Tetrahydroindazole inhibitors of CDK2/cyclin complexes

Abstract

Over 50 tetrahydroindazoles were synthesized after 7-bromo-3,6,6-trimethyl-1-(pyridin-2-yl)-5,6,7,7a-tetrahydro-1H-indazol-4(3aH)-one (3) was identified as a hit compound in a high throughput screen for inhibition of CDK2 in complex with cyclin A. The activity of the most promising analogues was evaluated by inhibition of CDK2 enzyme complexes with various cyclins. Analogues 53 and 59 showed 3-fold better binding affinity for CDK2 and 2- to 10-fold improved inhibitory activity against CDK2/cyclin A1, E, and O compared to screening hit 3. The data from the enzyme and binding assays indicate that the binding of the analogues to a CDK2/cyclin complex is favored over binding to free CDK2. Computational analysis was used to predict a potential binding site at the CDK2/cyclin E1 interface.

Keywords: CDK2; Cyclins; Kinase inhibitors; Tetrahydroindazoles.

Figure 1.

Structures of CDK2 inhibitors in clinical trials and structure of type II inhibitor K03861.

Figure 2.

Structures of hit compound 3, HSP90 inhibitor SNX-5422, and an opioid receptor agonist.

Figure 3.

Inhibition of CDK complexes by 3 and its analogues at 1 μM.

Figure 4.

FT-mapping of the protein-protein interface region of CDK2 interacting with cyclins: (a) CDK2 with ATP (PDB ID: 1B38) with the activation loop in the inactive conformation (orange, mapping solvents (magenta) in the blue circle) and (b) a complex of CDK2 with cyclin E1 (cyan) and dinaciclib (PDB ID: 5L2W) with the activation loop in the active conformation (blue, mapping solvents (red) in the yellow circle).

Figure 5.

Predicted binding modes of 3 (cyan), 53 (brown), and 59 (green) in CDK2 (a, b and c) and the complex of CDK2 (green) with cyclin E1 (gray) (d, e and f).

Scheme 1.

Synthesis of compounds 2-13. Reagents and conditions: 2: (a) 2-hydrazino-pyridine, EtOH, 80 °C, 4 h, 84%. 3: (b) N-bromosuccinimide, CHCl₃, 60 °C, 6 h, 61%. 4: c) AgF, CaF₂, acetonitrile, 80 °C, 22 h, 12%. 5: (c) AgNO₃. MeOH/acetone, rt, 5 days, 21%. 6: (c) AgNO₃, MeOH/H₂O/acetone, rt, 20 h, 70%; 7: (d) from 6 with SOCl₂, pyridine, rt, 2 h, 70%. 8: (e) from 2 with MeI, n-BuLi, −78 °C to rt, 16.5 h, 25%; 9: (c) dimethylamine, THF, microwave at 40 °C, 38 h, 21%. 10: (c) sodium thiophenate, DMF, rt, 7 h, 79%; (f) 11: from 10 with Oxone, H₂O/MeOH, rt, 3 h, 68%. 12: (c) NaN₃, 80% EtOH, 90–100 °C, 24 h, 43%; 13: (g) from 12 with H₂, 10% Pd/C, EtOH, rt, 4 h, 73%.

Scheme 2.

Synthesis of analogues 18a-18d. Reagents and conditions: (a) acetyl chloride, pyridine, CHCl₃, rt; (b) AlCl₃, CH₂Cl₂, rt; (c) 2-hydrazinopyridine, EtOH, 80 °C; (d) NBS, CHCl₃, 60 °C.

Scheme 3.

Synthesis of cyclic amides 19-21.Reagents and conditions: (a) sodium azide, polyphosphoric acid, 100 °C, 4.5 h, 17% for 19, 24% for 20; (b) NBS, CHCl₃, 60 °C, 6 h, 35% (20 to 21).

Scheme 4.

Synthesis of analogues 22 and 23. Reagents and conditions: (a) CeCl₃, NaBH₄, MeOH/THF, −40 to −10 °C, 30 min, 20%; (b) H₂NOH·HCl, NaOAc, 1,4-dioxane, reflux, 6 h, 48%.

Scheme 5.

Synthesis of analogue 25. Reagents and conditions: (a) AcOH/H₂O, microwave at 200 °C, 5 min, 54%; (b) NBS, CHCl₃, 60 °C, 4 h, 29%.

Scheme 6.

Introducing an isobutyl group at C3. Reagents and conditions: (a) isovaleryl chloride, pyridine, CHCl₃, rt, 2 h, 80%; (b) AlCl₃, CH₂Cl₂, rt, 2 h, 51%; (c) 2-hydrazinopyridine, EtOH, 80 °C, 2 h, 95%; (d) NBS, CHCl₃, 60 °C, 40 min, 58%.

Scheme 7.

Reagents and conditions: 30: (a) NH₂NH₂, pyridine, rt, 2.5 h, 41%; (b) EtOH, 80 °C, 2 h, 95%; (c) NBS, CHCl₃, 60 °C, 3 h, 50%. 33: (a) 35% NH₂NH₂·H₂O, 65 °C, 12 h, 75%; (b) EtOH, 80 °C, 2 h, 62%; (c) NBS, CHCl₃, 60 °C, 2.5 h, 59%. 36: (a) i) NaOH/H₂O, extraction with Et₂O, ii) NH₂NH₂·H₂O, EtOH, 100 °C, 16 h, 37%; (b) pyridine, EtOH, 80 °C, 2 h, 98%; (c) NBS, CHCl₃, 60 °C, 24 h, 93%.

Scheme 8.

Reagents and conditions: 39 and 40: (a) NH₂NH₂·H₂O, EtOH, 105 °C, 40 h, 36%; (b) EtOH, 80 °C, 2.5 h, 30%; (c) NBS, CHCl₃, 60 °C, 6.5 h, 39% for 39, 17% for 40. 43: (a) NH₂NH₂·H₂O, EtOH, 105 °C, 5 days, 79%; (b) EtOH, 80 °C, 2.5 h, 83%; (c) NBS, CHCl₃, 60 °C, 2 h, 81%. 46 and 47: (a) NH₂NH₂·H₂O, EtOH, 105 °C, 29 h, 53%; (b) pyridine, EtOH, 80 °C, 3 h, 22%; (c) NBS, CHCl₃, 60 °C, 2.5 h, 11% for 46, 10% for 47. 49: (b) EtOH, 80 °C, 3 h, 83%; (c) NBS, CHCl₃, 60 °C, 2 h, 60%. 51: b) EtOH, 80 °C, 3 h, 73%; (c) NBS, CHCl₃, 60 °C, 2 h, 23%.

Scheme 9.

Synthesis of N1-modified analogues. Reaction conditions: (a) EtOH, 80 °C, 2–3 h; (b) NBS, CHCl₃, 60 °C, 1.5–5 h; (c) SnCl₂-2H₂O, EtOH, 70 °C, 15 min, 62%.

Scheme 10.

Reagents and conditions: (a) NH₂NH₂·H₂O, EtOH, 80 °C, 4 h, 88%; (b) TEA, CH₂Cl₂, 0 °C to rt, 5 h, 75%; (c) NBS, CHCl₃, 60 °C, 4 h, 56%.

Scheme 11.

Reagents and conditions: 71: (a) monomethyl malonate, EDC·HCl, HOBT, DIPEA, CH₂Cl₂, rt, 24 h, 69%; 72: (a) sulfamoyl chloride, dimethylacetamide, rt, 20 h, 89%. 73: (a) methanesulfonyl chloride, pyridine, CH₂Cl₂, rt, 23 h, 88%; 74: (a) acetyl chloride, TEA, CH₂Cl₂, rt, 1.5 h, 92%.

Scheme 12.

Introducing a naphthalene-2-yl group at N1. Reagents and conditions: (a) pyridine, EtOH, 80 °C, 2 h, 79%; (b) NBS, CHCl₃, 60 °C, 3 h, 68%.