Resolution, configurational assignment, and enantiopharmacology at glutamate receptors of 2-amino-3-(3-carboxy-5-methyl-4-isoxazolyl)propionic acid (ACPA) and demethyl-ACPA

We have previously described (RS)-2-amino-3-(3-carboxy-5-methyl-4-isoxazolyl)propionic acid (ACPA) as a potent agonist at the (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptor subtype of (S)-glutamic acid (Glu) receptors. We now report the chromatographic resolution of ACPA and (RS)-2-amino-3-(3-carboxy-4-isoxazolyl)propionic acid (demethyl-ACPA) using a Sumichiral OA-5000 column. The configuration of the enantiomers of both compounds have been assigned based on X-ray crystallographic analyses, supported by circular dichroism spectra and elution orders on chiral HPLC columns. Furthermore, the enantiopharmacology of ACPA and demethyl-ACPA was investigated using radioligand binding and cortical wedge electrophysiological assay systems and cloned metabotropic Glu receptors. (S)-ACPA showed high affinity in AMPA binding (IC(50) = 0.025 microM), low affinity in kainic acid binding (IC(50) = 3.6 microM), and potent AMPA receptor agonist activity on cortical neurons (EC(50) = 0.25 microM), whereas (R)-ACPA was essentially inactive. Like (S)-ACPA, (S)-demethyl-ACPA displayed high AMPA receptor affinity (IC(50) = 0.039 microM), but was found to be a relatively weak AMPA receptor agonist (EC(50) = 12 microM). The stereoselectivity observed for demethyl-ACPA was high when based on AMPA receptor affinity (eudismic ratio = 250), but low when based on electrophysiological activity (eudismic ratio = 10). (R)-Demethyl-ACPA also possessed a weak NMDA receptor antagonist activity (IC(50) = 220 microM). Among the enantiomers tested, only (S)-demethyl-ACPA showed activity at metabotropic receptors, being a weak antagonist at the mGlu(2) receptor subtype (K(B) = 148 microM).     

Resolution, absolute stereochemistry, and enantiopharmacology of the GluR1-4 and GluR5 antagonist 2-amino-3-[5-tert-butyl-3-(phosphonomethoxy)-4-isoxazolyl]propionic acid

The phosphono amino acid, (RS)-2-amino-3-[5-tert-butyl-3-(phosphonomethoxy)-4-isoxazolyl+ ++]propio nic acid (ATPO), is a structural hybrid between the NMDA antagonist (RS)-2-amino-7-phosphonoheptanoic acid (AP7) and the AMPA and GluR5 agonist, (RS)-2-amino-3-(5-tert-butyl-3-hydroxy-4-isoxazolyl)propionic acid (ATPA). ATPO has been resolved into (S)-ATPO and (R)-ATPO using chiral HPLC, and the absolute stereochemistry of the two enantiomers was established by an X-ray crystallographic analysis of (R)-ATPO. (S)-ATPO and (R)-ATPO were characterized pharmacologically using rat brain membrane binding and electrophysiologically using the cortical wedge preparation as well as homo- or heteromeric GluR1-4, GluR5-6, and KA2 receptors expressed in Xenopus oocytes. (R)-ATPO was essentially inactive as an agonist or antagonist in all test systems. (S)-ATPO was an inhibitor of the binding of [(3)H]AMPA (IC(50) = 16 +/- 1 microM) and of [(3)H]-6-cyano-7-nitroquinoxaline-2,3-dione ([(3)H]CNQX) (IC(50) = 1.8 +/- 0.2 microM), but was inactive in the [(3)H]kainic acid and the [(3)H]-(RS)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid ([(3)H]CPP) binding assays. (S)-ATPO did not show detectable agonist effects at any of the receptors under study, but antagonized AMPA-induced depolarization in the cortical wedge preparation (IC(50) = 15 +/- 1 microM). (S)-ATPO also blocked kainic acid agonist effects at GluR1 (K(i) = 2.0 microM), GluR1+2 (K(i) = 3.6 microM), GluR3 (K(i) = 3.6 microM), GluR4 (K(i) = 6.7 microM), and GluR5 (K(i) = 23 microM), but was inactive at GluR6 and GluR6+KA2. Thus, although ATPO is a structural analog of AP7 neither (S)-ATPO nor (R)-ATPO are recognized by NMDA receptor sites.     

Resolution, configurational assignment, and enantiopharmacology of 2-amino-3-[3-hydroxy-5-(2-methyl-2H- tetrazol-5-yl)isoxazol-4-yl]propionic acid, a potent GluR3- and GluR4-preferring AMPA receptor agonist

We have previously shown that (RS)-2-amino-3-[3-hydroxy-5-(2-methyl-2H-tetrazol-5-yl)isoxazol -4-yl] propionic acid (2-Me-Tet-AMPA) is a selective agonist at (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) receptors, markedly more potent than AMPA itself, whereas the isomeric compound 1-Me-Tet-AMPA is essentially inactive. We here report the enantiopharmacology of 2-Me-Tet-AMPA in radioligand binding and cortical wedge electrophysiological assay systems, and using cloned AMPA (GluR1-4) and kainic acid (KA) (GluR5, 6, and KA2) receptor subtypes expressed in Xenopus oocytes. 2-Me-Tet-AMPA was resolved using preparative chiral HPLC. Zwitterion (-)-2-Me-Tet-AMPA was assigned the (R)-configuration based on an X-ray crystallographic analysis supported by the elution order of (-)- and (+)-2-Me-Tet-AMPA using four different chiral HPLC columns and by circular dichroism spectra. None of the compounds tested showed detectable affinity for N-methyl-D-aspartic acid (NMDA) receptor sites, and (R)-2-Me-Tet-AMPA was essentially inactive in all of the test systems used. Whereas (S)-2-Me-Tet-AMPA showed low affinity (IC(50) = 11 microM) in the [(3)H]KA binding assay, it was significantly more potent (IC(50) = 0.009 microM) than AMPA (IC(50) = 0.039 microM) in the [(3)H]AMPA binding assay, and in agreement with these findings, (S)-2-Me-Tet-AMPA (EC(50) = 0.11 microM) was markedly more potent than AMPA (EC(50) = 3.5 microM) in the electrophysiological cortical wedge model. In contrast to AMPA, which showed comparable potencies (EC(50) = 1.3-3.5 microM) at receptors formed by the AMPA receptor subunits (GluR1-4) in Xenopus oocytes, more potent effects and a substantially higher degree of subunit selectivity were observed for (S)-2-Me-Tet-AMPA: GluR1o (EC(50) = 0.16 microM), GluR1o/GluR2i (EC(50) = 0.12 microM), GluR3o (EC(50) = 0.014 microM) and GluR4o (EC(50) = 0.009 microM). At the KA-preferring receptors GluR5 and GluR6/KA2, (S)-2-Me-Tet-AMPA showed much weaker agonist effects (EC(50) = 8.7 and 15.3 microM, respectively). It is concluded that (S)-2-Me-Tet-AMPA is a subunit-selective and highly potent AMPA receptor agonist and a potentially useful tool for studies of physiological AMPA receptor subtypes.     

Optical resolution and absolute configuration of a nonsteroidal antiinflammatory drug, 2-(10,11-dihydro-10-oxodibenzo- [b,f]thiepin-2-yl)propionic acid

The title compound (+/-)-1 (CN-100) was efficiently resolved into a pair of enantiomers by fractional crystallization of the diastereomeric salts of (-)- and (+)-phenylethylamine. The purity of the enantiomers was determined using the chiral cellulose column (CHIRALCEL OJ) which was allowed direct separation of the enantiomers. A separation factor (alpha) of 1.73 was obtained. X-Ray crystallographic analysis of the (+)-isomer [salt of (-)-1-(4-bromophenyl)ethylamine] showed that this enantiomer has S-configuration. Biological studies have shown that only the (+)-isomer has antiinflammatory activity. Racemizaiton of (-)-isomer was carried out by heating its propionic acid solution in the presence of mineral acid, such as HBr.     

Synthesis and structural determination of stereoisomers of muscarinic ligands of the (3-propylthio-1,2,5-thiadiazol-4-yl)-1-azabicycloalkane type

Methods for the synthesis of each of the four stereoisomers of 6-(3-propylthio-1,2,5-thiadiazol-4-yl)-1-azabicyclo[3.2.1]octane ( 10, 11, 12 , and 13 ) and 3-(3-propylthio-1,2,5-thiadiazol-4-yl)-1-azabicyclo[2.2.1]heptane ( 18, 19, 20 , and 21 ), and the two stereoisomers of 3-(3-propylthio-1,2,5-thiadiazol-4-yl)-1-azabicyclo[2.2.2]octane ( 27 and 28 ) were developed. The relative configuration of the compounds was determined on the basis of previously described ¹H NOE experiments, and the absolute configuration of 6-(3-propylthio-1,2,5-thiadiazol-4-yl)-1-azabicyclo[3.2.1]octanes ( 10, 11, 12 , and 13 ) and 3-(3-propylthio-1,2,5-thiadiazol-4-yl)-1-azabicyclo[2.2.2]octane ( 27 and 28 ) was determined by single crystal X-ray crystallography. Optical purity was determined by capillary electrophoresis (CE) using chiral selectors as trimethyl-β-cyclodextrin and heparin dissolved in the running buffer. All the 3-(3-propylthio-1,2,5-thiadiazol-4-yl)-1-azabicycles had low nanomolar affinity for muscarinic receptors as determined by displacement of radiolabelled oxotremorine-M (³H-Oxo-M) and pirenzepine (³H-Pz) from cortical rat brain homogenates. The binding assay discriminated between diastereomers, but only a minor degree of enantioselectivity was observed in the binding assays. Chirality 9:739–749, 1997. © 1997 Wiley-Liss, Inc.     

3-(2-Ethoxy-4-{4-[3-hydroxy-2-methyl-4-(3-methylbutanoyl)phenoxy]butoxy}phenyl)propanoic acid: a brain penetrant allosteric potentiator at the metabotropic glutamate receptor 2 (mGluR2)

We have identified and synthesized a brain penetrant propanoic acid as an allosteric potentiator of the metabotropic glutamate receptor 2. Structure-activity relationship studies directed toward improving the potency, level of potentiation and brain penetration led to the discovery of 8 (EC50=1200 nM, 77% potentiation, 119% brain/plasma in rat, 20 mpk i.p., brain level of 5700 nM).     

3-(4-Aminobutyn-1-yl)pyridines: binding at alpha 4 beta 2 nicotinic cholinergic receptors

The binding of a series pyridylbutynylamines 6 was examined at alpha4beta2 nACh receptors. Structural modifications, comparing 6 with pyridyl ethers 2, did not consistently result in parallel effects on receptor affinity, suggesting possible differences in their modes of binding. Furthermore, the binding of amine 6a seemed to be accounted for by the newer vector pharmacophore models.     

Synthesis of base substituted 2-hydroxy-3-(purin-9-yl)-propanoic acids and 4-(purin-9-yl)-3-butenoic acids

Alkylation of 6-chloropurine and 2-amino-6-chloropurine with bromoacetaldehyde diethyl acetal afforded 6-chloro-9-(2,2-diethoxyethyl)purine (3a) and its 2-amino congener (3b). Treatment of compounds 3 with primary and secondary amines gave the N6-substituted adenines (5a-5c) and 2,6-diaminopurines (5d-5f). Hydrolysis of 3 resulted in hypoxanthine (6a) and guanine (6b) derivatives, while their reaction with thiourea led to 6-sulfanylpurine (7a) and 2-amino-6-sulfanylpurine (7b) compounds. Treatment with diluted acid followed by potassium cyanide treatment and acid hydrolysis afforded 6-substituted 3-(purin-9-yl)- and 3-(2-aminopurin-9-yl)-2-hydroxypropanoic acids (8-10). Reaction of compounds 3 with malonic acid in aqueous solution gave exclusively the product of isomerisation, 6-substituted 4-(purin-9-yl)-3-butenoic acids (15).     

Identification of metabolites of fluorine-18-labeled M2 muscarinic receptor agonist, 3-(3-[(3-fluoropropyl)thio]-1,2,5-thiadiazol-4-yl)-1,2,5,6-tetrahydro-1-methylpyridine, produced by human and rat hepatocytes

An accurate, rapid method for the determination of unmetabolized 3-(3-[(3-[18F]fluoropropyl)thio]-1,2,5-thiadiazol-4-yl)-1.2,5,6-tetrahydro-1-methylpyridine (FP-TZTP), a selective M2 muscarinic agonist, is necessary in order to obtain quantitative information from positron emission tomography (PET) imaging. Using LC-MS-MS to analyze products from cultured human and rat hepatocytes, we identified metabolites resulting from oxidation of the nitrogen in the tetrahydropyridine ring, sulfur-oxidation, demethylation of the tertiary amine, and oxidation of the tetrahydropyridine ring. From the knowledge of the structure of the metabolites, we have developed a two-step extraction sequence that allows rapid determination of the parent fraction in plasma without time-consuming chromatographic analysis.     

3-[2-Amino-2-imidazolin-4(5)-yl]alanine (enduracididine) and 2-[2-amino-2-imidazolin-4(5)-yl] acetic acid in seeds of Lonchocarpus sericeus

3-[2-Amino-2-imidazolin-4(5)-yl]alanine (enduracididine) and 2-[2-amino-2-imidazolin-4(5)-yl] acetic acid have been isolated from seeds of Lonchocarpus sericeus. The concentration of each compound was ca 0.5 % of the fresh seed weight.     

(2S)-2-Amino-5-chloro-4-hydroxy-5-hexenoic acid,a new chloroamino acid,and related compounds fromAmanita gymnopus

Combining different chromatography systems, unusual nonprotein amino acids were isolated and unequivocally identified from a small amount (less than 100 g fresh weight) ofAmanita gymnopus fruit body. Without obtaining crystals of these amino acids, on the basis of¹H-NMR determination, high resolution mass spectrometry, chlorine analysis and oxidation with L-amino acid oxidase, one of them proved to be a new chloroamino acid, (2S)-2-amino-5-chloro-4-hydroxy-5-hexenoic acid (G2). The other three were (2S)-2-amino-5-hexenoic acid (G1), (2S)-2-amino-4,5-hexadienoic acid (G3) and (2S)-2-amino-5-hexynoic acid (G4). Amino acid (G1) was also encountered for the first time in natural products. Amino acid (G3) has been reported from several kinds of fungi belonging toAmanita, subgenusLepidella. The occurrence of amino acid (G4) was already reported fromCortinarius claricolor.Part 23 in the series Biochemical studies of nitrogen compounds in fungi. Part 22, Hatanaka, S. I. et al. 1985. Trans. Mycol. Soc. Japan26: 61–68.     

Glutamate receptor ligands: synthesis, stereochemistry, and enantiopharmacology of methylated 2-aminoadipic acid analogs

Homologation and substitution on the carbon backbone of (S)-glutamic acid [(S)-Glu, 1], as well as absolute stereochemistry, are structural parameters of key importance for the pharmacological profile of (S)-Glu receptor ligands. We describe a series of methyl-substituted 2-aminoadipic acid (AA) analogs, and the synthesis, stereochemistry, and enantiopharmacology of 3-methyl-AA (4a-d), 4-methyl-AA (5a-d), 5-methyl-AA (6a-d), and (E)-Delta(4)-5-methyl-AA (7a and 7b) are reported. The compounds were resolved using chiral HPLC and the configurational assignments of the enantiomers were based on X-ray crystallographic analyses, chemical correlation, and CD spectral analyses. The effects of the individual stereoisomers at ionotropic and metabotropic (S)-Glu receptors (iGluRs and mGluRs) were characterized. Compounds with S-configuration at the alpha-carbon generally showed mGluR2 agonist activity of similar or slightly lower potencies than (S)-AA [e.g., EC(50) = 76 microM for (2S,4S)-4-methyl-AA (5a) as compared to EC(50) = 35 microM for (S)-AA]. The position of the methyl substituent had a profound effect on the observed pharmacology, whereas the absolute stereochemistry at the methylated carbon atom had a very limited effect on pharmacology. Structure-activity relationships at iGluRs in the rat cortical wedge preparation showed a complex pattern, some compounds being NMDA receptor agonists [e.g., EC(50) =110 microM for (2S,5RS)-5-methyl-AA (6a,b)] and some compounds showing NMDA receptor antagonist effects [e.g., IC(50) = 300 microM for (2R,4S)-4-methyl-AA (5d)]. The two unsaturated analogs (S)- (7a) and (R)-(E)-Delta(4)-5-methyl-AA (7b) turned out to be a weak AMPA receptor agonist and a weak mixed NMDA/AMPA receptor antagonist, respectively.     

AMPA receptor agonists: Resolution,configurational assignment,and pharmacology of (+)-(S)- and (-)-(R)-2-amino-3-[3-hydroxy-5-(2-pyridyl)-isoxazol-4-yl]-propionic acid (2-Py-AMPA)

We have previously shown that whereas (RS)-2-amino-3-(3-hydroxy-5-phenylisoxazol-4-yl)propionic acid (APPA) shows the characteristics of a partial agonist at (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) receptors, (S)-APPA is a full AMPA receptor agonist and (R)-APPA a weak competitive AMPA receptor antagonist. This observation led us to introduce the new pharmacological concept, functional partial agonism. Recently we have shown that the 2-pyridyl analogue of APPA, (RS)-2-amino-3-[3-hydroxy-5-(2-pyridyl)isoxazol-4-yl]propionic acid (2-Py-AMPA), is a potent and apparently full AMPA receptor agonist, and this compound has now been resolved into (+)- and (-)-2-Py-AMPA (ee ≥ 99.0%) by chiral HPLC using a Chirobiotic T column. The absolute stereochemistry of the enantiomers of APPA has previously been established by X-ray analysis, and on the basis of comparative studies of the circular dichroism spectra of the enantiomers of APPA and 2-Py-AMPA, (+)- and (-)-2-Py-AMPA were assigned the (S)- and (R)-configuration, respectively. In a series of receptor binding studies, neither enantiomer of 2-Py-AMPA showed detectable affinity for kainic acid receptor sites or different sites at the N-methyl-D-aspartic acid (NMDA) receptor complex. (+)-(S)-2-Py-AMPA was an effective inhibitor of [³H]AMPA binding (IC⁵⁰ = 0.19 ± 0.06 μM) and a potent AMPA receptor agonist in the rat cortical wedge preparation (EC₅₀ = 4.5 ± 0.3 μM) comparable with AMPA (IC₅₀ = 0.040 ± 0.01 μM; EC₅₀ = 3.5 ± 0.2 μM), but much more potent than (+)-(S)-APPA (IC₅₀ = 5.5 ± 2.2 μM; EC₅₀ = 230 ± 12 μM). Like (-)-(R)-APPA (IC₅₀ > 100 μM), (-)-(R)-2-Py-AMPA (IC₅₀ > 100 μM) did not significantly affect [³H]AMPA binding, and both compounds were week AMPA receptor antagonists (K_i = 270 ± 50 and 290 ± 20 μM, respectively). Chirality 9:274–280, 1997. © 1997 Wiley-Liss, Inc.     

2-(2,3-Dihydro-1H-indol-3-yl)ethanol: synthesis, separation of enantiomers, and assignment of absolute stereochemistry by X-ray structure analysis

The first direct resolution of racemic 2-(2,3-dihydro-lH-indol-3-yl)ethanol-prepared by catalytic hydrogenation of 2-(lH-indol-3-yl)ethanol-has been accomplished by chiral simulated moving bed (SMB) chromatography. The single enantiomers were isolated as their dihydrogen phosphate salts. Single-crystal X-ray analyses were successful, revealing that the (+)-enantiomer of 2-(2,3-dihydro-lH-indol-3-yl)ethanol has the (S) configuration. Chirality 16:126-130, 2004.     

3-[2-Amino-2-imidazolin-4-yl] alanine, 2-[2-amino-2-imidazolin-4-yl] acetic acid, 2-aminoimidazole and other guanidine derivatives in the tephrosieae

The distribution of enduracididine, 2-[2-amino-2-imidazolin-4-yl] acetic acid, 2-aminoimidazole, canavanine, homoarginine, γ-hydroxyhomoarginine and other unidentified guanidino compounds in seeds of spp. of the Tephrosieae is described. Within Lonchocarpus enduracididine and 2-[2-amino-2-imidazolin-4-yl] acetic acid were found only in American spp. and canavanine only in African spp.     

4-(Benzimidazol-2-yl)-1,2,5-oxadiazol-3-ylamine derivatives: Potent and selective p70S6 kinase inhibitors

We report herein the design and synthesis of 4-(benzimidazol-2-yl)-1,2,5-oxadiazol-3-amine derivatives as inhibitors of p70S6 kinase. Screening hits containing the 4-(benzimidazol-2-yl)-1,2,5-oxadiazol-3-ylamine scaffold were optimized for p70S6K potency and selectivity against related kinases. Structure-based design employing an active site homology model derived from PKA led to the preparation of benzimidazole 5-substituted compounds 26 and 27 as highly potent inhibitors (K_i <1 nM) of p70S6K, with >100-fold selectivity against PKA, ROCK and GSK3.     

Novel agonists of free fatty acid receptor 1 (GPR40) based on 3-(1,3,4-thiadiazol-2-yl)propanoic acid scaffold

1,3,4-Thiadiazole was explored as a more polar, heterocyclic replacement for the phenyl ring in the 3-arylpropionic acid pharmacophore present in the majority of GPR40 agonists. Out of 13 compounds synthesized using a flexible, three-step protocol (involving no chromatographic purification), four compounds were confirmed to activate the target in micromolar concentration range. While the potency of the series should be subject of further optimization, the remarkable aqueous solubility and microsomal stability observed for the lead compound (8g) apparently attests to this new scaffold’s high promise in the GPR40 agonist field.     

New broad-spectrum parenteral cephalosporins exhibiting potent activity against both methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa. Part 3: 7beta-[2-(5-Amino-1,2,4-thiadiazol-3-yl)-2-ethoxyiminoacetamido] cephalosporins bearing 4-[3-(aminoalkyl)-ureido]-1-pyridinium at C-3'

Among the prepared C-3' substituted-pyridinium cephalosporins, a series of 7beta-[2-(5-amino-1,2,4-thiadiazol-3-yl)-2-ethoxyiminoacetamido] cephalosporins bearing 4-[3-(aminoalkyl)-ureido]-1-pyridinium at C-3' showed highly potent antibacterial activity against MRSA and Pseudomonas aeruginosa.     

The formation of 2-hydroxy-4-hydroxymethyl-3-(indol-3-yl)-cyclopent-2-enone derivatives from ascorbigens

A facile preparation is described of 3-(indol-3-yl)-2-hydroxy-4-hydroxymethylcyclopent-2-enone and its N-derivatives in 15-40% yields by the degradation of ascorbigen or its N-derivatives in a warm solution of L-ascorbic acid through a sequential domino reaction. The same cyclopentenone derivatives were obtained in 30-40% yields by the condensation of (N-alkylindol-3-yl)glycolic acids with ascorbic acid. 2,6-Dihydroxy-1-(indol-3-yl)hexa-1,4-diene-3-one and 2-hydroxy-4-hydroxymethyl-5-(indol-3-yl)cyclopent-2-enone were identified as intermediates in this reaction.     

A concise,total synthesis and antibacterial evaluation of 2-hydroxy-1-(1H-indol-3-yl)-4-methylpentan-3-one

Treatment of racemic 2-hydroxy-3-(1H-indol-3yl)propionic acid methyl ester (5) with isopropyl magnesium chloride provided the title compound 1 and its isomer, 3-hydroxy-1-(indol-3-yl)-4-methylpentan-2-one (9). Both enantiomers (>96% ee) of each component were obtained via semi-preparative chiral supercritical fluid chromatography (SFC). In contrast to previous reports, these compounds, as well as their acetate derivatives, were not active or very weakly active against 16 bacterial strains, including Escherichia coli, Bacillus subtilis and Staphylococcus aureus.