Proline bis-amides as potent dual orexin receptor antagonists

A series of OX(2)R/OX(1)R dual orexin antagonists was prepared based on a proline bis-amide identified as a screening lead. Through a combination of classical and library synthesis, potency enhancing replacements for both amide portions were discovered. N-methylation of the benzimidazole moiety within the lead structure significantly reduced P-gp susceptibility while increasing potency, giving rise to good brain penetration. A compound from this series has demonstrated in vivo central activity when dosed peripherally in a pharmacodynamic model of orexin activity.     

A novel series of p38 MAP kinase inhibitors for the potential treatment of rheumatoid arthritis

A novel p38 MAP kinase inhibitor structural class was discovered through selectivity screening. The rational analogue design, synthesis and structure-activity relationship of this series of bis-amide inhibitors is reported. The inhibition in vitro of human p38alpha enzyme activity and lipopolysaccharide-induced tumour necrosis factor-alpha release is described for the series. The activity in vivo and pharmacokinetic properties are exemplified for the more potent analogues.     

Characterization of FdmV as an amide synthetase for fredericamycin A biosynthesis in Streptomyces griseus ATCC 43944

Fredericamycin (FDM) A is a pentadecaketide natural product that features an amide linkage. Analysis of the fdm cluster from Streptomyces griseus ATCC 43944, however, failed to reveal genes encoding the types of amide synthetases commonly seen in natural product biosynthesis. Here, we report in vivo and in vitro characterizations of FdmV, an asparagine synthetase (AS) B-like protein, as an amide synthetase that catalyzes the amide bond formation in FDM A biosynthesis. This is supported by the findings that (i) inactivation of fdmV in vivo afforded the ΔfdmV mutant strain SB4027 that abolished FDM A and FDM E production but accumulated FDM C, a biosynthetic intermediate devoid of the characteristic amide linkage; (ii) FdmV in vitro catalyzes conversion of FDM C to FDM B, a known intermediate for FDM A biosynthesis (apparent K(m) = 162 ± 67 μM and k(cat) = 0.11 ± 0.02 min(-1)); and (iii) FdmV also catalyzes the amidation of FDM M-3, a structural analog of FDM C, to afford amide FDM M-6 in vitro, albeit at significantly reduced efficiency. Preliminary enzymatic studies revealed that, in addition to the common nitrogen sources (L-Gln and free amine) of class II glutamine amidotransferases (to which AS B belongs), FdmV can also utilize L-Asn as a nitrogen donor. The amide bond formation in FDM A biosynthesis is proposed to occur after C-8 hydroxylation but before the carbaspirocycle formation.     

Synthesis and anticancer activity of novel amide derivatives of non-acetal deoxoartemisinin

Novel amide derivatives of C-12 non-acetal deoxoartemisinin were synthesized. Some of the derivatives had potent in vitro anticancer activity against major human cancer cell lines. The deoxoartemisinin amide trimer had potent in vivo antiangiogenic activity, according to the mouse matrigel plug assay.     

5-Chloroindoloyl glycine amide inhibitors of glycogen phosphorylase: synthesis, in vitro, in vivo, and X-ray crystallographic characterization

The synthesis, in vitro, and in vivo biological characterization of a series of achiral 5-chloroindoloyl glycine amide inhibitors of human liver glycogen phosphorylase A are described. Improved potency over previously reported compounds in cellular and in vivo assays was observed. The allosteric binding site of these compounds was shown by X-ray crystallography to be the same as that reported previously for 5-chloroindoloyl norstatine amides.     

N-alkyl-4-[(8-azabicyclo[3.2.1]-oct-3-ylidene)phenylmethyl]benzamides, micro and delta opioid agonists: a micro address

The tertiary amide delta opioid agonist 2 is a potent antinociceptive agent. Compound 2 was metabolized in vitro and in vivo to secondary amide 3, a potent and selective micro opioid agonist. The SAR of a series of N-alkyl-4-[(8-azabicyclo[3.2.1]-oct-3-ylidene)phenylmethyl]benzamides was examined.     

Coexpression of Glucagon-Like Peptide-1 (GLP-1) Receptor, Vasopressin, and Oxytocin mRNAs in Neurons of the Rat Hypothalamic Supraoptic and Paraventricular Nuclei

Abstract : This study was designed to gain better insight into the relationship between glucagon-like peptide-1 (GLP-1) (7-36) amide and vasopressin (AVP) and oxytocin (OX). In situ hybridization histochemistry revealed colocalization of the mRNAs for GLP-1 receptor, AVP, and OX in neurons of the hypothalamic supraoptic and paraventricular nuclei. To determine whether GLP-1(7-36)amide alters AVP and/or OX release, both in vivo and in vitro experimental study designs were used. In vivo, intravenous administration of 1 μg of GLP-1(7-36)amide into the jugular vein significantly decreased plasma AVP and OX concentrations. In vitro incubation of the neurohypophysis with either 0.1 or 1 μg of GLP-1(7-36)amide did not modify the release of AVP. However, addition of 1 μg of GLP-1(7-36)amide to the incubation medium increased slightly the secretion of OX. The coexpression of GLP-1 receptor and AVP mRNAs in hypothalamic supraoptic and paraventricular nuclei gives further support to the already reported central effects of GLP-1(7-36)amide on AVP. Our findings also suggest a dual secretory response of AVP and OX to the effect of GLP-1(7-36)amide, which most likely is related to the amount and/or the route of peptide administration.     

Multicomponent synthesis of α-acylamino and α-acyloxy amide derivatives of desmycosin and their activity against gram-negative bacteria

Bacterial resistance to the existing drugs requires constant development of new antibiotics. Developing compounds active against gram-negative bacteria thereby is one of the more challenging tasks. Among the many approaches to develop successful antibacterials, medicinal chemistry driven evolution of existing successful antibiotics is considered to be the most effective one. Towards this end, the C-20 aldehyde moiety of desmycosin was modified into α-acylamino and α-acyloxy amide functionalities using isonitrile-based Ugi and Passerini reactions, aiming for enhanced antibacterial and physicochemical properties. The desired compounds were obtained in 45–93% yield under mild conditions. The antibacterial activity of the resulting conjugates was tested against gram-negative Aliivibrio fischeri. The antibiotic strength is mostly governed by the amine component introduced. Thus, methylamine derived desmycosin bis-amide 4 displayed an enhanced inhibition rate vs. desmycosin (99% vs. 83% at 1 µM). Derivatives with long acyclic or bulky amine and isocyanide Ugi components reduced potency, whereas carboxylic acid reagents with longer chain length afforded increased bioactivity. In Passerini 3-component products, the butyric ester amide 22 displayed a higher activity (90% at 1 µM) than the parent compound desmycosin (2).     

New anti-malarial peroxides with in vivo potency derived from spongean metabolites

The structure-activity relationship of the anti-malarial substance 3 having a 6-carbomethoxymethyl-3-methoxy-1,2-dioxane structure was studied. The ester portion of the peroxide 3, showing little in vivo efficacy in malaria-infected mice in spite of the potent in vitro activity, was hydrolyzed in serum to afford an inactive free acid 4. The amide analogues (8 and 9) robust to mouse serum were disclosed to exhibit in vivo anti-malarial potency.     

Biochemical characterization of MelJ and MelK

Melithiazol and myxothiazol are two myxobacterial metabolites that are highly efficient electron transport inhibitors of the respiratory chain. MelJ and MelK encoded in the melithiazol biosynthetic gene cluster were recently shown to be involved in the formation of the methyl ester from a hypothetical amide intermediate. In vivo studies suggest that the structurally highly similar amide myxothiazol A can be used as a substrate mimic of the hypothetical melithiazol amide to characterize the hydrolase MelJ. Both enzymes were produced in Escherichia coli as intein chitin fusion proteins and were purified using affinity chromatography. MelJ was found to catalyse the conversion of the amide myxothiazol to free myxothiazol acid. The formerly unknown myxothiazol acid was purified and used as a substrate for the methyl transferase MelK which methylates the compound using S-adenosyl-methionine as cosubstrate. Sequence analyses suggest that MelJ and MelK are members of the amidase signature family and of a new subclass of methyltransferases, respectively. Kinetic analyses point at a very high substrate specificity for both enzymes. Furthermore, the in vitro reconstitution of a unique mechanism of methyl ester formation found in myxobacteria is reported.     

Pyridyl amides as potent inhibitors of T-type calcium channels

A novel series of amide T-type calcium channel antagonists were prepared and evaluated using in vitro and in vivo assays. Optimization of the screening hit 3 led to identification of the potent and selective T-type antagonist 37 that displayed in vivo efficacy in rodent models of epilepsy and sleep.     

Selection of novel analogs of thalidomide with enhanced tumor necrosis factor alpha inhibitory activity.

BACKGROUND: Tumor necrosis factor alpha (TNF alpha) is thought to mediate both protective and detrimental manifestations of the inflammatory response. Recently, thalidomide (alpha-N-phthalimidoglutarimide) was shown to partially inhibit monocyte TNF alpha production (by 50-70%) both in vivo and in vitro. More efficient inhibition of TNF alpha may, however, be necessary to rescue the host from more acute and extensive toxicities of TNF alpha-mediated inflammation. MATERIALS AND METHODS: Three structural analogues of thalidomide were selected for study based on increased activity against TNF alpha production. The parent drug and the analogs were tested in vitro in human peripheral blood mononuclear cell cultures for their effects on lipopolysaccharide (LPS) induced cytokine protein and mRNA production using ELISAs and Northern blot hybridization. The in vitro effects of the drugs were then confirmed in vivo in a mouse model of LPS induced lethality. RESULTS: The new compounds (two esters and one amide) showed increased inhibition of TNF alpha production by LPS-stimulated human monocytes, relative to the parent drug thalidomide. The analogs and the parent drug enhanced the production of interleukin 10 (IL-10), but had little effect on IL-6 and IL-1 beta protein and mRNA production. When tested in vivo, the amide analog protected 80% of LPS-treated mice against death from endotoxin induced shock. CONCLUSIONS: Analogs of thalidomide designed to better inhibit TNF alpha production in vitro have correspondingly greater efficacy in vivo. These finding may have therapeutic implication for the treatment of human diseases characterized by acute and extensive TNF alpha production such as tuberculous meningitis or toxic shock.     

Novel tumor-targeted RGD peptide–camptothecin conjugates: Synthesis and biological evaluation

Five RGD peptide–camptothecin (CPT) conjugates were designed and synthesized with the purpose to improve the therapeutic index of this antitumoral drug family. New RGD cyclopeptides were selected on the basis of their high affinity to α_v integrin receptors overexpressed by tumor cells and their metabolic stability. The conjugates can be divided in two groups: in the first the peptide was attached to the drug through an amide bond, in the second through a hydrazone bond. The main difference between the two spacers lies in their acid stability. Affinity to the receptors was maintained for all conjugates and their internalization into tumor cells was demonstrated. The first group conjugates showed lower in vitro and in vivo activity than the parent drug, probably due to the excessive stability of the amide bond, even inside the tumor cells. Conversely, the hydrazone conjugates exhibited in vitro tumor cell inhibition similar to the parent drug, indicating high conversion in the culture medium and/or inside the cells, but their poor solubility hampered in vivo experiments. On the basis of these results, information was acquired for additional development of derivatives with different linkers and better solubility for in vivo evaluation.     

Effect of potential amine prodrugs of selective neuronal nitric oxide synthase inhibitors on blood–brain barrier penetration

Several prodrug approaches were taken to mask amino groups in two potent and selective neuronal nitric oxide synthase (nNOS) inhibitors containing either a primary or secondary amino group to lower the charge and improve blood–brain barrier (BBB) penetration. The primary amine was masked as an azide and the secondary amine as an amide or carbamate. The azide was not reduced to the amine under a variety of in vitro and ex vivo conditions. Despite the decrease in charge of the amino group as an amide and as carbamates, BBB penetration did not increase. It appears that the uses of azides as prodrugs for primary amines or amides and carbamates as prodrugs for secondary amines are not universally effective for CNS applications.     

Synthesis of Biodegradable and Electroactive Tetraaniline Grafted Poly(ester amide) Copolymers for Bone Tissue Engineering

Biodegradable poly(ester amide)s have recently been used as biomaterials due to their desirable chemical and biological characteristics as well as their mechanical properties, which are amendable for material processing. In this study, electroactive tetraaniline (TA) grafted poly(ester amide)s were successfully synthesized and characterized. The poly(ester amide)s-graft-tetraaniline copolymers (PEA-g-TA) exhibited good electroactivity, mechanical properties, and biodegradability. The biocompatibility of the PEA-g-TA copolymers in vitro was systematically studied, which demonstrated that they were nontoxic and led to favorable adhesion and proliferation of mouse preosteoblastic MC3T3-E1 cells. Moreover, the PEA-g-TA copolymers stimulated by pulsed electrical signal could serve to promote the differentiation of MC3T3-E1 cells compared with TCPs. Hence, the biodegradable and electroactive PEA-g-TA copolymers possessed the properties in favor of the long-time potential application in vivo (electrical stimulation directly to the desired area) as bone repair scaffold materials in tissue engineering.     

Release of biotin from biotinylated proteins occurs enzymatically and nonenzymatically in human plasma

Studies in our laboratory and others indicate that biotin is released from biotinylated proteins in vivo and in vitro in human plasma. Using immunoglobulin G (IgG) as the model protein and four different biotinylating reagents, we investigated the mechanism of release. All of the biotin bonds shared an amide link to the carboxyl group of biotin but differed in the chemical links (amide, thioether, and hydrazone) between spacer arm and the various functional groups on IgG. Biotinylated IgG was incubated with phosphate-buffered saline, plasma, or plasma treated to either inactivate enzymes or remove all macromolecules. Released biotin was separated from bound biotin by ultrafiltration and quantitated by avidin-binding assay. As judged by high-performance liquid chromatography, greater than 95% of the released avidin-binding activity was biotin. We infer that the amide bond between the biotin and the spacer arm rather than the bond attaching the spacer to the protein was cleaved. Sodium dodecyl sulfate gel electrophoresis detected no proteolytic degradation of biotinylated IgG. Neither heat inactivation of plasma nor ultrafiltration of plasma to remove macromolecules completely eliminated biotin cleavage. We conclude that cleavage of biotin from protein occurs by both enzymatic and nonenzymatic mechanisms.     

The enzymatic inactivation of the fatty acid amide class of signaling lipids

The fatty acid amide (FAA) class of signaling lipids modulates a number of neurobehavioral processes in mammals, including pain, sleep, feeding, and locomotor activity. Representative FAAs include the endogenous cannabinoid anandamide and the sleep-inducing lipid oleamide. Despite activating several neuroreceptor systems in vitro, most FAAs produce only weak and transient behavioral effects in vivo, presumably due to their expeditious catabolism. This review focuses on one enzyme, fatty acid amide hydrolase (FAAH) that appears to play a major role in regulating the amplitude and duration of FAA signals in vivo. In particular, we will highlight a series of recent papers that have investigated the physiological functions of the mouse and human FAAH enzymes. Collectively, these studies promote FAAH as a central component of FAA signaling pathways, especially those mediated by the endocannabinoid anandamide, and suggest that this enzyme may represent an attractive pharmaceutical target for the treatment of pain and related neurophysiological disorders.     

Effects of malonate administration on renal ammoniagenesis in intact dogs

In the dog kidney in vivo, malonate augmented ammoniagenesis from both amide and nonamide nitrogen sources, similar to previous in vitro investigations using incubating canine renal tubules. This was highly significant in alkalotic dogs, where it was accompanied by decreased renal tissue concentrations of glutamate. Changes in renal ammonia metabolism were less evident in acidotic dogs where a markedly decreased glomerular filtration rate was noted following malonate administration. Under conditions of complete ureteric obstruction which effectively abolished glomerular filtration, malonate significantly augmented ammoniagenesis above baseline in acidotic dogs. These in vivo results with malonate have similarities to those seen in dogs subjected to an acid challenge alone and suggest that the adaptation in renal ammoniagenesis under both circumstances occurs via enhanced deamination of glutamate pools.     

Design, synthesis, and structure-activity relationships of indole-3-heterocycles as agonists of the CB1 receptor

Novel indole-3-heterocycles were designed and synthesized and found to be potent CB1 receptor agonists. Starting from a microsomally unstable lead 1, a bioisostere approach replacing a piperazine amide was undertaken. This was found to be a good strategy for improving stability both in vitro and in vivo. This led to the discovery of 24, which had an increased duration of action in the mouse tail flick test in comparison to the lead 1.