Characterization of (2S,2'R,3'R)-2-(2',3'-[3H]-Dicarboxycyclopropyl)glycine Binding in Rat Brain

Abstract: [(2S,2′R,3′R)-2-(2′,3′-[³H]Dicarboxycyclopropyl)glycine ([³H]DCG IV) binding was characterized in vitro in rat brain cortex homogenates and rat brain sections. In cortex homogenates, the binding was saturable and the saturation isotherm indicated the presence of a single binding site with a K_D value of 180 ± 33 nM and a B_max of 780 ± 70 fmol/mg of protein. The nonspecific binding, measured using 100 µM LY354740, was <30%. NMDA, AMPA, kainate, l (?)-threo-3-hydroxyaspartic acid, and (S)-3,5-dihydroxyphenylglycine were all inactive in [³H]DCG IV binding up to 1 mM. However, several compounds inhibited [³H]DCG IV binding in a concentration-dependent manner with the following rank order of potency: LY341495 = LY354740 > DCG IV = (2S,1′S,2′S)-2-(2-carboxycyclopropyl)glycine > (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid > (2S,1′S,2′S)-2-methyl-2-(2-carboxycyclopropyl)glycine > l -glutamate = ibotenate > quisqualate > (RS)-α-methyl-4-phosphonophenylglycine = l (+)-2-amino-3-phosphonopropionic acid > (S)-α-methyl-4-carboxyphenylglycine > (2S)-α-ethylglutamic acid > l (+)-2-amino-4-phosphonobutyric acid. N-Acetyl-l -aspartyl-l -glutamic acid inhibited the binding in a biphasic manner with an IC₅₀ of 0.2 µM for the high-affinity component. The binding was also affected by GTPγS, reducing agents, and CdCl₂. In parasagittal sections of rat brain, a high density of specific binding was observed in the accessory olfactory bulb, cortical regions (layers 1, 3, and 4 > 2, 5, and 6), caudate putamen, molecular layers of the hippocampus and dentate gyrus, subiculum, presubiculum, retrosplenial cortex, anteroventral thalamic nuclei, and cerebellar granular layer, reflecting its preferential (perhaps not exclusive) affinity for pre- and postsynaptic metabotropic glutamate mGlu2 receptors. Thus, the pharmacology, tissue distribution, and sensitivity to GTPγS show that [³H]DCG IV binding is probably to group II metabotropic glutamate receptors in rat brain.     

Syntheses of 2-Deoxy-2-[(2R,3S)-2-fluoro-3-hydroxytetradecanamido]-3-O-[(3R)-3-hydroxytetradecanoyl]-4-O-phosphono-D-glucopyranose and Its (2S,3R)-Isomer

2-Deoxy-2-[(2R,3S)-2-fluoro-3-hydroxytetradecanamido]-3-O-[(3R)-3-hydroxytetradecanoyl]-4-O-phosphono-D-glucopyranose and its (2S,3R)-isomer were respectively synthesized from allyl 2-[(2R,3S)-3-(benzyloxycarbonyloxy)-2-fluorotetradecanamido]-2-deoxy-4,6-O-isopropylidene-β-D-glucopyranoside and its corresponding (2S,3R)-isomer. Both target compounds did not activate macrophage, but the (2S,3R)-analogue strongly inhibited the binding of LPS to macrophage.     

Stereochemical action of mouse brain glutamate decarboxylase

4-[4-²H]Aminobutyrate was prepared by incubation in ²H₂O of glutamate with a partially purified glutamate decarboxylase from mouse brain. The 4R configuration was assigned to the compound on the basis of ¹H nmr analysis of the ω-camphanoylamide of its methyl ester in the presence of Eu(dpm)₃. Moreover 4-[4(S)4-³H,U-¹⁴C]aminobutyrate was shown to be formed from [2(S)2-³H,U-¹⁴C]glutamate by the same enzyme fraction. It is therefore demonstrated that glutamate decarboxylation catalyzed by this enzyme preparation occurs with retention of configuration.     

Biosynthesis of vitamin E and of the plastoquinones in chloroplasts: Steric course of the decarboxylation

Samples of (3R)- and (3S)-4′hydroxyphenyl[3-²H₁, 3-³H]pyruvate were prepared by taking advantage of the known stereospecificity of phenylpyruvate keto-enol isomerase (tautomerase). 4′-Hydroxyphenyl[3-¹⁴C]pyruvate was obtained by the action of l-amino acid oxidase on dl-[3-¹⁴C]tyrosine, whereas a simple base-catalyzed exchange procedure yielded samples of 4′-hydroxyphenyl[3-³H]- and 4′-hydroxyphenyl[3-²H₂]pyruvate. All labeled samples were converted in situ into the corresponding homogentisic acids on 4′-hydroxyphenyl-pyruvate dioxygenase that is known to catalyze the migration of the acetate side chain with retention of configuration. The isolated doubly labeled homogentisic acids were incubated with chloroplasts from Raphanus sativus cv. saxa Treib, and from the lipophilic products a fraction containing inter alia tocopherol, tocoquinone, and plastoquinone was obtained by chromatographic procedures. The incorporation of radioactivity was between 0.5 and 11% based on homogentisate. Reductive acetylation of the quinones yielded crystalline diacetylhydroquinones, which were submitted to Kuhn-Roth degradation. The radioactive acetate samples thus obtained were analyzed for chirality by an enzymatic procedure previously published. (2R)-[2-²H₁, 2-³H]Homogentisate gave mainly (S)-acetate, whereas (2S)-[2-²H₁, 2-³H]homogentisate was converted mainly into (R)-acetate. It is concluded that the decarboxylation of the side chain occurred with stereochemical retention during the biosynthetic process.     

(2R)-Catalponone,a biosynthetic intermediate for prenylnaphthoquinone congeners of the wood of Catalpa ovata

In accord with the results of experiments using callus tissues of Catalpa ovata, the simultaneous administration of (2R)- [1-¹⁴C]catalponone and (2S)- [8-³H]catalponone to the wood of the same plant demonstrated that the main pathway for the biosynthesis of naphthoquinone congeners, including catalpalactone, catalponol and 4,9-dihydroxy-α-lapachone, proceeds through the 2R-enantiomer of catalponone.     

Stereochemistry of Decarboxylation Reactions Catalyzed by a Constitutive Aromatic l-Amino Acid Decarboxylase

The stereochemistry of the decarboxylation reaction catalyzed by an aromatic l-amino acid decarboxylase, purified from Micrococcus percitreus, was studied using stereospecifically deuterium labelled phenylalanine (Phe). The ¹H NMR spectrum of [1,2-²H₂]-β-phenethylamine enzymatically derived from (2S, 3R)-[3-²H]-Phe in ²H₂O was compared with that of [1-²H]-β-phenethylamine from unlabelled Phe in ²H₂O. The results clearly indicate that the decarboxylation reaction of this enzyme proceeds exclusively through a course in which the configuration at C-2 of Phe is retained.     

Structure-activity relationship study towards non-peptidic positron emission tomography (PET) radiotracer for gastrin releasing peptide receptors: Development of [18F] (S)-3-(1H-indol-3-yl)-N-[1-[5-(2-fluoroethoxy)pyridin-2-yl]cyclohexylmethyl]-2-methyl-2-[3-(4-nitrophenyl)ureido]propionamide

Gastrin-releasing peptide receptors (GRP-Rs, also known as bombesin 2 receptors) are overexpressed in a variety of human cancers, including prostate cancer, and therefore they represent a promising target for in vivo imaging of tumors using positron emission tomography (PET). Structural modifications of the non-peptidic GRP-R antagonist PD-176252 ((S)-1a) led to the identification of the fluorinated analog (S)-3-(1H-indol-3-yl)-N-[1-[5-(2-fluoroethoxy)pyridin-2-yl]cyclohexylmethyl]-2-methyl-2-[3-(4-nitrophenyl)ureido]propionamide ((S)-1m) that showed high affinity and antagonistic properties for GRP-R. This antagonist was stable in rat plasma and towards microsomal oxidative metabolism in vitro. (S)-1m was successfully radiolabeled with fluorine-18 through a conventional radiochemistry procedure. [¹⁸F](S)-1m showed high affinity and displaceable interaction for GRP-Rs in PC3 cells in vitro.     

Identification of D3 and σ Receptors in the Rat Striatum and Nucleus Accumbens Using (±)-7-Hydroxy-N,N-Di-n-[3H]Propyl-2-Aminotetralin and Carbetapentane

Abstract: Cross-reactions between dopamine D₃ and σ receptor ligands were investigated using (±)-7-hydroxy-N,N-di-n-[³H]propyl-2-aminotetralin [(±)-7-OH-[³H]DPAT], a putative D₃-selective radioligand, in conjunction with the unlabeled σ ligands 1,3-di(2-tolyl)guanidine (DTG), carbetapentane, and R(?)-N-(3-phenyl-1-propyl)-1-phenyl-2-aminopropane [R(?)-PPAP]. In transfected CCL1.3 mouse fibroblasts expressing the human D₃ receptor, neither DTG nor carbetapentane (0.1 µM) displaced (±)-7-OH-[³H]DPAT binding. R(?)-PPAP (0.1 µM) displaced 39.6 ± 1.0% of total (±)-7-OH-[³H]DPAT binding. In striatal and nucleus accumbens homogenates, (±)-7-OH-[³H]DPAT labeled a single site (15–20 fmol/mg of protein) with high (1 nM) affinity. Competition analysis with carbetapentane defined both high- and low-affinity sites in striatal (35 and 65%, respectively) and nucleus accumbens (59 and 41%, respectively) tissue, yet R(?)-PPAP identified two sites in equal proportion. Carbetapentane and R(?)-PPAP (0.1 µM) displaced ～20–50% of total (±)-7-OH-[³H]DPAT binding in striatum, nucleus accumbens, and olfactory tubercle in autoradiographic studies, with the nucleus accumbens shell subregion exhibiting the greatest displacement. To determine directly (+)-7-OH-[³H]DPAT binding to σ receptors, saturation analysis was performed in the cerebellum while masking D₃ receptors with 1 µM dopamine. Under these conditions (+)-7-OH-[³H]DPAT labeled σ receptors with an affinity of 24 nM. These results suggest that (a) (±)-7-OH-[³H]DPAT binds D₃ receptors with high affinity in rat brain and (b) a significant proportion of (±)-7-OH-[³H]DPAT binding consists of σ₁ sites and the percentages of these sites differ among the subregions of the striatum and nucleus accumbens.     

Substrate stereochemistry of isovaleryl-CoA dehydrogenase elimination of the 2-pro-R hydrogen in biotin-deficient rats

(2R)-[³H]Isovaleric acid and (2S)-[³H]isovaleric acid (ammonium salts) have been synthesized. These substances, mixed with [1-¹⁴C]isovalerate, have been administered to biotin-deficient rats, which accumulate β-hydroxyisovaleric acid in their urine, the metabolite being formed via isovaleryl-CoA and β-methylcrotonyl-CoA. The results show that most of the tritium from (2R)-[³H]isovalerate was lost, and most of the tritium from (2S)-[³H]isovalerate retained in the conversion to β-hydroxyisovalerate. The stereochemistry of the isovaleryl-CoA dehydrogenase reaction is compared with the stereochemistry of other short-chain acyl-CoA dehydrogenase reactions.     

Biosynthesis of flavan-3-ols and other secondary plant products from (2S)phenylalanine

(2S)-Phenyl[2-¹⁴C,3R-³H₁]alanine and (2S)-phenyl[2-¹⁴C,3S-³H₁]alanine have been employed as substrates to study procyanidin and flavan-3-ol biosynthesis. Parallel studies with the cyanogenic glucosides prunasin and sambunigrin, Winterstein's acid [(3R)-3-dimethylaminophenylpropionic acid] and tropic acid show these to be derived by stereospecific processes from (2S)-phenylalanine. New proposals for procyanidin biosynthesis are briefly commented upon.     

Synthesis of hydantoin analogues of (2S,3R,4S)-4-hydroxyisoleucine with insulinotropic properties

The first synthesis of an optically pure (2R,3R,4S)-hydantoin 2, analogue of (2S,3R,4S)-4-hydroxyisoleucine, was achieved in two steps in un-optimized 35% overall yield from previously reported aldehyde synthon 1. (2R,3R,4S)-Hydantoin is stable at acidic pH. This solves the major drawback of (2S,3R,4S)-4-hydroxyisoleucine that easily cyclizes into inactive lactone. Furthermore, (2R,3R,4S)-hydantoin stimulates the insulin secretion by 150% at 25 μM compared with 4-hydroxyisoleucine and insulin secretagogue drug repaglinide. In view of its stability and biological activity, (2R,3R,4S)-hydantoin represents a good candidate for type-2 diabetes management and control.     

Biosynthesis of (10R)-Juvenile hormone III from farnesoic acid by Aedes aegypti ovary

Synthesis of (10R)-juvenile hormone III (JH III) outside the corpora allata (CA) was investigated in female Aedes aegypti. Intact females or ligated abdomens of blood-fed and sugar-fed females synthesized in vivo [12-³H]JH III-like molecules from [12-³H]-methyl farnesoate, indicating that an organ(s) in the female abdomen, other than the CA, converted methyl farnesoate into JH III. To find out the organ(s) that synthesized JH III-like molecules, ovaries, fat bodies, and midguts were incubated in vitro with [12-³H]methyl farnesoate and the synthesis of JH III-like molecules was compared with JH III synthesized by CA. To identify tissue(s) having both farnesoic acid methyl transferase and farnesoate epoxidase, enzymes that convert farnesoic acid into JH III, ovaries, and fat bodies were removed from sugar and blood-fed females and incubated with [12-³H]farnesoic acid. Chemical derivatization by methoxyhydrin formation followed by esterification with (+)-α-methoxy- α-trifluoromethyl phenylacetic (MTPA) acid chloride and reversed phase liquid chromatography identified (10R)-JH III methoxyhydrin (+)-MTPA ester as the sole JH III-like molecule produced in tissue culture incubation of ovaries. Since only (10R)-JH III is produced and not racemic JH III, the oxidation of farnesoic acid must be enzymatically mediated. Ovaries and corpora allata of female A. aegypti also synthesized [³H,¹⁴C]JH III from L-[methyl-³H]methionine and [¹⁴C]acetate which was characterized by HPLC and gas chromatography. These results suggest that mosquito ovary can synthesize (10R)-JH III from farnesoic acid, and that this tissue synthesizes JH III-like molecules from L-methionine and acetate. © 1994 Wiley-Liss, Inc.     

Mechanism and stereospecificity of alpha-hydroxylation of lignoceric acid in rat brain.

Cerebronic acid (2-hydroxytetracosanoic acid) is the major fatty acid component of cerebrosides and sulfatides in mammalian brain. Our previous communication demonstrated the synthesis of cerebronic acid from lignoceric acid (tetracosanoic acid) by a rat brain preparation in the presence of molecular oxygen and a reduced pyridine nucleotide (Hoshi, M., and Kishimoto, Y. (1973) J. Biol. Chem., 248, 4123–4130). The present'studies on the conversion of (RS)-[2-³H]-, (RS)-[3-³H]-, (R)-[2-³H]-, and (S)-[2-³H]lignoceric acids to cerebronic acid by rat brain preparations establish that the pro-R hydrogen at the α-carbon of lignoceric acid is replaced by a hydroxyl group with overall retention of configuration.     

Synthesis of Optically Active 1,4-Thiazane-3-carboxylic Acid via Optical Resolution by Preferential Crystallization of (RS)-2-Amino-3-[(2-chloroethyl)sulfanyl]propanoic Acid Hydrochloride

Optically active 1,4-thiazane-3-carboxylic acid [TCA] was synthesized from cysteine via optical resolution by preferential crystallization. The intermediate (RS)-2-amino-3-[(2-chloroethyl)sulfanyl]propanoic acid hydrochlo-ride [(RS)-ACS?HCl] was found to exist as a conglomerate based on its melting point, solubility and IR spectrum. (RS)-ACS?HCl was optically resolved by preferential crystallization to yield (R)- and (S)-ACS?HCl. (R)- and (S)-ACS?HCl thus obtained were recrystallized from a mixture of hydrochloric acid and 2-propanol, taking account of the solubility of (RS)-ACS?HCl, efficiently yielding both enantiomers in optically pure forms. (R)- and (S)-TCA were then respectively synthesized by the cyclization of (R)- and (S)-ACS?HCl in ethanol in the presence of triethylamine.     

Discovery of (1R,6S)-5-[4-(1-cyclobutyl-piperidin-4-yloxy)-phenyl]-3,4-diaza-bicyclo[4.1.0]hept-4-en-2-one (R,S-4a): Histamine H3 receptor inverse agonist demonstrating potent cognitive enhancing and wake promoting activity

A series of fused cyclopropyl-4,5-dihydropyridazin-3-one (3,4-diaza-bicyclo[4.1.0]hept-4-en-2-one) phenoxypiperidine analogs was designed and synthesized, leading to the identification of (1R,6S)-5-[4-(1-cyclobutyl-piperidin-4-yloxy)-phenyl]-3,4-diaza-bicyclo[4.1.0]hept-4-en-2-one (R,S-4a) as a second-generation pyridazin-3-one H₃R antagonist. Compound R,S-4a was a potent H₃R functional antagonist in vivo in the rat dipsogenia model, demonstrated potent wake activity in the rat EEG/EMG model, and enhanced short-term memory in the rat social recognition memory model at doses as low as 0.03–0.3 mg/kg po.     

Biosynthesis of Violacein: Origins of Hydrogen,Nitrogen and Oxygen Atoms in the 2-Pyrrolidone Nucleus

The biosynthetic origins of the hydrogen, nitrogen and oxygen atoms in the pyrrolidone ring of violacein'were established by an anaylses of the ¹H, ¹³C NMR and MS spectra of its isotope-enriched metabolites. Feeding experiments of [2-²H] and [3-²H₂]tryptophans have revealed that the hydrogen in the pyrrolidone ring was derived from the methylene protons at the 3-position of tryptophan. The stereochemical fate of the prochiral hydrogens was determined to be in the retention of the pro-S hydrogen by these feeding experiments using [3R-²H] and [3S”-²H]tryptophans. The incorporation experiment of [α-¹⁵N]tryptophan demonstrated that the nitrogen atom in the ring originated from the α-amino group of tryptophan. The incorporation experiment of ¹⁸O₂ gas verified that all the oxygen atoms of violacein were derived from the molecular oxygen.     

Development of selective inhibitors for the treatment of rheumatoid arthritis: (R)-3-(3-(Methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)pyrrolidin-1-yl)-3-oxopropanenitrile as a JAK1-selective inhibitor

A series of 3(R)-aminopyrrolidine derivatives were designed and synthesized for JAK1-selective inhibitors through the modification of tofacitinib’s core structure, (3R,4R)-3-amino-4-methylpiperidine. From the new core structures, we selected (R)-N-methyl-N-(pyrrolidin-3-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine as a scaffold for further SAR studies. From biochemical enzyme assays and liver microsomal stability tests, (R)-3-(3-(methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)pyrrolidin-1-yl)-3-oxopropanenitrile (6) was chosen for further in vivo test through oral administration. Compound 6 showed improved selectivity for JAK1 compared to that of tofacitinib (IC₅₀ 11, 2.4?×?10², 2.8?×?10³, and 1.1?×?10²?nM for JAK1, JAK2, JAK3, and TYK2, respectively). In CIA and AIA model tests, compound 6 exhibited similar efficacy to tofacitinib citrate.     

The enzymatic synthesis of S-adenosyl-l-[2(n)-H]homocysteine

A rapid, efficient method is described for the enzymatic conversion of S-adenosyl-l-[2(n)-³H]methionine to S-adenosyl-l-[2(n)-³H]homocysteine. Partially purified glycine N-methyltransferase is used in the reaction which yields 98% conversion. The product is purified using high-pressure liquid chromatography and is concentrated by lyophilization. S-Adenosyl-l-[2(n)-³H]homocysteine synthesized by this method is an active substrate for S-adenosylhomocysteine (SAH) hydrolase. A novel assay procedure for SAH hydrolase is also described, in which unreacted S-adenosyl-l-[2(n)-³H]homocysteine is removed by adsorption to dextran-coated charcoal.     

Identification ofS-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]-3-mercaptopyruvic acid with a metabolic intermediate betweenS-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]-l-cysteine andS-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]-3-mercaptolactic acid

Summary S-[2-Carboxy-1-(1H-imidazol-4-yl)ethyl]-3-mercaptopyruvic acid (I) was chemically synthesized in 15% yield by incubating a reaction mixture oftrans-urocanic acid and 3-fold excess of 3-mercaptopyruvic acid at 45°C for 6 days. The synthesized compound was characterized by fast-atom-bombardment mass spectrometry and high-voltage paper electrophoresis. CompoundI was identified with a product of an enzymatic reaction ofS-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]-l-cysteine (II) with rat liver homogenate in a phosphate buffer, pH 7.4. CompoundI was degraded toS-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]-3-mercaptolactic acid (III), a compound previously found in human urine [Kinuta et al. (1994) Biochem J 297: 475–478], by incubation with rat liver homogenate. From these results, we suggest that compoundI is a metabolic intermediate for the formation of compoundIII from compoundII. The present pathway follows a formation of compoundII fromS-[2-carboxy-1-(1H-imidazol-4-yl)ethyl] gluthathione [Kinuta et al. (1993) Biochim Biophys Acta 1157: 192–198], a proposed metabolite ofl-histidine.     

In Vitro Evaluation of 11C-Labeled (S)-Nicotine, (S)-3-Methyl-5-(1-Methyl-2-Pyrrolidinyl)isoxazole, and (R,S)-1-Methyl-2-(3-Pyridyl)azetidine as Nicotinic Receptor Ligands for Positron Emission Tomography Studies

Abstract: The binding characteristics of the novel ¹¹C-labeled nicotinic ligands (R,S)-1-methyl-2-(3-pyridyl) azetidine (MPA) and (S)-3-methyl-5-(1-methyl-2-pyrrolidinyl)isoxazole (ABT-418) were investigated in comparison with those of (S)-[¹¹C]nicotine in vitro in the rat brain to be able to predict the binding properties of the new ligands for positron emission tomography studies in vivo. The data from time-resolved experiments for all ligands indicated fast binding kinetics, with the exception of a slower dissociation of [¹¹C]MPA in comparison with (S)-[¹¹C]nicotine and [¹¹C]ABT-418. Saturation experiments revealed for all ligands two nicotinic receptor binding sites with affinity constants (K_D values) of 2.4 and 560 nM and binding site densities (B_max values) of 65.5 and 223 fmol/mg of protein for (S)-[¹¹C]nicotine, K_D values of 0.011 and 2.2 nM and B_max values of 4.4 and 70.7 fmol/mg of protein for [¹¹C]MPA, and K_D values of 1.3 and 33.4 nM and B_max values of 8.8 and 69.2 fmol/mg of protein for [¹¹C]ABT-418. In competing with the ¹¹C-ligands, epibatidine was most potent, followed by cytisine. A different rank order of potencies was found for (?)-nicotine, (+)-nicotine, MPA, and ABT-418 displacing each of the ¹¹C-ligands. Autoradiograms displayed a similar pattern of receptor binding for all ligands, whereby [¹¹C]MPA showed the most distinct binding pattern and the lowest nonspecific binding. We conclude that the three ¹¹C-labeled nicotinic ligands were suitable for characterizing nicotinic receptors in vitro. The very high affinity of [¹¹C]MPA to nicotinic acetylcholine receptors, its low nonspecific binding, and especially the slower dissociation kinetics of the [¹¹C]MPA from the putative high-affinity nicotinic acetylcholine receptor binding site compared with (S)-[¹¹C]nicotine and [¹¹C]ABT-418 raise the level of interest in [¹¹C]MPA for application in positron emission tomography.