Development of benzodioxine-heteroarylpiperazines as highly potent and selective α2c antagonists.

Here is reported the design and synthesis of a series of highly potent and selective α2C antagonists using benzodioxine methyl piperazine as a central scaffold by pharmacophoric analysis to improve the pharmacokinetics of suboptimal clinical candidate molecules.

Circadian entrainment in Arabidopsis.

Circadian clocks coordinate physiology and development as an adaption to the oscillating day/night cycle caused by the rotation of Earth on its axis and the changing length of day and night away from the equator caused by orbiting the sun. Circadian clocks confer advantages by entraining to rhythmic environmental cycles to ensure that internal events within the plant occur at the correct time with respect to the cyclic external environment. Advances in determining the structure of circadian oscillators and the pathways that allow them to respond to light, temperature, and metabolic signals have begun to provide a mechanistic insight to the process of entrainment in Arabidopsis (Arabidopsis thaliana). We describe the concepts of entrainment and how it occurs. It is likely that a thorough mechanistic understanding of the genetic and physiological basis of circadian entrainment will provide opportunities for crop improvement.

2,3-Dihydrobenzo-dioxine piperidine derivatives as potent and selective α2c antagonists.

In this manuscript, we report a series of benzodioxine methyl piperidine derivatives as highly potent and selective α2C antagonists by ligand design to improve the pharmacokinetics of a previous candidate molecule.

The FvCYP714C2 gene plays an important role in gibberellin synthesis in the woodland strawberry.

BACKGROUND: Fragaria vesca, the woodland strawberry, is a diploid relative of the cultivated strawberry. A GA-deficient mutant was found in ethyl methanesulfonate (EMS)-mutagenized lines of the Fragaria vesca accession 'Yellow Wonder'. OBJECTIVE: CYP714C2 was found to be differentially expressed using RNA-seq analysis. It is necessary to identify the function of this gene. METHODS: In order to identify the function of this gene, it was cloned and transformed into Arabidopsis thaliana. RESULTS: The DNA sequence of CYP714C2 was found to be 1940 bp in length, with an open reading frame (ORF) of 1539 bp that is predicted to encode a protein of 512 amino acids. The hydrophilicity of this protein is low and it is unstable. The highest relative expression of FvCYP714C2 was found in the leaves, followed by the pedicels, and low expression levels were found in the other tissues examined. Constitutive expression of FvCYP714C2 significantly promoted the growth of transgenic A. thaliana plants; transgenic Arabidopsis plants grew faster and grew well than wild type Col-0 plants. GA1+3 contents of the genetically modified Arabidopsis lines were significantly higher than that in the wild type. CONCLUSION: We conclude that FvCYP714C2 is a gene that functions in the gibberellin biosynthesis pathway in strawberry.

The types of skippers described by Shu-Iti Murayama (Lepidoptera: Hesperiidae).

Fifteen holotypes of Asian Hesperiidae taxa described by Shu-iti Murayama were examined, and their taxonomic status is discussed. We confirm that five are valid names while the rest are synonyms as indicated partially by previous authors. Valid species-level names are Aeromachus matudai (Murayama), Aeromachus bandaishanus Murayama Shimonoya, Coladenia pinsbukana (Shimonoya Murayama), and Sebastonyma suthepiana Murayama Kimura. Valid subspecies-level name is Ochlodes yuchingkina Murayama Shimonoya. In order to settle the taxonomic status of Pedesta masuriensis cuneomaculata Murayama, masuriensis and tali were studied morphologically and molecularly. As a result, we consider that masuriensis and tali are two different species and treat cuneomaculata as a junior subjective synonym of tali.

FveRGA1, encoding a DELLA protein, negatively regulates runner production in Fragaria vesca.

MAIN CONCLUSION: FveRGA1 was highly expressed in tender tissues such as young leaves and stem apices and was localized in the nucleus. RNAi silencing of FveRGA1 in non-runnering woodland strawberry produced many runners. FveRGA1 is thus a key gene controlling strawberry runner formation. The propagation of strawberry is mainly based on runners, while the genes controlling runner production have not been well characterized. Exogenous applications of optimum concentration gibberellins (GAs) promote runner formation in strawberry cultivation and GA can accelerate the degradation of DELLA proteins. To investigate whether DELLA proteins are responsible for runner production, we analyzed all the DELLA genes in Fragaria vesca and cloned a DELLA protein-encoding gene FveRGA1 in woodland strawberry using RT-PCR. Subcellular localization analysis indicated that FveRGA1 was localized in the nucleus. A transcription analysis suggested that FveRGA1 was expressed ubiquitously in all examined strawberry organs, especially in young leaves, petioles, and stem apices. RNA interference (RNAi) technology was carried out to investigate the function of FveRGA1 in woodland strawberry 'Yellow Wonder' (YW) and 'Ruegen' (RG) via an Agrobacterium-mediated transformation. Interestingly, the RNAi silencing transgenic plants in the naturally non-runnering YW and RG strains produced many runners, suggesting FveRGA1 as a key gene controlling strawberry runner formation. Our study lays a solid basis for unraveling the detailed molecular mechanism of runner formation in strawberry.

[Comparative Analysis of Different Inonotus obliquus].

OBJECTIVE: To screen optimal Inonotus obliquus strains that is suitable to cultivation, extracting effective components on industrialized production, genetics and breeding. METHODS: Inonotus obliquus mycelia from different countries were used. High quality strain was screened by comparing the antagonism between Inonotus obliquus and penicillium. The contents of crude fat, crude ash, crude protein and crude fiber were analyzed. RESULTS: It was showed that antagonism ability of Finnish birch was the strongest, its mycelia diameter was up to 53. 27 mm. It was also showed that nutritional ingredient was abundant in artificial cultured Inonotus obliquus strains. The contents of crude fat and crude protein in Finnish birch were higher, which was 1. 93% and 20. 23%, respectively. The contents of crude fat and crude protein in NBRC8681 strains were lower, which was 1. 55% and 19. 85%, respectively. The content of crude ash of JL04 strains was higher(8. 76%). The content of crude ash of NBRC8681 strains was lower(6. 8%). Crude fiber content was higher in HLJ01 strains, its content reached to 45. 09%. The least content appeared in MAFF420101 strains, whose crude fiber content was only 38. 27%. CONCLUSION: The strains of Finnish birch, HLJ01 and JL04 are suitable to exploitation.

[RUCAM scale-based diagnosis, clinical features and prognosis of 140 cases of drug-induced liver injury].

OBJECTIVE: To analyze the etiology, clinical features and prognosis of liver injuries caused by different drugs. METHODS: The types of suspected drugs related to liver injury, clinical manifestations, liver biochemical parameters, clinical outcomes and other associated data were retrospectively assessed for 140 patients with drug-induced liver injury (DILI). The Roussel Uclaf Causality Assessment Method (RUCAM) was used to assess the causality between drugs and liver injury. RESULTS: The most prevalent agents inducing DILI were Chinese traditional drugs (62.1%), followed by antipyretic analgesic drugs (10%) and antibiotics (5%). The ratio of male to female patients in the study cohort was 1:1.69, with 71 of the total patients (50.7%) being between the ages of 40 and 60 years-old. The RUCAM scale was not less than 3 points for any of the patients.In general, the clinical manifestations and biochemical results were not specific. The percentages of hepatocellular injury type, cholestatic injury type and mixed injury type were 51.4%, 30.7% and 17.9% respectively. The median age of patients with cholestatic liver injury was 55.6 years, which was older than that of patients with hepatocellular injury (47.1 years) or mixed injury (49.9 years). CONCLUSION: Although antipyretic analgesics and antibiotics are considered as common drugs that can induce DILI, Chinese traditional drugs have emerged as another important group of liver injurious agents. Cholestatic DILI was found to occur more often in elderly patients than in younger patients.

Structure based drug design: development of potent and selective factor IXa (FIXa) inhibitors.

On the basis of our understanding on the binding interactions of the benzothiophene template within the FIXa active site by X-ray crystallography and molecular modeling studies, we developed our SAR strategy by targeting the 4-position of the template to access the S1 beta and S2-S4 sites. A number of highly selective and potent factor Xa (FXa) and FIXa inhibitors were identified by simple switch of functional groups with conformational changes toward the S2-S4 sites.

Studies of benzothiophene template as potent factor IXa (FIXa) inhibitors in thrombosis.

FIXa is a serine protease enzyme involved in the intrinsic pathway of the coagulation cascade. The upstream intervention of the coagulation cascade in selectively inhibiting FIXa would leave hemostasis intact via the extrinsic pathway, leading to an optimum combination of efficacy and safety with low incidence of bleeding. We have identified 2-amindinobenzothiophene template as a lead scaffold for FIXa inhibiton based on its homology with urokinase plasminogen activator (uPA). Subsequent SAR work on the template revealed a number of highly potent FIXa inhibitors, though with moderate selectivity against FXa. X-ray study with one of the analogues demonstrated active site binding interaction with the induced opening of the S1 beta pocket and a secondary binding at the S2-S4 sites, which is in direct contrast with the previous finding.

XR5967, a novel modulator of plasminogen activator inhibitor-1 activity, suppresses tumor cell invasion and angiogenesis in vitro.

Recent reports suggest that elevated levels of plasminogen activator inhibitor (PAI)-1 may contribute to tumor progression. We have recently shown that antibodies to PAI-1 block the invasive and migratory potential of human fibrosarcoma cells and suppress angiogenesis in vitro. Here we report the in vitro evaluation of a low-molecular-weight modulator of PAI-1, XR5967, on invasion, migration and angiogenesis. XR5967, a diketopiperazine, dose-dependently inhibited the activity of human and murine PAI-1, towards urokinase plasminogen activator (uPA), with IC50 values of 800 nM and 8.3 microM, respectively. This was confirmed by SDS-PAGE, revealing that XR5967 inhibited complex formation between PAI-1 and uPA. This suppression may be caused by XR5967 promoting insertion of the reactive center loop within PAI-1. XR5967 dose-dependently inhibited the invasion of human HT1080 fibrosarcoma cells through Matrigel. Their invasion was reduced by 57% (p<0.001) at 5 microM. HT1080 cell migration was inhibited in a similar manner, indicating that PAI-1 may play an additional role in invasion, which is distinct to its role in the regulation of proteolysis. The potential of XR5967 to inhibit the invasion/migration of human endothelial cells was investigated in an in vitro model of angiogenesis. In this model XR5967 reduced tubule formation by 77% at 5 microM (p<0.001), highlighting a crucial role for PAI-1 in angiogenesis. These data stress the importance of a balanced proteolysis in the processes of invasion, migration and angiogenesis. Our results support the clinical findings and indicate that modulation of PAI-1 activity, with low-molecular-weight inhibitor of PAI-1 activity, may be of therapeutic benefit for the treatment of cancer.

Studies on pyrrolopyrimidines as selective inhibitors of multidrug-resistance-associated protein in multidrug resistance.

Multidrug resistance mediated by P-glycoprotein (Pgp) or multidrug-resistance-associated protein (MRP) remains a major obstacle for successful treatment of cancer. Inhibition of Pgp and MRP transport is important for high efficacy of anticancer drugs. While several Pgp inhibitors have entered clinical trials, the development of specific MRP1 inhibitors is still in its infancy. In our screening program, we have identified a pyrrolopyrimidine (4) as a novel and selective MRP1 inhibitor. Subsequent SAR work on the 4-position of the template revealed the phenethylpiperazine side chain as a potent replacement of the benzylthio group of the lead molecule. Introduction of groups at the 2-position seems to have no detrimental effect on activity. Modifications to the nitrile group at the 7-position resulted in the identification of analogues with groups, such as amides, with superior pharmacokinetic profiles. In vivo efficacy has been demonstrated by xenograft studies on selected compounds.

Design and synthesis of new templates derived from pyrrolopyrimidine as selective multidrug-resistance-associated protein inhibitors in multidrug resistance.

In our continued effort to identify selective MRP1 modulators, we have developed two novel templates, 3 and 4, through rational drug design by identifying the key pharmacophore interaction at the 7-position of the pyrrolopyrimidine template 1. Further synthesis and SAR work on these novel templates gave a number of potent MRP1 modulators with great selectivity against Pgp. Additional studies to reduce the CYP3A4 inhibition are also reported. Several compounds of these classes were subjected to in vivo xenograft studies and in vivo efficacies were demonstrated.

Novel inhibitors of plasminogen activator inhibitor-1: development of new templates from diketopiperazines.

Several isoquinoline-based templates were identified from the studies of the conformational effects of the diketopiperazine structures for PAI-1 inhibition. Moderate to good activity was retained with the elimination of unattractive characteristics in the diketopiperazine template.

Design, synthesis and in vitro evaluation of potent, novel, small molecule inhibitors of plasminogen activator inhibitor-1.

We have synthesized and evaluated a series of tetramic acid-based and hydroxyquinolinone-based inhibitors of plasminogen activator inhibitor-1 (PAI-1). These studies resulted in the identification of several compounds which showed excellent potency against PAI-1. The design, synthesis and SAR of these compounds are described.

Studies on quinazolinones as dual inhibitors of Pgp and MRP1 in multidrug resistance.

The syntheses and SAR studies of various quinazolinone compounds are described for the dual inhibition of Pgp and MRP1 in multidrug resistance.

Novel angular benzophenazines: dual topoisomerase I and topoisomerase II inhibitors as potential anticancer agents.

A series of substituted angular benzophenazines were prepared using a new synthetic route via a novel regiocontrolled condensation of 1,2-naphthoquinones and 2,3-diaminobenzoic acids. The synthesis and biological activity of this new series of substituted 8,9-benzo[a]phenazine carboxamide systems are described. The analogues were evaluated against the H69 parental human small cell lung carcinoma cell line and H69/LX4 resistant cell line which overexpresses P-glycoprotein. Selected analogues were evaluated against the COR-L23 parental human non small cell lung carcinoma cell line and the COR-L23/R resistant cell line which overexpresses multidrug resistance protein. This series of novel angular benzophenazines were potent cytotoxic agents in these cell lines and may be able to circumvent multidrug resistance mechanisms which result in the lack of efficacy of many drugs in cancer chemotherapy. These compounds show dual inhibition of topoisomerase I and topoisomerase II and thus target two key enzymes responsible for the topology of DNA that are active at different points in the cell cycle. The introduction of chirality into the carboxamide side chain of these novel benzophenazine carboxamides has resulted in the discovery of a potent enantiospecific series of cytotoxic agents, exemplified by 4-methoxy-benzo[a]phenazine-11-carboxylic acid (2-(dimethylamino)-1-(R)-methyl-ethyl)-amide, XR11576 ((R)-4j' '). In vivo activity has been demonstrated for 4-methoxy-benzo[a]phenazine-11-carboxylic acid (2-(dimethylamino)-1-(R)-methyl-ethyl)-amide, XR11576, after intravenous administration to female mice, and this compound has been selected as a development candidate for further evaluation.

Structure-activity relationships for analogues of the phenazine-based dual topoisomerase I/II inhibitor XR11576.

As part of a programme to identify further analogues of the dual topo I/II inhibitor XR11576, we describe here the syntheses and SAR studies of various 'minimal' and 3,4-benzofused phenazine chromophores of the phenazine template of XR11576.