RPR 119990, a novel alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid antagonist: synthesis, pharmacological properties, and activity in an animal model of amyotrophic lateral sclerosis.

Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptor antagonists are of potential interest for the treatment of certain acute and chronic neurodegenerative diseases, including amyotrophic lateral sclerosis. Here, we describe the synthesis and pharmacological properties of 9-carboxymethyl-4-oxo-5H,10H-imidazo[1,2-a]indeno[1,2-e]pyrazin-2-phosphonic acid (RPR 119990). The compound displaced [3H]AMPA from rat cortex membranes with a K(i) of 107 nM. In oocytes expressing human recombinant AMPA receptors, RPR 119990 depressed ion flux with a K(B) of 71 nM. The antagonist properties of this compound were confirmed on rat native AMPA receptors in cerebella granule neurons in culture and in hippocampal slices where it antagonized electrophysiological responses with IC50 values of 50 and 93 nM, respectively. RPR 119990 antagonized hippocampal evoked responses in vivo, demonstrating brain penetration at active concentrations. RPR 119990 is a potent anticonvulsant in the supramaximal electroshock in the mouse with an ED50 of 2.3 mg/kg 1 h post s.c. administration, giving it a workably long action. Pharmacokinetic studies show good passage into the plasma after subcutaneous administration, whereas brain penetration is low but with slow elimination. This compound was found active in a transgenic mouse model of familial amyotrophic lateral sclerosis (SOD1-G93A) where it was able to improve grip muscle strength and glutamate uptake from spinal synaptosomal preparations, and prolong survival with a daily dose of 3 mg/kg s.c.

Synthesis of anticonvulsive AMPA antagonists: 4-oxo-10-substituted-imidaz.

The overstimulation of excitatory amino acid receptors such as the glutamate AMPA receptor has been implicated in the physiopathogenesis of epilepsy as well as in acute and chronic neurodegenerative disorders. An original series of readily water soluble 4-oxo-10-substituted-imidazo[1,2-a]indeno[1,2-e]pyrazin-2-carboxylic acid derivatives was synthesized. The most potent derivative 6a exhibited nanomolar binding affinity (IC50 = 35nM) and antagonist activity (IC50 = 6nM) at ionotropic AMPA receptor. This compound also demonstrated potent anticonvulsant properties in MES in mice and rats with long durations of action with ED50 values in the 1-3 mg/kg dose range following ip and iv administration.

Bioisosteres of 9-carboxymethyl-4-oxo-imidazo[1,2-a]indeno-[1,2-e]pyrazin-2-carboxylic acid derivatives. Progress towards selective, potent in vivo AMPA antagonists with longer durations of action.

A novel series of 2- and 9-disubstituted heterocyclic-fused 4-oxo-indeno[1,2-e]pyrazin derivatives was synthesized. One of them, the 9-(1H-tetrazol-5-ylmethyl)-4-oxo-5,10-dihydroimidazo[1,2-a]indeno[1,2-e]pyrazin-2-yl phosphonic acid 4i exhibited a strong and a selective binding affinity for the AMPA receptor (IC50 = 13 nM) and demonstrated potent antagonist activity (IC50 = 6nM) at the ionotropic AMPA receptor. This compound also displayed good anticonvulsant properties against electrically-induced convulsions after ip and iv administration with ED50 values between 0.8 and 1 mg/kg. Furthermore, a strong increase in potency was observed when given iv 3 h before test (ED50 = 3.5 instead of 25.6 mg/kg for the corresponding 9-carboxymethyl-2-carboxylic acid analogue). These data confirmed that there is an advantage in replacing the classical carboxy substituents by their bioisosteres such as tetrazole or phosphonic acid groups.

8-Methylureido-10-amino-10-methyl-imidazo[1,2-a]indeno[1,2-e] pyrazine-4-ones: highly in vivo potent and selective AMPA receptor antagonists.

Water soluble 8-methylureido-10-amino-10-methyl-imidazo[1,2-a]indeno[1,2-e]pyraz ine-4-one 4 represents a novel class of highly potent and selective AMPA receptors antagonists with in vivo activity. The dextrorotatory isomer (+)-4 was found to display the highest affinity with an IC50 of 10 nM. It also exhibited very good anticonvulsant effects after i.p., s.c. and i.v. administration in mice subjected to electrical convulsions (MES) and i.p. in audiogenic seizure-e in DBA/2 mice (ED50's < or = 10 mg/kg).

4,10-Dihydro-4-oxo-4H-imidazo[1,2-a]indeno[1,2-e]pyrazin-2-carboxylic acid derivatives: highly potent and selective AMPA receptors antagonists with in vivo activity.

A novel series of 2-substituted-4,5-dihydro-4-oxo-4H-imidazo[1,2-a]indeno[1,2-e]pyrazine derivatives was synthesised. One of them, 4e-a highly water soluble compound exhibited a nanomolar affinity and demonstrated competitive antagonist properties at the ionotropic AMPA receptors. This compound also displayed potent anticonvulsant properties against electrically or sound-induced convulsions in mice after systemic administration, thus suggesting adequate brain penetration.

8-Methylureido-4,5-dihydro-4-oxo-10H-imidazo[1,2-a]indeno[1,2-e]pyrazines: highly potent in vivo AMPA antagonists.

A novel series of readily water soluble 8-methylureido-4,5-dihydro-4-oxo-10H-imidazo[1,2-a]indeno[1,2-e]++ +pyrazines were synthesized. The -10-yl acetic acid ((+)-3) and -10-carboxylidene (4) derivatives exhibit potent affinities (IC50=4 and 19 nM, respectively) and antagonist properties (IC50 = 2 and 3 nM, respectively) at the ionotropic AMPA receptor. These compounds also display anticonvulsant properties against both electrically and sound-induced convulsions in mice after ip, sc and iv administration with ED50 values between 0.9 and 11 mg/kg, thus suggesting adequate brain penetration.

RPR112378 and RPR115781: two representatives of a new family of microtubule assembly inhibitors.

A screening program aimed at the discovery of new antimicrotubule agents yielded RPR112378 and RPR115781, two natural compounds extracted from the Indian plant Ottelia alismoides. We report their isolation, structural determination, and mechanisms of action. RPR112378 is an efficient inhibitor of tubulin polymerization (IC(50) = 1.2 microM) and is able to disassemble preformed microtubules. Regarding tubulin activity, RPR115781 is 5-fold less active than RPR112378. Tubulin-RPR112378 complexes, when isolated by gel filtration, were able to block further tubulin addition to growing microtubules, a mechanism that accounts for the substoichiometric effect of the drug. RPR112378 was found to prevent colchicine binding but not vinblastine binding to tubulin. Although colchicine binding is known to induce an increase of tubulin GTPase activity, no such increase was observed with RPR112378. We show that RPR112378 is a highly cytotoxic compound and that RPR115781 is 10, 000-fold less active as an inhibitor of KB cell growth. Part of the cytotoxicity of RPR112378 is probably caused by a reaction of addition with sulfhydryl groups, an observation that has not been made with RPR115781. In conclusion, these molecules represent a new class of inhibitors of microtubule assembly with potential therapeutic value.

Synthesis and potent anticonvulsant activities of 4-oxo-imidazo[1,2-a]inden.

The over-stimulation of excitatory amino acid receptors such as the glutamate AMPA receptor has been suggested to be associated with neurodegenerative disorders. Here we describe an original series of readily water soluble 4-oxo-imidazo[1,2-a] indeno[1,2-e]pyrazin-8- and -9-carboxylic (acetic) acid derivatives. One of these compounds, 4f, exhibited nanomolar binding affinity, potent competitive antagonism at the ionotropic AMPA receptor and a long duration of anticonvulsant activity after administration by parenteral route in vivo.

Degradation pathways of salmon calcitonin in aqueous solution.

Salmon calcitonin (sCT), a 32-amino-acid peptide, is the active component in many pharmaceuticals used for the management of bone diseases. The degradation pathways of sCT were determined, and the structures of the major degradation products were identified. Aqueous solutions of sCT at pH values of 3, 4, 5, and 6 were degraded, and the major degradation products were detected using reversed phase and size-exclusion high-performance liquid chromatography (HPLC). The degradation rate and pathways of sCT are strongly dependent on pH in the pH range between 3 and 6. The major degradation products were isolated by semipreparative HPLC and identified using a variety of spectroscopic and bioanalytical techniques. The results show that sCT can undergo hydrolyses resulting in cleavage of the 1-2 amide bond and deamidation of the Gln14 and Gln20 residues, sulfide exchange that leads to an unusual trisulfide derivative, and dimerization to reducible and nonreducible dimers. The mechanisms for the pathways can be rationalized from known degradation pathways of peptide and proteins.

Identification and properties of a major plasma metabolite of irinotecan (CPT-11) isolated from the plasma of patients.

Irinotecan [7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin (CPT-11)] is a promising water-soluble analogue of camptothecin [S. Sawada et al., Chem. & Pharm. Bull. (Tokyo), 39: 1446-1454, 1991]. We have reported previously the presence of an important polar metabolite, in addition to 7-ethyl-10-hydroxycamptothecin (SN-38) beta-glucuronide, in samples of plasma taken from patients undergoing treatment with CPT-11 (L.P. Rivory and J. Robert, Cancer Chemother. Pharmacol. 36: 176-179, 1995; L. P. Rivory and J. Robert, J. Cromatogr., 661: 133-141, 1994). Plasma samples (0.5 ml) containing comparatively large amounts of this metabolite were extracted by solid-phase columns and subjected to high-performance liquid chromatography and mass spectrometry in parallel to fluorometric detection. The metabolite yielded [M + 1] ions with a m/z of 619, representing the addition of 32 atomic mass units to CPT-11. Purified fractions were subjected to proton nuclear magnetic resonance, and the structure determined, 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino]carbonyloxycampothecin (APC), was further validated following synthesis. Like CPT-11, APC was found to be only a weak inhibitor of the cell growth of KB cells in culture (IC50, 2.1 versus 5.5 micrograms/ml for CPT-11 and 0.01 microgram/ml for SN-38, the active metabolite of CPT-11) and was a poor inducer of topoisomerase I DNA-cleavable complexes (100-fold less potent than SN-38). In contrast to CPT-11, APC was not hydrolyzed to SN-38 by human liver microsomes or purified human liver carboxylesterase. Furthermore, APC did not inhibit the hydrolysis of CPT-11 in these preparations. Interestingly, APC was only a weak inhibitor of acetylcholinesterase in comparison to CPT-11 and neostigmine. It appears likely, therefore, that APC does not contribute directly to the activity and toxicity profile of CPT-11 in vivo.

Unusual transformation of the 3-hydroxy-picolinoyl residue of pristinamycin IA.

Pristinamycin IA was modified in a two-step procedure to give original derivatives possessing a tricyclic nucleus (8a, 8b, 8c) or a substituted pyrrole ring (10a, 10b) in place of the natural exocyclic 3-hydroxy-picolinoyl residue. This transformation involved firstly preparation of pyridinium betaines 5 from pristinamycin IA and secondly a 1-3 dipolar cycloaddition between 5 and N-substituted maleimides or diethyl acetylenedicarboxylate. The compounds obtained were evaluated as antibacterial agents alone and in association with pristinamycin IIA.

Primary structure control of recombinant proteins using high-performance liquid chromatography, mass spectrometry and microsequencing.

The conformity of two recombinant proteins (a von Willbrand factor fragment and human serum albumin, consisting of respectively 289 and 585 amino acids) has been examined by HPLC combined with mass spectrometry and microsequencing, on both intact material and fragment peptides obtained by proteolytic cleavage. These studies confirmed that the primary structure of the recombinant proteins corresponds to that predicted from their gene, particularly the integrity of their N and C termini, and, in the case of albumin, the agreement between the observed disulfide bond pattern and the published model. Furthermore, the structure of an albumin-related compound could be elucidated. Application of LC-MS for batch-to-batch quality control is also under discussion.

The origin of the diversity of crotoxin isoforms in the venom of Crotalus durissus terrificus.

Crotoxin, the main toxin from the venom of the South American rattlesnake Crotalus durissus terrificus, is a beta-neurotoxin which consists of the non-covalent association of two subunits: a phospholipase A2 subunit B (CB), and a non-enzymic subunit A (CA). We have previously purified and characterized several isoforms of each subunit of crotoxin in the venom collected from numerous snakes. Furthermore, three cDNAs encoding two CB isoforms and the precursor, pro-CA, of subunit A have been isolated from a cDNA library prepared from a single venom gland of Crotalus durissus terrificus. The aim of this study is to analyse an individual snake venom from an animal that has been used to construct a cDNA library. Several isoforms of subunit A and two isoforms of subunit B were isolated and compared to purified and characterized subunit isoforms from pooled venom. The result of this study showed that the multiplicity and the diversity of crotoxin isoforms result from post-translational modifications occurring on a precursor and from the expression of different messenger RNAs present in an individual snake. It allowed for the identification of the two CB isoforms encoding cDNAs expressed in the individual venom with two isoforms from pooled venom, CBc and probably CBa2, that belong to two classes of crotoxin complexes which can be distinguished biochemically and pharmacologically.

Solution structure of RP 71955, a new 21 amino acid tricyclic peptide active against HIV-1 virus.

The structure of RP 71955, a new tricyclic 21 amino acid peptide active against human immunodeficiency virus 1, was determined. Its amino acid composition was inferred from the results of fast atom bombardment mass spectrometry, nuclear magnetic resonance, Raman spectroscopy, and amino acid analysis. Its sequence could not be determined classically, using Edman degradation, given the lack of a free terminal NH2. It was deduced from the interpretation of interresidue nuclear Overhauser effects and confirmed by the sequencing of peptides obtained by limited chemical hydrolysis. It was found to be CLGIGSCNDFAGCGYAVVCFW. An internal amide bond between the NH2 of C1 and the gamma-COOH of D9 was observed, as well as two disulfide bridges, one between C1 and C13 and one between C7 and C19. The three-dimensional structure of RP 71955 was determined from nuclear magnetic resonance derived constraints using distance geometry, restrained molecular dynamics, nuclear Overhauser effect back calculation, and an iterative refinement using a full relaxation matrix approach. Analogies between the structure of RP 71955 and some functional domains of gp41, the transmembrane protein of human immunodeficiency virus 1, suggest hypotheses concerning the mode of action of RP 71955.

Four triterpenoid saponins from dried roots of Gypsophila species.

Four new triterpenoid saponins were isolated from the roots of Gypsophila paniculata and G. arrostii. Their structures were elucidated using a combination of homo- and heteronuclear 2D NMR techniques, without having recourse to chemical degradation or modification. The saponins investigated are: 3-O-beta-D-galactopyranosyl-(1----2)-[beta-D-xylopyranosyl-(1----3)]-bet a-D- glucuronopyranosyl quillaic acid 28-O-beta-D-glucopyranosyl-(1----3)-[beta-D-xylopyranosyl-(1----4)]-alph a- L-rhamnopyranosyl-(1----2)-beta-D-fucopyranoside; 3-O-beta-D-galactopyranosyl-(1----2)-[beta-D-xylopyranosyl-(1----3)]-bet a- D-glucuronopyranosyl quillaic acid 28-O-beta-D-arabinopyranosyl-(1----4)-beta-D-arabinopyranosyl++ +-(1----3)-beta-D- xylopyranosyl-(1----4)-alpha-L-rhamnopyranosyl-(1----2)-beta-D-fucopyran oside; 3-O-beta-D-glucopyranosyl-(1----2)-beta-D-glucuronopyranosyl gypsogenin 28-O-beta-D-glucopyranosyl-(1----3)-[beta-D-xylopyranosyl-(1----4)]-alph a- L-rhamnopyranosyl-(1----2)-beta-D-fucopyranoside; 3-O-beta-D-xylopyranosyl-(1----3)-[beta-D-galactopyranosyl-(1----2)]-bet a- D-glucuronopyranosyl gypsogenin 28-O-beta-D-glucopyranosyl-(1----3)-[beta-D-xylopyranosyl-(1----4)-alpha -L- rhamnopyranosyl-(1----2)-beta-D-fucopyranoside.

Sesquiterpene Lactones from Aegialophila pumila.

Cnicin was isolated as the major sesquiterpene lactone from Aegialophila pumila Jus. Boiss. The germacranolide salonitenolide was isolated as a minor constituent. Cnicin demonstrated pronounced cytotoxic activity when tested in vitro against TLX-5 mice lymphoma cells.