Discovery of tetrahydroisoquinoline-based bivalent heterodimeric IAP antagonists.

Bivalent heterodimeric IAP antagonists that incorporate (R)-tetrahydroisoquinoline in the P3' subunit show high affinity for the BIR2 domain and demonstrated potent IAP inhibitory activities in biochemical and cellular assays. Potent in vivo efficacy was observed in a variety of human tumor xenograft models. The bivalent heterodimeric molecule 3 with a P3-P3' benzamide linker induced pharmacodynamic markers of apoptosis and was efficacious when administered intravenously at a dose of 1mg/kg to mice harboring A875 human melanoma tumors. Analog 5, with a polyamine group incorporated at the P2' thiovaline side chain exhibited antiproliferative activity against the P-gp expressing HCT116/VM46 cell line.

A liquid chromatographic method for the determination of histamine in immunoglobulin preparation using solid phase extraction and pre-column derivatization.

An immunoglobulin (IgG) preparation with micro-amount of histamine fixed on the active protein fraction has been used to increase the resistance to allergic reactions. However, excessive histamine may cause hypo- or hypertension, headache, or anaphylactic shock and so the histamine content of the drug is strictly controlled by a regulation: 0.15 microg of histamine dihydrochloride is allowed for 12 mg of immunoglobulin. In this study, a liquid chromatographic method to determine micro-amount of histamine in the pharmaceutical was developed and validated. This method include a sample cleanup by a solid phase extraction (SPE) using a polystyrene-divinyl benzene (PS-DVB) polymeric sorbent and high-performance liquid chromatography after precolumn fluorescent labeling of the histamine with o-phthaldialdehyde. The drug sample was loaded to the SPE cartridge after adjusting to pH 9.5. After successive washings of the cartridge with water and 30% aqueous methanol, histamine was then eluted with 100 mM sodium acetate (pH 9.5)-methanol (20:80, v/v). An aliquot from the eluate was labeled with o-phthaldialdehyde-mercaptoethanol (OPA-ME) for fluorescence detection at the excitation maximum of 340 nm and emission maximum of 450 nm. HPLC analysis was performed on a phenyl-hexyl column with an acetonitrile-phosphate buffer (pH 6.8; 50 microM) (35:65, v/v) as the mobile phase. The retention times of histamine and 3-methylhistamine (IS) were approximately 7.2 and 9.5 min, respectively. The quantitation range was between 0.01-0.2 mg/mL of histamine showing good linearity (r=0.9996). This analytical method would provide a potential mean for the strict control of histamine content in the pharmaceutical product.

Selective growth-inhibiting effects of compounds identified in Tabebuia impetiginosa inner bark on human intestinal bacteria.

The growth-inhibiting activity of anthraquinone-2-carboxylic acid and lapachol identified in the inner bark of taheebo, Tabebuia impetiginosa, toward 10 human intestinal bacteria was evaluated by using a paper disk diffusion bioassay and compared to those of seven lapachol congeners (1,4-naphthoquinone, naphthazarin, menadione, lawsone, plumbagin, juglone, and dichlone) as well as two commercially available antibiotics, chloramphenicol and tetracycline. Anthraquinone-2-carboxylic acid exhibited very strong growth inhibition of Clostridium paraputrificum at 1 microg/disk while 100 microg/disk of lapachol was needed for moderate growth inhibition of the same organism. These two isolates exhibited weak inhibition of Clostridium perfringens and Escherichia coli at 100 microg/disk while no adverse effects were observed on the growth of Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium infantis, Lactobacillus acidophilus, and Lactobacillus casei at 1000 microg/disk. Structure-activity relationships indicate that a methyl group in the C-2 position of 1,4-naphthoquinone derivatives might play an important role in antibacterial activity.

Discovery of aminothiazole inhibitors of cyclin-dependent kinase 2: synthesis, X-ray crystallographic analysis, and biological activities.

High throughput screening identified 2-acetamido-thiazolylthio acetic ester 1 as an inhibitor of cyclin-dependent kinase 2 (CDK2). Because this compound is inactive in cells and unstable in plasma, we have stabilized it to metabolic hydrolysis by replacing the ester moiety with a 5-ethyl-substituted oxazole as in compound 14. Combinatorial and parallel synthesis provided a rapid analysis of the structure-activity relationship (SAR) for these inhibitors of CDK2, and over 100 analogues with IC(50) values in the 1-10 nM range were rapidly prepared. The X-ray crystallographic data of the inhibitors bound to the active site of CDK2 protein provided insight into the binding modes of these inhibitors, and the SAR of this series of analogues was rationalized. Many of these analogues displayed potent and broad spectrum antiproliferative activity across a panel of tumor cell lines in vitro. In addition, A2780 ovarian carcinoma cells undergo rapid apoptosis following exposure to CDK2 inhibitors of this class. Mechanism of action studies have confirmed that the phosphorylation of CDK2 substrates such as RB, histone H1, and DNA polymerase alpha (p70 subunit) is reduced in the presence of compound 14. Further optimization led to compounds such as water soluble 45, which possesses a favorable pharmacokinetic profile in mice and demonstrates significant antitumor activity in vivo in several murine and human models, including an engineered murine mammary tumor that overexpresses cyclin E, the coactivator of CDK2.

Anti-oxidant activities of the extracts from the herbs of Artemisia apiacea.

The anti-oxidant activities of the various fractions from the herbs of Artemisia apiacea were investigated. The n-hexane and n-butanol fractions were found to cause significant free radical scavenging effects on DPPH, their scavenging potencies as indicated in IC(50) values, being 230.1 and 183.7 microg/ml, respectively. The n-butanol fraction exhibited a significant decrease in serum transaminase activities elevated by hepatic damage induced by CCl(4)-intoxication in rats. All fractions tested exhibited a lipid peroxidation causing a significant decrease in MDA production in TBA-reactant assay. The n-butanol fraction was the strongest in the increase in the anti-oxidant enzymes such as hepatic cytosolic superoxide dismutase (SOD), catalase and glutathione peroxidase (GSH-px) activities in CCl(4)-intoxicated rats. These results suggest that the herbs of A. apiacea possess not only the anti-oxidant, but also the activities in CCl(4)-intoxicated rats. Especially, the n-butanol extract was found to cause significant increases in the rat liver cytosolic SOD, catalase, GSH-px activities as well as a significant decrease in the MDA production.

High-performance liquid chromatographic analysis of chrysin derivatives on a Nova-Pak C18 column.

A high-performance liquid chromatographic method has been developed for the separation and quantification of chrysin and synthetic chrysin derivatives (12 chrysin alkyl and 7 chrysin acyl derivatives). The chromatography was performed using a Nova-Pak C18 column. A RP-HPLC was performed by using a binary mixture (MeOH-10 mM H3PO4) as a mobile phase, and the column temperature was maintained at room temperature. A flow rate was 1.0 ml/min, and the effluent was monitored at a wavelenth of 280 nm. The retention times for chrysin acyl and alkyl derivatives were within 10 minutes and 20 minutes, respectively. The absolute recovery of samples were all over 96%. The detection limits were 0.1-18 ng at S/N = 3 ratio.

Constituents from the non-polar fraction of Artemisia apiacea.

Five compounds of terpenoids and coumarins were isolated from the non-polar fraction of Artemisia apiacea by open column chromatography. Their structures were elucidated as alpha-amyrin (1), beta-amyrin (2), beta-sitosterol (3), 5,6,7-trimethoxycoumarin (4) and 6-methoxy-7,8-methylenedioxycoumarin (5) by chemical and spectroscopic analysis. This is the first report of the isolation of alpha-amyrin, beta-amyrin, 5,6,7-trimethoxycoumarin and 6-methoxy-7,8-methylenedioxycoumarin from this plant.

Phytochemical constituents from the herba of Artemisia apiacea.

Four compounds of a coumarin, a steroid and two flavonoids were isolated from the herba of Artemisia apiacea by open column chromatography. Their structures were elucidated as artemicapin C, apigenin, daucosterol and cacticin by chemical and spectroscopic analysis. This is the first report of the isolation of these compounds from this plant.

In vivo anti-oxidant activities of tectochrysin.

The anti-oxidant activities of tectochrysin, a major compound of propolis, were investigated. Tectochrysin exhibited a significant decrease in serum transaminase activities elevated by hepatic damage induced by CCl4-intoxication in rats. Tectochrysin tested exhibited a lipid peroxidation causing a significant decrease in MDA production in TBA-reactant assay. Tectochrysin was strong in the increase in the anti-oxidant enzymes such as hepatic cytosolic superoxide dismutase, catalase and glutathione peroxidase activities in CCl4-intoxicated rats. These results suggest that tectochrysin possess not only the anti-oxidant, but also the activities in CCl4-intoxicated rats. Especially, tectochrysin was found to cause significant increases in the rat liver cytosolic SOD, catalase, GSH-px activities as well as a significant decrease in the MDA production.

Arteminin, a new coumarin from Artemisia apiacea.

The isolation of 6,7-dimethoxycoumarin (1), 6-methoxy-7,8-methylenedioxycoumarin (2), 5,6-dimethoxy-7,8-methylenedioxycoumarin (3), 6-hydroxy-7,8-methylenedioxycoumarin (4) and 5-hydroxy-6,8-dimethoxycoumarin (arteminin) (5) is reported.

Discovery of pyrrolopyridine-pyridone based inhibitors of Met kinase: synthesis, X-ray crystallographic analysis, and biological activities.

Conformationally constrained 2-pyridone analogue 2 is a potent Met kinase inhibitor with an IC50 value of 1.8 nM. Further SAR of the 2-pyridone based inhibitors of Met kinase led to potent 4-pyridone and pyridine N-oxide inhibitors such as 3 and 4. The X-ray crystallographic data of the inhibitor 2 bound to the ATP binding site of Met kinase protein provided insight into the binding modes of these inhibitors, and the SAR of this series of analogues was rationalized. Many of these analogues showed potent antiproliferative activities against the Met dependent GTL-16 gastric carcinoma cell line. Compound 2 also inhibited Flt-3 and VEGFR-2 kinases with IC50 values of 4 and 27 nM, respectively. It possesses a favorable pharmacokinetic profile in mice and demonstrates significant in vivo antitumor activity in the GTL-16 human gastric carcinoma xenograft model.

Synthesis and SAR of pyrrolotriazine-4-one based Eg5 inhibitors.

Synthesis and SAR of substituted pyrrolotriazine-4-one analogues as Eg5 inhibitors are described. Many of these analogues displayed potent inhibitory activities in the Eg5 ATPase and A2780 cell proliferation assays. In addition, pyrrolotriazine-4-one analogue 26 demonstrated in vivo efficacy in an iv P388 murine leukemia model. Both NMR and X-ray crystallographic studies revealed that these analogues bind to an allosteric site on the Eg5 protein.

Modulation of the activity of pro-inflammatory enzymes, COX-2 and iNOS, by chrysin derivatives.

Chrysin, a natural flavone compound found in plants, has anti-inflammatory activity that has been previously explained in part by the suppression of promoter activities of pro-inflammatory enzymes, cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). Here we present evidence that several chrysin derivatives modulate the activities, as well as the expression, of COX-2 and iNOS enzymes. Nitrate production triggered by lipopolysaccharide (LPS) was suppressed by treatment of cultured Raw264.7 cells (mice macrophage/monocyte) with chrysin, 5-hydroxy-7-methoxyflavone (Ch-2), and 5,7-diacetylflavone (Ch-4). Interestingly, COX-2 enzyme was strongly inhibited by Ch-4 (IC(50)=2.7 microM) but not by other derivatives. Furthermore, the inhibition of COX enzyme by Ch-4 was selective for COX-2 over COX-1. Three-dimensional modeling showed that Ch-4 fits well into the binding pocket of COX-2. The modeling suggested that a hydrogen bond exists between the oxygen of the ketone group at the 7-position of Ch-4 and the hydroxyl group of Tyr355. Docking Ch-4 into the V523I mutant of COX-2 indicated that Ile523 of COX-1 might contribute to the selectivity of COX-2 over COX-1. Ch-4 showed no effect on iNOS activity. Chrysin and Ch-2 weakly inhibited iNOS enzyme activity in the hemoglobin assay, but the underlying mechanisms of inhibition of iNOS by chrysin are not understood.