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  • Activity of compound in rat brain tissue highlights the


    Activity of compound 10 in rat Vacquinol-1 tissue highlights the potential to use this new class of allosteric sGC inhibitors to study the role of the NO—sGC—cGMP signalling pathway in the brain. Reducing amounts of cGMP in cells would have implications in downstream signalling proteins, such as cGMP-dependent protein kinases and phosphodiesterases.
    Conclusion We have identified a new type of small molecule inhibitors of sGC, which are thought to be the first class to act through allosteric regulation of the enzyme catalytic domain, rather than oxidation of the haem or through the purine p-site. It is possible that the inhibitors presented bind to the catalytic domain of sGC inducing a conformational change, or ‘locking’ the enzyme in a basal conformation, that is not favourable to activation by NO or GTP binding. The inhibitor also inhibits particulate GC but not the related adenylate cyclase. Compound 10 (D12), with an IC50 and KD in the micromolar range, may be used as a new tool to inhibit the NO—sGC—cGMP signalling pathway, and further study its implications and regulatory functions in the brain.
    Experimentals Full-length human recombinant guanylyl cyclase (soluble) and bovine lung guanylyl cyclase (soluble) were obtained from Enzo lifesciences (catalogue numbers ALX-201-177 and ALX-202-039-C005). Biacore consumables were purchased from GE Healthcare (UK), including buffer stock solutions. All other reagents used were obtained from Sigma. Starting materials were either commercially available or synthesized according to methods reported in the literature. 1H and 13C NMR spectra were recorded on a Bruker AMX-300 or a Bruker AMX-500 spectrometer. Chemical shifts are reported as ppm relative to TMS internal standard. Mass spectra were recorded on a Fisons VG70-SE spectrometer (EI, FAB) or an Agilent 6100 Series LC-mass spectrometer using C-18 or C-4 columns. Microwave reactions were carried out using a CEM Discover microwave.
    Acknowledgement This research was supported by the Medical Research Council UK with a studentship for F.M.
    Soluble guanylate cyclase (sGC) is a heterodimeric (α/β) heme-protein that converts guanosine-5′-triphosphate (GTP) to cyclic guanosine-3′5′-monophosphate (cGMP), an important messenger in signal transduction. Its natural stimulator is nitric oxide, which stimulates sGC via the formation of a nitrosyl-heme complex. Organic nitrates such as glycerol trinitrate or isosorbide dinitrate have been used as a treatment for angina pectoris. In vivo they are converted to NO which relaxes smooth muscles but develops tolerance on repeat dosing. Two mechanistic classes of direct stimulators of sGC have more recently been developed. One class of stimulators is NO-independent but heme-dependent, the other is both NO- and heme-independent. The first reported direct stimulator of NO was YC-1, followed by the discovery of more potent compounds such as BAY 41-8543 by Bayer and their more recent clinical compound BAY 63-2521 (). These compounds act synergistically with NO to potentiate stimulation of sGC and have been reported to bind to an allosteric binding site within the catalytic domain. BAY 41-8543 and related compounds have a co-planar arrangement of the two heterocycles as demonstrated by X-ray analysis, which seems a requisite for ligand function but could be detrimental to solubility. Neutral to weakly basic compounds can have a low volume of distribution which in turn could lead to short intrinsic half-life. Our medicinal chemistry strategy concentrated on keeping the potency and selectivity of these compounds while specifically addressing PK properties and solubility. Changing the pendant heterocycles such as the furan in YC-1 and the diaminopyrimidine in BAY 41-8543 to heterocycles containing weakly acidic hydrogens should improve solubility at physiological pH while keeping the required functionality required for potency. However, initial reports on acidic heterocycles such as tetrazole showed they were not particularly active. 1,2,4-Triazoles represented an attractive starting place because the p of NH on the triazole can be modulated depending on the C and C substituents. Overall volume of distribution () of these slightly acidic compounds, however, is likely to be low (0.2–1.0L/kg) so modifications to the core template and the northern fragment to vary physicochemical properties to address clearance is also key for a good ADMET profile.