Stereoselective synthesis of novel 2'-(S)-CCG-IV analogues as potent NMDA receptor agonists.

We developed a versatile stereoselective route for the synthesis of new 2'-(S)-CCG-IV analogues. The route allows for late stage diversification and thereby provides access to a great variety of conformationally restricted cyclopropyl glutamate analogues. A selection of the 2'-(S)-CCG-IV analogues were evaluated using two-electrode voltage-clamp electrophysiology at recombinant GluN1/GluN2A-D receptors, demonstrating that agonists can be developed with GluN2 subunit-dependent potency and agonist efficacy. We also describe a crystal structure of the GluN2A agonist binding domain in complex with 2'-butyl-(S)-CCG-IV that determines the position of 2'-substituents in (S)-CCG-IV agonists in the glutamate binding site and provides further insight to the structural determinants of their agonist efficacy. The stereoselective synthesis described here enables versatile and straight-forward modifications to diverse analogues of interest for the development of potent subtype-specific NMDA receptor agonists and other applications.

Synthesis and anti-TMV activity of novel ß-amino acid ester derivatives containing quinazoline and benzothiazole moieties.

Here, a series of ß-amino acid ester derivatives containing quinazoline and benzothiazoles was synthesized and evaluated for anti-tobacco mosaic virus (TMV) activity. The compounds 3n, 3o, 3p and 3q showed good antiviral activity against TMV at a concentration of 500 µg/mL, with curative rates of 55.55%, 52.32%, 52.77% and 50.91%, respectively, and protection rates of 52.33%, 55.96%, 54.21% and 50.98%, respectively. These values were close to those of the commercially available antiviral agent ningnanmycin (which has curative and protection rates of 55.27% and 52.16%, respectively). To our knowledge, this is the first report of the anti-TMV activity of ß-amino acid ester derivatives containing quinazoline and benzothiazoles moieties; the results indicate that these novel compounds can potentially be used as protective agents against TMV diseases.

Functional imaging of oxidative stress with a novel PET imaging agent, 18F-5-fluoro-L-aminosuberic acid.

UNLABELLED: Glutathione is the predominant endogenous cellular antioxidant, playing a critical role in the cellular defensive response to oxidative stress by neutralizing free radicals and reactive oxygen species. With cysteine as the rate-limiting substrate in glutathione biosynthesis, the cystine/glutamate transporter (system xc(-)) represents a potentially attractive PET biomarker to enable in vivo quantification of xc(-) activity in response to oxidative stress associated with disease. We have developed a system xc(-) substrate that incorporates characteristics of both natural substrates, L-cystine and L-glutamate (L-Glu). L-aminosuberic acid (L-ASu) has been identified as a more efficient system xc(-) substrate than L-Glu, leading to an assessment of a series of anionic amino acids as prospective PET tracers. Herein, we report the synthesis and in vitro and in vivo validation of a lead candidate, (18)F-5-fluoro-aminosuberic acid ((18)F-FASu), as a PET tracer for functional imaging of a cellular response to oxidative stress with remarkable tumor uptake and retention. METHODS: (18)F-FASu was identified as a potential PET tracer based on an in vitro screening of compounds similar to L-cystine and L-Glu. Affinity toward system xc(-) was determined via in vitro uptake and inhibition studies using oxidative stress-induced EL4 and SKOV-3 cells. In vivo biodistribution and PET imaging studies were performed in mice bearing xenograft tumors (EL4 and SKOV-3). RESULTS: In vitro assay results determined that L-ASu inhibited system xc(-) as well as or better than L-Glu. The direct comparison of uptake of tritiated compounds demonstrated more efficient system xc(-) uptake of L-ASu than L-Glu. Radiosynthesis of (18)F-FASu allowed the validation of uptake for the fluorine-bearing derivative in vitro. Evaluation in vivo demonstrated primarily renal clearance and uptake of approximately 8 percentage injected dose per gram in SKOV-3 tumors, with tumor-to-blood and tumor-to-muscle ratios of approximately 12 and approximately 28, respectively. (18)F-FASu uptake was approximately 5 times greater than (18)F-FDG uptake in SKOV-3 tumors. Dynamic PET imaging demonstrated uptake in EL4 tumor xenografts of approximately 6 percentage injected dose per gram and good tumor retention for at least 2 h after injection. CONCLUSION: (18)F-FASu is a potentially useful metabolic tracer for PET imaging of a functional cellular response to oxidative stress. (18)F-FASu may provide more sensitive detection than (18)F-FDG in certain tumors.

Synthesis and pharmacological characterization of 4-substituted-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylates: identification of new potent and selective metabotropic glutamate 2/3 receptor agonists.

As part of our ongoing interest in identifying novel agonists acting at metabotropic glutamate (mGlu) 2/3 receptors, we have explored the effect of structural modifications of 1S,2S,5R,6S-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylate (LY354740), a potent and pharmacologically balanced mGlu2/3 receptor agonist. Incorporation of relatively small substituents (e.g., F, O) at the C4 position of this molecule resulted in additional highly potent mGlu2/3 agonists that demonstrate excellent selectivity over the other mGlu receptor subtypes, while addition of larger C4-substituents (e.g., SPh) led to a loss of agonist potency and/or the appearance of weak mGlu2/3 receptor antagonist activity. Further characterization of the α-fluoro-substituted analogue (LY459477) in vivo revealed that this molecule possesses good oral bioavailability in rats and effectively suppresses phencyclidine-evoked locomotor activity at doses that do not impair neuromuscular coordination. This molecule therefore represents a valuable new addition to the arsenal of pharmacological tools competent to investigate mGlu2/3 receptor function both in vitro and in vivo.

Development of 2'-substituted (2S,1'R,2'S)-2-(carboxycyclopropyl)glycine analogues as potent N-methyl-d-aspartic acid receptor agonists.

A series of 2'-substituted analogues of the selective NMDA receptor ligand (2S,1'R,2'S)-2-(carboxycyclopropyl)glycine ((S)-CCG-IV) have been designed, synthesized, and pharmacologically characterized. The design was based on a docking study hypothesizing that substituents in the 2'-position would protrude into a region where differences among the NMDA receptor GluN2 subunits exist. Various synthetic routes were explored, and two different routes provided a series of alkyl-substituted analogues. Pharmacological characterization revealed that these compounds are NMDA receptor agonists and that potency decreases with increasing size of the alkyl groups. Variations in agonist activity are observed at the different recombinant NMDA receptor subtypes. This study demonstrates that it is possible to introduce substituents in the 2'-position of (S)-CCG-IV while maintaining agonist activity and that variation among NMDA receptor subtypes may be achieved by probing this region of the receptor.

Enantio- and diastereocontrolled conversion of chiral epoxides to trans-cyclopropane carboxylates: application to the synthesis of cascarillic acid, grenadamide and L-(-)-CCG-II.

An efficient high yielding improved method for the enantio- and diastereoselective cyclopropanation of chiral epoxides using triethylphosphonoacetate and base (Wadsworth-Emmons cyclopropanation) is reported. The utility of this protocol is illustrated by concise and practical synthesis of cascarillic acid, grenadamide and L-(-)-CCG-II, a cyclopropane containing natural products.

Counterion specificity of surfactants based on dicarboxylic amino acids.

The behavior in solution of a series of amino acid-based surfactants having two carboxyl groups separated by a spacer of one, two, or three carbon atoms has been investigated. All three surfactants precipitated on addition of acid, but the aspartate surfactant (with a two-carbon spacer) was considerably more resistant to precipitation than the aminomalonate surfactant (one-carbon spacer) and the glutamate surfactant (three-carbon spacer). The interactions with the monovalent counterions lithium, sodium, and potassium were investigated by conductivity. It was found that lithium ions bound the strongest and potassium ions the weakest to the surfactant micelles. These results were interpreted using the hard and soft acid-base theory. Comparing the three surfactants with respect to binding of one specific counterion, sodium, showed that the aminomalonate surfactant, which has the shortest spacer, bound sodium ions the strongest and the glutamate surfactant, which has the longest spacer, had the lowest affinity for the counterion. Also that could be explained by the hard and soft acid-base concept. The glutamate surfactant was found to be considerably more resistant to calcium ions than the two other surfactants. This was attributed to this surfactant forming an intermolecular complex with the calcium ion at the air-water interface while the aminomalonate and the aspartate surfactants, with shorter distance between the carboxylate groups could form six- and seven-membered intramolecular calcium complexes.

Synthesis and activity of N-oxalylglycine and its derivatives as Jumonji C-domain-containing histone lysine demethylase inhibitors.

N-Oxalylglycine (NOG) derivatives were synthesized, and their inhibitory effect on histone lysine demethylase activity was evaluated. NOG and compound 1 inhibited histone lysine demethylases JMJD2A, 2C and 2D in enzyme assays, and their dimethyl ester prodrugs DMOG and 21 exerted histone lysine methylating activity in cellular assays.

Synthesis of bis-armed amino acid derivatives via the alkylation of ethyl isocyanoacetate and the Suzuki-Miyaura cross-coupling reaction.

Two synthetic routes to bis-armed-alpha-amino acid derivatives are described. The first route involves alkylation of dibromo derivatives with ethyl isocyanoacetate under phase-transfer catalysis (PTC) conditions. The second route uses a palladium-mediated Suzuki-Miyaura cross-coupling reaction between a DL-4-boronophenylalanine derivative and aromatic diiodo (or dibromo) compounds.

Design, synthesis, potency, and cytoselectivity of anticancer agents derived by parallel synthesis from alpha-aminosuberic acid.

Chemotherapy in the last century was characterized by cytotoxic drugs that did not discriminate between cancerous and normal cell types and were consequently accompanied by toxic side effects that were often dose limiting. The ability of differentiating agents to selectively kill cancer cells or transform them to a nonproliferating or normal phenotype could lead to cell- and tissue-specific drugs without the side effects of current cancer chemotherapeutics. This may be possible for a new generation of histone deacetylase inhibitors derived from amino acids. Structure-activity relationships are now reported for 43 compounds derived from 2-aminosuberic acid that kill a range of cancer cells, 26 being potent cytotoxins against MM96L melanoma cells (IC50 20 nM-1 microM), while 17 were between 5- and 60-fold more selective in killing MM96L melanoma cells versus normal (neonatal foreskin fibroblasts, NFF) cells. This represents a 10- to 100-fold increase in potency and up to a 10-fold higher selectivity over previously reported compounds derived from cysteine (J. Med. Chem. 2004, 47, 2984). Selectivity is also an underestimate, because the normal cells, NFF, are rarely all killed by the drugs that also induce selective blockade of the cell cycle for normal but not cancer cells. Selected compounds were tested against a panel of human cancer cell lines (melanomas, prostate, breast, ovarian, cervical, lung, and colon) and found to be both selective and potent cytotoxins (IC50 20 nM-1 microM). Compounds in this class typically inhibit human histone deacetylases, as evidenced by hyperacetylation of histones in both normal and cancer cells, induce expression of p21, and differentiate surviving cancer cells to a nonproliferating phenotype. These compounds may be valuable leads for the development of new chemotherapeutic agents.

Synthesis, in vitro pharmacology, structure-activity relationships, and pharmacokinetics of 3-alkoxy-2-amino-6-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic acid derivatives as potent and selective group II metabotropic glutamate receptor antagonists.

Novel group II metabotropic glutamate receptor (mGluR) antagonists, 3-alkoxy-2-amino-6-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic acid derivatives 11 and 12, were discovered by the incorporation of a hydroxy or alkoxyl group onto the C-3 portion of selective and potent group II mGluR agonist 5, (1R,2S,5R,6R)-2-amino-6-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic acid. Among these compounds, (1R,2R,3R,5R,6R)-2-amino-3-(3,4-dichlorobenzyloxy)-6-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic acid (-)-11be (MGS0039) was a highly selective and potent group II mGluR antagonist with the best pharmacokinetic profile. Compound (-)-11be exhibited high affinities for mGlu 2 (Ki = 2.38 +/- 0.40 nM) and mGlu 3 (4.46 +/- 0.31 nM) but low affinity for mGluR 7 (Ki = 664 +/- 106 nM), and potent antagonist activities for mGlu 2 (IC50 = 20.0 +/- 3.67 nM) and mGluR 3 (IC50 = 24.0 +/- 3.54 nM) but much less potent antagonist activities for mGlu 4 (IC50 = 1740 +/- 1080 nM), mGlu 6 (IC50 = 2060 +/- 1270 nM), mGlu 1 (IC50 = 93300 +/- 14600 nM), and mGluR 5 (IC(50) = 117000 +/- 38600 nM). No significant agonist activities of (-)-11be were found for mGluRs 2, 3, 4, 6, 1, and 5 (EC50 > 100,000 nM). Furthermore, (-)-11be exhibited dose-dependent oral absorption (plasma C(max): 214 +/- 56.7, 932 +/- 235, and 2960 +/- 1150 ng/mL for 3 mg/kg, 10 mg/kg, and 30 mg/kg, po, respectively) and acceptable blood-brain barrier penetration (brain C(max): 13.2 ng/mL for 10 mg/kg, p.o. 6 h). In this paper, we report the synthesis, in vitro pharmacological profile, and structure-activity relationships (SARs) of 3-alkoxy-2-amino-6-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic acid derivatives 11 and 12, and pharmacokinetic profiles of several typical compounds.

Coordination modes vs. antitumor activity: synthesis and antitumor activity of novel platinum(II) complexes of N-substituted amino dicarboxylic acids.

The trans-(+/-)-1,2-diaminocyclohexaneplatinum(II) complexes of multidentate L-glutamate (Glu) and L-aspartate (Asp) were prepared and their antitumor activity was examined in relation with their coordination modes. All these complexes were obtained as a mixture of (O,O')- and (O,N)-chelate isomers due to rapid isomerization of the initially formed (O,O')-isomer to the thermodynamically more stable (O,N)-isomer. The (O,O')/(O,N)-isomeric mixture with the mole ratio of 80/20 exhibited excellent antitumor activity while the pure (O,N)-isomer was only marginally active. Therefore, in order to prevent the linkage isomerization of the active (O,O')-isomer to the inactive (O,N)-isomer, we have designed N-substituted amino dicarboxylic acids as a leaving group and prepared a new series of complexes, [Pt(dach)(RGlu)] and [Pt(dach)(RAsp)] (dach=trans-(+/-)-1,2-diaminocyclohexane; R=acetyl (Ac), propionyl (Pro), pivaloyl (Piv), carbobenzyloxy (Cbz) or phthaloyl (Phth)) and characterized by means of elemental analyses, and 1H NMR, 195Pt NMR and IR spectroscopies. The N-substituted amino dicarboxylate ligands were found to coordinate to platinum(II) ion through only the (O,O')-chelation mode, and their Pt(II) complexes were chemically stable in aqueous solution. The present Pt(II) complexes of N-substituted amino dicarboxylic acids showed excellent antitumor activity against both murine leukemia L1210 and human tumor cells. Especially, the highly hydrophobic N-phthaloylglutamate complex, [Pt(dach)(PhthGlu)], exhibited an outstanding in vitro activity (IC50=2.22 microM) on the human stomach cancer cells which are not responsive to cisplatin and carboplatin.

Enantioselective Synthesis of L-CCG-I.

Introduction of natural menthol as the chiral auxiliary in a gamma-Br-alpha,beta-unsaturated ester leads to enantioselective generation of three chiral centers in a single step on reaction with a glycine anion equivalent to provide L-CCG-I in 94% ee.

Synthesis and anticonvulsant activity of novel bicyclic acidic amino acids.

Bicyclic acidic amino acids (+/-)-6 and (+/-)-7, which are conformationally constrained homologues of glutamic acid, were prepared via a strategy based on a 1,3-dipolar cycloaddition. The new amino acids were tested toward ionotropic and metabotropic glutamate receptor subtypes; both of them behaved as antagonists at mGluR1,5 and as agonists at mGluR2. Furthermore, whereas (+/-)-6 was inactive at all ionotropic glutamate receptors, (+/-)-7 displayed a quite potent antagonism at the NMDA receptors. In the in vivo tests on DBA/2 mice, the compounds displayed an anticonvulsant activity. The interesting pharmacological profile of (+/-)-7 qualifies it as a lead of novel neuroprotective agents.

The peptides of alpha-aminosuberic acid I. New intermediates for synthetic studies.

The paper describes the synthesis of alpha-aminosuberic acid derivatives suitable for the synthesis of peptides. These include Z-, Boc- and Fmoc-protection on the alpha-amino group, benzyl ester, Boc-hydrazide and Z-hydrazide as well as the free carboxylic function in the side chain, and methyl ester, benzyl ester or free alpha-carboxylic group. Their use is demonstrated on the synthesis of the respective derivatives of Asu-Val-Leu. The enzyme catalyzed reaction was successfully used both as a route to L-Asu from the D,L-compound as well as for the direct synthesis of the optically active tripeptide derivative from the Z-D,L-Asu-OH.

Preparation of difluoro analogs of CCGs and their pharmacological evaluations.

All the stereoisomers of 2-(2-carboxy-3,3-difluorocyclopropyl)glycines (F2CCGs) were synthesized in enantiomerically pure forms using (R)-2,3-O-isopropyl-ideneglyceraldehyde as a chiral precursor. L-F2CCG-I, one of the stereoisomers corresponding to an extended form of L-glutamate was found to be a potent agonist for metabotropic glutamate receptors (mGluRs).

Tandem enzymatic resolution yielding L-alpha-aminoalkanedioic acid omega-esters.

The tandem action of serine protease (alpha-chymotrypsin or subtilisin BPN') and Aspergillus genus aminoacylase on racemic N-acetyl-alpha-aminoalkanedioic acid alpha,omega-diester produced L-alpha-aminoalkanedioic acid omega-ester in good yield and high optical purity. L-alpha-Aminosuberic acid omega-ester thus obtained was conveniently introduced into an oxytocin analog, [Asu1,6]oxytocin, by the solid-phase-synthesis and cyclization-cleavage method with oxime resin.

Definition of a pharmacophore for the metabotropic glutamate receptors negatively linked to adenylyl cyclase.

(2S,3S,4S)-alpha-Carboxycyclopropylglycine (L-CCG I) and trans-1-amino-(1S,3R)-cyclopentanedicarboxylic acid ((1S,3R)-ACPD), partially constrained L-glutamate analogs known to be agonists at the metabotropic glutamate receptors (mGluRs) adenylyl cyclase coupled, have been submitted to conformational analysis and the data obtained utilized to define a pharmacophore which takes into account the location of hydrogen bonding donating sites of the receptor. This pharmacophore has been utilized to define the agonist mGluRs decreases cAMP bioactive conformation of L-Glu.

Novel inhibitors of prolyl 4-hydroxylase. 3. Inhibition by the substrate analogue N-oxaloglycine and its derivatives.

N-Oxaloglycine (3) is an alpha-ketoglutarate (1) analogue that is a competitive inhibitor of prolyl 4-hydroxylase (EC 1.14.11.2). A study of the structure-activity relationships of some other oxalo derivatives shows that substitution on the glycine moiety modulates activity stereoselectively and that if the omega-carboxylate is homologated or replaced by either acylsulfonamides or anilide, then activity is sharply reduced. This sensitivity to these changes is contrasted with the relative insensitivity of another putative alpha-ketoglutarate analogue, pyridine-2,5-dicarboxylic acid (2), and the implication is discussed that compounds of both series are unlikely to bind to prolyl hydroxylase in the same way even though both inhibit the enzyme competitively.