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A series of isoquinuclidine benzamides as glycine uptake inhibitors for the treatment of schizophrenia are described. Potency, lipophilicity, and intrinsic human microsomal clearance were parameters for optimization. Potency correlated with the nature of the ortho substituents of the benzamide ring, and reductions in lipophilicity could be achieved through heteroatom incorporation in the benzamide and pendant phenyl moieties. Improvements in human CL int were achieved through changes in ring size and the N-alkyl group of the isoquinuclidine itself, with des-alkyl derivatives (40-41, 44) demonstrating the most robust microsomal stability. Dimethylbenzamide 9 was tested in a mouse MK801 LMA assay and had a statistically significant attenuation of locomotor activity at 3 and 10 μmol/kg compared to control., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The cytosolic portion of CD45, a major transmembrane glycoprotein found on nucleated hematopoietic cells, contains protein tyrosine phosphatase activity and is critical for T-cell receptor-mediated T-cell activation. CD45 inhibitors could have utility in the treatment of autoimmune disorders and organ graft rejection. A number of 9,10-phenanthrenediones were identified that reversibly inhibited CD45-mediated p-nitrophenyl phosphate (pNPP) hydrolysis. Chemistry efforts around the 9,10-phenanthrenedione core led to the most potent inhibitors known to date. In a functional assay, the compounds were also potent inhibitors of T-cell receptor-mediated proliferation, with activities in the low micromolar range paralleling their enzyme inhibition. It was also discovered that the nature of modification to the phenanthrenedione pharmacophore could affect selectivity for CD45 over PTP1B (protein tyrosine phosphatase 1B) or vice versa.

The cyclic peptide ANP 4-23 and the linear peptide analogue AP-811 have been shown to be selective ANP-CR antagonists. Via alanine scanning and truncation studies we sought to determine which residues in these molecules were important in their binding to the clearance receptor and the relationship between these two molecules. These studies show that several modifications to these compounds are possible which improve physical properties of these molecules while retaining high affinity for the ANP-CR.

This paper describes the development a series of peptidyl trifluoromethyl ketone inhibitors of human leukocyte elastase which are found to have excellent pharmacological profiles. Methods have been developed that allow for the synthesis of these inhibitors in stereochemically pure form. Two of these compounds, 1k and 1l, have high levels of oral bioavailability in several species. Compound 1l has entered development as ZD8321 and is presently undergoing clinical evaluation. These compounds demonstrate that peptidyl trifluoromethyl ketone inhibitors can achieve high levels of oral activity and bioavailability, and therefore they may prove useful as therapeutic agents in the treatment of diseases in which elastase is implicated.

Previously we had shown that tripeptidyl trifluoromethyl ketones (TFMKs) possessing an N-terminal diarylacylsulfonamide, such as ICI 200,880 and ICI 200,355, displayed unparalleled protection against the lung damage induced by human neutrophil elastase (HNE) when the inhibitors were administered intratracheally. Since the diarylacylsulfonamides were designed specifically to afford a long residence time in the lung, it was not unexpected that inhibitors from this class of TFMKs were not active when administered orally. Upon evaluating a large number of peptidyl TFMKs possessing a variety of N-terminal groups, several compounds were identified which demonstrated oral activity. Compounds were evaluated for their oral activity by measuring their ability to inhibit the increase in lung weight relative to body weight (Lw/Bw), the increase in red blood cells, and the increase in white blood cells induced by intratracheally administered HNE (100 micrograms/hamster). A number of tripeptidyl trifluoromethyl ketones containing neutral N-terminal groups displayed good oral activity, while those containing basic, acidic, or polar groups did not. Compound 50, possessing an N-terminal 4-(CH3O)C6H4CO group, was particularly effective, reducing Lw/Bw by 77%, red cells by 89%, and white cells by 91% when dosed at 37.5 mg/kg orally. Thus, by modifying the N-terminal group of tripeptidyl TFMKs, inhibitors can be designed which are effective in vivo when administered either orally or intratracheally.

The effects of changes in substitution in a series of 5-amino-2-pyrimidin-6-ones on both in vitro activity and oral activity in an acute hemorrhagic assay have been explored. These compounds contained either a trifluoromethyl ketone or a boronic acid moiety to bind covalently to the Ser-195 hydroxyl of human leukocyte elastase (HLE). Boronic acid-containing inhibitors were found to be more potent than the corresponding trifluoromethyl ketones in vitro but were less active upon oral administration. Compound 13b was found to offer the best combination of oral potency, duration of action, and enzyme selectivity and, as such, was selected for further biological testing. X-ray crystallography of a cocrystallized complex of compound 19m and porcine pancreatic elastase demonstrated that the inhibitor is bound to the enzyme in a manner similar to that found previously for a closely related series of pyridone-containing inhibitors of HLE.

A novel series of human leukocyte elastase (HLE) inhibitors containing the beta-carbolinone ring system are reported. The design of these trifluoromethyl ketone-based inhibitors used a combination of structural information obtained from X-ray crystallography and molecular modeling investigations. The beta-carbolinone ring in these compounds serves as a highly efficient peptidiomimetic for the P2-P3 region of peptidyl trifluoromethyl ketone inhibitors of HLE. Several of the beta-carbolinones exhibit significant in vitro potency, with Ki values in the nanomolar range. Using aqueous molecular dynamics simulations, realistic models for the molecular recognition of these inhibitors by HLE have been obtained and are discussed. This series of compounds are found to have excellent selectivity for HLE over a number of other proteolytic enzymes, including closely related enzymes such as porcine pancreatic elastase.

The biosynthesis of leukotrienes and lipoxins involves epoxide-containing intermediates which may be subject to several routes of transcellular metabolism. We have examined the capacity of leukotriene A4 (LTA4) and 15S-hydroxy-5,6-oxido-7,9,13-trans-11-cis-eicosatetraenoic acid [5(6)-epoxytetraene] to stimulate the mobilization of free cytosolic calcium [( Ca2+]i) in human blood neutrophils. To gain insight into structure-activity relationships, a putative intermediate in lipoxin biosynthesis, 5S-hydroxy-14,15-oxido-6,10,12-trans-8-cis-eicosatetraenoic acid [14(15)-epoxytetraene], was prepared by total synthesis. When added to fura-2 loaded neutrophils, each of these compounds provoked a rapid and transient increase in [Ca2+]i (maximum by 8 sec) which returned to baseline within 60-90 sec. Ca2+ mobilization with LTA4 was dose dependent and, at 1 microM, the efficacies of LTA4 and LTB4 were quantitatively similar. The 5(6)-epoxytetraene and 14(15)-epoxytetraene were less potent than LTA4. Prior exposure of the cells to ethyleneglycolbis(aminoethylether)tetra-acetate (EGTA) (60 sec, 3 mM) did not diminish either the amplitude or the extent of [Ca2+]i elicited by LTA4. Methyl esters of LTA4 and 5(6)-epoxytetraene were less potent than their corresponding free acids, whereas the free acid of 14(15)-epoxytetraene and its methyl ester were quantitatively similar. Results from alcohol trapping studies showed that these epoxides were intact during the initial phase of Ca2+i mobilization (t0-10 sec) stimulated by LTA4, 5(6)-epoxytetraene, and 14(15)-epoxytetraene. In addition, the individual mixtures of products formed upon aqueous hydrolysis of each of the epoxides did not stimulate changes in [Ca2+]i. In each case, the products formed were identified by physical methods including reverse phase high pressure liquid chromatography, ultraviolet spectroscopy and gas liquid chromatography-mass spectrometry. These results indicate that, when added to human neutrophils, LTA4, 5(6)-epoxytetraene and 14(15)-epoxytetraene each stimulate a rapid mobilization of [Ca2+]i. Moreover, they suggest that intermediates in the biosynthesis of leukotrienes and lipoxins possess intrinsic activities that may serve to amplify cellular responses within their cell of origin or act on adjacent cells during their transcellular metabolism.

Addition of (15S)-hydroxy-5,8,11-cis-13-trans-eicosatetraenoic acid (15-HETE) and the ionophore A23187 (2.5 microM) to human neutrophils led to the formation of both lipoxin A4 and lipoxin B4 as well as a novel 5,6,15-trihydroxyeicosatetraenoic acid. The new compound was identified using an improved isolation and detection system and its basic structure was determined by physical methods. On the basis of biosynthetic considerations, geometric isomers of lipoxin A4 and lipoxin B4 were prepared by total synthesis. Comparison of these synthetic materials with the neutrophil-derived product showed that the new compound is (5S,6R,15S)-trihydroxy-9,11,13-trans-7-cis-eicosatetraenoic acid or the 7-cis-11-trans-isomer of LXA4 (7-cis-11-trans-LXA4). LXA4, 11-trans-LXA4, 7-cis-LXA4 and 7-cis-11-trans-LXA4 all evoked dose-dependent (0.1-10 microM) contractions of the guinea pig lung strip, whereas 6-cis-LXB4 and 6-cis-8-trans-LXB4 relaxed this preparation. LXA4 and 7-cis-LXA4 were approx. 10-times more potent than the compounds with 11-trans geometry. However, all four double-bond isomers of LXA4 caused contractions which, based upon pharmacological evidence, appeared to involve specific activation of the same site as cysteinyl-containing leukotrienes. In conclusion, 7-cis-11-trans-LXA4 was isolated and identified as a novel biologically active eicosanoid formed by human neutrophils.

Lipoxins A4 (LxA4) and B4 (LxB4), two lipoxygenase-generated icosanoids of arachidonic acid metabolism, were found to have a distinct biological profile. Both LxA4 and LxB4 slowly contracted pulmonary parenchymal strips isolated from guinea pigs, rabbits, and rats in a concentration-dependent manner over the range 0.1-1 microM. This bronchoconstrictor effect was not associated with release of peptide leukotrienes or thromboxane A2, nor was it blocked by lipoxygenase inhibitors or thromboxane receptor antagonists, suggesting it is a direct effect of lipoxins. However, the leukotriene D4 (LTD4) receptor antagonist LY-171883 reduced the LxA4 response, indicating that LTD4 and LxA4 may share the same receptor. LxA4 and LxB4 also exerted an endothelium-dependent vasorelaxation in guinea pig, rat, and, to a lesser extent, rabbit aortic vascular smooth muscle. In contrast to other vasoactive icosanoids, LxA4 and LxB4 failed to aggregate rat, rabbit, or guinea pig platelets or to inhibit ADP-induced aggregation. LxA4 also enhanced the release of liver lysosomal hydrolases in a liver large granule fraction, indicating a lysosomal labilizing action of LxA4. LxA4 and LxB4 share a similar biological profile. It is not clear yet whether the lipoxins could be mediators of circulatory or pulmonary disease states.

Lipoxin A (LXA) was prepared by incubation of either (15S)-15-hydroxy-5,8,11-cis-13-trans-eicosatetraenoic acid (15-HETE) or (15S)-15-hydroperoxy-5,8,11-cis-13-trans-eicosatetraenoic (15-HPETE) with human leukocytes stimulated by either the ionophore A23187 or the chemotactic peptide fMet-Leu-Phe. Comparison with four trihydroxyeicosatetraenoic acids prepared by total synthesis showed that biologically derived LXA is 5S,6R,15S)-5,6,15-trihydroxy-7,9,13-trans-11-cis-eicosatetraenoic acid. Three isomers of LXA were also identified in extracts of leukocytes utilizing an improved isolation procedure. These were (5S,6S,15S)-5,6,15-trihydroxy-7,9,13-trans-11-cis-eicosatetraenoic acid (6S-LXA), (5S,6R,15S)-5,6,15-trihydroxy-7,9,11,13-trans-eicosatetraenoic acid (11-trans-LXA), and (5S,6S,15S)-5,6,15-trihydroxy-7,9,11,13-trans-eicosatetraenoic acid (6S-11-trans-LXA). 18O2-labeling studies indicated that formation of LXA and its isomers occurred with incorporation of 18O at their C-5 but not C-6 positions. These results suggest that 15-hydroxy-5,6-epoxy-7,9,13-trans-11-cis-eicosatetraenoic acid or its equivalent may serve as one intermediate in the biosynthesis of LXA and 6S-LXA. When added to guinea pig lung strips LXA provoked contractions which were slow in onset and long lasting. In addition, dose response studies showed that biologically derived LXA and synthetic LXA were indistinguishable in this bioassay whereas synthetic 6S-LXA and biologically derived 6S-LXA did not share this activity. Taken together, these results suggest that activated leukocytes utilize exogenous 15-HETE to generate lipoxins which in turn can modulate cellular responses.