Anthraquinones from the fruits of Vismia laurentii

Phytochemical study of the fruits of Vismia laurentii resulted in the isolation of five structurally related compounds. Three of them are constituents, namely, laurentiquinone A (1) (methyl 1,6,8-trihydroxy-3-methyl-7-(3-methylbut-2-enyl)-9,10-dioxo-9,10-dihydroanthracene-2-carboxylate), laurentiquinone B (2) (methyl 5,7-dihydroxy-2,2,9-trimethyl-6,11-dioxo-6,11-dihydro-2H-anthra[2,3-b]pyran-8-carboxylate) and laurentiquinone C (3) (methyl 9-(ethanoyloxymethyl)-5,7-dihydroxy-2,2-dimethyl-6,11-dioxo-6,11-dihydro-2H-anthra[2,3-b]pyran-8-carboxylate) and two are known compounds, emodin (4) and isoxanthorin (5). Their structures were elucidated by spectroscopic means. Crude extracts of hexane and EtOAc showed anti-plasmodial activity against the W2 strain of Plasmodium falciparum.     

Synthesis and absolute configuration of the stereoisomers of [2-(1-diethylaminopropyl)] 1-hydroxy-1,1′-bicyclohexyl-2-carboxylate,a muscarinic antagonist

The compound [2-(1-diethylaminopropyl)] 1-hydroxy-1,1′-bicyclohexyl-2-carboxylate 1 is a muscarinic antagonist characterized by the presence of three chiral atoms and eight possible stereoisomers. The binding affinities to the five cloned m₁–m₅ muscarinic receptors of the stereoisomers of 1 were previously investigated and proved to be related to the chirality of the molecules. The eight isomers are prepared through the synthesis of their racemates followed by chemical resolution as (+) and (−) tartrate or (+) and (−) dibenzoyltartrate salts. The isomers with cis-configuration of OH and COOH substituents of the cyclohexane are also obtained by coupling optically active (1S, 2S) or (1R,2R)-1-hydroxy-[1,1′-bicyclohexyl]-2-carboxylic acid with (S)- or (R)-1-diethylamino-2-propanol. Chiral GC and HPLC methods are used to determine their optical purity. The absolute configurations of the four cis- and four trans-isomers are established by stereospecific synthesis and X-ray crystallographic data. Chirality 9:713–721, 1997. © 1997 Wiley-Liss, Inc.     

Quantitative analysis of 6,11-dihydro-11-oxo-dibenz[b,e] oxepin-2-acetic acid (isoxepac) in plasma and urine by gas-liquid chromatography

A method is described for the quantitative analysis of 6,11-dihydro-11-oxo-dibenz[b,e] - oxepin-2-acetic acid (isoxepac) in plasma and urine. Isoxepac and internal standard are extracted from acidified plasma and urine, converted to the corresponding methyl esters and analysed by gas—liquid chromatography using a flame ionization detector. The method is accurate and precise over the range 0.1–30 μg/ml. The method has been applied to the analysis of plasma and urine from both healthy volunteers and patients receiving therapeutic oral doses of isoxepac.     

Synthesis of carbon-11-labeled 4-(phenylamino)-pyrrolo[2,1-f][1,2,4]triazine derivatives as new potential PET tracers for imaging of p38α mitogen-activated protein kinase

The reference standards methyl 4-(2-methyl-5-(methoxycarbamoyl)phenylamino)-5-methylpyrrolo[2,1-f][1,2,4]triazine-6-carboxylate (10a), methyl 4-(2-methyl-5-(ethoxycarbamoyl)phenylamino)-5-methylpyrrolo[2,1-f][1,2,4]triazine-6-carboxylate (10b) and corresponding precursors 4-(2-methyl-5-(methoxycarbamoyl)phenylamino)-5-methylpyrrolo[2,1-f][1,2,4]triazine-6-carboxylic acid (11a), methyl 4-(2-methyl-5-(ethoxycarbamoyl)phenylamino)-5-methylpyrrolo[2,1-f][1,2,4]triazine-6-carboxylic acid (11b) were synthesized from methyl crotonate and 3-amino-4-methylbenzoic acid in multiple steps with moderate to excellent yields. The target tracer [¹¹C]methyl 4-(2-methyl-5-(methoxycarbamoyl)phenylamino)-5-methylpyrrolo[2,1-f][1,2,4]triazine-6-carboxylate ([¹¹C]10a) and [¹¹C]methyl 4-(2-methyl-5-(ethoxycarbamoyl)phenylamino)-5-methylpyrrolo[2,1-f][1,2,4]triazine-6-carboxylate ([¹¹C]10b) were prepared from their corresponding precursors with [¹¹C]CH₃OTf under basic condition through O-[¹¹C]methylation and isolated by a simplified solid-phase extraction (SPE) method in 50–60% radiochemical yields at end of bombardment (EOB) with 185–555 GBq/μmol specific activity at end of synthesis (EOS).     

Radiolabeling and biodistribution of methyl 2-(methoxycarbonyl)-2-(methylamino) bicyclo[2.1.1] -hexane -5-carboxylate,a potential neuroprotective drug

Methyl 2-(methoxycarbonyl) -2-(methylamino) bicyclo[2.1.1] -hexane -5-carboxylate (MMMHC) is developed as a potential neuroprotective drug. It was labeled with C-11 from the desmethyl precursor methyl 2-(methoxycarbonyl)-2-amino bicyclo[2.1.1]-hexane-5-carboxylate with [11C]methyl triflate in acetone solution at 60 degrees C with labeling yield of 69% and with radiochemical purity of >99%. Positron Emission Tomography (PET) studies in a normal rat showed that Methyl 2-(methoxycarbonyl)-2-([11C]methylamino)bicyclo[2.1.1]-hexane-5-carboxylate ([11C] MMMHC) accumulated mainly in the cortical brain areas after iv administration. Frontal cortex/cerebellum ratios in a rat brain were 8.0/6.0, 6.8/4.2, 6.3/4.3, 5.5/4.2 and 5.2/4.5 percent of the injected dose in 100 ml at 2 min, 5 min, 10 min, 20 min and 40 min respectively after i.v. injection. During 20-40 min, 2.9+/-0.4% of the total activity stayed in the brain. These results showed that MMMHC could be labeled with C-11 with high yield, and it passed the brain-blood barrier and accumulated in several brain regions.     

Synthesis of the [8] gingerol enantiomers

(+)-(S)-5-Hydroxy-1-(4-hydroxy-3-methoxyphenyl)-3-dodecanone 1a commonly named (+)-(S)-[8] gingerol is a natural product known to have cardiotonic activity.^1–5 A total synthesis of both enantiomers is described with details for the first time using a general synthetic scheme which was recently outlined in the literature.⁶ This synthesis relies both on the separation of the diastereoisomers 4a and 4b by simple column chromatography on silica gel and on an HPLC analysis on a chiral phase to determine the optical purity of the enantiomers 8a and 8b of protected [8] gingerol. The gingerol isomers were thus obtained in good chemical yields in greater than 96% enantiomeric excess.     

Enzymatic Resolution of (±)-Epoxy-β-cyclogeraniol,a Synthetic Precursor for Abscisic Acid Analogs

Lipase-catalyzed optical resolution of (±)-epoxy-β-cyclogeraniol (1), a key synthetic intermediate for epoxy-β-ionylideneacetic acid, was achieved in high enantiomeric purity. Transesterification with vinyl acetate by using lipase P (Nagase) made enriched (-)-1, while hydrolysis of the corresponding acetate by using lipase P (Amano) afforded (+)-1 with a high E value (E=1600).     

Enantioselective enzymatic acetylation of racemic [4-[4α,6β (E)]]-6-[4,4-bis(4-fluorophenyl)-3-(1-methyl-1H-tetrazol-5-yl)-1,3-butadienyl]-tetrahydro-4-hydroxy-2H-pyran-2-one

Summary A chiral compound [4R-[4,6ß(E)]]-6-[4,4-bis(4-fluorophenyl)-3-(1-methyl-1H-tetrazol-5-yl)-1,3-butadienyl]-tetrahydro-4-hydroxy-2H-pyran-2-one (R-(+)-1) was prepared by the lipase-catalysed stereoselective acetylation of racemic 1 in an organic solvent. Chiral R-(+)-1 is a hydroxymethyl glutaryl coenzyme A (HMG CoA) reductase inhibitor and a potential anticholesterol drug candidate. Among various lipases evaluated, lipase PS-30 from Pseudomonas species efficiently catalysed acetylation of the undesired enantiomer of racemic 1 to yield the S-(–)-acetylated product 2 and unreacted desired R-(+)-1. A reaction yield of 48 mol% and an optical purity of 98% were obtained for R-(+)-1 when the reaction was conducted in toluence as solvent in the presence of isopropenyl acetate as acyl donor. Lipase PS-30 was immobilized on Accurel polypropylene (PP) and the immobilized enzyme was reused (five cycles) in the acetylation reaction without loss of enzyme activity, productivity, or optical purity of the R-(+)-1. The enzymatic acetylation process was scaled-up to 501 and a 640-l volume (preparative batches) at a substrate concentration of 4 g/l. R-(+)–1 was recovered from the preparative batches in 68–71% recovery yield with 98.5% gas chromatography homogeneity index and 98.5% optical purity. The S-(–) acetate 2 produced by the acetylation reaction was enzymatically hydrolysed by lipase PS-30 in a biphasic system to prepare the corresponding S-(–)-1.Correspondence to: R. N. Patel     

Synthesis and enantioselective rearrangement of (Z)-4-triphenylmethoxy-2,3-epoxybutan-1-ol enantiomers

Efficient enzyme catalyzed kinetic resolutions of a synthetically useful chiral building block, (Z)-4-triphenylmethoxy-2,3-epoxybutan-1-ol, are reported. The highest selectivities were achieved by Lipozyme TL IM and Amano Lipase PS enzymes in the presence of vinyl acetate. Enantiomeric enrichment of the optically active acetate isomer was accomplished by selective crystallization of the racemic part of the enantiomeric mixture. Enzyme catalyzed hydrolysis of the acetate also provided an optically pure epoxybutanol derivative. O-Benzylation of (+)-(Z)-1-hydroxy-4-triphenylmethoxy-2,3-epoxybutane followed by super base promoted diastereo- and enantio-selective rearrangement resulted in (+)-(2R,3R,1'R)-3-[1-hydroxy-2-(triphenylmethoxy)ethyl]-2-phenyloxetane in >98% ee and de. Configurations of the new optically active products were determined by chemical correlation.     

Enzymatic resolution of (+/-)-epoxy-beta-cyclogeraniol, a synthetic precursor for abscisic acid analogs

Lipase-catalyzed optical resolution of (+/-)-epoxy-beta-cyclogeraniol (1), a key synthetic intermediate for epoxy-beta-ionylideneacetic acid, was achieved in high enantiomeric purity. Transesterification with vinyl acetate by using lipase P (Nagase) made enriched (-)-1, while hydrolysis of the corresponding acetate by using lipase P (Amano) afforded (+)-1 with a high E value (E = 1600).     

The utility of 1-[18F]fluoro-3-iodopropane for the synthesis of certain dopamine D-1 and benzodiazepine receptor radioligands

No-carrier-added (NCA) R(+)-7-chloro-8-hydroxy-3-(3′-[¹⁸F]fluoropropyl)-1-phenyl-2,3,4,5-tetrahydro-3-benzazepine (2b) (an analog of dopamine D-1 receptor ligand SCH 23390), ethyl 8-fluoro-5,6-dihydro-5-(3′-fluoropropyl)-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylate (4b) and 3′-[¹⁸F]fluoropropyl 8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylate (6b) (analogs of the benzodiazepine RO 15-1788) were synthesized by alkylation of the corresponding nor-compound with NCA 1-[¹⁸F]fluoro-3-iodopropane in 10–15% yield (EOB) in ~110min and with a mass of 2–3nmol. Compound 2 is less potent (~ 12–14 times) than SCH 23390 in binding to rat striatal membranes in vitro. Compounds 2b, 4b and 6b exhibit no specific anatomical distribution to mouse brain. These results suggest that the substituent at position 3 of SCH 23390, and position 5 and carboxylate group of RO 15-1788 are critical determinants both of affinity and selectivity for receptor binding, and underscores the evaluation necessary when even minor changes (C1 to C3) are made in bioactive compounds.     

Stereoselective enzymatic hydrolysis of (exo,exo)-7-oxabicyclo[2.2.1]heptane-2,3-dimethanol diacetate ester in a biphasic system

Summary A key chiral intermediate lactol(3)[3aS (3a,4,7,7a)]-hexahydro-4,7-epoxy-isobenzofuran-1 (3H)-one was prepared for the total synthesis of a new thromboxane antagonist. The stereoselective hydrolysis of (exo,exo)-7-oxabicyclo[2.2.1]heptane-2,3-dimethanol, diacetate ester (1) to the corresponding chiral monoacetate ester (2) was carried out with lipases, among which Amano P-30 lipase from Pseudomonas sp. was most effective since it gave the desired enantiomer of monoacetate ester. A yield of 75 mol% and optical purity of >99% was obtained when the reaction was conducted in a biphasic system with 10% toluene at 5 g/l of the substrate. Lipase P-30 was immobilized on Accurel polypropylene (PP) and the immobilized enzyme was reused (five cycles) without loss of enzyme activity, productivity or optical purity. The reaction process was scaled-up to 80 1 (400 g substrate) and monoacetate (2) was isolated in 80 mol% yield with 99.3% optical purity as determined by chiral HPLC and nuclear magnetic resonance (NMR) analysis. A gas chromatography of 99.5% and specific rotation, []_D of -7.6° was obtained. The chiral monoacetate ester (2) was oxidized to its corresponding aldehyde and subsequently hydrolyzed to give lactol (3).     

Stereoselective enzymatic esterification of 3-benzoylthio-2-methylpropanoic acid

Summary A key intermediate, S-(–)-3-benzoylthio-2-methylpropanoic acid (1) was made in high optical purity by the lipase-catalyzed stereoselective esterification of racemic 1 with methanol in an organic solvent system. Among various lipases evaluated, Amano P-30 lipase from Pseudomonas sp. efficiently catalyzed the esterification of 1 to yield R-(+) methyl ester and unreacted S-(–) 1. A reaction yield of 40 mol% and an optical purity of 97.2% were obtained for compound 1 at a substrate concentration of 0.1 m (22 mg/ml). Lipase P-30 was immobilized on Accurel polypropylene (PP) and the immobilized enzyme was reused (23 cycles) in the esterification reaction without loss of enzyme acitivity, productivity or optical purity. Among various solvents evaluated, toluene was found to be the most suitable organic solvent and methanol was the best alcohol for the esterification of racemic 1 by immobilized lipase. Substrate concentrations as high as 1.0 m were used in the esterification reaction. When the temperature was increased from 28° C to 60° C, the reaction time required for the esterification of 0.1 m substrate decreased from 16 h to 2 h. On increasing the methanol to substrate molar ratio from 1:1 to 4:1, the rate of esterification decreased. A lipase fermentation using Pseudomonas sp. ATCC 21 808 was developed. In the batch-fermentation process, 56 units/ml of extracellular lipase activity was obtained. A fed-batch process using soybean oil gave a significant increase in the lipase activity (126 units/ml). Crude lipase recovered from the filtrate by ethanol precipitation and immobilized on Accurel PP was also effective: S-(–) compound 1 was obtained in 35 mol% yield and 95% optical purity. Offsprint requests to: R. N. Patel     

Quantitative analysis of oxepinac in human plasma,urine and saliva by gas chromatography—mass fragmentography

A sensitive and specific method is described for the quantitative analysis of 6,11-dihydro-11-oxo-dibenz[b,e]oxepin-3-acetic acid (oxepinac) in human plasma, urine and saliva. Oxepinac and internal standard are extracted from acidified plasma, urine or saliva, converted to the corresponding n-propyl esters and analysed by gas chromatography—mass fragmentography using selected ion monitoring. The method is accurate and precise over the range 100 μg/ml to 1.0 ng/ml. The method has been applied to the analysis of plasma, urine and saliva from healthy volunteers receiving therapeutic doses of oxepinac.     

Synthesis of (Rp,Rp)-P1,P4-Bis(5'-adenosyl)-1[17O,18O2],4[17O,18O2] tetraphosphate from (Sp,Sp)-P1,P4-Bis(5'-adenosyl)-1[thio-18O2], 4[thio-18O2]tetraphosphate with retention at phosphorus and the stereochemical course of hydrolysis by the unsymmetrical Ap4A phosphodiesterase from lupin seeds

The three stereoisomers of P1,P4-bis(5'-adenosyl)-1,4-dithiotetraphosphate have been synthesized and their 31P NMR spectra investigated. The effect of temperature on the circular dichroic spectrum of the (Sp,Sp)-stereoisomer shows that unstacking of the molecule occurs as the temperature is raised. Treatment of the (Sp,Sp)-stereoisomer with cyanogen bromide in [18O]water leads to substitution of sulfur by 18O with predominant retention of configuration at P1 and P4. (Sp,Sp)-P1,P4-Bis(5'-adenosyl)-1[thio-18O2],4[thio-18O2]tetraphosphate was synthesized and on treatment with cyanogen bromide in [17O]water gave (Rp,Rp)-P1,P4-bis(5'-adenosyl)-1[17O,18O2],4[17O,18O2]tetraphosphate. Hydrolysis by unsymmetrical Ap4A phosphodiesterase from lupin seeds gave (Rp)-5'-[16O,17O,18O]AMP. The reaction therefore proceeds with inversion of configuration at phosphorus, indicating that the enzyme-catalyzed displacement by water occurs by a direct "in-line" mechanism.     

Synthesis and evaluation of optically pure [3H]-(+)-pentazocine, a highly potent and selective radioligand for sigma receptors

Tritium-labeled (+)-pentazocine ([3H]-1b) of specific activity 26.6 Ci/mmol was synthesized in 3 steps starting with (+)-normetazocine (2) of defined optical purity. [3H]-1b has been characterized as a highly selective ligand for labeling of sigma receptors. Competition data revealed that [3H]-1b could be displaced from guinea pig brain membrane preparations with a number of commonly used sigma receptor ligands. [3H]-1b exhibited saturable, enantioselective binding with a Kd of 5.13 +/- 0.97 nM and a Bmax of 1146 +/- 122 fmol/mg protein. Phencyclidine (PCP) displaced [3H]-1b with low affinity while MK-801 was inactive, thus indicating insignificant activity at the PCP-binding site; apomorphine failed to displace [3H]-1b indicating lack of dopamine receptor cross-reactivity. Since the affinity of [3H]-1b is about 6 times that of the two commonly employed sigma ligands ((+)-3-[3H]PPP and [3H]DTG) and since it is more selective for sigma receptors than the benzomorphan [3H]SKF-10,047, it represents the first example of a highly selective benzomorphan based sigma receptor ligand. [3H]-1b should prove useful for further study of the structure and function of sigma receptors.     

(-)-Amarbellisine, a lycorine-type alkaloid from Amaryllis belladonna L. growing in Egypt

A new lycorine-type alkaloid, named (-)-amarbellisine, was isolated from the bulbs of Egyptian Amaryllis belladonna L. together with the well known alkaloids (-)-lycorine, (-)-pancracine, (+)-vittatine, (+)-11-hydroxyvittatine, and (+)-hippeastrine. The new alkaloid, containing the pyrrolo[de]phenanthridine ring system, was essentially characterised by spectroscopic and optical methods, and proved to be the 2-methoxy-3a,4,5,7,11b,11c-hexahydro-1H-[1,3]dioxolo[4,5-j]pyrrolo[3,2,1-de]phenanthridinol. By using HPTLC technique we also carried out a comparative study of the relative and total alkaloidal content at two different stages of plant growth. Finally, the antimicrobial activity of the isolated alkaloids was assayed.     

Radiolabeling and in vitro /in vivo evaluation of N‐(1‐adamantyl)‐8‐methoxy‐4‐oxo‐1‐phenyl‐1,4‐dihydroquinoline‐3‐carboxamide as a PET probe for imaging cannabinoid type 2 receptor

The cannabinoid type 2 (CB₂) receptor plays an important role in neuroinflammatory and neurodegenerative diseases such as multiple sclerosis, amyotrophic lateral sclerosis, and Alzheimer's disease and is therefore a very promising target for therapeutic approaches as well as for imaging. Based on the literature, we identified one 4‐oxoquinoline derivative (designated KD2) as the lead structure. It was synthesized, radiolabeled and evaluated as a potential imaging tracer for CB₂. [¹¹C]KD2 was obtained in 99% radiochemical purity. Moderate blood–brain barrier (BBB) passage was predicted for KD2 from an in vitro transport assay with P‐glycoprotein‐transfected Madin Darby canine kidney cells. No efflux of KD2 by P‐glycoprotein was detected. In vitro autoradiography of rat and mouse spleen slices demonstrated that [¹¹C]KD2 exhibits high specific binding towards CB₂. High spleen uptake of [¹¹C]KD2 was observed in dynamic positron emission tomography (PET) studies with Wistar rats and its specificity was confirmed by displacement study with a selective CB₂ agonist, GW405833. A pilot autoradiography study with post‐mortem spinal cord slices from amyotrophic lateral sclerosis (ALS) patients with [¹¹C]KD2 suggested the presence of CB₂ receptors under disease conditions. Specificity of [¹¹C]KD2 binding could also be demonstrated on these human tissues. In conclusion, [¹¹C]KD2 shows good in vitro and in vivo properties as a potential PET tracer for CB₂.

     

Analogs of 4-(3-bromo-8-methyl-10-methoxy-6,11-dihydro-5H-benzo[5,6]-cyclo hepta[1,2-b]pyridin-11-yl)-1-(4-pyridinylacetyl)piperidine N-oxide as inhibitors of farnesyl protein transferase.

A series of 3-substituted analogs 3 of 4-(3-bromo-8-methyl-10-methoxy-6,11-dihydro-5H-benzo[5,6]-cyclohepta[1,2 b]pyridin-11-yl)-1-(4-pyridinylacetyl)piperidine N-oxide 2 was prepared and evaluated as FPT inhibitors. The objective of this study was to identify other substituents at C3 in this series of FPT inhibitors that would have the FPT potency enhancement similar to that found for a C3 bromo substituent. The 3-methyl analog 17b was found to be tenfold less active than 2, and other C3 substituents having more steric bulk were found to cause a further reduction in activity.     

Synthesis and preliminary biological evaluation of a novel P2X7R radioligand [18F]IUR-1601

The reference standard IUR-1601 ((S)-N-(2-chloro-3-(trifluoromethyl)benzyl)-1-(2-fluoroethyl)-5-oxopyrrolidine-2-carboxamide) was synthesized from tert-butyl (S)-5-oxopyrrolidine-2-carboxylate, fluoroethylbromide, and 2-chloro-3-(trifluoromethyl)benzylamine with overall chemical yield 12% in three steps. The target tracer [¹⁸F]IUR-1601 ((S)-N-(2-chloro-3-(trifluoromethyl)benzyl)-1-(2-[¹⁸F]fluoroethyl)-5-oxopyrrolidine-2-carboxamide) was synthesized from desmethyl-GSK1482160 with 2-[¹⁸F]fluoroethyl tosylate, prepared from 1,2-ethylene glycol-bis-tosylate and K[¹⁸F]F/Kryptofix2.2.2, in two steps and isolated by HPLC combined with SPE in 1–3% decay corrected radiochemical yield. The radiochemical purity was >99%, and the molar activity at end of bombardment (EOB) was 74–370?GBq/μmol. The potency of IUR-1601 in comparison with GSK1482160 was determined by a radioligand competitive binding assay using [¹¹C]GSK1482160, and the binding affinity K_i values for IUR-1601 and GSK1482160 are 4.31 and 5.14?nM, respectively.