The discovery of (1R, 3R)-1-(3-chloro-5-fluorophenyl)-3-(hydroxymethyl)-1,2,3,4-tetrahydroisoquinoline-6-carbonitrile, a potent and selective agonist of human transient receptor potential cation channel subfamily m member 5 (TRPM5) and evaluation of as a potential gastrointestinal prokinetic agent.

This publication details the discovery of a series of selective transient receptor potential cation channel subfamily M member 5 (TRPM5) agonists culminating with the identification of the lead compound (1R, 3R)-1-(3-chloro-5-fluorophenyl)-3-(hydroxymethyl)-1,2,3,4-tetrahydroisoquinoline-6-carbonitrile (39). We describe herein our biological rationale for agonism of the target, the examination of the then current literature tool molecules, and finally the process of our discovery starting with a high throughput screening hit through lead development. We also detail the selectivity of the lead compound 39 versus related family members TRPA1, TRPV1, TRPV4, TRPM4 and TRPM8, the drug metabolism and pharmacokinetics (DMPK) profile and in vivo efficacy in a mouse model of gastrointestinal motility.

Potential hepatoprotective effect of Cheatomorpha gracilis extract against High Fat Diet (HFD)-Induced Liver Damage, and its characterization by HPLC / Efeito hepatoprotetor potencial do extrato de Cheatomorpha gracilis contra dano hepático induzido por dieta alta em gordura (HFD), e sua caracterização por HPLC

The current investigation was carried out to estimate the protective effect of aqueous extract of Cheatomorpha gracilis (AEC) against High fat Diet (HFD) induced liver damage in mice. The results of the in vitro study showed that AEC have higher antioxidant capacities in the DPPH and hydroxyl radical-scavenging assays. Indeed, many phenolic compounds (gallic acid, quercetin, naringenin, apigenin, kaempferol and rutin) were identified in the AEC. In the animal studies, during 6 weeks, HFD promoted oxidative stress with a rise level of malonaldehyde (MDA), protein carbonyls (PCOs) levels and a significant decrease of the antioxidant enzyme activities such as superoxide dismutase, catalase and glutathione peroxidase. Interestingly, the treatment with AEC (250 mg/kg body weight) significantly reduced the effects of HFD disorders on some plasmatic liver biomarkers (AST, ALT and ALP) in addition to, plasmatic proteins inflammatory biomarkers (α2 and β1 decreases / β2 and γ globulins increases). It can be suggest that supplementation of MECG displays high potential to quench free radicals and attenuates high fat diet promoted liver oxidative stress and related disturbances.

A presente investigação foi realizada para estimar o efeito protetor do extrato aquoso de Cheatomorpha gracilis (AEC) contra o dano hepático induzido por dieta rica em gordura (HFD) em camundongos. Os resultados do estudo in vitro mostraram que os AEC têm maiores capacidades antioxidantes nos ensaios DPPH e de eliminação de radicais hidroxila. De fato, muitos compostos fenólicos (ácido gálico, quercetina, naringenina, apigenina, kaempferol e rutina) foram identificados no AEC. Nos estudos em animais, durante 6 semanas, HFD promoveu estresse oxidativo com aumento do nível de malonaldeído (MDA), níveis de proteína carbonil (PCOs) e diminuição significativa das atividades de enzimas antioxidantes como superóxido dismutase, catalase e glutationa peroxidase. Curiosamente, o tratamento com AEC (250 mg / kg de peso corporal) reduziu significativamente os efeitos dos distúrbios de HFD em alguns biomarcadores hepáticos plasmáticos (AST, ALT e ALP), além de biomarcadores inflamatórios de proteínas plasmáticas (reduções α2 e β1 / β2 e γ aumenta as globulinas). Pode-se sugerir que a suplementação de MECG apresenta alto potencial para extinguir os radicais livres e atenua o estresse oxidativo do fígado promovido pela dieta rica em gordura e distúrbios relacionados.

Potential Hepatoprotective Effect of Cheatomorpha gracilis extract against High Fat Diet (HFD)-Induced Liver Damage, and its characterization by HPLC

Abstract The current investigation was carried out to estimate the protective effect of aqueous extract of Cheatomorpha gracilis (AEC) against High fat Diet (HFD) induced liver damage in mice. The results of the in vitro study showed that AEC have higher antioxidant capacities in the DPPH and hydroxyl radical-scavenging assays. Indeed, many phenolic compounds (gallic acid, quercetin, naringenin, apigenin, kaempferol and rutin) were identified in the AEC. In the animal studies, during 6 weeks, HFD promoted oxidative stress with a rise level of malonaldehyde (MDA), protein carbonyls (PCOs) levels and a significant decrease of the antioxidant enzyme activities such as superoxide dismutase, catalase and glutathione peroxidase. Interestingly, the treatment with AEC (250 mg/kg body weight) significantly reduced the effects of HFD disorders on some plasmatic liver biomarkers (AST, ALT and ALP) in addition to, plasmatic proteins inflammatory biomarkers (2 and 1 decreases / 2 and globulins increases). It can be suggest that supplementation of MECG displays high potential to quench free radicals and attenuates high fat diet promoted liver oxidative stress and related disturbances.

Resumo A presente investigação foi realizada para estimar o efeito protetor do extrato aquoso de Cheatomorpha gracilis (AEC) contra o dano hepático induzido por dieta rica em gordura (HFD) em camundongos. Os resultados do estudo in vitro mostraram que os AEC têm maiores capacidades antioxidantes nos ensaios DPPH e de eliminação de radicais hidroxila. De fato, muitos compostos fenólicos (ácido gálico, quercetina, naringenina, apigenina, kaempferol e rutina) foram identificados no AEC. Nos estudos em animais, durante 6 semanas, HFD promoveu estresse oxidativo com aumento do nível de malonaldeído (MDA), níveis de proteína carbonil (PCOs) e diminuição significativa das atividades de enzimas antioxidantes como superóxido dismutase, catalase e glutationa peroxidase. Curiosamente, o tratamento com AEC (250 mg / kg de peso corporal) reduziu significativamente os efeitos dos distúrbios de HFD em alguns biomarcadores hepáticos plasmáticos (AST, ALT e ALP), além de biomarcadores inflamatórios de proteínas plasmáticas (reduções 2 e 1 / 2 e aumenta as globulinas). Pode-se sugerir que a suplementação de MECG apresenta alto potencial para extinguir os radicais livres e atenua o estresse oxidativo do fígado promovido pela dieta rica em gordura e distúrbios relacionados.

Potential Hepatoprotective Effect of Cheatomorpha gracilis extract against High Fat Diet (HFD)-Induced Liver Damage, and its characterization by HPLC / Efeito Hepatoprotetor Potencial do Extrato de Cheatomorpha gracilis contra Dano Hepático Induzido por Dieta Alta em Gordura (HFD), e sua caracterização por HPLC

The current investigation was carried out to estimate the protective effect of aqueous extract of Cheatomorpha gracilis (AEC) against High fat Diet (HFD) induced liver damage in mice. The results of the in vitro study showed that AEC have higher antioxidant capacities in the DPPH and hydroxyl radical-scavenging assays. Indeed, many phenolic compounds (gallic acid, quercetin, naringenin, apigenin, kaempferol and rutin) were identified in the AEC. In the animal studies, during 6 weeks, HFD promoted oxidative stress with a rise level of malonaldehyde (MDA), protein carbonyls (PCOs) levels and a significant decrease of the antioxidant enzyme activities such as superoxide dismutase, catalase and glutathione peroxidase. Interestingly, the treatment with AEC (250 mg/kg body weight) significantly reduced the effects of HFD disorders on some plasmatic liver biomarkers (AST, ALT and ALP) in addition to, plasmatic proteins inflammatory biomarkers (α2 and ß1 decreases / ß2 and γ globulins increases). It can be suggest that supplementation of MECG displays high potential to quench free radicals and attenuates high fat diet promoted liver oxidative stress and related disturbances.

A presente investigação foi realizada para estimar o efeito protetor do extrato aquoso de Cheatomorpha gracilis (AEC) contra o dano hepático induzido por dieta rica em gordura (HFD) em camundongos. Os resultados do estudo in vitro mostraram que os AEC têm maiores capacidades antioxidantes nos ensaios DPPH e de eliminação de radicais hidroxila. De fato, muitos compostos fenólicos (ácido gálico, quercetina, naringenina, apigenina, kaempferol e rutina) foram identificados no AEC. Nos estudos em animais, durante 6 semanas, HFD promoveu estresse oxidativo com aumento do nível de malonaldeído (MDA), níveis de proteína carbonil (PCOs) e diminuição significativa das atividades de enzimas antioxidantes como superóxido dismutase, catalase e glutationa peroxidase. Curiosamente, o tratamento com AEC (250 mg / kg de peso corporal) reduziu significativamente os efeitos dos distúrbios de HFD em alguns biomarcadores hepáticos plasmáticos (AST, ALT e ALP), além de biomarcadores inflamatórios de proteínas plasmáticas (reduções α2 e ß1 / ß2 e γ aumenta as globulinas). Pode-se sugerir que a suplementação de MECG apresenta alto potencial para extinguir os radicais livres e atenua o estresse oxidativo do fígado promovido pela dieta rica em gordura e distúrbios relacionados.

Potential Hepatoprotective Effect of Cheatomorpha gracilis extract against High Fat Diet (HFD)-Induced Liver Damage, and its characterization by HPLC.

The current investigation was carried out to estimate the protective effect of aqueous extract of Cheatomorpha gracilis (AEC) against High fat Diet (HFD) induced liver damage in mice. The results of the in vitro study showed that AEC have higher antioxidant capacities in the DPPH and hydroxyl radical-scavenging assays. Indeed, many phenolic compounds (gallic acid, quercetin, naringenin, apigenin, kaempferol and rutin) were identified in the AEC. In the animal studies, during 6 weeks, HFD promoted oxidative stress with a rise level of malonaldehyde (MDA), protein carbonyls (PCOs) levels and a significant decrease of the antioxidant enzyme activities such as superoxide dismutase, catalase and glutathione peroxidase. Interestingly, the treatment with AEC (250 mg/kg body weight) significantly reduced the effects of HFD disorders on some plasmatic liver biomarkers (AST, ALT and ALP) in addition to, plasmatic proteins inflammatory biomarkers (α2 and ß1 decreases / ß2 and γ globulins increases). It can be suggest that supplementation of MECG displays high potential to quench free radicals and attenuates high fat diet promoted liver oxidative stress and related disturbances.

Effects of melatonin on aluminium-induced neurobehavioral and neurochemical changes in aging rats.

This study aimed to investigate the potential protective effects of melatonin (Mel) against aluminium-induced neurodegenerative changes in aging Wistar rats (24-28months old). Herein, aluminium chloride (AlCl3) (50mg/kg BW/day) was administered by gavage, and melatonin (Mel) was co-administered to a group of Al-treated rats by an intra-peritoneal injection at a daily dose of 10mg/kg BW for four months. The findings revealed that aluminium administration induced a significant decrease in body weight associated with marked mortality for the old group of rats, which was more pronounced in old Al-treated rats. Behavioural alterations were assessed by 'open fields', 'elevated plus maze' and 'Radial 8-arms maze' tests. The results demonstrated that Mel co-administration alleviated neurobehavioral changes in both old and old Al-treated rats. Melatonin was noted to play a good neuroprotective role, reducing lipid peroxidation (TBARs), and enhancing enzymatic (SOD, CAT and GPx) activities in the brain organs of old control and old Al-treated rats. Mel treatment also reversed the decrease of AChE activity in the brain tissues, which was confirmed by histological sections. Overall, the results showed that Mel administration can induce beneficial effects for the treatment of Al-induced neurobehavioral and neurochemical changes in the central nervous system (CNS).

Ciprofloxacin affects conformational equilibria of DNA gyrase A in the presence of magnesium ions.

The conformational equilibria of the A subunit of DNA gyrase (GyrA), of its 59 kDa N-terminal fragment (GyrA59) and of the quinolone-resistant Ser-Trp83 mutant (GyrATrp83), were investigated in the presence of mono- and divalent metal ions and ciprofloxacin, a clinically useful antibacterial quinolone. The stability of the proteins was estimated from temperature denaturation, monitoring unfolding with circular dichroism spectroscopy. Two transitions were observed in GyrA and GyrATrp83, which likely reflect unfolding of the N and C-terminal protein domains. Accordingly, one thermal transition is observed for GyrA59. The melting profile of the GyrA subunit is dramatically affected by monovalent and divalent metal ions, both transitions being shifted to lower temperature upon increasing salt concentration. This effect is much more pronounced with divalent ions (Mg(2+)) and cannot be accounted for by changes in ionic strength only. The presence of ciprofloxacin shifts the melting transitions of the wild-type subunit to higher temperatures when physiological concentrations of Mg(2+) are present. In contrast, both the mutant protein and the 59 kDa fragment do not show evidence for quinolone-driven changes. These data suggest that ciprofloxacin binds to the wild-type subunit in an interaction that involves Ser83 of GyrA and that both C and N-terminal domains may be required for effective drug-protein interactions. The bell-shaped dependence of the binding process upon Mg(2+) concentration, with a maximum centred at 3-4 mM [Mg(2+)], is consistent with a metal-ion mediated GyrA-quinolone-interaction. Affinity chromatography data fully support these findings and additionally confirm the requirement for a free carboxylate to elicit binding of the quinolone to GyrA. We infer that the Mg(2+)-GyrA interaction at physiological metal ion concentration could bear biological relevance, conferring more conformational flexibility to the active enzyme. The results obtained in the presence of ciprofloxacin additionally suggest that the Mg(2+)-mediated quinolone binding to the enzyme might be involved in the mechanism of action of this family of drugs.

A combinatorial approach to [1,5]benzothiazepine derivates as potential antibacterial agents.

[1,5]Benzothiazepines are widely used in a number of different therapeutic areas and therefore represent an interesting scaffold for de novo exploration. Recent literature reports suggest their value as antibacterial agents. The present paper reports the exploration of this scaffold for the generation of combinatorial libraries both in solution and on solid phase.

Synthesis and SAR of new 5-phenyl-3-ureido-1,5-benzodiazepines as cholecystokinin-B receptor antagonists.

A series of 5-phenyl-3-ureidobenzodiazepine-2,4-diones was synthesized and evaluated as cholecystokinin-B (CCK-B) receptor antagonists. Structure-activity relationship (SAR) studies revealed the importance of the N-1 substituent for potent and selective CCK-B affinity. Addition of substituents at the urea side chain provided in some cases more potent compounds. Moreover the introduction of bulky substituents such as adamantylmethyl at N-1 and resolution of the racemic ureas resulted in our lead compound GV150013.

Statistical analysis on a series of glycine antagonists.

Quantitative structure-activity relationships of two series of glycine antagonists, pyrido[2,3-b]pyrazines and pyrido[2,3-b]pyrazine N-oxides, was performed using PLS (Projection on Latent Variables) and traditional physico-chemical and topological descriptors. The effect of substitution on the heteroaromatic ring was investigated with the aim of further improving the affinity (expressed as pKi) of these derivatives towards the strychnine-insensitive glycine binding site associated with the NMDA receptor. A significant model was obtained for both series of compounds. Structure-activity implications are discussed.

Substituted analogues of GV150526 as potent glycine binding site antagonists in animal models of cerebral ischemia.

A series of analogues of the indole-2-carboxylate GV150526, currently in clinical trials as a potential neuroprotective agent for the control of the cerebral damage after stroke onset, was designed based on previous studies dealing with the electronic features of the north-east region of the glycine binding site associated with the NMDA receptor. In particular, the substitution of the para position of the terminal phenyl ring of GV150526 with suitable hydrophilic groups resulted in the identification of a new class of glycine antagonists. These compounds exhibited nanomolar in vitro affinity to the glycine binding site, high receptor selectivity, and outstanding in vivo potency. In particular, 3-[(E)-2-[(4-ureidomethylphenyl)aminocarbonyl]ethenyl]-4, 6-dichloroindole-2-carboxylic acid was found to be highly effective in the middle cerebral artery occlusion (MCAo) model in the rat, an animal model of focal ischemia, when given both prior to and after the occlusion of the middle cerebral artery. Notably, a significant neuroprotective effect was seen in this model postischaemia, when the administration of this compound was delayed up to 6 h from the occlusion of the middle cerebral artery, further confirming the wide therapeutic window seen for GV150526A.

Cycloalkyl indole-2-carboxylates as useful tools for mapping the "north-eastern" region of the glycine binding site associated with the NMDA receptor.

A novel series of indole-2-carboxylate analogues of GV 150526 (1) in which the terminal phenyl ring belonging to the side chain present in the position C-3 has been replaced with a bridged cycloalkyl group was synthesized and evaluated for its pharmacological profile. Modelling studies on this class of novel glycine antagonist allowed us to identify an asymmetric lipophilic pocket present in the "North-Eastern" region of the pharmacophoric model of the glycine binding site associated to the NMDA receptor. Among the derivatives prepared, 3-[2-(1-adamantylaminocarbonyl)ethenyl]-4,6-dichloroindole-2 -carboxylic acid 6b and 3-[2-(norbornylaminocarbonyl)ethenyl]-4,6-dichloroindole-2-c arboxylic acid 6l were found to be antagonists acting at the strychnine-insensitive glycine binding site, showing nanomolar affinity for the glycine binding site (Ki = 63 and 19 nM, respectively), coupled with high glutamate receptor selectivity (IC50 > 10(-5) M at the NMDA, AMPA, KA binding sites) and high in vivo potency after systemic administration by inhibition of convulsion induced by NMDA in mice.

(E)-3-(2-(N-phenylcarbamoyl)vinyl)pyrrole-2-carboxylic acid derivatives. A novel class of glycine site antagonists.

The synthesis and preliminary biological evaluation of novel (E)-3-(2-(N-phenylcarbamoyl)-vinyl)pyrrole-2-carboxylic acids bearing alkyl, acyl, alkoxy, phenyl, and halo substituents at the 4- and 5-positions of the pyrrole ring are reported. These compounds were studied for their in vitro affinity at the strychnine-insensitive glycine-binding site of the N-methyl-D-aspartate (NMDA) receptor complex. In the [3H]glycine binding assay (E)-4,5-dibromo-3-(2-(N-phenylcarbamoyl)vinyl)pyrrole-2-carboxylic acid 6w (pKi = 7.95 +/- 0.01) and the 4-bromo-5-methyl 6j (pKi = 7.24 +/- 0.01) and 4,5-dimethyl 6g (pKi = 6.70 +/- 0.03) analogues were the most active compounds of the series. Qualitative structure-activity analysis points to a negative correlation between bulk of the C-4 and C-5 substituents and affinity which is enhanced by halo-substituents. QSAR analysis by the Hansch descriptors F, R, pi, and MR, on a subset of compounds with pKi > or = 4, indicates that electron-withdrawing groups at C-4 and C-5 enhance the affinity. Bulk and lipophilicity are also relevant for the substituents at these positions. 6g was found to be a full antagonist (alpha = 0; enhancement of the [3H]TCP binding). The in vivo potency of 6g, 6j, and 6w was evaluated by the inhibition of NMDA-induced convulsions in mice by both the i.v. and po routes; 6w was the most active compound (ED50 = 3 x 10(-3) (0.8-10) g/kg, i.v. and 30 x 10(-3) (4.5-61) g/kg, p.o.). The results of this study indicate that the 3,4-disubstitutedpyrrole-2-carboxylate represents a novel template for the design of new glycine antagonists.

Substituted indole-2-carboxylates as in vivo potent antagonists acting as the strychnine-insensitive glycine binding site.

A series of indole-2-carboxylates bearing suitable chains at the C-3 position of the indole nucleus was synthesized and evaluated in terms of in vitro affinity using [3H]glycine binding assay and in vivo potency by inhibition of convulsions induced by N-methyl-D-aspartate (NMDA) in mice. 3-[2-[(Phenylamino)carbonyl]ethenyl]-4,6-dichloroindole-2-carboxyl ic acid (8) was an antagonist at the strychnine-insensitive glycine binding site (noncompetitive inhibition of the binding of [3H]TCP, pA2 = 8.1) displaying nanomolar affinity for the glycine binding site (pKi = 8.5), coupled with high glutamate receptor selectivity (> 1000-fold relative to the affinity at the NMDA, AMPA, and kainate binding sites). This indole derivative inhibited convulsions induced by NMDA in mice, when administered by both iv and po routes (ED50 = 0.06 and 6 mg/kg, respectively). The effect of the substituents on the terminal phenyl ring of the C-3 side chain was investigated. QSAR analysis suggested that the pKi value decreases with lipophilicity and steric bulk of substituents and increases with the electron donor resonance effect of the groups present in the para position of the terminal phenyl ring. According to these results the terminal phenyl ring of the C-3 side chain should lie in a nonhydrophobic pocket of limited size, refining the proposed pharmacophore model of the glycine binding site associated with the NMDA receptor.

Synthesis of 5-membered ring-type compounds as potential cholecystokinin receptor ligands.

Imidazolidine-2,4-diones and 1,5-diphenyl tetramic acid derivatives were selected in order to evaluate some 5-membered heterocyclic ring compounds as potential templates for the synthesis of CCK receptor ligands. All the compounds were evaluated in vitro towards both CCK-B and CCK-A receptors.

Cholinergic agents structurally related to furtrethonium. 2. Synthesis and antimuscarinic activity of a series of N-[5-[(1'-substituted-acetoxy) methyl]-2-furfuryl]dialkylamines.

In the first part of this study, devoted to the discovery of selective antimuscarinic agents, (+/-)- N-[5-[(1'-phenyl-1'-cyclohexylacetoxy)methyl]-2-furfuryl]dimeth yla mine (5a) proved to be at least 20 times more potent in the rat ileum and bladder than in guinea pig atria. Several (+/-)-N- [5-[(1'-substituted-acetoxy)methyl]-2-furfuryl]dialkylamine analogs of 5a were subsequently prepared. This involved exploration of the tertiary nitrogen substituents and modulation of the lipophilic side chain at position 5 of the furan ring, using the Hansch approach. A QSAR study was conducted to correlate activity with physicochemical properties of substituents. The possibility of describing all compounds in a single model indicates that variations of nitrogen and the lipophilic side chain contribute independently to activity. Compounds 5b, c,j, with bulky lipophilic substituents at the tertiary nitrogen, showed unprecedented selectivity between the two smooth muscle tissues, their antimuscarinic potency being from 10 to 90 times higher in the ileum than in the bladder. It is suggested that their interesting tissue selectivity is probably related to nonspecific phenomena involving the receptor environment, rather than real differences between the muscarinic receptors in the two tissues.

Synthesis and antimicrobial activity of DNA-gyrase inhibiting derivatives of 4-oxo-1,4-dihydro-3-pyridinecarboxylic acid.

A series of 6-substituted-1-aryl-4-oxo-1,4-dihydronicotinic acids were synthesised as monocyclic analogues of the quinolones. The 6-(2-aryl-1-methylethenyl)- and of the 6-(2-arylethenyl)-substituted compounds were shown to possess antibacterial properties that correlate with DNA gyrase inhibitory activity. Differently from the quinolones the antimicrobial activity of the compounds of this study is predominantly against Gram positive strains. The structure-activity relationships ascertained for these monocyclic compounds differ from those established for the quinolones.

A novel class of cholinergic agents structurally related to 2-morpholinol.

A series of esters and ethers of N-alkylmorpholin-2-ols, and their methiodides, which can be considered cyclic analogues of acetylcholine, were synthesized. The amines were obtained by acylation or etherification of morpholinols with the appropriate acyl chlorides and alcohols. All compounds were tested for their ability to interact with the muscarinic receptor M2 (guinea-pig atria) or M3 (rat ileum and urinary bladder) subtype. Some compounds, although endowed with relatively low potency, proved interesting for their organ selectivity. Some considerations on the structure-activity relationship are made and the results obtained with reference agonists and antagonists are also shown.