and DNA was stained by DAPI (blue). Haspin activity was quantified in prometaphase/metaphase cells measuring the H3T3ph and DAPI signals and representing the H3T3ph/DAPI ratio on a scatter plot (upper panel). The inserted dot plot allows the comparison of the 3 tested compounds on a more precise scale; n ≥ 15, *p ≤ 0.05; **p ≤ 0.01 (two-tailed unpaired t-test). Representative images are presented on the lower panel, Bar 5 µm.

Figure 9. Effect of compounds on the cell cycle. U-2 OS cells were treated for 24 h with 1 µM of each compound or 0.2% DMSO (CHR: CHR-6494). DNA content was measured by flow cytometry and the percentages of cells in each phase of the cell cycle is represented on a proportional bar graph. Representative profiles for each treatment are shown on the right panels.

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