Confluentic acid

Endo, Y., Hayashi, H., Sato, T., Maruno, M., Ohta, T., and Nozoe, S., 1994. Confluentic acid and 2 -0-methylperlatolic acid, monoamine oxidase B inhibitors in a Brazilian plant, Himatanthus sucuuba. Chem. Pharm. Bull. 42, 1198-1201. 

Confluentic acid Himatanthus sucuuba (Apocynaceae) [bark] MAO-B (0.2)... 

Fig. 22. A Chloroatranorin, Q. Bar lOjUm. B Confluentic acid, GE. Bar lOjim. C Confluentic acid, Py. Bar 100 jim. D Confluentic acid, oT. Bar 10 im.

Deriv Methyl confluentate, needles (MeOH-HjO), mp 123 °C, from confluentic acid with CH2N2 in EtjO at 20 °C in 1 min StL Lecidea confluens (G.Web.) Ach. 

Elix JA, Ferguson BA (1978) Synthesis of the lichen depsides, olivetoric acid, confluentic acid and 4-0-methylolivetoric acid. Aust J Chem 31 1041-1045 Elix JA, Gaul KL (1986) The interconversion of the lichen depsides para- and metascrobiculin, and the biosynthetic implications. Aust J Chem 39 613-624 Elix JA, Jayanthi VK (1977) 5-0-Methylhiascic acid, a new tridepside from Australian lichens. Aust J Chem 30 2695-2704... 

Depsides may be cleaved with either cone, sulphuric acid at 0°C or with KOH-H2O or KOH-MeOH. Prolonged boiling (5-20 hours) of a depside with terf.-butanol (TBA) yields the tert.-butyl ester of the S-part of the depside and the phenolic unit 23, 384, 542) (see Scheme 17). Depsides with a keto group in the 3-position of the side chain in the S-part of the molecule give with KOH at room temperature a y,6-unsaturated 6-lactone, e.g. confluentic acid — 4-D-methylolivetonide (see Scheme 18). 

Endo Y, Hayashi H, Sato T, Maruno M, Ohta T, Nozoe S (1994) Confluentic Acid and 2 -0-Methylperlatolic Acid, Monoamine Oxidase B Inhibitors in a Brazilian Plant, Himatanthiis sucuuba. Chem Pharm Bull 42 1198... 

Fig. 12. Inhibition of l25I-lys-plasminogen binding to human umbilical vein endothelial cells (HUVECs) by Lp(a) and apo(a). Confluent HUVEC monolayers were washed, treated with -aminocaproic acid, rewashed and incubated with 125I-lys-plasminogen (4.95 nM, specific activity 415.000cpm-pmo -1), for 30 min at 4°C in the presence of various excess amounts of unlabeled Lys-PLG (A) Lp(a) (0,0) apo(a) (x) LDL ( , ) or Lp(-) (V). [With permission of Hajjar el at. (HI 1).]...

For the in vitro test, the fibroblasts are allowed to form a half-confluent monolayer within 24 h. Different concentrations of the test chemical are then incubated for 1 h with two sets of cells in parallel (typically on 96-well plates, 104 cells per well, passage number <100). After the incubation with the test substances, one set is irradiated with a nontoxic dose of UVA light (5 J/cm2), while the other set is kept in the dark. Twenty hours after irradiation, cell viability is evaluated by measuring the uptake of NR for 3 h. After the end of the absorption process, excess NR is removed and the cells are treated with an NR desorption solution (ethanol/acetic acid) to extract the dye taken up by the cells. Subsequently, the optical density of the NR solution is measured at 540 nm. As positive control, a test with chlorpromazine is performed. 

Fibroblast cultures for each patient and control are grown to confluency in T-75 culture flasks. The assay is then performed in triplicate using six-well plates. First, 1 ml of 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) solution (Sigma T4049) is added to each T-75 flask. After the cells have lifted from the flask, they are resuspended in 6 ml complete MEM. A 1-ml aliquot of the cell suspension is transferred to a 50-ml conical tube containing 14 ml of MEM complete medium (see above). Following further resuspension, 4 mL is plated into each of three wells in a six-well culture plate and placed into an incubator. Once the cells have attached to the seeded plates and are approximately 90-100% confluent (3-4 days), complete MEM is replaced with 1.5 ml of complete IVPM (see above). 

Inspect the cells on d 3, they should be almost fully confluent on the flask base. Discard the medium, add 10 mL of cold sterile PBS containing 0.02% ethylene diamine tetraacetic acid and leave for a few minutes to allow the cells to detach. Split the cells equally between two flasks and add 75 mL of RPMI 1640 medium/5% FBS to each flask and return to the incubator. 

The Cytosensor Microphysiometer (CM) test method is currently under discussion (draft test guideline on the Cytosensor Microphysiometer) at OECD level [34], It is a cytotoxicity and cell-function based in vitro assay that is performed on a sub-confluent monolayer of adherent mouse L929 fibroblasts cultured in a sensor chamber using a pH-meter to detect changes in acidity [35]. The CM test method serves as an in vitro model system for the cytotoxic action of a test chemical on the cell membranes of the corneal and conjunctival epithelium where the irritant chemical would be... 

Answer The acidity of the sample carrier stream should be kept at 0.10 mol L-1 in order to avoid the establishment of undesirable pH gradients. Its contribution inside the main reactor can be calculated (Eq. 3.14) as 0.08 mol L 1. The pH to be adjusted matches the pKa value of the acetic acid acetate buffer system therefore, equimolar concentrations of acetate ion and acetic acid should be established inside the main reactor. Considering that the reaction between acetate and perchloric acid is stoichiometric, the acetate contribution from the confluent stream should be double or 0.16 mol L This corresponds (Eq. 3.14) to 0.80 mol L-1 in the reagent reservoir. It should be noted that this result does not depend on the inserted sample volume because a confluent flow system is used. 

Most spectrophotometric analytical procedures are pH-dependent, and the buffer system can be efficiently established in-line. In segmented flow systems and unsegmented flow systems with a chemically inert carrier stream, the main stream is characterised by a relatively steady acidity or alkalinity. The exact amount of the alkaline or acid component of the buffer system can then be added by confluence in order to attain a suitable pH value in the reaction medium. Moreover, if the main stream is initially too acidic or too alkaline, a strong base or acid can be added to the pH adjusting confluent stream. 

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