(Phosphinyloxy)acyl amino acid inhibitors of angiotensin converting enzyme. 2. Terminal amino acid analogues of (S)-1-[6-amino-2 [[hydroxy(4-phenylbutyl)phosphinyl]oxy]-1-oxohexyl]-L-proline.

Analogues of (S)-1-[6-amino-2[[hydroxy(4-phenylbutyl)phosphinyl] oxy]-1-oxohexyl]-L-proline (1, SQ 29,852) in which the terminal proline residue has been replaced by a variety of substituted and heteroatom-substituted prolines, N-arylglycines, N-cycloalkylglycines, and bicyclic amino acids have been synthesized and evaluated as inhibitors of angiotensin converting enzyme in vitro and in vivo. In general, the addition of lipophilic substituents to the 4-position of proline of the parent phosphonate 1 resulted in substantial increases in in vitro activity. The largest improvements were observed in the case of cis-benzyl (36-fold) and dithioketal (24-fold) analogues 2r and 2x, respectively. These enhancements of in vitro activity were accompanied by modest increases (2-3.5-fold) in in vivo (iv) activity. Among the various terminal amino acid replacements examined in this study, the indoline-based analogue 2i was by far the most potent compound on iv administration in the normotensive rat.

Potent, cell active, non-thiol tetrapeptide inhibitors of farnesyltransferase.

All previously reported CAAX-based farnesyltransferase inhibitors contain a thiol functionality. We report that attachment of the 4-imidazolyl group, via 1-, 2-, or 3-carbon alkyl or alkanoyl spacers, to Val-Tic-Met or tLeu-Tic-Gln provides potent FT inhibitors. (R*)-N-[[1,2,3,4-Tetrahydro-2-[N-[2-(1H-imidazol-4-yl)ethyl] -L-valyl]-3-isoquinolinyl]carbonyl]-L-methionine ([imidazol- 4-yl-ethyl]-Val-Tic-Met), with FT IC50 = 0.79 nM, displayed potent cell activity in the absence of prodrug formation (SAG EC50 = 3.8 muM).

Benzazepinone calcium channel blockers. 2. Structure-activity and drug metabolism studies leading to potent antihypertensive agents. Comparison with benzothiazepinones.

As part of a program to discover potent antihypertensive analogues of diltiazem (3a), we prepared 1-benzazepin-2-ones (4). Benzazepinones competitively displace radiolabeled diltiazem, and show the same absolute stereochemical preferences at the calcium channel receptor protein. Derivatives of 4 containing a trifluoromethyl substituent in the fused aromatic ring show potent and long-acting antihypertensive activity. Studies of the metabolism of 4 lead to the metabolically stable antihypertensive calcium channel blockers 5a and 5c. Benzazepinone 5a is a longer acting and more potent antihypertensive agent than the second generation diltiazem analogue TA-3090 (3e).

Benzazepinone calcium channel blockers. 3. Synthesis and structure-activity studies of 3-alkylbenzazepinones.

As part of a program aimed at identifying novel analogues of diltiazem, we developed several synthetic routes for 3-alkylbenzazepinones, both in racemic and nonracemic form. Structure-activity relationship studies in this series have led to identification of several analogues as potent calcium channel blocking agents, both in vitro and in vivo. Analogues containing a 6-trifluoromethyl substituent (17a and 17b) are the most potent vasorelaxants in vitro. The oral antihypertensive activity of these compounds is comparable to its 3-acetoxy derivative 1 (X = 6-CF3) and 8-chlorodiltiazem (2b). The 3-allyl analogue 17c is a more potent antihypertensive agent than 17a, 17b, or 8-chlorodiltiazem (2b), and has a longer duration of action in vivo.

Benzazepinone calcium channel blockers. 4. Structure-activity overview and intracellular binding site.

We have synthesized a series of benzazepinones (2) in order to determine the structure-activity relationships (SAR) for calcium channel blockers related to diltiazem. A prerequisite for calcium channel blocking activity in vitro and in vivo is the presence of two pharmacophores: a 4'-aryl methyl ether and a basic substituent appended to N1 with a pKa in the physiological range. When these constraints are satisfied, a wide variety of substitution is tolerated at C6, C7, and C3. The presence of an electron-withdrawing group at C6 appears to enhance potency in vitro and in vivo. For such benzazepinones, activity is primarily dependent upon lipophilicity, as measured by log P. We believe these compounds must partition into the cell membrane in order to access their receptor. The quaternary methiodide 15k was used to demonstrate that the binding site for benzazepinones is on the intracellular face of the membrane. This work represents the first comprehensive SAR of diltiazem-like calcium channel blockers.

Discovery and structure-activity relationships of sulfonamide ETA-selective antagonists.

Random screening of compounds in an ETA receptor binding assay led to the discovery of a class of benzenesulfonamide ligands. Optimization led to the development of 5-amino-N-(3,4-dimethyl-5-isoxazolyl)-1-naphthalenesulfonamides which were functional antagonists. Structural features which were important to activity included a 1,5-substitution pattern on the naphthalene ring; a sulfonamide NH with a pK value < 7; an amine, preferably with alkyl substituents, at the 5-position; and methyl groups on both the 3- and 4-positions of the isoxazole.

Biphenylsulfonamide endothelin antagonists: structure-activity relationships of a series of mono- and disubstituted analogues and pharmacology of the orally active endothelin antagonist 2'-amino-N- (3,4-dimethyl-5-isoxazolyl)-4'-(2-methylpropyl)[1, 1'-biphenyl]-2-sulfonamide (BMS-187308).

Substitution at the ortho position of N-(3,4-dimethyl-5-isoxazolyl) benzenesulfonamide led to the identification of the biphenylsulfonamides as a novel series of endothelin-A (ETA) selective antagonists. Appropriate substitutions on the pendant phenyl ring led to improved binding as well as functional activity. A hydrophobic group such as isobutyl or isopropoxyl was found to be optimal at the 4'-position. Introduction of an amino group at the 2'-position also led to improved analogues. Combination of the optimal 4'-isobutyl substituent with the 2'-amino function afforded an analogue (20, BMS-187308) with improved ETA binding affinity and functional activity. Compound 20 also has good oral activity in inhibiting the pressor effect caused by an ET-1 infusion in rats. Doses of 10 and 30 micromol/kg iv 20 attenuated the pressor responses due to the administration of exogenous ET-1 to conscious monkeys, indicating that the compound inhibits the in vivo activity of endothelin-1 in nonhuman primates.

The roles of iNOS in liver ischemia-reperfusion injury.

To determine the contribution of the inducible nitric oxide synthase (iNOS) to hepatic injury following warm ischemia-reperfusion, we developed a model of partial hepatic ischemia-reperfusion in mice and studied the injury response in iNOS knockout (KO) mice. Compared with wild types, iNOS KO animals exhibited lower plasma transaminase levels after 1 and 6 h of reperfusion following 1 h of ischemia. At the 3-h time point, enzyme levels were not different between the two groups. iNOS mRNA was not detectable in the ischemic hepatic lobes of wild-type mice until 3 h of reperfusion; however, perfusion studies identified a significant delay in reperfusion of the ischemic lobe in the iNOS KO mice at the 1-h time point with similar perfusion rates at 3 and 6 h compared with wild type. By way of comparison, mice deficient in the endothelial NOS (eNOS) were also assessed for the degree of hepatic damage 3 h post-reperfusion. Plasma transaminase levels were significantly increased in eNOS KO animals compared with wild-type controls. These data suggest that systemic as well as local sources of iNOS regulate reperfusion, and local iNOS contributes to hepatic injury, while eNOS is protective in warm hepatic ischemia-reperfusion.

Minimum requirements for inhibition of smooth-muscle myosin light-chain kinase by synthetic peptides.

Although the amino acid residues that are important for peptide substrates of myosin light-chain kinase have been reported, those that are important for peptide inhibitors of this enzyme have not previously been investigated. Synthetic peptides based on the sequence Lys11-Lys12-Arg13-Ala-Ala-Arg16-Ala-Thr-Ser19 -Asn-Val21-Phe22-Ala of the chicken gizzard myosin light chain were tested as inhibitors of pig carotid-artery myosin light-chain kinase. The basic amino acid residues of the known myosin light-chain kinase inhibitor Lys-Lys-Arg-Ala-Ala-Arg-Ala-Thr-Ser-NH2 (IC50 = 14 microM) [Pearson, Misconi & Kemp (1986) J. Biol. Chem. 261, 25-27] were shown to be the important residues that contribute to inhibitor potency, as evidence by the finding that the hexapeptide Lys-Lys-Arg-Ala-Ala-Arg-NH2 had an IC50 value of 22 microM. This indicates that binding of the phosphorylatable serine residue to myosin light-chain kinase, which is of obvious importance for a substrate, does not enhance the potency of an inhibitor. With the aim of preparing more potent inhibitors, peptides Lys-Lys-Arg-Ala-Ala-Arg-Ala-Ala-Xaa-NH2 were prepared with a variety of amino acids substituted for the phosphorylatable serine residue. None of these peptides was a more potent inhibitor than the serine peptide.

Venous smooth muscle contains vasoconstrictor ETB-like receptors.

Two endothelin (ET) receptor subtypes have been identified to date: the ETA receptor which preferentially binds ET-1 over ET-3, and the ETB receptor which is non-selective. This study characterized the ET receptor subtypes present in several vascular smooth muscle preparations using standard in vitro techniques. In all but one of the arteries tested, ET-3 was significantly less potent than ET-1. In contrast, the potency of ET-3 was very similar to that of ET-1 in all of the veins. The selective ETA receptor antagonist BQ-123 blunted the ET-1 contractions in rabbit carotid artery, but not in saphenous vein. The selective ETB receptor ligand sarafotoxin S6c contracted the rabbit saphenous vein, but not the carotid artery. These data suggest that vascular smooth muscle cells express ETA and ETB receptors. Stimulation of either receptor subtype can result in force development.

Hydroxyamino acid specificity of smooth muscle myosin light chain kinase.

Synthetic peptides corresponding to the phosphorylation site in the myosin regulatory light chain from smooth muscle, Lys-Lys-Arg-Ala-Arg-Ala-Thr-Ser-Asn-Val-Phe-Ala ([Ala14,15]MLC(11-23] and containing a variety of hydroxyamino acid analogs at position 19, were tested as substrates for the smooth muscle myosin light chain kinase. Peptide analogs containing either D-serine or cis-hydroxyproline were not phosphorylated. The corresponding trans-hydroxyproline containing peptide was poorly phosphorylated with a Km of 2.3 microM and a Vmax of 3 X 10(-3) mumol.min-1.mg-1 compared to a Km of 12.5 microM and a Vmax of 1.43 mumol.min-1.mg-1 for the parent peptide. All three hydroxyamino acid analog peptides acted as relatively potent inhibitors of myosin light chain phosphorylation with Ki values in the range 7.5-10 microM, comparable to 7 microM for the parent peptide. Thus the failure of the hydroxyamino acid analog peptides to act as effective substrates was not the result of poor binding to the enzyme. In contrast, the same substitutions made in the peptide substrate for the cAMP-dependent protein kinase resulted in poor inhibitors. It is likely that the hydroxyl group of the substituting amino acids in the myosin light chain peptide analogs is not presented in the correct orientation in the active site for transfer of the phosphate group.

Control of peptide disulfide regioisomer formation by mixed cysteine-penicillamine bridges. Application to endothelin-1.

While incorporation of penicillamine residues (Pen; beta,beta-dimethyl cysteine) into a peptide can cause dramatic changes in biological activity, the tendency of Pen to form mixed disulfides should also allow the exploitation of the steric bulk of the beta-methyls as a synthetic device to control the production of disulfide isomers. That is, oxidation of a peptide containing an equal number of Cys and Pen residues should predominantly form products which contain mixed Cys-Pen disulfides. Endothelin (ET) is a 21 amino acid peptide which contains Cys at positions 1, 3, 11 and 15. While oxidation of ET tetrathiol has been reported to produce a 3:1 ratio of the natural 1-15, 3-11 to the unnatural 1-11, 3-15 isomers, we show that oxidation of ET analogs containing two cysteines and two penicillamines predominantly formed products containing Cys-Pen disulfides. Random oxidation (air, aqueous NH4OH) of the tetrathiols of [Pen1,11, Nle7]-ET-1 or [Pen3,15, Nle7]-ET-1 produced the correct 1-15, 3-11 isomer in > 12:1 and > 22:1 ratios, respectively. Oxidation of the tetrathiol of [Pen1,15, Nle7]-ET-1 favored the unnatural 1-11, 3-15 isomer by a 4:1 ratio, indicating that a normally contrathermodynamic disulfide isomer can become the favored product as a result of the driving force for penicillamine mixed disulfide formation. Disulfide isomers were identified using ion-spray mass spectrometry in conjunction with enzymatic and acid hydrolysis. [Pen1,11, Nle7]-ET-1 was a partial agonist at the ETA receptor (EC50 = 7.5 nM in rabbit carotid artery rings; Kd = 4.5 nM in rat A10 cell membranes) while [Pen3,15, Nle7]-ET-1 (EC50 = 0.9 nM; Kd = 0.7 nM) was a full agonist with similar potency to ET-1.

The receptor binding affinity of monocyclic [Ala3,Xaa11]endothelin-1 analogs correlates with inducible helix length.

Endothelin-1, a bicyclic 21-amino acid peptide with disulfide bridges between cysteines 1 and 15 as well as between cysteines 3 and 11, has been reported to be partially helical based on both CD and NMR data. However, this remains an area of controversy with some claims that CD data indicate no alpha-helical structure (Calas, B.; Harricane, M.-C.; Gulmard, L.; Heitz, F.; Mendre, C.; Chabrier, P.E.; Bennes, R. Peptide Res. 1992, 5, 97) and a recent X-ray crystal structure placing the helix at a different locus (Janes, R.W.; Peapus, D.H.; Wallace, B.A. Structural Biology 1994, 1, 311). The CD studies reported herein indicate that the helical structures reported in NMR studies (e.g. Andersen, N.H.; Chen, C.; Marschner, T.M.; Krystek, Jr. S.R.; Bassolino, D.A. Biochemistry 1992, 31, 1280) apply to pure aqueous media as well. The helix located from Lys9 to the Cys15/His16 juncture is ca 75% populated in pH 4 aqueous buffer. Titration difference CDs reveal that the helix extent increases by one to two residues and that the 'helical conformation' is more completely populated upon addition of TFE to 50+ volume-%. Comparison with a more helical analog suggests that the helix propagates towards (but not to the end of) the C-terminus upon fluoroalcohol addition. A variety of monocyclic derivatives of [Nle7] ET-1 lacking the 3,11-disulfide were evaluated for biological activity and examined by TFE titration difference CD. The series included an Aib11 and a Pro11 analog. The helix promoting Aib analog was the most active while the Pro analog exhibited significantly lower vasoconstrictor activity and binding affinity for the ETA receptor. All of the monocyclic analogs became significantly more helical upon addition of fluoroalcohols. The inclusion of a proline residue at position 11 does not preclude helix formation upon addition of fluoroalcohols. Rather, helix formation is relatively easily induced but limited to a 5 residue span. Apparently this is insufficient to orient required side chains optimally for interaction with the ETA receptor. For the 1,15-monocyclic analogs differing only at position 11, ETA binding affinity and vasoconstrictor potency correlate with the facility which a 7-8 residue long helix can be induced. This presumably includes the segment Glu10-->Cys15 in all cases and may represent the full sequence from Lys9-->His16. CD studies also reveal that the C-terminal fragment of endothelins is not a fully disordered 'random coil' either alone or attached to the endothelin core.

Structure-activity studies of endothelin leading to novel peptide ETA antagonists.

With the goal of producing receptor antagonists, numerous monocyclic and bicyclic endothelin analogs were prepared and tested for vasoconstrictor activity, receptor affinity and functional antagonist activity. Bis-penicillamine endothelin analogs containing Ala or Asn at position 18 were functional antagonists, with Ki values of 20-40 nM but KB values of about 1 microM (e.g., [Pen1,11, Nle7, Ala18]-endothelin-1, Ki = 42 nM, KB = 1.2 microM). While these peptides are antagonists at the ETA receptor, they appear to be at least partial agonists at another receptor subtype.

1-Benzazepin-2-one calcium channel blockers--VI. Receptor-binding model and possible relationship to desmethoxyverapamil.

We have prepared a series of potent antihypertensive 1-benzazepin-2-one calcium channel blockers (CCBs) 1 that are structurally related to diltiazem 2. Structural studies and the preparation of conformationally constrained analogs of 1-benzazepin-2-ones have led us to postulate a receptor-bound conformation for both 1 and 2. We believe that these compounds bind to the calcium channel protein in an MI ("inboard") binding conformation in which the amine of the side chain is placed over the heptagonal benzazepione ring and in close proximity to the phenyl methyl ether pharmacophore. This receptor-bound conformation places the side chain amine and methyl ether pharmacophores in the same spatial relationship as 3-methoxyphenylethalamine. Combined with our SAR, this binding model rationalizes literature findings that desmethoxyverapamil can demonstrate pharmacology typical of both phenylalkylamine (PA) and benzothiazepinone (DTZ) calcium channel blockers. Simple experiments are proposed to test the hypothesis that desmethoxyverapamil can bind at the benzothiazepinone site on the calcium channel.