Tommy N. Johansen

Resolution, absolute stereochemistry and molecular pharmacology of the enantiomers of ATPA.

(RS)-2-Amino-3-(5-tert-butyl-3-hydroxy-4-isoxazolyl)propionic acid (ATPA), an analogue of (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA). has previously been shown to be a relatively weak AMPA receptor agonist and a very potent agonist at the GluR5 subtype of kainic acid-preferring (S)-glutamic acid ((S)-Glu) receptors. We report here the separation of (+)- and (-)-ATPA, obtained at high enantiomeric purity (enantiomeric excess values of 99.8% and > 99.8%, respectively) using chiral chromatography, and the unequivocal assignment of the stereochemistry of (S)-(+)-ATPA and (R)-(-)-ATPA. (S)- and (R)-ATPA were characterized in receptor binding studies using rat brain membranes, and electrophysiologically using the rat cortical wedge preparation and cloned AMPA-preferring (GluR1, GluR3, and GluR4) and kainic acid-preferring (GluR5, GluR6, and GluR6 + KA2) receptors expressed in Xenopus oocytes. In the cortical wedge, (S)-ATPA showed AMPA receptor agonist effects (EC50 = 23 microM) approximately twice as potent as those of ATPA. (R)-ATPA antagonized depolarizations induced by AMPA (Ki = 253 microM) and by (S)-ATPA (Ki = 376 microM), and (R)-ATPA antagonized the biphasic depolarizing effects induced by kainic acid (Ki = 301 microM and 1115 microM). At cloned AMPA receptors, (S)-ATPA showed agonist effects at GluR3 and GluR4 with EC50 values of approximately 8 microM and at GluR1 (EC50 = 22 microM), producing maximal steady state currents only 5.4-33% of those evoked by kainic acid. (R)-ATPA antagonized currents evoked by kainic acid at cloned AMPA receptor subtypes with Ki values of 33-75 microM. (S)-ATPA produced potent agonist effects at GluR5 (EC50 = 0.48 microM). Due to desensitization of GluR5 receptors, which could not be fully prevented by treatment with concanavalin A, (S)-ATPA-induced agonist effects were normalized to those of kainic acid. Under these circumstances, maximal currents produced by (S)-ATPA and kainic acid were not significantly different. (R)-ATPA did not attenuate currents produced by kainic acid at GluR5, and neither (S)- nor (R)-ATPA showed significant effects at GluR6. (S)-ATPA as well as AMPA showed weak agonist effects at heteromeric GluR6 + KA2 receptors, whereas (R)-ATPA was inactive. Thus, (S)- and (R)-ATPA may be useful tools for mechanistic studies of ionotropic non-NMDA (S)-Glu receptors, and lead structures for the design of new subtype-selective ligands for such receptors.

Molecular pharmacology of 4-substituted glutamic acid analogues at ionotropic and metabotropic excitatory amino acid receptors

The pharmacology of (2S,4R)-4-methylglutamic acid, (2S,4S)-4-methylglutamic acid and (S)- and (R)-4-methyleneglutamic acids (obtained in high chemical and enantiomeric purity from racemic 4-methyleneglutamic acid by chiral HPLC using a Crownpak CR(+) column), was examined in binding experiments using rat brain ionotropic glutamate receptors, and in functional assays using cloned metabotropic glutamate (mGlu) receptors. As a notable result of these studies, (2S,4R)-4-methylglutamic acid and (2S,4S)-4-methylglutamic acid were shown to be selective for kainic acid receptors and mGlu receptors (subtypes 1alpha and 2), respectively, whereas (S)-4-methyleneglutamic acid showed high but rather non-selective affinity for the (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA), kainic acid, NMDA and mGlu receptors (subtypes 1alpha and 2). Although none of the compounds were specific for any of the receptor subtypes, the results demonstrate that each of these structurally related compounds has a distinct pharmacological profile.

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.

Synthesis and Antitumor Effect in Vitro and in Vivo of Substituted 1,3-Dihydroindole-2-ones

Optimization of the anticancer activity for a class of compounds built on a 1,3-dihydroindole-2-one scaffold was performed. In comparison with recently published derivatives of oxyphenisatin the new analogues exhibited an equally potent antiproliferative activity in vitro and improved tolerability and activity in vivo. The best compounds from this series showed low nanomolar antiproliferative activity toward a series of cancer cell lines (compound (S)-38: IC(50) of 0.48 and 2 nM in MCF-7 (breast) and PC3 (prostate), respectively) and potent antitumor effects in well tolerated doses in xenograft models. The racemic compound (RS)-38 showed complete tumor regression at a dose of 20 mg/kg administered iv on days 1 and 7 in a PC3 rat xenograft.

A convenient synthesis of 4-substituted 3-ethoxy-5-methylisoxazoles by palladium-catalyzed coupling reactions

The coupling reactions were performed using 3-ethoxy-4-iodo-5-methylisoxazole (4) as the key intermediate. Coupling of 4 under Suzuki–Miyaura or Stille conditions using Pd(PPh3)2Cl2 and arylboronic acids or aryltin analogues, respectively, gave 4-aryl substituted isoxazoles in yields ranging from 49% for the 3-pyridyl analogue 14, to 96% for the 4-pyridyl analogue 12. Under Heck reaction conditions using Pd(PPh3)2Cl2 and 4, analogues of 3-ethoxy-5-methylisoxazole containing vinylic or acetylenic groups in the 4-position were synthesized in yields ranging from 58 to 98%. 3-Ethoxy-5-methylisoxazol-4-ylmagnesium bromide (19), prepared from 4 and isopropylmagnesium bromide, reacted smoothly with benzaldehyde or benzoyl chloride to give the desired 4-[hydroxy(phenyl)methyl] analogue 21 and 4-benzoyl-3-ethoxy-5-methylisoxazole (22), respectively. Transmetallation of 19 with ZnCl2 and subsequent treatment with Pd(PPh3)2Cl2 and 4-iodotoluene gave 3-ethoxy-5-methyl-4-(4-methylphenyl)isoxazole (23) in 80% yield.

Synthesis and Pharmacology of the Baclofen Homologues 5-Amino-4-(4-chlorophenyl)pentanoic Acid and the R- and S-Enantiomers of 5-Amino-3-(4-chlorophenyl)pentanoic Acid

(RS)-5-Amino-4-(4-chlorophenyl)pentanoic acid (10) and the R-form (11) and S-form (12) of (RS)-5-amino-3-(4-chlorophenyl)pentanoic acid, which are homologues of the 4-aminobutanoic acidB (GABAB) receptor agonist (RS)-4-amino-3-(4-chlorophenyl)butanoic acid (baclofen), were synthesized. Compound 10 was synthesized by homologation at the carboxyl end of baclofen using a seven-step reaction sequence. N-Boc-protected (4R, 5R)-4-(4-chlorophenyl)-5-hydroxy-2-piperidone (18) was deoxygenated via a modified Barton-McCombie reaction to give N-Boc-protected (R)-4-(4-chlorophenyl)-2-piperidone (20), which was ring opened and deprotected to give 11.HCl. The corresponding S-enantiomer, 12.HCl, was synthesized analogously from the 4S,5S-enantiomer of 18, compound 21. The enantiomeric purities of 11.HCl (ee = 99.8%) and 12. HCl (ee = 99.3%) were determined by chiral HPLC. Compound 10 did not show detectable affinity for GABAA or GABAB receptor sites and was inactive as an agonist or an antagonist at GABAB receptors in the guinea pig ileum. Like the enantiomers of baclofen, neither 11 nor 12 showed detectable affinity for GABAA receptor sites, and in agreement with the findings for (S)-baclofen, 12 did not interact significantly with GABAB receptor sites. Compound 11 (IC50 = 7.4 +/- 0.6 microM), a homologue of (R)-baclofen (2), was shown to be some 50 times weaker than 2 (IC50 = 0.14 +/- 0.01 microM) as an inhibitor of GABAB binding. Accordingly, 11 (EC50 = 150 +/- 23 microM) was shown to be weaker than 2 (EC50 = 11 +/- 1 microM) as an inhibitor of electrically induced contractions of the guinea pig ileum. However, whereas this effect of 2 was sensitive to the GABAB antagonist, CGP35348 (4), the inhibition by 11 was not significantly affected. Furthermore, 12 (EC50 = 310 +/- 16 microM) was shown to be one-half as potent as 11 in this test system, and this effect of 12 also was insensitive to 4. The dissimilarities of the pharmacological effects of 2 and compounds 11 and 12 were emphasized by the observation that whereas 2 only inhibits the ileum contraction by 59 +/- 5%, 11 as well as 12 were shown to inhibit this response by approximately 94%. Neither 11 nor 12 appeared to affect significantly cholinergic mechanisms in the ileum, and their mechanism(s) of action remain enigmatic.

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 > or = 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 microM) and a potent AMPA receptor agonist in the rat cortical wedge preparation (EC50 = 4.5 +/- 0.3 microM) comparable with AMPA (IC50 = 0.040 +/- 0.01 microM; EC50 = 3.5 +/- 0.2 microM), but much more potent than (+)-(S)-APPA (IC50 = 5.5 +/- 2.2 microM; EC50 = 230 +/- 12 microM). Like (-)-(R)-APPA (IC50 > 100 microM), (-)-(R)-2-Py-AMPA (IC50 > 100 microM) did not significantly affect [3H]AMPA binding, and both compounds were weak AMPA receptor antagonists (Ki = 270 +/- 50 and 290 +/- 20 microM, respectively).

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.

2-Amino-3-(3-hydroxy-1,2,5-thiadiazol-4-yl)propionic acid: resolution, absolute stereochemistry and enantiopharmacology at glutamate receptors

In order to identify new subtype-selective (S)-glutamate (Glu) receptor ligands we have synthesized (RS)-2-amino-3-(3-hydroxy-1,2,5-thiadiazol-4-yl)propionic acid [(RS)-TDPA]. Resolution of (RS)-TDPA by chiral chromatography was performed using a Crownpac CR(+) column affording (R)- and (S)-TDPA of high enantiomeric purity (enantiomeric excess=99.9%). An X-ray crystallographic analysis revealed that the early eluting enantiomer has R-configuration. Both enantiomers showed high affinity as well as high agonist activity at (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) receptors, determined using a [(3)H]AMPA binding assay and an electrophysiological model, respectively. The affinities and agonist activities obtained for (R)-TDPA (IC(50)=0.265 microM and EC(50)=6.6 microM, respectively) and (S)-TDPA (IC(50)=0.065 microM and EC(50)=20 microM, respectively) revealed a remarkably low AMPA receptor stereoselectivity, (S)-TDPA showing the highest affinity and (R)-TDPA the most potent agonist activity. In addition, (S)-TDPA was shown to interact with synaptosomal Glu uptake sites displacing [(3)H](R)-aspartic acid (IC(50 ) approximately 390 microM). An enantiospecific and subtype-selective agonist activity was observed for (S)-TDPA at group I metabotropic Glu (mGlu) receptors (EC(50)=13 microM at mGlu(5) and EC(50)=95 microM at mGlu(1)).

A New Phenylalanine Derivative Acts as an Antagonist at the AMPA Receptor GluA2 and Introduces Partial Domain Closure: Synthesis, Resolution, Pharmacology, and Crystal Structure

In order to map out molecular determinants for competitive blockade of AMPA receptor subtypes, a series of 2-carboxyethylphenylalanine derivatives has been synthesized and pharmacologically characterized in vitro. One compound in this series, (RS)-3h, showed micromolar affinity for GluA1(o) and GluA2(R)(o) receptors with an approximately 4-fold preference for GluA1/2 vs GluA3/4. In TEVC electrophysiological experiments (RS)-3h competitively antagonized GluA2(Q)(i) receptors. The X-ray structure of the active enantiomer (S)-3h in complex with GluA2-S1S2J showed a domain closure around 8°. Even though the nitro and the carboxyethyl groups of (S)-3h were both anchored to Tyr702 through a water H-bond network, these interactions only induced weak subtype selectivity. In spite of the fact that (S)-3h induced a domain closure close to that observed for partial agonists, it did not produce agonist responses at GluA2 receptors under nondesensitizing conditions. 2-Carboxyethylphenylalanine derivatives provide a new synthetic scaffold for the introduction of substituents that could lead to AMPA receptor subtype-selective ligands.