Contemporary Accounts in Drug Discovery and Development. Группа авторов

Scheme 3.5 Optimized scheme for the synthesis of acrylaldehyde derivative 35.

The synthesis of the acrylaldehyde derivative 35 started with activation of 2,2,3,3‐tetrafluoro‐1‐propanol 38 with trifluoromethanesulfonic anhydride and subsequent treatment with morpholine to yield 39. At 135 °C, 39 was alkylated with methyl methanesulfonate leading to quaternized derivative 40 which was converted into 41 using sodium hydroxide. Further reaction with morpholine and triethylamine yielded 35 after crystallization from toluene (Scheme 3.5).

3.5 Preclinical Studies

      The pathophysiology of CV diseases including HF includes endothelial cell dysfunction which impairs NO production leading to decreased NO availability and reduced cGMP tissue levels. This reduced NO availability and insufficient stimulation of sGC results in systemic, vascular dysregulation, affecting the coronary, pulmonary, and renal circulation. Moreover, impaired sGC signaling leads to organ damage and dysfunction of the heart. Therefore, sGC stimulation by vericiguat could provide a novel approach for the treatment of CV diseases and different forms of HF. Vericiguat has a unique mode of action and was extensively profiled preclinically in vitro, on the isolated sGC enzyme and on isolated vessels, ex vivo in isolated hearts and in vivo in a rat model of CV disease associated with cardiorenal syndrome.

      3.5.1 In vitro Effects on Recombinant sGC and sGC Overexpressing Cells

In vitro, vericiguat led to a concentration‐dependent stimulation of the sGC enzyme. Even in the absence of NO, vericiguat stimulated the sGC by 1.7‐fold to 57.6‐fold (Figure 3.5). When vericiguat was combined with the NO donor diethylamine/nitric oxide complex (DEA/NO), a synergistic effect on sGC activity and cGMP stimulation was observed over a wide range of concentrations (Figure 3.5). In addition, the stimulation of sGC by vericiguat was examined in vitro in recombinant CHO cells overexpressing sGC. Vericiguat stimulated these sGC reporter cell lines concentration dependently, with an EC₅₀ of 1005 ± 145 nM. In the presence of the NO donor S‐nitroso‐N‐acetyl‐D,L‐penicillamine (SNAP) (30 and 100 nM), the EC₅₀ value shifted to 39.0 ± 5.1 nM and 10.6 ± 1.7 nM, respectively (Table 3.6). Thus, vericiguat exhibits the characteristics of a potent and selective sGC stimulator, stimulating sGC NO independently and in synergy with NO.

Figure 3.5 Concentration‐dependent effects of vericiguat and NO (DEA/NO) on the stimulation of highly purified sGC.

      3.5.2 Ex vivo Effects on Isolated Blood Vessels and Hearts

      As shown in Table 3.6, vericiguat significantly inhibited phenylephrine‐induced contractions of rabbit saphenous artery rings, rabbit aortic rings and canine femoral vein rings concentration dependently, with IC₅₀ values of 798, 692 and 3072 nM, respectively. Accordingly, vericiguat inhibited the contractions induced by the thromboxane A2 agonist U46619 of porcine coronary artery rings concentration dependently, with an IC₅₀ of 956 nM. Since it is known that NO is leading to nitrate tolerance and tachyphylaxia, vericiguat was also investigated on isolated saphenous artery rings taken from nitrate‐tolerant rabbits. In contrast to the NO‐donor glycerol trinitrate (GTN) vericiguat also relaxed the nitrate‐tolerant saphenous artery rings, with IC₅₀ values of 5.6 and 5.8 nM, respectively (not shown).

      Ex vivo in an isolated rat heart preparation (Langendorff experiment), vericiguat reduced the coronary perfusion pressure in a concentration‐dependent manner with a maximal reduction of 30% at 10 μM (Table 3.6). In these rat heart experiments, no effects on HR and contractility (dp/dt, left ventricular diastolic pressure), were observed (Table 3.6).

      3.5.3 In vivo Effects in a Disease Model with CV Disease and HF and Kidney Failure

      Although predictive animal models for HF including HFrEF and HFpEF (heart failure with preserved ejection fraction) are scarce, there are models reflecting presenting with CV morbidity and some features of HF. The renin transgenic (RenTG) rats carrying the additional mouse renin gene (mRenR2)27 are characterized by hypertension. If these RenTG(mRenR2)27 rats were additionally treated with the NO synthase inhibitor N _𝝎‐nitro‐L‐arginine methyl ester (L‐NAME) in the drinking water, they suffer endothelial dysfunction, NO decoupling and rapidly progressing hypertension‐induced end‐organ damage resulting in high morbidity and mortality.

Table 3.6 Nonclinical pharmacodynamics of vericiguat in vitro, ex vivo and in vivo.

Experiments	Assay/Test system	Effects of vericiguat
In vitro effects on sGC activity in transfected cells	Recombinant CHO cell line overexpressing sGC(sGC reporter cell line)	sGC activity: vericiguat alone: EC50 = 1005 ± 145 nMvericiguat +30 nM SNAP: EC50 = 39.0 ± 5.1 nMvericiguat +100 nM SNAP: EC50 = 10.6 ± 1.7 nM
Ex vivo effects on relaxation of precontracted blood vessels of different vascular beds	Rabbit saphenous artery Rabbit aortic arteryPorcine coronary artery Canine femoral vein	Inhibition of contraction with vericiguat: rabbit saphenous artery: IC50 = 798 nM rabbit aorta: IC50 = 692 nM porcine coronary artery: IC50 = 956 nM canine femoral vein: IC50 = 3072 nM
Ex vivo effects in the Langendorff heart preparations	Langendorff‐perfused rat heart	Reduction of coronary perfusion pressure by up to 30% (10 μM); no influence on heart rate and contractility
In vivo effects on the cardiovascular system in a disease model	L‐NAME treated renin transgenic rats (RenTG)	Significant decrease of mortality rates: Placebo: 75% mortality, 3 mg/kg: 33%, 10 mg/kg: 7% Significant decrease of heart hypertrophy (% from placebo) 3 mg/kg: 8%, 10 mg/kg: 11% Significant decrease of plasma ANP (% from placebo) 3 mg/kg: 45%, 10 mg/kg: 64% Significant decrease of proteinuria (% from placebo) 3 mg/kg: 65%, 10 mg/kg: 88%

Chronic once daily oral treatment with 3 or 10 mg/kg vericiguat resulted in a significant attenuation of mortality. The mortality rate in the placebo arm was 75%, whereas mortality in the vericiguat treatment arms were 33 and 7% for the 3 and 10 mg/kg vericiguat dose arm, respectively (