Laboratory Scale Continuous Flow Systems for the Enantioselective Phase Transfer Catalytic Synthesis of Quaternary Amino Acids.

The use of stereoselective phase-transfer catalysis as a reliable method for the enantioselective synthesis of optically active α-amino acid derivatives using achiral Schiff base esters has been well-developed in batch in the last 40 years. Recently, continuous flow technology has become of great interest in the academy and industry, since it offers safer process operating conditions and higher efficiency compared to a traditional batch processing. Herein, we wish to report the first example of enantioselective phase transfer benzylation of alanine Schiff base ester, under continuous flow conditions. Two different methodologies were investigated: a liquid-solid phase transfer catalytic benzylation using a packed-bed reactor and a liquid-liquid phase transfer catalytic benzylation in continuous stirred-tank reactors. Liquid-liquid phase transfer process in flow showed slightly better productivity than the batch process, while solid-liquid phase transfer benzylation proved much more advantageous in terms of productivity and space-time yield. Furthermore, continuous flow system allowed the isolation of benzylated product without any work up, with a significant simplification of the process. In both cases, phase transfer asymmetric benzylation promoted by Maruoka catalyst demonstrated high enantioselectivity of target quaternary amino ester in flow, up to 93% ee.

Ruthenium(II)-N-alkyl phenothiazine complexes as potential anticancer agents.

In recent years, the search for effective anticancer compounds based on transition metal complexes has been the focus of medical investigations. The synergy between the ruthenium(II) and N-alkylphenothiazine counter-ions (chlorpromazine hydrochloride, thioridazine hydrochloride and trifluoperazine dihydrochloride, respectively) through the formation of three different complexes (1-3) was investigated. We explored whether the selected counter-ions and complexes might affect redox homeostasis and genome integrity of normal human blood cells, and induce an inhibition of Na+/K+-ATPase and AChE at pharmacologically relevant doses. Our results have shown that counter-ions and complexes did not affect the activity of Na+/K+-ATPase, while AChE activity was inhibited in a dose-dependent manner. All investigated compounds disturbed the viability and redox homeostasis of lymphocytes. Complexes 1 and 2 displayed potent cytotoxic and prooxidant action while complex 3 behaved as a weaker genotoxic inducer. Still, the tested complexes appeared to be less genotoxic and more cytostatic than the corresponding counter-ions. The effects of selected complexes were also tested in PC12 and U2OS cancer cells with special attention being given to the ability of phenothiazines to affect dopamine D2 receptors. Using the confocal laser scanning microscopy, we observed that all the complexes reduced cell viability. Although all investigated complexes have been bound to the dopamine receptor D2-eGFP, only complex 3 reduced its surface density and increased its lateral mobility in investigated cell lines. Albeit the role of alternative targets for complex 3 cannot be ruled out, its effects should be further examined as potential treatment strategy against cancer cells that overexpress D2.

Determination of heavy metals in muscle tissue of six fish species with different feeding habits from the Danube River, Belgrade-public health and environmental risk assessment.

The aim of this study was to investigate the presence of heavy metals/metalloids (Pb, Cd, Hg, Cu, Fe, Zn, As) in the muscle tissue of fish from the Danube River (two locations: Zemun and Grocka). For the purpose of heavy metal determination in fish muscle, 120 samples of six different fish species, Prussian carp, barbel, bream, carp, pike perch, and catfish were collected. For determining heavy metals, we used microwave oven digestion and atomic absorption spectrometer methods. The highest average content of Pb (0.084 ± 0.004 mg kg-1), Cd (0.082 ± 0.003 mg kg-1), Hg (0.466 ± 0.006 mg kg-1), and As (0.333 ± 0.007 mg kg-1) was found in the muscle of carp (an omnivorous fish) from Grocka, while the highest average level of Fe (13.60 ± 0.03 mg kg-1) was deposited in bream (also omnivorous) from Zemun. Also, the average Cu level (1.62 ± 0.13 mg kg-1) was the highest in catfish muscle (a carnivorous fish) from Grocka, while the highest Zn content (11.16 ± 0.17 mg kg-1) was determined in muscle of Prussian carp (an omnivorous fish) from Zemun. The highest content of heavy metals (Cu, Fe, and Zn, respectively) in muscle of the six different types of fish from both locations was symmetrically arranged by species (catfish, barbel, and Prussian carp, respectively). Concentrations of Pb, Hg, and As in the Danube River fish muscle were under the maximum residual levels prescribed by the European Union (EU) and the maximum allowed concentrations (MAC) for Serbia. On the other hand, in all fish muscle from both locations (Zemun and Grocka), higher concentrations of Cd than prescribed (MAC) were found, with the exception of bream and pike perch.

Prevalence of Borrelia burgdorferi sensu lato in Ixodes ricinus ticks and assessment of entomological risk index at localities in Belgrade.

Background/Aim: The first case of human Lyme borreliosis (LB) in Serbia was recorded in 1987. The number of reported LB cases has increased in the past decade. The aim of this study was to estimate the density of Ixodes ricinus (I. ricinus) ticks, the prevalence of Borrelia burgdorferi sensu lato (B. burgdorferi) in them, and entomological risk index (ERI) at 19 Belgrade localities which were grouped into three categories (forests, parkforests, parks). The values of ERI were compared with the number of tick bites in humans. Methods: Ticks were collected monthly by using the flag hours method and the infection rate was determined by using dark field microscopy. The ERI value was calculated for each locality where the ticks were collected. The related data about tick bites was obtained from the patient protocol of the Institute of Epidemiology, Military Medical Academy, Belgrade. Results: The total number of collected ticks, the number of nymphs and the infection rates of the nymphs were significantly higher in forests (p < 0.05) than park-forests and parks. Statistically, the ERI value was significantly higher in forests than parks of Belgrade (χ2 = 7.78, p < 0.01). In March and July, the ERI value was also significantly higher in forests, than park-forests (p < 0.01) and parks (p < 0.01). May was the month with the highest ERI value in each ecological category (forests p < 0.05; park-forests p < 0.01; parks p < 0.001). However, the number of tick bites in humans did not correlate with ERI values. Conclusion: The obtained results indicate that the risk of tick bite and human exposure to B. burgdorferi sensu lato is present at all selected localities in Belgrade. For a more comprehensive Lyme disease risk assessment the method of entomological risk index assessment should be combined with other methods, taking into consideration all tick stages and the behaviour and habits of people who may get infected B. burgdorferi sensu lato.

Synthesis, structural and spectroscopic characterization, in vitro cytotoxicity and in vivo activity as free radical scavengers of chlorido(p-cymene) complexes of ruthenium(II) containing N-alkylphenothiazines.

Three new ruthenium(II) complexes 1-3 containing N-alkylphenothiazine molecules were synthesized by reaction of [RuCl(2)(η(6)-p-cymene)](2) with chlorpromazine hydrochloride (1), trifluoperazine dihydrochloride (2) or thioridazine hydrochloride (3). The compounds of the general formula L[RuCl(3)(η(6)-p-cymene)] were characterized by elemental analysis and spectroscopic methods (FT-IR, UV-Vis, (1)H and (13)C NMR). Complex 2 was structurally characterized by single crystal X-ray diffraction. In vitro cytotoxic activity of complexes 1-3 were assayed in four human carcinoma cell lines MCF-7, MDA-MB-453 (breast carcinoma), SW-480 (colon carcinoma) and IM9 (myeloma multiple cells). The highest cytotoxicity (12.1 ≤ IC(50) ≤ 17.3 µM) and induced a total (SW-480) or almost total cell death (MCF-7, MDA-MB-453) at 25 µM in 48 h of treatment were observed for complex 2. The influence of three different doses (0.4, 4.5 and 90.4 µM/kg bw) of complex 2 on activities of antioxidants enzymes (superoxide dismutase (SOD) and catalase (CAT)) and lactate dehydrogenase (LDH) were investigated under physiological conditions. The effects on nitrite production (NO(2)(-)) and level of erythrocytes malondialdehyde (MDA) in rats blood were evaluated, too.

New ruthenium(II) complexes with N-alkylphenothiazines: synthesis, structure, in vivo activity as free radical scavengers and in vitro cytotoxicity.

Three new complexes of the general formula L[RuCl(3)(DMSO)(3)] (1-3), where L = chlorpromazine hydrochloride, trifluoroperazine dihydrochloride or thioridazine hydrochloride, were prepared and characterized by elemental analysis and spectroscopic methods (FT-IR, UV-Vis, (1)H NMR and (13)C NMR). In addition, the crystal structure of the complex 2 containing trifluoroperazine dihydrochloride was solved by single crystal X-ray diffraction. The complex crystallizes in the monoclinic system, space group P2(1)/n, with a = 10.4935(7) A, b = 18.6836(12) A, c = 19.9250(13) A, beta = 98.448(2) degrees, V = 3864.0(4) A(3). The structure was refined to the agreement factors of R = 4.79%, R(w) = 11.23%. The effect of three different doses (0.4, 4.5 and 90.4 microM/kg bw) of complex 2 on superoxide dismutase (SOD) and catalase (CAT) activity was investigated under physiological conditions. Influence on nitrite production (NO(2)(-)) and the level of erythrocytes malondialdehyde (MDA) in rats blood was also evaluated. Complex 2 did not affect the CAT enzyme activity in vivo and did not cause the hydroxyl radicals production. In the 0.4 and 4.5 microM/kg bw doses it showed almost the same or lower SOD activity and nitrite levels, while the dose of 90.4 microM/kg bw significantly increased these parameters. Finally, the cytotoxicity of complexes were assayed in four human carcinoma cell lines MCF-7, MDA-MB-453 (breast carcinoma), SW-480 (colon adenocarcinoma) and IM9 (myeloma multiple cells). Antiproliferative activity in vitro with low IC(50) during 48 h of treatment was observed.

[Risk of Lyme disease development after a tick bite].

BACKGROUND/AIM: Despite numerous research of Lyme disease (LD), there are still many concerns about environmental of infectious agent of LD, as well as its prophylaxis, diagnosis and treatment. The aim of this work was to determine the risk of LD in relation to the way of removing ticks and duration of tick attachment. METHODS: In the period from 2000 to 2007 a prospective study was conducted including persons with tick bite referred to the Institute of Epidemiology, Military Medical Academy, and followed for the occurrence of early Lyme disease up to six months after a tick bite. Epidemiological questionnaire was used to collect relevant information about the place and time of tick bites, the way of a removing tick, duration of tick attachment, remnants of a tick left in the skin (parts of the mouth device) and the signs of clinical manifestations of LD. Duration of tick attachment was determined on the basis of size of engorged tick and epidemiological data. Removed ticks were determined by the key of Pomerancev. Professional removing of attached tick was considered to be removing of tick with mechanical means by healthcare personnel. Fisher's exact test, Chi squares test and calculation of the relative risk (RR) were used for data analysis. RESULTS: Of 3 126 patients with tick bite, clinical manifestations of LD were demonstrated in 19 (0.61%). In the group of subjects (n = 829) in which a tick was not removed professionally there were 17 (2.05%) cases with LD, while in the group of respondents (n = 2 297) in who a tick was removed professionally there were 2 (0.09%) cases with LD after tick bite (RR, 23.55; p < 0.0001). The disease was most frequent in the group of respondents with incompletely and unprofessionally removed ticks (2.46%). In the groups of patients with unprofessionally but completely removed ticks LD occurred in 0.89%, while in the group of subjects with a tick removed by an expert, but incompletely in 0.78% cases. The disease occurred rarely in the group with a tick removed completely and professionally (0.05%). There was no case of LD in the group of patients with a tick removed within 24 hours. The longer time of exposure after 24 hours, the higher absolute risk of disease was reported. CONCLUSION: In prevention of Lyme disease it is important to urgent remove a tick, to use a correct procedure of removing and to remove the whole tick without any remnants.

[Risk for infection by lyme disease cause in green surfaces maintenance workers in Belgrade].

BACKGROUND/AIM: Lyme disease (LD) is a cosmopolitan disease from the group of zoonoses that in humans can affect skins, joints, heart and/or nervous system. Research conducted in endemic areas, shows not only that the population, which lives or works in tick habitats, is in a high risk for getting infected by LD cause, but also that every enviroment itself is specific in regard to both vectors and B. burgdoferi species. In our enviroment, research of seropositivity to B. bugdorferi in persons exposed to vectors of LD have not been conducted. The aim of this study was to determine whether there was a higher risk for getting B. burgdofei infection in the fieldworks of JKP "Zelenilo Beograd" than in other persons. METHODS: Risk for getting LD in the fieldworkers of JKP "Zelenilo Beograd" (park-maintanance company) was determined according to seropositivity, the number and infection rate of LD vectors, and entomological risk index (ERI) value. Serum samples of 34 fieldworkers ofJKP "Zelenilo Beograd", and 35 persons of the control group, were tested for the presence of specific IgM and IgG anti-B. burgdofedi antibodies by using ELISA test In the research localities, the number of ticks was estimated by using the flag hours method, and tick infection rate was determined by dark field microscopy, with magnification rate of 400x. The data were analyzed statistically using chi(2) test, and Pearson's correlation analysis. RESULTS: Out of 34 fieldworkes serum samples, 8 (23.5%) were positive on anti-B. buagdorfei antibody presence, while in the control group only one (2.9%/0) sample was positive (p < 0,05). Seropositivity rate varied from 0-66.6% among localities. Flag hours value among the localities ranged from 8.2-29.9, and tick infection rate ranged from 10.8-22.3%. ERI value was 0.03-0.15. Correlation between flag hour rate and seropositivity, tick infection rate and seropositivity, and also ERI and seropositivity was established. CONCLUSION: Frequency of B. burgdorferi seropositivity was significantly higher in green-surface maintenance workers, than in the control group not exposed to these habitats. A significant correlation was found between anti-B. burgdofedi antibodies findings and the number of ticks, tick infection rate, and ERI. The results obtained in our study indicate that there was a significantly higher risk for getting LD cause in the fieldworkers than in the control group.