Peroxynitrite induced formation of the neurotoxins 5-S-cysteinyl-dopamine and DHBT-1: implications for Parkinson's disease and protection by polyphenols

Mechanisms of nigral cell injury in Parkinson’s disease remain unclear, although a combination of increased oxidative stress, the formation of catecholamine-quinones and the subsequent formation of neurotoxic cysteinyl-catecholamine conjugates may contribute. In the present study, peroxynitrite was observed to generate both 2-S- and 5-S-cysteinyl-dopamine and a dihydrobenzothiazine species, DHBT-1, following the reaction of dopamine with l-cysteine. The formation of 5-S-cysteinyl-dopamine and DHBT-1 in the presence of peroxynitrite induced significant neuronal injury. Pre-treatment of cortical neurons with pelargonidin, quercetin, hesperetin, caffeic acid, the 4′-O-Me derivatives of catechin and epicatechin (0.1-3.0 μM) resulted in concentration dependant protection against 5-S-cysteinyl-dopamine-induced neurotoxicity. These data suggest that polyphenols may protect against neuronal injury induced by endogenous neurotoxins relevant to the aetiology of the Parkinson disease.     

Inhibition of the formation of the neurotoxin 5-S-cysteinyl-dopamine by polyphenols

The death of nigral neurons in Parkinson's disease is thought to involve the formation of the endogenous neurotoxin, 5-S-cysteinyl-dopamine. In the present study, we show that the polyphenols, (+)-catechin and caffeic acid, which contain a catechol moiety, inhibit tyrosinase-induced formation of 5-S-cysteinyl-dopamine via their capacity to undergo tyrosinase-induced oxidation to yield cysteinyl-polyphenol adducts. In contrast, the inhibition afforded by the flavanone, hesperetin, was not accompanied by the formation of cysteinyl-hesperetin adducts, indicating that it may inhibit via direct interaction with tyrosinase. Whilst the stilbene resveratrol also inhibited 5-S-cysteinyl-dopamine formation, this was accompanied by the formation of dihydrobenzothiazine, a strong neurotoxin. Our data indicate that the inhibitory effects of polyphenols against 5-S-cysteinyl-dopamine formation are structure-dependent and shed further light on the mechanisms by which polyphenols exert protection against neuronal injury relevant to neurodegenerative diseases.     

Determination of 5-S-cysteinyldopa in plasma and urine using a fully automated solid-phase extraction–high-performance liquid chromatographic method for an improvement of specificity and sensitivity of this prognostic marker of malignant melanoma

5-S-Cysteinyldopa (5-SCD) in plasma and urine was determined by means of a newly developed method. This method incorporates optimized conditions for blood collection and storage, as well as a new extraction and separation technique, required for the strong oxidation and light sensitive 5-SCD. The new aspects of the method are the following: immediate centrifugation and freezing of the samples after blood collection, fully automatical solid-phase extraction (SPE) with phenylboronic acid (PBA) cartridges and immediate HPLC injection of the eluate, nearly complete exclusion of light and air–oxygen during extraction, constant sample cooling, use of the more suitable internal standard 5-S- -cysteinyldopa and easy, sensitive and selective HPLC conditions (RP18-column with isocratic separation and electrochemical detection). The method has a linear range from 0.25 to 50 μg l⁻¹ and 25 to 5000 μg l⁻¹ for plasma and urine samples, respectively, a limit of detection of 0.17 μg l⁻¹, intra-assay variabilities from 1.7 to 3.6%, inter-assay variabilities from 4.0 to 18.3% and an average relative recovery of 103.5% for plasma and 105.4% for urine samples. In our study the measured 5-SCD concentrations of patients with melanomas at various stages correlated better with their clinical pictures than described in literature up to date. The results were obtained in comparison to patients with other skin tumors and in comparison to healthy control persons.     

Discovery of 5-substituent-N-arylbenzamide derivatives as potent,selective and orally bioavailable LRRK2 inhibitors

Leucine-rich repeat kinase 2 (LRRK2) has been suggested as a potential therapeutic target for Parkinson’s disease. Herein we report the discovery of 5-substituent-N-arylbenzamide derivatives as novel LRRK2 inhibitors. Extensive SAR study led to the discovery of compounds 8e, which demonstrated potent LRRK2 inhibition activity, high selectivity across the kinome, good brain exposure, and high oral bioavailability.     

Induction of gentisic acid 5-O-beta-D-xylopyranoside in tomato and cucumber plants infected by different pathogens

Tomato plants infected with the citrus exocortis viroid exhibited strongly elevated levels of a compound identified as 2,5-dihydroxybenzoic acid (gentisic acid, GA) 5-O-beta-D-xylopyranoside. The compound accumulated early in leaves expressing mild symptoms from both citrus exocortis viroid-infected tomato, and prunus necrotic ringspot virus-infected cucumber plants, and progressively accumulated concomitant with symptom development. The work presented here demonstrates that GA, mainly associated with systemic infections in compatible plant-pathogen interactions [Bellés, J.M., Garro, R., Fayos, J., Navarro, P., Primo, J., Conejero, V., 1999. Gentisic acid as a pathogen-inducible signal, additional to salicylic acid for activation of plant defenses in tomato. Mol. Plant-Microbe Interact. 12, 227-235], is conjugated to xylose. Notably, this result contrasts with those previously found in other plant-pathogen interactions in which phenolics analogues of GA as benzoic or salicylic acids, are conjugated to glucose.     

Design and synthesis of potent, orally-active DGAT-1 inhibitors containing a dioxino[2,3-d]pyrimidine core

A novel series of potent DGAT-1 inhibitors was developed originating from the lactam-based clinical candidate PF-04620110. Incorporation of a dioxino[2,3-d]pyrimidine-based core afforded good alignment of pharmacophore features and resulted in improved passive permeability. Development of an efficient, homochiral synthesis of these targets facilitated confirmation of predictions regarding the stereochemical-dependence of DGAT-1 inhibition for this series. Compound 10 was shown to be a potent inhibitor of human DGAT-1 (10 nM) and to suppress triglyceride synthesis at oral doses of <3mg/kg.     

Crystal structure of Plasmodium falciparum spermidine synthase in complex with the substrate decarboxylated S-adenosylmethionine and the potent inhibitors 4MCHA and AdoDATO

Plasmodium falciparum is the causative agent of the most severe type of malaria, a life-threatening disease affecting the lives of over three billion people. Factors like widespread resistance against available drugs and absence of an effective vaccine are seriously compounding control of the malaria parasite. Thus, there is an urgent need for the identification and validation of new drug targets. The enzymes of the polyamine biosynthesis pathway have been suggested as possible targets for the treatment of malaria. One of these enzymes is spermidine synthase (SPDS, putrescine aminopropyltransferase), which catalyzes the transfer of an aminopropyl moiety from decarboxylated S-adenosylmethionine (dcAdoMet) to putrescine, leading to the formation of spermidine and 5'-methylthioadenosine. Here we present the three-dimensional structure of P. falciparum spermidine synthase (pfSPDS) in apo form, in complex with dcAdoMet and two inhibitors, S-adenosyl-1,8-diamino-3-thio-octane (AdoDATO) and trans-4-methylcyclohexylamine (4MCHA). The results show that binding of dcAdoMet to pfSPDS stabilizes the conformation of the flexible gatekeeper loop of the enzyme and affects the conformation of the active-site amino acid residues, preparing the protein for binding of the second substrate. The complexes of AdoDATO and 4MCHA with pfSPDS reveal the mode of interactions of these compounds with the enzyme. While AdoDATO essentially fills the entire active-site pocket, 4MCHA only occupies part of it, which suggests that simple modifications of this compound may yield more potent inhibitors of pfSPDS.     

Thermodynamic stabilities and conformational analyses of 4,6-O-acetalized 1,5-anhydro-5-thio-dl-threo-2-enitols

4,6-O-Methylidene and 4,6-O-neopentylidene derivatives of 1,5-anhydro-2,3-dideoxy-5-thio-dl-thero-hex-2-enitol having the C-inside form were found to be thermodynamically more stable than the corresponding O-inside conformers. Thermodynamic stabilities, as well as the conformation of sulfoxide and sulfone derivatives of the 4,6-O-neopentylidene compound were examined by experiment and ab initio MO and DFT calculations. These thermodynamic stabilities, and the most stable conformations determined by NMR data, were corroborated by calculations.     

5′-Uridyl derivatives of N-glycosyl allophanic acid and biuret

(2′,3′-O-Isopropylidene-5′-uridyl) 4-(2,3,4,6-tetra-O-acetyl-β-d-glycopyranosyl)allophanates were obtained in the reactions of 2′,3′-O-isopropylidene-uridine and O-peracetylated β-d-gluco-, galacto- and xylopyranosylamines, and OCNCOCl. 2,3,4,6-Tetra-O-acetyl-β-d-glucopyranosyl isocyanate and N-(2′,3′-O-isopropylidene-5′-uridyl)urea gave 1-(2,3,4,6-tetra-O-acetyl-β-d-glucopyranosyl)-5-(2′,3′-O-isopropylidene-5′-uridyl)biuret. Deprotection of the β-d-gluco configured allophanate and biuret was carried out by standard methods.     

Pyrimido[4,5-d]azepines as potent and selective 5-HT2C receptor agonists: design, synthesis, and evaluation of PF-3246799 as a treatment for urinary incontinence

New pyrimido[4,5-d]azepines 7 are disclosed as potent 5-HT_2C receptor agonists. A preferred example, 7b had minimal activation at either the 5-HT_2A or 5-HT_2B receptors combined with robust efficacy in a preclinical canine model of stress urinary incontinence (SUI) and attractive pharmacokinetic and safety properties. Based on this profile, 7b (PF-3246799) was identified as a candidate for clinical development for the treatment of SUI. In addition, it proved to be critical to build an understanding of the translation between recombinant cell-based systems, native tissue preparations and in vivo preclinical models. This was a significant undertaking and proved to be crucial in compound selection.     

Discovery of 4-ethoxy-7H-pyrrolo[2,3-d]pyrimidin-2-amines as potent,selective and orally bioavailable LRRK2 inhibitors

Inhibition of LRRK2 kinase activity with small molecules has emerged as a potential novel therapeutic treatment for Parkinson’s disease. Herein we disclose the discovery of a 4-ethoxy-7H-pyrrolo[2,3-d]pyrimidin-2-amine series as potent LRRK2 inhibitors identified through a kinase-focused set screening. Optimization of the physicochemical properties and kinase selectivity led to the discovery of compound 7, which exhibited potent in vitro inhibition of LRRK2 kinase activity, good physicochemical properties and kinase selectivity across the kinome. Moreover, compound 7 was able to penetrate into the CNS, and in vivo pharmacology studies revealed significant inhibition of Ser935 phosphorylation in the brain of both rats (30 and 100?mg/kg) and mice (45?mg/kg) following oral administration.     

Electronic effects on the interactions of complexes [Ru(phen)2(p-L)] (L=MOPIP, HPIP, and NPIP) with DNA

In order to explore the electronic effects of Ru(II) complexes binding to DNA, a series of Ru(II) complexes [Ru(phen)₂ (p-MOPIP)]²⁺ (1), [Ru(phen)₂ (p-HPIP)]²⁺ (2), and [Ru(phen)₂(p-NPIP)]²⁺ (3) were synthesized and characterized by elementary, ¹H NMR, and ES-MS analysis. The binding properties of these complexes to CT-DNA were investigated with spectroscopic methods and viscosity experiments. Furthermore, the computations for these complexes applying the density functional theory (DFT) method have also been performed. The results show that all of these complexes can well bind to DNA in intercalation mode and DNA-binding affinity of these complexes is greatly influenced by electronic effects of intercalating ligands. The intrinsic binding constants for 1, 2, and 3 are 0.20, 0.69, and 1.56 × 10⁵ M⁻¹, respectively. This order is in accordance with that of the electron-withdrawing ability of substituent [-OR < -OH < -NO₂]. Such a trend in electronic effects of Ru(II) complexes binding to DNA can be reasonably explained by the DFT calculations.     

Stereospecific ligands and their complexes. V. Synthesis, characterization and antimicrobial activity of palladium(II) complexes with some alkyl esters of (S,S)-ethylenediamine-N,N′-di-2-propanoic acid

Three new ligands and their palladium(II) complexes of general formula [PdCl₂(R₂-S,S-eddp)] (R = n-propyl, n-butyl and n-pentyl) have been synthesized and characterized by microanalysis, infrared and ¹H and ¹³C NMR spectroscopy. Antimicrobial activity of these ligands and complexes was tested by microdilution method and both minimal inhibitory and microbicidal concentration were determined. These tested complexes demonstrated the significant antifungal activity against pathogenic fungi Aspergillus flavus and Aspergillus fumigatus. On the other hand, these complexes demonstrated moderate antibacterial activity.     

Isomerization and/or racemization at Asp23 of Abeta42 do not increase its aggregative ability, neurotoxicity, and radical productivity in vitro

Aggregation of the 42-mer amyloid β peptide (Aβ42) plays a pivotal role in the pathogenesis of Alzheimer’s disease. Recent investigations suggested the isomerization and/or racemization of Asp at position 1, 7, or 23 to be associated with the pathological role of Aβ42. Our previous study indicated that the turn at positions 22 and 23 of Aβ42 is closely related to its neurotoxicity through the formation of radicals. To clarify the contribution of these modifications at Asp23 to the pathology, three isomerized and/or racemized Aβ42 mutants were prepared. l-isoAsp23- and d-Asp23-Aβ42 showed moderate aggregative ability similar to the wild type. However, d-Asp23-Aβ42 was less neurotoxic than the wild type, while l-isoAsp23-Aβ42 was as toxic as the wild type. In contrast, d-isoAsp23-Aβ42 showed weak aggregative ability without neurotoxicity. These results suggest the isomerization and/or racemization of Asp23 not to be related to the pathogenesis, but to be a consequence of chemical reactions during the long-term deposition of fibrils.     

Efficient synthesis of 5-thio-D-arabinopyranose and 5-thio-D-xylopyranose from the corresponding D-pentono-1,4-lactones

5-Thio-D-arabinopyranose (5) and 5-thio-D-xylopyranose (10) were synthesized from the corresponding D-pentono-1,4-lactones. After regioselective bromination at C-5, transformation into 5-S-acetyl-5-thio derivatives, reduction into lactols and deprotection afforded the title compounds in 49 and 42% overall yield, respectively.     

Toward the synthesis of fine chemicals from lactose: preparation of d-xylo and l-lyxo-aldohexos-5-ulose derivatives

The transformation of (5R)-2,6-di-O-benzyl-5-C-methoxy-β-d-galactopyranosyl-(1→4)-2,3:5,6-di-O-isopropylidene-aldehydo-d-glucose dimethyl acetal (8) into partially protected derivatives of d-xylo- and l-lyxo-aldohexos-5-ulose has been reported, applying appropriate epimerisation methods to its 3′-O- and 4′-O-protected alcoholic derivatives.     

Lactose as an inexpensive starting material for the preparation of aldohexos-5-uloses: synthesis of l-ribo and d-lyxo derivatives

Partially protected derivatives of l-ribo- and d-lyxo-aldohexos-5-ulose have been prepared starting from triacetonlactose dimethyl acetal derivatives. Key steps of the synthetic sequences are (a) the synthesis of 4′-deoxy-4′-eno- and 6′-deoxy-5′-eno lactose derivatives, and (b) the epoxidation-methanolysis of the above-mentioned enol ethers to give 1,5-bis-glycopyranosides, masked form of the target 1,5-dicarbonyl hexoses.     

Repair efficiency of (5′S)-8,5′-cyclo-2′-deoxyguanosine and (5′S)-8,5′-cyclo-2′-deoxyadenosine depends on the complementary base

5′-R and 5′-S diastereoisomers of 8,5′-cyclo-2′-deoxyadenosine (cdA) and 8,5′-cyclo-2′-deoxyguanosine (cdG) containing a base-sugar covalent bond are formed by hydroxyl radicals. R-cdA and S-cdA are repaired by nucleotide excision repair (NER) in mammalian cellular extracts. Here, we have examined seven purified base excision repair enzymes for their ability to repair S-cdG or S-cdA. We could not detect either excision or binding of these enzymes on duplex oligonucleotide substrates containing these lesions. However, both lesions were repaired by HeLa cell extracts. Dual incisions by human NER on a 136-mer duplex generated 24–32 bp fragments. The time course of dual incisions were measured in comparison to cis-anti-B[a]P-N²-dG, an excellent substrate for human NER, which showed that cis-anti-B[a]P-N²-dG was repaired more efficiently than S-cdG, which, in turn, was repaired more efficiently than S-cdA. When NER efficiency of S-cdG with different complementary bases was investigated, the wobble pair S-cdG·dT was excised more efficiently than the S-cdG·dC pair that maintains nearly normal Watson-Crick base pairing. But S-cdG·dA mispair with no hydrogen bonds was excised less efficiently than the S-cdG·dC pair. Similar pattern was noted for S-cdA. The S-cdA·dC mispair was excised much more efficiently than the S-cdA·dT pair, whereas the S-cdA·dA pair was excised less efficiently. This result adds to complexity of human NER, which discriminates the damaged base pairs on the basis of multiple criteria.     

Discovery of tricyclic 5,6-dihydro-1H-pyridin-2-ones as novel, potent, and orally bioavailable inhibitors of HCV NS5B polymerase

A novel series of non-nucleoside small molecules containing a tricyclic dihydropyridinone structural motif was identified as potent HCV NS5B polymerase inhibitors. Driven by structure-based design and building on our previous efforts in related series of molecules, we undertook extensive SAR studies, in which we identified a number of metabolically stable and very potent compounds in genotype 1a and 1b replicon assays. This work culminated in the discovery of several inhibitors, which combined potent in vitro antiviral activity against both 1a and 1b genotypes, metabolic stability, good oral bioavailability, and high C₁₂ (PO)/EC₅₀ ratios.