New benzomorphan derivatives of MPCB as MOP and KOP receptor ligands.

There is considerable interest in developing KOP Opioid receptor ligands as clinically useful analgesics. Moreover, compounds with mixed KOP receptor and mu-opioid peptide (MOP) receptor agonist/antagonist properties could have a better therapeutic potential. The benzomorphan-based synthetic ligands MPCB and CCB have been shown to bind KOP receptors with high affinity and selectivity. We report here a series of compounds synthesized to perform structure-affinity relationship (SAR) studies on MPCB. The aim of this study was to optimise KOP receptor-ligand interaction and to modulate MOP receptor selectivity. In the benzylamide analogue of MPCB (compound 9) the presence of a third aromatic nucleus, at an appropriate distance and conformation with respect to aromatic pharmacophoric residues, increased KOP receptor affinity by about 6-fold compared to MPCB (Ki = 35 nM and Ki = 240 nM, respectively). Instead, compound 28 with a tertiary amino group in the nitrogen substituent displayed a comparable KOP receptor affinity (Ki = 179 nM) but also high MOP receptor affinity (Ki = 45 nM). Thus, the present study shows that in benzomorphan-based ligands the presence of different functional groups in the nitrogen substituent, ranging from a positive charged amine to an additional aromatic ring, is able to promote the correct aligment of aromatic pharmacophoric residues with MOP and KOP receptor types. Evaluation of docking simulations of compounds 9 and 28 into the KOP and MOP receptor displayed selective ligand interactions with the important amino acid residues Tyr320 (TMVII) and Trp318 (TMVII), respectively.

The in vitro effect of a lyophilized extract of wine obtained from Jacquez grapes on human chondrocytes.

The present work was aimed at evaluating the in vitro effects of a lyophilized extract of wine (JW-E) obtained from Jacquez grapes (Vitis aestivalis-cinereaxVitis vinifera grapes) on the production of key molecules released in inflammatory disease utilising interleukin-1beta (IL-1beta) activated chondrocytes. The extract contains large amounts of phenolic components, in particular some flavonoids (flavan-3-ols, also known as catechins) and proanthocyanidins, as hydroxycinnamic acids and anthocyanins, that possess several biological features such as antiinflammatory and antioxidant effects and a "radical scavenger" activity too. In this study, we assayed the effect of JW-E on the production of key molecules released during chronic inflammatory events as nitric oxide (NO), prostaglandins E(2) (PGE(2)) and reactive oxygen species (ROS) in human chondrocytes culture, stimulated with proinflammatory cytokine interleukin-1beta. The JW-E proved to possess good ability against the harmfull effects of IL-1beta. Our data showed the protective effects of JW-E in cartilage alteration, that appears greater than that elicited by indomethacin, a not steroidal antiinflammatory drug (NSAID), commonly employed in joint diseases.

Protective effect of Capparis spinosa on chondrocytes.

The aim of the present study was to evaluate the in vitro chondroprotective effects of the lyophilised methanolic extract from flowering buds of Capparis Spinosa L (LECS). This plant, common to the Mediterranean basin, has been used by the traditional medicine for its diuretic and antihypertensive effects and also in certain pathological conditions related to uncontrolled lipid peroxidation. The extract contains many constituents, in particular some flavonoids (kaempferol and quercetin derivatives) and hydrocinammic acids with several known biological effects such as the anti-inflammatory and the antioxidant ones. In this study, we assayed the effect of LECS on human chondrocytes cultures stimulated by proinflammatory cytokine interleukin-1beta (IL-1beta) and we determined the production of key molecules released during chronic inflammatory events (nitric oxide, glycosaminoglycans, prostaglandins and reactive oxygen species). We observed that LECS was able to counteract the harmful effects induced by IL-1beta. This protection appeared to be greater than that elicited by indomethacin, which is usually employed in joint diseases. Since LECS possess a chondroprotective effect, it might be used in the management of cartilage damage during the inflammatory processes.

Protective effects of benzisothiazolylamidines on IL-1 beta induced alterations in human articular chondrocyte metabolism.

The in vitro effects on human articular chondrocytes were evaluated for a series of N-benzo[d]isothiazol-3-yl-amidines, bearing as pharmacophoric moiety the nonacidic isosteric nitrogen analogue of the carboxylic group. The aim was to verify their effectiveness in articular diseases, such as arthritis. Human chondrocytes were treated with IL-1beta in the presence of a series of N-benzo[d]isothiazol-3-yl-amidines at a concentration of 100 microg/mL. After 120 h, the amount of glycosaminoglycans (GAGs), the production of nitric oxide (NO) and the inhibition of metalloproteinases (MMP-3) and prostaglandin (PGE2) were measured. Nitrite production induced by inflammatory IL-1beta on cultured chondrocytes was inhibited by the N-benzo[d]isothiazol-3-yl-amidines tested, in particular by N-benzo[d]isothiazol-3-yl-benzamidine, which was the most active. Concerning the effects on GAGs, all the tested benzisothiazolylamidines, and in particular N-benzo[d]isothiazol-3-yl-acetamidine, prevented the depletion of proteoglycan induced by IL-1beta. Inhibitory effects of the tested compounds on MMP-3 activity and on PGE2 production were also observed.

Synthesis and anti-measles virus activity of new isoquinolin-4-one derivatives.

Despite intense efforts to increase vaccine coverage, measles virus (MV) still causes significant morbidity and mortality in the world sometimes as a results of severe, chronic and lethal diseases. In an effort to develop therapies to supplement immunization strategies a number of 1-oxo-2-[[(1E)-phenylmethylene]amino]-1,2-dihydroisoquinoline-4-carboxylic acid derivatives were synthesized and evaluated for anti-measles activity. The substituents on the aromatic ring were chosen in order to evaluate the influence of electron-withdrawing or electron-donating effects on the electronic density of the aromatic moiety. We also evaluated the introduction of a vinyl chain between the exocyclic nitrogen and phenyl moiety. The biological results allow to outline some preliminary considerations on structure-activity relationship.

Different in vitro activity of flurbiprofen and its enantiomers on human articular cartilage.

The 2-arylpropionic acid derivatives or 'profens' are an important group of non-steroidal anti-inflammatory drugs that have been used for the symptomatic treatment of various forms of arthritis. These compounds are chiral and the majority of them are still marketed as racemate although it is known that the (S)- form is the principal effective in the cyclooxygenase inhibition. However, recent findings suggest that certain pharmacological effect of 2-arylpropionic acids cannot be attributed exclusively to the (S)-(+) enantiomer. To obtain further insights into the pharmacological effect of profens, the present study investigated the influence of racemic and pure enantiomers of flurbiprofen on the production of nitric oxide and glycosaminoglycans, key molecules involved in cartilage destruction. The culture of human articular cartilage stimulated by interleukin-1beta (IL-1beta), which plays an important role in the degradation of cartilage, has been established, as a profit experimental model, for reproducing the mechanisms involved in the pathophysiology of arthritic diseases. Our results show that mainly (S)-(+)-flurbiprofen decreases, at therapeutically concentrations, the IL-1beta induced cartilage destruction.

Determination of undecylenic and sorbic acids in cosmetic preparations by high performance liquid chromatography with electrochemical detection.

A highly sensitive and selective method for the determination of sorbic (SA) and undecylenic acid (UA) in cosmetic formulations by a high performance liquid chromatography method with electrochemical detection (ECD) is described. The pre-column derivatizations of SA and UA and the internal standard (cyclohexanoic acid (cHA)) were carried out using 1-(2,5-dihydroxyphenyl)-2-bromoethanone (2,5-DBE) as an electroactive labeling reagent previously synthesized in our lab. The resulting electroactive esters were separated by isocratic elution of a 5 micrometer Hypersil CN column with acetonitrile-acetate buffer eluent. The compounds were detected by a porous graphite electrode set at an oxidation potential of +0.45 V. The analytical method developed in this study is suitable for quality control assays of complex cosmetic formulations containing sorbic and/or UA.

Analysis of aliphatic amines in air samples by HPLC with electrochemical detection.

A method was developed for the analysis of primary aliphatic amines by high performance liquid chromatography coupled with electrochemical detector. The electrochemical oxidation of aliphatic amines derivatized with 2,5-dihydroxybenzaldehyde was investigated at porous graphite electrodes. The derivatization reactions were performed off-line, before the chromatographic separation. The compounds were separated on a reversed phase column with a methanol-acetonitrile-phosphate buffer and detected setting at an oxidation potential of +0.5 V. The influence of the mobile phase buffer concentration and pH on the detector response was also studied. The derivatization was shown to be quantitative and the response linear between 50 and 200 ng/ml. The method is sensitive, selective and could be applicable for the assay of volatile amines in the field of environmental toxicology and also for biological monitoring after occupational exposure.

In vitro evaluation of thiazolyl and benzothiazolyl Schiff bases on pig cartilage.

A series of anti-inflammatory agents known as Schiff bases, combining thiazolyl and benzothiazolyl ring and vanillin moieties in the same molecule, was synthesized and evaluated for screening anti-degenerative activity on nasal pig cartilage cultures treated with interleukin 1beta, (IL-1beta). The amount of glycosaminoglycans (GAGs), the production of nitric oxide (NO) and prostaglandin E2 (PGE2), released into the culture medium, were detected. The tested Schiff bases decreased, dose-dependently, the NO and PGE2 production and the GAGs release with respect to samples treated with IL-1beta alone, showing a different behavior correlated to their structure. These results suggest that thiazolyl and benzothiazolyl Schiff bases in general, and particularly the Schiff base with bromine and methoxyl group in position three would protect cartilage matrix from degenerative factors induced by IL-1beta.

Synthesis and pharmacological evaluation of potent and enantioselective sigma 1, and sigma 2 ligands.

In a previous study we found that substitutions of the (+)-cis-N-normetazocine nucleus of (+)-MPCB with 1-adamantanamine provide the compound (+/-)-10 with high affinity and selectivity for sigma receptors. Starting with this result we have synthesized a new series of eight 1-phenyl-2-cyclopropylmethylamines structurally related to (+/-)-10, and binding affinities, with respect to sigma 1, sigma 2, opioid and dopaminergic D2 receptors, have been reported. All compounds showed a negligible opioid and dopaminergic affinity and high selectivity for sigma receptors. Modifications on the amino moiety and methylcarboxyester group of 10 provide compounds with different sigma 1 and sigma 2 binding affinity and selectivity. Moreover, we have also synthesized the respective enantiomers of componds (+/-)-10 and (+/-)-18 in order to evaluate the enantioselectivity for sigma 1 and sigma 2 receptors. The binding data showed that carboxymethylester on the cyclopropane ring was more critical for enantioselectivity than the hydroxymethylenic group. In fact, the (-)-10 enantiomer showed a preference for sigma 1 whereas (+)-10 showed a preference for sigma 2.

Specific kappa opioid receptor agonists.

The results of studies on the design of a heterocyclic scaffold for the dynorphin A pharmacophore and on structure-affinity relationships in the MPCB/CCB series are described. The representative ligands provide insights to binding modes of benzomorphan derivatives to the kappa opioid receptor.

Synthesis and binding affinity of cis-(-)- and cis-(+)-N-ethyleneamino-N-nordeoxymetazocine and cis-(-)-N-normetazocine analogues at sigma1, sigma2 and kappa opioid receptors.

The synthesis of cis-(+)- and cis-(-)-N-ethyleneamino-N-nordeoxymetazocine and cis-(-)-N-normetazocine analogues is described and their affinities to sigma1, sigma2 and kappa opioid receptors are evaluated. The cis-(+)-deoxy compounds displayed high sigma/kappa selectivity with nanomolar K(i) values for sigma1 receptors, whereas in the cis-(-)-N-normetazocine series the compound (-)-7b was found to bind with nanomolar affinity to the kappa opioid receptor (K(i)=21.5 nM). Compound (-)-7b showed good selectivity for the kappa opioid receptor in comparison to the sigma1 and sigma2 sites and to the mu and delta opioid receptors. A correlation of the binding affinities between cis-(-)- and cis-(+)-N-deoxynormetazocine derivatives show that both isomers of the deoxy analogs have similar sigma1 and sigma2 binding profiles as the cis-(+)-N-normetazocine derivatives.

Nonpeptide analogues of dynorphin A(1-8): design, synthesis, and pharmacological evaluation of kappa-selective agonists.

Two novel series of kappa opioid receptor agonist analogues of MPCB-GRRI and MPCB-RRI, hybrid ligands of MPCB ((-)-cis-N-(2-phenyl-2-carbomethoxy)cyclopropylmethyl-N-normetazocine ) and of the C-terminal fragments of dynorphin A(1-8), have been synthesized. The critical functional groups of the peptide fragments of hybrid compounds were maintained, and the binding affinities and selectivities for compounds 1-40 to mu, delta, and kappa opioid receptors were analyzed. Compounds 15 and 16, MPCB-Gly-Leu-NH-(CH(2))(n)()-NH-C(=NH)-C(4)H(9) (n = 5, 6), displayed high affinity and selectivity for kappa opioid receptors (K(i)(kappa) = 6.7 and 5.3 nM, K(i)(mu)/K(i)(kappa) = 375 and 408, and K(i)(delta)/K(i)(kappa) = 408 and 424, respectively). Since kappa agonists may also cause psychotomimetic effects by interaction with sigma sites, binding assays to sigma(1) sites were performed where compounds 15 and 16 showed negligible affinity (K(i) > 10 000). Compounds 15 and 16 were further characterized in vivo and showed potent antinociceptive activity in mouse abdominal constriction tests (ED(50) = 0.88 and 1.1 mg/kg, respectively), fully prevented by nor-BNI. Thus, these novel analogues open an exciting avenue for the design of peptidomimetics of dynorphin A(1-8).

Substituted 1-phenyl-2-cyclopropylmethylamines with high affinity and selectivity for sigma sites.

A series of 1-phenyl-2-cyclopropylmethylamines structurally related to (+)- and (-)-MPCB were synthesized and their binding affinities for sigma1, sigma2, opioid and dopamine (D2) receptors were evaluated. Substitution of the cis-N-normetazocine with different aminic moieties provided compounds with high affinity and selectivity for sigma binding sites with respect to opioid and dopamine (D2) receptors. The observed increase in sigma2 affinity as compared to the parent (+)-MPCB, supports the idea that the particular stereochemistry of (+)-cis-N-normetazocine affects sigma1 selectivity but does not affect sigma1 affinity. The (+/-)-cis isomers of methyl 2-[(1-adamantylamino)methyl]-1-phenylcyclopropane-1-carboxyl ate (18) displayed a higher affinity and selectivity for the sigma1 and sigma2 receptor subtypes compared to the (+/-)-trans 19. Interestingly, the enantiomer (-)-cis 18 displayed a preference for sigma1 receptor subtype whereas the (+)-cis 18 did for sigma2. These results prompt us to synthesize compounds with modification of nitrogen and carboxyl groups. The compounds obtained showed high affinities and selectivity for sigma sites. Moreover, modifications of carboxyl groups provided compounds with the highest affinities in the series. In particular, compound 25 with reverse-type ester showed a Ki of 0.6 and 4.05 nM for sigma1 and sigma2 binding sites, respectively.

Modeling of kappa-opioid receptor/agonists interactions using pharmacophore-based and docking simulations.

The interaction of the kappa-opioid receptor with arylacetamide and benzomorphan derivatives acting as agonists was modeled through pharmacophore-based and docking calculations. Potentially bioactive conformations of representative ligands (U-50,488 and its benzo-fused analogues 4 and 6 for arylacetamides and MPCB for benzomorphans) were identified by systematic conformational analysis and docked into a 3D model of the kappa-receptor. The obtained complexes, refined by energy-minimization and molecular dynamics, were evaluated for their consistency with structure-activity relationships and site-directed mutagenesis data. The following interactions are hypothesized to govern the ligand-receptor recognition process: (i) a salt bridge between the Asp138 carboxylate and the protonated nitrogen of the bound agonist; (ii) a hydrogen bond donated by the Tyr312 hydroxyl to the carbonyl oxygen of arylacetamides and MPCB; (iii) hydrophobic interactions established by the dichlorophenyl moiety of arylacetamides and the pendant phenyl ring of MPCB with the surrounding side chains of Tyr312, Leu224, Leu295, and Ala298; (iv) a pi-stacking contact between the Tyr312 side chain and the phenyl ring of arylacetamides; (v) a hydrogen bond linking the His291 imidazole ring to the phenolic hydroxy group featured by typical benzomorphans and the arylacetamides 4 and 6.

4-quinazolinones: synthesis and reduction of prostaglandin E2 production.

We synthesized and evaluated the anti-inflammatory activity of a series of 4-quinazolinone derivatives. Two approaches were used to yield the title compounds. A first group of quinazolinone derivatives was obtained by the appropriate substituted anthranilates. A second group of quinazolinone compounds was prepared through the benzoxazin-4-ones intermediate. The pharmacological results reveal that the synthesized derivatives exhibit a significant anti-inflammatory effect in an experimental ocular inflammation model. In fact, all the tested compounds lowered the prostaglandin E2 (PGE2) production with respect to the control group (P < 0.05). The 3-cyclohexyl-6-chloro-quinazolin-4(3H)-one and 3-cyclohexyl-quinazolin-4(3H)-one derivatives were the most active compounds. These compounds significantly reduced PGE2 levels even more than the reference drug tolmetin and significantly lower protein concentration and polymorphonuclear leukocytes number compared to the control group (P < 0.05). Therefore, these compounds may be useful to prevent ocular inflammatory reactions.

(+)-cis-N-ethyleneamino-N-normetazocine derivatives. Novel and selective sigma ligands with antagonist properties.

A series of (+)-cis-N-normetazocine derivatives has been described, and their affinities for sigma1, sigma2, and phencyclidine (PCP) sites and opioid, muscarinic (M2), dopamine (D2), and serotonin (5-HT2) receptors were evaluated. The effect of the N-substitution with a substituted ethylamino spacer was investigated. Compounds 8c-11c displayed high affinities for sigma1 sites and for opioid receptors. Substitution of the second basic nitrogen either with alkyl or cycloalkyl substituents give compounds (1a-6a) with high affinity and selectivity for sigma1 binding sites. Compounds 1a-5a were further characterized in vivo, and their agonist/antagonist activity was evaluated. In mouse, compound 1a and 2a as well as haloperidol suppressed in a dose-related manner the stereotyped behavior induced by (+)-SKF 10,047. Compounds 3a-5a and (+)-pentazocine do not affect the stereotyped behavior induced by ip injection of (+)-SKF 10,047. Therefore, from this series of compounds we identified potent and selective sigma1 ligands which might prove useful to unveil the functional role of sigma1 sites.

Synthesis of (+)-(1'R,2'S) and (1'S,2'R)-6,11-dimethyl-1,2,3,4,5,6 -hexahydro-3-[[2'-(alkoxycarbonyl)-2'-phenylcyclopropyl]methyl]-2 ,6 -methano-3-benzazocin-8-ol. Comparison of the affinities for sigma 1 and opioid receptors with in the diastereoisomeric MPCB and CCB.

The synthesis and the in vitro receptor affinity for sigma 1 and opiod receptors of the two diastereoisomers of (+)-cis-MPCB namely, (+)-cis-(1'S,2'R)-6,11-Dimethyl-1,2,3,4,5,6 -hexahydro-3-[[2'-(methoxycarbonyl)-2'-phenylcyclopropyl]methyl]-2 ,6 -methano-3-benzazocin-8-ol, (1'S,2'R)6a and (+)-cis-(1'R,2'S)-6,11-Dimethyl-1,2,3,4,5,6-hexahydro-3- [[2-(methoxycarbonyl)-2'-phenylcyclopropyl]methyl]-2,6-methano-3-+ ++benzazocin-8 -ol, (1'R,2'S)6a are reported. Affinities of (1'S,2'R)6a and (1'R,2'S)6a were compared with those of the (-)-cis-diastereoisomers of MPCB(1), and of its p-Cl phenyl derivative CCB(2). The (+)-cis-N-normetazocine derivatives showed higher affinity for the sigma 1 sites, labeled with [3H]-(+)-pentazocine than the corresponding (-)-cis- analogs. In particular, compound (1'S,2'R)6a showed a Ki = 66.7 nM for sigma 1 receptor, associated with a good selectivity for sigma 1 with respect to kappa, mu, delta opioid receptors subtypes (Ki = > 1,000 nM). Analysis of the data seem to support the hypothesis that the (+)-cis-N-normetazocine nucleus posses a specific enantioselectivity for sigma 1 sites, when supporting bulkier N-substituents functionalized with a carboxy ester group.

Synthesis and antimicrobial evaluation of 4-phenyl-3-isoquinolinoyl-hydrazones.

2-Methyl-1-oxo-1,2-dihydro-3-carbazoyl-4-phenylisoquinoline 2, 1-methoxy-7 and 1-chloro-3-carbazoyl-4-phenylisoquinoline 12 as well as a series of their 2-hydrazono-derivatives 3 a-i, 8 a-i and 14 a-i were synthesized and evaluated for their antibacterial and antifungal activities, in vitro. Compound 3 h was fairly active against Staphylococcus aureus, Staphylococcus epidermidis and streptococci group B.