Zika virus (ZIKV, genus Flavivirus) has emerged as a major mosquito-transmitted human pathogen, with recent outbreaks associated with an increased incidence of neurological complications, particularly microcephaly and the Guillain-Barré syndrome. Because the virus has only very recently emerged as an important pathogen, research is being hampered by a lack of reliable molecular tools. Here we report an infectious cDNA (icDNA) clone for ZIKV isolate BeH819015 from Brazil, which was selected as representative of South American ZIKV isolated at early stages of the outbreak. icDNA clones were assembled from synthetic DNA fragments corresponding to the consensus sequence of the BeH819015 isolate. Virus rescued from the icDNA clone had properties identical to a natural ZIKV isolate from South America. Variants of the clone-derived virus, expressing nanoluciferase, enhanced green fluorescent or mCherry marker proteins in both mammalian and insect cells and being genetically stable for multiple in vitro passages, were obtained. A ZIKV subgenomic replicon, lacking a prM- and E glycoprotein encoding region and expressing a Gaussia luciferase marker, was constructed and shown to replicate both in mammalian and insect cells. In the presence of the Semliki Forest virus replicon, expressing ZIKV structural proteins, the ZIKV replicon was packaged into virus-replicon particles. Efficient reverse genetic systems, genetically stable marker viruses and packaged replicons offer significant improvements for biological studies of ZIKV infection and disease, as well as for the development of antiviral approaches.
Pincer complexes featuring readily tunable tridentate ligand frameworks comprise one of the most actively studied classes of organometallic and metalorganic compounds and find extensive use in catalysis, organic synthesis, materials science, and other fields of chemistry and allied disciplines. Currently growing attention is devoted to non-classical ligand scaffolds, such as functionalized carboxamides, which offer multiple options for directed structural modifications. In this study, the reactions of (methylsulfanyl)acetyl and propanoyl chlorides with 2-(aminomethyl)pyridine, 2-(2-aminoethyl)pyridine, 8aminoquinoline and 2-(diphenylthiophosphoryl)aniline afford a series of new pincer-type ligands based on functionalized carboxamides. The ligands obtained readily undergo direct cyclopalladation under the action of PdCl 2 (NCPh) 2 in dichloromethane at room temperature, resulting in Pd(II) pincer complexes with N,N,S-and S,N,S-donor sets. Importantly, some of the cyclopalladated derivatives can also be produced efficiently under solvent-free conditions according to the approach recently developed by our group. The complexes obtained have been tested for cytotoxicity against several human cancer cell lines and catalytic activity in the model Suzuki reaction. The results have been compared to those for the related Pd(II) pincer complexes to define the main structure-activity relationships and to outline the most promising structures for further investigations. Yield: 87 mg (95%). M.p. > 225°C (dec.). 1 H NMR (400.13 MHz, CDCl 3 , δ, ppm): 2.73 (ddd, 1H, CH 2 , 2 J HH = 1.9 Hz, 3 J HH = 16.8 Hz, 3 J HH = 7.3 Hz), 3.15 (ddd, 1H, CH 2 S(O), 2 J HH = 2.2 Hz, 3 J HH = 16.8 Hz, 3 J HH = 11.4 Hz), 3.36 (ddd, 1H, CH 2 , 2 J HH = 1.9 Hz, 3 J HH = 13.0 Hz, 3 J HH = 11.4 Hz), 3.47 (ddd, 1H, CH 2 S(O), 2 J HH = 2.2 Hz, 3 J HH = 13.0 Hz, 3 J HH = 7.3 Hz), 3.51 (s, 3H, Me), 5.03 and 5.22 (ABq, 2H, CH 2 Py, J AB = 20.3 Hz), 7.35-7.39 (m, 1H, H(C4)), 7.48 (d, 1H, H(C2), 3 J HH = 7.9 Hz), 7.91 (dt, 1H, H(C3), 3 J HH = 7.9 Hz, 4 J HH = 1.6 Hz), 9.05 (d, 1H, H(C5), 3 J HH = 5.4 Hz).13
The reactions of picolinyl and 4-chloropicolinyl chlorides with methyl esters of S-methyl-l-cysteine, l- and d-methionine, and l-histidine afforded a series of functionalized carboxamides, which readily formed pincer-type complexes upon interaction with PdCl(NCPh) in solution under mild conditions. The direct cyclopalladation of the ligands derived was also accomplished in the solid phase, in particular, mechanochemically, although it was complicated by the partial deactivation of the starting amides. The resulting complexes with 5,5- and 5,6-membered fused metallocycles were fully characterized by IR and NMR spectroscopy, including variable-temperature and 2D-NMR studies. In the case of some cysteine- and methionine-based derivatives, the realization of κ-N,N,S-coordination was supported by X-ray diffraction. The cytotoxic effects of these complexes were examined on HCT116, MCF7, and PC3 human cancer cell lines as well as HEK293 as a representative of normal cells. The comparative studies allowed us to determine that the presence of the sulfide ancillary donor group is crucial for cytotoxic activity of this type of Pd(II) complexes. The main structure-activity relationships and the most promising palladocycles were outlined. The additional studies by gel electrophoresis revealed that 4-chloropicolinyl derivatives, despite the nature of an amino acid, can bind with DNA and inhibit topoisomerase I activity.
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