Nitric oxide (NO) is a widespread, potent, biological mediator that has many physiological and pathophysiological roles. Research in the field of NO appears to have followed a straightforward path, and the findings have been progressive: NO and cyclic GMP are involved in vasodilatation; glycerol trinitrate relaxes vascular smooth muscles by bioconversion to NO; mammalian cells synthesize NO; and last, NO mediates vasodilatation by stimulating the soluble guanylate cyclase (sGC), a heterodimeric (alpha/beta) haem protein that converts GTP to cGMP2-4. Here we report the discovery of a regulatory site on sGC. Using photoaffinity labelling, we have identified the cysteine 238 and cysteine 243 region in the alpha1-subunit of sGC as the target for a new type of sGC stimulator. Moreover, we present a pyrazolopyridine, BAY 41-2272, that potently stimulates sGC through this site by a mechanism that is independent of NO. This results in antiplatelet activity, a strong decrease in blood pressure and an increase in survival in a low-NO rat model of hypertension, and as such may offer an approach for treating cardiovascular diseases.

1 Soluble guanylyl cyclase (sGC) is the only proven receptor for the ubiquitous biological messenger nitric oxide (NO) and is intimately involved in many signal transduction pathways, most notably in regulating vascular tone and platelet function. sGC is a heterodimeric (a/û) protein that converts GTP to cyclic GMP; NO binds to its prosthetic haem group. Here, we report the discovery of a novel sGC activating compound, its interaction with a previously unrecognized regulatory site and its therapeutic implications. 2 Through a high-throughput screen we identi®ed BAY 58-2667, an amino dicarboxylic acid which potently activates sGC in an NO-independent manner. In contrast to NO, YC-1 and BAY 41-2272, the sGC stimulators described recently, BAY 58-2667 activates the enzyme even after it has been oxidized by the sGC inhibitor ODQ or rendered haem de®cient. 3 Binding studies with radiolabelled BAY 58-2667 show a high anity site on the enzyme. 4 Using photoanity labelling studies we identi®ed the amino acids 371 (a-subunit) and 231 ± 310 (û-subunit) as target regions for BAY 58-2667. 5 sGC activation by BAY 58-2667 results in an antiplatelet activity both in vitro and in vivo and a potent vasorelaxation which is not in¯uenced by nitrate tolerance. 6 BAY 58-2667 shows a potent antihypertensive eect in conscious spontaneously hypertensive rats. In anaesthetized dogs the hemodynamic eects of BAY 58-2667 and GTN are very similar on the arterial and venous system. 7 This novel type of sGC activator is a valuable research tool and may oer a new approach for treating cardiovascular diseases.

Direct stimulation of soluble guanylate cyclase (sGC) represents a promising therapeutic strategy for the treatment of a range of diseases, including the severely disabling pulmonary hypertension (PH). Optimization of the unfavorable DMPK profile of previous sGC stimulators provided riociguat, which is currently being investigated in phase III clinical trials for the oral treatment of PH.magnified imageSoluble guanylate cyclase (sGC) is a key signal‐transduction enzyme activated by nitric oxide (NO). Impairments of the NO–sGC signaling pathway have been implicated in the pathogenesis of cardiovascular and other diseases. Direct stimulation of sGC represents a promising therapeutic strategy particularly for the treatment of pulmonary hypertension (PH), a disabling disease associated with a poor prognosis. Previous sGC stimulators such as the pyrazolopyridines BAY 41‐2272 and BAY 41‐8543 demonstrated beneficial effects in experimental models of PH, but were associated with unfavorable drug metabolism and pharmacokinetic (DMPK) properties. Herein we disclose an extended SAR exploration of this compound class to address these issues. Our efforts led to the identification of the potent sGC stimulator riociguat, which exhibits an improved DMPK profile and exerts strong effects on pulmonary hemodynamics and exercise capacity in patients with PH. Riociguat is currently being investigated in phase III clinical trials for the oral treatment of PH.

1 BAY 41-8543 is a novel non-NO-based stimulator of sGC. This study investigates the acute e ects of BAY 41-8543 on haemodynamics in anaesthetized rats and dogs, its long-term e ects in conscious hypertension rat models and its antiplatelet e ects. 2 In anaesthetized dogs, intravenous injections of BAY 41-8543 (3 ± 100 mg kg 71 ) caused a dosedependent decrease in blood pressure and cardiac oxygen consumption as well as an increase in coronary blood¯ow and heart rate. 3 In anaesthetized normotensive rats, BAY 41-8543 produced a dose-dependent and long-lasting blood pressure lowering e ect after intravenous (3 ± 300 mg kg 71 ) and oral (0.1 ± 1 mg kg 71 ) administration. A dose-dependent and long-lasting decrease in blood pressure was also observed in conscious spontaneously hypertensive rats with a threshold dose of 0.1 mg kg 71 p.o. After 3 mg kg 71 the antihypertensive e ect lasted for nearly 24 h. After multiple dosages, BAY 41-8543 did not develop tachyphylaxis in SHR. 4 BAY 41-8543 prolonged the rat tail bleeding time and reduced thrombosis in the FeCl 3 thrombosis model after oral administration. 5 In a low NO, high renin rat model of hypertension, BAY 41-8543 prevented the increase in blood pressure evoked by L-NAME and reveals a kidney protective e ect. In this model, the overall bene®cial e ects of BAY 41-8543 manifested as both antiplatelet e ect and vasodilatation were re¯ected in a signi®cant reduction in mortality. 6 The pharmacological pro®le of BAY 41-8543 suggests therefore that this compound has the potential to be an important research tool for in vivo investigations in the sGC/cGMP ®eld and it also has the potential of being a unique clinical utility for treatment of cardiovascular diseases.

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