Novel hydroxystearoyl-, heterocyclic and carbocyclic derivatives: synthesis and applications in biological field

This PhD project has been mainly focused on the synthesis of novel organic compounds containing heterocyclic and/or carbocyclic scaffold and on the study of stearic acid derivatives and their applications in biological field. The synthesis of novel derivatives of 9-hydroxystearic acid (9-HSA) evidenced how the presence of substituents on C9, able to make hydrogen bonds is of crucial importance for the biological activity. Also the position of the hydroxy group along the chain of hydroxystearic acids was investigated: regioisomers with the hydroxy group bound to odd carbons resulted more active than those bearing the hydroxy group on even carbons. Further, the insertion of (R)-9-HSA in magnetic nanoparticles gave a novel material which characterization remarked its suitability for drug delivery. Structural hybrids between amino aza-heterocycles and azelaic acid have been synthesized and some of them showed a selective activity towards osteosarcoma cell line U2OS. Several Apcin analogues bearing indole, benzothiazole, benzofurazan moieties connected to tryptaminyl-, amino pyridinyl-, pyrimidinyl- and pyrazinyl ring through a 1,1,1-trichloroethyl group were synthesized. Biological tests showed the importance of both the tryptaminyl and the pyrimidinyl moieties, confirming the effectiveness against acute leukemia models. The SNAr between 2-aminothiazole derivatives and 7-chlorodinitrobenzofuroxan revealed different behaviour depending from amino substituent of the thiazole. The reaction with 2-N-piperidinyl-, 2-N-morpholinyl-, or 2-N-pyrrolidinyl thiazole gave two isomeric species derived from the attack on C-5 of thiazole ring. Thiazoles substituted with primary- or not-cyclic secondary amines reacted with the exocyclic amino nitrogen atom giving a series of compounds whose biological activity have highlighted as they might be promising candidates for further development of antitumor agents. A series of 9-fluorenylidene derivatives, of interest in medical and optoelectronic field as organic scintillators, was synthesized through Wittig or Suzuky reaction and will be analyzed to test their potential scintillatory properties.

1-Benzyl-2,5-dioxopyrrolidine-3,4-diyl diacetate

The pyrrolidine-2,5-dione ring in the title compound, C(15)H(15)NO(6), is in a twisted conformation with the acetyl C atoms projecting to opposite sides of the ring. The acetyl groups lie to opposite sides of the five-membered ring. The benzene ring is roughly perpendicular to the heterocyclic ring, forming a dihedral angle of 76.57 (14)degrees with it. In the crystal, molecules are connected through a network of C-H center dot center dot center dot O and C-H center dot center dot center dot pi interactions.

Hydrogen bonding in 2-(2-oxothiazolidin-3-yl)-4,5-dihydrothiazolium hydrogen sulfate monohydrate

The asymmetric unit of the title compound, C(6)H(9)N(2)OS(2)(+)center dot-HSO(4)(-)center dot H(2)O, contains a heterocyclic cation, a hydrogen sulfate anion and a water molecule. There are strong hydrogen bonds between the hydrogen sulfate anions and water molecules, forming an infinite chain along the [010] direction, from which the cations are pendent. The steric, electronic and geometric features are compared with those of similar compounds. In this way, structural relationships are stated in terms of the influence of the sulfate group on the protonation of the heterocycle and on the tautomeric equilibrium in the solid state.

Thermodynamics of aqueous solutions of polyelectrolytes: Experimental results for the activity of water in aqueous solutions of some single synthetic polyelectrolytes

Experimental results for the activity of water in aqueous solutions of 10 single polyelectrolytes (two polysodium acrylates, two polysodium methacrylates, three polyammonium acrylates, two polysodium ethylene sulfonates, and one polysodium styrene sulfonate) at (298.2 and 323.2) K are reported. The isopiestic method was employed in these experiments with aqueous solutions of sodium chloride as references. The polyelectrolytes were characterized by three averaged molecular masses determined by gel permeation chromatography. Furthermore, the density and the refractive index increments of the aqueous polyelectrolyte solutions are reported. Although a similar pattern for the activity of water was observed for all systems (i.e., the osmotic coefficient increases with rising polyelectrolyte concentration), the experimental results show that this property depends on the monomer type as well as on the size of the polymer chain. The temperature (varied from (298.2 to 323.2) K) has only a small influence on the activity of water.

Electrospray MS-based characterization of beta-carbolines - mutagenic constituents of thermally processed meat

The beta-carbolines 1-methyl-9H-pyrido [3,4-b]indole and 9H-pyrido[3,4b]indole have been implicated as having causative roles in a number of human diseases, such as Parkinson`s disease and cancer. As they can be formed during the heating of protein-rich food, a number of analytical methodologies have been proposed for their detection and quantification in foodstuff For this purpose, LC-MS and LC-MS/MS have emerged as the most specific analytical methods, and the quantification is based on the occurrence of unusual ions, such as [M+H-(H(center dot))](+) and [M + H-2H](+). In this study, we have investigated the formation of these ions by accurate-mass electrospray MS/MS and demonstrated that these ions are formed from gas-phase ion-molecule reactions between water vapor present in the collision cell and the protonated molecule of 1-methyl-9H-pyrido [3,4-b]indole and 9H-pyrido[3,4b]indole. Although this reaction has been previously described for heterocyclic amine ions, it has been overlooked in the most of recent LC-MS and LC-MS/MS studies, and no complete data of the fragmentation are reported. Our results demonstrate that additional attention should be given with respect to eliminating water vapor residues in the mass spectrometer when analysis of beta-carbolines is performed, as this residue may affect the reliability in the results of quantification.

Functional polymorphism of the human arylamine N-acetyltransferase type 1 gene caused by (CT)-T-190 and G(560)A mutations

Human N-acetyltransferase type 1 (NAT1) catalyses the N- or O-acetylation of various arylamine and heterocyclic amine substrates and is able to bioactivate several known carcinogens. Despite wide inter-individual variability in activity, historically, NAT1 was considered to be monomorphic in nature. However, recent reports of allelic variation at the NAT1 locus suggest that it may be a polymorphically expressed enzyme. In the present study, peripheral blood mononuclear cell NAT1 activity in 85 individuals was found to be bimodally distributed with approximately 8% of the population being slow acetylators. Subsequent sequencing of the individuals having slow acetylator status showed all to have either a (CT)-T-190 or G(560)A base substitution located in the protein encoding region of the NAT1 gene. The (CT)-T-190 base substitution changed a highly conserved Arg(64), which others have shown to be essential for fully functional NAT1 protein. The (CT)-T-190 mutation has not been reported previously and we have named it NAT1*17. The G(560)A mutation is associated with the base substitutions previously observed in the NAT1*10 allele and this variant (NAT1*14) encodes for a protein with reduced acetylation capacity. A novel method using linear PCR and dideoxy terminators was developed for the detection of NAT1*14 and NAT1*17. Neither of these variants was found in the rapid acetylator population. We conclude that both the (CT)-T-190 (NAT1*17) and G(560)A (NAT1*14) NAT1 structural variants are involved in a distinct NAT1 polymorphism. Because NAT1 can bioactivate several carcinogens, this polymorphism may have implications for cancer risk in individual subjects. (C) 1998 Chapman & Hall Ltd.

mRNA differential display of 2-amino-1-methyl-6-phenylinlidazo[4,5-b]pyridine-induced rat mammary gland tumors

The mRNA differential display technique was used to compare mRNAs between normal mammary gland and turner-derived epithelial cells from female Sprague-Dawley rat mammary gland tumors induced by the heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and promoted by a high-fat diet (23.5% corn oil). Two genes, beta-casein and transferrin, were identified as differentially expressed. The expression of these genes was examined across a bank of rat mammary gland tumors derived from animals on a low-fat diet (5% corn oil) or the high-fat diet. Carcinomas had over a 10- and 50-fold lower expression of beta-casein and transferrin, respectively than normal mammary gland. In addition, carcinomas from animals on the high-fat diet showed on average a 5-fold higher expression of beta-casein, and transferrin than carcinomas from animals on the low-fat diet. The results indicate the process of mammary gland tumorigenesis alters the expression of certain genes in the mammary gland, and that the level of dietary fat further modulates the expression of these genes.

Water diffusion in hydroxyethyl methacrylate (HEMA)-based hydrogels formed by gamma-radiolysis

Polymer hydrogels based upon methacrylates are used extensively in the pharmaceutical industry, particularly as controlled release drug delivery systems. These materials are generally prepared by chemically initiated polymerization, but this can lead to the presence of unwanted initiator fragments in the polymer matrix. In the present work, initiation of polymerization by gamma-irradiation of hydroxyethyl methacrylate, with and without added crosslinkers, has been investigated, and the diffusion coefficients for water in the resulting polymers have been measured through mass uptake by the polymers. The diffusion of water in poly(hydroxyethyl methacrylate) at 310 K was found to be Fickian, with a diffusion coefficient of 1.96 +/- 0.1 x 10(11) m(2) s(-1) and an equilibrium water content of 58%, NMR imaging analyses confirmed the adherance to a Fickian model of the diffusion of water into polymer cylinders. The incorporation of small amounts (0.2-0.5 wt%) of added ethyleneglycol-dimethacrylate-based crosslinkers was found to have only a small effect on the diffusion coefficient and the equilibrium water content for the copolymers. (C) 1999 Society of Chemical Industry.

Base-catalysed rearrangements of 5-oxodihydroisoxazoles

Five distinct pathways for the reaction of isoxazol-5(2H)-ones with bases or nucleophiles have been reported, and are detailed herein. That investigated in greatest detail, and of greatest application in heterocyclic synthesis, is that of isoxazolones unsubstituted at C-3, in which the sequentially formed ketenimine, alpha-lactone and ketene may all react with a nucleophile.

The biologically active iron chelators 2-pyridylcarboxaldehyde isonicotinoylhydrazone, 2-pyridylcarboxaldehyde benzoylhydrazone monohydrate and 2-furaldehyde isonicotinoylhydrazone

In the crystal structures of the respective title compounds, C12H10N4O, C13H11N3O . H2O and C11K9N3O2, variations in the torsion angles of the aromatic pyridyl and benzoyl groups are observed, and the disposition of the heterocyclic aldehyde is shown to be influenced by the ring size of this group.

Cyclic octapeptides containing thiazole. Effect of stereochemistry and degree of flexibility on calcium binding properties

Solution conformation and calcium binding properties have been investigated for the two cyclic octapeptides cyclo(-D-Thr-D-Val(Thz)-Ile-)(2) (4) and cyclo(-Thr-Gly(Thz)-Ile-Ser-Gly(Thz)-Ile-)(5) and the results are compared to those for the cyclic octapeptides previously studied; ascidiacyclamide (1), patellamide D (2), cyclo(-Thr-D-Val(Thz)-Ile-)(2) (3), and cyclo(-Thr-D-Val-alphaAbu-Ile-)2 (6). Both 4 and 5 contain two heterocyclic thiazole ring constraints but the latter has a larger degree of flexibility as a consequence of the glycine residues within the cyclic framework. The solution conformation of 4 and 5 was determined from H-1 NMR spectra and found to be a twisted figure of eight similar to that for 2. Complexation studies using H-1 NMR and CD spectroscopy yielded 1 : 1 calcium-peptide binding constants (logK) for the two peptides (2.3 (4) and 5.7 (5)). For 5 the magnitude of the binding constant was verified by a competition titration using CD. The different calcium-binding affinities of 3 (logK = 4.0) and 4 is attributed to the stereochemistry of the threonine residue. The magnitude of the binding constant for 5 compared to 3 and 4 (all peptides containing two thiazole ring constrains) demonstrates that the increase in flexibility of the cyclic peptide has a dramatic effect on the Ca2+ binding ability. The affinity for Ca2+ thus decreases in the order (6 similar to 5 > 3 > 2 similar to 1 > 4). The number of carbonyl donors available on each peptide has only a limited effect on calcium binding. The most important factor is the flexibility, which allows for a conformation of the peptide capable of binding calcium efficiently.

The role of xenobiotic metabolizing enzymes in arylamine toxicity and carcinogenesis: Functional and localization studies

In both animal models and humans, the first and obligatory step in the activation of arylamines is N-hydroxylation. This pathway is primarily mediated by the phase-I enzymes CYP1A1, CYP1A2 and CYP4B1. In the presence of flavonoids such as alpha-naphthoflavone and flavone, both CYP3A4 and CYP3A5 have also been shown to play a minor role in the activation of food-derived heterocyclic amines. The further activation of N-hydroxyarylamines by phase-II metabolism can involve both N,O-acetylation and N,O-sulfonation catalyzed by N-acetyltransferases (NAT1 and NAT2) and sulfotransferases, respectively. Using an array of techniques, we have been unable to detect constitutive CYP1A expression in any segments of the human gastrointestinal tract. This is in contrast to the rabbit where CYP1A1 protein was readily detectable on immunoblots in microsomes prepared from the small intestine. In humans, CYP3A3/3A4 expression was detectable in the esophagus and all segments of the small intestine. Northern blot analysis of eleven human colons showed considerable heterogeneity in CYP3A mRNA between individuals, with the presence of two mRNA species in same subjects. Employing the technique of hybridization histochemistry (also known as in situ hybridization), CYP4B1 expression was observed in some human colons but not in the liver or the small intestine. Hybridization histochemistry studies have also demonstrated variable NAT1 and NAT2 expression in the human gastrointestinal tract. NAT1 and NAT2 mRNA expression was detected in the human liver, small intestine, colon, esophagus, bladder, ureter, stomach and lung. Using a general aryl sulfotransferase riboprobe (HAST1), we have demonstrated marked sulfotransferase expression in the human colon, small intestine, lung, stomach and liver. These studies demonstrate that considerable variability exists in the expression of enzymes involved in the activation of aromatic amines in human tissues. The significance of these results in relation to a role for heterocyclic amines in colon cancer is discussed.

Antileishmanial activity of ruthenium(II)tetraammine nitrosyl complexes

The complexes trans-[Ru(NO)(NH(3))(4)L](X)(3) (X = BF(4)(-), PF(6)(-) or Cl(-) and L = N-heterocyclic ligands, P (OEt)(3), SO(3)(-2)), and [Ru(NO)Hedta)] were shown to exhibit IC(50pro) in the range of 36 (L = imN) to 5000 mu M (L = imC). The inhibitory effects of trans-[Ru(NO)(NH(3))(4)imN](BF(4))(3) and of the Angeli`s salt on the growth of the intramacrophage amastigote form studied were found to be similar while the trans-[Ru(NH(3))(4)imN(H(2)O)](2+) complex was found not to exhibit any substantial antiamastigote effect. The trans-[Ru(NO)(NH(3))(4)imN](BF(4))(3) compound, administered (500 nmol kg(-1) day(-1)) in BALB/c mice infected with Leishmania major, was found to exhibit a 98% inhibition on the parasite growth. Furthermore, this complex proved to be at least 66 times more efficient than glucantime in in vivo experiments. (C) 2010 Elsevier Masson SAS. All rights reserved.

Network structures of Bis-GMA/TEGDMA resins differ in DC, shrinkage-strain, hardness and optical properties as a function of reducing agent

Objectives. To evaluate the influence of different tertiary amines on degree of conversion (DC), shrinkage-strain, shrinkage-strain rate, Knoop microhardness, and color and transmittance stabilities of experimental resins containing BisGMA/TEGDMA (3: 1 wt), 0.25wt% camphorquinone, 1wt% amine (DMAEMA, CEMA, DMPT, DEPT or DABE). Different light-curing protocols were also evaluated. Methods. DC was evaluated with FTIR-ATR and shrinkage-strain with the bonded-disk method. Shrinkage-strain-rate data were obtained from numerical differentiation of shrinkage-strain data with respect to time. Color stability and transmittance were evaluated after different periods of artificial aging, according to ISO 7491: 2000. Results were evaluated with ANOVA, Tukey, and Dunnett`s T3 tests (alpha = 0.05). Results. Studied properties were influenced by amines. DC and shrinkage-strain were maximum at the sequence: CQ < DEPT < DMPT <= CEMA approximate to DABE < DMAEMA. Both DC and shrinkage were also influenced by the curing protocol, with positive correlations between DC and shrinkage-strain and DC and shrinkage-strain rate. Materials generally decreased in L* and increased in b*. The strong exception was the resin containing DMAEMA that did not show dark and yellow shifts. Color varied in the sequence: DMAEMA < DEPT < DMPT < CEMA < DABE. Transmittance varied in the sequence: DEPT approximate to DABE < DABE approximate to DMPT approximate to CEMA < DMPT approximate to CEMA approximate to DMAEMA, being more evident at the wavelength of 400 nm. No correlations between DC and optical properties were observed. Significance. The resin containing DMAEMA showed higher DC, shrinkage-strain, shrinkage-strain rate, and microhardness, in addition to better optical properties. (C) 2011 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.