Amide Analogues Research Articles

Diacylglycerol metabolism in neonatal rat liver: characterization of cytosolic diacylglycerol lipase activity and its activation by monoalkylglycerols

Diacylglycerol lipase (glycerol ester hydrolase, EC 3.1.1.3) activities were investigated in subcellular fractions from neonatal and adult rat liver in order to determine whether one or more different lipases might provide the substrate for the developmentally expressed, activity monoacylglycerol acyltransferase. The assay for diacylglycerol lipase examined the hydrolysis of sn-1- stearoyl,2-[ 14C] oleoylglycerol to labeled monoacylglycerol and fatty acid. Highest specific activities were found in lysosomes (pH 4.8) and cytosol and microsomes (pH 8). The specific activity from plasma membrane from adult liver was 5.8-fold higher than the corresponding activity in the neonate. In other fractions, however, no developmental differences were observed in activity or distribution. In both lysosomes and cytosol, 75 to 90% of the labeled product was monoacylglycerol, suggesting that these fractions contained relatively little monoacylglycerol lipase activity. In contrast, 80% of the labeled product from microsomes was fatty acid, suggesting the presence of monoacylglycerol lipase in this fraction. Analysis of the reaction products strongly suggested that the lysosomal and cytosolic diacylglycerol lipase activities hydrolyzed the acyl-group at the sn-1 position. The effects of serum and NaCl on diacylglycerol lipase from each of the subcellular fractions differed from those effects routinely observed on lipoprotein lipase and hepatic lipase, suggesting that the hepatic diacylglycerol lipase activities were not second functions of these triacylglycerol lipases. Cytosolic diacylglycerol lipase activity from neonatal liver and adult liver was characterized. The apparent K m for 1-stearoyl,2-oleoylglycerol was 115 μM. There was no preference for a diacylglycerol with arachidonate in the sn-2 position. Bovine serum albumin stimulated the activity, whereas dithiothreitol, N-ethylmaleinide, and ATP inhibited the activity. Both sn-1(3)- and 2-monooleylglycerol ethers stimulated cytosolic diacylglycerol lipase activity 2–3-fold. The corresponding amide analogs stimulated 28 to 85%, monooleoylglycerol itself had little effect, and 1-alkyl- or 1-acyl-lysophosphatidylcholine inhibited the activity. These data provide the first characterization of hepatic subcellular lipase activities from neonatal and adult rat liver and suggest that independent diacylglycerol and monoacylglycerol lipase activities are present in microsomal membranes and that the microsomal and cytosolic diacylglycerol lipase activities may describe an ambipathic enzyme. The data also suggest possible cellular regulation by monoalkylglycerols.

Chemical Structure-Activity Relationships: Peroxisome Proliferation and Lipid Regulation in Rats

Studies described here address structure-activity relationships of novel hypolipidemic agents that induce peroxisome proliferation. Male rats were given equivalent doses of three well-studied fibrates, fibrate amides, and structurally dissimilar agents. Aryloxyalkanoic acids, amide analogs, and thio, benzimidazole, phenylpiperazine, and oxazole derivatives induced peroxisome proliferation and decreased plasma cholesterol and triglyceride levels. These compounds contain an acidic function or appear to be readily metabolized to a derivative with an acidic function. Substitution of this substituent with an adamantyloxy eliminated peroxisome proliferation and induced contrasting effects on the lipid profile, substantially increasing triglycerides. A direct correlation was established between hepatocellular peroxisome proliferation and plasma high-density lipoprotein (HDD-cholesterol levels.

Amino acids and peptides. XVI. Synthesis of N-terminal tetrapeptide analogs of fibrin alpha-chain and their inhibitory effects on fibrinogen/thrombin clotting.

N-Terminal tetrapeptide analogs of fibrin alpha-chain were synthesized by the solution method using a new active ester, the ester of the oxime of p-nitroacetophenone, and by the solid-phase method. Their inhibitory effects on fibrinogen/thrombin clotting were examined. Of the synthetic peptides, amide analogs of Gly-Pro-Arg-Pro exhibited a more potent inhibitory effect.

Discovery and preliminary pharmacology of Sch 37370, a dual antagonist of PAF and histamine

From a series of amide analogs of the histamine H1 antagonist, azatadine, a potent, orally active, dual platelet-activating factor (PAF) and histamine antagonist, Sch 37370, namely 1-acetyl-4-(8-chloro-5,6-dihydro-11H-benzo- [5,6]cyclohepta[1,2-b]pyridin-11-ylidine)piperidine, was discovered. Sch 37370 selectively inhibits PAF-induced aggregation of human platelets in vitro (IC50 = 0.6 microM), and in vivo inhibits PAF- and histamine-induced bronchospasm in guinea pigs with ED50 values of 6.0 and 2.4 mg/kg p.o., respectively. Sch 37370 is expected to be more efficacious than single mediator antagonists in allergic diseases, such as asthma.

Parathyroid Hormone (1-34) and Nleu8,18Tyr34-Parathyroid Hormone, (3-34) Amide Increase Diacylglycerol in Neonatal Mouse Calvaria

Previously, we and others have presented evidence that a calcium second messenger system is involved in the action of parathyroid hormone (PTH) on bone. In the present report, the effects of PTH(1-34) and PTH(3-34)amide treatment on diacylglycerol (DG) in neonatal mouse calvaria are described. PTH(1-34) produced a rapid (within 5 minutes) increase in calvarial incorporation of 3H-arachidonic acid into DG. The effect was maximal at 0.1 nMPTH(1-34), the lowest concentration tested. The 3-34 amide analogue of PTH increased DG to the same extent as PTH(1-34). The effect was maximal at 10 nM PTH(3-34)amide, the lowest concentration tested. These concentrations were lower than those required to elicit maximal effects on bone resorption. In contrast to effects on cyclic AMP, where the 3-34 amide inhibited the increase elicited by PTH, combined treatment of calvaria with PTH(1-34) and PTH(3-34)amide did not inhibit effects on resorption or diacylglycerol.

Structure-Activity Relationship Studies with Mosquito Repellent Amides

A series of amide analogues of N, N-diethyl-m-toluamide (DEET) and N, N-diethylphenylacetamide (DEPA) were synthesized to study their mosquito repellency in relation to chemical structure and physical properties. None of these parameters (vapor pressure, lipophilicity, and molecular length) could be related to protection time. However, when the protection time of all five amides derived from the same carboxylic acid was expressed in terms of all three parameters together, excellent correlation was obtained.

Critical role of a hydrogen bond in the interaction of phospholipase A2 with transition-state and substrate analogues.

The inhibition of phospholipase A2 by an amide substrate analogue, 1-hexadecylthio-2-hexadecanoyl-amino-1,2-dideoxy-sn-glycero-3-phos phocholine, and a phosphonate transition-state analogue, 1-hexadecylthio-1-deoxy-2-hexadecylphosphono-sn-glycero-3-ph osphocholine, is dramatically influenced by pH. However, these two inhibitors show opposite pH dependencies. The amide analogue acts more potently under basic conditions, whereas the phosphonate acts more potently under acidic conditions. In both cases, ligand binding is perturbed by protonation of an enzyme functional group with an apparent pKa of 6.1, which corresponds to that of a histidine residue. Thus, His-48, which has previously been implicated in catalysis, appears to be critically involved in the hydrogen bond interactions between the enzyme and these two inhibitors. The amide analogue binds most effectively to the enzyme when His-48 is deprotonated. Upon protonation of the histidine residue, the amide cannot form a critical hydrogen bond and loses its ability to interact effectively with the enzyme. In contrast, the phosphonate analogue binds much tighter to the protonated form of the enzyme than to the deprotonated form. The phosphonate analogue needs a bridging hydrogen between the oxygen on its phosphorus atom and the N delta 1 of His-48 to form a strong hydrogen bond. At optimal pH values for inhibitor binding, both the amide and the phosphonate analogues are potent competitive inhibitors of cobra (Naja naja naja) venom phospholipase A2. The IC50 for the amide was 4.4 x 10(-4) mol fraction and for the phosphonate was 1.6 x 10(-5) mol fraction. Under the experimental conditions used, this corresponds to a bulk concentration of 2 microM and 70 nM, respectively.

Active-site-directed specific competitive inhibitors of phospholipase A2: novel transition-state analogs

More than 100 amphiphilic phosphoesters, possible tetrahedral transition-state analogues capable of coordinating to the calcium ion at the active site of phospholipase A2, were designed, synthesized, and tested as inhibitors for the hydrolysis of 1,2-dimyristoyl-sn-glycero-3-phosphomethanol vesicles in the scooting mode. This assay system permits the study of structurally diverse inhibitors with phospholipase A2S from different sources, and it is not perturbed by factors that change the quality of the interface. As a prototype, 1-hexadecyl-3-trifluoroethylglycero-2-phosphomethanol (MJ33) was investigated in detail. Only the (S)-(+) analogue of MJ33 is inhibitory, and it is as effective as the sn-2 phosphonate or the sn-2 amide analogues of sn-3 phospholipids. The inhibitory potencies of the various phosphoesters depended strongly on the stereochemical and structural features, and the mole fractions of inhibitors required for 50% inhibition, X1(50), ranged from more than 1 to less than 0.001 mole fraction. The affinity of certain inhibitors for enzymes from different sources differed by more than 200-fold. The inhibitors protected the catalytic site residue His-48 from alkylation in the presence of calcium but not barium as expected if the formation of the EI complex is supported only by calcium. The equilibrium dissociation constant for the inhibitor bound to the enzyme at the interface was correlated with the XI(50) values, which were different if the inhibition was monitored in the pseudo-zero-order or the first-order region of the progress curve. These results show that the inhibitors described here interfered only with the catalytic turnover by phospholipase A2's bound to the interface, their binding to the enzyme occurred through calcium, and the inhibitors did not have any effect on the dissociation of the enzyme bound to the interface.

Affinity labeling of the membrane protein-binding component of human polymorphonuclear leukocyte receptors for leukotriene B4.

A radiolabeled N-(3-aminopropyl)-leukotriene B4 amide ([3H]LTB4-APA) analog of the potent leukocyte chemotactic factor leukotriene B4 (LTB4) binds to receptors for LTB4 in plasma membrane-enriched preparations from human blood polymorphonuclear leukocytes (PMNL) and intact PMNL with respective mean dissociation constants of 2.3 nM and 69 nM at 4 degrees C. The [3H]LTB4-APA bound to plasma membrane-enriched preparations from PMNL was covalently cross-linked to membrane proteins with disuccinimidyl suberate. Solubilization and resolution by SDS-PAGE of proteins from [3H]LTB4-APA-labeled PMNL membranes revealed predominant labeling of a 60-kDa protein. Labeling of the PMNL membrane protein was inhibited by LTB4 and its analogs at concentrations similar to those inhibiting the binding of [3H]LTB4 to its receptor, with an identical rank order of potency of LTB4 greater than 20-hydroxy-LTB4 greater than LTB4-APA = 5(S),12(R)-dihydroxy-eicosa-14-cis-6,8,10-trans-tetraenoic acid much greater than LTD4 = LTC4. GTP suppressed the labeling of the 60-kDa PMNL membrane protein to an extent consistent with the decrease in receptor affinity for LTB4 induced by GTP. The stereospecificity of the affinity cross-linking reaction and the regulation by GTP support the identification of an approximately 60-kDa protein as the binding component of the PMNL receptor for LTB4.

Molecular Biology of Serotonin Receptors

Serotonin (S-hydroxytryptamine; SHT) is a biogenic amine that functions as both a neurotransmitter and a hormone in the mammalian central nervous system (CNS) and in the periphery. Within the brain, serotonergic neurons originate primarily in the raphe nuclei of the brainstem and project to most areas of the CNS, where they regulate a wide variety of sensory, motor, and cortical functions (Osborne 1982). In the periphery, serotonin is involved in such diverse functions as the regulation of enteric reflexes, the modulation of platelet shape change and aggregation, the modulation of vascular smooth muscle contraction, the initiation of activity in primary afferent nociceptors, and the regulation of lymphocyte cytotoxicity and phagocytosis (for review see Peroutka 1988, Richardson & Engel 1986). Pharmacological and physiological studies have shown that the multiple actions of serotonin are mediated by several distinct cell surface receptor subtypes, designated SHTl a, Ib, lc, Id, SHT2, SHT3, and SHT4 (Bradley et aI1986). For example, the hallucinatory actions of lysergic acid diethyl­ amide (LSD) and other psychotropic serotonin analogues are probably elicited by activation of cortical 5HT2 (Rasmussen & Aghajanian 1988) or 5HTIc receptors. In contrast, the pain that is produced from applying serotonin to a blister base results from activation of 5HT3 receptors on primary sensory nerve endings (Richardson & Engel 1986). Individual serotonin receptor subtypes exhibit characteristic ligand­ binding profiles and couple to different intracellular signaling systems.

ChemInform Abstract: Synthesis and X‐Ray Crystal Structures of Isonipecotinamide Derivatives as Reverse Amide Analogues of Fentanyl.

AbstractThe isonipecotinamide derivatives (II), (VIII) and (IX) (obtained similarly to (VIII)), which represent reverse amide derivatives of the potent analgetic fentanyl (III), are evaluated for analgetic activity.

The interaction of phospholipase A2 with phospholipid analogues and inhibitors.

A series of structurally modified phospholipids have been used to delineate the structural features involved in the interaction between cobra venom (Naja naja naja) phospholipase A2 and its substrate. Special emphasis has been placed on sn-2 amide analogues of the phospholipids. These studies have led to a very potent, reversible phospholipase A2 inhibitor. A six-step synthesis of this compound, 1-palmitylthio-2-palmitoylamino-1,2-dideoxy-sn-glycero-3- phosphorylethanolamine (thioether amide-PE), was developed. Other analogues studied included 1-palmitylthio-2-palmitoylamino-1,2-dideox-sn- glycero-3-phosphorylcholine, 1-palmityl-2-palmitoylamino-2- deoxy-sn-glycero-3-phosphorylcholine, 1-palmitoyl-2-palmitoylamino-2-deoxy-sn-glycero-3- phosphorylcholine, 1-palmitylthio- 2([(tetradecyloxy)carbonyl]amino)-1,2-dideoxy-sn-glycero-3- phosphorylcholine, 1-palmitoyl- 2([(octadecylylamino)carbonyl]amino)-2-deoxy-sn-glycero-3- phosphorylcholine, and sphingomyelin. Inhibition studies used the well defined Triton X-100 mixed micelle system and the spectroscopic thio assay. The phospholipid analogues showed varying degrees of inhibition. The best inhibitor was the thioether amide-PE which had an IC50 of 0.45 microM. In contrast, sphingomyelin, a natural phospholipid that resembles the amide analogues, did not inhibit but rather activated phosphatidylcholine hydrolysis. This systematic study of phospholipase A2 inhibition led to the following conclusions about phospholipid-phospholipase A2 interactions: (i) sn-2 amide analogues bind tighter than natural phospholipids, presumably because the amide forms a hydrogen bond with the water molecule in the enzyme active site, stabilizing its binding. (ii) Inhibitor analogues containing the ethanolamine polar head group appear to be more potent inhibitors than those containing the choline group. This difference in potency may be due solely to the fact that the cobra venom phospholipase A2 is activated by choline-containing phospholipids. Thus, choline-containing non-hydrolyzable analogues both inhibit and activate this enzyme. Both of these effects must be taken into account when studying phosphatidylcholine inhibitors of the cobra venom enzyme. (iii) The potency of inhibition of these analogues is significantly enhanced by increasing the hydrophobicity of the sn-1 functional group.(ABSTRACT TRUNCATED AT 400 WORDS)

Comparative studies on the antirhinovirus activity and the mode of action of the rhinovirus capsid binding agents, chalcone amides

Studies of various analogs related to the antirhinovirus agent 4′-ethoxy-2′-hydroxy-4,6′-dimethoxychalcone (Chalcone Ro 09-0410) led to the identification of amide analogs that are 4.5 to 10 times more active against human rhinovirus (HRV) in tissue culture as measured by chemotherapeutic indices. Chalcone amides Ro 09-0535, Ro 09-0696 and Ro 09-0881 inhibited viral replication at concentrations as low as <2–3 ng/ml and were cytotoxic between 30 to 50 μg/ml. These compounds bind to HRV and reduce the virus infectivity titers by 3 log 10 or greater at 0.5 μg/ml for 60 min similar to Ro 09-0410. These amide analogs competitively inhibited the binding of [ 3H]Ro 09-0410 to the viral capsid similar to capsid binding antirhinovirus agents, Ro 09-0410, 4′,6-dichloroflavan and WIN-51711. Furthermore, strains of HRV type 2 resistant to each of the above agents showed cross-resistance to all the other agents. These results indicate that the chalcone amides also bind to the same or close-proximity site for the capsid binding antirhinovirus agents, which is on the specific site within the viral capsid protein. However, differences in the degree of the inhibition of [ 3H]Ro 09-0410 binding, cross-resistance of strains of HRV resistant to the agents and HRV serotype specificity were observed not only between the chalcone amides and the other antivirus agents (Ro 09-0410, 4′,6-dichloroflavan and WIN-51711) but also among the chalcone amides, particularly between Ro 09-0535 and Ro 09-0696. These differences are presumably due to alterations in the binding affinities of compounds as a consequence of variations in the shape and size of the hydrophobic pocket that exists between serotypes including resistant strains.

Synthesis and x‐ray crystal structures of isonipecotinamide derivatives as reverse amide analogues of fentanyl

AbstractSeveral isonipecotinamide derivatives which represent reverse amide derivatives of the potent analgetic agent fentanyl were prepared and evaluated for analgetic activity. The synthetic approaches utilized and stereochemical assignments are discussed. The most potent compound, 3, displayed much weaker analgetic activity than fentanyl itself.

Desmethionine alkylamide bombesin analogs: a new class of bombesin receptor antagonists with potent antisecretory activity in pancreatic acini and antimitotic activity in Swiss 3T3 cells

Bombesin-related peptides have a large number of physiological functions as well as having an autocrine growth mechanism for the regulation of small cell lung cancer cells. In the present study we have synthesized 21 des-Met amide or alkylamide analogues of bombesin and compared their abilities to function as bombesin receptor antagonists in guinea pig pancreatic acini and Swiss 3T3 cells with those of the previously most potent antagonist described, [Leu13 psi(CH2NH)Leu14]bombesin (analogue I). All des-Met analogues functioned as antagonists. Bn(1-13)NH2 was approximately equipotent to I (Ki = 60-80 nM) whereas Bn(6-13)NH2 was 30-fold less potent (Ki = 1800 nM). Formation of an ethylamide, Bn(6-13)ethylamide, increased the potency 30-fold such that this octapeptide was equipotent to I. The addition of a D-Phe6 moiety to I did not change potency but caused a 30-fold increase in potency of Bn(6-13)NH2 and a 8-fold increase in the potency of Bn(6-13)ethylamide (Ki = 16 nM). Additional studies of both NH2- and COOH-terminal alterations in Bn(6-13)NH2 demonstrated that the most potent antagonist was [D-Phe6]Bn(6-13)propylamide (PA), having IC50's of 1.6 nM and 0.8 nM for bombesin-stimulated amylase release and Swiss 3T3 cell growth, respectively. Detailed studies of the most potent amide analogue, [D-Phe6]Bn(6-13)NH2, and alkylamide analogue, [D-Phe6]Bn(6-13)PA, demonstrated that these analogues functioned as competitive antagonists and that their action was selective for the bombesin receptor. These results demonstrate that, as with CCK- and gastrin-related peptides, the C-terminal amino acid is important for initiating a biologic response but not essential for determining receptor affinity.(ABSTRACT TRUNCATED AT 250 WORDS)

Carbene complexes in intramolecular Diels-Alder reactions

Diene-dienophile functionalized Fischer type carbene complexes [(CO) 5W=C(R)]R']are accessible from hexacarbonyl tungsten by a tandem nucleophilic addition/ substitution sequence. Either the diene or the dienophile functionality can be bonded directly to the carbene carbon atom. The synthetic potential of these complexes is demonstrated by furfurylamino(prop-2-enyl)-carbene and diallylamino(furyl) carbene complexes 2 and 11 which - in contrast to their amide analogues - under-go an intramolecular [4+2]cycloaddition under mild conditions. The cycloaddition occurs in a trans mode as is established by X-ray analyses.

Synthesis and structure-activity relationships of dynorphin A-(1-8) amide analogs

In order to study the structure-activity relationships of dynorphin A-(1-8) amide [Dyn(1-8)-NH2], 20 analogues were synthesized by the solution method. Their biological activities were determined in the three bioassays [guinea pig ileum (GPI), mouse vas deferens (MVD), and rabbit vas deferens (RVD)] and in the mouse tail-pinch test after intravenous administration. Some analogues that showed interesting activity in the bioassays and/or in the analgesic tests were further characterized in mu-, delta-, and kappa-representative binding assays. The obtained data indicate that modification of the enkephalin segment to give metabolically stable analogues with high affinity and selectivity for the kappa receptor is strictly limited and that introduction of MeArg in position 7 protects the Arg6-Arg-7 bond from enzymatic degradation without potency drop and change of opioid receptor selectivity. [MeTyr1,MeArg7,D-Leu8]Dyn(1-8)-NHEt (18) [IC50 (nM) = 0.3 (GPI), 7.4 (MVD), and 2.6 (RVD); tail pinch ED50 (mg/kg) = 0.75] showed opioid activity similar to that of dynorphin A in the three bioassays and relatively high kappa-receptor selectivity in the binding assays and produced a 2.5-fold more potent analgesic effect than morphine. [D-Cys2-Cys5,MeArg7,D-Leu8]Dyn(1-8)-NHEt (20) showed a 40-60-fold more potent opioid activity than 18 in the three bioassays and produced a 3.4-fold more potent analgesic effect than 18. In the binding assays, however, 20 showed higher affinity for mu and delta receptors than for the kappa receptor.

Protein stability and electrostatic interactions between solvent exposed charged side chains

To investigate the contribution to protein stability of electrostatic interactions between charged surface residues, we have studied the effect of substituting three negatively charged solvent exposed residues with their side-chain amide analogs in bovine calbindin D9k--a small (Mr 8,500) globular protein of the calmodulin superfamily. The free energy of urea-induced unfolding for the wild-type and seven mutant proteins has been measured. The mutant proteins have increased stability towards unfolding relative to the wild-type. The experimental results correlate reasonably well with theoretically calculated relative free energies of unfolding and show that electrostatic interactions between charges on the surface of a protein can have significant effects on protein stability.

Pharmacokinetic screening of o-naphthoquinone 5-lipoxygenase inhibitors.

The o-naphthoquinone derivative, CGS 8515 (I), is a potent inhibitor (IC50, 0.1 microM) of 5-lipoxygenase, but its therapeutic potential is compromised by a short plasma half-life (22 min) and extremely poor oral bioavailability (less than 2%). Poor biopharmaceutical properties of CGS 8515 were attributed to poor aqueous solubility and rapid in vivo hydrolysis of its methyl ester function to an inactive metabolite (IC50, 100 microM). An active amide analogue (II) was synthesized to prevent rapid hydrolysis. While analogue II appeared to be stable in vivo, its plasma half-life was also short (10 min), possibly because of rapid tissue distribution rather than metabolic elimination. Therefore, three potent analogues with increased aqueous solubilities were synthesized and compared with respect to their pharmacokinetic properties. The analogue with the highest aqueous solubility (V) demonstrated a plasma concentration vs time profile with the largest area under the curve (AUC) and the smallest distribution (alpha) phase of all the analogues studied. The percentage AUC of the terminal phase (beta) for three analogs paralleled their aqueous solubilities. The oral bioavailability of V was improved to 27%, compared to 2% for the parent compound, CGS 8515.

C-Terminal peptide alcohol, acid and amide analogs of desulfato hirudin 54–65 as antithrombin agents

Analogs of the antithrombin peptide hirudin 54–65 with C-terminal modifications have been synthesized in order to examine the requirements for α-thrombin inhibition. The C-terminal residue, Gln 65, could be replaced with L -amino acids or amino alcohols with neutral or charged hydrophilic side chains without greatly affecting the peptide's antithrombin potency as determined by inhibition of thrombin-induced clot formation in human plasma in vitro . Derivatives with D- or L -amino carboxamides at position 65-had significantly reduced potency, but still retained activity. Deletion of residue 65 with conversion of residue 64 to the amide or alcohol derivative resulted in a three-fold loss of potency. In addition to these results the solid-phase synthesis of peptide alcohols via direct displacement of p-nitrobenzhydrylideneisonitroso resin attached peptides with the desired C-terminal amino alcohol is reported.