Contemporary Accounts in Drug Discovery and Development. Группа авторов

2.3 1.21 0.1 2 (74) 14 0.2 2.05 2.1 n.d.^b 15 0.2 2.09 1.4 n.d.^b

      ^a MEC, minimal effective concentration to achieve stimulation of cGMP formation (≥3‐fold increase in basal luminescence) in a recombinant sGC‐overexpressing cell line [36].

      ^b n.d., not determined.

At first, changes were made to the pyrazolo portion of the molecule, leading to the 1H‐pyrazolo[4,3‐b]pyridine derivative 16. In addition to the benefit of a shorter synthetic route than that for compound 10, 16 proved to be a moderately potent sGC stimulator with an MEC of 1.2 μM and had high metabolic stability when tested in rat hepatocytes. The introduction of substituents at the 6‐position of the 1H‐pyrazolo[4,3‐b]pyridine core was, in general, not well tolerated leading to a dramatic loss of potency (not shown), with the exception of the 6‐fluoro derivative 17 which exhibited an MEC of 0.5 μM and good metabolic stability. To our surprise, compounds 16 and 17 had very different in vivo clearances when compared in a rat pharmacokinetic experiment (i.v. dosing): fluoro derivative 17 had a low clearance of 0.3 l/h/kg versus 1.0 l/h/kg for compound 16. As metabolite identification did not point to metabolism occurring at the pyridine core, the rationale for this threefold reduction in blood clearance remains unclear.

Table 3.4 Properties of the core variation compounds 16–20, 2.

Compound	Core	cGMP formation MEC a (μM)	In vitro clearance (rat hepatocytes) CLb (l/h/kg)	In vivo clearance (rat) CLb (l/h/kg)
16		1.2	<0.1	1.0
17		0.5	0.1	0.3
18		2.0	<0.1	3.8
19		1.7	n.d.b	1.8
20		0.7	<0.1	0.9
2		0.3	0.1	0.3

      ^a MEC, minimal effective concentration to achieve stimulation of cGMP formation (≥3‐fold increase in basal luminescence) in a recombinant sGC‐overexpressing cell line [36].

      ^b n.d., not determined.

      Novel 1H‐pyrazolo[3,4‐c]pyridazine 18 proved to be a reasonably potent sGC stimulator and seemed to be stable in rat hepatocytes (although solubility was limited and that might have impacted the assay) but exhibited an unacceptable high clearance of 3.8 l/h/kg when tested in vivo in rats and thus was not further profiled, along with imidazo[1,5‐a]pyrimidine 19 for similar reasons. In contrast, imidazo[1,5‐b]pyridazine 20 exhibited potent sGC stimulator properties (MEC = 0.7 μM), good metabolic stability in rat hepatocytes and a low to moderate clearance of 0.9 l/h/kg after i.v. dosing to rats. Finally, revisiting 1H‐pyrazolo[3,4‐b]pyridine 10 with an additional fluorine at the 5‐position resulted in the potent sGC stimulator 2 (vericiguat) (MEC = 0.3 μM) with good metabolic stability in rat hepatocytes and a surprisingly low clearance of 0.3 l/h/kg after i.v. dosing to rats (Table 3.4). Thus, the blood clearance of derivative 10 in rats (1.2 l/h/kg) was reduced fourfold by fluorination at position 5.

Finally, compounds 17, 20 and 2 were selected for further pharmacokinetic profiling across different species (Table 3.5). Fluoropyrazolo[3,4‐b]pyridine derivative 2 exhibited the best overall pharmacokinetic profile by far [40], with a low clearance and long half‐life in rats and dogs after i.v. dosing, as well as high oral bioavailability. In addition, 2 had no inhibitory effects on major CYP isoforms (1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4), as indicated by IC₅₀ values of >50 μM. Metabolite identification in human hepatocytes was characterized by a low turnover, with the glucuronide (major) and the debenzylated compound (minor) being the only metabolites found. Thus, the main biotransformation pathway shifted from predominantly phase 1 oxidative CYP‐mediated metabolism (1) to primarily phase 2 UGT‐mediated conjugation with glucuronic acid.