Reaction of enzyme-bound 5-deazaflavin with peroxides. Pyrimidine ring contraction via an epoxide intermediate

Reaction of peroxides with 5-deazaflavin bound to glucose oxidase, lactate oxidase, or D-amino acid oxidase results in the formation of 5-deazaflavin 4a, 5-epoxide. The reaction of D-amino acid oxidase with m-chloroperoxybenzoate is an exception since the reagent reacts rapidly with the protein moiety to form m-chlorobenzoate which then binds noncovalently near the unmodified coenzyme. Epoxide bound to glucose oxidase is converted to deazaFAD X X in a reaction similar to that observed previously with oxynitrilase and glycolate oxidase. With lactate oxidase the epoxide is quite stable in the absence of light. With D-amino acid oxidase, denaturation of the protein is accompanied by the release of the epoxide into solution where it decomposes in a manner similar to that observed with model epoxide compounds at neutral pH. Reaction of deazaFAD X X with phosphodiesterase and alkaline phosphatase yields deazariboflavin X X. The same compound has been formed in model studies by exposing 5-deazariboflavin 4a,5-epoxide to alkaline conditions. Structural studies indicate that this reaction involves contraction of the pyrimidine ring to yield 4-ribityl-6,7-dimethyloxazolo[ 4,5-b ]quinolin-2(4H)-one. Model reaction studies are consistent with a mechanism initiated by alkaline hydrolysis of the pyrimidine ring at position 4 followed by two additional steps which proceed at neutral pH. A similar mechanism for the enzyme reactions appears likely since analogous intermediates are detected in the glycolate oxidase and the model reactions. The results suggest that position 4 of the coenzyme in oxynitrilase, glycolate oxidase, and glucose oxidase must be accessible to solvent and that the protein moiety must facilitate the initial hydrolysis of the pyrimidine ring since the enzyme reactions occur at neutral pH. Failure to observe formation of deazaFMN X X with lactate oxidase is attributed, at least in part, to the inaccessibility of the pyrimidine ring to solvent.     

Reaction of thiol nucleophiles with 1,2-epoxy- and 4,5-epoxy-estrene-3-one-17 beta-ols

Four ring A steroidal epoxyenones as probable intermediate in the formation of catechol estrogens were synthesized. The isomeric 1 alpha,2 alpha-epoxy-17 beta-hydroxyestr-4-en-3-one (9) and 1 beta,2 beta-epoxy-17 beta-hydroxyestr-4-en-3-one (8) were synthesized from 17 beta-hydroxy-5 alpha-estra-3-one. The isomeric 4 alpha,5 alpha-epoxy-17 beta-hydroxyestr-1-en-3-one (11) and 4 beta,5 beta-epoxy-17 beta-hydroxyestr-1-en-3-one (10) were prepared from 19-nortestosterone. The reaction of 9 and 10 with sodium/ethanethiol resulted in the formation of three types of reactions leading to multiple products: 1,4-addition, opening of epoxide, and epoxide opening followed by dehydration. Reaction of 8 with ethanethiol gave only one compound identified as 2-ethanethio-1,4-estradien-17 beta-ol-3-one, while reaction of 9 with ethanethiol gave an unusual product identified as 4-estren-1 alpha,17 beta-diol-3-one. Unlike reaction of ethanethiol with 9 and 10, reaction with N-acetylecysteine or glutathione results in epoxide opening followed by dehydration leading to the formation of estradiol-4-thioethers.     

Comparison of the substrate specificities of human liver cytochrome P450s 2C9 and 2C18: application to the design of a specific substrate of CYP 2C18.

A series of 2-aroylthiophenes derived from tienilic acid by replacement of its OCH2COOH substituent with groups bearing various functions have been synthesized and studied as possible substrates of recombinant human liver cytochrome P450s 2C9 and 2C18 expressed in yeast. Whereas only compounds bearing a negative charge acted as substrates of CYP 2C9 and were hydroxylated at position 5 of their thiophene ring at a significant rate, many neutral 2-aroylthiophenes were 5-hydroxylated by CYP 2C18 with kcat values of >2 min-1. Among the various compounds that were studied, those bearing an alcohol function were the best CYP 2C18 substrates. One of them, compound 3, which bears a terminal O(CH2)3OH function, appeared to be a particularly good substrate of CYP 2C18. It was regioselectively hydroxylated by CYP 2C18 at position 5 of its thiophene ring with a KM value of 9 +/- 1 microM and a kcat value of 125 +/- 25 min-1, which are the highest described so far for a CYP 2C. A comparison of the oxidations of 3, by yeast-expressed CYP 1A1, 1A2, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4, and 3A5, showed that only CYP 2C8, 2C18, and 2C19 were able to catalyze the 5-hydroxylation of 3. However, the catalytic efficiency of CYP 2C18 for that reaction was considerably higher (kcat/KM value being 3-4 orders of magnitude larger than those found for CYP 2C8 and 2C19). Several human P450s exhibited small activities for the oxidative O-dealkylation of 3. The four recombinant CYP 2Cs were the best catalysts for that reaction (kcat between 1 and 5 min-1) when compared to all the P450s that were tested, even though it is a minor reaction in the case of CYP 2C18. All these results show that compound 3 is a new, selective, and highly efficient substrate for CYP 2C18 that should be useful for the study of this P450 in various organs and tissues. They also suggest some key differences between the active sites of CYP 2C9 and CYP 2C18 for substrate recognition.     

Synthesis of (2R,5S)-dihydroxymethyl-(3R,4R)-dihydroxypyrrolidine (DGDP) via stereoselective amination using chlorosulfonyl isocyanate

A stereoselective approach for synthesizing (2R,5S)-dihydroxymethyl-(3R,4R)-dihydroxypyrrolidine 1 (2,5-dideoxy-2,5-imino-d-glucitol, DGDP) was achieved using a seven-step approach starting from 2,3,4,6-tetra-O-benzyl-d-mannose (7). Key steps for the preparation of the title compound 1 involved the regioselective and diastereoselective amination of the cinnamyl anti-1,2-polybenzyl ethers 5 and 6 using chlorosulfonyl isocyanate (CSI) and ring cyclization to form the pyrrolidine ring. The reaction between anti-1,2-polybenzyl ether 5 and CSI in toluene at 0 degrees C afforded the corresponding anti-1,2-amino alcohol 4 as a major product with a diastereoselectivity of 16:1 in 76% yield. The mechanism underlying these reactions may be explained by the neighboring-group effect leading to the retention of stereochemistry.     

Biodegradation of 4-methyl-5-nitrocatechol by Pseudomonas sp. strain DNT.

Pseudomonas sp. strain DNT degrades 2,4-dinitrotoluene (DNT) by a dioxygenase attack at the 4,5 position with concomitant removal of the nitro group to yield 4-methyl-5-nitrocatechol (MNC). Here we describe the mechanism of removal of the nitro group from MNC and subsequent reactions leading to ring fission. Washed suspensions of DNT-grown cells oxidized MNC and 2,4,5-trihydroxytoluene (THT). Extracts prepared from DNT-induced cells catalyzed the disappearance of MNC in the presence of oxygen and NADPH. Partially purified MNC oxygenase oxidized MNC in a reaction requiring 1 mol of NADPH and 1 mol of oxygen per mol of substrate. The enzyme converted MNC to 2-hydroxy-5-methylquinone (HMQ), which was identified by gas chromatography-mass spectrometry. HMQ was also detected transiently in culture fluids of cells grown on DNT. A quinone reductase was partially purified and shown to convert HMQ to THT in a reaction requiring NADH. A partially purified THT oxygenase catalyzed ring fission of THT and accumulation of a compound tentatively identified as 3-hydroxy-5-(1-formylethylidene)-2-furanone. Preliminary results indicate that this compound is an artifact of the isolation procedure and suggest that 2,4-dihydroxy-5-methyl-6-oxo-2,4-hexadienoic acid is the actual ring fission product.     

Dantrolene analogues revisited: general synthesis and specific functions capable of discriminating two kinds of Ca2+ release from sarcoplasmic reticulum of mouse skeletal muscle

The general synthesis of dantrolene analogues with various substituents on its phenyl ring has been developed via palladium-catalyzed cross-coupling reactions, the Stille or Suzuki reaction, as the key step. The effects of synthesized analogues have been evaluated by two kinds of Ca(2+) release modes from sarcoplasmic reticulum (SR) of mouse skeletal muscle fibers based on: (1) the measurement of twitch contraction caused by the physiological Ca(2+) release (PCR) of intact skeletal muscle and (2) the rate of Ca(2+)-induced Ca(2+) release (CICR) in saponin-treated skinned muscle fibers. Although dantrolene, a lead compound, inhibits both twitch contraction and CICR, some structurally modified analogues exhibit one or the other of these effects. The methoxy congener, GIF-0185, potently inhibits the twitch contraction without affecting the CICR, while GIF-0166 and GIF-0248, the ortho-nitro regioisomer and ortho, ortho-dinitro substituted analogues, respectively, doubly potentiate the CICR exclusively.     

Biosynthesis of riboflavin. Enzymatic formation of 6,7-dimethyl-8-ribityllumazine from pentose phosphates

The xylene ring of riboflavin originates by dismutation of the precursor, 6,7-dimethyl-8-ribityllumazine. The formation of the latter compound requires a 4-carbon unit as the precursor of carbon atoms 6 alpha, 6, 7, and 7 alpha of the pyrazine ring. The formation of riboflavin from GTP and ribose phosphate by cell extract from Candida guilliermondii has been observed by Logvinenko et al. (Logvinenko, E. M., Shavlovsky, G. M., Zakal'sky, A. E., and Zakhodylo, I. V. (1982) Biokhimiya 47, 931-936). We have studied this enzyme reaction in closer detail using carbohydrate phosphates as substrates and synthetic 5-amino-6-ribitylamino-2,4-(1H,3H)-pyrimidinedione or its 5'-phosphate as cosubstrates. Several pentose phosphates and pentulose phosphates can serve as substrate for the formation of riboflavin with similar efficiency. The reaction requires Mg2+. Various samples of ribulose phosphate labeled with 14C or 13C have been prepared and used as enzyme substrates. Radioactivity was efficiently incorporated into riboflavin from [1-14C]ribulose phosphate, [3,5-14C]ribulose phosphate, and [5-14C]ribulose phosphate, but not from [4-14C]ribulose phosphate. Label from [1-13C]ribose 5-phosphate was incorporated into C6 and C8 alpha of riboflavin. [2,3,5-13C]Ribose 5-phosphate yielded riboflavin containing two contiguously labeled segments of three carbon atoms, namely 5a, 9a, 9 and 8, 7, 7 alpha. 5-Amino-6-[1'-14C] ribitylamino-2,4 (1H,3H)-pyrimidinedione transferred radioactivity exclusively to the ribityl side chain of riboflavin in the enzymatic reaction. It follows that the 4-carbon unit used for the biosynthesis of 6,7-dimethyl-8-ribityllumazine consists of the pentose carbon atoms 1, 2, 3, and 5 in agreement with earlier in vivo studies.     

Enzymatic separation of hardly separable mixtures of structural isomers

Lipase-meditated separation of six hardly separable mixtures of structural isomers was carried out. It has become apparent that the hydrolysis rate of the C(5)-acetoxyl group of methyl (±)-5-acetoxy-4-aryl-(2E)-pentenoate possessing the ortho-substituents in the aromatic ring was slower than that of the corresponding compound with no substituents at the ortho-position. The hydrolysis rate of the C(5)-acetoxyl group of methyl (±)-5-acetoxy-2-aryl-(3E)-pentenoate possessing a remote reaction site from the ortho-substituents in the aromatic ring was proved to be faster than that of methyl (±)-5-acetoxy-4-aryl-(2E)-pentenoate.     

A reporter group delivery system with both absolute and selective specificity for thiol groups and an improved fluorescent probe containing the 7-nitrobenzo-2-oxa-1,3-diazole moiety.

1. 4-(N-2-Aminoethyl2'-pyridyl disulphide)-7-nitrobenzo-2-oxa-1,3-diazole (compound I) was synthesized and evaluated as a fluorescent labelling reagent for thiol groups. 2. The design of compound (I) as one example of a general type of reporter group delivery reagent (2-pyridyl-S-S-X, where X contains an environmentally sensitive spectroscopic probe) is discussed. 3. The electronic absorption spectrum of compound (I) was determined over a wide range of pH and the spectral changes that accompany its reaction with low-molecular-weight thiols, e.g. L-cysteine, and with papain (EC 3.4.22.2) and bovine serum albumin are discussed. 4. A new value of epsilon343 for 2-thiopyridone (Py-2-SH) was determined as 8.08 X 10(3) +/- 0.08 X 10(3)M-1-cm-1. 5. Spectral analysis of the reactions of compound (I) with L-cysteine and with papain (in the pH range 3.5-8.0) showed that even under equimolar conditions the reaction (thiol-disulphide interchange to release Py-2-SH) is essentially stoicheimoetric and probably proceeds by specific attack at the sulphur atom distal from the pyridyl ring of compound (I). 6. The fluorescence-emission spectra of compound (I) and of the products of its reaction with papain and with ficin (EC 3.4.22.3) were determined. Compound (I) is highly fluorescent in aqueous solution. Excitation within the intense visible absorption band (lambda max. 481 nm, epsilon max. 2.52 X 10(4)M-1-cm-1) provides green fluorescence with an emission maximum at 540 nm. Both papain and ficin labelled by reaction with compound (I) are characterized by fluorescence-emission maxima (535 nm and 530 nm respectively) of even higher intensity. The fluorescence emission of the product of the reaction of papain with compound (I) was shown to be 25 times more intense than that of the product of the reaction of papain with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (Nbd chloride). 7. The second-order rate constants (k2) for the reactions of compound (I) and of Nbd chloride with GSH, papain, albumin, ficin, 2-benzimidazolylmethanethiol and 2-benzimidazolylethanethiol were determined at 25.0 degrees C and various pH values. At pH4 the values of k2(compound I)/k2(Nbd chloride) are: GSH, 288; albumin, 36; papain 3 X 10(3); ficin, 3 X 10(4). 8. The pH-k2 profiles for the reactions of compound (I) and of Nbd chloride with the two 2-benzimidazolylalkanethiols were determined. Of the four profiles only that for the reaction of compound (I) with 2-benzimidazolylmethanethiol is characterized by a striking rate maximum in acidic media.     

Photolabile precursors of inositol phosphates. Preparation and properties of 1-(2-nitrophenyl)ethyl esters of myo-inositol 1,4,5-trisphosphate

1-(2-Nitrophenyl)ethyl esters of D-myo-inositol 1,4,5-trisphosphate (InsP3) have been synthesized and shown to have suitable properties for use as photolabile precursors of InsP3. Synthesis was accomplished by treatment of InsP3 with 1-(2-nitrophenyl)diazoethane in a CHCl3/water mixture. This resulted in esterification of each of the three phosphate residues in InsP3, the 1-phosphate being more reactive than the 4- or 5-phosphate. Singly esterified P-1, P-4, and P-5 esters, termed P-1, P-4, and P-5 caged InsP3, were isolated from the reaction mixture by anion-exchange HPLC and characterized by 500-MHz 1H NMR spectroscopy. Each of these caged InsP3 esters exists as a pair of diastereoisomers and was identified by examining the effects of pH and nitrophenyl ring current shielding on the chemical shifts of nonexchangeable inositol protons. 1H NMR spectra of InsP3 were analyzed for comparison. On photolysis the compounds released InsP3 with rate constants of 175 (P-1), 225 (P-4), and 280 s-1 (P-5) as determined by monitoring the aci-nitro decay reaction at pH 7.1, 0.2 M ionic strength, 21 degrees C. Quantum yields determined by steady-state near-UV photolysis were 0.65 +/- 0.08 for each compound. P-4 and P-5 caged InsP3 were the most promising biologically inactive InsP3 precursors since at concentrations up to 50 microM they did not release Ca2+ from smooth muscle sarcoplasmic reticulum (SR) and were not metabolized by vascular smooth muscle InsP3 5-phosphatase or bovine brain InsP3 3-kinase.(ABSTRACT TRUNCATED AT 250 WORDS)     

5-Phenyl substituted 1-methyl-2-pyridones and 4'-substituted biphenyl-4-carboxylic acids. synthesis and evaluation as inhibitors of steroid-5alpha-reductase type 1 and 2.

The synthesis of a series of 5-phenyl substituted 1-methyl-2-pyridones (I) and 4'-substituted biphenyl-4-carboxylic acids (II) as novel A-C ring steroidomimetic inhibitors of 5alpha-reductase (5alphaR) is described. Compounds 1-4 (I) were synthesized by palladium catalyzed cross coupling (Ishikura) reaction between diethyl(3-pyridyl)borane and aryl halides (1b-4b) followed by alpha-oxidation with sodium ferrocyanate of the 1-methyl-pyridinium salt. Inhibitors II (5-18) were obtained either by two successive Friedel-Crafts acylations from biphenyl (5a-10a) followed by saponification to yield the corresponding carboxylic acids (5-10) or by Suzuki cross coupling reaction to give the 4'-substituted biphenyl-4-carbaldehydes 11a-18a. The latter compounds were subjected to a Lindgren oxidation to yield compounds 11-18. The compounds were tested for inhibitory activity toward human and rat 5alphaR1 and 2. The test compounds inhibited 5alphaR, showing a broad range of inhibitory potencies. The best compound in series I was the N-(dicyclohexyl)-4-(1,2-dihydro-1-methyl-2-oxopyrid-5-yl)benzamide 4 exhibiting an IC(50) value for the human type 2 enzyme of 10 microM. In series II, the most active compound toward human type 2 isozyme was the 4'-(dicyclohexyl)acetyl-4-biphenyl carboxylic acid (10; IC(50)=220nM). Both series showed only marginal activity toward the human type 1 isozyme. In conclusion, the biphenyl carboxylic acids (II) are more appropriate for 5alphaR inhibition than the 5-phenyl-1-methyl-2-pyridones (I). Especially the 4'-carbonyl compounds 5-10 represent new lead structures for the development of novel human type 2 inhibitors.     

Chemical modification of aryl-1,2,3,6-tetrahydropyridinopyrimidine derivative to discover corticotropin-releasing factor(1) receptor antagonists: aryl-1,2,3,6-tetrahydropyridino-purine, -3H-1,2,3-triazolo[4,5-d)pyrimidine, -purin-8-one, and -7H-pyrrolo[2,3-d]pyrimidine derivatives

Structure-affinity relationships (SARs) of non-peptide CRF(1) antagonists suggest that such antagonists can be constructed of three units: a hydrophobic unit (Up-Area), a proton accepting unit (Central-Area), and an aromatic unit (Down-Area). Recently, various non-peptide corticotropin-releasing factor(1) (CRF(1)) receptor antagonists obtained by modification of the Central-Area have been reported. In contrast, we modified the Up-Area and presented 4- or 5-aryl-1,2,3,6-tetrahydropyridinopyrimidine derivatives including potent CRF receptor ligands 1a-c, and proposed that the 4- or 5-aryl-1,2,3,6-tetrahydropyridino moiety might be useful as a substituent in the Up-Area. Our interest shifted to the chemical modification in which the pyrimidine ring of 1a-c was replaced by other heterocycles, purine ring of 2, 3H-1,2,3-triazolo[4,5-d]pyrimidine ring of 3, purin-8-one ring of 4 and 7H-pyrrolo[2,3-d]pyrimidine ring of 5. Among them, 5-aryl-1,2,3,6-tetrahydropyridinopurine compound 6j (CRA0186) had the highest affinity for CRF(1) receptors (IC(50)=20nM). We report here the synthesis and SARs of derivatives 6-9.     

1-Acylthiosemicarbazides, 1,2,4-triazole-5(4H)-thiones, 1,3,4-thiadiazoles and hydrazones containing 5-methyl-2-benzoxazolinones: synthesis, analgesic-anti-inflammatory and antimicrobial activities

Acetic acid hydrazide containing 5-methyl-2-benzoxazolinone (4) was synthesized by the condensation of 2-(5-methyl-2-benzoxazolinone-3-yl)acetate with hydrazine hydrate. Thiosemicarbazide derivatives (5a-5d) were afforded by the reaction of corresponding compound 4 with substituted isothiocyanates. The cyclization of compounds 5a-5d in the presence of triethylamine resulted in the formation of compounds 6a-6d containing 1,2,4-triazole ring. On the other hand, the treatment of compounds 5a-5d with orthophosphoric acid caused the conversion of side chain of compounds 5a-5d into 1,3,4-thiadiazole ring: thus, compounds 7a-7c were obtained. The treatment of compound 4 with aromatic aldehydes resulted in the formation of arylidene hydrazides as cis-trans conformers (8a-8e). The structures of the compounds were elucidated by spectral and elemental analysis. While most compounds were exhibiting high activity in the analgesic-anti-inflammatory field, most of them were found to be inactive against bacteria and fungi.     

Synthesis and biological activities of novel nonpeptide angiotensin II receptor antagonists based on benzimidazole derivatives bearing a heterocyclic ring

A series of benzimidazole derivatives bearing a heterocyclic ring imidazole (1), 5-chloroimidazole (2), 1,2,4-triazol (3), and imidazoline (4) were synthesized and evaluated for angiotensin II antagonistic activities. The synthetic compounds 1-4 were biologically evaluated in vitro using an AT(1) receptor binding assay, where compounds 1 and 3 provided weak binding affinity, compound 2 showed moderate binding affinity, and compound 4 showed good binding affinity. Moreover, compound 4 was found to be almost equipotent with telmisartan in vivo biological evaluation study.     

Synthesis, antimicrobial activity and cytotoxicity of novel oxadiazole derivatives

In the present study, 5-substituted-1,3,4-oxadiazolin-2-thiones (1a-b) were synthesized via the ring closure reactions of appropriate acid hydrazides with carbon disulphide. N-(Benzothiazol-2-yl)-2-[[5-substituted-1,3,4-oxadiazol-2-yl]sulfanyl]acetamide derivatives (3a-j) were obtained by the nucleophilic substitution reactions of 5-substituted-1,3,4-oxadiazolin-2-thiones (1a-b) with N-(benzothiazol-2-yl)-2-chloroacetamides. The chemical structures of the compounds were elucidated by IR, (1)H NMR, (13)C NMR and FAB(+)-MS spectral data and elemental analyses. The synthesized compounds were screened for their antimicrobial activities against Micrococcus luteus, Bacillus subtilis, Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Listeria monocytogenes and Candida albicans. All compounds except compound 3h exhibited the highest antibacterial activity against P. aeruginosa. Among all compounds (3a-j), the compounds bearing 4-methoxyphenoxymethyl moiety on oxadiazole ring (3a-e) exhibited the highest inhibitory activity against C. albicans. Although compound 3j did not possess 4-methoxyphenoxymethyl moiety on oxadiazole ring, this derivative also exhibited the same level of anti-candidal activity. The compounds were also investigated for their cytotoxic effects using MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Compound 3a exhibited the highest cytotoxic activity, whereas compound 3g possessed the lowest cytotoxic activity against NIH/3T3 cells.     

Novel carbohydrate-induced modification of peptides: crystal structure and NMR analysis of ester-bridged bicyclic galactose-enkephalin adduct containing imidazolidinone moiety.

Comparative studies based on x-ray crystallography and NMR spectroscopy were used for structural characterization of the novel minor, imidazolidinone moiety containing, product 2b of the Maillard reaction obtained in vitro by using the galactose-modified endogenous opioid pentapeptide leucine-enkephalin (Tyr-Gly-Gly-Phe-Leu) 1. The x-ray analysis uniquely defined the molecular structure as cyclo-(N-(12-[-4)-D-galacto-pentitol-1-yl]-4-(4-hydroxybenzyl)-5-oxoimidazolidin-1-yl-(1 --> O]acetyl]glycyl-L-phenylalanyl-L-leucyl-] (3), having an 18-membered ring with an ester bond between the secondary (C4') hydroxyl group of a D-galacto-pentitolyl residue and the C-terminal carboxy group of leucine-enkephalin. The absolute configuration of the new chiral centre at the imidazolidinone moiety was established as C2(S), indicating a cis arrangement of C2 and C4 substituents at the 5-membered heterocyclic ring. The NMR analysis of compound 2b carried out in CH3CN-d3 and DMSO-d6, indicated the existence of two isomers in solution, differing only in the position of the ester group in the molecule. NMR data for the minor isomer (13%-16%) are in agreement with structure 3. The migratory tendency of the peptidyl group from the primary (2b) to the secondary hydroxyl group (3) of a D-galacto-pentitolyl residue in methanol/water solution was confirmed by RP HPLC analysis.     

Synthesis and conformation of four 16,17-diols in the 3-methoxy-13alpha-estra-1,3,5(10)-triene series

All four diasteromeric 16,17-diols in the 3-methoxy-13alpha-estra-1,3,5(10)-triene series have been synthesized. The trans-diols 1 and 2 can be obtained by hydroborating the 17-enol acetate 6 (61%, ratio 27:73, preferred alpha attack). OsO(4) dihydroxylation of the olefin 7 yielded the cis-diols 3 and 4 (ratio 13:87). The dihydroxylation proceeds with preference for beta attack caused by a C-ring twist-boat form of 7. The conformations of the diols 2 and 4, the 17-benzyl-17-hydroxy compounds 9 and 10 (obtained by Grignard reaction), and the 16alpha-bromo-17beta-hydroxy compound 8 were determined by X-ray analysis and by 1H NMR spectroscopy in solution. Some compounds, in spite of a 17beta-hydroxy group, had a conformation with a ring C chair form (4, 8, 9) caused by intermolecular interaction in the solid state. The rest of the compounds studied here (2, 10) possessed a conformation with a ring C twist-boat form, which has been also found for all 17beta-substituted compounds in solution. The preferred conformation of the D-ring with 17beta-substituents seems to be the 16beta-envelope form or near this form, but the existence of the 16alpha-envelope form (inversion of the ring D) for some compounds showed great variance in the conformation of ring D, which is substituent dependent.     

Ring opening is not rate-limiting in the GTP cyclohydrolase I reaction

GTP cyclohydrolase I catalyzes a mechanistically complex ring expansion affording dihydroneopterin triphosphate and formate from GTP. Single turnover quenched flow experiments were performed with the recombinant enzyme from Escherichia coli. The consumption of GTP and the formation of 5-formylamino-6-ribosylamino-2-amino-4(3H)-pyrimidinone triphosphate, formate, and dihydroneopterin triphosphate were determined by high pressure liquid chromatography analysis. A kinetic model comprising three consecutive unimolecular steps was used for interpretations where the first intermediate, 5-formylamino-6-ribosylamino-2-amino-4(3H)-pyrimidinone 5'-triphosphate, was formed in a reversible reaction. The rate constant k(1) for the reversible opening of the imidazole ring of GTP was 0.9 s(-1), the rate constant k(3) for the release of formate from 5-formylamino-6-ribosylamino-2-amino-4(3H)-pyrimidinone triphosphate was 2.0 s(-1), and the rate constant k(4) for the formation of dihydroneopterin triphosphate was 0.03 s(-1). Thus, the hydrolytic opening of the imidazole ring of GTP is rapid by comparison with the overall reaction.     

Alpha 1 adrenergic receptor function in senescent Fischer 344 rat aorta

There have been numerous conflicting reports concerning alpha 1 adrenergic receptor-mediated blood vessel contraction during aging and possible changes in alpha 1 receptor transduction mechanisms have not been investigated. These studies assess capacity of the aging vascular alpha 1 receptor to stimulate production of inositol phosphates, which are its intracellular second messengers, and to elicit a contractile response via this pathway. Aortic ring segments from mature adult (6 month old) and senescent (24 month old) Fischer 344 rats were incubated with [3H]myo-inositol and then stimulated with the alpha 1 agonist norepinephrine (NE, 10(-7)M-3 x 10(-5)M) in the presence of LiCl (10mM), an inhibitor of inositol phosphate metabolism. There was a substantial increase in inositol phosphate accumulation throughout the dose range in aortic rings from 24 month old rats compared to 6 month old rats. This is an alpha 1 receptor response since it is blocked by the alpha 1 antagonist prazosin but not by the alpha 2 antagonist yohimbine. Aortic inositol phosphate accumulation in response to serotonin did not change with age. To assess second messenger stimulated contraction, aortic ring segments were placed in Ca++ free buffer and then stimulated with NE. Under these conditions Ca++ influx is eliminated and contraction depends on the actions of intracellular second messengers. There is an age-related reduction in aortic contraction in Ca++ free buffer. These results suggest that aortic alpha 1 receptor-mediated formation of inositol phosphate intracellular second messengers is enhanced during aging. Despite this, the capacity of senescent arteries to elicit contraction utilizing second messenger pathways seems to be deficient.     

Synthesis of novel Schiff base analogues of 4-amino-1,5-dimethyl-2-phenylpyrazol-3-one and their evaluation for antioxidant and anti-inflammatory activity

4-Aminoantipyrine (4-amino-1,5-dimethyl-2-phenylpyrazole-3-one) and its analogues have been found to be compounds of interest for their anti-inflammatory, analgesic, antiviral, antipyretic, antirheumatic and antimicrobial activities. In the present study, Schiff base analogues of 4-aminoantipyrine were synthesized by the condensation reaction with substituted benzaldehydes and then evaluated for their antioxidant and anti-inflammatory activities. From among the synthesized compounds (3a-m, 4 and 5), 3 k and 3f exhibited the highest antioxidant activity followed by 3g, 3l, 3c, 3i, 5, 3m and 3h. The IC(50) values for compounds 3 k and 3f were found to be 0.44 and 0.93 μM, respectively, comparable to that of ascorbic acid (IC(50) 0.41 μM), a standard antioxidant agent. From the comparisons between the hydroxylated and methoxylated compounds, the rank order of antioxidant activity for the products resulting from benzylidene phenyl ring substitution was 2,4,6-OH>3,4-OH>3-OMe-4-OH>3,5-OMe-4-OH>2,4-OH>3-Me-4-OMe>3,4-OMe>4-OMe>4-OH. The structure-activity relationship study revealed that the position and nature of the substituted group on the benzylidene phenyl ring of the Schiff base analogues of 4-aminoantipyrine play an important role in their antioxidant activity. The anti-inflammatory activity of 3f, which also exhibited excellent antioxidant activity, was evaluated in terms of its inhibition of NO production, an inflammatory modulator, in LPS pretreated RAW 264.7 cells using the Griess method. We also examined whether or not this compound had effect on iNOS and COX-2 mRNA expression in RAW 264.7 cells. It was observed that compound 3f significantly reduced NO production and inhibited LPS-stimulated iNOS and COX-2 mRNA levels in a dose-dependent manner. Overall, 3f showed promising antioxidant and anti-inflammatory activities and may be used as the lead compound in a future study.