More than an Amide Bioisostere: Discovery of 1,2,4-Triazole-containing Pyrazolo[1,5- a]pyrimidine Host CSNK2 Inhibitors for Combatting β-Coronavirus Replication

Abstract

The pyrazolo[1,5-a]pyrimidine scaffold is a promising scaffold to develop potent and selective CSNK2 inhibitors with antiviral activity against β-coronaviruses. Herein, we describe the discovery of a 1,2,4-triazole group to substitute a key amide group for CSNK2 binding present in many potent pyrazolo[1,5-a]pyrimidine inhibitors. Crystallographic evidence demonstrates that the 1,2,4-triazole replaces the amide in forming key hydrogen bonds with Lys68 and a water molecule buried in the ATP-binding pocket. This isosteric replacement improves potency and metabolic stability at a cost of solubility. Optimization for potency, solubility, and metabolic stability led to the discovery of the potent and selective CSNK2 inhibitor 53. Despite excellent in vitro metabolic stability, rapid decline in plasma concentration of 53 in vivo was observed and may be attributed to lung accumulation, although in vivo pharmacological effect was not observed. Further optimization of this novel chemotype may validate CSNK2 as an antiviral target in vivo.

Figure 1

Exemplar CSNK2 inhibitors of the previously reported pyrazolo[1,5-a]pyrimidine series.

Figure 2

Co-crystal structure of 1 (left, PDB ID: 6Z83) and 14 (right, PDB ID: 8P07) with CSNK2A1. Part of the kinase P-loop is hidden for clarity. Water molecules are represented as red spheres. Hydrogen bonds are denoted as yellow dashed lines.

Figure 3

Co-crystal structure of 50 (left, PDB ID: 8P06) and 53 (right, PDB ID: 9EZG) with CSNK2A1. Part of the kinase P-loop is hidden for clarity. Water molecules are represented as red spheres. Hydrogen bonds are denoted as yellow dashed lines.

Figure 4

Selectivity profile of 53 determined at 10 μM using the NanoBRET K192 panel. Only CSNK2A1 and CSNK2A2 had 97% occupancy (red). Kinases with 50–75% occupancy are shown in yellow. Kinases with <50% occupancy are shown in green. Kinases not in the NanoBRET K192 panel are shown in gray. Image generated using CORAL. No kinases had occupancy >75% except CSNK2A1 and CSNK2A2. Detailed % occupancy values for each kinase are described in Table S3.

Figure 5

(A) Structure of 53. (B) Dose-dependent effect of 53 against SARS-CoV-2 in A549-ACE2 cells and cell viability of A549-ACE2 cells as measured by the LDH assay.

Figure 6

(A) Concentration over time of 53 in CD-1 mice, dosed i.p. at 10 mg/kg at t = 0 h. Plasma concentrations (light blue line) and lung concentrations (dark blue line) of the compound were measured at 0.5, 1, 2, 4, 8, 12, and 24 h, and the lung/plasma ratio is calculated (black dashed line). Data points are shown as mean ± s.d. (n = 3). The SARS-CoV-2 IC₅₀ of 53 determined in A549-ACE2 cells is plotted as a yellow dotted line. (B) Pharmacokinetic parameters.

Figure 7

(A) Viral titer measured in mouse lungs 24 h post inoculation when treated with vehicle or with compound 53 (10 mg/kg i.p. b.i.d.) (n = 5). (B) Western blot for total EIF2S2 expression in mouse lung samples after treatment with vehicle or 53 (10 mg/kg i.p. b.i.d.) (n = 3). (C) Western blot for EIF2S2 (phospho-Ser2) in mouse lung samples after treatment with vehicle or 53 (10 mg/kg i.p. b.i.d.) (n = 3). (D) EIF2S2 (phospho-Ser2) levels normalized to total EIF2S2 levels in mouse lung. (E) Western blot for total AKT expression in mouse lung samples after treatment with vehicle or 53 (10 mg/kg i.p. b.i.d.) (n = 3). (F) Western blot for AKT (phospho-Ser129) in mouse lung samples after treatment with vehicle or 53 (10 mg/kg i.p. b.i.d.) (n = 3). (G) AKT (phospho-Ser129) levels normalized to total AKT levels in mouse lung.

Scheme 1. Synthesis of Analogues Varying the meta-Position of the Aniline 3–29

Reagents and conditions: (a) cyclopropylamine, EtOH, 25 °C, 2 h; (b) Pd(OAc)₂, BINAP, Cs₂CO₃ or ^tBuOLi or ^tBuOK, dioxane, μW, 130 °C, 0.5 h or 100 °C, 2 h; (c) Brettphos Pd G3, Cs₂CO₃, dioxane, μW, 130 °C, 0.5 h; (d) TFA, DCM, 25 °C, 2 h.

Scheme 2. Synthesis of Aniline Intermediates 79–81, 83, 87, 89, 92, 93, 95, 96, 99, 101

Reagents and conditions: (a) NaSMe, MeOH, 0–25 °C, 3 h; (b) mCPBA, DCM, 0 °C, 1 h; (c) Rh(OAc)₂, MgO, iodobenzene diacetate, tert-butyl carbamate, DCM, 25 °C, 16 h; (d) Fe, NH₄Cl, EtOH, H₂O, 65–90 °C, 1–3 h; (e) Me₂P(=O)H, NaHMDS, THF, −30 °C, 1 h then 25 °C, 12 h; (f) TFA, DCM, 25 °C, 2–10 h; (g) H₂, Pd/C, MeOH, 25 °C, 2–12 h; (h) CbzCl, DIPEA, DCM, 25 °C, 16 h; (i) MeMgBr, THF, 25 °C, 16 h; (j) TMSN₃, CuI, DMF, MeOH, 100 °C, 12 h; (k) acetohydrazide, DBU, EtOH, 85 °C, 10 h; (l) NaNO₂, AcOH, H₂O, 0–100 °C, 5 h; (m) SnCl₂·H₂O, HCl, EtOH, 70 °C, 10 h; (n) NH₂CH₂CH(OMe)₂, DCM, 0–25 °C, 3 h; (o) 4H-1,2,4-triazole, K₂CO₃, MeCN, 100 °C, 10 h; (p) ArBr, Pd(dppf)Cl₂, Cs₂CO₃, dioxane, H₂O, 100 °C, 5 h; (q) ^tBuONO, HBF₄, EtOH, 0–25 °C, 1 h; (r) TMSCHN₂, (CF₃CO₂)Ag, Et₃N, THF, −78 °C, 1 h; (s) CsF, MeOH, rt, 0.5 h.

Scheme 3. Synthesis of Analogues Varying the 7-Position Substituent of the Pyrazolo[1,5-a]pyrimidine 30–37

Reagents and conditions: (a) RNH₂, EtOH, rt, 2 h; (b) 86, Pd(OAc)₂, BINAP, Cs₂CO₃ or ^tBuOLi, dioxane, μW, 130 °C, 0.5 h; (c) 86, Brettphos Pd G3, Cs₂CO₃, dioxane, μW, 130 °C, 0.5 h; (d) TFA, DCM, 35 °C, 2 h.

Scheme 4. Synthesis of Analogues Varying the 7-Position Substituent of the Pyrazolo[1,5-a]pyrimidine 38–46

Reagents and conditions: (a) H₂, Pd/C, MeOH or THF, 20–40 °C, 2–12 h; (b) 74, EtOH, 25 °C, 2–5 h; (c) 86, Pd(OAc)₂, BINAP, Cs₂CO₃, dioxane, μW, 130 °C, 0.5–6 h; (d) 2-bromoethanol, K₂CO₃, MeCN, 60 °C, 10 h; (e) BocNHCH₂CH₂Br, K₂CO₃, DMF, 90 °C, 3 h; (f) SOCl₂, DCM, DMF, 25 °C, 2 h; (g) MeNH₂, K₂CO₃, NaI, μW, 80 °C, 4 h; (h) Boc₂O, K₂CO₃, THF, 25 °C, 3 h; (i) MeI, NaH, THF, 0–25 °C, 10 h; (j) TFA, DCM, 25–35 °C, 2–6 h; (k) RCH₂CH₂Cl, K₂CO₃, MeCN or DMF, 60–120 °C, 4–12 h; (l) RCH₂CH₂Cl, NaH, DMF, 15 °C, 3.5 h; (m) 86, Brettphos Pd G3, Cs₂CO₃, dioxane, μW, 130 °C, 4 h.

Scheme 5. Synthesis of Compounds 47–50

Reagents and conditions: (a) 1,2-diformylhydrazine, Me₃SiCl, Et₃N, pyridine, 100 °C, 12 h; (b) for 179, 180, and 182, H₂, Pd/C, MeOH, 25 °C, 10 h; (c) for 181, NH₃, H₂O, 100 °C, 72 h; (d) 75, Brettphos Pd G3, ^tBuOLi, dioxane, μW, 130 °C, 0.5 h; (e) 75, Pd(OAc)₂, BINAP, Cs₂CO₃ or ^tBuOLi, dioxane, μW, 130 °C, 0.5 h.

Scheme 6. Synthesis of Non-fluorinated Analogues Varying the para-Position of the Aniline 51, 53, 55–60, 62–66, 68–70, 72–73

Reagents and conditions: (a) R-H, K₂CO₃, MeCN, 100 °C, 10–12 h; (b) H₂, Pd/C, MeOH or THF, 25–35 °C, 2–12 h; (c) 75, Pd(OAc)₂, BINAP, Cs₂CO₃, dioxane, μW, 130 °C, 0.5 h; (d) 75, Brettphos Pd G3, Cs₂CO₃ or ^tBuOLi, dioxane, μW, 130 °C, 0.5 h; (e) TFA, DCM, 25–35 °C, 1–2 h; (f) for 60, TBAF, THF, 25 °C, 10 h.

Scheme 7. Synthesis of Fluorinated Analogues Varying the para-Position of the Aniline 52, 54, 61, 67, 71

Reagents and conditions: (a) R-H, K₂CO₃, MeCN, 60–100 °C, 10–12 h; (b) for 234, 2-(methylamino)ethan-1-ol, K₂CO₃, 60 °C, 2 h, then TBDPSCl, imidazole, DMF, 25 °C, 12 h; (c) CbzCl, K₂CO₃, THF, 25 °C, 2–12 h; (d) Fe, NH₄Cl, EtOH, H₂O, 80–100 °C, 2–12 h; (e) 1,2-diformylhydrazine, Me₃SiCl, Et₃N, pyridine, 100 °C, 12 h; (f) H₂, Pd/C, MeOH, 25 °C, 2–5 h; (g) 75, Pd(OAc)₂, BINAP, Cs₂CO₃, dioxane, μW, 130 °C, 0.5–6 h; (h) TFA, DCM, 25 °C, 1–3 h; (i) for 61, TBAF, THF, 25 °C, 2 h.