Assay of Δ1-piperideine-2-carboxylate and synthesis of l-[14C]pipecolate from dl-[14C]pipecolate

Four assay methods were tested for the measurement of Δ¹-piperideine-2-carboxylate, a proposed alicyclic ketimino acid intermediate in the pathway of lysine metabolism to l-pipecolate, and the product of d-amino acid oxidase on d-pipecolate. The method using Δ¹-piperideine-2-carboxylate reductase from Pseudomonas putida was found to be most sensitive and specific. Measurement of Δ¹-piperideine-2-carboxylate by reduction with NaBH₄ and ninhydrin assay of the resultant pipecolate, by direct acidic ninhydrin assay, and by o-aminobenz-aldehyde assay were less desirable because of lower sensitivity and specificity. Two synthetic methods for preparing l-[¹⁴C]pipecolate from the racemic dl-[¹⁴C]pipecolate were investigated. Incubation of dl-[¹⁴C]pipecolate with a combination of d-amino acid oxidase and Δ¹-piperideine-2-carboxylate reductase or d-amino acid oxidase and NaBH₄ totally inverted the d-isomer to the l-isomer, with $\text{Δ}{}^1\text{-[}{}^{14}\text{C]piperideine-2-carboxylate}$ as an intermediate in each cycle of interconversion. No purification except desalting through a Dowex 50 (H⁺) column was necessary in order to recover l-[¹⁴C]pipecolate in pure form. The yield was 95–97% compared to <50% in the conventional method.     

Synthesis of novel spiro[pyrazolo[4,3-d]pyrimidinones and spiro[benzo[4,5]thieno[2,3-d]pyrimidine-2,3′-indoline]-2′,4(3H)-diones and their evaluation for anticancer activity

An efficient and novel method for the preparation of spiro[pyrazolo[4,3-d]pyrimidin]-7′(1′H)-ones by the condensation of 4-amino-1-methyl-3-propylpyrazole-5-carboxamide with ketones under mild conditions using catalytic InCl₃ was reported. This method has been extended for the synthesis of novel spiro[benzo[4,5]thieno[2,3-d]pyrimidine-2,3′-indoline]-2′,4(3H)-dione which are having potential applications in medicinal chemistry. All the synthesized compounds were evaluated for their anti-proliferative properties in vitro against cancer cell lines and several compounds were found to be active. Further in vitro studies revealed that inhibition of sirtuins could be the possible mechanism of action of these molecules.     

Incorporation of [14C]glucosamine and [14C]leucine into mouse kidney β-glucuronidase induced by gonadotrophin

Male BALB/C mice were injected intraperitoneally with 2.5 i.u. of gonadotrophin. After the injection, increase of β-glucuronidase activity was first observed in the microsomal fraction. By 36h 45–50% of the total homogenate activity was found in the microsomal fraction compared with 20–25% in the control microsomal fraction. From 36 to 80h not only microsomal β-glucuronidase but also lysosomal β-glucuronidase increased progressively. After 69h stimulation with 2.5 i.u. of gonadotrophin, d-[1-¹⁴C]glucosamine or l-[U-¹⁴C]leucine was injected intraperitoneally. After a further 3h the kidneys were homogenized and five particulate fractions were prepared by differential centrifugation. The β-glucuronidase in the microsomal and lysosomal fractions was released respectively by ultrasonication and by freezing and thawing treatment. The enzyme was purified by organic-solvent precipitation and by sucrose-density-gradient centrifugation. The results demonstrated the incorporation of these two labels into the mouse renal β-glucuronidase. The microsomal β-glucuronidase was much more radioactive than the lysosomal enzyme and approx. 80% of the newly synthesized enzyme appeared in microsomes and approx. 20% of that was found in lysosomes at this period. These results suggest that the mouse renal β-glucuronidase is a glycoprotein and that the newly synthesized enzyme is transported from endoplasmic reticulum to lysosomes.     

Microwave-assisted synthesis and structure–activity relationships of neuroactive pyrazolo[3,4-b]pyrrolo[3,4-d]pyridine derivatives

We described herein the optimization of the synthetic methodology exploited to obtain the pyrazolo[3,4-b]pyrrolo[3,4-d]pyridine sedative prototype 1a and novel analogues designed by successive molecular simplifications. By applying microwave irradiation during the hetero Diels-Alder key-step to obtain the heterotricyclic scaffold, under solvent-free conditions, we were able to obtain the desired compounds in drastically shorter times and better yields. Additionally, in vivo evaluation of the sedative effects of these heterocyclic derivatives showed that 1a and the novel structurally-related analogue 1e were the most efficient compounds to impair the locomotor activity in mice at the dose of 10 μmol/kg.     

Synthesis and physicochemical characterization of the lanthionine analog of somatostatin[1–14]

Summary The synthesis of the lanthionine analog of somatostatin[1–14] on a Kaiser-oxime resin is described. The 12-residue peptide segment [3–14] was assembled and cyclized on the resin by using the method of peptide cyclization on an oxime resin (PCOR); the product was obtained with good yield (41%) and purity (94%). The Fmoc protecting group on the N-terminus was cleaved with DBU, followed by a 2+12 segment condensation in solution. The chromatographic (HPLC, CZE) and spectral (UV, NMR) properties of the lanthionine and the natural somatostatins have been studied and compared. Preliminary biological tests show that the lanthionine and the natural somatostatins exhibit similar binding affinities to somatostatin receptor SSTR2.Abbreviations Ala_L one end of a lanthionine unit - Boc tert-butyloxycarbonyl - BOP benzotriazol-l-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate - Bzl benzyl - Cbz benzyloxycarbonyl - DQF-COSY double-quantum-filtered correlated NMR spectroscopy - CZE capillary zone electrophoresis - DBU 1,8-diazabicyclo[5.4.0]undec-7-ene - DCC N,N-dicyclohexylcarbodiimide - DCM dichloromethane - DIEA N,N-diisopropylethylamine - DMF N,N-dimethylformamide - DMSO-d₆ hexadeuterated dimethylsulfoxide - EDC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide - Fmoc 9-florenylmethoxycarbonyl - For formyl - HMPA hexamethylphosphoramide - HOBt N-hydroxybenzotriazole - HOHAHA homonuclear Hartmann-Hahn experiment - HPLC high-performance liquid chromatography - ROESY rotating frame nuclear Overhauser enhancement spectroscopy - TFA trifluoroacetic acid - PCOR peptide cyclization on an oxime resin - Tmac₂O trimethylacetic or pivalic anhydride - Tos p-toluenesulfonyl     

Syntheses and highly enantioselective fluorescent recognition of α-aminocarboxylic acid anions using chiral oxacalix[2]arene[2]bisbinaphthes

The triazine-based bisbinaphthyl crown ethers oxacalix[2]arene[2]bisbinaphthes R-1, R-2, R-3 and S-1, S-2, S-3 were synthesized. The interactions of these compounds with various α-aminocarboxylic acid anions were studied. The crown ethers were found to carry out highly enantioselective fluorescent recognition of α-aminocarboxylic acid anions. It is observed that within a certain concentration range, one enantiomer of the chiral α-aminocarboxylic acid anions can increase the fluorescence intensity of the crown ethers by fivefold to sixfold, whereas the other enantiomer scarcely enhances the fluorescence. Such unusually high enantioselective responses make these crown ethers very attractive as fluorescent sensors in determining the enantiomeric composition of α-aminocarboxylic acid anions.     

Kinetics of [3H]prazosin and [3H]dihydroalprenolol binding to α1- and β-adrenoreceptors in rat brain cortex membranes

The binding of nonselective α₁- and β-adrenoreceptor antagonists [³H]prazosin and [³H]dihydroalprenolol ([³H]DHA) to rat cerebral cortex synaptosomal membranes has been studied. It is found that ligand-receptor interactions of α₁-adrenoreceptors fit into a single receptor pool model, which assumes the binding of two ligand molecules to one receptor molecule. The parameters of [³H]prazosin binding to α₁-adrenoreceptors are as follows: K _d = 2.58 ± 0.20 nM; B _m = 2.95 ± 1.12 fmol/mg protein; Hill coefficient, n = 2. For β-adrenoreceptors, ligand-receptor interactions fit into a model assuming the presence of two receptor pools in the same effector system and binding of two ligand molecules to one receptor molecule. The corresponding parameters of the [³H]DHA binding to β-adrenoreceptors are as follows: K _d1 = 0.74 ± 0.09 nM; K _d2 = 7.63 ± 0.70 nM; B _m1 = 25 ± 2 fmol/mg, B _m2 = 48 ± 2 fmol/mg, n ₁ = 2; n ₂ = 2. We suggest that in rat cerebral cortex membranes α-and β-adrenoreceptors exist as dimers.     

Metabolic Characteristics of the Mutant Yarrowia lipolytica Strain 1 Producing α-Ketoglutaric and Citric Acids from Ethanol and the Effect of [NH+4] and [O2] on Yeast Respiration and Acidogenesis

The comparative studies performed in this work showed that overproduction of -ketoglutaric acid (KGA) and citric acid (CA) from ethanol by the mutantYarrowia lipolyticastrain 1 requires both a deficiency of thiamine and a relatively high concentration of ammonium ions in the medium, whereas CA overproduction requires an almost zero concentration of ammonium ions. The threshold value of the dissolved oxygen concentration in the medium, pO₂, for CA overproduction is considerably higher than for KGA overproduction. The respiration rate of CA-overproducing cells was 2–3.5 times higher than that of KGA-overproducing cells. The main terminal electron carrier functioning in the KGA-overproducing cells was cytochrome oxidase. In the CA-overproducing cells, the main terminal oxidase was presumably o-type cytochrome.     

One-pot microwave assisted stereoselective synthesis of novel dihydro-2′H-spiro[indene-2,1′-pyrrolo-[3,4-c]pyrrole]-tetraones and evaluation of their antimycobacterial activity and inhibition of AChE

An efficient one-pot microwave assisted stereoselective synthesis of novel dihydro-2′H-spiro[indene-2,1′-pyrrolo[3,4-c]pyrrole]-tetraone derivatives through three-component 1,3-dipolar cycloaddition of azomethine ylides generated in situ from ninhydrin and sarcosine with a series of 1-aryl-1H-pyrrole-2,5-diones is described. The synthesised compounds were screened for their antimycobacterial and AChE inhibition activities. Compound 4b (IC₅₀ 1.30 µM) has been found to display twelve fold antimycobacterial activity compared to cycloserine and it is thirty seven times more active than pyrimethamine. Compound 4h displays maximum AchE inhibitory activity with IC₅₀ value of 0.78 ± 0.01 µmol/L.     

Synthesis and cytotoxic activity of new azepino[3′,4′:4,5]pyrrolo[2,1-a]isoquinolin-12-ones

A series of azepino[3′,4′:4,5]pyrrolo[2,1-a]isoquinolin-12-ones (3a–f), that were conformationally restricted analogs of lead compound 2, were designed as potential cytotoxic compounds and synthesized using a radical oxidative aromatic substitution reaction as the key step. Compounds 3a–f were tested on five tumor cell lines to determine the conformational requirements for biological activity of compound 2. The results show that conformational restrictions on compound 2, generating the derivatives 3a–f, do not appreciably reduce the cytotoxic activity of 2, although compound 3d (R = Br) showed good activity against U-251 cells. Preliminary structure–activity relationship studies with these compounds revealed the importance of halogens bonded to the isoquinoline moiety. Additionally, derivatives 3f (R = NO₂) and 3b (R = F) were cytotoxic to PC-3 and K-562 cells. However, none of the azepino[3′,4′:4,5]pyrrolo[2,1-a]isoquinolinones inhibited the enzymatic activity of CDK1/cyclin B, CDK5/p25, or GSK-3.     

In vitro metabolism of a mixture of [4-C]pregnenolone and [17α-H]pregnenolone by polycystic ovary: Pathways of testosterone biosynthesis

When minced polycystic ovarian tissue was incubated with a mixture of [4-¹⁴C]-pregnenolone and [17α-³H]pregnenolone² and added cofactors for 25, 40 and 60 min, the following metabolites were isolated and characterized: progesterone, 17-hydroxyprogesterone, 17-hydroxypregnenolone, dehydroepiandrosterone, 4-androstenedione, and testosterone; unmetabolized substrates were also recovered. There were increased amounts of progesterone, 17-hydroxyprogesterone, dehydroepiandrosterone, and 4-androstenedione formed as the incubation time increased.     

Synthesis of 1-[3-deoxy-β-D-psicofuranosyl]uracil and related compounds

D-Fructose affords on treatment with cyanamide 2-amino-beta-D-fructofuro[2',3':3,4]-oxazoline which is not isolated but transformed directly by the reaction with ethyl propiolate into O(2,3)-anhydro-2-[beta-D-fructofuranosyl]uracil. This compound is benzoylated to the 1',4',6'-tri-O-benzoyl derivative by the action of benzoyl cyanide and triethylamine. On treatment with hydrogen chloride/dimethylformamide, the latter intermediate is converted to the 1-[1,4,6-tri-O-benzoyl-3-chloro-3-deoxy-beta-D-psicofuranosyl]uracil 1',4',5'-tribenzoate from which the title nucleoside derivative is obtained by methanolysis.     

Antagonists of 5-HT₆ receptors. Substituted 3-(phenylsulfonyl)pyrazolo[1,5-a]pyrido[3,4-e]pyrimidines and 3-(phenylsulfonyl)pyrazolo[1,5-a]pyrido[4,3-d]pyrimidines-Synthesis and 'structure-activity' relationship

Synthesis and biological evaluation of a new series of structurally unrestricted and intramolecular hydrogen bond restricted derivatives of 3-(phenylsulfonyl)pyrazolo[1,5-a]pyrido[3,4-e]pyrimidines (angular tricyclics) and 3-(phenylsulfonyl)pyrazolo[1,5-a]pyrido[4,3-d]pyrimidines (linear tricyclics) are described. Structurally restricted derivatives are highly potent and selective blockers of 5-HT(6) receptors with little difference between angular or linear shape of the tricyclic core, the angular species being only slightly more potent. The angular representative of 3-(phenylsulfonyl)pyrazolo[1,5-a]pyrido[3,4-e]pyrimidines, 5, can be considered as more favorable candidate for further development as it shows only weak 5-HT(2B) blocking activity (IC(50)=6.16 μM as compared with IC(50)=1.8 nM for 5-HT(6) receptors) and very low hERG potassium channel blocking potency (IC(50)=54.2 μM). The linear analog, 11, is less favorable as while showing no binding to the 5-HT(2B) receptor at concentrations of up to 10 μM, it exhibits quite a high potency to block the hERG channel (IC(50)=0.5 μM).     

H2-dependent azoreduction by Shewanella oneidensis MR-1: involvement of secreted flavins and both [Ni–Fe] and [Fe–Fe] hydrogenases

In this paper, the hydrogen (H₂)-dependent discoloration of azo dye amaranth by Shewanella oneidensis MR-1 was investigated. Experiments with hydrogenase-deficient strains demonstrated that periplasmic [Ni–Fe] hydrogenase (HyaB) and periplasmic [Fe–Fe] hydrogenase (HydA) are both respiratory hydrogenases of dissimilatory azoreduction in S. oneidensis MR-1. These findings suggest that HyaB and HydA can function as uptake hydrogenases that couple the oxidation of H₂ to the reduction of amaranth to sustain cellular growth. This constitutes to our knowledge the first report of the involvement of [Fe-Fe] hydrogenase in a bacterial azoreduction process. Assays with respiratory inhibitors indicated that a menaquinone pool and different cytochromes were involved in the azoreduction process. High-performance liquid chromatography analysis revealed that flavin mononucleotide and riboflavin were secreted in culture supernatant by S. oneidensis MR-1 under H₂-dependent conditions with concentration of 1.4 and 2.4 μmol g protein^-1, respectively. These endogenous flavins were shown to significantly accelerate the reduction of amaranth at micromolar concentrations acting as electron shuttles between the cell surface and the extracellular azo dye. This work may facilitate a better understanding of the mechanisms of azoreduction by S. oneidensis MR-1 and may have practical applications for microbiological treatments of dye-polluted industrial effluents.     

The interplay of hybridization and clonal reproduction in the evolution of willows – Experiments with hybrids of S. eriocephala[R] & S. exigua[X] and S. eriocephala & S. petiolaris[P].

Clonal reproduction may contribute to plant evolution either by affecting population biology or by allowing partially sterile individuals (e.g., hybrids) repeated opportunities to reproduce sexually. The interplay of hybridization and clonal reproduction was first proposed by Stebbins (1950), but previously has not been tested experimentally. Alternative models for the effects of hybridization include speciation, introgression, and swamping. Salix spp. were chosen to test comparatively these hypotheses because they are easily hybridized and cloned. Over 500 separate crosses and backcrosses were made and over 6000 separate plants were measured in field experiments and statistically compared for significance to both evolutionary theory and plant breeding for biomass production. The F₁ hybrids in this study always equalled, and in the case of the hybrid PR, outperformed their parents in vegetative parameters. It seems likely that even without reproducing sexually, these F₁ hybrids could exist as successful individuals (sensu Stebbins 1950). However, it also seems likely that they would sexually reproduce: three of four F₁ hybrids studied (RX, RP, and PR) equalled or surpassed their parents in sexual parameters when crossing with at least one other accession. Of the alternative models, experimental data suggest that introgression would be the most likely outcome of a hybridization event. The hybrid XR, however, was partially sterile and performed poorly when crossing with all other accessions in its group except S. exigua (pistillate parent). Thus, this hybrid may fit Stebbins' model of a partially sterile yet vegetatively vigorous plant that can exist as a successful individual and make some contribution to interspecific gene flow over time. This is the first experimental study to confirm the evolutionary importance of clonal reproduction coupled with hybridization. However, distinguishing any of these evolutionary pathways would be difficult in nature using morphological techniques, as interspecific hybrids tend to resemble their pistillate parents in terms of leaf shape.     

Synthesis and antimicrobial activity of some new substituted pyrido[3′,2′:4,5]thieno[3,2-d]-pyrimidinone derivatives

A series of new 3-substituted-7-(2-chloro-6-ethoxypyridin-4-yl)-9-(2,4-dichlorophenyl)-2-methylpyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin-4(3H)-one derivatives were synthesized as antimicrobial agents using 7-(2-chloro-6-ethoxypyridin-4-yl)-9-(2,4-dichlorophenyl)-2-methyl-4H-pyrido[3′,2′:4,5]thieno[3,2-d]-[1,3]oxazin-4-one as a starting compound. Its condensation with substituted aniline derivatives or phenyl hydrazine gave the corresponding N-substituted derivatives. Treatment of the starting compound with hydrazine hydrate afforded the corresponding N-amino derivative, which was reacted with substituted phenylisocyanate and phenylisothiocyanate derivatives to give the corresponding semicarbazides and thiosemicarbazide derivatives. All the newly synthesized compounds were evaluated for their antimicrobial activities in comparison to streptomycin and fusidic acid as positive controls. The structure assignments of the new compounds are based on chemical and spectroscopic evidence.     

Dynamic NMR structures of [Rp]- and [Sp]-phosphorothioated DNA-RNA hybrids: is flexibility required for RNase H recognition?

Chemically modified DNA oligonucleotides have been crucial to the development of antisense therapeutics. High-resolution structural studies of pharmaceutically relevant derivatives have been limited to only a few molecules. We have used NMR to elucidate the structure in solution of two DNA-RNA hybrids with the sequence d(CCTATAATCC).r(GGAUUAUAGG). The two hybrids contain an unmodified RNA target strand, whereas the DNA strand contains one of two different stereoregular sugar-phosphate backbone linkages at each nucleotide: 1), [Rp]-phosphorothioate or 2), [Sp]-phosphorothioate. Homonuclear two-dimensional spectroscopy afforded nearly complete nonlabile proton assignments. Distance bounds, calculated from the nuclear Overhauser effect (NOE) crosspeak intensities via a complete relaxation matrix approach with the program MARDIGRAS, were used to restrain the structure of the two hybrids during simulations of molecular dynamics. Analysis of restrained molecular dynamics trajectories suggests that both hybrids are flexible, requiring the use of molecular dynamics with time-averaged restraints (MDtar) to generate ensembles of structures capable of satisfying the NMR data. In particular, the deoxyribose sugars of the DNA strand show strong evidence of repuckering. Furthermore, deoxyribose sugar repuckering is accompanied by increased flexibility of overall helical geometry. These observations, together with the analysis of the crystal structure of a hybrid duplex in complex with ribonuclease H (RNase H), suggested that this flexibility may be required for recognition by RNase H.     

Analysis of [2H7]methionine, [2H4]methionine,methionine, [2H4]homocysteine and homocysteine in plasma by gas chromatography–mass spectrometry to follow the fate of administered [2H7]methionine

Homocysteine plays a key role in several pathophysiological conditions. To assess the methionine–homocysteine kinetics by stable isotope methodology, we developed a simultaneous quantification method of [²H₇]methionine, [²H₄]methionine, methionine, [²H₄]homocysteine and homocysteine in rat plasma by gas chromatography–mass spectrometry (GC–MS). [¹³C]Methionine and [¹³C]homocysteine were used as analytical internal standards to account for losses associated with the extraction, derivatization and chromatography. For labeled and non-labeled homocysteine measurements, disulfide bonds between homocysteine and other thiols or proteins were reduced by dithiothreitol. The reduced homocysteine and methionine species were purified by cation-exchange chromatography and derivatized with isobutyl chlorocarbonate in water–ethanol–pyridine. Quantification was carried out by selected ion monitoring of the molecular-related ions of N(O,S)-isobutyloxycarbonyl ethyl ester derivatives on the chemical ionization mode. The intra- and inter-day precision of the assay was less than 6% for all labeled and non-labeled methionine and homocysteine species. The method is sensitive enough to determine pharmacokinetics of labeled methionine and homocysteine.     

An enduring association? Microfilariae and immunosuppression [correction of immunosupression] in lymphatic filariasis

Human filarial infection is characterized by a defect in T-cell proliferative responses which is most pronounced among actively infected individuals. This article reviews the immunomodulatory potential of the first larval stage, the blood-borne microfilariae, which has long been associated with the most profound suppression of cellular responses. In particular, we focus on the induction of host cell apoptosis following murine infection with microfilariae. Promoting the apoptotic elimination of potentially reactive T cells could represent an important means of both facilitating parasite survival and limiting inflammatory pathology.     

Synthesis and in vitro and in vivo anticancer activity of novel 3-methyl-5H-isoxazolo[5′,4′:5,6]pyrido[2,3-b]indoles

A series of novel isoxazolo[5′,4′:5,6]pyrido[2,3-b]indoles 7a–h were synthesized and tested for their in vitro and in vivo anticancer activities. The analogs 7d and 7g have shown potential anticancer activity as compared with the reference compound Cisplatin.