Synthesis and biological activity of piperazine-based dual MMP-13 and TNF-alpha converting enzyme inhibitors

A series of novel MMP-13 and TNF-alpha converting enzyme inhibitors based on piperazine 2-hydroxamic acid scaffolds are described. The TACE, MMP-1 and MMP-13 activity of these inhibitors as well as the effect of substitution of the piperazine nitrogen and the P-1' benzyloxy tailpiece is discussed. Moderate in vivo activity is observed with several members of this group.     

Design and identification of selective HER-2 sheddase inhibitors via P1' manipulation and unconventional P2' perturbations to induce a molecular metamorphosis

In an effort to obtain a MMP selective and potent inhibitor of HER-2 sheddase (ADAM-10), the P1' group of a novel class of (6S,7S)-7-[(hydroxyamino)carbonyl]-6-carboxamide-5-azaspiro[2.5]octane-5-carboxylates was attenuated and the structure-activity relationships (SAR) will be discussed. In addition, it was discovered that unconventional perturbation of the P2' moiety could confer MMP selectivity, which was hypothesized to be a manifestation of the P2' group effecting global conformational changes.     

Synthesis of new glycyrrhetinic acid derived ring A azepanone, 29-urea and 29-hydroxamic acid derivatives as selective 11β-hydroxysteroid dehydrogenase 2 inhibitors

Glycyrrhetinic acid, the metabolite of the natural product glycyrrhizin, is a well known nonselective inhibitor of 11β-hydroxysteroid dehydrogenase (11β-HSD) type 1 and type 2. Whereas inhibition of 11β-HSD1 is currently under consideration for treatment of metabolic diseases, such as obesity and diabetes, 11β-HSD2 inhibitors may find therapeutic applications in chronic inflammatory diseases and certain forms of cancer. Recently, we published a series of hydroxamic acid derivatives of glycyrrhetinic acid showing high selectivity for 11β-HSD2. The most potent and selective compound is active against human 11β-HSD2 in the low nanomolar range with a 350-fold selectivity over human 11β-HSD1. Starting from the lead compounds glycyrrhetinic acid and the hydroxamic acid derivatives, novel triterpene type derivatives were synthesized and analyzed for their biological activity against overexpressed human 11β-HSD1 and 11β-HSD2 in cell lysates. Here we describe novel 29-urea- and 29-hydroxamic acid derivatives of glycyrrhetinic acid as well as derivatives with the Beckman rearrangement of the 3-oxime to a seven-membered ring, and the rearrangement of the C-ring from 11-keto-12-ene to 12-keto-9(11)-ene. The combination of modifications on different positions led to compounds comprising further improved selective inhibition of 11β-HSD2 in the lower nanomolar range with up to 3600-fold selectivity.     

Discovery of benzo[g]indoles as a novel class of non-covalent Keap1-Nrf2 protein-protein interaction inhibitor

The Keap1-Nrf2 system is an attractive target for drug discovery regarding various unmet medical needs. Only covalent inhibitors for protein-protein interaction (PPI) between Keap1 and Nrf2 to activate Nrf2 have been approved or are under clinical trials, but such electrophilic compounds lack selectivity. Therefore, specific non-covalent Keap1-Nrf2 PPI inhibitors are expected to be safer Nrf2 activators. We found a novel class of non-covalent Keap1-Nrf2 PPI inhibitor that has a benzo[g]indole skeleton and an indole-3-hydroxamic acid moiety and that exhibits significant PPI inhibitory activity. Additionally, the benzo[g]indole-3-carbohydrazide derivatives were newly prepared. The benzo[g]indole derivatives showed a stronger Keap1-Nrf2 PPI inhibitory activity than Cpd16, a previously reported non-covalent PPI inhibitor. Moreover, most of the PPI inhibitors showed a high metabolic stability in a human microsome system with a low cytotoxicity against HepG2 cell lines, which suggests that novel benzo[g]indole-type Keap1-Nrf2 PPI inhibitors are expected to be biological tools or lead compounds for Nrf2 activators.     

Isoxazole-3-hydroxamic acid derivatives as peptide deformylase inhibitors and potential antibacterial agents

A series of isoxazole-3-hydroxamic acid derivatives has been identified as a new class of small, nonpeptidic inhibitors of peptide deformylase (PDF). The synthesis, enzyme inhibition and preliminary investigation of the binding mode of this potential antibacterial compounds are reported.     

Design, synthesis and biological evaluation of β-carboline derivatives as novel inhibitors targeting B-Raf kinase

β-Carboline family of compounds is a large group of alkaloids widely distributed in nature and exhibits broad-spectrum anti-tumor activities. We designed and synthesized two series of novel 1-carboxamide- and 6-sulfonamide-substituted β-carboline derivatives 7a-p and 12a-b, and their wild type B-Raf kinase inhibitory activities were described. Most compounds showed moderate to excellent inhibitory activities. Among them, 1-carboxamide-6-(N-(3-(dimethylamino)propyl)-sulfamoyl)-β-carboline, 7e exhibited potent activity (IC(50)=1.62 μM), showing the potential for further investigation as a lead compound.     

Identification and optimisation of a series of substituted 5-pyridin-2-yl-thiophene-2-hydroxamic acids as potent histone deacetylase (HDAC) inhibitors

Further investigation of a series of thienyl-based hydroxamic acids that included ADS100380 and ADS102550 led to the identification of the 5-pyridin-2-yl-thiophene-2-hydroxamic acid 3c, which possessed modest HDAC inhibitory activity. Substitution at the 5- and 6-positions of the pyridyl ring of compound 3c provided compounds 5a-g, 7a, b, 9, and 13a. Compound 5b demonstrated improved potency, in vitro DMPK profile, and rat oral bioavailability, compared to ADS102550. Functionalisation of the pendent phenyl group of compounds 5b, 5e and 13a provided analogues that possessed excellent enzyme inhibition and anti-proliferative activity.     

Identification and optimisation of a series of substituted 5-(1H-pyrazol-3-yl)-thiophene-2-hydroxamic acids as potent histone deacetylase (HDAC) inhibitors

Optimisation of ADS100380, a sub-micromolar HDAC inhibitor identified using a virtual screening approach, led to a series of substituted 5-(1H-pyrazol-3-yl)-thiophene-2-hydroxamic acids (6a-i), that possessed significant HDAC inhibitory activity. Subsequent functionalisation of the pendent phenyl group of compounds 6f and 6g provided analogues 6j-w with further enhanced enzyme and anti-proliferative activity. Compound 6j demonstrated efficacy in a mouse xenograft experiment.     

Formation and structure in aqueous solution of complexes between vanadium(V) and aminohydroxamic acids that potentiates vanadium’s insulinomimetic activity: l-glutamic γ-hydroxamic and l-aspartic-β-hydroxamic acids

The full speciation of the vanadium(V) complexation systems with two aminohydroxamic acids, aspartic-β- and glutamic-γ-hydroxamic acid, has been determined using potentiometric and spectroscopic techniques. Formation constants were calculated in a systematic study at different ligand to metal molar ratios and the coordination types are proposed. An almost constant value of the ⁵¹V NMR signal in neutral medium can be attributed to two (1:1 and 1:2 metal to ligand ratios) similar structures, both of which can be either protonated or deprotonated. The two ligands have a carboxylic group in the structure and show comparable biological activities. In this work analogous complexation behavior at physiological conditions was found despite the presence of two or three methylenic groups between the amino and hydroxamate groups. The carboxylic groups are quite distant from the hydroxamic groups and are not involved directly in the coordination process. Therefore the coordination structures are related to that found in the vanadium(V)-β-alaninehydroxamic acid in which there is not a carboxylic group.     

5-Aryl-4-carboxamide-1,3-oxazoles: potent and selective GSK-3 inhibitors

5-Aryl-4-carboxamide-1,3-oxazoles are a novel, potent and selective series of GSK-3 inhibitors. The optimization of the series to yield compounds with cell activity and brain permeability is described.     

5-Aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside-induced AMP-activated protein kinase phosphorylation inhibits basal and insulin-stimulated glucose uptake, lipid synthesis, and fatty acid oxidation in isolated rat adipocytes

The objective of this study was to investigate the effects of 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR)-induced AMP-activated protein kinase (AMPK) activation on basal and insulin-stimulated glucose and fatty acid metabolism in isolated rat adipocytes. AICAR-induced AMPK activation profoundly inhibited basal and insulin-stimulated glucose uptake, lipogenesis, glucose oxidation, and lactate production in fat cells. We also describe the novel findings that AICAR-induced AMPK phosphorylation significantly reduced palmitate (32%) and oleate uptake (41%), which was followed by a 50% reduction in palmitate oxidation despite a marked increase in AMPK and acetyl-CoA carboxylase phosphorylation. Compound C, a selective inhibitor of AMPK, not only completely prevented the inhibitory effect of AICAR on palmitate oxidation but actually caused a 2.2-fold increase in this variable. Compound C also significantly increased palmitate oxidation in the presence of inhibitory concentrations of malonyl-CoA and etomoxir indicating an increase in CPT1 activity. In contrast to skeletal muscle in which AMPK stimulates fatty acid oxidation to provide ATP as a fuel, we propose that AMPK activation inhibits lipogenesis and fatty acid oxidation in adipocytes. Inhibition of lipogenesis would conserve ATP under conditions of cellular stress, although suppression of intra-adipocyte oxidation would spare fatty acids for exportation to other tissues where their utilization is crucial for energy production. Additionally, the stimulatory effect of compound C on long chain fatty acid oxidation provides a novel pharmacological approach to promote energy dissipation in adipocytes, which may be of therapeutic importance for obesity and type II diabetes.     

Oleic acid uptake by in vitro English sparrow skeletal muscle

Studies of prolonged avian flight have shown it to require large amounts of energy supplied mainly by free fatty acids (FFA). In the present study, the high levels of plasma ketone bodies found in sparrows (2.58 mmol l(-1)) are supportive of the use of fatty acids for flight. To determine the nature of fatty acid (oleic acid, OA) uptake, various pharmacological agents were used. The uptake of OA was examined using the extensor digitorum communis (EDC) muscle of English sparrows incubated in vitro. Initial studies demonstrated that radiolabeled OA uptake decreased in the presence of increasing unlabeled OA, suggesting that uptake occurred by a facilitative transport process. To further characterize OA uptake, EDC muscles were incubated with either: insulin (2 ng ml(-1)), insulin-like growth factor isoform-1 (IGF-I; 48 ng ml(-1)), 5'-Aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR; 2 mmol) or caffeine (5 mmol). Insulin, but not IGF-I, significantly increased OA uptake by avian EDC (P < 0.01). Caffeine and AICAR were ineffective at increasing OA uptake. A specific inhibitor of FFA transport by fatty acid transporters (FAT/CD36), sulfo-N-succinimidyl oleate (SSO; 500 micromoles), significantly decreased OA uptake at 2.5 min. The effectiveness of SSO suggests that a FAT/CD36-like protein is expressed in avian tissues. As uptake of OA was not completely blocked by SSO, it is likely that other mechanisms for FFA movement across membranes, such as diffusion, may be present.     

Salmonella enterica serovar Typhimurium growth is inhibited by the concomitant binding of Zn(II) and a pyrrolyl-hydroxamate to ZnuA,the soluble component of the ZnuABC transporter

BackgroundUnder conditions of Zn(II) deficiency, the most relevant high affinity Zn(II) transport system synthesized by many Gram-negative bacteria is the ZnuABC transporter. ZnuABC is absent in eukaryotes and plays an important role in bacterial virulence. Consequently, ZnuA, the periplasmic component of the transporter, appeared as a good target candidate to find new compounds able to contrast bacterial growth by interfering with Zn(II) uptake.MethodsAntibacterial activity assays on selected compounds from and in-house library against Salmonella enterica serovar Typhimurium ATCC14028 were performed. The X-ray structure of the complex formed by SeZnuA with an active compound was solved at 2.15 Å resolution.ResultsTwo di-aryl pyrrole hydroxamic acids differing in the position of a chloride ion, RDS50 ([1-[(4-chlorophenyl)methyl]-4-phenyl-1 H-pyrrol-3-hydroxamic acid]) and RDS51 (1-[(2-chlorophenyl)methyl]-4-phenyl-1 H-pyrrol-3-hydroxamic acid) were able to inhibit Salmonella growth and its invasion ability of Caco-2 cells. The X-ray structure of SeZnuA containing RDS51 revealed its presence at the metal binding site concomitantly with Zn(II) which is coordinated by protein residues and the hydroxamate moiety of the compound.ConclusionsTwo molecules interfering with ZnuA-mediated Zn(II) transport in Salmonella have been identified for the first time. The resolution of the SeZnuA-RDS51 X-ray structure revealed that RDS51 is tightly bound both to the protein and to Zn(II) thereby inhibiting its release. These features pave the way to the rational design of new Zn(II)-binding drugs against Salmonella.General SignificanceThe data reported show that targeting the bacterial ZnuABC transporter can represent a good strategy to find new antibiotics against Gram-negative bacteria.     

Carbonic anhydrase inhibitors. The X-ray crystal structure of human isoform II in adduct with an adamantyl analogue of acetazolamide resides in a less utilized binding pocket than most hydrophobic inhibitors

We investigated the inhibitory activity of several 1,3,4-thiadiazole-sulfonamides against all catalytically active CA (EC 4.2.1.1), CA I–XV. The tail derivatizing the 5-position in the 1,3,4-thiadiazole-2-sulfonamide scaffold was observed to be critical as an inhibitory determinant of these compounds. The high resolution X-ray crystal structure of hCA II in complex with 5-(1-adamantylcarboxamido)-1,3,4-thiadiazole-2-sulfonamide, showed the adamantyl moiety of the inhibitor residing in a less utilized binding pocket than that of most hydrophobic inhibitors, lined by the amino acid residues Ile91, Val121 and Phe131. This binding site may explain the diverse inhibition profiles of 5-carboxamide- and sufonamide-derivatized 1,3,4-thiadiazole-2-sulfonamides and offers a hot spot for designing isoform selective inhibitors, considering that residues 91 and 131 are highly variable among the 13 catalytically active isoforms.     

Aging-associated reductions in AMP-activated protein kinase activity and mitochondrial biogenesis

Recent studies have demonstrated a strong relationship between aging-associated reductions in mitochondrial function, dysregulated intracellular lipid metabolism, and insulin resistance. Given the important role of the AMP-activated protein kinase (AMPK) in the regulation of fat oxidation and mitochondrial biogenesis, we examined AMPK activity in young and old rats and found that acute stimulation of AMPK-alpha(2) activity by 5'-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) and exercise was blunted in skeletal muscle of old rats. Furthermore, mitochondrial biogenesis in response to chronic activation of AMPK with beta-guanidinopropionic acid (beta-GPA) feeding was also diminished in old rats. These results suggest that aging-associated reductions in AMPK activity may be an important contributing factor in the reduced mitochondrial function and dysregulated intracellular lipid metabolism associated with aging.     

A Methanocaldococcus jannaschii archaeal signature gene encodes for a 5-formaminoimidazole-4-carboxamide-1-beta-D-ribofuranosyl 5'-monophosphate synthetase. A new enzyme in purine biosynthesis

We have identified and characterized a new member of the ATP-grasp enzyme family that catalyzes the ATP- and formate-dependent formylation of 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranosyl 5'-monophosphate (AICAR) to 5-formaminoimidazole-4-carboxamide-1-beta-D-ribofuranosyl 5'-monophosphate (FAICAR) in the absence of folates. The enzyme, which we designate as PurP, is the product of the Methanocaldococcus jannaschii purP gene (MJ0136), which is a signature gene for Archaea. As is characteristic of reactions catalyzed by this family of enzymes, the other products of the reaction, ADP and P(i), were produced stoichiometrically with the amount of ATP, formate, and AICAR used. Formyl phosphate was found to substitute for ATP and formate in the reaction, yet the methylene analog, phosphonoacetaldehyde, was not an inhibitor or substrate for the reaction. The enzyme, along with PurO, which catalyzes the cyclization of FAICAR to inosine 5'-monophosphate, catalyzes the same overall transformation in purine biosynthesis as is accomplished by PurH in bacteria and eukaryotes. No homology exists between PurH and either PurO or PurP. 1H NMR and gas chromatography-mass spectrometry analysis of an M. jannaschii cell extract showed the presence of free formate that can be used by the enzyme for purine biosynthesis. This formate arises by the reduction of CO2 with hydrogen; this was demonstrated by incorporating 13C into the formate when M. jannaschii cell extracts were incubated with H13CO3- and hydrogen gas. The presence of this signature gene in all of the Archaea indicates the presence of a purine biosynthetic pathway proceeding in the absence of folate coenzymes.     

Quantitative colorimetric and gas chromatoographic determination of arecaidine propargyl ester

Arecaidine propargyl ester (APE) is a potent muscarinic agonist often used in pharmacological studies. To date, no sensitive quantitative analytical method for APE has been published. In this study, two methods for the quantitative determination of APE are compared: a colorimetric assay, based on the formation of the corresponding ferric(III)-hydroxamic acid complex, and a direct gas chromatographic method, using arecoline as the internal standard. The latter method was found to be more precise. The utility of the gas chromatographic assay was further demonstrated in a stability study of the drug in the biological fluid aqueous humor of rabbits.     

Adiponectin activates adenosine monophosphate-activated protein kinase and decreases luteinizing hormone secretion in LbetaT2 gonadotropes

Metabolic dysregulation is associated with reproductive disorders, but the underlying mechanisms are not clearly understood. Adiponectin is an adipocyte-derived secretory factor that improves insulin sensitivity. Results from animal models indicate that overexpression of adiponectin impairs female fertility. We hypothesized that adiponectin regulates reproduction by altering the hypothalamic-pituitary axis. Mouse LbetaT2 immortalized gonadotrope cells express both adiponectin receptors 1 and 2. Adiponectin increases phosphorylation of AMP-activated protein kinase (AMPK), a downstream target of adiponectin receptors, and reduces basal and GnRH-stimulated LH secretion, acutely. The repression of LH secretion can be mimicked by 5-aminoimidazole-4-carboxamide-1-beta-riboside, an AMP analog, suggesting the involvement of AMPK. A dominant-negative AMPK mutant or compound C, a selective AMPK inhibitor, potentiates basal LH secretion and abolishes the inhibitory effect of adiponectin. Chronic activation of AMPK by 5-aminoimidazole-4-carboxamide-1-beta-riboside decreases cellular LH levels, and expression of dominant-negative AMPK increases cellular LH levels, suggesting a second effect of AMPK to regulate LH synthesis. Lastly, intravenous injection of an adenovirus expressing adiponectin into male mice reduces serum LH levels without changing FSH levels. In conclusion, our results suggest that adiponectin decreases LH secretion in pituitary gonadotropes in an AMPK-dependent manner.