Caliciviruses are contagious pathogens of humans and various animals. They are the most common cause of viral gastroenteritis in humans, and can cause lethal diseases in domestic animals such as cats, rabbits and immunocompromised mice. In this study, we conducted cytopathic effect-based screening of 2080 selected compounds from our in-house library to find antiviral compounds against three culturable caliciviruses: feline calicivirus, murine norovirus (MNV) and porcine sapovirus (PoSaV). We identified active six compounds, of which two compounds, both related to theaflavins, showed broad antiviral activities against all three caliciviruses; three compounds (abamectin, a mixture of avermectin B1a and B1b; avermectin B1a; and (− )-epigallocatechin gallate hydrate) were effective against PoSaV only; and a heterocyclic carboxamide derivative (BFTC) specifically inhibited MNV infectivity in cell cultures. Further studies of the antiviral mechanism and structure-activity relationship of theaflavins suggested the following: (1) theaflavins worked before the viral entry step; (2) the effect of theaflavins was time-and concentration-dependent; and (3) the hydroxyl groups of the benzocycloheptenone ring were probably important for the anti-calicivirus activity of theaflavins. Theaflavins could be used for the calicivirus research, and as potential disinfectants and antiviral reagents to prevent and control calicivirus infections in animals and humans.

There is an urgent need for structurally novel anti-norovirus agents. In this study, we describe the synthesis, anti-norovirus activity, and structure-activity relationship (SAR) of a series of heterocyclic carboxamide derivatives. Heterocyclic carboxamide 1 (50% effective concentration (EC 50 ) 37 µM) was identified by our screening campaign using the cytopathic effect reduction assay. Initial SAR studies suggested the importance of halogen substituents on the heterocyclic scaffold and identified 3,5-di-boromo-thiophene derivative 2j (EC 50 24 µM) and 4,6-di-fluoro-benzothiazole derivative 3j (EC 50 5.6 µM) as more potent inhibitors than 1. Moreover, their hybrid compound, 3,5-di-bromo-thiophen-4,6-di-fluoro-benzothiazole 4b, showed the most potent anti-norovirus activity with a EC 50 value of 0.53 µM (70-fold more potent than 1). Further investigation suggested that 4b might inhibit intracellular viral replication or the late stage of viral infection.Key words norovirus; heterocyclic carboxamide; antiviral activity; murine norovirus Human norovirus causes acute nonbacterial gastroenteritis. Although the gastroenteritis symptoms are generally self-limiting, the elderly, infants, and immunocompromised individuals have a higher risk of mortality. Moreover, norovirus has a low infectious dose and spreads very easily from infected persons, contaminated food, water, or a contaminated environment. 1)Noroviruses are single-stranded, positive-sense RNA viruses that belong to the family Caliciviridae and genus Norovirus, and species Norwalk virus. The norovirus genome contains three open reading frames (ORFs): ORF1, ORF2, and ORF3. ORF1 encodes six or seven nonstructural proteins, including RNA-dependent RNA polymerase (RdRp) and 3C-like cysteine protease (3CLpro), responsible for viral replication. ORF2 and ORF3 encode the major and minor structural proteins, respectively. 1)Establishment of an efficient cell culture system for human norovirus is still challenging.2,3) However, murine norovirus (MNV) can replicate efficiently in the murine macrophage cell line RAW264.7. MNV belongs to the genus Norovirus and shares common biological and molecular properties with human norovirus.4,5) Therefore, it is frequently used for identifying antiviral compounds based on the cell culture assay. [6][7][8][9][10][11][12] Several anti-norovirus agents have been reported that target viral attachment/entry, viral protein translation, or viral replication. 7,8) The RdRp inhibitor used as an anti-hepatitis C virus agent 2′-C-methylcytidine (2′-CMC) 9,10) and anti-influenza agent favipiravir 11) also have anti-MNV activity, and the broad spectrum 3CLpro inhibitor, dipeptidyl inhibitor GC376 also inhibits MNV protease activity in vitro.12) Currently, there are no vaccines or drugs for clinical use, and there is an urgent need for new anti-norovirus compounds.We describe here the discovery and synthesis of anti-norovirus thienyl benzothiazolyl carboxamide compounds bearing multiple halogen substituents, and discuss their structure-activity ...

Using chiral liquid chromatography-mass spectrometry (LC/MS), a simple and rapid identification test was developed for aminotadalafil [(6R,12aR)-2-amino-6-(1,3-benzodioxol-5-yl)-2,3,6,7,12,12a-hexahydropyrazino[1 ,2 : 1,6]pyrido [3,4-b]indole-1,4-dione; RR-ATDF] and its stereoisomers contained in health foods, e.g. herbal products. A sample solution was prepared using methanol extraction. Analysis was performed on a chiral column with the mixture of 0.1% formic acid/acetonitrile (7 : 3) as mobile phase at 30• C. Each resolution value of four stereoisomers of ATDF was greater than 1.3. A mass spectrometer was used as a detector to enhance specificity by excluding the effects of general components derived from the sample. The four individual stereoisomers of ATDF in the health foods were identified based on their respective retention times. Results showed that structural conversions of RR-ATDF into (6R,12aS)-2-amino-6-(1,3-benzodioxol-5-yl)-2,3,6,7,12,12a-hexahydropyrazino[1 ,2 : 1,6]pyrido [3,4-b]indole-1,4-dione (RS-ATDF) and (6S ,12aS)-2-amino-6-(1,3-benzodioxol-5-yl)-2,3,6,7,12,12a-hexahydropyrazino[1 ,2 : 1,6]pyrido [3,4-b]indole-1,4-dione (SS-ATDF) into (6S ,12aR)-2-amino-6-(1,3-benzodioxol-5-yl)-2, 3,6,7,12,12a-hexahydropyrazino[1 ,2 : 1,6]pyrido [3,4-b]indole-1,4-dione (SR-ATDF) occurred under strongly basic conditions, which indicates that such conditions must be avoided during sample preparation. Using this method, RR-ATDF and SR-ATDF were detected in a health food; this is the first report describing that ATDF diastereomers are present in health foods.

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A rapid, simple, and reliable liquid chromatographic method has been developed for the simultaneous determination of nicotinamide (niacinamide), thiamine, riboflavin, riboflavin sodium phosphate, pyridoxine, caffeine, and sodium benzoate in commercial oral liquid tonics. The 7 components are separated on a reverse phase C18 column using a mobile phase of acetonitrile-O.OlM potassium dihydrogen phosphate-triethylamine (8 + 91.5 + 0.5 v/v/v) containing 5mM sodium octanesulfonate and adjusted to pH 2.8 with phosphoric acid. Components are detected at 254 nm with attenuation 0.02 AUFS. Acetanilide is used as an internal standard. In addition to the 7 components mentioned, nicotinic acid (niacin), cyanocobalamin, and folic acid are also separated under the same conditions. Sample preparation involves only addition to internal standard solution and dilution with mobile phase and then filtration. Recoveries of the 7 components and cyanocobalamin from spiked preparations ranged from 97 to 104% with coefficients of variation of 0.9-4.2%.