Determination of 6,4′-bis-(2-imidazolinylhydrazone)-2-phenylimidazo[1,2-a]pyridine in plasma and whole blood by high-performance liquid chromatography

A selective and sensitive HPLC assay for the quantitative determination of a new antifilarial drug, 6,4′-bis-(2-imidazolinylhydrazone)-2-phenylimidazo[1,2-a]pyridine (CDR 101) is described. After extraction from plasma and blood, CDR 101 was analysed using a C₁₈ Nucleosil ODS column (250×4.6 mm, 5 μm particle size) and mobile phase of acetonitrile-0.05 M ammonium acetate adjusted to pH 3.0, with UV detection at 318 nm. The mean recoveries of CDR 101 in plasma and blood over a concentration range of 25–500 ng/ml were 95.5±2.01% and 83.3±1.87%, respectively. The within-day and day-to-day coefficient of variations for plasma were 3.23-6.21% and 2.59-9.90%, respectively, those for blood were 2.59-5.92% and 2.89-6.82%, respectively. The minimum detectable concentration for CDR 101 was 1 ng/ml in plasma and 2.5 ng/ml in whole blood. This method was found to be suitable for clinical pharmacokinetic studies.     

Synthesis and anticancer evaluation of certain indolo[2,3-b]quinoline derivatives

The present report describes the synthesis and anticancer evaluation of certain 11-substituted 6H-indolo[2,3-b]quinolines and their methylated derivatives. These 6H-indolo[2,3-b]quinoline derivatives 11–13 were prepared from the commercially available 1,4-dihydroxyquinoline through alkylation, chlorination, nucleophilic reaction, and ring cyclization. Depending on the ratio of 11, (MeO)₂SO₂, and K₂CO₃, alkylation occurred primarily on N-5 (1:0.8:0.8) or N-6 (1:1.5:1.5) leading to the isolation of 14a or 14b as a major product. Accordingly, major product 15a (2/(MeO)₂SO₂/K₂CO₃ = 1:2:2) or 15b (1:1:1), respectively, was obtained by alkylation of 12 while 16a (13/(MeO)₂SO₂/K₂CO₃ = 1:2:2) or 16b (1:1:1), respectively, was obtained by alkylation of 13. The in vitro anticancer assay indicated 5-methylated derivatives 14a, 15a, 16a are more cytotoxic than their respective 6-methylated counterparts 14b, 15b, 16b and 6H-indolo[2,3-b]quinoline precursors 11, 12, 13. Among them, 11-(4-methoxyanilino)-6-methyl-6H-indolo[2,3-b]quinoline (16a) was the most cytotoxic with a mean GI₅₀ value of 0.78 μM and also exhibited selective cytotoxicities for HL-60 (TB), K-562, MOLT-4, RPMI-8226, and SR with GI₅₀ values of 0.11, 0.42, 0.09, 0.14, and 0.19 μM, respectively.     

Binding of [H][D-Ala, MePhe, Gly-ol] Enkephalin, [H][D-Pen, D-Pen]Enkephalin, and [H]U-69,593 to Airway and Pulmonary Tissues of Normal and Sensitized Rats

Bhargava, H. N., V. M. Villar, J. Cortijo and E. J. Morcillo. Binding of [³H][D-Ala², MePhe⁴, Gly-ol⁵]enkephalin, [³H][D-Pen², D-Pen⁵]enkephalin, and [³H]U-69,593 to airway and pulmonary tissues of normal and sensitized rats. Peptides 18(10) 1603–1608, 1997.—The role of endogenous opioid peptides in the regulation of bronchomotor tone, as well as in the pathophysiology of asthma is uncertain. We have studied the binding of highly selective [³H]labeled ligands of μ-([D-Ala², MePhe⁴, Gly-ol⁵]enkephalin; DAMGO), δ ([D-Pen², D-Pen⁵]enkephalin; DPDPE), and κ-(U-69,593) opioid receptors to membranes of trachea, main bronchus, lung parenchyma and pulmonary artery obtained from normal (unsensitized) and actively IgE-sensitized rats acutely challenged with the specific antigen. [³H]DAMGO, [³H]DPDPE and [³H]U-69,593 bound to membranes of normal and sensitized tissues at a saturable, single high-affinity site. The rank order of receptor densities in normal tissues was δ- ≥ κ- ≥ μ-, with lung parenchyma exhibiting the greatest binding capacity for δ- and μ- receptors compared to the other regions examined. The K_d values showed small differences between ligands and regions tested. The μ- and δ-opioid receptor densities were decreased in sensitized main bronchus and lung parenchyma, respectively, compared to normal tissues. By contrast, κ-opioid receptor density was augmented in sensitized lung parenchyma but an increase in K_d values was also observed. These differential changes in the density and affinity of opioid receptor types may be related to alterations in endogenous opioid peptides during the process of sensitization.     

Synthesis, spectral studies and in vitro assessment for antiamoebic activity of new cyclooctadiene ruthenium(II) complexes with 5-nitrothiophene-2-carboxaldehyde thiosemicarbazones

We report here the synthesis, characterization and in vitro antiamoebic activity of 5-nitrothiophene-2-carboxaldehyde thiosemicarbazones (TSC), 1–5, and their bidentate complexes [Ru(η⁴-C₈H₁₂)(TSC)Cl₂] 1a–5a. The biological studies of these compounds were investigated against HK-9 strain of Entamoeba histolytica and the concentration causing 50% cell growth inhibition (IC₅₀) was calculated in the micromolar range. The ligands exhibited antiamoebic activity in the range (2.05–5.29 μM). Screening results indicated that the potencies of the compounds increased by the incorporation of ruthenium(II) in the thiosemicarbazones. The complexes 1a–5a showed antiamoebic activity with an IC₅₀ of 0.61–1.43 μM and were better inhibitors of growth of E. histolytica, based on IC₅₀ values. The most promising among them is Ru(II) complex 2a having 1,2,3,4-tetrahydroquinoline as N⁴ substitution.     

Photosynthetic response of Myriophyllum salsugineum A.E. orchard to photon irradiance, temperature and external free CO2

The photosynthetic capacity of Myriophyllum salsugineum A.E. Orchard was measured, using plants collected from Lake Wendouree, Ballarat, Victoria and grown subsequently in a glasshouse pond at Griffith, New South Wales. At pH 7.00, under conditions of constant total alkalinity of 1.0 meq dm⁻³ and saturating photon irradiance, the temperature optimum was found to be 30–35°C with rates of 140 μmol mg⁻¹ chlorophyll a h⁻¹ for oxygen production and 149 μmol mg⁻¹ chlorophyll a h⁻¹ for consumption of CO₂. These rates are generally higher than those measured by other workers for the noxious Eurasian water milfoil, Myriophyllum spicatum L., of which Myriophyllum salsugineum is a close relative. The light-compensation point and the photon irradiance required to saturate photosynthetic oxygen production were exponentially dependent on water temperature. Over the temperature range 15–35°C the light-compensation point increased from 2.4 to 16.9 μmol (PAR) m⁻² s⁻¹ for oxygen production while saturation photon irradiance increased from 41.5 to 138 μmol (PAR) m⁻² s⁻¹ for oxygen production and from 42.0 to 174 μmol (PAR) m⁻² s⁻¹ for CO₂ consumption. Respiration rates increased from 27.1 to 112.3 μmol (oxygen consumed) g⁻¹ dry weight h⁻¹ as temperature was increased from 15 to 35°C. The optimum temperature for productivity is 30°C.     

Thermodynamics of the disproportionation of adenosine 5'-diphosphate to adenosine 5'-triphosphate and adenosine 5'-monophosphate, II. Experimental data

High-pressure liquid-chromatography and microcalorimetry have been used to determine equilibrium constants and enthalpies of reaction for the disproportionation reaction of adenosine 5′-diphosphate (ADP) to adenosine 5′-triphosphate (ATP) andadenosine 5′-monophosphate (AMP). Adenylate kinase was used to catalyze this reaction. The measurements were carried out over the temperature range 286 to 311 K, at ionic strengths varying from 0.06 to 0.33 mol kg⁻¹, over the pH range 6.04 to 8.87, and over the pMg range 2.22 to 7.16, where pMg = -log a(Mg²⁺). The equilibrium model developed by Goldberg and Tewari (see the previous paper in this issue) was used for the analysis of the measurements. Thus, for the reference reaction: 2 ADp³⁻ (ao) AMp²⁻ (ao)+ ATp⁻ (ao), K° = 0.225 ± 0.010, ΔG° = 3.70 +- 0.11 kJ mol ⁻¹, ΔH° = −1.5 ± 1. 5 kJ mol ⁻¹, °S ° = −17 ± 5 J mol⁻¹ K⁻¹, and ACP_p°≈ = −46 J mo1l⁻¹ K⁻¹ at 298.15 K and 0.1 MPa. These results and the thermodynamic parameters for the auxiliary equilibria in solution have been used to model the thermodynamics of the disproportionation reaction over a wide range of temperature, pH, ionic strength, and magnesium ion morality. Under approximately physiological conditions (311.15 K, pH 6.94, [Mg²⁺] = 1.35 × 10⁻³ mol kg⁻¹, and I = 0.23 mol kg⁻¹) the apparent equilibrium constant (K_A′ = m(ΣAMP)m(ΣATP)/[ m(ΣADP)]²) for the overall disproportionation reaction is equal to 0.93 ± 0.02. Thermodynamic data on the disproportionation reaction and literature values for this apparent equilibrium constant in human red blood cells are used to calculate a morality of 1.94 × 10⁻⁴ mol kg⁻¹ for free magnesium ion in human red blood cells. The results are also discussed in relation to thermochemical cycles and compared with data on the hydrolysis of the guanosine phosphates.     

Synthesis and cytotoxic activity of a new potent daunomycinone derivative

The preparation and cytotoxic activity of 4′-azido-3′-bromo-3′-deamino-4′-deoxydaunorubicin is described. The new compound was found to be less active in vitro than adriamycin against L1210 and the sensitive cell lines KB-3-1 and MES-SA, but retained interesting cytotoxicity against the adriamycin resistant subline KB-A1 and the multidrug resistant MES-SA/Dx5 subline.     

Purification and characterization of alanine racemase from hepatopancreas of black-tiger prawn, Penaeus monodon

Alanine racemase has been purified to homogeneity from the hepatopancreas of the black tiger prawn, Panaeus mondon. The enzyme depends on pyridoxal 5′-phosphate and consists of two subunits with an identical molecular weight of 41,000. V_max and K_m values for -alanine are 460 μmol/min/mg and 50 mM, and those for -alanine are 94 μmol/min/mg and 24 mM, respectively. The enzyme is highly specific toward alanine. Among other amino acids examined, only serine served as a substrate: -serine was racemized at a rate of approximately 0.5% of that of -alanine. The prawn enzyme is immunochemically distinguishable from the enzymes of Bacillus stearothermophilus and Schizosaccharomyces pombe, which resemble each other. The prawn enzyme is activated and stabilized by the presence of monovalent anions including chloride. This is consistent with the previous hypothesis (e.g. E. Fujita, E. Okuma, H. Abe, Comp. Biochem. Physiol. 116A (1997) 83–87) that -alanine serves as an osmoregulator in marine and euryhaline animals.     

New series of potent delta-opioid antagonists containing the H-Dmt-Tic-NH-hexyl-NH-R motif

Heterodimeric compounds H-Dmt-Tic-NH-hexyl-NH-R (R = Dmt, Tic, and Phe) exhibited high affinity to δ- (K_iδ = 0.13–0.89 nM) and μ-opioid receptors (K_iμ = 0.38–2.81 nM) with extraordinary potent δ antagonism (pA₂ = 10.2–10.4). These compounds represent the prototype for a new class of structural homologues lacking μ-opioid receptor-associated agonism (IC₅₀ = 1.6–5.8 μM) based on the framework of bis-[H-Dmt-NH]-alkyl (Okada, Y.; Tsuda, Y.; Fujita, Y.; Yokoi, T.; Sasaki, Y.; Ambo, A.; Konishi, R.; Nagata, M.; Salvadori, S.; Jinsmaa, Y.; Bryant, S. D.; Lazarus, L. H. J. Med. Chem. 2003, 46, 3201), which exhibited both high μ affinity and bioactivity.     

Efficient synthesis of functionalized oligodeoxyribonucleotides with base-labile groups using a new silyl linker

In this study, we developed new 3′-terminal deoxyribonucleoside-loading reagents 1 with a new silyl-type linker. These reagents could increase the efficiency of introduction of 3′-terminal deoxyribonucleoside components into polymer supports to a level of 17–29 μmol/g. The efficiency was higher than that of previous T-loading reagents because reagents 1 contain a 4-aminobutyryl residue as a spacer. Moreover, we could synthesize not only unmodified DNA oligomers but also a base-labile modified DNA oligomer using resins 9a–d in the activated phosphite method without base protection.     

Synthesis, radiolabeling and receptor binding of [3H][(1S,2R)ACPC2]endomorphin-2

Previously, we have shown that substitution of Pro² for cis-2-aminocyclopentanecarboxylic acid, ACPC in endomorphin-2 results in an analogue with greatly augmented proteolytic stability, high μ-opioid receptor affinity and selectivity. We now report the synthesis and biochemical characterization of [³H][(1S,2R)ACPC²]endomorphin-2 with a specific activity of 1.41 TBq/mmol (38.17 Ci/mmol). Specific binding of [³H][(1S,2R)ACPC²]endomorphin-2 was saturable and of high affinity with an equilibrium dissociation constant, K_d = 1.80 ± 0.21 nM and receptor density, B_max = 345 ± 27 fmol × mg protein⁻¹ at 25 °C in rat brain membranes. Similar affinity values were obtained in kinetic and displacement assays. Both Na⁺ and Gpp(NH)p decreased the affinity proving the agonist character of the radioligand. [³H][(1S,2R)ACPC²]endomorphin-2 retained the μ-specificity of the parent peptide. The new radioligand will be a useful tool to map the topographical requirements of μ-opioid peptide binding due to its high affinity, selectivity and enzymatic stability.     

Synthesis, structure, and heterogeneous catalytic activity of tungsten chalcogenide cubane cluster containing alkaline earth metal complex

A new compound containing a cubane tungsten chalcogenide cluster [W₄(μ₃-Te)₄(CN)₁₂]⁶⁻ and Ca²⁺ complex units has been prepared by the reaction of aqueous solution of K₆[W₄(μ₃-Te)₄(CN)₁₂] · 5H₂O with the solution of a Ca(NO₃)₂ and phen(1,10-phenanthroline) (1:2 molar ratio) in a solvent mixture of H₂O/EtOH. The structure of [{Ca(phen)₂(H₂O)}{Ca(phen)(H₂O)₄}{Ca(phen)₂(H₂O)₃}][W₄Te₄(CN)₁₂] · 5H₂O 1 has been determined by X-ray crystallography. Compound 1 contains [{Ca(phen)(H₂O)₄}{Ca(phen)₂(H₂O)₃}][W₄(μ₃- Te)₄(CN)₁₂] units bridged by {Ca(phen)₂(H₂O)}²⁺ units to form an one-dimensional zigzag chain structure. Interestingly, compound 1 showed a heterogeneous catalytic activity in the transesterification of a range of esters with methanol under the mild conditions. Moreover, it can be reused without any loss of activity through 10 runs with ester.     

Immobilization of RNase Rs via its carbohydrate moiety to aminoethyl-Bio-Gel P-2 and its application for the hydrolysis of RNA to 2′,3′ cyclic nucleotides

Purified RNase Rs, from Rhizopus stolonifer, when covalently coupled to aminoethyl (AE) Bio-Gel P-2, via its carbohydrate moiety, retained 35–40% activity of the soluble enzyme. Optimization of coupling conditions showed that the most active immobilized preparations are obtained when 400 units of 100 μM periodate oxidized enzyme are allowed to react with 1 ml (packed volume) of AE-Bio-Gel P-2 at 6±1°C for 15 h. Immobilization did not change the pH and temperature optima of the enzyme but it increased the temperature stability. Immobilization did not bring about a change in the K_m but resulted in a 2·5-fold decrease in the V_max. Substrate concentrations as high as 25 mg of RNA could be converted to more than 80% 2′,3′ cyclic nucleotides in 14 h, at pH 5·5 and 37°C. On repeated use, the bound enzyme retained 70% of its initial activity after six cycles of use. The bound enzyme could be stored in wet state for 60 days without any significant loss in its initial activity.     

Synthesis and in vitro anti-hepatitis B and C virus activities of ring-expanded ('fat') nucleobase analogues containing the imidazo[4,5-e][1,3]diazepine-4,8-dione ring system

As part of our structure–activity relationship studies, we report here the synthesis and in vitro anti-HBV and anti-HCV activities of a number of ring-expanded (‘fat’) nucleobases containing the imidazo[4,5-e][1,3]diazepine-4,8-dione ring system. One of the compounds, ZP-88, exhibited a good activity/toxicity profile against HBV by inhibition of the synthesis of extracellular virion release (EC₅₀ = 1.7 μM, CC₅₀ = 286 μM, SI = 168) and intracellular HBV replication intermediates (EC₅₀ = 8.4 μM, CC₅₀ = 286 μM, SI = 34) in cultured human hepatoblastoma 2.2.15 cells. By contrast, most of the compounds tested against HCV had only marginal activity/toxicity profile, although that was still better than that of the reference compound ribavirin.     

Beta-endorphin-like peptide SLTCLVKGFY reduces the production of 11-oxycorticosteroids by rat adrenal cortex through nonopioid beta-endorphin receptors

β-Endorphin-like peptide immunorphin (SLTCLVKGFY), a selective agonist of nonopioid β-endorphin receptor, was labeled with tritium to specific activity of 24 Ci/mmol. It was used for the detection and characterization of nonopioid β-endorphin receptors on rat adrenal cortex membranes (K_d=31.6±0.2 nM, B_max=37.4±2.2 pmol/mg protein). Immunorphin at concentrations of 10⁻⁹ to 10⁻⁶ M was found to inhibit the adenylate cyclase activity in adrenal cortex membranes, while intramuscular injection of immunorphin at doses of 10–100 μg/kg was found to reduce the secretion of 11-oxycorticosteroids from the adrenals to the bloodstream.     

Production d'NH4 par la crevette Crangon crangon L. dans deux écosystémes côtiers. approche expérimentale et étude de l'influence du sédiment sur le taux d'excrétion

Estimation of the ammonia production of the shrimp C. crangon in two littoral ecosystems (oligotrophic sand and eutrophic mud) was determined in winter and summer conditions from laboratory observations in experimental microcosms. The ammonia excretion rate of C. crangon was not influenced by either the sediment type or the ammonia concentration of the overlying water; on the other hand, the mean excretion rate and the response to initial handling stress increased markedly as shrimp were deprived of soft substratum.

The daily ammonia production of C. crangon was 16 μmol NH₃ · g ⁻¹ wet wt · day ⁻¹ in winter and 40 μmol in summer. A gross production of 12 μmol NH₃ · m⁻² · day ⁻¹ and 300–700 μmol μ m⁻² · day⁻¹, respectively, could be expected in the two ecosystems studied. This would account for 5% (winter) and 2–4% (summer) of the total NH⁺₄ flux at the sediment-water interface. The contribution of the excretion of all macrofauna to the NH⁺₄ flux from the sediment is discussed.     

A novel one-pot three-component reaction: synthesis of triheterocyclic 4H-pyrimido[2,1-b]benzazoles ring systems

A novel one-pot three-component condensation reaction of an aldehyde, β-ketoester and 2-aminobenzimidazole or 2-aminobenzothiazole in 1,1,3,3-N,N,N′,N′-tetramethylguanidinium trifluoroacetate as an ionic liquid is described. During the course of this reaction 4H-pyrimido[2,1-b]benzimidazoles or 4H-pyrimido[2,1-b]benzothiazoles are formed in high yields at 100 °C. The ionic liquid can be recovered conveniently and reused efficiently.     

Synthesis, tuberculosis inhibitory activity, and SAR study of N-substituted-phenyl-1,2,3-triazole derivatives

The aim of this work was to describe the synthesis, the in vitro anti-Mycobacterium tuberculosis profile, and the structure–activity relationship (SAR) study of new N-substituted-phenyl-1,2,3-triazole-4-carbaldehydes (3a–l). The reactions of aromatic amine hydrochlorides with diazomalonaldehyde (1) produced several N-substituted-phenyl-1,2,3-triazole-4-carbaldehydes (3a–l) in moderate-to-good yields. In order to investigate the influence of the difluoromethylene group on the anti-Mycobacterium activity of these compounds, fluorination of triazoles with DAST converted the corresponding carbaldehyde compounds into new difluoromethyl derivatives (4a–l) in excellent yield. Characterization of all compounds was achieved by spectroscopic means and additional for 1-(4-methylphenyl)-1,2,3-triazole-4-carbaldehyde, 3k by X-ray crystallography. Compounds (3a–l) and (4a–l) have been screened for the inhibitory activity against Mycobacterium tuberculosis H37Rv strain (ATCC 27294) and all of them were able to inhibit the growth of the bacterium. Interestingly, 3a and 3k exhibited the best inhibition with MIC values of 2.5 μg/mL, similar to pharmaceuticals currently used in the treatment of tuberculosis. Our SAR study indicated the importance of the hydrogen bond acceptor subunit (3a–l), the position in the aromatic ring, the planarity of triazole and phenyl rings in these compounds, and a correlation between the uniform HOMO coefficient distribution and the anti-tubercular activity. The significant activity of 3a and 3k pointed them as promising lead molecules for further synthetic and biological exploration.     

Anti-proliferative lichen compounds with inhibitory activity on 12(S)-HETE production in human platelets

Several lichen compounds, i.e. lobaric acid (1), a β-orcinol depsidone from Stereocaulon alpinum L., (+)-protolichesterinic acid (2), an aliphatic -methylene-γ-lactone from Cetraria islandica Laur. (Parmeliaceae), (+)-usnic acid (3), a dibenzofuran from Cladonia arbuscula (Wallr.) Rabenh. (Cladoniaceae), parietin (4), an anthraquinone from Xanthoria elegans (Link) Th. Fr. (Calaplacaceae) and baeomycesic acid (5), a β-orcinol depside isolated from Thamnolia vermicularis (Sw.) Schaer. var. subuliformis (Ehrh.) Schaer. were tested for inhibitory activity on platelet-type 12(S)-lipoxygenase using a cell-based in vitro system in human platelets. Lobaric acid (1) and (+)-protolichesterinic acid (2) proved to be pronounced inhibitors of platelet-type 12(S)-lipoxygenase, whereas baeomycesic acid (5) showed only weak activity (inhibitory activity at a concentration of 100 μg/ml: 1 93.4±6.62%, 2 98,5±1.19%, 5 14.7±2.76%). Usnic acid (3) and parietin (4) were not active at this concentration. 1 and 2 showed a clear dose–response relationship in the range of 3.33–100 μg/ml. According to the calculated IC₅₀ values the highest inhibitory activity was observed for the depsidone 1 (IC₅₀=28.5 μM) followed by 2 (IC₅₀=77.0 μM). The activity of 1 was comparable to that of the flavone baicalein, which is known as a selective 12(S)-lipoxygenase inhibitor (IC₅₀=24.6 μM).     

Synergistic effects of sequential treatment with methyl jasmonate, salicylic acid and yeast extract on benzophenanthridine alkaloid accumulation and protein expression in Eschscholtzia californica suspension cultures

To develop an optimal bioprocess for secondary metabolite production and explain the bioprocess at the molecular level, we examine the synergistic effects of sequential treatment with methyl jasmonate (MJ), salicylic acid (SA) and yeast extract (YE) on benzophenanthridine alkaloid accumulation and protein expression in Eschscholtzia californica suspension cultures. Serial treatment of MJ, SA and YE at 24 h intervals enhanced the accumulation of dihydrosanguinarine (2.5 times) and sanguinarine (5.5 times). This sequential treatment using different signal elicitors was more effective than single elicitor or simultaneous treatment of the elicitors; it induced benzophenanthridine alkaloid accumulation to 917.7 ± 42.0 mg/L. Also, (S)-methylcoclaurine-3′-hydroxylase (CYP80B1) and 3′-hydroxy-(S)-N-methylcoclaurine-4′-O-methyltransferase (4′OMT) expressions among enzymes in sanguinarine biosynthetic pathway explained the synergistic effects by sequential treatment of the elicitors. The sequential treatment strategy using elicitors related to different signal transduction pathways can be used to design better processes to increase accumulation of secondary metabolites in plant cell culture. Analysis of protein expression provides the detailed information about metabolite accumulation through the correlated results.