The 'azirine/oxazolone method' in peptaibol synthesis: preparation of a derivative of trichotoxin A-50 (G)

The synthesis of a mixture of epimeric derivatives of the peptaibol trichotoxin A-50 (G) is described. The 'azirine/oxazolone method' has been used as a superior method for the introduction of the Aib as well as the Iva units into the peptide chain. In this protocol, 2,2-disubstituted 2H-azirin-3-amines are the synthons for 2,2-disubstituted glycines, which undergo coupling with N-protected amino or peptide acids in high yield, and without any need of coupling reagents. The problem of the instability of the amide function of the Gln side chain under the conditions of the acid-catalyzed hydrolysis of Z-Gln-(Aib)(n)-N(Me)Ph was solved by using an appropriate protecting group for the amide function of the Gln side chain, e.g., the triphenylmethyl (trityl; Tr) group. The structures of two intermediate peptides, i.e., the segments comprising residues 1-5 and 10-13, resp., were established by X-ray crystallography.     

A novel and facile preparation of bremazocine enantiomers through optically pure N-norbremazocines

In order to provide ready access to multigram quantities of the optically pure bremazocines [(-)- and (+)-9,9-dimethyl-5-ethyl-2-hydroxy-2-(1-hydroxy-cyclopropylmethyl)-6,7-benzomorphan)], we have developed an improved non-chromatographic synthesis, and determined the optical purity of their N-nor precursors using a rapid and relatively simple 1H NMR method based on diastereomeric derivatization with optically pure 1-phenylethylisocyanate. This method of determining optical purity should be readily amenable to similar systems containing phenolic amino functionalities. Finally, a greatly simplified methodology for introduction of the N-(1-hydroxycyclopropylmethyl) substituent in bremazocine is described. The improved synthetic method-the overall yield was increased about 3-fold-combined with the practical methodology to determine optical purity will considerably facilitate the employment of these enantiomers as pharmacological tools for examination of the kappa-opioid receptor system, as well as their evaluation as drug abuse treatment agents. This synthesis will also enable the study of these enantiomers for other, non-classical applications (e.g., treatment agents for HIV).     

Fluorescent phosphonate labels for serine hydrolases. Kinetic and spectroscopic properties of (7-nitrobenz-2-oxa-1,3-diazole)aminoalkyl methylphosphonofluoridates and their conjugates with acetylcholinesterase molecular forms

The synthesis, kinetic, and spectral characterization of (7-nitrobenz-2-oxa-1,3-diazole)aminoethyl and (7-nitrobenz-2-oxa-1,3-diazole)aminopentyl methylphosphonofluoridate are described. These homologous organophosphorous agents contain the environmentally sensitive 7-nitrobenz-2-oxa-1,3-diazole chromophore. They inhibit acetylcholinesterase from Torpedo at rates exceeding 10(7) M-1 min-1 to form long-lived conjugates with one chromophore/80-kilodalton subunit. The intensity, position, and line width of the absorption spectra of the conjugates and reactivation kinetics in the presence and absence of the bisquaternary oxime 1,1'-trimethylene-bis(4-formylpyridinium bromide) dioxime indicate that these agents form conjugates in which the NBD-aminoalkyl moieties experience distinctive microscopic environments within the active center. NBD-aminoethyl methylphosphono-acetylcholinesterase undergoes oxime-induced as well as spontaneous reactivation at rates that are 3.6 and 35 times faster, respectively, than the corresponding rates measured for the NBD-aminopentyl conjugate. Hence, reactivation exhibits a marked dependence on structure of the methylphosphonate. Fluorescence emission at wavelengths greater than 520 nm is highly quenched and exhibits quantum efficiencies of less than 5%. Absorption maxima for the covalent NBD-aminoethyl methylphosphono-acetylcholinesterase appear at 475-480 nm while those for the corresponding NBD-aminopentyl methylphosphono-acetylcholinesterase appear at 485-490 nm. Bandwidths of the absorption maxima are substantially broader for the acetylcholinesterase adduct with NBD-aminoethyl methylphosphonofluoridate (3870 cm-1) than for the enzyme adduct with NBD-aminopentyl methylphosphonofluoridate (2870 cm-1). The CD spectrum of NBD-aminopentyl methylphosphono-acetylcholinesterase shows optical activity coincident with the shape and position of the absorption spectrum. In contrast, in addition to optically active transitions at the absorption maxima, the CD spectrum of NBD-aminoethyl methylphosphono-acetylcholinesterase shows intense optical activity at 430 nm, a wavelength region coincident with the region of spectral broadening. The spectral properties of alpha-chymotrypsin conjugates formed by reaction with the two probes are different, and the respective spectra differ also from those observed for the acetylcholinesterase conjugates. These results indicate that there is a reciprocal relationship between the structure of the probe and the structure of the active center.(ABSTRACT TRUNCATED AT 400 WORDS)     

The gastric ulcer protective effect of boswellic acids, a leukotriene inhibitor from Boswellia serrata, in rats

Aim of the study is to evaluate the anti-ulcer efficacy of the boswellic acids (BA), a triterpenoid known as anti-inflammatory/anti-arthritic agent, which is in clinical use. The reason for the study is that, the known non-steroidal anti-inflammatory drugs (NSAIDs) are full of side effects especially ulceration which is at the top. BA, although, used as an anti-arthritic agent yet it is not only devoid of ulcer production but protective also. The activity evaluation was done by the following universally accepted animal models viz., pyloric ligation, ethanol–HCl, acetylsalicylic acid, indomethacin and cold restrained stress-induced ulceration in rats. Results of the present study revealed that BA possess a dose dependent antiulcer effect against different experimental models. It showed different degree of inhibition of the ulcer score towards different ulcerogenic agents. The ulcer score against various ulcer inducing agents viz., pyloric ligation, ethanol/HCl, (acute and chronic) acetylsalicylic acid, indomethacin and cold restraint stress, was inhibited by 39%, 38%, 51%, 31%, 37% and 42% respectively at 250 mg/kg. From the data it is concluded that BA inhibited ulcer production non-specifically in all the experimental models, whereby, it is not possible to propose a single specific mechanism. Nevertheless it is possible that BA might be acting by increasing the gastric mucosal resistance and local synthesis of cytoprotective prostaglandins and inhibiting the leukotriene synthesis.     

Involvement of tyrosyl residues in the structure-function relationships of D-beta-hydroxybutyrate dehydrogenase: a phospholipid-requiring enzyme

The involvement of tyrosyl residues in the function of D-beta-hydroxybutyrate dehydrogenase, a lipid-requiring enzyme, has been investigated by using several tyrosyl modifying reagents, i.e., N-acetylimidazole, a hydrophilic reagent, and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and tetranitromethane, two hydrophobic reagents. Modification of the tyrosyl residues highly inactivates the derived enzyme: Treatment of the enzyme with 7-chloro-4-nitro[14C]benzo-2-oxa-1,3-diazole leads to an absorbance at 380 nm and to an incorporation of about 1 mol of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole per polypeptide chain for complete inactivation. Inactivation by N-acetylimidazole induces a decrease in absorbance at 280 nm which can be reversed by hydroxylamine treatment. On the other hand, the ligands of the active site, such as methylmalonate, a pseudosubstrate, and NAD+ (or NADH), do not protect the enzyme against inactivation. In contrast, the presence of phospholipids strongly protects the enzyme against hydrophobic reagents. Finally, previous modification of the enzyme with N-acetylimidazole does not affect the incorporation of 7-chloro-4-nitro[14C]benzo-2-oxa-1,3-diazole while modification with tetranitromethane does. These results indicate the existence of two classes of tyrosyl residues which are essential for enzymatic activity, and demonstrate their location outside of the active site. One of these residues appears to be located close to the enzyme-phospholipid interacting sites. These essential residues may also be essential for maintenance of the correct active conformation.     

Cell wall anionic polymers of Streptomyces sp. MB-8, the causative agent of potato scab

The cell wall of Streptomyces sp. MB-8 contains a major teichoic acid, viz., 1,3-poly(glycerol phosphate) substituted with N-acetyl-alpha-D-glucosamine (the degree of substitution is 60%), a minor teichoic acid, viz., non-substituted poly(glycerol phosphate), and a family of Kdn (3-deoxy-D-glycero-D-galacto-non-2-ulopyranosonic acid)-containing oligomers of the following general structure: [carbohydrate structure: see text]. The composition of the oligomers was established using MALDI-TOF mass spectroscopy. The present study provides the second example of the identification of Kdn as a component of cell wall polymers of streptomycetes, which are the causative agents of potato scab.     

Discovery and structure-activity relationship of 2,6-disubstituted pyrazines, potent and selective inhibitors of protein kinase CK2

We report the discovery and structure-activity relationship of 2,6-disubstituted pyrazines, which are potent and selective CK2 inhibitors. Lead compound 1 was identified, and derivatives were prepared to develop potent inhibitory activity. As a result, we obtained compound 7, which was the smallest unit that retained potency. Then, introducing an aminoalkyl group at the 6-position of the indazole ring resulted in improved efficacy in both enzymatic and cell-based CK2 inhibition assays. Moreover, compound 13 showed selectivity against other kinases and in vivo efficacy in a rat nephritis model. These results show that 2,6-disubstituted pyrazines have potential as therapeutic agents for nephritis.     

Synthesis of constrained phenylalanine derivatives via a [2+2+2] cycloaddition strategy

A simple synthesis of dialkyne building blocks (6, 7, 8 and 9) embodying amino acid moiety is described. The dialkyne 6 participated in a [2+2+2] cycloaddition reaction with various monoalkynes in presence of Wilkinson's catalyst to give 5- and 5,6-disubstituted indan-based -amino acid derivatives. Cobalt catalyst [e.g., CpCo(CO)2] has also been employed in the synthesis of various 2-indanyl glycine derivatives via co-trimerization reaction of the diyne building blocks 6 and 7 with several monoalkynes.     

Larvicidal isoxazoles: Synthesis and their effective susceptibility towards Aedes aegypti larvae

Twenty 3,5-disubstituted isoxazoles have been synthesized and tested against fourth instar Aedes aegypti larvae. In the synthesis of title compounds, modifications have been made in the C-5 side-chain with a view to test their larvicidal activity. These isoxazoles have been obtained by 1,3-dipolar cycloaddition of arylnitrile oxides to terminal alkynes which furnished the desired products in 20% to 79% yields. A comparative study of the larvicidal activity between 3-(3-aryl-isoxazol-5-yl)-propan-1-ols and 3-(3-aryl-isoxazol-5-yl)-propionic acids clearly demonstrated that the latter compounds possess much better larvicidal activity than the former. We also tested two esters, viz., methyl 3-[3-(phenyl)-isoxazole-5-yl] propionate and methyl 3-[3-(4-chlorophenyl)-isoxazole-5-yl] propionate, where the latter presented an excellent larvicidal profile.     

Reaction of chicken egg white lysozyme with 7-chloro-4-nitrobenz-2-oxa-1,3-diazole. II. Sites of modification.

1. Procedure for the isolation of peptides from proteins bearing the chemically labile aromatic ether, O-tyrosyl-4-nitrobenz-2-oxa-1,3-diazole group, is described. 2. The tyrosyl residue reactive towards 7-chloro-4-nitrobenz-2-oxa-1,3-diazole in chicken egg white lysozyme (Aboderin, A. A., Boedefeld, E. and Luisi, P. L., (1973) Biochim. Biophys. Acta 328. 20-30) is tyrosine-23. The amino group in the protein whose reaction with the reagent is dependent on the prior reaction of tyrosine-23 is the epsilon-amino group of lysine-33.     

Design, synthesis and insight into the structure-activity relationship of 1,3-disubstituted indazoles as novel HIF-1 inhibitors

Design, synthesis and insight into the structure-activity relationship (SAR) of 1,3-disubstituted indazoles as novel HIF-1 inhibitors are described. In particular, the substituted furan moiety on indazole skeleton as well as its substitution pattern turns out crucial for the high HIF-1 inhibition.     

Protection by superoxide dismutase, catalase, and poly(ADP-ribose) synthetase inhibitors against alloxan- and streptozotocin-induced islet DNA strand breaks and against the inhibition of proinsulin synthesis

We have shown previously that alloxan and streptozotocin, two major diabetogenic agents, cause DNA strand breaks in rat pancreatic islets and stimulate nuclear poly(ADP-ribose) synthetase, thereby depleting intracellular NAD level and inhibiting proinsulin synthesis (Okamoto, H. (1981) Mol. Cell. Biochem. 37, 43-61; Yamamoto, H., Uchigata, Y., and Okamoto, H. (1981) Nature 294, 284-286). In the present study, superoxide dismutase and catalase, scavengers of radical oxygens, were found to protect against islet DNA strand breaks and inhibition of proinsulin synthesis induced by alloxan. The radical scavengers did not affect islet DNA strand breaks or inhibition of proinsulin synthesis induced by streptozotocin. On the other hand, compounds that inhibit islet nuclear poly(ADP-ribose) synthetase were found to protect against alloxan- as well as streptozotocin-induced inhibition of proinsulin synthesis. The poly(ADP-ribose) synthetase inhibitors were ineffective in protection against DNA strand breaks induced by the agents. These results may provide an important clue for elucidating the prevention of insulin-dependent diabetes as well as for understanding the cause of diabetes.     

Identification of a novel 3,5-disubstituted pyridine as a potent, selective, and orally active inhibitor of Akt1 kinase

Based on lead compounds 2 and 3 a series of 3,5-disubstituted pyridines have been designed and evaluated for inhibition of AKT/PKB. Modifications at the 3 position of the pyridine ring led to a number of potent compounds with improved physical properties, resulting in the identification of 11g as a promising, orally active Akt inhibitor. The synthesis, structure-activity relationship studies, and pharmacokinetic data are presented in this paper.     

Effect of various aqueous extracting agents on fluorescence properties of waste tea residue derived carbon dots (WTR-CDs): Comparative spectroscopic analysis

Fluorescent carbon dots (CDs) are one of the important carbonaceous nanomaterials in the area of nanoscience and nanotechnology because of their interesting physical as well as chemical properties. Herein we studied the effect of various aqueous extracting agents on fluorescence properties of waste tea residue-based carbon dots (WTR-CDs). WTR-CDs are firstly synthesized by utilizing kitchen waste-based carbonaceous biomass. To check the role of various aqueous media during the course of WTR-CDs synthesis from carbonized carbon powder, extraction of WTR-CDs was carried out in various kinds of aqueous media viz., only aqueous (100% water, WT), aqueous-alcoholic (10% ethanol, ET), aqueous-acidic (10% acetic acid, AA), and aqueous-basic (10% ammonia, AM). The consequences of extracting agents on the photophysical properties of final WTR-CDs-WT, WTR-CDs-ET, WTR-CDs-AA and WTR-CDs-AM were also discussed in detail. We have observed interesting blue shift fluorescence spectra in acidic medium for WTR-CDs-AA and polar protic solvents compared to polar aprotic medium. The solvatochromic behaviour of WTR-CDs-WT in model polar and non-polar solvent was also studied. The effect of cationic, anionic and non-anionic surfactants on the fluorescence of WTR-CDs-WT was also evaluated. The proposed findings may help researchers in the near future to obtain fast, easy and direct synthesize CDs from a variety of biomass-based precursors under different aqueous conditions.     

Design, synthesis and the effect of 1,2,3-triazole sialylmimetic neoglycoconjugates on Trypanosoma cruzi and its cell surface trans-sialidase

This work describes the synthesis of a series of sialylmimetic neoglycoconjugates represented by 1,4-disubstituted 1,2,3-triazole-sialic acid derivatives containing galactose modified at either C-1 or C-6 positions, glucose or gulose at C-3 position, and by the amino acid derivative 1,2,3-triazole fused threonine-3-O-galactose as potential TcTS inhibitors and anti-trypanosomal agents. This series was obtained by Cu(I)-catalysed azide-alkyne cycloaddition reaction ('click chemistry') between the azido-functionalized sugars 1-N(3)-Gal (commercial), 6-N(3)-Gal, 3-N(3)-Glc and 3-N(3)-Gul with the corresponding alkyne-based 2-propynyl-sialic acid, as well as by click chemistry reaction between the amino acid N(3)-ThrOBn with 3-O-propynyl-GalOMe. The 1,2,3-triazole linked sialic acid-6-O-galactose and the sialic acid-galactopyranoside showed high Trypanosoma cruzitrans-sialidase (TcTS) inhibitory activity at 1.0mM (approx. 90%), whilst only the former displayed relevant trypanocidal activity (IC(50) 260μM). These results highlight the 1,2,3-triazole linked sialic acid-6-O-galactose as a prototype for further design of new neoglycoconjugates against Chagas' disease.     

Experimental skeletal teratogenesis: a disturbance of relative osteo-neural growth]

The previously suggested concept of the closest growth relations existing between the bony and the nervous tissue at the organ level of the spinal cord and the peripheral (including the facial) nervous trunks is experimentally buttressed. It is shown that the normal gross-morphological features of the vertebrae as well as of the tubular bones (viz., their length, physiological curvatures and terminal expansions) result from the adaptation of the bone growth to the slower proceeding and vulnerable neural extensive growth, viz., from a physiological osteo-neural growth disproportion. The more or less conspicuous growth in length of the facial skeleton depends upon the phylogenetically established, more or less evolved extensive-growth potentially of the facial nervous trunks as well. The growth relation existing between the developing brain and its bony case applies essentially even for the axial organ, the extremities as well as for the facial skeleton. The experimental findings speak in favour of the theoretical expectation that the typical teratogenic deformities of the extremities (micromelia), of the spine (scoliosis, defects of the vertebrae and of the ribs) as well as of the beak (jaws) which may be produced by a great number of most diverse teratogens, result from the adaptation of the bone growth to the growth-insufficient nervous trunks, viz., from the pathologically enhanced osteo-neural growth disproportion. The cleft palate and the digital defects (syndactylia, oligodactylia) may be readily explained by the growth-inhibition of the palatal and digital nervous structures as well. The vertebrate body may be thus conceived as composed of 2 growth types, viz., the neural-extensive and the cellular-divisional (mitotic). The former is represented by an extremely dense feltwork of nerve fibers and trunks (the DONALDSON'S "nervous skeleton") which is "stuffed" with the other, mostly mitotically growing tissues. The 2 growth types are closely related partly at the macro-(organ-) level concerning the normal and teratogenic morphogenesis of the skeleton, partly at the micro-level of the utmost periphery, viz., of the terminal extensive meshwork and the individual cells or groups of cells. The cells which escape from the extensive feltwork (i.e. from the "nervous skeleton") such as the superficial cells of the epidermis or mucous membranes and, in all probability, the elements of the haemopoetic organs, perish under normal conditions, suffer a planned, highly purposeful death. With regard to the lack of normal nerves within malignant tumours, the malignant cell may be conceived as the one which escaped from the limiting confines of the extensive feltwork and, inspite of that, continues to live instead of "committing suicide."     

Discovery of novel trans-3,5-disubstituted pyrrolidinylthio-1beta-methylcarbapenems

Novel trans-3,5-disubstituted pyrrolidinylthio-1beta-methylcarbapenems were designed and synthesized to provide J-111,347 (1a) as the first example of an exceptionally broad-spectrum antibiotic, showing activity against methicillin-resistant Staphyloccocus aureus (MRSA) as well as Pseudomonas aeruginosa. Further derivation of 1a afforded J-111,225 (2a), J-114,870 (3a), and J-114,871 (3b). which showed improved safety profiles and retained broad-spectrum antibacterial activities.     

Synthesis and biological activity of 22-oxa CD-ring modified analogues of 1alpha,25-dihydroxyvitamin D3: cis-perhydrindane CE-ring analogues

The synthesis and biological activity of novel CD-ring modified analogues of 22-oxa-1alpha,25-dihydroxyvitamin D(3), lacking the D-ring and featuring a connection between C-12 and C-21 (cis-perhydrindane CE-ring analogues), is described. The synthesis of the CE-ring system follows Meyers' methodology for the preparation of enantiomerically pure hydrinden-2-ones. The analogues show a complete lack of binding affinity for the vitamin D receptor (pig nVDR) and of antiproliferative activity (MCF-7 cells), as compared to calcitriol.     

Interspecies differences in the enantioselectivity of epoxide hydrolases in Cryptococcus laurentii (Kufferath) C.E. Skinner and Cryptococcus podzolicus (Bab'jeva & Reshetova) Golubev

Isolates representing Cryptococcus laurentii and Cryptococcus podzolicus, originating from soil of a heathland indigenous to South Africa, were screened for the presence of enantioselective epoxide hydrolases for 2,2-disubstituted epoxides. Epoxide hydrolase activity for the 2,2-disubstituted epoxide (+/-)-2-methyl-2-pentyl oxirane was found to be abundantly present in all isolates. The stereochemistry of the products formed by the epoxide hydrolase enzymes from isolates belonging to the two species (11 isolates representing C. laurentii and 23 isolates representing C. podzolicus) was investigated. The enantiopreferences of the epoxide hydrolases for 2,2-disubstituted epoxides of these two species were found to be opposite. All strains of C. laurentii preferentially hydrolysed the (S)-epoxides while all C. podzolicus isolates preferentially hydrolysed the (R)-epoxides of (+/-)-2,2-disubstituted epoxides. These findings indicate that the stereochemistry of the products formed from 2,2-disubstituted epoxides by the epoxide hydrolase enzymes of these yeasts should be evaluated as additional taxonomic criterion within the genus Cryptococcus. Also, the selectivity of some epoxide hydrolases originating from isolates of C. podzolicus was high enough to be considered for application in biotransformations for the synthesis of enantiopure epoxides and vicinal diols.