Anti-proliferative, Morphological and Molecular Docking Studies of New Thiophene Derivatives and their Strategy in Ionic Liquids Immobilized Reactions.

BACKGROUND: A number of research were conducted on the pyran and thiophene derivatives, which were attributed to have a wide range of biological activities, including anti-plasmodial, as well as acting as caspase, hepatitis C and cancer inhibitors. OBJECTIVE: The multicomponent reactions of the 5-acetyl-2-amino-4-(phenylamino)-thiophene-3-carbonitrile produced biologically active target molecules like pyran and their fused derivatives. Comparison between regular catalytic multi-component reactions and solvent-free ionic liquids immobilized multicomponent was studied. METHODS: The multicomponent reactions in this work were carried out not only under the reflux conditions using triethylamine as a catalyst but also in solvent-free ionic liquids immobilized magnetic nanoparticles (MNPs) catalysts. RESULTS: Through this work, thirty-one new compounds were synthesized and characterized and were evaluated toward the six cancer cell lines, namely A549, HT-29, MKN-45, U87MG, and SMMC-7721 and H460. The most active compounds were further screened toward seventeen cancer cell lines classified according to the disease. In addition, the effect of compound 11e on the A549 cell line was selected to make further morphological changes in the cell line. The Molecular docking studies of 11e and 11f were carried and promising results were obtained. CONCLUSION: The synthesis of heterocyclic compounds derived from thiophene derivatives has been receiving significant attention. After a detailed optimizing study, it has been found that the solvent-free ionic liquids immobilized multi-component syntheses afforded a high yield of compounds, opening a greener procedure for this synthetically relevant transformation. Many of the synthesized compounds can be considered anticancer agents, enhancing further studies.

Synthesis and Biological Evaluation of New Fluorescent Probe BPN-01: A Model Molecule for Fluorescence Image-guided Surgery.

Fluorescence image-guided surgery (FIGS) can serve as a tool to achieve successful resection of tumour tissues during surgery, serving as a surgical navigator for surgeons. FIGS relies on the use of fluorescent molecules that can specifically interact with cancer cells. In this work, we developed a new model of fluorescent probe based on benzothiazole-phenylamide moiety featuring the visible fluorophore nitrobenzoxadiazole (NBD), namely BPN-01. This compound was designed and synthesised for potential applications in the tissue biopsy examination and ex-vivo imaging during FIGS of solid cancers. The probe BPN-01 exhibited favourable spectroscopic properties, particularly in nonpolar and alkaline solvents. Moreover, in vitro fluorescence imaging revealed that the probe appeared to recognise and be internalised in the prostate (DU-145) and melanoma (B16-F10) cancer cells, but not in the normal cells (myoblast C2C12). The cytotoxicity studies revealed that probe BPN-01 was not toxic to the B16 cells, suggesting excellent biocompatibility. Furthermore, the computational analysis showed that the calculated binding affinity of the probe to both translocator protein 18 kDa (TSPO) and human epidermal growth factor receptor 2 (HER2) was considerably high. Hence, probe BPN-01 displays promising properties and may be valuable for visualising cancer cells in vitro. Furthermore, ligand 5 can potentially be labelled with NIR fluorophore and radionuclide, and serves as a dual imaging agent for in vivo applications.

Separation and characterization of phenylamides from Piper kadsura using preparative supercritical fluid chromatography and ultra-high-performance supercritical fluid chromatography-tandem mass spectrometry.

A preparative supercritical fluid chromatography method for the separation of Piper kadsura obtained five phenylamide compounds, which had the same structural skeleton, but changed in the number and position of methoxyl substituents. To improve the separation selectivity of these structural analogues, silica, phenyl, and chiral stationary phases were screened. Only through the combination of Chiral C and phenyl columns could the separation of the five phenylamides be solved. The two-step strategy using preparative supercritical fluid chromatography presented good orthogonality that ensured the purity of the phenylamides. Then, an ultra-high-performance supercritical fluid chromatography hyphened tandem mass spectrometry method was developed, and the fragmentation pattern of phenylamides was summarized. It mainly cleaved in the amide bond to produce the fragment ion, which could help to judge the substituent positions. Twenty-eight possible molecular weights of hydroxyl and methoxyl substituted phenylamides were calculated and screened. Nine compounds were extracted in three [M + H]+ ions at m/z 284.13, 314.13, and 344.13, including five purified compounds and the other four positional or trans-cis phenylamide isomers in low content. The methods developed in this research were useful in the separation and characterization of phenylamide analogues.

Biosynthesis of Phenylamide Phytoalexins in Pathogen-Infected Barley.

Phytoalexins are inducible antimicrobial metabolites in plants, and have been indicated to be important for the rejection of microbial infection. HPLC analysis detected the induced accumulation of three compounds 1-3 in barley (Hordeum vulgare) roots infected by Fusarium culmorum, the causal agent of Fusarium root rot. Compounds 1-3 were identified as cinnamic acid amides of 9-hydroxy-8-oxotryptamine, 8-oxotryptamine, and (1H-indol-3-yl)methylamine, respectively, by spectroscopic analysis. Compounds 1 and 2 had been previously reported from wheat, whereas 3 was an undescribed compound. We named 1-3 as triticamides A-C, respectively, because they were isolated from barley and wheat, which belong to the Triticeae tribe. These compounds showed antimicrobial activities, indicating that triticamides function as phytoalexins in barley. The administration of deuterium-labeled N-cinnamoyl tryptamine (CinTry) to barley roots resulted in the effective incorporation of CinTry into 1 and 2, which suggested that they were synthesized through the oxidation of CinTry. Nine putative tryptamine hydroxycinnamoyl transferase (THT)-encoding genes (HvTHT1-HvTHT9) were identified by database search on the basis of homology to known THT gene sequences from rice. Since HvTHT7 and HvTHT8 had the same sequences except one base, we measured their expression levels in total by RT-qPCR. HvTHT7/8 were markedly upregulated in response to infection by F. culmorum. The HvTHT7 and HvTHT8 enzymes preferred cinnamoyl- and feruloyl-CoAs as acyl donors and tryptamine as an acyl acceptor, and (1H-indol-3-yl)methylamine was also accepted as an acyl acceptor. These findings suggested that HvTHT7/8 are responsible for the induced accumulation of triticamides in barley.

Identification of phenylamide phytoalexins and characterization of inducible phenylamide metabolism in wheat.

Changes in specialized metabolites were analyzed in wheat leaves inoculated with Bipolaris sorokiniana, the causal agent of spot blotch of Poaceae species. HPLC analysis detected the accumulation of six compounds in B. sorokiniana-infected leaves. Of these, we purified two compounds by silica gel and ODS column chromatography and preparative HPLC, and identified them as cinnamic acid amides, N-cinnamoyl-9-hydroxy-8-oxotryptamine and N-cinnamoyl-8-oxotryptamine, by spectroscopic analyses. The remaining four compounds were predicted to be p-coumaric acid amides of hydroxyputrescine, hydroxyagmatine, hydroxydehydroagmatine, and agmatine by mass spectrometry. The accumulation of two cinnamic acid amides was also induced by Fusarium graminearum infection, and by treatment with CuCl2, jasmonic acid, and isopentenyladenine. Antifungal activity of these amides was shown by inhibition of conidial germination and germ tube elongation of F. graminearum and Alternaria brassicicola, indicating that they act as phytoalexins. The accumulation of these amides also detected in barley leaves treated with CuCl2. We examined the accumulation of 25 phenylamides in B. sorokiniana-infected wheat leaves using LC-MS/MS. Hydroxycinnamic acid amides of tryptamine, serotonin, putrescine, and agmatine, were induced after infection with B. sorokiniana. Thus, the induced accumulation of two groups of phenylamides, cinnamic acid amides with indole amines, and p-coumaric acid amides with putrescine and agmatine related amines, represents a major metabolic response of wheat to pathogen infection.

Induced phenylamide accumulation in response to pathogen infection and hormone treatment in rice (Oryza sativa).

Rice plants accumulate various specialized metabolites, including phenylamides, in response to pathogen attack. We prepared 25 phenylamides, and developed a method of analyzing them by multiple reaction monitoring with liquid chromatography coupled with tandem mass spectrometry. We analyzed phenylamides in rice leaves infected with Cochliobolus miyabeanus and Xanthomonas oryzae. The phenylamides induced included benzoyltryptamine, cinnamoyl-, p-coumaroyl-, feruloyl-, and benzoylserotonins, cinnamoyl and benzoyltyramines, feruloylagmatine, and feruloylputrescine. Some of the phenylamides exhibited antimicrobial activity against C. miyabeanus and X. oryzae, indicating that they are phytoalexins. Treatment with jasmonic acid, salicylic acid, 6-benzylaminopurine, and ethephone also induced phenylamide accumulation. The compositions of the induced amides varied depending on the plant hormone used, and cinnamoyltryptamine, cinnamoylserotonin, and cinnamoyltyramine were not induced by the plant hormones. These findings suggest that several plant hormones and additional factors are involved in phenylamide accumulation in response to pathogen infection in rice.

Phenolic Phytoalexins in Rice: Biological Functions and Biosynthesis.

Phytoalexins are inducible secondary metabolites possessing antimicrobial activity against phytopathogens. Rice produces a wide array of phytoalexins in response to pathogen attacks and environmental stresses. With few exceptions, most phytoalexins identified in rice are diterpenoid compounds. Until very recently, flavonoid sakuranetin was the only known phenolic phytoalexin in rice. However, recent studies have shown that phenylamides are involved in defense against pathogen attacks in rice. Phenylamides are amine-conjugated phenolic acids that are induced by pathogen infections and abiotic stresses including ultra violet (UV) radiation in rice. Stress-induced phenylamides, such as N-trans-cinnamoyltryptamine, N-p-coumaroylserotonin and N-cinnamoyltyramine, have been reported to possess antimicrobial activities against rice bacterial and fungal pathogens, an indication of their direct inhibitory roles against invading pathogens. This finding suggests that phenylamides act as phytoalexins in rice and belong to phenolic phytoalexins along with sakuranetin. Phenylamides also have been implicated in cell wall reinforcement for disease resistance and allelopathy of rice. Synthesis of phenolic phytoalexins is stimulated by phytopathogen attacks and abiotic challenges including UV radiation. Accumulating evidence has demonstrated that biosynthetic pathways including the shikimate, phenylpropanoid and arylmonoamine pathways are coordinately activated for phenolic phytoalexin synthesis, and related genes are induced by biotic and abiotic stresses in rice.

Sequence diversity in the large subunit of RNA polymerase I contributes to Mefenoxam insensitivity in Phytophthora infestans.

Phenylamide fungicides have been widely used for the control of oomycete-incited plant diseases for over 30 years. Insensitivity to this chemical class of fungicide was recorded early in its usage history, but the precise protein(s) conditioning insensitivity has proven difficult to determine. To determine the genetic basis of insensitivity and to inform strategies for the cloning of the gene(s) responsible, genetic crosses were established between Mefenoxam sensitive and intermediate insensitive isolates of Phytophthora infestans, the potato late blight pathogen. F1 progeny showed the expected semi-dominant phenotypes for Mefenoxam insensitivity and suggested the involvement of multiple loci, complicating the positional cloning of the gene(s) conditioning insensitivity to Mefenoxam. Instead, a candidate gene strategy was used, based on previous observations that the primary effect of phenylamide compounds is to inhibit ribosomal RNA synthesis. The subunits of RNA polymerase I (RNApolI) were sequenced from sensitive and insensitive isolates and F1 progeny. Single nucleotide polymorphisms (SNPs) specific to insensitive field isolates were identified in the gene encoding the large subunit of RNApolI. In a survey of field isolates, SNP T1145A (Y382F) showed an 86% association with Mefenoxam insensitivity. Isolates not showing this association belonged predominantly to one P. infestans genotype. The transfer of the 'insensitive' allele of RPA190 to a sensitive isolate yielded transgenic lines that were insensitive to Mefenoxam. These results demonstrate that sequence variation in RPA190 contributes to insensitivity to Mefenoxam in P. infestans.

Transcriptomic analysis of UV-treated rice leaves reveals UV-induced phytoalexin biosynthetic pathways and their regulatory networks in rice.

Rice produces diterpenoid and flavonoid phytoalexins for defense against pathogen attack. The production of phytoalexins in rice is also induced by UV-irradiation. To understand the metabolic networks involved in UV-induced phytoalexin biosynthesis and their regulation, phytochemical and transcriptomic analyses of UV-treated rice leaves were performed. In response to UV treatment, the accumulation of flavonoids was observed in rice leaves, which may serve as antioxidants against UV-induced oxidative stress. The phytochemical analysis confirmed sakuranetin accumulation and also demonstrated the induction of phenylamide synthesis in rice leaves by UV-irradiation. Transcriptomic analysis established that aromatic amino acid biosynthetic genes were immediately up-regulated after UV treatment. The genes involved in the phenylpropanoid pathway and flavonoid biosynthesis were also up-regulated. These findings suggest that the aromatic amino acid and flavonoid biosynthetic pathways are coordinately activated for the production of flavonoids and phenolic phytoalexins such as sakuranetin and phenylamides. An in silico analysis of UV-induced O-methyltransferase and acyltransferase genes suggested that these genes may be implicated in sakuranetin and phenylamide synthesis, respectively. The transcriptomic analysis also showed up-regulation of both methylerythritol phosphate pathway and the diterpenoid phytoalexin biosynthetic genes in response to UV treatment. A functional gene network analysis of phytoalexin biosynthetic and UV-induced genes for signaling components and transcription factors using RiceNet suggested that regulatory networks comprising signal perceiving receptor kinases, G-proteins, signal transducing mitogen-activated protein kinases and calcium signaling components, and transcription factors control flavonoid and phytoalexin biosynthesis in rice leaves under UV-C stress conditions.