Tetra- and hexahydro derivatives of aldosterone and 18-hydroxycorticosterone by chemical and microbial reductions

Preparative methods were developed for reduction with NaBH4 at 0 of 3 beta, 5 alpha- and 3 alpha, 5 beta-tetrahydroaldosterone (1) and (12) to their respective 20 alpha-ol derivatives 2a and 13a. Corroboration of structures was obtained by periodate oxidations to the lactols 3b and 14b and thence, by further oxidation, to the lactones 4 and 15 respectively; these lactones were also independently obtained from 1 and 12. Reduction with NaBH4 at 80 degrees C converted 1 and 12 into 18-hydroxy-3 beta, 5 alpha, 20- and 18-hydroxy-3 alpha, 5 beta, 20-hexahydrocorticosterone 6a and 17a respectively, which were mixtures of epimers at C-20. Compound 17a could also be prepared by reduction of the lactone 21 with sodium aluminum bis-(methoxyethoxy) hydride. Again, periodate oxidations of 6a and 17a gave the lactols 7b and 22b and thence, by Jones oxidation, the diketolactones 8 and 23, which were also prepared from 18-hydroxy-11-dehydrocorticosterone (10) and 18-hydroxycorticosterone (24) respectively. Improved conditions for reduction with Clostridium paraputrificum permitted convenient conversion of aldosterone (11), the corresponding 18 leads to 11 lactone 18a and 18-hydroxycorticosterone (24) into their 3 alpha, 5 beta-tetrahydro derivatives.     

Isolation of urinary C-20 alpha- and C-20 beta-hydroxy-C21-steroid metabolites in cases of congenital adrenal hyperplasia, postpubertal virilizing syndrome and polycystic ovary syndrome.

The proportion of 20 alpha/20 beta-epimers of the urinary C21-steroid metabolites was estimated in normal volunteers and in patients with congenital adrenal hyperplasia (CAH), postpubertal virilizing syndrome (PVS) and polycystic ovary syndrome (POS). In the normal individuals 20 alpha- and 20 beta-epimers of 5 beta-pregnane-3 alpha, 17 alpha, 20-triol (pregnanetriol) and 5-pregnene-3 beta, 17 alpha, 20-triol (5-pregnenetriol) were measured. In the other case 20 alpha- and 20 beta-epimers of the characteristic, representative metabolites were also investigated. In 26 of 27 normal persons and in all the patients with normotensive CAH, PVS and POS the 20 beta-epimer comprised 0-10% of the total pregnanetriol. The 20 beta-epimer of 5-pregnenetriol was found in only one normal case (6% of the total). The percentage of 20 beta-epimers of 3 alpha, 17 alpha, 20-trihydroxy-5 beta-pregnan-11-one, 5 beta-pregnane-3 alpha, 11 beta, 17 alpha, 20-tetrol (in the normotensive CAH, PVS and POS) and 5 alpha- or 5 beta-pregnane-3 alpha, 17 alpha, 20, 21-tetrol (in the hypertensive CAH) varied from nil to 76%. The effect of functional groups and the stereochemistry of the molecule on the direction of C-20-keto group reduction is discussed; the existence of additional factors determining this reduction in certain pathological conditions is suggested.     

Comparison of vasodilatory properties of 14,15-EET analogs: structural requirements for dilation

Epoxyeicosatrienoic acids (EETs) are endothelium-derived eicosanoids that activate potassium channels, hyperpolarize the membrane, and cause relaxation. We tested 19 analogs of 14,15-EET on vascular tone to determine the structural features required for activity. 14,15-EET relaxed bovine coronary arterial rings in a concentration-related manner (ED(50) = 10(-6) M). Changing the carboxyl to an alcohol eliminated dilator activity, whereas 14,15-EET-methyl ester and 14,15-EET-methylsulfonimide retained full activity. Shortening the distance between the carboxyl and epoxy groups reduced the agonist potency and activity. Removal of all three double bonds decreased potency. An analog with a Delta8 double bond had full activity and potency. However, the analogs with only a Delta5 or Delta11 double bond had reduced potency. Conversion of the epoxy oxygen to a sulfur or nitrogen resulted in loss of activity. 14(S),15(R)-EET was more potent than 14(R),15(S)-EET, and 14,15-(cis)-EET was more potent than 14,15-(trans)-EET. These studies indicate that the structural features of 14,15-EET required for relaxation of the bovine coronary artery include a carbon-1 acidic group, a Delta8 double bond, and a 14(S),15(R)-(cis)-epoxy group.     

Etoposide: a new approach to the synthesis of 4-O-(2-amino-2-deoxy-4,6-O-ethylidene-beta-D-glucopyranosyl)-4'-O- demethyl-4-epipodophyllotoxin

Synthesis of 3-O-acetyl-2-benzyloxycarbonylamino-2-deoxy-4,6-O-ethylidene- alpha-(7 alpha) and-beta-D-glucopyranose (7 beta) and their 3-O-chloroacetyl analogues (11 alpha and 11 beta) are described. Condensation (BF3-etherate, ethyl acetate, -20 degrees) of 7 alpha with 4'-O-benzyloxycarbonyl-4'-O-demethyl-4-epipodophyllotoxin (8) afforded mainly the beta-glycoside 9 beta (alpha, beta-ratio 1:9). Condensation of 11 alpha beta with 8 or the 4'-O-chloroacetyl analogue 13 gave mainly the 4-O-(2-benzyloxycarbonylamino-3-O-chloroacetyl-2-deoxy-4,6-O-ethyl idene-beta-D- glucopyranosyl)-epipodophyllotoxin 12 beta or 15 beta. Glycosidation of podophyllotoxin (14) with 11 alpha beta (during which the aglycon epimerized at C-4 under the action of BF3-etherate) afforded alpha- (16 alpha) and beta-glycoside (16 beta) in the ratio 1:5. Removal of the chloroacetyl groups from 12 beta, its alpha analogue 12 alpha, and 15 beta gave the 4-O-(2-benzyloxycarbonylamino-2-deoxy-4,6-O-ethylidene-alpha-(17 alpha) and -beta-D-glucopyranosyl)-4'-O-demethyl-epipodophyllotoxins (17 beta and 20 beta), respectively. Hydrogenolysis of the benzyloxycarbonyl groups then gave 4-O-(2-amino-2-deoxy-4,6-O-ethylidene-alpha- (18 alpha) and -beta-D-glucopyranosyl)-4'-O-demethyl-4-epipodophyllotoxin (18 beta). Reductive alkylation of 18 beta and 18 alpha afforded the 2"-deoxy-2"-dimethylamino-etoposide 3 and its alpha analogue 19 alpha.     

小金梅草化学成分研究(英文)

从小金梅草乙醇提取物中分离得到了12个化合物,分别为3-O-β-D-槲皮素葡萄糖苷(1)、3-O-β-D-山柰酚葡萄糖苷(2)、5-O-β-D-芹菜素葡萄糖苷(3)、α-菠甾醇(4)、2,6-二甲氧基苯甲酸(5)、3-吲哚甲酸(6)、(2S,3R,4E,8E)-1-(β-D-吡喃葡萄糖苷)-N-[(R)-2’-羟基-二十碳酰基]-9-甲基-4,8-二烯-1,3-二醇-2-氨基-十八烷(7)、正三十二烷醇(8)、14,15-二十碳烯酸(9)、木腊酸(10)、β-谷甾醇(11)、胡萝卜苷(12)。以上化合物均为首次从该植物中得到。     

Synthesis of (25R)-26-hydroxy-15-ketosterols

(25R)-3beta,26-Dihydroxy-5alpha-cholest-8(14)-en-15-one (1) and (25R)-3beta,26-dihydroxy-5alpha,14beta-cholest-16-en-1 5-one (2) were synthesized from (25R)-3beta,26-dibenzoyloxy-5alpha,14alpha-chole st-16-ene (4). Oxidation of 4 with CrO3-3,5-dimethylpyrazole at -20 degrees C gave (25R)-3beta,26-dibenzoyloxy-5alpha,14alpha-chole st-16-en-15-one (5) along with (25R)-3beta,26-dibenzoyloxy-5alpha-cholest-16alpha+ ++,17alpha-epoxide (6). Oxidation of 5 with selenium dioxide afforded (25R)-3beta,26-dibenzoyloxy-5alpha-cholest-8(14),16-++ +dien-15-one (7) and (25R)-3beta,26-dibenzoyloxy-5alpha,14beta-choles t-16-en-15-one (8). Selective hydrogenation of 7 followed by hydrolysis in alcoholic potassium hydroxide yielded (25R)-3beta,26-dihydroxy-5alpha-cholest-8(14)-en-15-one (1). Hydrolysis of 5 and 8 in alcoholic potassium hydroxide provided (25R)-3beta,26-dihydroxy-5alpha,14beta-cholest-16-en-1 5-one (2).     

Syntheses of 19-[O-(carboxymethyl)oxime] haptens of epipregnanolone and pregnanolone

O-(Carboxymethyl)oximes 1 and 2 derived from two epimeric 5beta-pregnanolones (3beta-hydroxy-5beta-pregnan-20-one and 3alpha-hydroxy-5beta-pregnan-20-one) in position 19 were prepared. Two synthetic routes were employed, both using protection of the 20-keto group after reduction into the (20R)-alcohol in the form of acetate. In the first route, (20R)-19-hydroxy-5beta-pregnan-3beta,20-diyl diacetate (3) was transformed into the corresponding 19-[O-(carboxymethyl)oxime] methyl ester 6, then deacetylated by acid and partially silylated with tert-butyldimethylsilyl chloride. The desired 3-O-silylated derivative 8 was separated, oxidized to the 20-ketone and protecting groups were sequentially removed to give the first title hapten 1. The second route started from (20R)-19-hydroxy-3-oxopregn-4-en-20-yl acetate (11), which was hydrogenated in the presence of base to the 5beta-pregnan-3-one derivative 12, protected in position 19 with tert-butyldimethylsilyl group and reduced with borohydride. The prevailing 3alpha-alcohol 15 was separated, protected in position 3 with a methoxymethyl group, deprotected in position 19 and transformed into the 19-[O-(carboxymethyl)oxime] 19. After deacetylation, esterification with diazomethane and oxidation in position 20, the pregnanolone skeleton was regenerated. Final deprotection steps gave the second title hapten 2. Both haptens, i.e., (19E)-3beta- and -3alpha-hydroxy-20-oxo-5beta-pregnan-19-al 19-[O-(carboxymethyl)oxime], were designed for the development of immunoassays of the corresponding parent neuroactive steroids.     

Mechanism and signal transduction of 14 (R), 15 (S)-epoxyeicosatrienoic acid (14,15-EET) binding in guinea pig monocytes

14(R), 15(S)-epoxyeicosatrienoic acid (14,15-EET) is a cytochrome P-450 monooxygenase (epoxygenase) metabolite of arachidonic acid (AA). In this study, we have identified a population of specific high affinity binding sites for 14,15-EET in the guinea pig mononuclear (GPM) cells. The results of competition studies showed that 14(R), 15(S)-EET was an effective competing ligand with a Ki of 226.3 nM followed by 11(R), 12(S)-EET, 14(S), 15(R)-EET, 14,15 thia(S)-ET, and 14,15-aza(N)-ET. The binding was sensitive to various protease treatments suggesting that the binding site is protein in nature. Cholera toxin (CT) and dibutyryl cAMP attenuated 14,15-EET binding in GPM cells. Mean binding site density (Bmax), decreased 32.0% and 19.1% by the pretreatment with cholera toxin (200 micrograms/ml) and dibutyryl cAMP (100 nM), respectively, without changing the dissociation constant. A specific protein kinase A (PKA) inhibitor, H-89, but not the PKC inhibitor K252a reversed the down regulation of 14,15-EET receptor binding caused by dibutyryl cAMP in GPM cells. Thus, the results sug-gest that the specific binding site of 14,15-EET in GPM cells be associated with a receptor that could be down regulated through an increase in intracellular cAMP and activation of a PKA signal trans-duction. We propose that the signal transduction mechanism begins with the binding of 14,15-EET to its receptor that leads to increase intracellular cAMP levels and the activation of PKA, and finally, with the down regulation of 14,15-EET receptor binding.     

Taxanes with C-5-amino-side chains from the needles of Taxus canadensis

Five taxanes with an amino-side chain on C-5 were identified for the first time in the needles of the Canadian yew, Taxus canadensis. Their structures were characterized as 2alpha,7beta,9alpha,10beta,13-pentaacetoxy-11beta-hydroxy-5alpha-(3'-N,N-dimethylamino-3'-phenyl)-propionyloxytaxa-4(20),12-diene (1), 2alpha,9alpha-dihydroxy-10beta,13alpha-diacetoxy-5alpha-(3'-methylamino-3'-phenyl)-propionyloxytaxa-4(20),11-diene (2), 2alpha17-dihydroxy-9alpha,10beta,13alpha-triacetoxy-5alpha-(3'-N,N-dimethylamino-3'-phenyl)-propionyloxytaxa-4(20),11-diene (3), 2alpha-hydroxy-7beta,9alpha,10beta,13alpha-tetraacetoxy-5alpha-(2'-hydroxy-3'-N,N-dimethylamino-3'-phenyl)-propionyloxytaxa-4(20),11-diene (4), and 9alpha-hydroxy-2alpha,10beta,13alpha-triacetoxy-5alpha-(3'-N,N-dimethylamino-3'-phenyl)-propionyloxytaxa-4(20),11-diene (5) on the basis of 1D-, 2D-NMR spectroscopic data and high-resolution fast atom bombardment MS analyses. Metabolite (1) was isolated from the needles of the Canadian yew for the first time but had previously been detected in the stems of the Japanese yew, whereas taxanes (2-5) are only now reported. Metabolite (3) is the first reported nitrogen-containing taxane with a 17-hydroxyl substitution.     

11,12,20-Trihydroxy-eicosa-8(Z)-enoic acid: a selective inhibitor of 11,12-EET-induced relaxations of bovine coronary and rat mesenteric arteries

Arachidonic acid is metabolized to four regioisomeric epoxyeicosatrienoic acids (EETs) by cytochrome P-450. 5,6-, 8,9-, 11,12-, and 14,15-EET are equipotent in relaxing bovine coronary arteries (BCAs). Vasorelaxant effects of EETs are nonselectively antagonized by 14,15-epoxyeicosa-5(Z)-enoic acid. The 11,12-EET analogs, 20-hydroxy-11,12-epoxyeicosa-8(Z)-enoic acid (20-H-11,12-EE8ZE) and 11,12,20-trihydroxyeicosa-8(Z)-enoic acid (11,12,20-THE8ZE) were synthesized and tested for antagonist activity against EET-induced relaxations in BCAs. In U-46619-preconstricted arterial rings, 5,6-, 8,9-, 11,12-, and 14,15-EET caused concentration-dependent relaxations with maximal relaxations ranging from 80 to 96%. Preincubation of arteries with 20-H-11,12-EE8ZE (10(-5) M) inhibited relaxations to 14,15- and 11,12-EET, but not 5,6- and 8,9-EET; however, greatest inhibitory effect was against 11,12-EET (maximal relaxation = 80.6 ± 4.6 vs. 26.7 ± 7.4% without and with 20-H-11,12-EE8ZE, respectively). Preincubation with the soluble epoxide hydrolase inhibitor (tAUCB, 10(-6) M) significantly enhanced the antagonist effect of 20-H-11,12-EE8ZE against 14,15-EET-induced relaxations (maximal relaxation = 86.6 ± 4.4 vs. 27.8 ± 3.3%, without and with 20-H-11,12-EE8ZE and tAUCB) without any change in its effect against 11,12-EET-induced relaxations. In contrast to the parent compound, the metabolite, 11,12,20-THE8ZE (10(-5) M), significantly inhibited relaxations to 11,12-EET and was without effect on other EET regioisomers. Mass spectrometric analysis revealed conversion of 20-H-11,12-EE8ZE to 11,12,20-THE8ZE by incubation with BCA. The conversion was blocked by tAUCB. 14,15-Dihydroxy-eicosa-5Z-enoic acid (a 14,15-EET antagonist), but not 11,12,20-THE8ZE (an 11,12-EET antagonist), inhibited BCA relaxations to arachidonic acid and flow-induced dilation in rat mesenteric arteries. These results indicate that 11,12,20-THE8ZE is a selective antagonist of 11,12-EET relaxations and a useful pharmacological tool to elucidate the function of 11,12-EET in the cardiovascular system.     

Mechanisms of leukotriene formation: hemoglobin-catalyzed transformation of 15-HPETE into 8,15-DiHETE and 14,15-DiHETE isomers

Four isomers of 8,15-diHETE as well as 14,15-diHETEs are isolated and characterized after exposure of 15-HPETE to hemoglobin. It is found that 83% of the C-8 oxygen atoms in 8(R), 15(S)-diHETE and 8(S), 15(S)-diHETE, and 41% of the C-8 oxygen atoms in 8(R), 15(S)-11Z-diHETE and 8(S), 15(S)-11Z-diHETE are derived from H2(18)O. These results suggest that hemoglobin catalyzes the transformation of 15-HPETE into these products via a free radical process, possibly involving the intermediacy of 14,15-LTA. Intact human leukocytes contain a distinct enzyme system for catalyzing the conversion of 15-HPETE into 14,15-LTA. This enzyme activity is inhibited by ETYA and is rapidly denatured upon homogenization of the intact leukocytes.     

Steric analysis of 8-hydroxy- and 10-hydroxyoctadecadienoic acids and dihydroxyoctadecadienoic acids formed from 8R-hydroperoxyoctadecadienoic acid by hydroperoxide isomerases

8-Hydroxyoctadeca-9Z,12Z-dienoic acid (8-HODE) and 10-hydroxyoctadeca-8E,12Z-octadecadienoic acid (10-HODE) are produced by fungi, e.g., 8R-HODE by Gaeumannomyces graminis (take-all of wheat) and Aspergillus nidulans, 10S-HODE by Lentinula edodes, and 10R-HODE by Epichloe typhina. Racemic [8-(2)H]8-HODE and [10-(2)H]10-HODE were prepared by oxidation of 8- and 10-HODE to keto fatty acids by Dess-Martin periodinane followed by reduction to hydroxy fatty acids with NaB(2)H(4). The hydroxy fatty acids were analyzed by chiral phase high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) with 8R-HODE and 10S-HODE as standards. 8R-HODE eluted after 8S-HODE on silica with cellulose tribenzoate (Chiralcel OB-H), and 10S-HODE eluted before 10R-HODE on silica with an aromatic chiral selector (Reprosil Chiral-NR). 5S,8R-Dihydroxyoctadeca-9Z,12Z-dienoic acid (5S,8R-DiHODE) is formed from 18:2n-6 by A. nidulans and 8R,11S-dihydroxyoctadeca-9Z,12Z-dienoic acid (8R,11S-DiHODE) by Agaricus bisporus. 8R-Hydroperoxylinoleic acid (8R-HPODE) can be transformed to 5S,8R-DiHODE and 8R,11-DiHODE by Aspergillus spp., and 8R,13-dihydroxy-9Z,11E-dienoic acid (8R,13-DiHODE) can also be detected. We prepared racemic [5,8-(2)H(2)]5,8- and [8,11-(2)H(2)]8,11-DiHODE by oxidation and reduction as above and 8R,13S- and 8R,13R-DiHODE by oxidation of 8R-HODE by S and R lipoxygenases. The diastereoisomers were separated and identified by normal phase HPLC-MS/MS analysis. We used the methods for steric analysis of fungal oxylipins. Aspergillus spp. produced 8R-HODE (>95% R), 10R-HODE (>70% R), and 5S,8R- and 8R,11S-DiHODE with high stereoselectivity (>95%), whereas 8R,13-DiHODE was likely formed by nonenzymatic hydrolysis of 8R,11S-DiHODE.     

Synthesis of some epoxy and/or N-oxy 17-picolyl and 17-picolinylidene-androst-5-ene derivatives and evaluation of their biological activity

Steroidal epoxy and/or N-oxy 17-picolyl and 17-picolinylidene-androst-5-ene derivatives have been prepared using 3beta,17beta-dihydroxy-17alpha-picolyl-androst-5-ene (1), 3beta-acetoxy-17-picolinylidene-androst-5-ene (2), and 3beta-hydroxy-17-picolinylidene-androst-5-ene (3) as synthetic precursors. The compounds 2 and/or 3 were reacted with m-chloroperoxybenzoic acid (MCPBA). The compounds synthesized from 2 were 17-picolinylidene-N-oxide 4, 5alpha,6alpha-epoxy and 5beta,6beta-epoxy-17-picolinylidene-N-oxide 5 and 6, and 5alpha,6alpha:17alpha,20alpha- and 5beta,6beta:17alpha,20alpha-diepoxy-N-oxide 7 and 8. Starting from compound 3, a mixture of 5alpha,6alpha-epoxy and 5beta,6beta-epoxy-17-picolinylidene 9 and 10, 5alpha,6alpha-epoxy and 5beta,6beta-epoxy-17-picolinylidene-N-oxide 11 and 12, and 5alpha,6alpha:17alpha,20alpha- and 5beta,6beta:17alpha,20alpha-diepoxy-N-oxide 13 and 14 were obtained. From compounds 15 and 18, obtained from 1 and 3 by the Oppenauer oxidation, the 4alpha,5alpha-epoxy and 4beta,5beta-epoxy derivatives 16, 17 and 20, 21 were prepared by oxidation with 30% H(2)O(2). Oxidation of 18 with MCPBA yielded only the N-oxide 19. The structures of compounds 15 and 18 were proved by the X-ray analysis. Compounds 1-6, 9, 15, 17, 18, and 21 were tested on activity against the enzyme aromatase. Antitumor activity against three tumor cell lines (human breast adenocarcinoma ER+, MCF-7, human breast adenocarcinoma ER-, MDA-MB-231, and prostate cancer PC3) was evaluated. Three tested compounds (1, 4, and 19) showed strong activity against PC3, the IC(50) values being in the range 0.55-10microM, whereas compound 17 showed strong activity against MDA-MB-231 (IC(50) 10.4microM).     

CXCL8((3-73))K11R/G31P antagonizes ligand binding to the neutrophil CXCR1 and CXCR2 receptors and cellular responses to CXCL8/IL-8

We recently reported that CXCL8((3-73))K11R is a high affinity agonist of neutrophil activation and chemotactic responses. In this report we employed CXCL8((3-73))K11R as a template to generate CXCL8/IL-8 analogues with antagonist activities, using site-directed mutagenesis to introduce conservative amino acid substitutions into the first turn within the molecule's beta-pleated sheet region (G31P, P32G) and, in association with these, into the putative receptor-recognition site (T12S, H13F, F17S). We then examined their impact on the analogues' biological activities and found that a G31P substitution rendered CXCL8((3-73))K11R a high affinity antagonist of CXCL8/IL-8. The ranking (in the order of decreasing CXCL8/IL-8 antagonist activities) of the CXCL8((3-73))K11R analogues we generated was, G31P>T12S/G31P>H13F/G31P>T12S/H13F/G31P>P32G approximately T12S/P32G approximately H13F/P32G>T12S/H13F/P32G; CXCL8((3-73))K11R/F17S did not inhibit CXCL8/IL-8-dependent responses. CXCL8((3-73))K11R/G31P had no discernible agonist (beta-glucuronidase release, chemotactic) activity, but at 12.5 ng/ml it bound to purified neutrophils more avidly than did 1.25 microg/ml CXCL8/IL-8. Furthermore, CXCL8((3-73))K11R/G31P competitively antagonized the binding of CXCR1- and CXCR2-specific antibodies to these receptors. Taken together, these data thus provide further impetus to the study of the potential efficacy of CXCL8((3-73))K11R/G31P as a broad-spectrum antagonist of the ELR-CXC chemokines in experimental and clinical settings.     

Differential effects of (R)-, (R, S)- and (S)-8-hydroxy-2-(di-n-propylamino)tetralin on hippocampal serotonin release and induction of hypothermia in awake rats

The effects of (R)- and (S)-optical isomers of 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT) and of the racemate (R,S)-8-OH-DPAT on serotonin (5-HT) release in the ventral hippocampus of awake rats and on induction of the whole-body hypothermia were studied. Extracellular 5-HT levels were determined by a newly developed high-sensitive HPLC method based on derivatization with benzylamine and fluorescence detection. The basal levels of 5-HT in 20 min microdialysates from rats perfused with Ringer solution or with Ringer solution containing 1 microM citalopram were 6.3 +/- 1.3 fmol/20 microl and 36.1 +/- 4.2 fmol/20 microl (n=20), respectively. The reduction of hippocampal 5-HT levels induced by subcutaneous (s.c.) administration of (R,S)-8-OH-DPAT (0.3 mg/kg) was significantly attenuated by the presence of 5-HT reuptake inhibitor citalopram in Ringer solution only at its peak value at 40 min (maximal reduction to 60% compared to 46% of control values in Ringer-perfused rats), whereas the overall effects were comparable at both experimental conditions. Injection of (R)-8-OH-DPAT (0.3 mg/kg s.c.) caused further reduction of 5-HT levels, to 49% and 41%, respectively, whereas (S)-8-OH-DPAT (0.3 mg/kg s.c.) caused maximal reduction of 5-HT levels only to 74% of controls in both perfusion groups. Similar pattern and time-courses were observed in rats with hypothermia induced by injection of 8-OH-DPAT enantiomers, where (R,S), (R)-forms were about two-times more potent than the (S)-isomer. It is concluded that the acute systemic dose of (R)-, (S)- and (R,S)-8-OH-DPAT enantiomers exerted enantiomer-specific effects on 5-HT(1A) receptor-mediated function both at the presynaptic and postsynaptic sites as revealed by monitoring hippocampal 5-HT levels and body temperature.     

Synthesis of 17 beta-[(1S)-1-hydroxy-2-propynyl]- and 17 beta-[(1R)-1-hydroxy-2-propynyl]androst-4-en-3-one. Potential suicide substrates of 20 alpha- and 20 beta-hydroxysteroid dehydrogenases.

The title compounds have been synthesized for evaluation as potential suicide substrates of 20 alpha- and 20 beta-hydroxysteroid dehydrogenases. Synthesis was achieved by the following route. Acetylenedimagnesium bromide was reacted with 3 beta-hydroxyandrost-4-ene-17 beta-carboxaldehyde to give 17 beta-[(1R,S)-1-hydroxy-2-propynyl] androst-4-en-3 beta-ol. Separation of the R and S diols was achieved by HPLC (high pressure liquid chromatography). Selective oxidation of the 3 beta-hydroxyl group with Jones reagent at 0 degrees gave the title compounds. Further oxidation with Jones reagent converted each acetylenic alcohol to the conjugated acetylenic ketone, 17 beta-(1-oxo-2-propynyl)androst-4-en-3-one.     

Arachidonate 12-lipoxygenase purified from porcine leukocytes by immunoaffinity chromatography and its reactivity with hydroperoxyeicosatetraenoic acids

Arachidonate 12-lipoxygenase was purified to near homogeneity from the cytosol fraction of porcine leukocytes by ammonium sulfate fractionation, DEAE-cellulose chromatography, and immunoaffinity chromatography using a monoclonal antibody against the enzyme. The purified enzyme was unstable (half-life of about 24 h at 4 degrees C) but was markedly protected from the inactivation by storage in the presence of ferrous ion or in the absence of air. The lag phase which was observed before the start of the enzyme reaction was abolished by the presence of 12-hydroperoxy-5,8,10,14-eicosatetraenoic acid. An apparent substrate inhibition was observed with arachidonic acid and other active substrates; however, the substrate concentration curve was normalized by the presence of 0.03% Tween 20. Arachidonic acid was transformed to the omega-9 oxygenation product 12-hydroperoxy-5Z,8Z,10Z,14Z-eicosatetraenoic acid. C-12 oxygenation also occurred with 5-hydroxy- and 5-hydroperoxyeicosatetraenoic acids; the respective maximal velocities were 60 and 150% of the rate with arachidonic acid. Octadecaenoic acids were also good substrates. gamma-Linolenic acid was oxygenated in the omega-9 position (C-10), while linoleic and alpha-linolenic acids were subject to omega-6 oxygenation (C-13). A far more complex reaction was observed using 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid as substrate. Reaction occurred at 70% of the rate with arachidonic acid. The dihydroperoxy and dihydroxy products were identified by their UV absorption spectra, high performance liquid chromatography, and gas chromatography-mass spectrometry. Among these products, (8S,15S)-dihydroperoxy-5Z,9E,11Z,13E-eicos atetraenoic acid and (14R,15S)-erythro-dihydroperoxy-5Z,8Z,10E, 12E-eicosatetraenoic acid were produced in larger amounts than the (8R)- and (14S,15S)-threo isomers, respectively; these products were attributed to 8- and 14-oxygenation of the 15-hydroperoxy acid. Furthermore, formation of 14,15-leukotriene A4 was inferred from the characteristic pattern of its hydrolysis products comprised of equal amounts of (8R,15S)- and (8S,15S)-dihydroxy-5Z,9E,11E,13E-eicosatetraenoi c acids together with smaller amounts of (14R,15S)-erythro- and (14S,15S)-threo-dihydroxy-5Z,8Z,10E,12E-eicosate traenoic acids. Thus, both lipoxygenase and leukotriene synthase activities were demonstrated with the homogeneous preparation of porcine leukocyte 12-lipoxygenase.     

11(R),12(S),15(S)-trihydroxyeicosa-5(Z),8(Z),13(E)-trienoic acid: an endothelium-derived 15-lipoxygenase metabolite that relaxes rabbit aorta

Previous studies indicate that 11,12,15-trihydroxyeicosatrienoic acid (11,12,15-THETA), an endothelium-derived hyperpolarizing factor in the rabbit aorta, mediates a portion of the relaxation response to acetylcholine by sequential metabolism of arachidonic acid by 15-lipoxygenase, hydroperoxide isomerase, and epoxide hydrolase. To determine the stereochemical configuration of the endothelial 11,12,15-THETA, its activity and chromatographic migration were compared with activity and migration of eight chemically synthesized stereoisomers of 11,12,15(S)-THETA. Of the eight isomers, only 11(R),12(S),15(S)-trihydroxyeicosa-5(Z),8(Z),13(E)-trienoic acid comigrated with the biological 11,12,15-THETA on reverse- and normal-phase HPLC and gas chromatography. The same THETA isomer (10(-7)-10(-4) M) relaxed the rabbit aorta in a concentration-related manner (maximum relaxation = 69 +/- 5%). These relaxations were blocked by apamin (10(-7) M), an inhibitor of small-conductance Ca2+-activated K+ channels. In comparison, 11(S),12(R),15(S),5(Z),8(Z),13(E)-THETA (10(-4) M) relaxed the aorta by 22%. The other six stereoisomers were inactive in this assay. With use of the whole cell patch-clamp technique, it was shown that 10(-4) M 11(R),12(S),15(S),5(Z),8(Z),13(E)-THETA increased outward K+ current in isolated aortic smooth muscle cells by 119 +/- 36% at +60 mV, whereas 10(-4) M 11(R),12(R),15(S),5(Z),8(Z),13(E)-THETA increased outward K+ current by only 20 +/- 2%. The 11(R),12(S),15(S),5(Z),8(Z),13(E)-THETA-stimulated increase in K+ current was blocked by pretreatment with apamin. These studies suggest that 11(R),12(S),15(S)-trihydroxyeicosa-5(Z),8(Z),13(E)-trienoic acid is the active stereoisomer produced by the rabbit aorta. It relaxes smooth muscle by activating K+ channels. The specific structural and stereochemical requirements for K+ channel activation suggest that a specific binding site or receptor of 11,12,15-THETA is involved in these actions.     

Synthesis and biological evaluation in vitro of selective,high affinity peptide antagonists of human melanin-concentrating hormone action at human melanin-concentrating hormone receptor 1

Human melanin-concentrating hormone (hMCH) and many of its analogues are potent but nonspecific ligands for human melanin-concentrating hormone receptors 1 and 2 (hMCH-1R and hMCH-2R). To differentiate between the physiological functions of these receptors, selective antagonists are needed. In this study, analogues of Ac-Arg(6)-cyclo(S-S)(Cys(7)-Met(8)-Leu(9)-Gly(10)-Arg(11)-Val(12)-Tyr(13)-Arg(14)-Pro(15)-Cys(16))-NH(2), a high affinity but nonselective agonist at hMCH-1R and hMCH-2R, were prepared and tested in binding and functional assays on cells expressing these receptors. In the new analogues, 5-aminovaleric acid (Ava) was incorporated in place of the Leu(9)-Gly(10) and/or Arg(14)-Pro(15) segments of the disulfide ring. Several of these compounds turned out to be high affinity antagonists selective for hMCH-1R. Moreover, even at micromolar concentrations, they were devoid of agonist potency at both hMCH receptors and not effective as hMCH-2R antagonists. For example, peptide 14, Gva(6)- cyclo(S-S)(Cys(7)-Met(8)-Leu(9)-Gly(10)-Arg(11)-Val(12)-Tyr(13)-Ava(14,15)-Cys(16))-NH(2), (Gva = 5-guanidinovaleric acid), was a full competitive hMCH-1R antagonist (IC(50) = 14 nM, K(B) = 0.9 nM) with more than 1000-fold selectivity over hMCH-2R. Examination of various compounds with Ava in positions 9,10 and/or 14,15 revealed that the Leu(9)-Gly(10) and Arg(14)-Pro(15) segments of the disulfide ring are the principal structural elements determining hMCH-1R selectivity and ability to act as a hMCH-1R antagonist.     

Microbiological degradation of bile acids. The preparation of some hypothetical metabolites involved in cholic acid degradation.

1. To identify the intermediates involved in the degradation of cholic acid, the further degradation of (4R)-4-[4alpha-(2-carboxyethyl)-3aalpha-hexahydro-7abeta-methyl-5-oxoindan-1beta-yl]valeric acid (IVa) by Arthrobacter simplex was attempted. The organism could not utilize this acid but some hypothetical intermediate metabolities of compound (IVa) were prepared for later use as reference compounds. 2. The nor homologue (IIIa) and the dinor homologue (IIIb) of compound (IVa) were prepared by exposure of 3-oxo-24-nor-5beta-cholan-23-oic acid (I) and (20S)-3beta-hydroxy-5-pregnene-20-carboxylic acid (II) to A. simplex respectively. These compounds correspond to the respective metabolites produced by the shortening of the valeric acid side chain of compound (IVa) in a manner analogous to the conventional fatty acid alpha- and beta-oxidation mechanisms. Their structures were confirmed by partial synthesis. 3. The following authentic samples of reduction products of the oxodicarboxylic acids (IIIa), (IIIb) and (IVa) were also synthesized as hypothetical metabolities: (4R)-4-[3aalpha-hexahydro-5alpha-hydroxy-4alpha-(3-hydroxypropyl)-7abeta-methylindan-1beta-yl]valeric acid (Vb) and its nor homologue (VIIa) and dinor homologue (IXa);(4R)-4-[3Aaalpha-hexahydro-5alpha-hydroxy-4alpha-(3-hydroxypropyl)-7abeta-methylindan-1beta-yl]-pentan-1-ol (Vc); and their respective 5beta epimers (Ve), (VIIc), (IXc) and (Vf). 4. In connexion with the non-utilization of compound (IVa) by A. simplex, the possibility that not all the metabolites formed from cholic acid by a certain micro-organism can be utilized by the same organism is considered.