Novel Benzotriazole-ß-lactam Derivatives as Antimalarial Agents: Design, Synthesis, Biological Evaluation and Molecular Docking Studies.

Many people around the world suffer from malaria, especially in tropical or subtropical regions. While malaria medications have shown success in treating malaria, there is still a problem with resistance to these drugs. Herein, we designed and synthesized some structurally novel benzotriazole-ß-lactams using 2-(1H-benzo[d][1,2,3]triazol-1-yl)acetic acid as a key intermediate. To synthesize the target molecules, the ketene-imine cycloaddition reaction was employed. First, The reaction of 1H-benzo[d][1,2,3]triazole with 2-bromoacetic acid in aqueous sodium hydroxide yielded 2-(1H-benzo[d][1,2,3]triazol-1-yl)acetic acid. Then, the treatment of 2-(1H-benzo[d][1,2,3]triazol-1-yl)acetic acid with tosyl chloride, triethyl amine, and Schiff base provided new ß-lactams in good to moderate yields.The formation of all cycloadducts was confirmed by elemental analysis, FT-IR, NMR and mass spectral data. Moreover, X-ray crystallography was used to determine the relative stereochemistry of 4a compound. The in vitro antimalarial activity test was conducted for each compound against P. falciparum K1. The IC50 values ranged from 5.56 to 25.65 µM. A cytotoxicity profile of the compounds at 200 µM final concentration revealed suitable selectivity of the compounds for malaria treatment. Furthermore, the docking study was carried out for each compound into the P. falciparum dihydrofolate reductase enzyme (PfDHFR) binding site to analyze their possible binding orientation in the active site.

In vitro evaluation of cytotoxic effects of di (2-ethylhexyl) phthalate (DEHP) produced by Bacillus velezensis strain RP137 isolated from Persian Gulf.

Phthalates are widely used in polymer science and have potential toxicity related to their chemical structures. However, lots of evidence indicate that phthalate derivatives are undoubtedly produced as secondary metabolites by organisms, including plants, animals, and microorganisms. In the present study, Bacillus velezensis strain RP137 was cultured under optimized conditions. Its biomass was extracted with ethyl acetate with one fraction showing cytotoxic properties. A pure compound was isolated from the active fraction using combined silica gel and LH20 size exclusion column chromatography. Structural evaluation including FT-IR, 1H NMR, 13C NMR, HR-MS and CHN analysis identified the purified compound as di(2-ethylhexyl)phthalate (DEHP) with the formula C24H38O4 and the molecular weight of 389.29 Da. The microorganism-derived (stereospecific) DEHP was strongly reduced the proliferation and induced cytotoxic effects on various eukaryotic cell lines in compare to the synthetic racemic mixture of the compound when assessed by MTT assay. Furthermore, crystal violet assay and morphological changes confirmed the cytotoxic effect of DEHP. Interestingly, non-malignant SV40-immortalized fibroblast cells were less affected by the purified DEHP. Further evaluation on the antibacterial activity of DEHP documented no effect toward Gram-positive (S. aureus) and Gram-negative (E. coli and P. aeruginosa) pathogens even at a high concentration of 100 µM. In conclusion, existence of DEHP as byproduct of microorganism's metabolism can seriously be considered as a warning to human health.

Optimization of Fermentation Conditions to Enhance Cytotoxic Metabolites Production by Bacillus velezensis Strain RP137 from the Persian Gulf.

BACKGROUND: Isolation, introduction and producing bioactive compounds from bacteria, especially marine bacteria, is an attractive research area. One of the main challenges of using these metabolites as drug and their industrialization is the optimization of production conditions. METHODS: In the present study, the response surface methodology was applied to optimize the production of a cytotoxic extract (C-137-R) by Bacillus velezensis (B. velezensis) strain RP137. Initially, among the three carbon and three nitrogen sources, rice starch and potassium nitrate were selected as the best, with cell toxicity equal to IC50=54.4 and 45.1 µg/ml in human lung and liver cancer cell lines, respectively (A549 and HepG2). In the next step, fractional factorial design was performed to survey effect of seven physical and chemical factors on the amount of production, and the most important factors including carbon and nitrogen sources with the positive effect and the sea salt with negative effect were determined. Finally, using the central composite design with 20 experiments, the best concentrations of rice starch and potassium nitrate (1.5%) and sea salt (1%) were obtained. RESULTS: The average amount of dried extract produced in the optimum conditions was 131.1 mg/L and the best response was 71.45%, which is more than 28-fold better than the pre-optimized conditions. CONCLUSION: In general, it can be suggested that the use of modern statistical methods to optimize environmental conditions affecting the growth and metabolism of bacteria can be a highly valuable tool in industrializing the production of bioactive compounds.

Microindoline 581, an Indole Derivative from Microbacterium Sp. RP581 as A Novel Selective Antineoplastic Agent to Combat Hepatic Cancer Cells: Production, Optimization and Structural Elucidation.

Screening of bioactive compounds with potential binding affinity to DNA as one of the target molecules in fighting against cancer cells has gained the attention of many scientists. Finding such compounds in the cellular content of microorganisms, especially marine bacteria as valuable and rich natural resources, is of great importance. Microbacterium sp. RP581, as a member of Actinobacteria phylum, was isolated from the Persian Gulf coastal area and the production of the target compound was optimized using statistical methods in cheap culture ingredients. The purification of the target compound was performed by flash chromatography and preparative HPLC. Both molecular and structural analyses indicated that the compound was an indole derivate which was tentatively named as Microindoline 581. Interaction of Microindoline 581 with genomic and circular DNA revealed that this compound can cause double- strand breaks through binding to the DNA. The analysis of cellular growth and proliferation of various cancer cell lines suggested proper and specific effect of the Microindoline 581 towards HepG2 cells with an IC50 of 172.2 ± 1.7 µM. Additional studies on cell migration inhibition and cell-death induction indicated a concentration-dependent inhibitory effect on proliferation and induction of death of HepG2 cells. The selective action of Microindoline 581 which was isolated from the Microbacterium sp. RP581 in killing HepG2 cells might be due to its specific metabolism in those cells as a precursor.

An Aminoglycoside Antibacterial Substance, S-137-R, Produced by Newly Isolated Bacillus velezensis Strain RP137 from the Persian Gulf.

Given antibiotic resistance in pathogens, finding antibiotics from new sources is always a topic of interest to scientists. In the present study, among various isolates from the Persian Gulf coastal area, the strain RP137 was selected as producer of antibacterial compound. Morphological and biochemical studies along with 16S rDNA sequencing showed that strain RP137 belongs to Bacillus genus and was tentatively named Bacillus velezensis strain RP137. The effect of various carbon and nitrogen sources on optimizing the production of antibacterial compound showed that the low-cost rice starch and potassium nitrate supply to the strain RP137 caused producing of 86.0 ± 8.7 µg/mL extract having the antibacterial activity. The fractionation of the primary methanol extract in different solvents followed by reversed-phase HPLC obtained a pure antibacterial-active sample, S-137-R. Structural analysis of the purified S-137-R with the help of FTIR, HR-MS, 1H-NMR, and 13C-NMR showed that the S-137-R compound is classified as aminoglycoside. Minimum inhibition concentration (MIC) of the pure compound for Gram-positive bacteria, Staphylococcus aureus and methicillin resistant Staphylococcus aureus, showed an average antibacterial effect of about 80 µg/mL and 150 µg/mL, respectively and for Pseudomonas aeruginosa (100 µg/mL), while having very little toxic effect on E. coli. Moreover, low cytotoxicity effect of the S-137-R on cancerous and normal cells as well as the low intensity of the hemolysis of red blood cells in higher concentrations of S-137-R make it an ideal candidate for further structure-activity relationship assessments towards its medical applications.

Synthesis and biological evaluation of some novel diastereoselective benzothiazole ß-lactam conjugates.

Highly diastereoselective synthesis of some novel benzothiazole-substituted ß-lactam hybrids was achieved starting from (benzo[d]thiazol-2-yl)phenol as an available precursor. This is the first time (benzo[d]thiazol-2-yl)phenoxyacetic acid has been used as ketene source in synthesizing monocyclic 2-azetidinones. These compounds were evaluated for their antimicrobial activities against a large panel of Gram-positive and Gram-negative bacterial strains and moderate activities were encountered. Antimalarial data revealed that adding methoxyphenyl or ethoxyphenyl group on the ß-lactam ring makes compounds that are more potent. Moreover, hemolytic activity and mammalian cell toxicity survey of the compounds showed their potential as a medicine.

Investigation of the complex structure, comparative DNA-binding and DNA cleavage of two water-soluble mono-nuclear lanthanum(III) complexes and cytotoxic activity of chitosan-coated magnetic nanoparticles as drug delivery for the complexes.

Two water-soluble mono-nuclear macrocyclic lanthanum(III) complexes of 2,6-diformyl-4-methylphenol with 1,3-diamino-2-propanol (C1) or 1,3-propylenediamine (C2) were synthesized and characterized by UV-Vis, FT-IR, 13C and 1H NMR spectroscopy and elemental analysis. C1 complex was structurally characterized by single-crystal X-ray diffraction, which revealed that the complex was mononuclear and ten-coordinated. The coordination sites around lanthanum(III) were occupied with a five-dentate ligand, two bidentate nitrates, and one water molecule. The interaction of complexes with DNA was studied in buffered aqueous solution at pH7.4. UV-Vis absorption spectroscopy, emission spectroscopy, circular dichroism (CD) and viscometric measurements provided clear evidence of the intercalation mechanism of binding. The obtained intrinsic binding constants (Kb) 9.3×103 and 1.2×103M-1 for C1 and C2, respectively confirmed that C1 is better intercalator than C2. The DNA docking studies suggested that the complexes bind with DNA in a groove binding mode with the binding affinity of C1>C2. Moreover, agarose gel electrophoresis study of the DNA-complex for both compounds revealed that the C1 intercalation cause ethidium bromide replacement in a competitive manner which confirms the suggested mechanism of binding. Finally, the anticancer experiments for the treated cancerous cell lines with both synthesized compounds show that these hydrophilic molecules need a suitable carrier to pass through the hydrophobic nature of cell membrane efficiently.

Secretion of recombinant archeal lipase mediated by SVP2 signal peptide in Escherichia coli and its optimization by response surface methodology.

Towards the targeting of recombinant Thermoanaerobacter thermohydrosulfuricus lipase (TtL) for secretion into the culture medium of Escherichia coli, we have investigated a combination of the archeal lipase gene with a Salinovibrio metalloprotease (SVP2) signal peptide sequence. The SVP2 signal peptide has shown all necessary features of a leader sequence for high level secretion of a recombinant target protein in E. coli. Two sets of primers were designed for amplification of the corresponding gene fragments by PCR. Firstly, the PCR product of the TtL gene with designed restriction sites of SacI and HindIII was cloned into pQE-80L plasmid, named as pQE80L-TtL. Afterwards, the amplified fragment of SVP2 signal peptide with EcoRI and SacI restriction sites was also cloned into pQE80L-TtL and the final construct pQE-STL was obtained. A study on the extracellular expression of recombinant STL revealed that most of the enzyme activity was located in the periplasmic space. Glycine and Triton X-100 were investigated to determine whether the leakage of recombinant STL from the outer membrane was promoted, and it was revealed that glycine has a positive effect. Statistical media optimization design was then applied to optimize the effect of seven factors including glycine, Triton X-100, IPTG, yeast extract concentration, incubation time, induction time, and temperature on the extracellular expression of STL. The optimum conditions for the secretion of the lipase was obtained by incubating recombinant E. coli BL21 cells in the medium supplemented by 1.27% glycine and 24h of incubation in the presence of 0.2mM IPTG concentration.