Mechanism of Action of the Fungicide Thiabendazole, 2-(4′-Thiazolyl) Benzimidazole

Thiabendazole, 2-(4'-thiazolyl) benzimidazole (TBZ) inhibited the growth of Penicillium atrovenetum at 8 to 10 mug/ml. Oxygen consumption with exogenous glucose was inhibited at 20 mug/ml, but endogenous respiration required more than 100 mug/ml. TBZ inhibited completely the following systems of isolated heart or fungus mitochondria: reduced nicotinamide adenine dinucleotide oxidase, succinic oxidase, reduced nicotinamide adenine dinucleotide-cytochrome c reductase, and succinic-cytochrome c reductase at concentrations of 10, 167, 10, and 0.5 mug/ml, respectively. Cytochrome c oxidase was not inhibited. Antimycin A and sodium azide caused the usual inhibition patterns for both fungus and heart terminal electron transport systems. In the presence of antimycin, the fungicide inhibited completely succinate-dichloro-phenolindophenol reductase and succinate-2, 2-di-p-nitrophenyl-(3, 3-dimethoxy-4, 4-biphenylene-5, 5-diphenylditetrazolium)-reductase at 2 and 4 mug of TBZ per ml, respectively. Coenzyme Q reductase required 15 mug/ml. TBZ reduced the uptake by P. atrovenetum of glucose and amino acids and decreased the synthesis of various cell components. At 120 mug/ml, the incorporation of labeled carbon from amino acids-U-(14)C was decreased: lipid, 73%; nucleic acids, 80%; protein, 80%; and a residual fraction, 89%. TBZ did not inhibit peptide synthesis in a cell-free protein-synthesizing system from Rhizoctonia solani. Probably the primary site of inhibition is the terminal electron transport system and other effects are secondary.     

Mechanism of killing of HeLa cells by the antitumor sulfonylurea,N-(4-methylphenylsulfonyl)-N′-(4-chlorophenyl)urea (LY 181984)

Summary The antitumor sulfonylureas appear to inhibit both mitochondrial activity in susceptible human colon lines and to inhibit the oxidation of NADH by isolated plasma membrane vesicles from HeLa cells. The results reported here describe the morphological appearance of HeLa cells treated with the antitumor sulfonylurea N-(4-methylphenylsulfonyl)-N-(4-chlorophenyl)urea (LY181984). The cells remain viable for several days although the rate of increase in cell number is slowed especially at high concentrations of the drug. Cells become smaller with normal nuclei or maintain a normal size but contain multiple or enlarged nuclei. The morphological observations suggest that the drug may somehow interfere with the ability of the cells to enlarge following cytokinesis. Between 72 and 96 h, the cells begin to die. Cell death is accompanied by a condensed and fragmented appearance of the nuclear DNA as revealed by fluorescence microscopy with 4,6-diamidino-2-phenylindole suggestive of apoptosis. Early transients in loss of pH control (4 min after sulfonylurea addition) and an increase in cytoplasmic calcium (4 h after sulfonylurea addition) were observed but were small and perhaps secondary to the mechanism responsible for the failure of the cells to grow and ensuing cell death.     

Identification of the Metabolite (MΔ1) of 2-tert-Butyl-4-(2,4-dichloro-5-isopropoxyphenyl)-Δ21,3, 4-oxadiazolin-5-one (Oxadiazon) in Rice Plants

Out of metabolites of 2-tert-butyl-4-(2,4-dichloro-5-isopropoxyphenyl)-Δ²-1,3,4-oxadiazolin-5-one (oxadiazon) in rice plants one of the unidentified compounds nominated as M–1 was found much in head parts as compared with the parent compound and other metabolites. Identification of M–1 was made by means of thin-layer chromatography, gas-liquid chromatography, mass spectrometry and coincidence by the synthetic compound.

M–1 was identified as 1-(2,4-dichloro-5-isopropoxyphenyl)-1-methoxycarbonyl-2-trimethyl-acetyl-hydrazine and a pathway of cleavage of oxadiazolin ring of oxidiazon in rice plants was confirmed.     

Synthesis of the Fully Protected Phosphoramidite of the Benzene-DNA Adduct,N 2-(4-Hydroxyphenyl)-2′-Deoxyguanosine and Incorporation of the Later into DNA Oligomers

N²- (4-Hydroxyphenyl)-2 ′-deoxyguanosine-5 ′-O-DMT-3 ′-phosphoramidite has been synthesized and used to incorporate the N²-(4-hydroxyphenyl)-2 ′-dG (N²-4-HOPh-dG) into DNA, using solid-state synthesis technology. The key step to obtaining the xenonucleoside is a palladium (Xantphos-chelated) catalyzed N²-arylation (Buchwald-Hartwig reaction) of a fully protected 2 ′-deoxyguanosine derivative by 4-isobutyryloxybromobenzene. The reaction proceeded in good yield and the adduct was converted to the required 5 ′-O-DMT-3 ′-O-phosphoramidite by standard methods. The latter was used to synthesize oligodeoxynucleotides in which the N²-4-HOPh-dG adduct was incorporated site-specifically. The oligomers were purified by reverse-phase HPLC. Enzymatic hydrolysis and HPLC analysis confirmed the presence of this adduct in the oligomers.     

Identification of biaryl sulfone derivatives as antagonists of the histamine H₃ receptor: discovery of (R)-1-(2-(4'-(3-methoxypropylsulfonyl)biphenyl-4-yl)ethyl)-2-methylpyrrolidine (APD916)

The design of a new clinical candidate histamine-H(3) receptor antagonist for the potential treatment of excessive daytime sleepiness (EDS) is described. Phenethyl-R-2-methylpyrrolidine containing biphenylsulfonamide compounds were modified by replacement of the sulfonamide linkage with a sulfone. One compound from this series, 2j (APD916) increased wakefulness in rodents as measured by polysomnography with a duration of effect consistent with its pharmacokinetic properties. The identification of a suitable salt form of 2j allowed it to be selected for further development.     

Behavioral effects of the novel tropane analog, 2β-propanoyl-3β-(4-toluyl)-tropane (PTT)

In vitro studies have demonstrated that a novel tropane analog, PTT, in which both of the esters of cocaine have been removed is 20 times more potent than cocaine and more selective than cocaine in binding to dopamine transporters. The present studies compared the ability of PTT and cocaine to stimulate locomotor activity in rats. The intraperitoneal administration of PTT and cocaine to male Fisher-344 rats produced dose-dependent increases in spontaneous locomotor activity and stereotypic behaviors. PTT was 10–20 times more potent than cocaine in this behavioral assay, closely paralleling its potency relative to cocaine in dopamine transporter binding and uptake assays in vitro. PTT, however, elicited a qualitatively different profile of stereotypic behaviors, and PTT had a longer duration of action than cocaine. These results show how changes in kinetics and selectivity of tropanes can affect stimulant-elicited behaviors.     

Synthesis, structure and reactivity of the homoleptic iron(II) complex of the novel 4′-(4?-pyridyl-N-oxide)-2,2′:6′,2″-terpyridine ligand

The new ligand 4′-(4?-pyridyl-N-oxide)-2,2′:6′,2″-terpyridine (pyNoxterpy) and its homoleptic iron(II) complex have been synthesised, and structural and spectroscopic studies have been carried out. The obtained results have been compared with the reported data for the parent ligand 4′-(4?-pyridyl)-2,2′:6′,2″-terpyridine (pyterpy) and its homoleptic iron(II) complex. Significant differences between the spectral and electrochemical properties of the metal complexes have been found, derived from the changes in the electronic properties of the coordinated ligands.     

Effects of cAMP,theophylline, imidazole,and 4-(3,4-dimethoxybenzyl)-2-imidazolidone on the leaf movement rhythm of Trifolium repens—a test of the cAMP-hypothesis of circadian rhythms

The period length of the leaf movement rhythm of Trifolium repens L. is lengthened by continuously offered cAMP (0.5–1.0 mol m^-3) and theophylline (0.5–4 mol m^-3). At the higher concentrations this effect is more pronounced and the rhythm damps out faster. Imidazole (0.5 and 1 mol m^-3) has no effect on the period length; however, after 5 mol m^-3 the rhythm is abolished. Offered as 4 h pulses the resulting phase response curves for cAMP and imidazole are similar and show delays of up to 4 h during the day position of the leaves. Theophylline pulses lead to delays of up to 5 h during closure and advances of up to 3 h during opening. No phase shift is brought about by 4-(3,4-dimethoxybenzyl)-2-imidazolidone. The results do not support the cAMP-model of the circadian clock which has been proposed by Cummings (J. theor. Biol. 55, 455–470; 1975). The effect of the substances tested could, however, be based upon influences on the transport of Ca²⁺.Abbreviations ATP adenosine triphosphate - cAMP cyclic adenosine 35 monophosphate - AMP adenosine 5 monophosphate - AC adenyl cyclase - PDE phosphodiesterase - LL continuous light     

Inhibition of the 1-(o-chlorophenyl)-1-(p-chlorophenyl)-2,2,2-trichloroethane (o,p′DDT)- and estradiol-mediated induction of rat uterine ornithine decarboxylase by prior treatment with o,p′DDT,estradiol, and tamoxifen

This study was designed to determine whether prior administration of inducers of rat uterine ornithine decarboxylase (ODC), such as 1-(o-chlorophenyl)-1-(p-chlorophenyl)-2,2,2-trichloroethane (o,p′DDT), estradiol-17β (E₂), or tamoxifen, inhibits the elevation of ODC by subsequently administered o,p′ DDT or estradiol-17β. o,p′ DDT (10 mg/day) was injected for 2 days to ovariectomized rats. One or two days later, when the levels of ODC returned to basal levels, o,p′ DDT (10 mg) and E₂ (0.05 μg) were administered intraperitoneally and, 6 or 5 h after these injections, uterine ODC was analyzed. The pretreatment with o,p′ DDT almost entirely blocked the induction of ODC by E₂ or o,p′ DDT. In another experiment, pretreatment with o,p′ DDT for 5 or 6 days eliminated the induction of ODC after injection of o,p′ DDT on Day 7. Similarly, the treatment of rats with the antiestrogen-tamoxifen (0.1 mg/day) for 4 days completely inhibited the subsequent elevation of ODC by either E₂ or o,p′ DDT administered on the fifth day. However, attempts to block the E₂-mediated elevation of ODC by prior treatment with E₂ yielded variable results. Two possibilities were considered to attempt to explain the mechanism of inhibition of induction of ODC by o,p′ DDT and tamoxifen: (a) induction of hepatic monooxygenase by these compounds, resulting in increased metabolism of the subsequently administered o,p′ DDT and E₂ into biologically less active components; (b) involvement of putrescine, the product of ODC action, in inhibiting ODC formation at the pretranslational level or at the post-translational level. It appears unlikely that the o,p′ DDT- and tamoxifen-mediated inhibition of ODC induction was due to an increase in hepatic biotransformation of o,p′ DDT and E₂. Pretreatment with tamoxifen or E₂ did not appear to induce the formation of hepatic microsomal cytochrome P-450, a component of the monooxygenase system. Furthermore, pretreatment with 2,2-bis-(p-chlorophenyl)-1,1-dichloroethylene (a compound with a structure similar to o,p′ DDT), which is not estrogenic but like o,p′ DDT elevates hepatic monooxygenase activity, did not inhibit E₂-or o,p′ DDT-mediated induction of uterine ODC. Concerning the possibility of putrescine inhibitory action, our observations that uterine diamine oxidase activity is negligible and that o,p′ DDT administration has no effect on this enzymatic activity suggested that elevation of ODC may result in higher levels of uterine putrescine or spermidine and spermine. The finding that the administration of putrescine to ovariectomized rats inhibited uterine ODC induction by o, p′ DDT supports the treatise that inhibition of ODC elevation after initial induction of ODC by antiestrogens and o,p′ DDT is due to putrescine- or polyamine-mediated inhibition of ODC. The possible mechanism of such product inhibition of ODC is disucssed.     

Leucocyanidin: Synthesis and properties of (2R,3S,4R)-(+)-3,4,5,7,3′,4′-hexahydroxyflavan

An isomer of leucocyanidin, (2R,3S,4R)-(+)-3,4,5,7,3′,4′-hexahydroxyflavan has been synthesized from (+)-taxifolin, isolated in its phe     

An Easy Synthesis of (E)-9-Oxo-2-decenoic Acid,the Queen Substance of the Honey Bee,and (Z)-6-Heneicosen-ll-one,the Sex Pheromone of the Douglas Fir Tussock Moth†

We investigated the effect of dietary phospholipid (PL) concentrate from bovine milk on the epidermis. Thirteen-week-old hairless male and female mice (Hos:HR-1) were separated into two experimental groups, each fed two experimental diets: the control group and the PL group. The mice were given the experimental diets for 6 weeks. Stratum corneum hydration and transepidermal water loss (TEWL) were measured using Corneometer CM825 and Tewameter TM300 (Courage and Khazaka Electronics, Cologne, Germany) at 3 weeks and 6 weeks. After the feeding period, ceramides in stratum corneum were analyzed. We found that stratum corneum hydration and ceramides in the PL group were significantly higher than those in the control group and that TEWL in the PL group tended to decrease.

These results indicate that dietary PL concentrate improves epidermal function by increasing the amount of ceramides, resulting in higher hydration.     

Structural Basis for Small Molecule NDB (N-Benzyl-N-(3-(tert-butyl)-4-hydroxyphenyl)-2,6-dichloro-4-(dimethylamino) Benzamide) as a Selective Antagonist of Farnesoid X Receptor α (FXRα) in Stabilizing the Homodimerization of the Receptor

Farnesoid X receptor α (FXRα) as a bile acid sensor plays potent roles in multiple metabolic processes, and its antagonist has recently revealed special interests in the treatment of metabolic disorders, although the underlying mechanisms still remain unclear. Here, we identified that the small molecule N-benzyl-N-(3-(tert-butyl)-4-hydroxyphenyl)-2,6-dichloro-4-(dimethylamino) benzamide (NDB) functioned as a selective antagonist of human FXRα (hFXRα), and the crystal structure of hFXRα ligand binding domain (hFXRα-LBD) in complex with NDB was analyzed. It was unexpectedly discovered that NDB induced rearrangements of helix 11 (H11) and helix 12 (H12, AF-2) by forming a homodimer of hFXRα-LBD, totally different from the active conformation in monomer state, and the binding details were further supported by the mutation analysis. Moreover, functional studies demonstrated that NDB effectively antagonized the GW4064-stimulated FXR/RXR interaction and FXRα target gene expression in primary mouse hepatocytes, including the small heterodimer partner (SHP) and bile-salt export pump (BSEP); meanwhile, administration of NDB to db/db mice efficiently decreased the gene expressions of phosphoenolpyruvate carboxykinase (PEPCK), glucose 6-phosphatase (G6-pase), small heterodimer partner, and BSEP. It is expected that our first analyzed crystal structure of hFXRα-LBD·NDB will help expound the antagonistic mechanism of the receptor, and NDB may find its potential as a lead compound in anti-diabetes research.     

Occurrence of Endogenous Pollen Growth Inhibitors, 4-Hydroxybenzoic Acid and 4-(β-D-Glucopyranosyloxy)benzoic Acid,in the Pollen of Pinus densiflora†

A Pseudomonas strain capable of using pyrazinamide as the sole source of nitrogen was isolated from soil. An aromatic amidase from the bacterium was purified 400-fold to homogeneous on polyacrylamide gel electrophoresis. The enzyme had a molecular weight of 43,000 by gel filtration on Sephadex G-150 and consisted of two identical subunits. The isoelectric point was at 4.45. Among the compounds tested, pyrazinamide (relative activity, 100%), nicotinamide (60%), and 5-methylpyrazinamide (3.4%) were hydrolyzed at considerable rates. Benzamide, picolinamide, and isonicotinamide were not substrates. Apparent Km of the enzyme for pyrazinamide and nicotinamide were 5.6 × 10 ^?5 m and below 5 × 10^?6 m, respectively. The enzyme was not able to hydrolyze aliphatic amides. The enzyme was most active between pH 6.5 and 10 and 75°C, and was stable between pH 5.5 and 8.5 and below 45°C.     

Discovery of (3S,4S)-3-methyl-3-(4-fluorophenyl)-4-(4-(1,1,1,3,3,3-hexafluoro-2-hydroxyprop-2-yl)phenyl)pyrrolidines as novel RORγt inverse agonists

A novel series of cis-3,4-diphenylpyrrolidines were designed as RORγt inverse agonists based on the binding conformation of previously reported bicyclic sulfonamide 1. Preliminary synthesis and structure–activity relationship (SAR) study established (3S,4S)-3-methyl-3-(4-fluorophenyl)-4-(4-(1,1,1,3,3,3-hexafluoro-2-hydroxyprop-2-yl)phenyl)pyrrolidine as the most effective scaffold. Subsequent SAR optimization led to identification of a piperidinyl carboxamide 31, which was potent against RORγt (EC₅₀ of 61 nM in an inverse agonist assay), selective relative to RORα, RORβ, LXRα and LXRβ, and stable in human and mouse liver microsomes. Furthermore, compound 31 exhibited considerably lower PXR Y_max (46%) and emerged as a promising lead. The binding mode of the diphenylpyrrolidine series was established with an X-ray co-crystal structure of 10A/RORγt.     

Thiolytic cleavage of the anti-tumour compound 4′-(9-acridinylamino)-methanesulphon-m-anisidine (m-AMSA,NSC 156 303) in blood

4′-(9-acridinylamino)methanesulphon-m-anisidide (m-AMSA), a compound with a broad spectrum of experimental anti-tumour activity, was found to have a short biological half-life in mice bearing L1210 leukaemia. The fate of m-AMSA [³H]-labelled in the acridine nucleus, was determined following injection into mice. There was rapid formation of covalent adducts with plasma proteins. Adducts were also formed in freshly isolated blood samples following incubation at 37°C, and were found to be highly fluorescent. The formation of adducts was accompanied by a decrease in the free thiol concentration in plasma, and the concomitant addition of radioactivity from [³H]acridine nuclei. Acid or alkaline hydrolysis of the plasma protein adduct liberated acridone, while digestion with a protease produced unstable fluorescent compounds. A comparison of the rates of acid hydrolysis of the adducts and of model compounds suggested that the adducts were produced as a result of nucleophilic attack at the C-9 position of m-AMSA by protein thiol groups. The side chain of m-AMSA was liberated as 4-amino-3-methoxymethanesulphonanilide. Several congeners of m-AMSA were shown to form similar or identical adducts both in vivo and in vitro, and at rates which correlated with their reactivity towards simple organic thiols.     

Synthesis,Protonation Behavior,Conformational Analysis,and Regioselective Enzymatic Acylation of the Novel Diamino Analogue of (E)-5-(2-Bromovinyl)-2′-deoxyuridine (BVDU)

Abstract

(E)-3′,5′-Diamino-5-(2-bromovinyl)-2′,3′,5′-trideoxyuridine (5), the diamino analogue of BVDU (1), was synthesized from BVDU. The protonation behavior of 5 has been studied by means of pH-metric measurements and NMR spectroscopy. This study allows the determination of the basicity constants and the stepwise protonation sites. Thus, the main species at physiological pH is the monoprotonated form. The conformational analysis of this nucleoside analogue was also carried out through ¹H NMR spectroscopy. In addition, a convenient synthesis of N-3′ and N-5′ acylated derivatives was developed by regioselective enzymatic acylation. Thus, Candida antarctica lipase B (CAL-B) selectively acylated the 5′-amino group, thus furnishing nucleosides 8. On the other hand, immobilized Pseudomonas cepacia lipase (PSL-C) exhibited the opposite selectivity, conferring acylation at the 3′-amino group, thus affording derivatives 9.     

Chemical analysis of new water-soluble (1→6)-, (1→4)-α, β-glucan and water-insoluble (1→3)-, (1→4)-β-glucan (Calocyban) from alkaline extract of an edible mushroom, Calocybe indica (Dudh Chattu)

Two different glucans (PS-I, water-soluble; and PS-II, water-insoluble) were isolated from the alkaline extract of fruit bodies of an edible mushroom Calocybe indica. On the basis of acid hydrolysis, methylation analysis, periodate oxidation, and NMR analysis ((1)H, (13)C, DEPT-135, TOCSY, DQF-COSY, NOESY, ROESY, HMQC, and HMBC), the structure of the repeating unit of these polysaccharides were established as: PS-I: →6)-β-D-Glcp-(1→6)-β-D-glcp-(1→6)-)-β-D-Glcp-(1→ α-D=Glcp (Water-soluble glucan). PS-II: →3)-β-D-Glcp-(1→3)-β-D-glcp-(1→3)-)-β-D-Glcp-(1→3)-β-D-Glcp-(1→ β-D-Glcp (Water-insoluble glucan, Calocyban).