A series of azaphilones produced by Penicillium sclerotiorum (Xenova culture collection number XI 1 853) active in assays for the detection of antagonists of the endothelin-A (ETA) and endothelin-B (ETB) receptors has been identified. The series includes two novel sclerotiorin analogues, ($S,%a-R)-7-deacetyl-l ,O 8,8,8a-tetrahydro-7-à¬>/?/-sclerotiorin, 1, and its 5-dechloro analogue, 2. It also includes 5-chloroisorotiorin, 6, previously unreported as a natural product, in addition to the major product of these fermentations, (+)-sclerotiorin, 5. Data for the inhibition of endothelin-1 (ET-1) and endothelin-3 (ET-3) binding in the ETAand ETBreceptor assays respectively are reported for this series. Compounds1 and 2 were more selective for the rabbit ETAreceptor than for the rat ETB receptor. The IC50 values for 1 and 2 were 9 and 28/*m respectively in an assay based on binding of ET-1 to rabbit ETAreceptors. In an assay based on the binding of ET-3 to the rat ETBreceptor The endothelins (which exist as three isoforms: ET-1, ET-2 and ET-3) are a family of potent vasoconstricting peptides with a variety of biological activities including bronchoconstriction, positive inotropic and chronotropic effects, mitogenesis and potent renal effects. Endothelins are implicated in several human disease states including hypertension, congestive heart failure, renal failure, pulmonary hypertension, ischemia and cerebral vasospasm1 "40. Recent results obtained with mice deficient in endothelin-1 suggest that it is essential for normal mouse development and may also play a physiological role in cardiovascular homeostasis5). non-selective receptor, recognising the ET isopeptides with equal affinity, was originally identified as the non-vascular smooth muscle receptor. This receptor is localised on endothelial cells in certain tissues and has been associated with vasodilatory activity, perhaps through the release of the endothelin-derived relaxing factor (EDRF). It has been reported, however, that the ETBreceptor is also localised on vascular smooth muscle and mediates a vasoconstrictor response in certain tissues/species. The use of endothelin receptor antagonists is furthering the understanding of the pathophysiological role of the endothelins17~22).The endothelin antagonists discovered to date from microbial sources are predominantly actinomycete metabolites. These include the ETA receptor specific cyclic pentapeptide BE-18257B from Streptomyces misakiensis23\ the benzarlthraquinones WS009Aand WS009B from a Streptomyces sp.24), and the depsipeptide cochinmicins from a Microbispora sp.2 5) There have been fewer reports of endothelin receptor
S = 112 excited state, the three fits are each quite different. The first gives an S = 7 / 2 ground state with an S = 9 / 2 excited state only 9 cm-' higher in energy. The second fit has an S = 9 / 2 ground state and an S = 7 / 2 excited state, separated by only 0.5 cm-' with seven other spin states lying within 50 cm-' of the ground state; whereas, the third fit has an S = 9 / 2 ground state with the closest excited state being S = 7 / 2 at 226 cm-' above the ground state. This third fit was used for the calculation of the unbroken curve in Figure 2a.To our surprise, magnetization data for complex 2, collected over the temperature range 1.8 to 40 K at field values from 2.5 to 4.5 T, conclusively show an isolated S=9/2 ground state (Fig. 2b). This is in agreement with the third set of parameters obtained from the susceptibility data. Additional support for the isolation of this S = 9 / 2 level is found in the fact that, although there are some changes and loss of resolution at elevated temperatures, the EPR spectrum of 2 (powder or glass) is readily seen at both liquid-helium and liquid-nitrogen temperatures. Such behavior would not be expected from the manifold of spin states found in the second fit.It is likely that all the features in the glass EPR spectrum of 2 at g z 2, 6, and 9 (similar to those seen for 1 ,@I) are attributable to transitions between components of the S = 9/2 ground state, which, as a detailed analysis of the M versus H/T data (Fig. 2b) shows, experiences an axial zero-field splitting (0s:) with D = 0.25-0.35 cm-'. Whether the S, state of PS I1 contains a cluster similar to that of 2 is still unclear, but the described work does suggest that the possibility that the &-state EPR spectral features might also arise from components of a large-spin ground state should be seriously considered. Interestingly, we have just characterized"" a dinuclear Mn"Mn"' complex which is also ferromagnetically coupled and has an S = 912 ground state (verified by M versus H/T data). The EPR spectrum for this Mn"Mn"' complex is similar to those for 1 and 2.
We here report the discovery and early characterization of Compound 3, a representative of a novel class of small molecule bradykinin (BK) B 2 receptor antagonists, and its superior profile to the prior art B 2 receptor antagonists Compound 1 and Compound 2. Compound 3, Compound 2, and Compound 1 are highly potent antagonists of the human recombinant B 2 receptor (K b values 0.24, 0.95, and 1.24 nM, respectively, calcium mobilization assay). Compound 3 is more potent than the prior art compounds and icatibant in this assay (K b icatibant 2.81 nM). The compounds also potently inhibit BK-induced contraction of endogenous B 2 receptors in a human isolated umbilical vein bioassay. The potencies of Compound 3, Compound 2, Compound 1, and icatibant are (pA 2 values) 9.67, 9.02, 8.58, and 8.06 (i.e. 0.21, 0.95, 2.63, and 8.71 nM), respectively. Compound 3 and Compound 2 were further characterized. They inhibit BK-induced c-Fos signaling and internalization of recombinant human B 2 receptors in HEK293 cells, and do not antagonize the venous effects mediated by other G protein-coupled receptors in the umbilical vein model, including the bradykinin B 1 receptor. Antagonist potency of Compound 3 at cloned cynomolgus monkey, dog, rat, and mouse B 2 receptors revealed species selectivity, with a high antagonist potency for human and monkey B 2 receptors, but several hundred-fold lower potency for the other B 2 receptors. The in vitro off-target profile of Compound 3 demonstrates a high degree of selectivity over a wide range of molecular targets, including the bradykinin B 1 receptor. Compound 3 showed a lower intrinsic clearance in the microsomal stability assay than the prior art compounds. With an efflux ratio of 1.0 in the Caco-2 permeability assay Compound 3 is predicted to be not a substrate of efflux pumps. In conclusion, we discovered a novel chemical class of highly selective and very potent B 2 receptor antagonists, as exemplified by Compound 3. The compound showed excellent absorption in the Caco-2 assay, predictive of good oral bioavailability, and favourable metabolic stability in liver microsomes. Compound 3 has provided a significant stepping stone towards the discovery of the orally bioavailable B 2 antagonist PHA-022121, currently in phase 1 clinical development.
A series of azaphilones produced by Penicillium sclerotiorum (Xenova culture collection number XI 1 853) active in assays for the detection of antagonists of the endothelin-A (ETA) and endothelin-B (ETB) receptors has been identified. The series includes two novel sclerotiorin analogues, ($S,%a-R)-7-deacetyl-l ,O 8,8,8a-tetrahydro-7-à¬>/?/-sclerotiorin, 1, and its 5-dechloro analogue, 2. It also includes 5-chloroisorotiorin, 6, previously unreported as a natural product, in addition to the major product of these fermentations, (+)-sclerotiorin, 5. Data for the inhibition of endothelin-1 (ET-1) and endothelin-3 (ET-3) binding in the ETAand ETBreceptor assays respectively are reported for this series. Compounds1 and 2 were more selective for the rabbit ETAreceptor than for the rat ETB receptor. The IC50 values for 1 and 2 were 9 and 28/*m respectively in an assay based on binding of ET-1 to rabbit ETAreceptors. In an assay based on the binding of ET-3 to the rat ETBreceptor compounds 1 and 2 exhibited IC50's of 77 and 172/zm. Members of this series of compounds demonstrated antagonist behavior in a secondary assay based on blockade of ET-1 stimulated arachidonic acid release from rabbit renal artery smoothmuscle cells, whenpresent at concentrations of>30jum. The endothelins (which exist as three isoforms: ET-1, ET-2 and ET-3) are a family of potent vasoconstricting peptides with a variety of biological activities including bronchoconstriction, positive inotropic and chrono-tropic effects, mitogenesis and potent renal effects. Endothelins are implicated in several human disease states including hypertension, congestive heart failure, renal failure, pulmonary hypertension, ischemia and cerebral vasospasm1 "40. Recent results obtained with mice deficient in endothelin-1 suggest that it is essential for normal mouse development and may also play a physiological role in cardiovascular homeostasis5). Two subtypes of endothelin receptors, classified as ETAand ETBreceptors, have been cloned and charac-terised in mammalian systems6~9). Both receptor sub-types are rhodopsin-like in structure and are coupled to G-proteins. A third endothelin receptor subtype has been cloned from Xenopusdermal melanophores and heart10~11.) although this subtype has not yet been described in mammaliantissues. Vasoconstriction in a wide variety of animal tissues can clearly occur via activation of ETA and/or ETB receptors, depending upon the species and vascular bed 913 under study12~ 16). The ETAreceptor mediates vasocon-striction and mitogenic responses and is widely localised in vascular smooth muscle in most tissues. The ETB, or non-selective receptor, recognising the ET isopeptides with equal affinity, was originally identified as the non-vascular smooth muscle receptor. This receptor is localised on endothelial cells in certain tissues and has been associated with vasodilatory activity, perhaps through the release of the endothelin-derived relaxing factor (EDRF). It has been reported, however, that the ETBreceptor is also localised on vas...
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