Synthesis and LTD4 antagonist activity of 2-norleukotriene analogues.

A series of structural analogues of 4(R)-hydroxy-5(S)-cysteinylglycyl-6(Z)-nonadecenoic acid [4R,5S,6Z)-2-nor-LTD1 (10b), SK&F 101132) has been synthesized and pharmacologically characterized. (4R,5S,6Z)-2-nor-LTD1 significantly antagonized LTD4-induced contractile responses on isolated guinea pig trachea. The cis double-bond geometry appears to be critical for antagonist activity, whereas the trans isomer 17 exhibited weak contractile activity. Replacement of the cysteinylglycyl moiety with cysteine afforded 20, which retained significant antagonist activity, while lengthening or shortening the lipid tail by five methylene groups resulted in complete loss of activity. The eicosanoid amide 15, glycinamide 14, and C-1 carbinol 18 analogues all possessed antagonist activity, whereas the diol derivative 19 exhibited increased intrinsic agonist activity.

Orally active and potent inhibitors of gamma-aminobutyric acid uptake.

3-Pyrrolidineacetic acid (1a), certain piperidinecarboxylic acids--i.e., 3-piperidinecarboxylic acid (2a), 1,2,5,6-tetrahydro-3-pyridinecarboxylic acid (3a), and cis-4-hydroxy-3-piperidinecarboxylic acid (4a)--cis-3-aminocyclohexanecarboxylic acid (5a, cis-3-ACHC), and gamma-aminobutyric acid (6a, GABA) itself are among the most potent inhibitors of [3H]GABA uptake by neurons and glia in vitro. These hydrophilic amino acids, however, do not readily enter the central nervous system in pharmacologically significant amounts following peripheral administration. We now report that N-(4,4-diphenyl-3-butenyl)-3-piperidinecarboxylic acid (2b) is a specific GABA-uptake inhibitor that is more potent, more lipophilic and, in limited testing, as selective as 2a. Similar results were obtained with the N-(4,4-diphenyl-3-butenyl) derivatives of 1a, 3a, and 4a. By contrast, N-(4,4-diphenyl-3-butenyl) derivatives of 5a and 6a were not more potent than the parent amino acids and appear to inhibit GABA uptake, at least in part, by a nonselective mechanism of action. The N-(4,4-diphenyl-3-butenyl)amino acids 1b-4b exhibit anticonvulsant activity in rodents following oral or intraperitoneal administration [Yunger, L.M.; et al. J. Pharmacol. Exp. Ther. 1984, 228, 109].

Conformationally constrained peptides and semipeptides derived from RGD as potent inhibitors of the platelet fibrinogen receptor and platelet aggregation.

Structure-activity studies have been pursued on cyclo-S,S-[Ac-Cys-(N alpha-Me)Arg-Gly-Asp-Pen]-NH2, 2 (SK&F 106760), a potent inhibitor of platelet aggregation, in an effort to improve potency and affinity for the GPIIb/IIIa receptor. Modifications on the N- and C-termini of 2 produced a series of peptides which indicate that the C-terminal carboxylate group may be a secondary receptor-binding element. Further modification by replacing the disulfide tether N alpha-acetylcysteine/penicillamineamide with the novel, inexpensive, achiral, constrained, and more lipophilic tether 2-mercaptobenzoyl/2-mercaptoaniline (Mba/Man) afforded the semipeptide cyclo-S,S-[Mba-(N alpha-Me)Arg-Gly-Asp-Man], 18 (SK&F 107260), which exhibited significant enhancement in both affinity and potency. To further investigate the effect of the phenyl ring at the C-terminus, peptides bearing the novel (2R,3S)- and (2R,3R)-beta-phenylcysteines were synthesized, which culminated in the cyclo-S,S-[Ac-Cys-(N alpha-Me)Arg-Gly-Asp-(2R,3S)-beta-phenylCys]-OH peptide, 22, which displayed substantial affinity and potency. We describe, herein, the development of both 18 and 22 and the additional structural modifications within the constrained cyclic disulfide ring to probe the stereochemical and steric requirements for receptor interaction.

Conformational preferences in a benzodiazepine series of potent nonpeptide fibrinogen receptor antagonists.

Previously, we reported the direct design of highly potent nonpeptide 3-oxo-1,4-benzodiazepine fibrinogen receptor antagonists from a constrained, RGD-containing cyclic semipeptide. The critical features incorporated into the design of these nonpeptides were the exocyclic amide at the 8-position which overlaid the Arg carbonyl, the phenyl ring which maintained an extended Gly conformation, and the diazepine ring which mimicked the gamma-turn at Asp. In this paper, we investigate conformational preferences of the 8-substituted benzodiazepine analogues by examining structural modifications to both the exocyclic amide and the seven-membered diazepine ring and by studying the conformation of the benzodiazepine ring using molecular modeling, X-ray crystallography, and NMR. We found that the directionality of the amide at the 8-position had little effect on activity and the (E)-olefin analogue retained significant potency, indicating that the trans orientation of the amide, and not the carbonyl or NH groups, made the largest contribution to the observed activity. For the diazepine ring, with the exception of the closely analogous 3-oxo-2-benzazepine ring system described previously, all of the modifications led to a significant reduction in activity compared to the potent 3-oxo-1, 4-benzodiazepine parent ring system, implicating this particular type of ring system as a desirable structural feature for high potency. Energy minimizations of a number of the modified analogues revealed that none could adopt the same low-energy conformation as the one shared by the active (S)-isomer of the 3-oxo-1, 4-benzodiazepines and 3-oxo-2-benzazepines. The overall data suggest that the features contributing to the observed high potency in this series are the orientation of the 3-4 amide and the conformational constraint imposed by the seven-membered ring, both of which position the key acidic and basic groups in the proper spatial relationship.

Aminoglycosides. V. Synthesis of glucopyranoside derivatives of neamine modified in the 2-deoxystreptamine ring.

trans-4-Aminocyclohexanol-2'-amino-alpha-D-glucopyranosides were prepared which are derivatives of neamine having the 3-amino and 5 and 6 hydroxyl groups of the 2-deoxystreptamine ring replaced with hydrogen. The 2'-amino-alpha-glycosides were synthesized by the method of LEMIEUX using a chloro nitroso dimer of a glucal and appropriately substituted cyclohexanols. Reductive deblocking of the intermediate 2-oximino derivatives afforded paromamine and neamine analogues. Two examples of 2'-amino-alpha-glycosides with ring-opened variations of the 2-deoxystreptamine aglycone are described. None of the compounds exhibited better in vitro antibacterial activity than neamine when compared against Gram-positive and Gram-negative bacteria.

Calpain immunoreactivity in baker's yeast, lobster and wheat germ.

The immunoreactivities of mu-calpain and m-calpain in wheat germ, lobster tail meat, and three strains of yeast were analysed by Western blotting using mouse anti-mu-calpain and rabbit anti-m-calpain. The occurrence of multiple bands may be due to either autolyses or the interactions between the calpains and other molecules. The results suggest not only a ubiquitous distribution and a universal regulatory role of calpain in eukaryotes, but also an evolutional conservation of calpain.

Immunoreactivity of S-100 protein in baker's yeast, lobster, and wheat germ.

The immunoreactivity of S-100 proteins in three strains of yeast and wheat germ was observed by analysis of Western blotting with rabbit anti-bovine S-100. A high level of activity was exhibited in wheat germ, whereas a very low level was found in lobster tail meat. The occurrence of multiple bands may be due to the interactions between S-100A and S-100B and/or other molecules. The highly evolutionally conserved S-100 may play an important role in cellular signal transduction and cell growth in yeast and wheat germ.

Protein kinase C alpha, beta immunoreactivity in baker's yeast, lobster and wheat germ.

The immunoreactivity of PKC alpha (protein kinase C alpha) and PKC beta in wheat germ, lobster tail muscle and three strains of yeast was analysed by Western blotting with mouse anti-PKC active fragments. The potency of the immunoreactivity of PKC alpha activity was much greater than that of PKC beta. The occurrence of multiple bands may be due to PKC self-interactions and/or the interactions between PKC and other molecules. The evolutionary conservation of PKC alpha and PKC beta implies that these PKC isoenzymes may play important roles in Ca2+/lipid-dependent signal transduction and cell growth in these eukaryotes.

Immunoreactivity of PKC gammalambda and RACK1 in baker's yeast, lobster and wheat germ.

Varied patterns of immunoreactive bands of protein kinase C gamma (PKC gamma) and receptor for activated C-kinase-1 (RACK1) were detected by analysis of Western blots in crude extracts of wheat germ, lobster tail meat, and three strains of baker's yeast. Anti-PKC lambda also reacted with wheat germ and yeast extracts, but failed to react with the lobster extract. The findings may implicate a regulatory role and an evolutionary conservation of these PKC isoenzymes and their receptor proteins in eukaryotes.

Immunoreactivities of PKC delta, eta, zeta in baker's yeast, lobster and wheat germ.

Varied immunoreactive bands of protein kinase C delta (PKC delta), PKC eta, and PKC zeta were detected in crude extracts of wheat germ, lobster tail meat, and three strains of baker's yeast by analysis of Western blots. Protease-deficient and Fleischmann's Active Dry yeasts exhibited immunoreactivity of PKC delta, whereas wheat germ and Fleischmann's RapidRise yeast displayed immunoreactivities of both PKC delta and PKC zeta. Lobster tail meat showed immunoreactivities of PKC eta and PKC zeta. These positive and negative immunoreactivities reflected evolutionary conservation and divergence, respectively, of these PKC isozymes in eukaryotes.

A Korean population study of the nine STR loci FGA, VWA, D3S1358, D18S51, D21S11, D8S1179, D7S820, D13S317 and D5S818.

DNA typing was performed on 379 randomly selected unrelated Koreans using the nine short tandem repeat loci FGA, VWA, D3S1358, D18S51, D21S11, D8S1179, D7S820, D13S317 and D5S818 present in the AmpF/STR Profiler Plus PCR amplification kit. Allele frequencies, heterozygosity, power of discrimination, mean exclusion chance, and polymorphism information content of each locus were calculated by statistical analysis. All nine loci were in Hardy-Weinberg equilibrium. The combined discrimination index and the combined mean exclusion chance in Koreans was 2.31 x 10(-12) and 0.99983, respectively. By evaluation of 297 children from 128 families, 2 mutations were found at the FGA locus and 1 each at the D18S51 and D13S317 loci. This study demonstrates that this multiplex system is a useful and convenient tool for forensic identification and parentage testing in Korea.

2-nor-leukotriene analogs: antagonists of the airway and vascular smooth muscle effects of leukotriene C4, D4 and E4.

A structural analog of LTD4, 4R-hydroxy-5S-cysteinylglycyl-6Z-nonadecenoic acid (4R, 5S, 6Z-2-nor-LTD1) has been synthesized and pharmacologically characterized. It significantly antagonized the contractile action of LTD4, LTC4 and LTE4 in guinea pig airways. In addition, this compound antagonized the in vitro vasoconstrictive effects of LTD4 in the guinea pig pulmonary artery. The study of a series of structural analogs of 4R, 5S, 6Z-2-nor-LTD1 suggests that the spatial separation of the C-1 (eicosanoid) carboxyl relative to the hydroxyl is a critical determinant in LTD4 agonist/antagonist activity.

Antagonism of the pulmonary effects of the peptidoleukotrienes by a leukotriene D4 analog.

4R-hydroxy-5S-cysteinylglycine-6-Z-nonadecenoic acid (4R,5S, 6Z-2-nor-LTD1), a structural analog of leukotriene (LT) D4 (LTD4), significantly antagonized the pulmonary actions of LTD4 in several guinea pig models of LT-mediated bronchoconstriction and edema formation. In vitro, 4R,5S,6Z-2-nor-LTD1 (10(-5) and 10(-4) M) antagonized the LTD4-induced contraction of tracheal spirals and lung parenchymal strips. This antagonist action of 4R,5S,6Z-2-nor-LTD1 was specific for the LTs, in that LTC4- and LTE4-induced contractions of the trachea were also antagonized, whereas the contractions elicited by other spasmogens, e.g., histamine, carbachol, prostaglandin F2 alpha and KCl, were not antagonized. In vivo, the LTD4-induced bronchoconstriction in anesthetized, spontaneously breathing guinea pigs as reflected by decreases in dynamic lung compliance and airway conductance were attenuated significantly by a 1-min pretreatment with 4R,5S,6Z-2-nor-LTD1 at 5 mg/kg i.v. Similarly, the LTD4-induced increase in tracheal microvascular permeability, as assessed by extravasation of [125I]bovine serum albumin, was blocked by pretreatment with 4R,5S,6Z-2-nor-LTD1. These results provide the first demonstration that a structural analog of the peptidoleukotrienes can pharmacologically antagonize the potent actions of these LTs.

Binding of [3H]-SK&F 107260 and [3H]-SB 214857 to purified integrin alphaIIbbeta3: evidence for a common binding site for cyclic arginyl-glycinyl-aspartic acid peptides and nonpeptides.

The aggregation of activated platelets is mediated by the binding of fibrinogen to its cell surface receptor, the integrin alphaIIbbeta3. The recognition of fibrinogen by alphaIIbbeta3 depends, in part, on the tripeptide sequence Arg-Gly-Asp (RGD) in the adhesive protein. The interactions of a cyclic RGD-containing pentapeptide, [3H]-SK&F-107260, and a 1,4-benzodiazepine-based nonpeptide [3H]-SB-214857, with purified alphaIIbbeta3 have been investigated. Both compounds potently inhibit platelet aggregation at submicromolar concentrations. Binding of both [3H]-SK&F-107260 (Kd = 1.19 nM) and [3H]-SB-214857 (Kd = 1.85 nM) to alphaIIbbeta3 is of high affinity and fully reversible. The binding is monophasic, indicating a single class of noncooperative binding sites. The two radioligands exhibited similar values in binding to alphaIIbbeta3 purified on an RGD-affinity column (Bmax = 0.2 mol/mol alphaIIbbeta3) or to alphaIIbbeta3 purified over a lentil lectin column (Bmax = 0.03 mol/mol alphaIIbbeta3), suggesting that SK&F-107260 and SB-214857 interact with the same population of receptors. Binding of [3H]-SK&F-107260 and [3H]-SB-214857 to alphaIIbbeta3 require divalent cations, Mg++, Ca++ and Mn++ are able to support binding, with Mn++ being the most effective. Thirteen alphaIIbbeta3 antagonists, including four linear and three cyclic RGD peptides, five peptidomimetics, the fibrinogen gamma-chain dodecapeptide (HHLGGAKQAGDV) and the snake venom protein, echistatin, complete for [3H]-SK&F-107260 or [3H]-SB-214857 binding to alphaIIbbeta3. The affinity constants (Ki) of these compounds, determined by the two radioligand binding assays, are similar. Furthermore, these compounds exhibit the same rank order of potency in inhibiting biotinylated-fibrinogen binding to alphaIIbbeta3. Scatchard plot analyses of the [3H]-SK&F-107260 binding isotherms in the presence of unlabeled SB-214857 and gamma-chain dodecapeptide reveal competitive-type antagonism, indicating that SB-214857, gamma-chain dodecapeptide and SK&F-107260 interact with mutually exclusive binding sites on alphaIIbbeta3.

Synthesis and LTD4-antagonist activity of desamino-2-nor-leukotriene analogs.

A series of desamino-2-nor-leukotriene analogs has been prepared by the reaction of various thiols with several methyl trans-4,5-epoxy-6Z-alkenoates, followed by deprotection. The products were assessed for their ability to antagonize the LTD4-induced contraction of the isolated guinea pig trachea. Several compounds displayed potent leukotriene antagonist activity, i.e., KB values in the sub-micromolar range, while only minimally affecting basal airway tone. The most potent analog, 4-hydroxy-5-(2-carboxyethylthio)-6Z-nonadecenoic acid, antagonized both LTD4- and LTE4-induced contractions of the trachea in an apparently competitive fashion. These agents possess increased potency relative to SK&F 101132, the first leukotriene analog identified as having LT-antagonist activity. Thus, these results demonstrate that deletion of the peptide amino group can produce leukotriene analogs which have minimal intrinsic contractile activity on the isolated guinea pig trachea, yet possess potent leukotriene-antagonistic effects.

Preclinical pharmacokinetics and interspecies scaling of a novel vitronectin receptor antagonist.

Allometric scaling may be used in drug development to predict the pharmacokinetics of xenobiotics in humans from animal data. Although allometry may be successful for compounds that are excreted unchanged or that are oxidatively metabolized (with corrections for metabolic capacity), it has been more challenging for compounds excreted primarily as conjugates in bile. (S)-10, 11-Dihydro-3-[3-(pyridin-2-ylamino)-1-propyloxy]-5H-dibenzo[ a, d]cycloheptene-10-acetic acid (SB-265123) is a novel alphavbeta3 ("vitronectin receptor") antagonist. In this study, the in vivo pharmacokinetics and in vitro plasma protein binding of SB-265123 were examined in four species: mice, rats, dogs, and monkeys. In monkeys and dogs, SB-265123 exhibited moderate clearance, whereas low clearance (<20% hepatic blood flow) was observed in the rat, and high clearance (>70% hepatic blood flow) was seen in the mouse. The concentration-time profiles indicated the possibility of enterohepatic recirculation; subsequent studies in bile duct-cannulated rats demonstrated extensive biliary excretion of an acyl-glucuronide of SB-265123. In allometric scaling to predict the disposition of SB-265123 in humans, various standard correction factors were applied, including protein binding, maximum lifespan potential, and brain weight; each failed to produce adequate interspecies scaling of clearance (r(2) < 0.72). Consequently, a novel correction factor incorporating bile flow and microsomal UDP-glucuronosyltransferase activity in each species was applied, demonstrating substantial improvement in the correlation of the allometric plot (r(2) = 0.96). This study demonstrates a novel allometric correction that may be applicable to compounds that undergo conjugation and biliary excretion.