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, 59 (4), 1388-409

8-Substituted Pyrido[3,4-d]pyrimidin-4(3H)-one Derivatives As Potent, Cell Permeable, KDM4 (JMJD2) and KDM5 (JARID1) Histone Lysine Demethylase Inhibitors

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8-Substituted Pyrido[3,4-d]pyrimidin-4(3H)-one Derivatives As Potent, Cell Permeable, KDM4 (JMJD2) and KDM5 (JARID1) Histone Lysine Demethylase Inhibitors

Vassilios Bavetsias et al. J Med Chem.

Abstract

We report the discovery of N-substituted 4-(pyridin-2-yl)thiazole-2-amine derivatives and their subsequent optimization, guided by structure-based design, to give 8-(1H-pyrazol-3-yl)pyrido[3,4-d]pyrimidin-4(3H)-ones, a series of potent JmjC histone N-methyl lysine demethylase (KDM) inhibitors which bind to Fe(II) in the active site. Substitution from C4 of the pyrazole moiety allows access to the histone peptide substrate binding site; incorporation of a conformationally constrained 4-phenylpiperidine linker gives derivatives such as 54j and 54k which demonstrate equipotent activity versus the KDM4 (JMJD2) and KDM5 (JARID1) subfamily demethylases, selectivity over representative exemplars of the KDM2, KDM3, and KDM6 subfamilies, cellular permeability in the Caco-2 assay, and, for 54k, inhibition of H3K9Me3 and H3K4Me3 demethylation in a cell-based assay.

Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Pyridine-based histone demethylase inhibitors.
Scheme 1
Scheme 1
Reagents and conditions: (a) EDCI, 1-hydroxybenzotriazole, CH2Cl2 or CH2Cl2/DMF, room temp, 3.5–36 h, (7a = 33%, 7b = 25%, 7c = 40%, 7d = 50%, 9a = 28%, 9b = 16%).
Scheme 2
Scheme 2
Reagents and conditions: (a) Pd(PPh3)4, 1,4-dioxane, reflux, 18 h, 65%; (b) N-bromosuccinimide, 10% water in THF, room temp, 1 h, 61%; (c) N-Boc-thiourea, Et3N, EtOH, reflux, 1 h, 56%; (d) (i) 4 M HCl in dioxane, room temp, 1 h, (ii) THF/H2O, NaOH, room temp, 1 h, 55%; (e) (i) 7 M NH3 in MeOH, 80 °C, 18 h, (ii) 4 M HCl in dioxane, room temp, 1 h, 91%; (f) 4 M HCl in dioxane, room temp, 1 h, 83%; (g) benzoyl chloride, CH2Cl2, Et3N, room temp, 2 h, 35%; (h) THF/MeOH, H2O, NaOH, room temp, 1 h, 40%.
Scheme 3
Scheme 3
Reagents and conditions: (a) Pd(PPh3)4, Ag2O, DMF, 100 °C, 1 h, 64%; (b) MeOH/H2O, NaOH, 1 h, 46%; (c) AcOH, 48% aqueous HBr, Br2, 0 °C to room temp for 1 h, then 75 °C for 1.5 h, 38%; (d) (i) thioacetamide, EtOH, Et3N, reflux, 1 h, (ii) EtOH, conc HCl (2 drops), reflux, 20 min, 23%; (e) MeOH/H2O, 1 M NaOH, room temp, 50 min, 45%.
Scheme 4
Scheme 4
Reagents and conditions: (a) PTSA, EtOH, 80 °C, 79%; (b) DIBAL, THF, PhMe, −78 °C, 39%; (c) benzylamine or 2-aminomethylfuran, NaBH(OAc)3, AcOH, CH2Cl2, (29a = 99%, 29b = 93%); (d) LiOH (aq), MeCN, (30a = quant, 30b = quant).
Scheme 5
Scheme 5
Reagents and conditions: (a) MeOH/H2O, 1 M NaOH, room temp, 45 min, 77%; (b) (i) SOCl2, DMF, reflux, 2 h, (ii) ammonium hydroxide, THF, room temp, 1.5 h, 89%; (c) CH(OEt)3, reflux 24 h, 82%; (d) SEM-Cl, K2CO3, DMF, 60 °C, 4 h, 84%; (e) 4-(tributylstannyl)thiazole, Pd(PPh3)4, Ag2O, DMF, 100 °C, 2 h, 32%; (f) THF, 6 M HCl, 50 °C, 4 h, 94%; (g) Pd(PPh3)4, 1,4-dioxane, reflux, 2 h, 64%; (h) 10% H2O in THF, N-bromosuccinimide, room temp, 1 h, 72%; (i) thiourea, Et3N, EtOH, reflux, 1 h, 13%.
Scheme 6
Scheme 6
Reagents and conditions: (a) Pd(PPh3)4, Na2CO3, DMA, (1H-pyrazol-3-yl)boronic acid hydrochloride or ((N-methyl)pyrazol-3-yl)boronic pinacol ester, 100 °C, 1 h, (41a = 12%, 41b = 21%).
Scheme 7
Scheme 7
Reagents and conditions: (a) Pd(PPh3)4, K2CO3, trimethylboroxine, DME, 90 °C, 1 h, 59%; (b) SeO2, 1,4-dioxane, 90 °C, 8 h, 14%; (c) NaBH(OAc)3, R1R2NH, MeOH/CH2Cl2, room temp, 1–3 h, 12–55%.
Scheme 8
Scheme 8
Reagents and conditions: (a) Cs2CO3, anhydrous MeCN, reflux, 18 h, 71%; (b) 1 M HCl, MeOH, 0 °C, 5 min, 99%; (c) Dess–Martin periodinane, anhydrous CH2Cl2, room temp; (d) (i) methanesulfonic anhydride, Et3N, anhydrous CH2Cl2, 0 °C, 15 min, workup, (ii) Cs2CO3, R1R2NH, anhydrous DMF, 90 °C, 15 h or Et3N, anhydrous DMF, 50 °C, 15 h, 17–84%; (e) R1R2NH, anhydrous 1,2-dichloroethane, NaBH(OAc)3, room temp, overnight, 24–69%; (f) 6 M HCl, THF, 50–60 °C, 3 to 8 h or 4 M HCl in dioxane, dioxane/H2O, 50 °C, 16–97%; (g) pyrazole, Cs2CO3, anhydrous MeCN, reflux, 3 h, 80%; (h) THF, 6 M HCl, 50 °C, 4 h, (56 = 70%, 57 = 75%).
Figure 2
Figure 2
Overlay of a view from crystal structure of 2 (light-blue ball-and-sticks) bound to KDM4A (displayed in gray lines or gray surface; PDB code 3PDQ) and a model of 5 (magenta) docked into the protein structure from 3PDQ. (A) Interaction network. (B) Surface representation. Ni(II) shown in place of the active site Fe(II).
Figure 3
Figure 3
Views from crystal structures of 15, 16, and 30b bound to KDM4A and of 30a bound to KDM4D. (A) KDM4A-bound 15 (gray), showing a close-up view of ligand–metal coordination and interaction with Y132 and K206. (B) Overlay of KDM4A-bound 15 (gray) and 16 (green). (C) 2D interaction map of 15 with key residues in KDM4A. (D) Overlay of KDM4A-bound 15 (gray) and 30b (purple for chain A, cyan for chain B of the asymmetric unit). (E) Overlay of KDM4A-bound 15 (gray) and KDM4D-bound 30a (orange). Indicated residues correspond to KDM4A numbering. Zn(II) and Ni(II) are shown at the active site.
Figure 4
Figure 4
Comparison of binding modes of 15, 37, 40, and 58 to KDM4A and 44a bound to KDM4D. (A) Overlay of KDM4A-bound 15 (gray) and 58 (yellow). (B) Overlay of KDM4A-bound 15 (gray), 37 (light-green), and 40 (orange). (C) Overlay of KDM4A-bound 15 (gray) and KDM4D-bound 44a (light blue). The indicated residue numbers correspond to KDM4A numbering. Zn(II) and Ni(II) are shown in place of the active site Fe(II).
Figure 5
Figure 5
(A) Overlay of views from crystal structure of KDM4A–37 (light-green) and −52d (two main conformations: dark-brown for conformation A and light-brown for conformation B). (B) Overlay of 52d bound to KDM4A (brown) and KDM5B (cyan), the indicated residue numbers correspond to KDM5B numbering. Zn (II) or Ni(II) are shown in place of the active site Fe (II).
Figure 6
Figure 6
Binding mode of 53a in KDM4A. Zn(II) is shown in place of the active site Fe(II).
Figure 7
Figure 7
(A) Overlay of KDM4A-bound 54a (orange) and 54j (cyan). (B) 2D interaction diagram of 54j showing interactions with key residues in KDM4A. Zn(II) is shown for the active site Fe(II).

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