Discovery of Molidustat (BAY 85-3934): A Small-Molecule Oral HIF-Prolyl Hydroxylase (HIF-PH) Inhibitor for the Treatment of Renal Anemia

Abstract

Small-molecule inhibitors of hypoxia-inducible factor prolyl hydroxylases (HIF-PHs) are currently under clinical development as novel treatment options for chronic kidney disease (CKD) associated anemia. Inhibition of HIF-PH mimics hypoxia and leads to increased erythropoietin (EPO) expression and subsequently increased erythropoiesis. Herein we describe the discovery, synthesis, structure-activity relationship (SAR), and proposed binding mode of novel 2,4-diheteroaryl-1,2-dihydro-3H-pyrazol-3-ones as orally bioavailable HIF-PH inhibitors for the treatment of anemia. High-throughput screening of our corporate compound library identified BAY-908 as a promising hit. The lead optimization program then resulted in the identification of molidustat (BAY 85-3934), a novel small-molecule oral HIF-PH inhibitor. Molidustat is currently being investigated in clinical phase III trials as molidustat sodium for the treatment of anemia in patients with CKD.

Keywords: BAY 85-3934; HIF-PH; inhibitors; metalloenzymes; molidustat.

Figure 1

A promising novel therapeutic approach for the treatment of anemia: stabilization of HIF via inhibition of HIF‐PH translates into EPO induction in the kidney and formation of red blood cells in the bone marrow. EPO: erythropoietin; HIF: hypoxia‐inducible transcription factor; HIF‐PH: HIF‐prolyl hydroxylase; HO‐P: hydroxylated proline residues.

Figure 2

Other advanced small‐molecule oral HIF‐PH inhibitors currently in clinical development.

Figure 3

Concentration‐response curves of HIF‐PH activity at Fe²⁺ concentrations of 3 μm and 30 μm (30 μm indicated by dotted lines). While IC₅₀ values of BAY‐908 (1) remained below the concentration of Fe²⁺, concentrations of compound 2 beyond the free iron concentration were required to achieve inhibition of the enzyme (IC₅₀ shifts indicated by arrows).

Figure 4

Pyrazolone–pyrazolol tautomerism of compound 1.11

Figure 5

Proposed binding mode: structure of BAY‐908 (1) docked into X‐ray structure of PHD2. Docking was performed with the electron‐neutral pyrazolol tautomer for technical reasons. Dotted lines indicate iron coordination (additional coordination to His313 and H₂O not shown).

Figure 6

Compounds 44 and 45 (molidustat, BAY 85‐3934), combining imidazole/1,2,3‐triazole (left‐hand side) with cyclic amino‐substituted pyrimidines (right‐hand side).

Figure 7

Plasma erythropoietin levels in rats at 4 h after oral dosing of compounds 1, 44 and 45 (molidustat).

Figure 8

Docked structure of molidustat (BAY 85‐3934, 45, green carbon atoms) into the X‐ray crystal structure of PHD2 (PDB ID: https://www.rcsb.org/structure/2G19, grey carbon atoms) superimposed with a recently published X‐ray crystal structure of PHD2 with the pyrazolol derivative 6‐[5‐hydroxy‐4‐(1H‐1,2,3‐triazol‐1‐yl)‐1H‐pyrazol‐1‐yl]nicotinic acid (PDB ID: https://www.rcsb.org/structure/5A3U, yellow carbon atoms). Dotted lines indicate iron coordination (additional coordination to His313 and H₂O not shown). Docking was performed with the electron‐neutral pyrazolol tautomer of molidustat.

Scheme 1

Synthesis of compound 1: a) NaH, 18‐K‐6, toluene, RT→85 °C, then HC(O)OEt, RT→90 °C, 67 %; b) 2‐hydrazinopyridine, EtOH, RT, 93 %; c) NaOEt, EtOH, RT; then HCl, 85 %; d) (Me)₂NCH(OEt)₂, 100 °C, 70 %; e) 2‐hydrazinopyridine, CSA, EtOH, reflux; then NaOEt, RT; then HCl, 83 %. CSA=camphorsulfonic acid.

Scheme 2

Synthesis of compound 4: a) pyridin‐2‐amine, CDI, acetone, RT, 56 %; b) NaOEt, 4‐acetamidobenzenesulfonyl azide, EtOH, 0 °C→RT, 3 %. CDI=N,N′‐dicyclohexylcarbodiimide.

Scheme 3

Synthesis of compounds 5 and 6: a) Lawesson's reagent, toluene, reflux, 17 %; b) 2‐hydrazinopyridine, EtOH, CSA, reflux, 4 %; c) NaH, 18‐K‐6, toluene, RT→90 °C, then HC(O)OEt, RT→90 °C, 46 %; d) 2‐hydrazinopyridine, EtOH, RT→80 °C, then NaOEt, RT, 13 %.

Scheme 4

Synthesis of compound 7: a) 3‐isocyanatopyridine, CHCl₃, 55 °C, 42 %; b) NaOH, MeOH, RT→45 °C, 19 %. Compound 55 was obtained from 2‐hydrazinopyridine under the following conditions: HCO₂H, RT, 79 %.

Scheme 5

Synthesis of compounds 8 (29 %) and 9 (35 %): a) PPh₃, MeOH, DIAD, RT, then separation of isomers via column chromatography on silica gel. DIAD=diisopropyl azodicarboxylate.

Scheme 6

Synthesis of derivative 10: a) 2‐(tributylstannyl)pyridine, Pd(PPh₃)₄ toluene, reflux, 34 %; b) pyridin‐3‐ylboronic acid, Pd(PPh₃)₂Cl₂, NaHCO₃, dioxane, 80 °C, 43 %; c) NaOH, diethylene glycol, 200 °C, 95 %.

Scheme 7

Synthesis of selected compounds of Table 3: a) 2‐hydrazino‐isonicotinonitrile, HOAc, RT, 83 %; b) NaOH, EtOH/H₂O, reflux, 84 %; c) MeOH, H₂SO₄, reflux, 74 %; d) NaBH₄, CaCl₂, EtOH, 0 °C→RT, then 1 n HCl, 63 %; e) 4‐chloro‐6‐hydrazinopyrimidine, HOAc, RT, 77 %; f) MeOH, NaH, THF, TBAI, RT→reflux, 52 %; g) ethyl 6‐hydrazinonicotinate, CSA, EtOH, reflux, 54 %; h) LiOH, 1,4‐dioxane, reflux, 88 %; i) (6‐hydrazinopyridin‐3‐yl)methanol, CSA, EtOH, reflux, 66 %. CSA=camphorsulfonic acid, TBAI=tetrabutylammonium iodide.

Scheme 8

Synthesis of compounds 34 and 35: a) 2‐hydrazinopyridine, HOAc, RT, 59 %; b) Pd(PPh₃)₄, Zn(CN)₂, DMF, microwave, 190 °C, 12 %; c) 2‐hydrazinopyridine, HOAc, RT, 31 %; d) Pd(PPh₃)₄ Zn(CN)₂, DMF, microwave, 190 °C, 48 %. For syntheses of 64 and 65 see Supporting Information.

Scheme 9

Synthesis of compounds 44 and 45 (molidustat, BAY 85‐3934): a) imidazole, NaOEt, EtOH, RT, 81 %; b) 1,2,3‐1H‐triazole, NaOEt, EtOH, RT, 50 % for 70 and 28 % for 71, followed by separation via high‐vacuum distillation; c) (Me)₂NCH(OEt)₂, 100 °C, 42 %; d) (Me)₂NCH(OEt)₂, 100 °C, 100 %; e) 77, PTS, EtOAc, reflux, 34 %; f) 78, TFA, EtOAc, reflux, 71 %; g) piperidine/morpholine, H₂O, 115 °C/90 °C, 47 % for 75 and 85 % for 76; h) hydrazine hydrate, EtOH, RT→80 °C/reflux, 69 % for 77 and 68 % for 78. PTS=4‐toluenesulfonic acid.

Scheme 10

Large‐scale synthesis of molidustat (BAY 85‐3934, 45) and molidustat sodium (84): a) NEt₃, hydrazine hydrate, H₂O, no work‐up; b) morpholine, NaHCO₃, H₂O, heat, 72 % (one‐pot/two steps); c) 1,2,3‐triazole, N,N‐diisopropylethylamine (Hünig's base), EtOAc, filtration; d) N,N‐dimethylformamide dimethyl acetal, heat, acetone, crystallization from 2‐propanol, 76 % of 81; e) EtOAc, TFA, heat, H₂O/HOAc, filtration, 85 %; f) NaOH, MeOH/H₂O, NEt₃, 98 %.

Scheme 11

Biocatalytic transformation of molidustat sodium (84) into the N‐glucuronide 85 (BAY‐348, M‐1 metabolite of molidustat): a) S. griseochromogenes, sterile aqueous nutrient solution, DMF, 27 °C, 21 %.