Highly stereoselective acid-catalyzed homonucleophilic substitution reactions

Enantiomeric 3-O-methyltemazepam and 3-O-ethyltemazepam were highly stereoselectively substituted by the 3-methoxy group of methanol in acidic anhydrous methanol and by the 3-ethoxy group of ethanol in acidic anhydrous ethanol, respectively. The stereoselectivity of the homonucleophilic substitution reactions was determined by circular dichroism spectropolarimetry and gas chromatography-mass spectrometry. In anhydrous solutions containing 0.5 M D2SO4 at 50 degrees C, for example, the stereoselectivity was approximately 63:1 for enantiomeric 3-O-methyltemazepam in CD3OD and approximately 94:1 for enantiomeric 3-O-ethyltemazepam in C2D5OD. The high stereoselectivity at C3 position was primarily due to the presence of a methyl group at N1 position.     

Transamination as a side-reaction catalyzed by alanine racemase of Bacillus stearothermophilus

The pyridoxal form of alanine racemase of Bacillus stearothermophilus was converted to the pyridoxamine form by incubation with its natural substrate, D- or L-alanine, under acidic conditions: the enzyme loses its racemase activity concomitantly. The pyridoxamine form of the enzyme returned to the pyridoxal form by incubation with pyruvate at alkaline pH. Thus, alanine racemase catalyzes transamination as a side function. In fact, the apo-form of the enzyme abstracted tritium from [4'-3H]pyridoxamine in the presence of pyruvate. A mutant enzyme containing alanine substituted for Lys39, whose epsilon-amino group forms a Schiff base with the C4' aldehyde of pyridoxal 5'-phosphate in the wild-type enzyme, was inactive as a catalyst for racemization as well as transamination. However, when methylamine was added to the mutant enzyme, it became active in both reactions. These results suggest that the epsilon-amino group of Lys39 participates in both racemization and transamination when catalyzed by the wild-type enzyme.     

The T cell-B cell interaction via OX40-OX40L is necessary for the T cell-dependent humoral immune response

Recent in vitro studies have established that activated B cells express OX40 ligand (L), a member of the tumor necrosis factor/nerve growth factor family of cytokines, and become stimulated to proliferate and secrete immunoglobulin (Ig) after cross-linking of OX40L by its counterreceptor OX40, which is expressed on activated T cells. In the present study we investigated the in vivo role of this receptor-ligand pair for the interaction of T and B cells in the course of the T-dependent B cell response against 2,4,6 trinitro-phenyl-keyhole limpet hemocyanin. First, we showed that OX40 is maximally expressed by T cells in the periarteriolar lymphoid sheath (PALS) 3 d after primary immunization. These OX40+ cells are located in close proximity to antigen-specific, activated B cells. Second, we demonstrated that blocking of OX40-OX40L interaction with polyclonal anti-OX40 antibody or with antibodies against certain peptide sequences within its extracellular domain resulted in a profound decrease of the anti-hapten IgG response, whereas the antihapten IgM response was grossly unchanged. Third, we showed that this antibody treatment leads to an inhibition of the development of PALS-associated B cell foci, whereas the formation of germinal centers remained intact. Finally, our data suggest that, whereas B cell memory development was not impaired by anti-OX40 administration, OX40-OX40L interaction seems to be crucial in the secondary immune response. We conclude from these data that the OX40-OX40L interaction in vivo is necessary for the differentiation of activated B cells into highly Ig-producing cells, but is not involved in other pathways of antigen-driven B cell differentiation such as memory cell development in the germinal centers.     

Active-site Arg --> Lys substitutions alter reaction and substrate specificity of aspartate aminotransferase

Arg386 and Arg292 of aspartate aminotransferase bind the alpha and the distal carboxylate group, respectively, of dicarboxylic substrates. Their substitution with lysine residues markedly decreased aminotransferase activity. The kcat values with L-aspartate and 2-oxoglutarate as substrates under steady-state conditions at 25 degrees C were 0.5, 2.0, and 0.03 s-1 for the R292K, R386K, and R292K/R386K mutations, respectively, kcat of the wild-type enzyme being 220 s-1. Longer dicarboxylic substrates did not compensate for the shorter side chain of the lysine residues. Consistent with the different roles of Arg292 and Arg386 in substrate binding, the effects of their substitution on the activity toward long chain monocarboxylic (norleucine/2-oxocaproic acid) and aromatic substrates diverged. Whereas the R292K mutation did not impair the aminotransferase activity toward these substrates, the effect of the R386K substitution was similar to that on the activity toward dicarboxylic substrates. All three mutant enzymes catalyzed as side reactions the beta-decarboxylation of L-aspartate and the racemization of amino acids at faster rates than the wild-type enzyme. The changes in reaction specificity were most pronounced in aspartate aminotransferase R292K, which decarboxylated L-aspartate to L-alanine 15 times faster (kcat = 0.002 s-1) than the wild-type enzyme. The rates of racemization of L-aspartate, L-glutamate, and L-alanine were 3, 5, and 2 times, respectively, faster than with the wild-type enzyme. Thus, Arg --> Lys substitutions in the active site of aspartate aminotransferase decrease aminotransferase activity but increase other pyridoxal 5'-phosphate-dependent catalytic activities. Apparently, the reaction specificity of pyridoxal 5'-phosphate-dependent enzymes is not only achieved by accelerating the specific reaction but also by preventing potential side reactions of the coenzyme substrate adduct.     

Stereo- and regioselective hydroxylation of alpha-ionone by Streptomyces strains

A total of 215 Streptomyces strains were screened for their capacity to regio- and stereoselectively hydroxylate beta- and/or alpha-ionone to the respective 3-hydroxy derivatives. With beta-ionone as the substrate, 15 strains showed little conversion to 4-hydroxy- and none showed conversion to the 3-hydroxy product as desired. Among these 15 Streptomyces strains, S. fradiae Tü 27, S. arenae Tü 495, S. griseus ATCC 13273, S. violaceoniger Tü 38, and S. antibioticus Tü 4 and Tü 46 converted alpha-ionone to 3-hydroxy-alpha-ionone with significantly higher hydroxylation activity compared to that of beta-ionone. Hydroxylation of racemic alpha-ionone [(6R)-(-)/(6S)-(+)] resulted in the exclusive formation of only the two enantiomers (3R,6R)- and (3S, 6S)-hydroxy-alpha-ionone. Thus, the enzymatic hydroxylation of alpha-ionone by the Streptomyces strains tested proceeds with both high regio- and stereoselectivity.     

Stereoselective HPLC bioanalysis of atenolol enantiomers in plasma: application to a comparative human pharmacokinetic study

An enantioselective HPLC bioassay has been developed relying on extraction of (R)- and (S)-atenolol from alkalinized plasma or serum (pH > 12) into dichloromethane containing 5% (v/v) 1-butanol followed by an achiral derivatization of the drug with phosgene leading to (R)- and (S)-oxazolidine-2-one derivatives. Under these conditions there was quantitative conversion of the acetamido group to the corresponding nitrile. These stable derivatives were separated on a (R,R)-diaminocyclohexane-dinitrobenzoyl chiral stationary phase [(R,R)-DACH-DNB] using dichloromethane/methanol 98/2 as mobile phase. Determination limits of 0.5 ng for (R)- and 0.6 ng for (S)-atenolol could be achieved using fluorimetric detection. The assay was applied to a human pharmacokinetic study which was performed in a randomized cross-over, double-blind fashion in 12 healthy volunteers, administering single oral doses of 100 mg (R,S)-, 50 mg (R)-, and 50 mg (S)-atenolol. AUC0-24 and Cmax values of (R)-atenolol were slightly but significant higher than those of (S)-atenolol. The R/S ratios were 1.09 for AUC(R)/AUC(S) and 1.03 for Cmax (R)/Cmax(S) (P < 0.01) respectively after administration of the racemic drug. However, there were no difference between AUC, Cmax, and t1/2 values of each enantiomer, whether they were administered as single enantiomers or in the form of its racemic mixture.     

Involvement of OX40-OX40L interactions in the intestinal manifestations of the murine acute graft-versus-host disease

BACKGROUND & AIMS: The intestinal histology of murine semiallogeneic graft-versus-host (GVH) disease is characterized by lymphocytic infiltrates, crypt hyperplasia, and villous atrophy. Mechanisms of T cell-mediated changes of the mucosal architecture were investigated. METHODS: The rate of cellular apoptosis and proliferation, changes in the composition of extracellular matrix (ECM), and the role of OX40-OX40L interactions in the pathogenesis of villous atrophy and crypt hyperplasia were examined. RESULTS: The rate of apoptosis and the number of proliferating cells were significantly increased in GVH animals compared with control animals. In addition, expression of tenascin, an ECM component, was down-regulated in GVH animals. Inhibition of OX40-OX40L interactions in GVH animals by administration of an OX40-Ig fusion protein completely prevented the development of crypt hyperplasia and villous atrophy in GVH animals. Tenascin expression was up-regulated in OX40-Ig-treated mice compared with GVH animals, suggesting an important function of this ECM component in mucosal repair. CONCLUSIONS: The OX40-OX40L interaction is crucial in the pathogenesis of GVH, a T cell-mediated intestinal disease. The data suggest that the ECM component tenascin is probably relevant for the regeneration and maintenance of intestinal tissue architecture.     

2-substituted (2SR)-2-amino-2-((1SR,2SR)-2-carboxycycloprop-1-yl)glycines as potent and selective antagonists of group II metabotropic glutamate receptors. 2. Effects of aromatic substitution, pharmacological characterization, and bioavailability

In this paper we describe the synthesis of a series of alpha-substituted analogues of the potent and selective group II metabotropic glutamate receptor (mGluR) agonist (1S,1'S,2'S)-carboxycyclopropylglycine (2, L-CCG 1). Incorporation of a substitutent on the amino acid carbon converted the agonist 2 into an antagonist. All of the compounds were prepared and tested as a series of four isomers, i.e., two racemic diastereomers. On the basis of the improvement in affinity realized for the alpha-phenylethyl analogue 3, in this paper we explored the effects of substitution on the aromatic ring as a strategy to increase the affinity to these compounds for group II mGluRs. Affinity for group II mGluRs was measured using [3H]glutamic acid (Glu) binding in rat forebrain membranes. Antagonist activity was confirmed for these compounds by measuring their ability to antagonize (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid-induced inhibition of forskolin stimulated cyclic-AMP in RGT cells transfected with human mGluR2 and mGluR3. Meta substitution on the aromatic ring of 3 with a variety of substituents, both electron donating (e.g., methyl, hydroxy, amino, methoxy, phenyl, phenoxy) and electron withdrawing (e.g., fluorine, chlorine, bromine, carboxy, trifluoromethyl) gave from 1.5- to 4.5-fold increases in affinity. Substitution with p-fluorine, as in 97 (IC50 = 0.022 +/- 0.002), was the exception. Here, a greater increase in affinity was realized than for either the ortho- or meta-substituted analogues; 97 was the most potent compound resulting from monosubstitution of the aromatic. At best, only modest increases in affinity were realized for certain compounds bearing either two chlorines or two fluorines, and two methoxy groups gave no improvement in affinity (all examined in a variety of substitution patterns). Three amino acids, 4, 5, and 104, were resolved into their four constituent isomers, and affinity and functional activity for group II mGluRs was found to reside solely in the S,S,S-isomers of each, consistent with 1. With an IC50 = 2.9 +/- 0.6 nM, the resolved xanthylmethyl compound 168 was the most potent compound from this SAR. Amino acid 168 demonstrated high plasma levels following intraperitoneal (i.p.) administration and readily penetrated into the brain. This compound, however, had only limited (approximately 5%) oral bioavailability. Systemic administration of 168 protected mice from limbic seizures produced by the mGluR agonist 3,5-dihydroxyphenylglycine, with an ED50 = 31 mg/kg (i.p., 60 min preinjection). Thus, 168 represents a valuable tool to study the role of group II mGluRs in disease.     

Synthesis and serotonin receptor affinities of a series of trans-2-(indol-3-yl)cyclopropylamine derivatives

A series of four racemic ring-substituted trans-2-(indol-3-yl)cyclopropylamine derivatives was synthesized and tested for affinity at the 5-HT1A receptor, by competition with [3H]-8-OH-DPAT in rat hippocampal homogenates, and for affinity at the agonist-labeled cloned human 5-HT2A, 5-HT2B, and 5-HT2C receptor subtypes. None of the compounds had high affinity for the 5-HT1A receptor, with the 5-methoxy substitution being most potent (40 nM). At the 5-HT2A and 5-HT2B receptor isoforms, most of the compounds lacked high affinity. At the 5-HT2C receptor, however, affinities were considerably higher. The 5-fluoro-substituted compound was most potent, with a Ki at the 5-HT2C receptor of 1.9 nM. In addition, the 1R,2S-(-) and 1S,2R-(+) enantiomers of the unsubstituted compound were also evaluated at the 5-HT2 isoforms. While the 1R,2S enantiomer had higher affinity at the 5-HT2A and 5-HT2B sites, the 1S,2R isomer had highest affinity at the 5-HT2C receptor. This reversal of stereoselectivity may offer leads to the development of a selective 5-HT2C receptor agonist. The cyclopropylamine moiety therefore appears to be a good strategy for rigidification of the ethylamine side chain only for tryptamines that bind to the 5-HT2C receptor isoform.     

Comparative repellency of commercial formulations of deet, permethrin and citronellal against the mosquito Aedes aegypti, using a collagen membrane technique compared with human arm tests

The plasma concentrations and urinary excretions of bisoprolol enantiomers in four Japanese male healthy volunteers after a single oral administration of 20 mg of racemic bisoprolol were evaluated. The AUC(infinity) and elimination half-life of (S)-(-)-bisoprolol were slightly larger than those of (R)-(+)-bisoprolol in all subjects. The metabolic clearance of (R)-(+)-bisoprolol was significantly (P < 0.05) larger than that of (S)-(-)-bisoprolol (S/R ratio: 0.79+/-0.03), although the difference was small. In contrast, no stereoselective in vitro protein binding of bisoprolol in human plasma was found. An in vitro metabolic study using recombinant human cytochrome P450 (CYP) isoforms indicated that oxidation of both bisoprolol enantiomers was catalyzed by the two isoforms, CYP2D6 and CYP3A4. CYP2D6 metabolized bisoprolol stereoselectively (R > S), whereas the metabolism of bisoprolol by CYP3A4 was not stereoselective. The S/R ratio of the mean clearance due to renal tubular secretion was 0.68, indicating a moderate degree of stereoselective renal tubular secretion. These findings taken together suggest that the small differences in the pharmacokinetics between (S)-(-)- and (R)-(+)-bisoprolol are mainly due to the stereoselectivity in the intrinsic metabolic clearance by CYP2D6 and renal tubular secretion.     

Synthesis and biological evaluation of substituted flavones as gastroprotective agents

Flavone (1) was found to protect against ethanol-induced gastric damage in rats; however, it is known that certain compounds in the flavone class, including flavone itself, are inducers of hepatic drug metabolizing enzymes. With the hope of identifying gastroprotective flavones that have minimal effects on drug metabolizing enzymes, we have synthesized and evaluated selected flavone analogs. Gastroprotective potency in the ethanol model was retained by methoxy substitution in the 5-position (4) and by methoxy (12) or methyl (14) substitution in the 7-position. A number of substituted analogs of the potent molecule 5-methoxyflavone (4) were also synthesized, and in many cases, these substitutions provided gastroprotective molecules. In order to assess liver enzyme induction potential, two of the gastroprotective flavones, 7-methoxyflavone (12) and 5-methoxy-4'-fluoroflavone (26), were examined for their effect on liver microsomal cytochrome P450 and 7-ethoxyresorufin O-dealkylase (CYP1A) activity. These two compounds caused minimal changes in the cytochrome P450 concentration and were considerably less potent than beta-naphthoflavone as inducers of CYP1A enzyme activity. Furthermore, following oral administration to rats, 5-methoxy-4'-fluoroflavone (26) was found to protect against indomethacin-induced gastric damage. These results indicate that, through appropriate substitution, flavones can be obtained that are gastroprotective but have minimal effects on drug-metabolizing enzymes.     

Synthesis and thermodynamics of oligonucleotides containing chirally pure R(P) methylphosphonate linkages

Methylphosphonate (MP) oligodeoxynucleotides (MPOs) are metabolically stable analogs of conventional DNA containing a methyl group in place of one of the non-bonding phosphoryl oxygens. All 16 possible chiral R(P) MP dinucleotides were synthesized and derivatized for automated oligonucleotide synthesis. These dimer synthons can be used to prepare (i) all-MP linked oligonucleotides having defined R(P) chirality at every other position (R(P) chirally enriched MPOs) or (ii) alternating R(P) MP/phosphodiester backbone oligonucleotides, depending on the composition of the 3'-coupling group. Chirally pure dimer synthons were also prepared with 2'-O-methyl sugar modifications. Oligonucleotides prepared with these R(P) chiral methylphosphonate linkage synthons bind RNA with significantly higher affinity than racemic MPOs.     

Activation of OX40 signal transduction pathways leads to tumor necrosis factor receptor-associated factor (TRAF) 2- and TRAF5-mediated NF-kappaB activation

We investigated the intracellular signaling of OX40, a member of the tumor necrosis factor receptor family. Activation of NF-kappaB in OX40-transfected HSB-2 cells was detected by electrophoretic mobility shift assay within 30 min after the binding of the ligand gp34. In vitro binding experiments showed that tumor necrosis factor receptor-associated factor (TRAF) 1, TRAF2, TRAF3, and TRAF5 but not TRAF4 associated with glutathione S-transferase-OX40 fusion protein. The cotransfection experiments using human embryo kidney cell derived HEK 293T cells showed that TRAF2, TRAF3, and TRAF5 associated with OX40 in vivo. Studies with OX40 deletion mutants demonstrated that the cytoplasmic portion consisting of amino acid sequence 256-263 (GGSFRTPI) was required for the association with TRAFs and NF-kappaB activation. The introduction of the dominant negative mutants of TRAF2 and TRAF5 into HSB-2-OX40 cells suppressed NF-kappaB activation in a dose-dependent manner. In addition, the introduction of TRAF3 together with the dominant negative mutants of TRAF2 or TRAF5 further reduced NF-kappaB activation. These results indicate that the NF-kappaB activation resulting from OX40 stimulation is mediated by both TRAF2 and TRAF5, and is likely to be negatively modulated by TRAF3.     

Studies towards the synthesis of the hypermodified nucleoside of rat liver phenylalanine transfer ribonucleic acid: improved synthesis of the base beta-hydroxywybutine

An improved synthesis of the key intermediates (3 and 8) for the synthesis of beta-hydroxywybutines [[R-(R*,S*)]- and [S-(R*,R*)]-4], the most probable structures for the minor base from rat liver tRNA(Phe), has been achieved by the Wittig reaction between 1-benzyl-7-formylwye (1) and the phosphorane derived from (R)-2-[(methoxycarbonyl)amino]-3-(triphenylphosphonio)propanoate (10), followed by methylation, OsO4 oxidation, and cyclocondensation with COCl2 in the presence of pyridine. The racemic forms of beta-hydroxywybutines [(R*,S*)- and (R*,R*)-4], which were required for the determination of the optical purity of [R-(R*,S*)]- and [S-(R*,R*)]-4 by means of chiral HPLC, were conveniently prepared through pyrolysis of the cyclic carbonate 3 followed by NaBH4 reduction and catalytic hydrogenolysis. The samples of [R-(R*,S*)]- and [S-(R*,R*)]-4 were thus shown to be optically pure.     

Role of the colchicine ring A and its methoxy groups in the binding to tubulin and microtubule inhibition

The roles of the methoxy substituents on ring A of two ring colchicine (COL) analogues were probed by the synthesis of a number of drugs and the examination of their effect on binding to tubulin, inhibition of microtubule assembly, and induction of GTPase activity. Selective elimination of ring A methoxy groups at positions 2, 3, and 4 weakened all three processes. The effects on binding and inhibition were independent of the nature of ring C (or C'). Specifically, excision of the 2- or 3-methoxy groups weakened binding by ca. 0.4 kcal mol-1, while that of the 4-methoxy group of ring A was weakened by 1.36 +/- 0.15 kcal mol-1. The effect on the inhibition of microtubule assembly, expressed as the equilibrium constant for the binding of the tubulin-drug complex to the end of a microtubule, was more complex and strongly dependent on the nature of ring C (or C'). This was attributed to the abilities of various groups on ring C' to overcome the wobbling in the tubulin-drug complex introduced by the weakening of the anchoring provided by ring A. It is concluded that ring A of COL is not germane to the mechanism of the inhibition of tubulin self-assembly. It serves only as a complex-stabilizing anchor. The control of this process resides in the interactions that key oxygen atoms of ring C of COL or C' of structural analogues establish with the protein. It is proposed that the 4-methoxy group of ring A serves as a key attachment point for immobilization of the drugs on the protein.     

Pharmacokinetics and bioavailability of stereoisomeric analogues of ifosfamide

Analogues of ifosfamide (IPA): racemic bromofosfamide (+/-)-(R,S)-KM 135, racemic chlorobromofosfamide (+/-)-(R,S)-CBM 4a and levorotatory enantiomer of chlorobromofosfamide (-)-(S)-CBM 11 belong to the known group of oxazaphosphorines. Antitumor activity of those three selected compounds, investigated in the Institute of Immunology and Experimental Therapy, Polish Academy of Sciences in Wroc?aw against L1210 leukemia, Lewis lung carcinoma and B16 melanoma tumor model system showed cytostatic activity higher than the referential - ifosfamide [1]. These interesting data prompted us to conduct the preclinical pharmacokinetic studies in rats.     

Pharmacological characterization of the novel discriminative stimulus effects of a low dose of cocaine

Twelve rats were trained to press one lever after cocaine injection (3 mg/kg i.p.) and another lever after saline injection. Once rats were reliably discriminating cocaine from saline, other drugs were examined for their efficacies in substituting for cocaine. The dopamine uptake inhibitors WIN 35,428 [2-beta-carbomethoxy-3-beta-(4-fluorophenyl)tropane-1,5-naphthalene - disulfonate] and GBR 12909 (1-[2-bis(4-fluorophenyl)methoxy]ethyl]-4-[3- phenylpropyl]piperazine dihydrochloride) fully substituted for cocaine (cocaine responding > 80%), whereas the peripherally active cocaine methiodide and the 5-hydroxytryptamine uptake inhibitor fluoxetine did not substitute at all. Pentobarbital also failed to produce any cocaine-appropriate responding. Two selective norepinephrine uptake inhibitors were tested: tomoxetine fully substituted for the 3-mg/kg dose of cocaine and nisoxetine approached full substitution (79.7% cocaine responding). The direct-acting dopamine D-1 agonists SKF 38393 [(+-)-7-bromo-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-be nzazepin e HCl], SKF 77434 [(+-)-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1H-benzazepine HCl] and SKF 75670 [3-methyl-7,8-dihydroxyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benza zep ine HBr] fully substituted for cocaine, whereas the peripherally active dopamine D-1 agonist fenoldopam did not. Of four dopamine D-2 agonists tested, only quinpirole fully substituted; the others (N-0434 [(+-)-2-(N-propyl-N-phenylethylamino)-5-hydroxytetralin], (-)-NPA [R(-)-propylnorapomorphine HCl] and SDZ 208-912 (N-[(8-)-2,6-dimethylergoline-8-yl]-2,2-dimethyl-propanamide)) produced very limited partial substitution (cocaine responding < 32%).(ABSTRACT TRUNCATED AT 250 WORDS)     

The Stimulated Raman Spectrum of Symmetric 13C Cyanogen, 13C2N2

BACKGROUND: The authors examined the interaction of ketamine with recombinant mu, kappa, and delta opioid receptors and recombinant orphan opioid receptors expressed in Chinese hamster ovary cells (CHO-mu, CHO-kappa, CHO-delta, and CHO(ORL1), respectively). METHODS: CHO-mu, CHO-kappa, and CHO-delta membranes were incubated with the opioid receptor radioligand [3H]diprenorphine at room temperature. Ketamine (racemic, R(-) and S(+)) was included at concentrations covering the clinical range. CHO(ORL1) membranes were incubated with [125I]Tyr(14)nociceptin and racemic ketamine at room temperature. The effects of racemic ketamine and selective opioid receptor agonists (mu: [D-Ala2, MePhe4, Gly(ol)5] enkephalin (DAMGO); kappa: spiradoline or delta: [D-pen2, D-pen5] enkephalin (DPDPE)) on forskolin-stimulated cyclic adenosine monophosphate formation also were examined. Data are mean +/- SEM. RESULTS: Racemic ketamine increased the radioligand equilibrium dissociation constant for [3H]diprenorphine from 85+/-5 to 273+/-11, 91+/-6 to 154+/-16, and 372+/-15 to 855+/-42 pM in CHO-mu, CHO-kappa, and CHO-delta, respectively. The concentration of radioligand bound at saturation was unaffected. In CHO-mu and CHO-kappa cells, racemic ketamine did not slow the rate of naloxone-induced [3H]diprenorphine dissociation. Ketamine and its isomers also displaced [3H]diprenorphine binding to mu, kappa, and delta receptors in a dose-dependent manner, with pKi values for racemic ketamine of 4.38+/-0.02, 4.55+/-0.04, and 3.57+/-0.02, respectively. S(+)-ketamine was two to three times more potent than R(-)-ketamine at mu and kappa receptors. Racemic ketamine displaced [125I]Tyr(14)nociceptin with an estimated affinity constant of 0.5 mM. Racemic ketamine inhibited the formation of cyclic adenosine monophosphate (naloxone insensitive) in a dose-dependent manner (concentration producing 50% inhibition approximately 2 mM) in all cell lines, including untransfected CHO cells. Ketamine (100 microM) reversed DAMGO (mu) and spiradoline (kappa) inhibition of formation of cyclic adenosine monophosphate. CONCLUSIONS: Ketamine interacts stereoselectively with recombinant mu and kappa opioid receptors.     

Enantioseparation of beta-blockers labelled with a chiral fluorescent reagent, R (-)-DBD-PyNCS, by reversed-phase liquid chromatography

A fluorescent chiral tagging reagent, 4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfony l)-2,1, 3-benzoxadiazole [R(-)-DBD-PyNCS], has been used for the liquid chromatographic resolution of racemic pairs of beta-blockers. The reagents reacts with beta-blockers at 65 degrees C for 90 min in aqueous acetonitrile containing 0.05% triethylamine to produce the corresponding pair of diastereomers. No racemization occurs during the tagging reaction under these conditions. From results of the time-course study of oxprenolol the reactivities of the enantiomers of beta-blockers with R(-)-DBD-PyNCS are comparable. The optimum excitation and emission wavelengths of the resulting derivatives were ca. 460 and 550 nm, respectively. The derivatives of beta-blockers were efficiently resolved by a reversed-phase column with water-acetonitrile containing 0.1% trifluoroacetic acid as the eluent. The resolution (Rs) values of the diastereomers derived from 10 beta-blockers were in the range of 1.54-4.80. The Rs value for timolol was 0.643. The detection limits (signal-to-noise ratio of 2) were one or two orders of magnitude lower with beta-blockers having the iso-propylamino structure (15-300 fmol) than with those having the tert-butylamino structure (1.25-8.00 pmol). The proposed procedure was applied to the determination of R(+)- and S(-)-propranolol in rat plasma and saliva after oral administration of R(+)-propranolol hydrochloride or S(-)-propranolol hydrochloride.     

Stereochemistry of methylene transfer involving 5,10-methylenetetrahydrofolate

The stereochemistry of the transfer catalyzed by rabbit liver serine transhydroxymethylase (EC 2.1.2.1) of the prochiral hydroxymethyl group from serine to tetrahydrofolate to form 5,10-methylenetetrahydrofolate was studied. Initial kinetic studies on labeled 5,10-methylenetetrahydrofolate showed that nonenzymatic racemization of the prochiral methylene center was buffer dependent and was slow under the conditions employed. Specifically tritiated (3R)- and (3S)-serines were employed to study the transfer reaction. Reactions were carried out under various conditions and the stereochemistry of the methylene carbon of the 5,10-methylenetetrahydrofolate produced was determined. The enzyme was shown to be partially stereospecific for this transfer reaction, proceeding with a loss of about 50% of the stereochemical purity of the transferred carbon center. Possible mechanistic interpretations of this finding are discussed.