The metabolism of the isomeric decalones

1. The metabolism of (+/-)-cis-1-, (+/-)-trans-1-, (+/-)-cis-2- and (+/-)-trans-2-decalone in the rabbit has been investigated. 2. (+/-)-cis-2- and (+/-)-trans-2-Decalone were both reduced to racemic secondary alcohols, conformationally related to the ketones administered, and possessing an equatorial hydroxyl group. These alcohols were excreted in the urine as glucuronides in amounts equal to about half the dose administered. The glucuronides were isolated both as triacetyl methyl esters and as sodium salts. The ester obtained after feeding with (+/-)-cis-2-decalone proved to be methyl (cis-cis-2-decalyl tri-O-acetyl-beta-d-glucosid)uronate, whereas (+/-)-trans-2-decalone yielded methyl (trans-cis-2-decalyl tri-O-acetyl-beta-d-glucosid)uronate. The sodium salts were shown to be sodium (cis-cis-2-decalyl glucosid)uronate and sodium (trans-cis-2-decalyl glucosid)uronate. 3. Enzyme hydrolysis of the sodium salts and acid hydrolysis of the esters derived from (+/-)-cis-2-decalone yielded (+/-)-cis-cis-2-decalol, and of those from (+/-)-trans-2-decalone yielded (+/-)-trans-cis-2-decalol. 4. (+/-)-cis-1-Decalone was reduced mainly to (-)-cis-cis-1-decalol and excreted as [(-)-cis-cis-1-decalyl glucosid]-uronic acid. A small amount of the corresponding (+)-isomer was produced, yielding [(+)-cis-cis-1-decalyl glucosid]uronic acid on isolation. Enzyme hydrolysis of these compounds gave the corresponding aglycones; both alcohols possessed an equatorial hydroxyl group. 5. (+/-)-trans-1-Decalone was reduced to (+)-trans-trans-1-decalol, with an equatorial hydroxyl group, and in smaller amount to (+)-trans-cis-1-decalol possessing an axial group. The former alcohol was excreted as [(+)-trans-cis-1-decalyl glucosid]uronic acid, and the latter as [(+)-trans-cis-1-decalyl glucosid]uronic acid. 6. From a knowledge of the conformations, and in some cases the absolute configurations, of the compounds administered and excreted, and by making the assumption that the coenzyme concerned in the reductions is NADH (or NADPH), an explanation of the above findings in terms of steric hindrance and thermodynamic stability is given.     

Chiroptical properties and synthesis of enantiopure cis and trans pterocarpan skeleton

The first enantioselective synthesis of trans-(6aS,11aR)-pterocarpan [(+)-2] and its conversion to cis-(6aS,11aS)-pterocarpan [(+)-1] was achieved starting from racemic 2'-benzyloxyflavanone (rac-3). Their stereochemistry was deduced by X-ray analysis of the ketal intermediate (-)-5a. The CD study of (+)-1 and (+)-2 allows the configurational assignment of similar pterocarpan derivatives by CD spectroscopy.     

Neodictyoprolenol and dictyoprolenol, the possible biosynthetic intermediates of dictyopterenes, in the Japanese brown algae Dictyopteris

Neodictyoprolenol [(-)-(3S)-(1,5Z,8Z)-undecatrien-3-ol] and dictyoprolenol [(-)-(3S)-(1,5Z,8Z)-undecadien-3-ol]), which had been proposed as possible biosynthetic intermediates of the sex pheromones of marine brown algae such as dictyopterene B [(-)-trans-1-((1'E,3'Z)-hexadienyl)-2-vinylcyclopropane], D' [(+)-6-((1'Z)-butenyl)-1,4-cycloheptadiene] and C' [(+)-6-butyl-1,4-cycloheptadiene], were again identified in the essential oils from Dictyopteris prolifera, D. latiscula, and in D. undulata, together with the C11-related volatile compounds such as neodictyoprolene, dictyoprolene and dictyopterenes. Incubation of D. prolifela preparation with racemic neodictyoprolenol and dictyoprolenol as substrates showed (S)-enantioselective decreases of the added substrates and increases in dictyopterenes. From these results, a possible pathway to form dictyopterenes is discussed.     

Therapeutic agent for treating dysuria (NC-1800): absolute configuration of the enantiomers

The absolute configuration of an enantiomer of the title compound, (+/-)-1. fumaric acid salt, was determined by X-ray crystallographic analysis of (+)-1 [salt of (+)-tartaric acid [(+)-2]]. This salt for X-ray crystallographic analysis was prepared by a simple method. The analysis of (+)-1 [salt of (+)-2] showed that this enantiomer has the 5-S and 1-R absolute configuration. The final R and Rw values were 0.0614 and 0.0713, respectively.     

The metabolism of the isomeric tert.-butylcyclohexanones

1. (+/-)-2-, (+/-)-3- and 4-tert.-Butylcyclohexanone are reduced in the rabbit to secondary alcohols, which are excreted extensively conjugated with glucuronic acid. 2. The major metabolite of (+/-)-2-tert.-butylcyclohexanone is (+)-cis-2-tert.-butylcyclohexanol, which has been isolated from the urine as [(+)-cis-2-tert.-butylcyclohexyl beta-d-glucosid]uronic acid. The minor metabolite is (+)-trans-2-tert.-butylcyclohexanol. 3. (+/-)-3-tert.-Butylcyclohexanone is reduced mainly to (+/-)-cis-3-tert.-butylcyclohexanol, and to a smaller extent to (+/-)-trans-3-tert.-butylcyclohexanol. 4. 4-tert.-Butylcyclohexanone yields mainly the trans-alcohol, which is excreted in conjugated form and has been recovered from the urine as (trans-4-tert.-butylcyclohexyl beta-d-glucosid)uronic acid. The cis-alcohol is formed to a minor extent and excreted in conjugated form. 5. The ratios of the amounts of cis- to trans-alcohols produced by the three ketones differed from the relative amounts of cis- and trans-alcohols produced by the corresponding methylcyclohexanones. 6. From these findings the suggestion is made that two orientations of ketone relative to coenzyme occur: alcohols with an equatorially orientated hydroxyl group are thought to be produced as a result of a ;face-to-face' interaction with NADH, and alcohols with axially orientated hydroxyl groups as a result of a ;perpendicular' interaction. Which will predominate is thought to depend on steric factors, particularly the size and position of alkyl substituents in the substrate.     

Comparison of psychic emergence reactions after (+/-)-ketamine and (+)-ketamine in mice

Ketamine is a racemic mixture containing equal parts of (+)-ketamine and (-)-ketamine. The ketamine enantiomorphs are different in anesthesia and psychic emergence reactions after anesthesia. Therefore, (+)-ketamine was compared with racemic ketamine in a number of randomized studies in volunteers and patients. However, their relations remain controversial. In the present studies, the psychic emergence reactions after injection of (+/-)-ketamine and (+)-ketamine were compared in mice. At equimolar doses, the (+)-isomers elicited episodes of hypnosis nearly 1.4-fold more potent than those of the racemic ketamine. After the administration of equihypnotic doses of (+)-ketamine and (+/-)-ketamine, the posthypnotic stimulation of locomotor activity, stereotype behavior and 5-HT-induced head-twitch response by the (+)-enantiomorph was significantly less intense than that of racemic ketamine. In receptor binding test, (+)-ketamine showed a higher affinity for NMDA receptor than that of (+/-)-ketamine, while (+)-ketamine and (+/-)-ketamine showed no affinity for dopamine D2 and serotonin 5-HT2 receptor. These results suggest that the (+)-ketamine has fewer posthypnotic side effects than (+/-)-ketamine when (+)-ketamine and (+/-)-ketamine were administered at equihypnotic dosages and that dopamine D2 and serotonin 5-HT2 receptor were not involved in the effects of (+)-ketamine and (+/-)-ketamine.     

Ionization states of the complex formed between 2-benzyl-3-phosphonopropionic acid and carboxypeptidase A.

The binding to carboxypeptidase A of two phosphonic acid analogues of 2-benzylsuccinate, 2-DL-2-benzyl-3-phosphonopropionic acid (inhibitor I) and 2-DL-2-benzyl-3-(-O-ethylphosphono)propionic acid (inhibitor II) was studied by observing their 31P resonances when free and bound to the enzyme in the range of pH from 5 to 10. The binding of I by co-ordination to the active-site Zn(II) lowered the highest pKa of I from a value of 7.66(+/- 0.10) to a value of 6.71(+/- 0.17). No titration of any protons on II occurred over the pH range studied. The enzyme-bound inhibitor II also did not titrate over the pH range 6.17-7.60. The pH-dependencies of the apparent inhibition constants for I and II were also investigated by using N-(-2-(furanacryloyl)-L-phenylalanyl-L-phenylalanine as substrate. Two enzymic functional groups with pKa values of 5.90(+/- 0.06) and 9.79(+/- 0.14) must be protonated for binding of inhibitor I, and two groups with pKa values of 6.29(+/- 0.10) and 9.19(+/- 0.15) for binding of inhibitor II. Over the pH range from 6.71 to 7.66, inhibitor I binds to the enzyme in a complex of the enzyme in a more protonated form, and the inhibitor in a less protonated form than the predominant unligated forms at this pH. Mock & Tsay [(1986) Biochemistry 25, 2920-2927] made a similar finding for the binding of L-2-(1-carboxy-2-phenylethyl)-4-phenylazophenol over a pH range of nearly 4 units. The true inhibition constant for the dianionic form of inhibitor I (racemic) was calculated to be 54.0(+/- 5.9) nM and that of the trianionic form to be 5.92(+/- 0.65) nM. The true inhibition constant of the fully ionized II (racemic) was calculated to be 79.8(+/- 6.4) nM.     

Partial resolution of racemic trans-4-[5-(4-alkoxyphenyl)-2,5-dimethylpyrrolidine-1-oxyl-2-yl]benzoic acids by the diastereomer method with (R)- or (S)-1-phenylethylamine

The partial resolution is described of a series of racemic trans-4-[5-(4-alkoxyphenyl)-2,5-dimethylpyrrolidine-1-oxyl-2-yl]benzoic acids (1), which are the key intermediates for the synthesis of chiral organic radical liquid crystalline compounds and are crystallized to give racemic compounds. Racemic acid 1 [(+/-)-1] with a long alkyl chain (C7 to C13) could be resolved by the conventional diastereomeric salt formation using (R)- or (S)-1-phenylethylamine (2) as the resolving agent, whereas resolution of (+/-)-1 with a short alkyl chain (C4 to C6) was unsuccessful. Use of six equiv of (R)- or (S)-2 for the initial diastereomeric salt formation of (+/-)-1 with a C7-C13 alkyl chain, followed by recrystallization of the resulting salts once or twice, gave 2S,5S- or 2R,5R-enriched 1, respectively, in an ee range of 75-92% and with an overall recovery of 11-27%, based on the original quantity of (+/-)-1.     

Genotype effects on ABA consumption and somatic embryo maturation in interior spruce (Picea glaucaxengelmanni)

Abscisic acid (ABA) plays an important role during somatic embryo development and maturation in coniferous species. The purpose of this research was to study ABA utilization by genotypes with different embryo maturation capabilities in interior spruce. Cell lines ISP11 and ISP48 were of high embryo maturation capability. By contrast, the tissue of line ISP16 contained numerous immature embryos, but only a few mature embryos developed. Exogenous ABA, i.e. S-ABA [(+)-cis, trans-ABA], racemic ABA, or ABA isomers were added into suspension cultures at a final concentration of 30 microM. In comparison to racemic ABA and ABA isomers, S-ABA reduced tissue proliferation the most. In all cell lines, about half of the racemic ABA was used within 2 weeks; the remaining ABA was (-)-cis, trans-ABA. The concentration of ABA showed little change thereafter. In the cultures supplied with ABA isomers, about half of (+/-)-cis, trans-ABA was utilized during 22 d. By contrast, (+/-)-trans, trans-ABA was hardly used, especially in line ISP16. S-ABA was almost completely metabolized by line ISP11. However, approximately 28% and 22% of the S-ABA remained in the culture of cell lines ISP16 and ISP48, respectively. Cell line ISP16 grew the fastest in culture. By 3 weeks, S-ABA consumption by ISP11 and ISP48 on the basis of tissue growth was, respectively, 2.2-fold and 3.4-fold greater than that of ISP16. A higher ratio of dihydrophaseic acid to phaseic acid existed with cell lines of higher embryo maturation capability, especially when the exogenously supplied ABA was chemically synthesized.     

Transfer of chirality by dithiophosphate ligands and chiral discrimination in the stereoselective formation of square-planar Ni(II) complexes

In the formation reaction of Ni(2+) with the chiral racemic ligand, (R)(R)bdtp(-)/(S)(S)bdtp(-), bdtp(-) = [SSPOCH)CH(3))CH(CH(3))O](-), cyclo- O,O'-[1,2-dimethylethylene] dithiophosphato ion, the meso-complex Ni[(R)(R)(lambda)bdtp][(S)(S)(delta)-bdtp] is stereoselectively produced. The meso-complex was compared with the enantiopure crystals of (+)(589)Ni[(R)(R)(lambda)bdtp](2) or (-)(589)Ni[(S)(S)(delta)bdtp](2), as well as racemic crystals, rac-(+/-)Ni[bdtp](2), which were prepared from the solution containing the two enantiomers in a 1:1 ratio. Dissociation constants in solutions indicate different stability of the meso and enantiopure complexes depending on the solvent, whereas a more efficient crystal packing, weak H-bonding, and nonbonding interactions contribute to stabilization of the meso-species over the racemic one. Molecular structures show that the outer five-membered ligand ring adopts the half-chair conformation C(2) with either the lambda or the delta chirality and the methyl groups are in equatorial (e) positions. Enantiopure ligands of (+)(589)Ni[(R)(R)(lambda)bdtp](2) and (-)(589)Ni[(S)(S)(delta)bdtp](2) induce chirality into the symmetric SSNiSS chromophore with slightly helical distortion. Thus, their CD spectra exhibit weak negative or positive Cotton effects at 662 nm. CD spectra in L(+)- and D(-)diethyltartrate of the meso-complex and racemic crystal, rac-(+/-)Ni[bdtp](2), exhibit different weak Cotton effects of opposite sign. Complexes dissociate in methanol; rac-(+/-)Ni[bdtp](2) in methanol undergoes a crystallization-induced second-order asymmetric transformation which finally yields crystals of the meso-Ni[(R)(R)(lambda)bdtp][(S)(S)(delta)bdtp] complex.     

Preferential enrichment: significant influence of minor molecular modification on the mode of polymorphic transition during crystallization

The relationship between the molecular structure and the occurrence of Preferential Enrichment, an unusually symmetry-breaking enantiomeric resolution phenomenon observed upon simple recrystallization of a certain kind of racemic crystals from organic solvents, has been investigated with respect to a new series of racemic ammonium sulfonate compounds. Racemic [2-[4-(3-ethoxy-2-hydroxypropoxy)phenylcarbamoyl]ethyl]trimethylammonium p-iodobenzenesulfonate [(+/-)-1a] can show preferential enrichment by simple recrystallization from EtOH, whereas its terminal methoxy and propoxy derivatives [(+/-)-1b and (+/-)-1c] are unable to do so. The influence of such a minor molecular modification on Preferential Enrichment has been rationalized in terms of the change of the mode of polymorphic transition during crystallization, which has been confirmed by in situ ATR-FTIR (ReactIR) spectroscopy in solution and in the solid state and by X-ray crystallographic analysis of their single crystals.     

Effects of isoflurane on voltage-dependent calcium fluxes in rabbit T-tubule membranes: comparison with alcohols

The effects of racemic (+/-) and (+)- and (-)-stereoisomers of isoflurane on depolarization-induced (45)Ca(2+) fluxes mediated by voltage-dependent Ca(2+) channels were investigated in transverse tubule membrane vesicles from rabbit skeletal muscle. In the concentration range 0.5 to 2 mM, (+/-)-isoflurane inhibited (45)Ca(2+) fluxes and functionally modulated the effects of the Ca(2+) channel antagonist nifedipine (1-10 microM). Isoflurane-induced inhibition of (45)Ca(2+) fluxes was not significantly affected by pretreatment with either pertussis toxin (5 microg/ml) or phorbol 12-myristate 13-acetate (50 nM). Further experiments indicated that there were no significant differences between (+)- and (-)-stereoisomers of isoflurane with respect to the extent of inhibition of (45)Ca(2+) fluxes. Radioligand binding studies indicated that racemic and (+)- and (-)-isoflurane were equally effective in displacing the specific binding of [(3)H]PN 200-110 to transverse tubule membranes. There were no apparent differences between the effects of (+)- and (-)-isoflurane on the characteristics of [(3)H]PN 200-110 binding. Although the concentrations of isoflurane for the inhibitions of (45)Ca(2+) fluxes and radioligand bindings were similar, the concentrations of n-alcohols required for the inhibition of (45)Ca(2+) fluxes were lower than those for the displacement of radioligand. Comparison of the data for the displacement of [(3)H]PN 200-110 binding and the inhibition of (45)Ca(2+) fluxes by isoflurane and by n-alcohols suggested that both isoflurane and n-alcohols may have more than a single binding site. In conclusion, results indicate that isoflurane, independent of intracellular Ca(2+) levels, nonstereospecifically inhibits the function of voltage-dependent Ca(2+) channels and this effect is mediated through multiple binding sites.     

The mechanism of binding of dihydropyridine calcium channel blockers to rat brain membranes

Detailed kinetic and equilibrium studies of the binding of two radiolabeled 1,4-dihydropyridine calcium antagonists to putative calcium channels in rat brain membranes were performed. (+/-)-[3H]Nitrendipine, a racemic ligand, and (+)-[3H]isopropyl 4-(2,1,3-benzoxadiazol-4-yl)-1, 4-dihydro-2,6-dimethyl-5-methoxycarbonylpyridine-3-carboxylate (PN200-110), a pure isomer, were used and their binding properties were quantitated and compared. Analysis of equilibrium binding revealed a single high affinity component for each radioligand with the same density of binding sites for both ligands. Association rates were determined over a 60-fold range of concentration of each radioligand. For both radioligands, the pseudo-first order association time courses were biphasic with the rate of the faster component dependent on radioligand concentration and the rate of the slower component independent of both the structure of the radioligand and the concentration of the radioligand. Dissociation rates were determined after various times of association. The dissociation of the optically pure radioligand, (+)-[3H]PN200-110, was monophasic at all association times, consistent with a single bound species being present throughout association. However, (+/-)-[3H]nitrendipine dissociation was biphasic after short association times (1-10 min). The biphasic dissociation observed with (+/-)-[3H]nitrendipine is consistent with the two optical isomers binding with approximately the same association rate but having different dissociation rates. These results appear to reflect the existence of two interconvertible binding states of the putative calcium channel in the membrane, one which binds the radioligands with high affinity in a simple bimolecular reaction and one which has no detectable affinity for the ligands. This mechanism of isomerization before ligand binding has been modeled by numerical solution of the differential equations of the scheme providing estimates of the rate constants for each reaction in the scheme.     

Molecular modeling, structure--activity relationships and functional antagonism studies of 4-hydroxy-1-methyl-4-(4-methylphenyl)-3-piperidyl 4-methylphenyl ketones as a novel class of dopamine transporter inhibitors.

We previously disclosed the discovery of 4-hydroxy-1-methyl-4-(4-methylphenyl)-3-piperidyl 4-methylphenyl ketone (3) as a novel class of dopamine transporter (DAT) inhibitors and showed that (+/-)-3 has a significant functional antagonism against cocaine in vitro. Our previous preliminary structure-activity relationship study led to identification of a more potent DAT inhibitor [(+/-)-4] but this compound failed to show any significant functional antagonism. To search for more potent analogues than 3 but still displaying significant functional antagonism, further SARs, molecular modeling studies and in vitro pharmacological evaluation of this novel class of DAT inhibitors were performed. Sixteen new analogues were synthesized in racemic form and evaluated as DAT inhibitors. It was found that seven new analogues are reasonably potent DAT inhibitors with K(i) values of 0.041--0.30 and 0.052--0.16 microM in [(3)H]mazindol binding and inhibition of DA reuptake. Chiral isomers of several potent DAT inhibitors were obtained through chiral HPLC separation and evaluated as inhibitors at all the three monoamine transporter sites. In general, the (-)-isomer is more active than the (+)-isomer in inhibition of DA reuptake and all the (-)-isomers are selective inhibitors at the DAT site. Evaluation of cocaine's effect on dopamine uptake in the presence and absence of (+)-3 and (-)-3 showed that (-)-3 is responsible for the functional antagonism obtained with the original lead (+/-)-3. Out of the new compounds synthesized, analogue (+/-)-20, which is 8- and 3-fold more potent than (+/-)-3 in binding and inhibition of DA reuptake, appeared to have improved functional antagonism as compared to (+/-)-3.     

Resolution of 4-amino-cyclopentanecarboxylic acid methyl esters using hydrolytic enzymes.

A number of esterases (EC 3.1.1.1) and lipases (EC 3.1.1.3) of microbial and mammalian origin were screened for the ability to resolve racemic 4-amino-cyclopentanecarboxylic acid methyl ester derivatives as potential intermediates in the production of carbocyclic nucleosides. Surprisingly, functionalization of the remote amino group had a profound effect on both the rate and enantioselectivity of hydrolysis of the methyl ester. 4-(Benzoylamino)-2-cyclopentenecarboxylic acid, methyl ester (V) with pig liver esterase gave the highest enantioselectivity. The residual ester, which was of the correct absolute stereochemistry [(+) 1S, 4R] for carbocyclic nucleoside synthesis, could be obtained in high optical purity. Optimization of pH, solvent type, and concentration improved the enantioselectivity of the process by a further twofold.     

Resolution and synthesis of optically active alcohols with immobilized water-soluble proteins from green pea, soybean and buckwheat as new bio-catalysts

Kinetic resolution of racemic alcohols, (+/-)-1-(4-substituted phenyl)ethanol and (+/-)-1-(2-naphthyl)ethanol, was done with immobilized green pea, soybean, or buckwheat proteins. The resolution was done stereoselectively by oxidizing only one enantiomer of a racemic alcohol to leave an optically active alcohol with a high purity. In addition, each protein could be reused consecutively at least three times without any decrease of yield or optical purity.     

Metabolism of deuterium-labeled jasmonic acid and OPC 8:0 in the potato plant (Solanum tuberosum L.)

The metabolism of deuterium-labeled (+/-)-jasmonicacid and 3-oxo-2-[(Z)pent-2'-enyl]-cyclopentan-1- octanoic acid in potato (Solanum tuberosum L.) was examined by using cultures of potato single-node stems. Deuterium-labeled (+/-)-jasmonic acid and 3-oxo-2-[(Z) pent-2'-enyl]cyclopentan-1-octanoic acid, which had been prepared from commercially available methyl (+/-)-jasmonate, were fed to the cultures, and the metabolites were extracted from the plants and analyzed by a liquid chromatography-selected ion monitoring system. The metabolism of deuterium-labeled (+/-)-jasmonic acid and 3-oxo-2-[(Z)-2'-pentenyl]cyclopentan-1-octanoic acid to 5' and 4'-O-glucopyranosyloxyjasmoic acids was strongly suggested.     

Induction of apoptosis by pterocarpans from Platymiscium floribundum in HL-60 human leukemia cells

(+)-2,3,9-Trimethoxy-pterocarpan (1) (+)-3,9-dimethoxy-pterocarpan [(+)-homopterocarpin] (2), (+)-3-hydroxy-9-methoxy-pterocarpan [(+)-medicarpin] (3) and (+)-3,4-dihydroxy-9-methoxy-pterocarpan [(+)-vesticarpan] (4) are cytotoxic pterocarpans isolated from the native Brazilian plant Platymiscium floribundum. The purpose of the present study was to examine whether induction of apoptosis and/or inhibition of DNA synthesis is involved in the cytotoxicity of these pterocarpans in human leukemia cells. The effect on cell viability determined using the trypan exclusion assay revealed that all compounds tested reduced the number of viable cells, while only in the presence of 3 and 4, there was an increase of nonviable cells. The analysis of membrane integrity and morphological modifications by flow cytometry in the presence of these two compounds indicated that treated cells undergo necrosis, while 1 and 2 trigger apoptosis. DNA synthesis seemed to be affected since BrdU incorporation was inhibited in a dose-dependent manner in the presence of all tested compounds. Pterocarpan treatment also induced an increase in the amount of subdiploid DNA, indicating internucleosomal DNA breakdown, mitochondrial depolarization and caspase-3 activation, which indicate apoptosis induction.     

Further exploration of 1-[2-[Bis-(4-fluorophenyl)methoxy]ethyl]piperazine (GBR 12909): role of N-aromatic,N-heteroaromatic,and 3-oxygenated N-phenylpropyl substituents on affinity for the dopamine and serotonin transporter

A series of N-aromatic, N-heteroaromatic, and oxygenated N-phenylpropyl derivatives of 1-(2-benzhydryloxyethyl)-piperazine and 1-[2-[bis-(4-fluorophenyl)methoxy]ethyl]piperazine, analogues of GBR 12909 (1a) and 12935 (1b), was synthesized and examined for their dopamine (DAT) and serotonin (SERT) transporter binding properties. One of these compounds, racemic 3-[4-(2-benzhydryloxyethyl)piperazin-1-yl]-1-(3-fluorophenyl)-propan-1-ol (33), had DAT affinity as good as, or better than, GBR 12909 and 12935, and was more selective for DAT over SERT than the GBR compounds. Both trans- (43) and cis- (47) (+/-)-2-(4-[2-[bis-(4-fluorophenyl)-methoxy]ethyl]piperazin-1-ylmethyl)-6-methoxy-1,2,3,4-tetrahydronaphthalen-1-ol had relatively good SERT selectivity and, as well, showed high affinity for SERT.     

Characterization and photoaffinity labeling of receptor sites for the Ca2+ channel inhibitors d-cis-diltiazem, (+/-)-bepridil, desmethoxyverapamil, and (+)-PN 200-110 in skeletal muscle transverse tubule membranes

In order to further understand the molecular nature of the voltage-sensitive Ca2+ channel in skeletal muscle, we have performed classical radioligand binding studies and photoaffinity labeling with different types of tritiated inhibitors of the Ca2+ channel. The equilibrium dissociation constants (KD) for (-)-[3H]desmethoxyverapamil, d-cis-[3H]diltiazem, and (+/-)-[3H]bepridil at their receptor sites in skeletal muscle transverse tubule membranes are: 1.5 +/- 0.5, 50 +/- 5, and 20 +/- 5 nM, respectively. Maximum binding capacities in picomoles/milligram of protein were: 70 +/- 10 for (-)-[3H]desmethoxyverapamil, 50 +/- 15 for d-cis-[3H]diltiazem, and 75 +/- 15 for (+/-)-[3H]bepridil. The kinetics of association at 10 degrees C for the three types of tritiated compounds were relatively slow (3 X 10(5) M-1 S-1 for (-)-[3H]desmethoxyverapamil, 8 X 10(3) M-1 S-1 for d-cis-[3H]diltiazem, and 4.2 X 10(5) M-1 S-1 for (+/-)-[3H]bepridil). The dissociation of (-)-[3H]desmethoxyverapamil and d-cis-[3H]diltiazem from their receptor sites was also a slow process with half-lives of dissociation of 33 and 36 min, respectively. Competition studies using the three tritiated ligands suggest that they bind to the same receptor site which appears to be in a 1:1 stoichiometry with the dihydropyridine receptor. Photoaffinity labeling with high intensity ultraviolet light in the presence of (+/-)-[3H]bepridil or d-cis[3H]diltiazem resulted in the specific covalent incorporation of radioactivity into a polypeptide of Mr 170,000 +/- 10,000. A polypeptide of Mr 170,000 was also specifically labeled in photoaffinity labeling experiments using the high affinity dihydropyridine derivative (+)-[3H]PN 200-100.