Nonpeptide Ut Ligands

magnitude greater UT affinity (pECso = 11.3 and 11.0 at the human and rat UT respectively). 15 was UT selective and did not bind to a number of other dopaminergic, adrenergic and serotonergic GPCRs or peptide GPCRs. Interestingly, 15 was also selective for UT over opioid (k) and somatostatin (sst-2, sst-2 and sst-5) receptors which are the most closely related to UT having some, albeit relatively limited, sequence similarity to UT.

Antagonists - Several reports have now appeared in both the scientific and patent literature describing non-peptide UT antagonists. In general, most of the compounds reported contain features typical of GPCR binding small molecules such as a basic amino group believed to form a charged interaction with an Asp acid (D130 in hUT) within TM3 of the receptor and multiple aromatic moieties.

Positive ionizable

Figure 1. Pharmacophore query used for virtual screening mapped onto bioactive fragment of NMR structure of (a) U-ll and (b) Ac-CFwKYC-NH2. The light blue spheres represent two hydrophobic aromatic features; the red sphere represents positive ionizable features of the three point 3D pharmacophore. The gray spheres display the shape requirements linked to the pharmacophore query (Reprinted with permission from 34. Copyright 2003 American Chemical Society)

Positive ionizable

Hydrophobic aromatic

\ Positive tonuabie

Hydrophobic aromatic

\ Positive tonuabie

Hydrophobic aromatic

In an elegant study, a combination of structure-based design and high throughput virtual screening has been used to discover potent, small molecule UT antagonists (34). Using a classical and systematic investigation of the SAR of U-ll, the identity of the key residues in the cyclic hexapeptide core of the peptide was established and the analysis was used to generate a solution 1H-NMR structure for the binding conformation U-ll.

Hydrophobic aromatic

Figure 1. Pharmacophore query used for virtual screening mapped onto bioactive fragment of NMR structure of (a) U-ll and (b) Ac-CFwKYC-NH2. The light blue spheres represent two hydrophobic aromatic features; the red sphere represents positive ionizable features of the three point 3D pharmacophore. The gray spheres display the shape requirements linked to the pharmacophore query (Reprinted with permission from 34. Copyright 2003 American Chemical Society)

The resulting 3-point pharmacophore for UT binding was then used as the basis for computational searching of a small molecule library to identify a number of templates which antagonized the U-ll-induced intracellular Ca2+ mobilization (FLIPR) with IC5o values in the 0.4 - 7 uM range. The most potent class was substituted indoles such as S6716 (16) which had an EC5o in the functional assay of 400 nM. The authors recognized that these benzamidine containing compounds are likely to suffer from poor oral absorption based on earlier work on related factor Xa inhibitor compounds.

To date, however, the patent literature has documented a much larger number of UT antagonists. This no doubt is a reflection of the increased level of interest in the potential therapeutic application of such compounds. In a series of published patent applications, diarylsulfonamides with a variety of substitution patterns have been claimed for diverse therapeutic indications including hypertension, CHF, ischemic heart disease and stroke. The initial reports in this series of compounds center around ortho substituted (dialkylaminoalkoxy)arylsulfonamldes in which the basic amine center is linked to the biarylsulfonamide by means of a linear three atom chain (41). More elaborate variation of the sulfonamide to incorporate, for example, biaryethers and heterocycles such as pyrimidines, are claimed in later applications from the same group (42 - 44). The biological activity of UT antagonists was determined in a radioligand competition binding assay using [125l]-hU-ll and cell membranes containing stable, cloned hUT and typical compounds in the series such as the analog 17, had a Ki in this assay of 1,300 nM. In the most recent patent applications covering this series, the early compounds have been further investigated and constraint of the amine side chain by incorporation into a heterocyclic ring together with additional manipulation of the aryl groups is reported(45 - 48). This has resulted in the identification of compounds of higher UT affinity such as the pyrrolidine sulfonamide, 18 (Ki = 590 nM).

17 18

17 18

Japanese researchers have claimed fused 4-aminoquinolines such as 19 - 21 as having UT antagonism and hence of utility in the treatment of cardiovascular diseases (49). Only human UT affinity is reported. The reported in vitro biological activity, however, shows that UT affinity is sensitive to the substituent at the quinoline 6-position with the presence of either a methyl or bromine group being associated with an ~10-fold higher activity relative the parent unsubstituted system. Interestingly, the compounds of this class have also been claimed more recently (50) to be useful remedies for central nervous system diseases based on the amyloid (340 secretion-inhibitory effect of UT antagonism. This was demonstrated in a cellular assay measuring the U-ll induced secretion of amyloid p-protein (Ap) from the human neuroblastoma cell line IMR32 where analog 20 (3 - 10 uM) was shown to reduce AP40 secretion to control levels.

ICW nM

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