A New triterpenoid in the fruits of Phellodendron chinense Schneid

A new triterpenoid, phellochin, C31H52O4 (HRMS 488.3747), mp. 173–175℃, [α]25D -84.8 (C=0.10, methanol), was isolated from the fruits of Phellodendron chinense Schneid, and its structure was established by spectroscopic methods (IR, MS, NMR and COSY).     

The lactam of alpha-guanidinoglutaric acid (1-amidino-2-pyrrolidone-5-carboxylic acid)

alpha-Guanidinoglutaric acid (alpha-GGA) has been reported to occur in the cerebral cortex after epileptic seizures. No physical characteristics of alpha-GGA have been given. A practical procedure for the preparation of alpha-GGA is reported here. alpha-GGA forms a lactam in aqueous solution at 80 degrees C. It is proposed to substitute this lactam, 1-amidino-2-pyrrolidone-5-carboxylic acid (pAGlu), for pyroglutamic acid (pGlu) at the N-terminal position in neuropeptides to modify their biological characteristics. L(+)-Glutamic acid was reacted with S-methylisothiourea (I) at pH 10 in aqueous solution to form L(-)-alpha-guanidinoglutaric acid: mp 165-168 degrees C, [alpha]22D = -22.7 (C = 4, 2 M HCl). alpha-GGA reacted promptly with excess reagent to form a salt, S-methylisothiourea-alpha-guanidinoglutarate: mp 209-210 degrees C, [alpha]22D = -13.0 (C = 4, 2 M HCl). I was removed from the salt with aqueous picric acid, since I readily formed an insoluble picrate, S-methylisothiourea picrate (mp 225-228 degrees C). Alternatively, the salt was added to a cation exchange column, and the alpha-GGA was eluted with molar ammonium acetate buffer, pH 9.5. Its lactam, 1-amidino-2-pyrrolidone-5-carboxylic acid, mp 248-249 degrees C, [alpha]22D = +2.1 (C = 4, 2 M HCl), formed a picrate (mp 196-199 degrees C).     

A new crystal form of beta-cyclodextrin-ethanol inclusion complex: channel-type structure without long guest molecules

A new crystal form of beta-cyclodextrin (beta-CD)[bond]ethanol[bond]dodecahydrate inclusion complex [(C(6)H(10)O(5))(7).0.3C(2)H(5)OH.12H(2)O] belongs to monoclinic space group C2 (form II) with unit cell constants a=19.292(1), b=24.691(1), c=15.884(1) A, beta=109.35(1) degrees. The beta-CD macrocycle is more circular than that of the complex in space group P2(1) [form I: J. Am. Chem. Soc. 113 (1991) 5676]. In form II, a disordered ethanol molecule (occupancy 0.3) is placed in the upper part of beta-CD cavity (above the O-4 plane) and is sustained by hydrogen bonding to water site W-2. In form I, an ethanol molecule located below the O-4-plane is well ordered because it hydrogen bonds to surrounding O-3[bond]H, O-6[bond]H groups of the symmetry-related beta-CD molecules. In the crystal lattice of form I, beta-CD macrocycles are stacked in a typical herringbone cage structure. By contrast, the packing structure of form II is a head-to-head channel that is stabilized at both O-2/O-3 and O-6 sides of each beta-CD by direct O(CD)...O(CD) and indirect O(CD)...O(W)...(O(W))...O(CD) hydrogen bonds. The 12 water molecules are disordered in 18 positions both inside the channel-like cavity of beta-CD dimer (W-1[bond]W-6) and in the interstices between the beta-CD macrocycles (W-7[bond]W-18). The latter forms a cluster that is hydrogen bonded together and to the neighboring beta-CD O[bond]H groups.     

PGR-08理化性质及生物活性的研究

PGR-0 8是从某种土壤放线菌 (Actinomyces)的发酵液中分离提纯得到的白色结晶 ,纸层析和熔点检测证明它是化学单体 ,mp 1 80～ 1 81℃ ,[α]2 1D+ 4 3°(H2 O)。溶解性试验表明它易溶于 H2 O(≥ 6 0℃ )、HCl、Na OH、(CH3 ) 2 CO、CH3 OH和 C2 H5OH等 ,但不溶于 Na HCO3 。Tollen、Fehling和 Ag NO3 -溴酚兰等专性试剂的颜色反应表明其组成成分中有嘌呤和糖。生物试验证明 :PGR-0 8具有细胞分裂素的活性 ,例如它能减缓蒲公英离体叶圆片 Chl.的降解 ,延长叶圆片寿命 1～ 2倍以上 ,诱导叶圆片分化出不定根或不定芽 ,甚至建成小植株。它还能促进小麦种子萌发。     

Studies on the Constituents of Aristolochia versicolar S. M. Hwang

Twenty-four constituents were isolated from the root of Aristolochia versicolar S. M. Hwang (collected from Guangxi province, China). Ten of them (two novel sesquiterpene lacrones and eight known compounds) were reported recently. This paper deals with the other eight compounds. From the petroleum ether extraction a sesquiterpene lactone, C15H20O2, mp. 94–95℃ [α]D36 –49˚ (C=1.8, EtOH), confirmed to be isoaristolactone, was obtained from plants for the first time. On the basis of spectral analysis (UV, IR, 1HNMR, 13CNMR, MS) and chemical reactions its structure was determined as B2. From the petroleum enter unsoluble sec- tion 6-methoxyaristolic acid methyl ester (D1), aristolic acid (D3), 6-methoxyaristolic acid (ari- stolinic acid) (D4f), 6-methoxyaristolochic acid methyl ester (D4y), aristolochic acid A(D5), β-sitosterol-D-glucoside (D10) and allantoin (C2) were isolated and identified. Dx, Ds, and Ds show ed antifertility activity in mice.     

Novel synthesis of cholesterol analogs: condensation of pregnenolone with dihydropyran or dihydrofuran.

Pregnenolone 3-(2'-tetrahydropyranyl) ether (1) was condensed with 3,4-[2H]dihydropyran to mainly give (20R)-[6'-(3',4'-[2'H]dihydropyranyl)]-pregn-5-ene-3 beta,20-diol 3-(2'-tetrahydropyranyl) ether (20R-3), according to nuclear magnetic resonance (NMR). Cold, dilute HCl in ethanol removed the tetrahydropyranyl group at C-3 and also opened the dihydropyranyl ring at the C-20 position of 20R-3 to give (20R)-27-norcholest-5-en-22-one-3 beta,20,26-triol (20R-5). Analogous results were obtained by condensing pregnenolone 3-acetate with 3,4-[2H]dihydropyran to provide (20R)-[6'-(3',4'-[2'H]dihydropyranyl)]-pregn-5-ene-3 beta,20-diol 3-acetate (20R-4). Acid-catalyzed opening of the dihydropyranyl ring at C-20 in 20R-4 yielded 20R-7, which, on acetylation followed by crystallization, provided (20R)-27-norcholest-5-en-22-one-3 beta,20,26-triol 3,26-diacetate (20R-8), identical to the diacetate made from 20R-5. Varying the reaction sequence beginning with 20(R,S)-4 gave an 84:16 ratio of 20R to 20S in a mixture of 20(R,S)-8, according to NMR analysis. Crystallization of the mixture from methanol provided pure 20R-8. Condensing 2,3-dihydrofuran and 1 for producing (20R)-[5'-(2',3'-dihydrofuranyl)]-pregn-5-ene-3 beta,20-diol 3-(2'-tetrahydropyranyl) ether (6) gave instead (20R)-26,27-bisnorcholest-5-en-22-one-3 beta,20,25-triol 3-(2'-tetrahydropyranyl) ether (20R-9) by partial hydrolysis during workup. Treating 20R-9 briefly with dilute HCl produced (20R)-26,27-bisnorcholest-5-en-22-one-3 beta,20,25-triol (20R-10).(ABSTRACT TRUNCATED AT 250 WORDS)     

Two γ-Methyl Biosides from Dregea volubilis (L.) Benth

From the hydrolysate of the crude glycosides from the roots, of Dregea volubilis(L.) Benth in Dehong, Yunnan, two α-methyl biosides Ⅰ and Ⅱ (yields: 0.016%and 0.0097%, respectively) were isolated by silica gel column chromatography. Theirchemical structures were established by interpretation of MS, IR,1H,13C-NMR, andgas chromatographic analysis of their degradation products, and comparison of thephysical properties of Ⅰ, Ⅱ and their acetates which were reported in literatures asfollows: α-methyl-pachybioside for Ⅰ, and α-methyl-[3-O-methyl-6-deoxy-D-allose(1→4)-D-olivoside] for Ⅱ. Ⅱ named α-methyl-dredehongbioside, is reported for the first time. Ⅱ, α-methyl-dredehongbioside, colorless needles (from MeOH), bitter, mp. 184–186℃,[α]D22 +74.5˚~(c= 0.52, MeOH). Anal. Cald(%) for C14H26O8:C52.17, H8.07;Found; C52.23,H8.22. Irvmaxkbr: 3370, 1443, 1419, 1375, 1268, 1218, 1168,1127,1060cm-1. MS(m/e,%): 322(M+,3),291(M+-OCH3,15), 273(M+-OCH3-H2O,12), 258,246,232, 222, 159, 145, 141, 128, 95, 87, 85, 74 (base peak, 100), 59. 1H NMRδ(CDCl3): 4.73(1H, dd, J= 4.0 Hz, J= 1.5Hz, C-1-H), 4.55(1H, d, J= 8.0Hz,C-1′-H), 3.79(1H, dd, J=3.0Hz, J= 3.0Hz, C-3′-H), 3.00(1H, dd, J= 9.0Hz,J= 9.0Hz, C-4-H), 2.22(1H, m, C-2-Ha), 1.60(1H, m, C-2-He), 1.33(3H, d,J= 6.0Hz,C-5-CH,), 1.31(3H,d,,J= 6.5Hz, C-5′-CH), 3.68(3H,s,,C-3′-OCH),3.31(3H,s,C-1-OCH). 13C NMR data were seen in Table 1. Ⅳ, tri-acetyl-α-methyl-dredehongbioside, colorless granular (from MeOH),mp. 135--137℃, [a]D22+ 88.2˚(c= 0.50, MeOH). MS(m/e, %): 488 (M+, 2), 388(M+-HOAc,2), 357(M+-OCH3-HOAc,33), 288, 187, 127, 116, 85, 74, 59, 43(basepeak, 100). 1H NMR,δ(CDCl3): 5.25(1H, ddd,.J= 11.0Hz, J=9.0Hz,.J= 5.5Hz,C-3-H), 4.86(1H, d, J= 8.0Hz, C-1′-H), 4.69 (1H, dd, J= 4.0Hz, J= 1.5Hz,C-1-H), 4.58(1H,m,C-2′-H),3.94(1H, dd, J= 3.0Hz,J= 3.0Hz,C-3′-H),3.64(1H,m,C-5-H), 3.22(1H,dd, J= 9.5Hz, J= 8.5Hz, C-4-H), 2.30(1H, m, C-2-Ha),1.67(1H,m,C-2-He), 1.31(3H, d, .J= 6.5Hz, C-5-CH3), 1.17(3H, d, J= 6.0Hz,C-5-CH3), 3.47(3H, s, C-3′-OCH3), 3.30(3H,s, C-1-OCH3), 2.10(6H, s, C-2′, C- 4′ -OCH3), 2.03(3H,s,C-3-OCH3).     

Identification of the cytokinins in red pine seedlings

Zeatin-9-riboside was identified in shoots and roots of Pinus resinosa by GC-MS analysis of its permethyl derivative. Based on their chromatographic properties on Sephadex LH-20 and C₁₈ HPLC, and their susceptibility to enzymatic degradation, several other cytokinins have been tentatively identified. The basic fraction of both the roots and shoots contained zeatin, whereas the shoots contained dihydrozeatin-O-glucoside and the roots contained zeatin-O-glucoside. Zeatin-9-riboside monophosphate, isopentenyladenosine monophosphate ([9R-5P]iP) and glucosyl phosphate derivatives were detected in the acidic fractions from both roots and shoots. There were equivalent amounts of [9R-5P]iP in both roots and shoots. The presence of equivalent amounts of [9R-5P]iP in both the roots and shots suggests that cytokinin biosynthesis may be occurring in both locations.Abbreviations AMP adenosine-5-monophosphate - BAP benzylaminopurine - BSA bovine serum albumin - BuOH butan-1-ol - CK cytokinin - (diH)Z dihydrozeatin - (diH OG)Z dihydrozeatin-O-glucoside - (diH OG)[9R]Z dihydrozeatin-9-riboside-O-glucoside - DW dry weight - EtOH ethanol - FW fresh weight - GC-MS gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - [9R]iP isopentenyladenosine - [9R-5P]iP isopentenyladenosine monophosphate - MeOH methanol - PVP polyvinylpyrrolidone - RFE rotary film evaporation - TEAB triethyl ammonium bicarbonate - Z zeatin - [9R]Z zeatin-9-riboside - (OG)Z zeatin-O-glucoside - [7G]Z zeatin-7-glucoside - [9R-5P]Z zeatin-9-riboside monophosphate     

Enantiomeric Neolignans and a Sesquiterpene from Solanum erianthum and Their Absolute Configuration Assignment

One pair of new C‐8–C‐3′/C‐7–O–C‐4′ linked neolignan enantiomers ( 1a / 1b ) and one new guaiane sesquiterpene ( 2 ) first featuring the 1(2),9(10)‐conjugated double bond were isolated from the stems of Solanum erianthum (Solanceae). Their structures were characterized on the basis of extensive spectroscopic analyses, especially from their 2D nuclear magnetic resonance (NMR) spectra. The absolute configurations of 1a / 1b were rigorously elucidated by electronic circular dichroism (ECD) experiments combined with the reversed helicity rule for the 2,3‐dihydrobenzo[b]furan chromophore, and compound 2 is the first report on the sterochemical assignment of a guaiane sesquiterpene by using the allylic axial chirality rule for the conjugated diene chromophore in combination with the calculated ECD spectrum. Chirality 28:259–263, 2016. © 2016 Wiley Periodicals, Inc.     

Shapes of benz[a]anthracenes: the crystal and molecular structure of 6-methylbenz[a]anthracene

The crystal structure of 6-methylbenz[a]anthracene (6-MBA), a more potent carcinogen than the other K-region monomethyl-substituted benz[a]anthracene (5-MBA), has been determined by application of direct methods to single-crystal X-ray diffractometric data and refined by least squares to R = 0.047 (Rw = 0.053). Deviations of the carbon atoms from planarity are very small with even the methyl carbon displaced by only 0.05 A from the mean molecular plane. The benzo-ring A is inclined at only about 1 1/2 degrees to each of the three rings in the anthracene moiety, i.e. 6-MBA is one of the most nearly planar benz[a]anthracenes. The K-region bond C(5)-C(6) = 1.328(6) A and two other short bonds are C(8)-C(9) = 1.341(7) and C(10)-C(11) = 1.361(7) A in the anthracene D ring.     

Regio- and stereo-selective metabolism of 4-methylbenz[a]anthracene by the fungus Cunninghamella elegans.

Metabolism of 4-methylbenz[a]anthracene by the fungus Cunninghamella elegans was studied. C. elegans metabolized 4-methylbenz[a]anthracene primarily at the methyl group, this being followed by further metabolism at the 8,9- and 10,11-positions to form trans-8,9-dihydro-8,9-dihydroxy-4-hydroxymethylbenz[a]anthracene and trans-10,11-dihydro-10,11-dihydroxy-4-hydroxymethylbenz[a]anthracene. There was no detectable trans-dihydrodiol formed at the methyl-substituted double bond (3,4-positions) or at the 'K' region (5,6-positions). The metabolites were isolated by reversed-phase high-pressure liquid chromatography and characterized by the application of u.v.-visible-absorption-, 1H-n.m.r.- and mass-spectral techniques. The 4-hydroxymethylbenz[a]anthracene trans-8,9- and -10,11-dihydrodiols were optically active. Comparison of the c.d. spectra of the trans-dihydrodiols formed from 4-methylbenz[a]anthracene by C. elegans with those of the corresponding benz[a]anthracene trans-dihydrodiols formed by rat liver microsomal fraction indicated that the major enantiomers of the 4-hydroxymethylbenz[a]anthracene trans-8,9-dihydrodiol and trans- 10,11-dihydrodiol formed by C. elegans have S,S absolute stereochemistries, which are opposite to those of the predominantly 8R,9R- and 10R,11R-dihydrodiols formed by the microsomal fraction. Incubation of C. elegans with 4-methylbenz[a]anthracene under 18O2 and subsequent mass-spectral analysis of the metabolites indicated that hydroxylation of the methyl group and the formation of trans-dihydrodiols are catalysed by cytochrome P-450 mono-oxygenase and epoxide hydrolase enzyme systems. The results indicate that the fungal mono-oxygenase-epoxide hydrolase enzyme systems are highly stereo- and regio-selective in the metabolism of 4-methylbenz[a]anthracene.     

Phospholipids chiral at phosphorus. Absolute configuration of chiral thiophospholipids and stereospecificity of phospholipase D

Separate diastereomers of 1,2-dipalmitoyl-sn-glycero-3- thiophosphoethanolamine ( DPPsE ) were prepared in 97% diastereomeric purity and characterized by 31P, 13C, and 1H nuclear magnetic resonance (NMR). The isomers hydrolyzed by phospholipases A2 and C specifically were designated as isomer B (31P NMR delta 59.13 in CDCl3 + Et3N ) and isomer A (59.29 ppm), respectively, analogous to the isomers B and A of 1,2-dipalmitoyl-sn-glycero-3- thiophosphocholine ( DPPsC ) [ Bruzik , K., Jiang , R.-T., & Tsai, M.-D. (1983) Biochemistry 22, 2478-2486]. Phospholipase D from cabbage was shown to be specific to isomer A of DPPsC in transphosphatidylation . The product DPPsE was shown to be isomer A. The absolute configuration of chiral DPPsE at phosphorus was elucidated by bromine-mediated desulfurization in H2 18O to give chiral 1,2-dipalmitoyl-sn-glycero-3-[18O]phosphoethanolamine ( [18O]DPPE) followed by 31 P NMR analysis [ Bruzik , K., & Tsai, M.-D. (1984) J. Am. Chem. Soc. 106, 747-754]. The absolute configuration of chiral DPPsC was elucidated by desulfurization in H2 18O mediated by bromine or cyanogen bromide to give chiral 1,2-dipalmitoyl-sn-glycero-3-[18O]phosphocholine ( [18O]DPPC), which was then converted to [18O]DPPE by phospholipase D with retention of configuration [ Bruzik , K., & Tsai, M.-D. (1984) Biochemistry (preceding paper in this issue)]. The results indicate that isomer A of both DPPsE and DPPsC is SP whereas isomer B is RP.     

Effect of ethanol on ATP-induced phospholipases C and D and serine base exchange in glioma C6 cells

The effect of extracellular ATP, a nucleotide receptor agonist in the central nervous system, was investigated in glioma C6 cells on the intracellular Ca2+ level and the formation of phosphatidylethanol and phosphatidic acid in the presence and absence of ethanol (150 mM). In the cells prelabeled with [14C]palmitic acid, 100 microM ATP induced both the hydrolysis and the transphosphatidylation reactions leading to the formation of [14C]phosphatidic acid; addition of ethanol generated [14C]phosphatidylethanol. However, ATP-mediated increase in the level of [14C]phosphatidic acid was not inhibited by ethanol. Furthermore, ethanol augmented ATP-induced transient and sustained increase in the intracellular Ca2+ concentration, whereas ethanol alone did not produce any change in the intracellular Ca2+ level. These results indicate that in glioma C6 cells, ATP induces activation of polyphosphoinositide-specific phospholipase C and phospholipase D and that ethanol enhances this effect. In the present investigation we have also shown that long-term (2 days) ethanol treatment, at concentration relevant to chronic alcoholism (100 mM), decreased the incorporation of [14C]serine into phosphatidylserine. Since the effect of ethanol on ATP-induced activities of phospholipase C and phospholipase D and on serine base-exchange in glioma C6 cells differs significantly from that in cultured neuronal cells, these results may contribute to a better understanding of the mechanisms of ethanol action in cells of glial origin.     

Fatty acid metabolism in Atlantic salmon (Salmo salar L.) hepatocytes and influence of dietary vegetable oil

Isolated hepatocytes from Atlantic salmon (Salmo salar), fed diets containing either 100% fish oil or a vegetable oil blend replacing 75% of the fish oil, were incubated with a range of seven (14)C-labelled fatty acids. The fatty acids were [1-(14)C]16:0, [1-(14)C]18:1n-9, 91-(14)C]18:2n-6, [1-(14)C]18:3n-3, [1-(14)C]20:4n-6, [1-(14)C]20:5n-3, and [1-(14)C]22:6n-3. After 2 h of incubation, the hepatocytes and medium were analysed for acid soluble products, incorporation into lipid classes, and hepatocytes for desaturation and elongation. Uptake into hepatocytes was highest with [1-(14)C]18:2n-6 and [1-(14)C]20:5n-3 and lowest with [1-(14)C]16:0. The highest recovery of radioactivity in the cells was found in triacylglycerols. Of the phospholipids, the highest recovery was found in phosphatidylcholine, with [1-(14)C]16:0 and [1-(14)C]22:6n-3 being the most prominent fatty acids. The rates of beta-oxidation were as follows: 20:4n-6>18:2n-6=16:0>18:1n-9>22:6n-3=18:3n-3=20:5n-3. Of the fatty acids taken up by the hepatocytes, [1-(14)C]16:0 and [1-(14)C]18:1n-9 were subsequently exported the most, with the majority of radioactivity recovered in phospholipids and triacylglycerols, respectively. The major products from desaturation and elongation were generally one cycle of elongation of the fatty acids. Diet had a clear effect on the overall lipid metabolism, with replacing 75% of the fish oil with vegetable oil resulting in decreased uptake of all fatty acids and reduced incorporation of fatty acids into cellular lipids, but increased beta-oxidation activity and higher recovery in products of desaturation and elongation of [1-(14)C]18:2n-6 and [1-(14)C]18:3n-3.     

N,N-disubstituted aminomethyl benzofuran derivatives: synthesis and preliminary binding evaluation

A series of new N-substituted 2,3-dihydro-2-aminomethyl-2H-1-benzofuran derivatives was prepared and evaluated for affinity at 5-HT1A, 5-HT2A, 5-HT2C, 5-HT3, D2, and D3 receptors. Compound 9, 8-[4-[N-propyl-N-(7-hydroxy-2,3-dihydro -2H-1-benzofuran-2-yl)methyl]aminobutyl]-8-azaspiro[4,5]decane-7,9 -dione, bound at 5-HT1A sites with nanomolar affinity (IC50= 1.5 nM) and high selectivity over 5-HT2A, 5-HT2C, 5-HT3, D2, and D3 receptors.     

Inhibitory monoclonal antibodies against rat liver alcohol dehydrogenase

Eleven hybridoma clones which secrete monoclonal antibodies against purified rat liver alcohol dehydrogenase (EC 1.1.1.1) were isolated. Antibodies (R-1-R-11) were identified by their ability to bind to immobilized pure alcohol dehydrogenase in an enzyme-linked immunoadsorbent assay, in which antibody R-9 showed the highest binding capacity. Except for R-1 and R-7, all antibodies inhibited catalytic activity of the enzyme isolated from inbred (Fischer-344) or outbred (Sprague-Dawley) strains (R-11 greater than R-9 greater than R-4 greater than R-6 greater than R-10 greater than R-8 greater than R-2 = R-3 = R-5). The inhibition of enzyme activity by antibodies was noncompetitive for ethanol and NAD+, and was dependent on antibody concentration and incubation time. Antibodies R-4, R-9, and R-11 were most effective when enzyme activity was assayed below pH 7.7-7.8, a condition thought to protonate the enzyme's active center. These three antibodies did not inhibit horse liver alcohol dehydrogenase activity, indicating their species specificity. Such antibodies will be useful to delineate structural and functional roles of rat liver alcohol dehydrogenase.     

Binding of [3H]SCH23390 in Rat Brain: Regional Distribution and Effects of Assay Conditions and GTP Suggest Interactions at a D1-Like Dopamine Receptor

The compound [9-3H]SCH23390 [R-(+)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepine-7- ol] was synthesized, and the binding of this purportedly selective antagonist of D1 3,4-dihydroxyphenylethylamine (dopamine) receptors was characterized. The regional distribution of high-affinity, specific [3H]SCH23390 binding sites in the rat brain correlated well with levels of endogenous dopamine. Receptor densities were greatest in corpus striatum, nucleus accumbens, and olfactory tubercle; intermediate levels were found in several limbic and cortical areas, whereas few sites were detectable in cerebellum, brainstem, and ol-factory bulb. Specific binding in caudate-putamen was found to be both temperature- and pH-dependent, with optima at 25-30 degrees C and pH 7.8-8.0. Scatchard or Woolf analyses of binding in caudate-putamen suggest that most of the sites are either of a single class or of classes with similar characteristics (KD = 0.7 +/- 0.1 nM; Bmax = 347 +/- 35 fmol/mg of protein). Both dopamine and cis-flupenthixol altered the slope but not the intercept of lines generated by Scatchard analysis, suggesting a competitive mode of inhibition of [3H]SCH23390 binding. Competition for binding by dopamine or the D1 agonist SKF38393 was inhibited by guanine nucleotides, whereas GTP had little effect on the competition for binding by the antagonist cis-flupenthixol. The competition for [3H]SCH23390 binding sites by dopamine was much more sensitive to GTP than was competition for [3H]spiperone binding. These data support the hypotheses that [3H]SCH23390 binds to recognition sites that differ from those previously described using other radiolabeled dopamine antagonists and that these sites have the characteristics expected of dopamine receptors.     

Stereoselective metabolism of 7-methylbenz[a]anthracene: absolute configuration of five dihydrodiol metabolites and the effect of dihydrodiol conformation on circular dichroism spectra

7-Methylbenz[a]anthracene (7-MBA) was metabolized stereoselectively by rat liver microsomes to form five optically active dihydrodiols as the predominant metabolites. The dihydrodiols were purified by a combination of reversed-phase and normal-phase high performance liquid chromatography (HPLC). By comparison of their circular dichroism (CD) spectra with the corresponding benz[a]anthracene (BA) dihydrodiols of known absolute stereochemistry, the major dihydrodiol enantiomers of 7-MBA have been determined to have 1R,2R-, 3R,4R- and 10R , 11R - absolute configurations, respectively. Due to their quasi- diaxial conformations, the absolute configuration of trans-5,6- and trans-8,9-dihydrodiols, the two most abundant metabolites of 7-MBA, could not be determined by simple comparisons of their circular dichroism spectra with those of the quasidi -equatorial BA 5R, 6R - and 8R , 9R -dihydrodiols. The major enantiomers of the quasi- diaxial trans-5,6- and trans-8,9-dihydrodiol metabolites of 7-MBA were determined by comparison to the CD spectrum of 7-bromo-BA 5R, 6R -dihydrodiol and by the exciton chirality method to have R,R absolute stereochemistry. This study also revealed that the circular dichroism Cotton effects of an enantiomeric dihydrodiol of polycyclic aromatic hydrocarbons can be drastically altered if the conformation (quasi- diaxial vs. quasi di-equatorial ) of the dihydrodiol is changed.     

Design of oxytocin antagonists, which are more selective than atosiban.

We report the solid phase synthesis of four pairs of L- and D-thienylalanine (Thi/D-Thi) position two modified analogues of the following four oxytocin (OT) antagonists: des-9-glycinamide [1-(beta-mercapto-beta,beta-pentamethylene propionic acid), 2-O-methyltyrosine, 4-threonine]ornithine-vasotocin (desGly(NH2)9,d (CH2)5[Tyr(Me)2,Thr4]OVT) (A); the Tyr-(NH2)9 analogue of (A), d(CH2)5[Tyr(Me)2,Thr4,Tyr-(NH2)9]OVT (B); the Eda9 analogue (where Eda = ethylenediamine) of (A), d(CH2)5[Tyr(Me)2, Thr4, Eda9]OVT (C); and the retro Tyr10 modified analogue of (C), d(CH2)5[Tyr(Me)2, Thr4, Eda9<--Tyr10]OVT (D). The eight new analogues of A-D are (1) desGly(NH2),d(CH2)5[Thi2,Thr4]OVT, (2) desGly(NH2),d(CH2)5[D-Thi2,Thr4]OVT, (3) d(CH2)5[Thi2, Thr4,Tyr-(NH2)9]OVT, (4) d(CH2)5[D-Thi2,Thr4,Tyr-(NH2)9]OVT (5) d(CH2)5[Thi2,Thr4Eda9]OVT, (6) d(CH2)5[D-Thi2,Thr4,Eda9]OVT, (7) d(CH2) [Thi2,Thr4,Eda9<--Tyr10]OVT, (8) d(CH2),[D-Thi2,Thr4,Eda9<--Tyr10]OVT. We also report the synthesis of (C). Peptides 1-8 and C were evaluated for agonistic and antagonistic activities in in vitro and in vivo OT assays, in in vivo vasopressor (V1a receptor) assays and in in vivo antidiuretic (V2 receptor) assays. None of the eight peptides nor C exhibit oxytocic or vasopressor agonism. Peptides 1-8 are extremely weak V2 agonists (antidiuretic activities range from < 0.0005 to 0.20 U/mg). Peptide C is a weak mixed V2 agonist/antagonist. Peptides 1-8 and C exhibit potent in intro (no Mg2+) OT antagonism (anti-OT pA2 values range from 7.76 to 8.05). Peptides 1-8 are all OT antagonists in vivo (estimated in vivo anti-OT pA2 values range from 6.54-7.19). With anti-V1a pA2 values of approximately 5-5.80, peptides 1-8 exhibit marked reductions in anti-V1a potencies relative to those of the parent peptides A-D (anti-V1a pA2 range from 6.48 to 7.10) and to l-deamino[D-Tyr(Et)2, Thr4]OVT (Atosiban, trade name Tractocile) (anti-V1a pA2-6.14). Atosiban has recently been approved in Europe for clinical use for the prevention of premature labour (Pharm. J. 264(7-100): 871). Peptides 1-8 exhibit striking gains in in vitro anti-OT/anti-V1a selectivities with respect to the parent peptides A, B, C and D and to Atosiban. Peptides 1-8 exhibit anti-OT (in vitro)/anti-V1a selectivities of 450, 525, 550, 450, approximately 1080, 116, 355, 227 respectively. The corresponding values for A-D and Atosiban are 30, 4.2, 4.3, 2.6 and 37. With the exception of peptide 6, the remaining seven peptides exhibit 3-18-fold gains in anti-OT (in vivo)/anti-V1a selectivity with respect to Atosiban, peptides 1-8 exhibit anti-OT (in vivo)/anti-V1a selectivities of 22, approximately 82, approximately 82, 147, approximately 83, 11, 31 and 42. By comparison, Atosiban exhibits an anti-OT (in vivo)/anti-V1a selectivity = 8. With an estimated in vivo anti-OT pA2 value = 7.19+/-0.06, peptide 4 is equipotent with Atosiban (pA2 = 7.05+/-0.05). However, with its significantly reduced anti-vasopressor potency, pA2 = approximately 5, it is approximately 18 times more selective for OT receptors with respect to VP V1a receptors than Atosiban. Since we have shown that V1a antagonism could be an unwanted side-effect in tocolytics, peptide 4 and some of the OT antagonists reported here have advantages over Atosiban and thus may be suitable candidates for evaluation as potential tocolytic agents for the treatment of preterm labour.     

Structure and characterization of platinum(II) and platinum(IV) complexes with protonated nucleobase ligands

The reaction of H₂[PtCl₆] · 6H₂O and (H₃O)[PtCl₅(H₂O)] · 2(18C6) · 6H₂O (18C6 = 18-crown-6) with 9-methylguanine (MeGua) proceeded with the protonation of MeGua forming 9-methylguaninium hexachloroplatinate(IV) dihydrate (MeGuaH)₂[PtCl₆] · 2H₂O (1).The same compound was obtained from the reaction of Na₂[PtCl₆] with (MeGuaH)Cl.On the other hand, the reaction of guanosine (Guo) with (H₃O)[PtCl₅(H₂O)] · 2(18C6) · 6H₂O in methanol at 60 °C proceeded with the cleavage of the glycosidic linkage and with ligand substitution to give a guaninium complex of platinum(IV), [PtCl₅(GuaH)] · 1.5(18C6) · H₂O (2).Within several weeks in aqueous solution a slow reduction took place yielding the analogous guaninium platinum(II) complex, [PtCl₃(GuaH)] · (18C6) · 2Me₂CO (3).H₂[PtCl₆] · 6H₂O and guanosine was found to react in water, yielding (GuoH)₂[PtCl₆] (4) and in ethanol at 50 °C, yielding [PtCl₅(GuoH)] · 3H₂O (5).Dissolution of complexes 2 and 5 in DMSO resulted in the substitution of the guaninium and guanosinium ligands, respectively, by DMSO forming [PtCl₅(DMSO)]⁻.Reactions of 1-methylcytosine (MeCyt) and cytidine (Cyd) with H₂[PtCl₆] · 6H₂O and(H₃O)[PtCl₅(H₂O)] · 2(18C6) · 6H₂O resulted in the formation of hexachloroplatinates with N3 protonated pyrimidine bases as cation (MeCytH)₂[PtCl₆] · 2H₂O (6) and (CydH)₂[PtCl₆] (7), respectively. Identities of all complexes were confirmed by ¹H, ¹³C and ¹⁹⁵Pt NMR spectroscopic investigations, revealing coordination of GuoH⁺ in complex 5 through N7 whereas GuaH⁺ in complex 3 may be coordinated through N7 or through N9. Solid state structure of hexachloroplatinate 1 exhibited base pairing of the cations yielding (MeGuaH⁺)₂, whereas in complex 6 non-base-paired MeCytH⁺ cations were found. In both complexes, a network of hydrogen bonds including the water molecules was found. X-ray diffraction analysis of complex 3 exhibited a guaninium ligand that is coordinated through N9 to platinum and protonated at N1, N3 and N7. In the crystal, these NH groups form hydrogen bonds N–HO to oxygen atoms of crown ether molecules.