Norepinephrine release from the ischemic heart is greatly enhanced in mice lacking histamine H3 receptors

We previously reported that histamine H(3) receptors (H(3)Rs) are present in cardiac sympathetic nerve endings (cSNE) of animals and humans, where they attenuate norepinephrine (NE) release in normal and hyperadrenergic states, such as myocardial ischemia. The recent creation of a transgenic line of mice lacking H(3)R provided us with the opportunity to assess the relevance of H(3)R in the ischemic heart. We isolated SNE from hearts of wild-type (H(3)R(+/+)) and knockout (H(3)R(-/-)) mice and found that basal NE release from H(3)R(-/-) cSNE was approximately 60% greater than that from H(3)R(+/+) cSNE. NE exocytosis evoked by K(+)-induced depolarization of cSNE from H(3)R(+/+) mice was attenuated by activation of either H(3)R or adenosine A(1) receptors (A(1)R). In contrast, NE release from cSNE of H(3)R(-/-) was unaffected by H(3)R agonists, but it was still attenuated by A(1)R activation. When isolated mouse hearts were subjected to ischemia for 20 min, NE overflow into the coronaries was 2-fold greater in the H(3)R(-/-) hearts than in those from H(3)R(+/+) mice. Furthermore, whereas stimulation of H(3)R or A(1)R reduced ischemic NE overflow from H(3)R(+/+) hearts by 50%, only A(1)R, but not H(3)R activation, reduced NE release in H(3)R(-/-). Our data demonstrate that NE release from cSNE can be modulated by various heteroinhibitory receptors (e.g., H(3)R and A(1)R) and that H(3)Rs are particularly important in modulating NE release in myocardial ischemia. Inasmuch as excessive NE release is clinically recognized as a major cause of arrhythmic cardiac dysfunction, our findings reveal a significant cardioprotective role of H(3)R on cSNE.     

A1 receptor deficiency causes increased insulin and glucagon secretion in mice

Adenosine influences metabolism and the adenosine receptor antagonist caffeine decreases the risk of type 2 diabetes. In this study the metabolic role of one adenosine receptor subtype, the adenosine A(1)R, was evaluated in mice lacking this receptor [A(1)R (-/-)]. The HbA1c levels and body weight were not significantly different between wild type [A(1)R (+/+)] and A(1)R (-/-) mice (3-4 months) fed normal lab chow. At rest, plasma levels of glucose, insulin and glucagon were similar in both genotypes. Following glucose injection, glucose tolerance was not appreciably altered in A(1)R (-/-) mice. Glucose injection induced sustained increases in plasma insulin and glucagon levels in A(1)R (-/-) mice, whereas A(1)R (+/+) control mice reacted with the expected transient increase in insulin and decrease in glucagon levels. Pancreas perfusion experiments showed that A(1)R (-/-) mice had a slightly higher basal insulin secretion than A(1)R (+/+) mice. The first phase insulin secretion (initiated with 16.7 mM glucose) was of the same magnitude in both genotypes, but the second phase was significantly enhanced in the A(1)R (-/-) pancreata compared with A(1)R (+/+). Insulin- and contraction-mediated glucose uptake in skeletal muscle were not significantly different between in A(1)R (-/-) and A(1)R (+/+) mice. All adenosine receptors were expressed at mRNA level in skeletal muscle in A(1)R (+/+) mice and the mRNA A(2A)R, A(2B)R and A(3)R levels were similar in A(1)R (-/-) and A(1)R (+/+) mice. In conclusion, the A(1)R minimally affects muscle glucose uptake, but is important in regulating pancreatic islet function.     

(R)- and (S)-5,6,7,8-tetrahydro-1-hydroxy-N,N-dipropyl-9H-benzocyclohepten- 8-ylamine. Stereoselective interactions with 5-HT1A receptors in the brain

The enantiomers of 5,6,7,8-tetrahydro-1-hydroxy-N,N-dipropyl-9H-benzocyclohepten-8-++ +ylamine (3) have been synthesized and evaluated for central 5-hydroxytryptamine (5-HT) and dopamine (DA) receptor activity by use of behavioral and biochemical tests in rats. In addition, the ability of the compounds to displace [3H]-8-OH-DPAT from 5-HT1A binding sites was evaluated. The absolute configuration of the enantiomers of 3 was determined indirectly by X-ray diffraction of (+)-(8R,alpha R)-5,6,7,8-tetrahydro-1-methoxy-N-(alpha-phenethyl)-9H- benzocyclohepten-8-ylamine hydrochloride (9.HCl), a resolved synthetic precursor. The stereoselectivity of the interaction of 3 with 5-HT1A receptors was more pronounced than that of 8-hydroxy-2-(dipropylamino)tetralin (1; 8-OH-DPAT); only (R)-3 displayed 5-HT activity. However, (R)-3 was of lower potency than any of the enantiomers of 1. The enantiomer (S)-3, which was found to be inactive as a 5-HT-receptor agonist, appeared to be a weakly potent DA-receptor agonist whereas (R)-3 seemed to be devoid of dopaminergic activity. The conformational preferences of 3 were studied by use of NMR spectroscopy and molecular mechanics calculations. Preferred conformations of (R)-3 are similar in shape to those of the stereoselective 5-HT1A-receptor agonist (2R,3S)-8-hydroxy-3-methyl-2-(dipropylamino)tetralin.     

Two new monoterpene glycosides and a new (+)-jasmololone glucoside from Bidens parviflora Willd

Two new monoterpene glycosides named bidensmenthosides A, B and a new (+)-jasmololone glucoside, were isolated from the air-dried whole plant of Bidens parviflora Willd. Their structures were determined as (1S, 3S, 4R)-3-hydroxy-p-menth-6-one 3-O-beta-D-glucopyranoside (1), (3R, 4R)-3-hydroxy-p-menth-1 (2)-en-6-one 3-O-beta-D-glucopyranoside (2) and (4R)-hydroxy-3-methyl-2-(2 Z-pentenyl)-cyclopent-2-enone 4-O-beta-D-glucopyranoside (3) based on spectroscopic analysis and physiochemical properties, respectively. The bidensmenthosides A, B and aglycone of 3 were found to reduce 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radicals.     

Norsesquiterpenoid glucosides and a rhamnoside of pyrrolizidine alkaloid from Tephroseris kirilowii

Four new glycosylated compounds have been isolated from the whole plant of Tephroseris kirilowii, including (-)-(1R,5R,6S,7R,8S)-8-O-beta-D-glucopyranosyloxy-7-hydroxy-6-(2-hydroxypropan-2-yl)-9-methylenebicyclo[4.3.0]non-3-one (tephroside A, 1), (-)-(1R,5R,6R,8R)-6-(2-O-beta-D-glucopyranosyloxypropan-2-yl)-8-hydroxy-9-methylenebicyclo[4.3.0]non-3-one (tephroside B, 2), thesinine-4'-O-alpha-L-rhamnoside (3), and p-coumaric acid 4-O-alpha-L-rhamnoside (4), together with the known roseoside. The structures of the new compounds were established by means of spectroscopic analysis.     

Pyrimidine derivatives. 4. Synthesis and antihypertensive activity of 4-amino-2-(4-cinnamoylpiperazino)-6,7-dimethoxyquinazoline derivatives

A series of 30 4-amino-2-(4-cinnamoylpiperazino)-6,7-dimethoxyquinazoline derivatives was prepared and tested for their ability to reduce blood pressure in conscious, spontaneously hypertensive rates (SHR). A number of these compounds, notably 4-amino-2-(4-cinnamoylpiperazino)-6,7-dimethoxyquinazolines 3a (R1 = H; R2 = Ph), 3j (R1 = H; R2 = 4-EtOPh), and 5a (R1 = H; R2 = 2-furyl), showed activity at oral doses of 0.3-10 mg/kg. The effects of the 4-substituents of the piperazino group on activity are discussed. Compounds 3a, 3j, and 5a were effective in renal hypertensive rats at oral doses of 3 and 10 mg/kg and showed alpha-adrenoceptor blocking effects in isolated aortas of rats. A 5-day consecutive oral administration of 3a and 3j in SHR did not lead to development of tolerance.     

CYP3A induction by N-hydroxyformamide tumor necrosis factor-alpha converting enzyme/matrix metalloproteinase inhibitors use of a pregname X receptor activation assay and primary hepatocyte culture for assessing induction potential in humans.

A series of N-hydroxyformamide tumor necrosis factor-alpha converting enzyme (TACE)/matrix metalloprotease (MMP) inhibitors were evaluated for their potential to induce human cytochrome P450 3A (CYP3A). Two in vitro assays were used: 1) a cell-based reporter gene assay for activation of the pregnane X receptor (PXR), and 2) a primary "sandwich" culture of human hepatocytes. Approximately 50 TACE/MMP inhibitors were evaluated in the human PXR assay. A range of PXR activation was observed, 0 to 150% of the activation of the known human CYP3A inducer rifampicin. Three TACE/MMP inhibitors were evaluated in rat and human hepatocytes. Significantly higher PXR activation/CYP3A induction was observed in PXR/hepatocyte models, respectively, for (2R,3S) 3-(formyl-hydroxyamino)-2-(2-methyl-1-propyl)-4-methylpentanoic acid [(1S,2S)-2-methyl-1-(2-pyridylcarbamoyl)-1-butyl]amide (GW3333) compared with (2R,3S)-6,6,6-trifluoro-3-[formyl(hydroxy)amino]-2-isobutyl-N-[(1S,2R)-2-methoxy-1-[(1,3-thiazol-2-ylamino)carbonyl]propyl]hexanamide (GW6495) and (2R)-N-[(1S)-2,2-dimethyl-1-[(methylamino)carbonyl]-propyl]-2-[(1S)-1-[formyl(hydroxy)amino]ethyl]-5-phenylpentanamide (GI4023). The CYP3A induction level achieved with GW3333 at a concentration of approximately 10 microM in human hepatocytes was comparable to that achieved with rifampicin at a concentration of 10 microM. The extent of rodent CYP3A induction caused by GW3333 was confirmed in vivo after daily oral administration for 14 days to rats. In conclusion, GW3333 is a potential inducer of CYP3A expression in vivo in humans, but other N-hydroxyformamides are less likely to induce CYP3A.     

Mutations of arginine 222 in pre-transmembrane domain I of mouse 5-HT(3A) receptor abolish 20(R)- but not 20(S)-ginsenoside Rg(3) inhibition of 5-HT-mediated ion currents

Ginsenosides, active ingredients of Panax ginseng, exist as stereoisomers depending on the position of the hydroxyl group on carbon-20; i.e. 20(R)-ginsenoside and 20(S)-ginsenoside are epimers. We previously investigated the structure-activity relationship of the ginsenoside Rg(3) stereoisomers, 20-R-protopanaxatriol-3-[O-beta-D-glucopyranosyl (1-->2)-beta-glucopyranoside], (20(R)-Rg(3)) and 20-S-protopanaxatriol-3-[O-beta-D-glucopyranosyl (1-->2)-beta-glucopyranoside], (20(S)-Rg(3)) in regulating 5-HT(3A) receptor-mediated ion currents (I(5-HT)) expressed in Xenopus oocytes and found that 20(S)-Rg(3) rather than 20(R)-Rg(3) was more stronger inhibitor of I(5-HT). In the present study, we further investigated the effects of 20(R)-Rg(3) and 20(S)-Rg(3) on mouse 5-HT(3A) receptor channel activity after site-directed mutations of 5-HT(3A) receptor facilitation site, which is located at pre-transmembrane domain I (pre-TM1). 5-HT(3A) receptor was expressed in Xenopus oocytes, and I(5-HT) was measured using two-electrode voltage clamp technique. In wild-type, both 20(R)-Rg(3) and 20(S)-Rg(3) inhibited I(5-HT) with concentration-dependent and reversible manner. Induction of 5-HT(3A) receptor facilitation by point mutations of pre-TM1 amino acid residue R222 to R222A, R222D, R222E or R222T not only decreased EC(50) values for I(5-HT) compared to wild-type but also abolished 20(R)-Rg(3)-induced inhibition of I(5-HT). Those mutations also shifted the IC(50) values by 20(S)-Rg(3) into right direction by 2- to 4-folds compared with wild-type. These results indicate that 5-HT(3A) receptor facilitation differentially affects 20(R)-Rg(3)- and 20(S)-Rg(3)-mediated 5-HT(3A) receptor channel regulation.     

a-glucosidase Inhibitors From Paraguayan Natural Medicine, Ñangapiry,The Leaves Of Eugenia Uniflora

The water-soluble extract from a Paraguayan natural medicine, Nangapiry, the leaves of Eugenia uniflora L. (Myrtaceae), which has been used as an antidiabetic agent, was found to show inhibitory activities on the increase of plasma glucose level in the sucrose tolerance test (STT) conducted with mice. The portion adsorbed on a cation exchange resin was also found to inhibit a-glucosidases. From the active portion, two new active compounds named uniflorines A ( 1 ) and B ( 2 ) and known (+)-(3a, 4a, 5ß)-1-methylpiperidine-3, 4, 5-triol ( 3 ) were isolated. The structures of uniflorines A and B were determined as (-)-(1S, 2R, 6S, 7R, 8R, 8aR)-1,2,6,7,8–pentahydroxyindolizidine and (+)-(1S, 2R, 5R, 7R, 8S, 8aS)-1,2,5,7,8-pentahydroxyindolizidine by spectral means, respectively.     

Synthesis of novel 5-substituted 3-amino-3,4-dihydro-2H-1-benzopyran derivatives and their interactions with the 5-HT1A receptor

A series of new enantiomerically pure 3-amino-3,4-dihydro-2H-1-benzopyrans (3-aminochromans) has been synthesized from (R)- and (S)-5-methoxy-3-amino-3,4-dihydro-2H-1-benzopyran. The absolute configuration of the respective (R)- and (S)-enantiomers was deduced from X-ray crystallography of (R)-3-(N-isopropylamino)-5-methoxy-3,4-dihydro-2H-1-benzopyran, (R)-9a. Various 5-substituents were introduced via palladium-catalyzed carbonylation of N-substituted 3-amino-5-trifluoromethanesulfonyloxy-3,4-dihydro-2H-1-benzopyran. The effect of N- and 5-substitution on affinity for the 5-HT1A receptor was evaluated in competition experiments using rat hippocampal membranes and [3H]8-OH-DPAT as radioligand. Selected compounds were also tested for their affinity to the D1 (rat striatum), D2 (rat striatum), D2A (human cloned), and 5-HT2A (rat cortex) receptors. The intrinsic activity of the compounds was evaluated by measuring their effect on VIP-stimulated cAMP production in GH4ZD10 cells stably transfected with the 5-HT1A receptor. High-affinity compounds with high selectivity for the 5-HT1A receptor were found among structures substituted with carboxylate esters, amides, and ketones in the 5-position. Primary and secondary amines bound with lower affinity than tertiary amines. Larger substituents were well-tolerated by the receptor, but the smaller N-ethyl-N-isopropyl bound with lower affinity. Generally, the (R)-enantiomers displayed higher affinity for the 5-HT1A receptor than the corresponding (S)-enantiomers. In the present series of compounds, both full and partial agonists were found.     

a-glucosidase Inhibitors From Paraguayan Natural Medicine, Ñangapiry, The Leaves Of Eugenia Uniflora

The water-soluble extract from a Paraguayan natural medicine, Nangapiry, the leaves of Eugenia uniflora L. (Myrtaceae), which has been used as an antidiabetic agent, was found to show inhibitory activities on the increase of plasma glucose level in the sucrose tolerance test (STT) conducted with mice. The portion adsorbed on a cation exchange resin was also found to inhibit a-glucosidases. From the active portion, two new active compounds named uniflorines A ( 1 ) and B ( 2 ) and known (+)-(3a, 4a, 5?)-1-methylpiperidine-3, 4, 5-triol ( 3 ) were isolated. The structures of uniflorines A and B were determined as (-)-(1S, 2R, 6S, 7R, 8R, 8aR)-1,2,6,7,8-pentahydroxyindolizidine and (+)-(1S, 2R, 5R, 7R, 8S, 8aS)-1,2,5,7,8-pentahydroxyindolizidine by spectral means, respectively.     

N-phosphonocarbonylpyrrolidine derivatives of guanine: a new class of bi-substrate inhibitors of human purine nucleoside phosphorylase

A complete series of pyrrolidine nucleotides, (3R)- and (3S)-3-(guanin-9-yl)pyrrolidin-1-N-ylcarbonylphosphonic acids and (3S,4R)-, (3R,4S)-, (3S,4S)-, and (3R,4R)-4-(guanin-9-yl)-3-hydroxypyrrolidin-1-N-ylcarbonylphosphonic acids, were synthesized and evaluated as potential inhibitors of purine nucleoside phosphorylase (PNP) isolated from peripheral blood mononuclear cells (PBMCs) and cell lines of myeloid and lymphoid origin. Two compounds, (S)-3-(guanin-9-yl)pyrrolidin-1-N-ylcarbonylphosphonic acid (2a) and (3S,4R)-4-(guanin-9-yl)-3-hydroxypyrrolidin-1-N-ylcarbonylphosphonic acid (6a), were recognized as nanomolar competitive inhibitors of PNP isolated from cell lines with K(i) values within the ranges of 16-100 and 10-24 nM, respectively. The low (MESG)K(i) and (Pi)K(i) values of both compounds for PNP isolated from PBMCs suggest that these compounds could be bisubstrate inhibitors that occupy both the phosphate and nucleoside binding sites of the enzyme.     

Chemical constituents of the rhizomes of Coeloglossum viride var. bracteatum

Seven new compounds, named coelovirins A-G (1-7), along with fourteen known constituents were isolated from the rhizomes of Coeloglossom viride var. bracteatum (Orchidaceae). On the basis of chemical and spectroscopic methods, including 2D-NMR techniques, the structures of new compounds were elucidated as 1-(4-beta-D-glucopyranosyloxybenzyl)-(2R,3S)-2-isobutyltartrate (1), 4-(4-beta-glucopyranosyloxybenzyl)-(2R,3S)-2-isobutyltartrate (2), 1-(4-beta-D-glucopyranosyloxybenzyl)-(2R,3S)-2-beta-D-glucopyranosyl-2-isobutyltartrate (3), 4-(4-beta-D-glucopyranosyloxybenzyl)-(2R,3S)-2-beta-D-glucopyranosyl-2-isobutyltartrate (4), (2R,3S)-2-beta-D-glucopyranosyl-2-isobutyltartaric acid (5), bis(4-beta-D-glucopyranosyloxybenzyl)-(2R,3S)-2-[beta-D-glucopyranosyl-(1 --> 4)-beta-D-glucopyranosyl]-2-isobutyltartrate (6) and bis(4-beta-D-glucopyranosyloxybenzyl)-(2R)-2-[beta-D-glucopyranosyl-(1 --> 4)-beta-D-glucopyranosyl]-2-isobutylmalate (7). The known compounds are 4-hydroxybenzaldehyde, 4-hydroxybenzyl alcohol, 4,4'-dihydroxydibenzyl ether, 4,4'-dihydroxydiphenylmethane, 4-(4-hydroxybenzyloxy)benzyl alcohol, gastrodin, quercetin-3,7-diglucoside, thymidine, loroglossin, militarine, dactylorhin A, dactylorhin B, beta-sitosterol and daucosterol.     

New spirostanol glycosides from Solanum nigrum and S. jasminoides

A new characteristic steroidal glycoside possessing a hydroxyl group at C-23, inunigroside A (1), was isolated from the withered berries of Solanum nigrum L. On the basis of spectroscopic analysis, the structure of 1 was characterized as (5α,22S,23S,25R)-3β,23-dihydroxyspirostane 3-O-β-lycotetraoside. Next, a major steroidal sapogenol, (22R, 25S)-3β,15α-dihydroxy-spirost-5-ene (3), was obtained from the acid hydrolysate of the methanolic extract of the aerial parts of Solanum jasminoides L. A new bisdesmoside, 3-O-β-D-glucopyranosyl-(1→4)-β-D-glucopyranosyl-(1→4)-β-D-glucopyranosyl (22R,25S)-3β,15α-dihydroxyspirost-5-ene 15-O-α-L-rhamnopyranoside (4), named jasminoside A, was isolated from the methanolic extract of S. jasminoides.     

Lipid peroxidation as a possible cause of ochratoxin A toxicity

Addition of the mycotoxin ochratoxin A (OA), a nephrotoxic carcinogen, to rat liver microsomes greatly enhanced the rate of NADPH or ascorbate-dependent lipid peroxidation as measured by malondialdehyde formation. NADPH-dependent lipid peroxidation in kidney microsomes was similarly enhanced by OA. The process required the presence of trace amounts of iron but cytochrome P-450 and free active oxygen species appeared not to be involved. The efficiency of several ochratoxins (ochratoxins A, B, C, alpha and O-methyl-ochratoxin C) to enhance lipid peroxidation was related to the presence and reactivity of the phenolic hydroxyl group. Furthermore, the ability of these ochratoxins to enhance lipid peroxidation in microsomes correlated precisely with their known toxicities in chicks. Administration of ochratoxin A to rats also resulted in enhanced lipid peroxidation in vivo as evidenced by a seven-fold increase in the rate of ethane exhalation. These results suggest that lipid peroxidation may play a role in the observed toxicity of ochratoxin A in animals; a mechanism is proposed. (Formula: see text). Ochratoxin A: X = Cl; R1 = R2 = R3 = R4 = H Ochratoxin B: X = H; R1 = R2 = R3 = R4 = H Ochratoxin C: X = Cl; R1 = R2 = R3 = H; = R4 = CH3 O-Methyl-ochratoxin C: X = Cl; R2 = R3 = H; R1 = R4 = CH3 (4R)-4-hydroxyochratoxin A: X = Cl; R1 = R3 = R4 = H; R2 = OH (4S)-4-hydroxyochratoxin A: X = Cl; R1 = R2 = R4 = H; R3 = OH Fig. 1. Chemical structures of the various ochratoxins.     

Three new phloroglucinol derivatives from Hypericum scabrum

A chemical investigation on the aerial parts of Hypericum scabrum L. resulted in the isolation of three new phloroglucinol derivatives, hyperscabrins A (1), B (2), and C (3), together with one known compound, (2R,3R,4S,6R)-3-methyl-4,6-di(3-methyl-2-butenyl)-2-(2-methyl-1-oxopropyl)-3-(4-methyl-3-pentenyl)-cyclohexanone (4). The structures were elucidated by means of spectroscopic methods, including MS, IR, 1D NMR, and 2D NMR, and the absolute configurations of chiral centers in these phloroglucinol derivatives were determined for the first time by studying their circular dichroism spectra.     

Synthetic studies of 1-beta-methylcarbapenem antibiotics

The diastereoselective synthesis of the 1-beta-methylcarbapenems, (1R,5R,6S)-6-((R)-1-hydroxyethyl)-2-((S)-1-acetimidoylpyrrolidin-3 - ylthio)-1- methyl-1-carbapen-2-em-3-carboxylic acid and sodium (1R,5R,6S)-6-((R)-1-hydroxyethyl)-2-(5-chloro-2-oxobenzoxazolin-3- y l)-1- methyl-1-carbapen-2-em-3-carboxylate has been achieved. The key step was an aldol reaction between the achiral boron enolate which was generated from dibutylboron triflate and 3-propionyl-2-oxobenzoxazoline, and (3R,4R)-4-acetoxy-3-((R)-1-hydroxyethyl)azetizin-2-one.     

法罗培南钠的合成

（3R，4R）-4-乙酰氧基-3-[（1R）-（叔丁基二甲基硅氧基）乙基]氮杂环丁-2-酮与（R）-四氢呋喃-2-硫代羧酸经缩合、酰化、Wittig反应、脱保护、水解等反应制得青霉烯类抗生素法罗培南钠，总收率约37％。     

N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR141716A) interaction with LYS 3.28(192) is crucial for its inverse agonism at the cannabinoid CB1 receptor

In superior cervical ganglion neurons, N-(piperidiny-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR141716A) competitively antagonizes the Ca(2+) current effect of the cannabinoid (CB) agonist (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone (WIN55212-2), and behaves as an inverse agonist by producing opposite current effects when applied alone. In contrast, in neurons expressing CB1 with a K-->A mutation at residue 3.28(192) (i.e., K3.28A), SR141716A competitively antagonizes the effects of WIN55212-2, but behaves as a neutral antagonist by producing no current effects itself. Receptor modeling studies suggested that in the CB1 inactive (R) state, SR1417A16A stabilizes transmembrane helix 6 in its inactive conformation via aromatic stacking with F3.36/W6.48. In this binding site, SR141716A would exhibit higher affinity for CB1 R due to a hydrogen bond between the SR141716A C3 substituent and K3.28(192), a residue available to SR141716A only in R. To test this hypothesis, a "mutant thermodynamic cycle" was constructed that combined the evaluation of SR141716A affinity at WT CB1 and K3.28A with an evaluation of the wild-type CB1 and K3.28A affinities of an SR141716A analog, 5-(4-chlorophenyl)-3-[(E)-2-cyclohexylethenyl]-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole (VCHSR), that lacks hydrogen bonding potential at C3. Binding affinities suggested that K3.28 is involved in a strong interaction with SR141716A in WT CB1, but does not interact with VCHSR. Thermodynamic cycle calculations indicated that a direct interaction occurs between the C3 substituent of SR141716A and K3.28 in WT CB1. Consistent with these results, VCHSR acted as a neutral antagonist at WT CB1. These results support the hypothesis that hydrogen bonding of the SR141716A C3 substituent with K3.28 is responsible for its higher affinity for the inactive R state, leading to its inverse agonism.     

Synthesis of stereospecifically deuterated 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) iastereomers and metabolism by A/J mouse lung microsomes and cytochrome p450 2A5

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a lung carcinogen in mice and rats and is a putative human lung carcinogen. NNK undergoes cytochrome p450-mediated metabolic activation to DNA-binding intermediates but is also extensively reduced to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in vivo. Because NNAL is also tumorigenic, the carcinogenicity of NNK may actually be governed by the metabolic activation of NNAL, rather than direct activation of NNK. Metabolism of NNK and NNAL at the 4-position generates the same critical DNA lesion, O(6)-methylguanine, the levels of which are correlated to tumorigenicity in the A/J mouse model. In an effort to better understand the bioactivation of NNAL and the effect of carbinol-carbon stereochemistry on prochiral selectivity at the 4-position, (R)- and (S)-NNAL, along with the stereospecifically 4-deuterated diastereomers (1R,4R)-[4-(2)H(1)]NNAL, (1R,4S)-[4-(2)H(1)]NNAL, (1S,4R)-[4-(2)H(1)]NNAL, and (1S,4S)-[4-(2)H(1)]NNAL, were synthesized. The in vitro metabolism of these compounds was investigated using A/J mouse lung microsomes and Spodoptera frugiperda-expressed mouse cytochrome p450 2A5. Carbinol-carbon stereochemistry did not appreciably influence stereoselectivity at the 4-position in the metabolism of these compounds by mouse lung microsomes or p450 2A5 but did influence the regiochemistry of metabolism. The ratio of 4- to N-methyl hydroxylation was approximately 1:1 for the A/J mouse lung microsome-mediated metabolism of all substrates, but this ratio was higher for (1S) substrates than for their (1R) counterparts when p450 2A5 was used. Interestingly, p450 2A5 converted substrates with (1S) stereochemistry to the respective N-oxides, but this metabolite was not formed from substrates with (1R) stereochemistry. Furthermore, p450 2A5 catalyzed the formation of NNK from (1S) substrates at significantly greater maximal rates than from (1R) substrates. The implications of these differences in metabolism for the tumorigenic mechanism of NNAL are discussed.