Probing the active site of aromatase with 2-methyl-substituted androstenedione analogs

To gain insight into the spatial nature of the androstenedione (AD) binding (active) site of aromatase in relation to the catalytic function of the enzyme, we synthesized 2,2-dimethylAD (4), 2beta- and 2alpha-methylADs (5 and 6), 19-oxygenated derivatives of compounds 4 and 6, and 2-methyleneAD (17), and we then tested their inhibitory activity as well as their aromatase reaction (aromatization for 2-methyl and 2-methylene analogs or 19-oxygenation for 2,2-dimethyl steroids) with human placental aromatase. 2-Methyl and 2-methylene steroids 5, 6, and 17 were good competitive inhibitors of aromatase (K(i)=22-68nM), but less effective compared to the 2,2-dimethyl analog 4 (K(i)=8.8nM), indicating that a combination of 2beta- and 2alpha-methyl moieties is essential for the formation of a thermodynamically stable inhibitor-aromatase complex. A series of 2alpha-methyl steroids were good substrates for aromatase, whereas 2beta-methyl steroid 5 was an extremely poor substrate, and a series of 2,2-dimethyl steroids did not serve as substrate, suggesting that a 2beta-methyl moiety of the 2,2-dimethyl and 2beta-methyl steroids would prevent the aromatase reaction probably due to steric hindrance in each case. The 2-methylene compound 17 was also aromatized to produce 2-methylestrogen with a low conversion rate where the 1,4-diene structure may have been created before the C(10)-C(19) bond cleavage. Kinetic analysis of the aromatization of androgens revealed that a good substrate was not essentially a good inhibitor for aromatase.     

Synthesis and human leukocyte elastase inhibitory evaluation of phosphate triesters and acyl phosphates of penam sulfides and sulfones

The synthesis of 6,6-dibromo-3alpha-(diphenylphosphate)oxymethyl-2,2-dimethyl penam sulfone (3a), 6alpha-chloro-3alpha-(diphenylphosphate)oxymethyl-2,2-dimethyl penam sulfone (3b), benzyl 6alpha-(diphenyl-phosphate)oxypenicillanate sulfone (4) and 6,6-dibromo-3alpha-(methylphosphate)carbonyl-2,2-dimethylpenam sulfone (12) are reported. When tested as inhibitors of human leukocyte elastase, the compound 4 proved to be the most active.     

Ion-transport Activity of Phenylpentanoic Acids Occurring in the Roots of Athyrium yokoscense

5-(3′-Hydroxyphenyl)pentanoic acid (1) and 5-(3′-methoxyphenyl)pentanoic acid (2) occurring in the roots of Athyrium yokoscense showed transport activity to alkaline and alkaline earth metal ions and heavy divalent metal ions.     

Inhibition by prostacyclin and carbacyclins of endothelin-induced DNA synthesis in cultured vascular smooth muscle cells

Effects of prostacyclin and carbacyclins on endothelin-induced DNA synthesis were investigated in vascular smooth muscle cells. DNA synthesis was estimated by [3H]thymidine incorporation. Five carbacyclins used in this report were 5-[(1S, 5S, 6R, 7R)-7-hydroxy-6-[(E)-(S)-3-hydroxy-1-octenyl]bicyclo [3.3.0]oct-2-en-3-yl) pentanoic acid (TEI-7165), methyl 5-[(1S, 5S, 6R, 7R)-7-hydroxy-6-[(E)-(S)-3-hydroxy-1-octenyl]bicyclo[3.3.0]oct-2-en-3- yl]pentanoate (TEI-9090), 5-[(1S, 5S, 6R, 7R)-7-hydroxy-6-[(E)-(3S, 5S)-3-hydroxy-5-methyl-1-nonenyl]bicyclo[3.3.0]oct-2-en-3-yl)penta noic acid (TEI-9063), methyl 5-[(1S, 5S, 6R, 7R)-7-hydroxy-6-[(E)-(3S, 5S)-3-hydroxy-5-methyl-1- nonenyl]bicyclo[3.3.0]oct-2-en-3-yl)pentanoate (TEI-1324), 5-[(1S, 5S, 6R, 7R)-7-hydroxy-6-[(E)-(S)-4-hydroxy-4-methyl-1- octenyl]bicyclo[3.3.0]oct-2-en-3-yl] pentanoic acid (TEI-3356). Prostacyclin and the carbacyclins inhibited the endothelin-induced DNA synthesis within the nanomolar range. These results suggest that prostacyclin and carbacyclins are possibly effective in inhibiting the proliferation of vascular smooth muscle cells under some situations in vivo.     

Synthesis of (S)-2-Hydroxy-β-ionone,Employing (S)-3-Hydroxy-2,2-dimethylcyclohexanone as the Chiral Starting Material

(S)-2-Hydroxy-β-ionone of 96% e.e. was synthesized from (S)-3-hydroxy-2,2-dimethyl cyclohexanone, which was easily obtained by the baker’s yeast reduction of 2,2-dimethylcyclohexane-l,3-dione.     

Synthesis of a phosphono analogue of cardiolipin and study of its silicic acid column chromatographic behaviour

The synthesis is reported of the phosphono analogue of cardiolipin namely of 1,2-dipalmitoyloxypropyl-3-(2'-hydroxy propyl-3'-(1",2"-dipalmitoyl glycerol]biphosphonate. This was prepared by the condensation of 1,2-dipalmitoyl glycerol with 1,2-dipalmitoyl-oxypropyl-3-(2'-hydroxy propyl-3')-biphosphonate catalyzed by tri-isopropylbenzene sulfonyl chloride in pyridine. The final product was characterized by elemental analyses, phosphono phosphorus determinations, thin-layer chromatography and IR spectroscopy. The silicic acid column chromatographic behaviour of the phosphono analogue of cardiolipin was also studied.     

Studies of Chrysanthemic Acid

(±) -cis and trans-2,2-dimethyl-3- (2′-cyano-l′-propenyl) cyclopropane carboxylic acids and their optically active forms were synthesized starting from chrysanthemic acids via oximes of 2,2-dimethyl3- (2′-formyl-l′-propenyl) cyclopropane carboxylates. Their allethrolone esters were also prepared which showed insecticidal activity.     

Synthesis of 3',5'-cyclic diguanylic acid (cdiGMP) using 1-(4-chlorophenyl)-4-ethoxypiperidin-4-yl as a protecting group for 2'-hydroxy functions of ribonucleosides

We herein report a convenient synthesis of 3',5'-cyclic diguanylic acid via the modified H-phosphonate approach. The 1-(4-chlorophenyl)-4-ethoxypiperidin-4-yl (Cpep) group was used as protecting group for the 2'-hydroxy functions of ribonucleosides. Complete unblocking of the fully protected 3',5'-cyclic diguanylic acid gave cdiGMP as a homogeneous compound in an excellent yield.     

Metabolism of 2,2'-dihydroxybiphenyl by Pseudomonas sp. strain HBP1: production and consumption of 2,2',3-trihydroxybiphenyl.

Cells of Pseudomonas sp. strain HBP1 grown on 2-hydroxy- or 2,2'-dihydroxybiphenyl contain NADH-dependent monooxygenase activity that hydroxylates 2,2'-dihydroxybiphenyl. The product of this reaction was identified as 2,2',3-trihydroxybiphenyl by 1H nuclear magnetic resonance and mass spectrometry. Furthermore, the monooxygenase activity also hydroxylates 2,2',3-trihydroxybiphenyl at the C-3' position, yielding 2,2',3,3'-tetrahydroxybiphenyl as a product. An estradiol ring cleavage dioxygenase activity that acts on both 2,2',3-tri- and 2,2',3,3'-tetrahydroxybiphenyl was partially purified. Both substrates yielded yellow meta-cleavage compounds that were identified as 2-hydroxy-6-(2-hydroxyphenyl)-6-oxo-2,4-hexadienoic acid and 2-hydroxy-6-(2,3-dihydroxyphenyl)-6-oxo-2,4-hexadienoic acid, respectively, by gas chromatography-mass spectrometry analysis of their respective trimethylsilyl derivatives. The meta-cleavage products were not stable in aqueous incubation mixtures but gave rise to their cyclization products, 3-(chroman-4-on-2-yl)pyruvate and 3-(8-hydroxychroman-4-on-2-yl)pyruvate, respectively. In contrast to the meta-cleavage compounds, which were turned over to salicylic acid and 2,3-dihydroxybenzoic acid, the cyclization products are not substrates to the meta-cleavage product hydrolase activity. NADH-dependent salicylate monooxygenase activity catalyzed the conversions of salicylic acid and 2,3-dihydroxybenzoic acid to catechol and pyrogallol, respectively. The partially purified estradiol ring cleavage dioxygenase activity that acted on the hydroxybiphenyls also produced 2-hydroxymuconic semialdehyde and 2-hydroxymuconic acid from catechol and pyrogallol, respectively.     

Biochemical effects of the hypoglycaemic compound pent-4-enoic acid and related non-hypoglycaemic fatty acids. Oxidative phosphorylation and mitochondrial oxidation of pyruvate, 3-hydroxybutyrate and tricarboxylic acid-cycle intermediates

1. The effects of the hypoglycaemic compound pent-4-enoic acid, and of four structurally related non-hypoglycaemic compounds (pent-2-enoic acid, pentanoic acid, cyclopropanecarboxylic acid and cyclobutanecarboxylic acid), on several reactions in rat liver mitochondria were determined. 2. The use of manometric techniques for measurements of oxidations and of phosphorylation is critically discussed. 3. Pent-4-enoic acid and pentanoic acid uncoupled oxidative phosphorylation at low concentrations, but usually by not more than about 50%. 4. All the compounds, except cyclobutanecarboxylic acid, strongly inhibited the oxidation of pyruvate and 2-oxoglutarate, but the oxidations of succinate, citrate and 3-hydroxybutyrate were not strongly inhibited. 5. All the compounds, except cyclobutanecarboxylic acid, inhibited decarboxylation of [1-(14)C]pyruvate with ferricyanide as electron acceptor. 6. All the compounds, except pent-2-enoic acid, caused mitochondrial swelling after a time-lag.     

云南金钱槭叶子中的酚性成分

利用各种色谱技术从云南金钱槭(Dipteronia dyeriana)叶片中分离得到13个化合物。通过波谱学方法鉴定为11,12-诃子裂酸二甲酯(1),12,13-诃子裂酸二甲酯(2),11-诃子裂酸甲酯(3),12-诃子裂酸甲酯(4),13-诃子裂酸甲酯(5),鞣料云实素(6),类叶升麻苷(7),短叶苏木酚酸甲酯(8),3-O-没食子酰基莽草酸(9),樱桃苷(10),山萘酚-3-O-β-D-木糖基-(1→2)-β-D-葡萄糖苷(11),没食子酸(12),莽草酸(13)。所有化合物均首次从金钱槭属植物中分离得到。     

Acylated flavonoids and phenol glycosides from Veronica thymoides subsp. pseudocinerea

A new acylated flavone glucoside, 3'-hydroxyscutellarein 7-O-(6'-O-protocatechuoyl)-beta-glucopyranoside (1), and a new phenol glucoside, 3,5-dihydroxyphenethyl alcohol 3-O-beta-glucopyranoside (6) were isolated from Veronica thymoides subsp. pseudocinerea together with seven known flavone, phenol and lignan glycosides; 3'-hydroxyscutellarein 7-O-(6'-O-trans-feruloyl)-beta-glucopyranoside (2), 3'-hydroxy, 6-O-methylscutellarein 7-O-beta-glucopyranoside (3), luteolin 7-O-beta-glucopyranoside (4), isoscutellarein 7-O-(6'-O-acetyl)-beta-allopyranosyl (1' --> 2')-beta-glucopyranoside (5), 3,4-dihydroxyphenethyl alcohol 8-O-beta-glucopyranoside (7), benzyl alcohol 7-O-beta-xylopyranosyl (1" --> 2')-beta-glucopyranoside (8), and (+)-syringaresinol 4'-O-beta-glucopyranoside (9). Compounds 2, 3 and 7-9 were reported for the first time in the genus Veronica. The structures of the isolates were determined by means of spectroscopic (UV, IR, 1D and 2D NMR, HR ESI-MS) methods. Isolated compounds (1-7) exhibited potent radical scavenging activity against the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical.     

A chromene and prenylated benzoic acid from Piper aduncum.

In addition to nerolidol, 2',6'-dihydroxy-4'-methoxydihydrochalcone, methyl 2,2-dimethyl-8-(3'-methyl-2'-butenyl)-2H-1-chromene-6-carboxylate, methyl 2,2-dimethyl-2H-1-chromene-6-carboxylate and methyl 8-hydroxy-2,2-dimethyl-2H-1-chromene-6-carboxylate, two new natural products were isolated from the leaves of Piper aduncum, 2,2-dimethyl-2H-1-chromene-6-carboxylic acid and 3-(3',7'-dimethyl-2',6'-octadienyl)-4-methoxybenzoic acid. The structures of the isolates were established based on analysis of spectroscopic data, including ES-MS. The DNA-damaging activity of the isolated compounds was also investigated against mutant strains of Saccharomyces cerevisiae.     

Microbial Hydrocarbon Co-oxidation: I. Oxidation of Mono- and Dicyclic Hydrocarbons by Soil Isolates of the Genus Nocardia

Nocardia cultures, isolated from soil by use of n-paraffins as the sole carbon source, have been shown to bring about significant oxidation of several methyl-substituted mono- and dicyclic aromatic hydrocarbons. Oxygen uptake by washed cell suspensions was not a reliable indicator of oxidation. Under co-oxidation conditions in shaken flasks, o- and p-xylenes were oxidized to their respective mono-aromatic acids, o-toluic and p-toluic acids. In addition, a new fermentation product, 2, 3-dihydroxy-p-toluic acid, was found in the p-xylene oxidation system. Of 10 methyl-substituted naphthalenes tested (1-methyl, 2-methyl, 1, 3-dimethyl, 1, 4-dimethyl, 1, 5-dimethyl, 1, 8-dimethyl, 1, 6-dimethyl, 2, 3-dimethyl, 2, 6-dimethyl, 2, 7-dimethyl), only those containing a methyl group in the β position were oxidized at this position to the mono acid.     

Identification of csypyrone B2 and B3 as the minor products of Aspergillus oryzae type III polyketide synthase CsyB

Since our first report on the identification of the fungal type III polyketide synthase (PKS) genes csyA～D in Aspergillus oryzae RIB40, type III PKS homologues have also been found in other fungal species. We previously reported the isolation and structural determination of csypyrone B1 as the main product of CsyB when inductively expressed in Aspergillus oryzae. Herein we report the isolation and identification of the two minor products of the csyB transformant in addition to csypyrone B1 as 4-(3-acetyl-4-hydroxy-2-oxo-2H-pyran-6-yl)butyric acid and 5-(3-acetyl-4-hydroxy-2-oxo-2H-pyran-6-yl)pentanoic acid. These compounds were named csypyrone B2 and B3, respectively, and both are homologues of main product csypyrone B1 with different side chain lengths. This result suggests that the carbon skeleton of the csypyrone B precursor is constructed by the condensation of fatty acyl-CoA and acetylmalonyl-CoA followed by pyrone formation. The alkyl side chain of the precursor may be oxidatively cleaved by enzyme(s) in the host fungus to give variations of csypyrone B with propanoic acid, butyric acid, or pentanoic acid side chains.     

Bianthraquinones from Cassia siamea

The isolation of two bianthraquinones, 1,1',3,8,8'-pentahydroxy-3',6-dimethyl[2,2'-bianthracene]-9,9',10,10'-tetrone and 7-chloro-1,1',6,8,8'-pentahydroxy-3,3'-dimethyl[2,2'-bianthracene]-9,9',10,10'-tetrone, from the root bark of Cassia siamea is reported. The structures were established by analysis of spectroscopic data and 7-chloro-1,1',6,8,8'-pentahydroxy-3,3'-dimethyl[2,2'-bianthracene]-9,9',10,10'-tetrone was determined on the direct comparison with synthetic compound.     

Studies of the inhibitory activity of MK-0591 (3-[1-(4-chlorobenzyl)-3-(t-butylthio)-5-(quinolin-2-yl-methoxy)-indol- 2-yl]-2,2-dimethyl propanoic acid) on arachidonic acid metabolism in human phagocytes.

We have investigated the inhibitory activity of compound MK-0591 (3-[1-(4-chlorobenzyl)-3-(t-butylthio)-5-(quinolin-2-yl-methoxy)-i ndol-2- yl]-2,2-dimethyl propanoic acid) on 5-lipoxygenase (5-LO) product synthesis in various human phagocytes stimulated with either the ionophore A23187, opsonized zymosan (OPZ), platelet-activating factor (PAF), or formyl-methionyl-leucyl-phenylalanine (fMLP). The lipoxygenase products were analyzed by reversed-phase HPLC. MK-0591 inhibited the formation of 5-hydroxyeicosatetraenoic acid, leukotriene (LT) B4, its omega-oxidation products, and 6-trans-isomers with IC50 values of 2.8-4.8 nM in A23187-stimulated neutrophils. In these conditions, arachidonic acid at a concentration of 10 microM had no effect on MK-0591 inhibitory activity. In neutrophils stimulated with OPZ, the synthesis of LTB4, its omega-oxidation products, and 6-trans-isomers was inhibited with IC50 values of 9.5-11.0 nM. MK-0591 inhibited 5-LO product synthesis in A23187-stimulated blood monocytes, eosinophils, and alveolar macrophages with IC50 values of 0.3-0.9, 3.7-5.3, and 8.5-17.3 nM, respectively. In neutrophils primed with granulocyte--macrophage colony-stimulating factor and stimulated with PAF, lipoxygenase product synthesis was inhibited with IC50 values of 7.7-8.7 nM. At the concentration of 1 microM, MK-0591 had no inhibitory effect on 15-lipoxygenase activity in human polymorphonuclear leukocytes, nor on human platelet 12-lipoxygenase and cyclooxygenase. In conclusion, MK-0591 is a very potent and specific inhibitor of 5-LO product synthesis in various types of human phagocytes.     

Aerobic biodegradation of 2,2'-dithiodibenzoic acid produced from dibenzothiophene metabolites

Dibenzothiophene is a sulfur heterocycle found in crude oils and coal. The biodegradation of dibenzothiophene through the Kodama pathway by Pseudomonas sp. strain BT1d leads to the formation of three disulfides: 2-oxo-2-(2-thiophenyl)ethanoic acid disulfide, 2-oxo-2-(2-thiophenyl)ethanoic acid-2-benzoic acid disulfide, and 2,2'-dithiodibenzoic acid. When provided as the carbon and sulfur source in liquid medium, 2,2'-dithiodibenzoic acid was degraded by soil enrichment cultures. Two bacterial isolates, designated strains RM1 and RM6, degraded 2,2'-dithiodibenzoic acid when combined in the medium. Isolate RM6 was found to have an absolute requirement for vitamin B12, and it degraded 2,2'-dithiodibenzoic acid in pure culture when the medium was supplemented with this vitamin. Isolate RM6 also degraded 2,2'-dithiodibenzoic acid in medium containing sterilized supernatants from cultures of isolate RM1 grown on glucose or benzoate. Isolate RM6 was identified as a member of the genus Variovorax using the Biolog system and 16S rRNA gene analysis. Although the mechanism of disulfide metabolism could not be determined, benzoic acid was detected as a transient metabolite of 2,2'-dithiodibenzoic acid biodegradation by Variovorax sp. strain RM6. In pure culture, this isolate mineralized 2,2'-dithiodibenzoic acid, releasing 59% of the carbon as carbon dioxide and 88% of the sulfur as sulfate.     

HCO3-secretion by bullfrog duodenum: dependence on nutrient Na+ during secretory stimulation

HCO3(-) secretion across in vitro duodenal mucosa of Rana catesbeiana was investigated under baseline conditions and during secretory stimulation. Baseline secretion was abolished by removal of CO2-HCO3(-)and reduced approximately 60% by removal of nutrient Na+, but was not sensitive to changes in Cl- or K+. Baseline secretion was not directly altered by exposure to 10(-3) M amiloride or 10(-3) M H2DIDS (dihydro-4,4'-diisothiocyanostilbene-2,2'-disulfonic acid) in the nutrient solution and only mildly reduced by acetazolamide. Following removal and restoration of Na+, recovery of secretion was impaired by exposure to acetazolamide (5 x 10(-4) M) or H2DIDS (5 x 10(-4) M) in the nutrient solution. Secretion stimulated by glucagon (10(-6) M) or 16,16-dimethyl prostaglandin E2 (10 microg.mL(-1)) was markedly attenuated by removal of Na+ or by exposure to H2DIDS, but secretion was not altered by acetazolamide (5 x 10(-4) M) or nutrient amiloride (1 mM). Thus, the HCO3(-) that is secreted under nonstimulated conditions derives partly from basolateral Na(+)-dependent uptake and partly from cellular CO2 hydration. Secretagogue-stimulated secretion by duodenal surface epithelium depends on stilbene-sensitive Na+(HCO3(-))n uptake across the basolateral membrane.     

Photoisomerization and photo-hydration of 3-hydroxystyrylnaphthalenes.

Fluorescence and trans-->cis photoisomerization are the main deactivation paths following excitation of trans-1-(2'-naphthyl)-2-(3'-hydroxyphenyl)ethene (trans-2,3NOH) in cyclohexane, methanol and acetonitrile. The quantum yield of both processes is wavelength dependent: this is due to the presence of conformational isomers deriving from rotation of the naphthyl group around the single bond with ethene. Addition of water to acetonitrile quenches the fluorescence (lambda(max)= 380 nm). In CH(3)CN/H(2)O (4/6, v/v) the emission spectrum displays a broad band with maximum at approximately 550 nm besides the original quenched fluorescence. This indicates that 2,3-NOH undergoes acid-base equilibration in the singlet excited state as supported by the enhancement of the fluorescence quantum yield with increasing acidity of the medium. Ground and excited state acidity constants have been determined. The main photochemical process is photo-hydration, i.e. water addition to the ethene bond. Fluorescence and photo-hydration have the same sigmoidal dependence on the acid concentration, which indicates that the undissociated form of singlet excited 2,3NOH is the photoreactive species. Laser flash photolysis experiments allowed identification of the reactive intermediates. The photophysics of trans-1-(1'-naphthyl)-2-(3'-hydroxyphenyl)ethene (trans-1,3NOH) is similar to that of 2,3NOH as regards the effect of water on fluorescence and the acid-base behaviour in the ground and first excited singlet state; the main photochemical process is trans-->cis photoisomerization together with photo-cylization to hydroxychrysene in neutral water/acetonitrile, but with lower yield compared to cyclohexane, and photo-hydration in strongly acidic medium.