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MDL 105,519, ( E )-3-(2-phenyl-2-carboxyethenyl)-4,6-dichloro-1 H -indole-2-carboxylic acid, is a potent and selective inhibitor of [ 3 H]glycine binding to the NMDA receptor. MDL 105,519 inhibits NMDA ( N -methyl- d -aspartate)-dependent responses including elevations of [ 3 H] N -[1,(2-thienyl)cyclohexyl]-piperidine ([ 3 H]TCP) binding in brain membranes, cyclic GMP accumulation in brain slices, and alterations in cytosolic Ca 2+ and Na + -Ca 2+ currents in cultured neurons. Inhibition was non-competitive with respect to NMDA and could be nullified with d -serine. Intravenously administered MDL 105,519 prevented harmaline-stimulated increases in cerebellar cyclic GMP content, providing biochemical evidence of NMDA receptor antagonism in vivo. This antagonism was associated with anticonvulsant activity in genetically based, chemically induced, and electrically mediated seizure models. Anxiolytic activity was observed in the rat separation-induced vocalization model, but muscle-relaxant activity was apparent at lower doses. Higher doses impair rotorod performance, but were without effect on mesolimbic dopamine turnover or prepulse inhibition of the startle reflex. This pattern of activities differentiates this compound from (5 R ,10 S )-(+)-5-methyl-10,11-dihydro-5 H -dibenzo[ a , d ]cyclohepten-5,10-imine (MK-801) and indicates a lower psychotomimetic risk. © 1997 Elsevier Science B.V. All rights reserved.

NMDA receptors play a critical role in neurotransmission and are also involved in the occurrence of excitotoxic nerve cell death. Synthetic halogenated analogs of the endogenous broad spectrum excitatory amino acid receptor blocker kynurenic acid are among the most potent and selective antagonists of the glycine co-agonist site of the NMDA receptor complex. Pharmacological blockade of this site provides neuroprotection in animal models of cerebral ischemia, epilepsy and neurodegenerative disorders, and does not appear to be associated with some of the undesirable side effects linked to classic competitive and non-competitive NMDA receptor antagonists. Here we demonstrate the neuroprotective quantities of 7-chloro-kynurenic acid (7-Cl-KYNA), one of the most selective and well-studied glycine site antagonists, can be synthesized in the brain from its bioprecursor L-4-chlorokynurenine (4-Cl-KYN). Intracerebral infusion of 4-Cl-KYN dose-dependently reduced quinolinate neurotoxicity in the rat hippocampus after enzymatic conversion to 7-Cl-KYNA by kynurenine aminotransferase. In accordance with previous studies demonstrating that kynurenine aminotransferase is preferentially localized in astrocytes, both the enzymatic formation of 7-Cl-KYNA and the neuroprotective potency of 4-Cl-KYN were substantially reduced following an intrahippocampal injection of the gliotoxin fluorocitrate. In situ produced 7-Cl-KYNA offers a novel neuroprotective strategy for targeting the glycine/NMDA site while avoiding excessive receptor blockade and reducing the clinical risks associated with conventional NMDA receptor antagonism.

L'acide 3-(4,6-dichloro-2-carboxyindol-3-yl) propionique ainsi que d'autres derives de l'acide indolepropionique representent une nouvelle classe d'antagonistes de l'acide N-methyl-D-aspartique, agissant au niveau d'un site de liaison de la glycine insensible a la strychnine

The syntheses and molecular modeling of several 3-acylamino-2-carboxyindole antagonists of the glycine binding site associated with the NMDA receptor complex is described. Tertiary amide 8 is a potent antagonist; this result is rationalized by a preferred cis amide bond conformation. The potency of the trans secondary oxalamide 10 is explained by binding to the receptor in its enolic form.

L'acide 3-(4,6-dichloro-2-carboxyindol-3-yl) propionique ainsi que d'autres derives de l'acide indolepropionique representent une nouvelle classe d'antagonistes de l'acide N-methyl-D-aspartique, agissant au niveau d'un site de liaison de la glycine insensible a la strychnine

Steroidal antiestrogens appear to have at least two major modes of action in breast cancer cells, direct antagonism of estrogen binding to its receptor and depletion of estrogen receptors (ER) due to inhibition of dimerization of the receptor and a resultant destabilization of the receptor protein. In a search for other classes of compounds which would act as dimerization inhibitors, a novel substituted indole (8-{2-[1-(4-chlorobenzoyl)-5-hydroxy-2-methyl-1H-indol-3-yl]-acetylamino} octanoic acid butyl-methyl amide, MDL 101,906) was synthesized. Binding of the ER to its consensus response element (ERE) was apparently decreased in nuclear extracts from MCF-7 human breast cancer cell treated with MDL 101,906. This decreased binding was found to be due to depletion of ER based on direct measurement of ER using an enzyme-linked immunoassay. Other transcription factors were apparently unaffected by MDL 101,906 treatment. Whereas depletion of ER with a steroidal antiestrogen was almost complete after 3 h of treatment of MCF-7 cells, the effect of MDL 101,906 took significantly longer to occur, suggesting a fundamental difference in the mechanisms of action of the two drugs. This was also evident in the lack of binding of MDL 101,906 to the hormone binding domain of ER. MDL 101,906 treatment also caused depletion of ER mRNA in MCF-7 cells. Depletion of ER mRNA was noted by 3 h of drug treatment and was apparently almost complete after 24 h of treatment. Depletion of ER from MCF-7 cells led to a dose-dependent decrease in the expression of luciferase by an ERE-driven luciferase reporter gene assay system. The mechanism of MDL 101,906 appears to be unique and additional studies with this chemical class seem to be warranted to assess the potential for therapeutic utility.

Glycine receptor antagonists have been proposed to have multiple therapeutic applications, including the treatment of stroke, epilepsy, and anxiety. The present study compared the biochemical and behavioral profiles of two strychnine-insensitive glycine receptor antagonists, MDL 100,458 (3-(benzoylmethylamino)-6-chloro-1H-indole-2- carboxylic acid) and MDL 102,288 (5,7-dichloro-1,4-dihydro-4-[[[4- [(methoxycarbonyl)amino]phenyl]sulfonyl]imino]-2-quinolinecarboxylic acid monohydrate). Both compounds potently inhibited [3H]glycine binding to rat cortical/hippocampal membranes (Ki = 136, 167 nM, respectively) without showing significant activity in 18 other receptor binding assays. In an in vitro functional assay, both compounds completely antagonized N-methyl-D-aspartate (NMDA)-stimulated cGMP accumulation in rat cerebellar slices. However, in contrast to their equipotency in the glycine receptor assay, MDL 100,458 was approximately 6-fold more potent than MDL 102,288 in the cGMP assay (IC50 values = 1.25, 7.8 microM, respectively). Behavioral tests demonstrated that MDL 102,288 and MDL 100,458 exhibited strikingly different in vivo profiles. MDL 100,458 antagonized audiogenic seizures in DBA/2J mice (ED50 = 20.8 mg/kg i.p.), whereas MDL 102,288 was without effect in the dose range tested (ED50300 mg/kg i.p.). Central nervous system penetration did not appear to account for this difference. For example, MDL 102,288 was not active following direct intracerebroventricular administration (ED5016 micrograms; vs. 0.78 microgram for MDL 100,458). In a test of anxiolytic activity, MDL 102,288 reduced separation-induced ultrasonic vocalizations in rat pups (ED50 = 6.3 mg/kg i.p.) whereas MDL 100,458 was only weakly active (ED50 = 80.8 mg/kg i.p.). Furthermore, the anxiolytic effect of MDL 102,288 was selective in that it occurred at doses that did not produce motoric disruption as measured by an inclined-plane test (ED50160 mg/kg; therapeutic index25.4). In contrast, the anxiolytic activity of MDL 100,458 was non-selective in that it occurred at doses that also produced motoric disruption (ED50 = 57.7 mg/kg; therapeutic index = 0.7). Thus, two glycine receptor antagonists which have similar in vitro binding profiles as selective ligands for the strychnine-insensitive glycine receptor, demonstrate different in vitro and in vivo functional profiles. The reason for these differences is not clear, though one possibility could be that the compounds may act on different NMDA receptor subtypes. These data support the possibility that different glycine receptor antagonists may have different therapeutic targets.

A series of substituted 3-(2-carboxyindol-3-yl)propionic acids was synthesized and tested as antagonists for the strychnine-insensitive glycine binding site of the NMDA receptor. Chlorine, and other small electron-withdrawing substituents in the 4- and 6-positions of the indole ring, greatly enhanced binding and selectivity for the glycine site over the glutamate site of the NMDA receptor; one of the most potent compounds is 3-(4,6-dichloro-2-carboxyindol-3-yl)propionic acid (IC50 = 170 nM; greater than 2100-fold selective for glycine). The importance of a heteroatom NH and the enhancing effect of the propionic acid side chain were demonstrated and are consistent with previous results which suggest the presence of a pocket on the receptor which can accept an acidic side chain. Substitution of a sulfur at C3 led to the most potent compound 3-[(carboxymethyl)thio]-2-carboxy-4,6-dichloroindole (IC50 = 100 nM).

A series of pepstatin analogues having structural variations in the P2', P1', and P2 positions have been synthesized and tested for inhibition of porcine pepsin. The standard peptide for this study was Iva-Sta-Val-Ala-Iaa. Structural variations in the P2' and P1' positions have relatively little effect on Ki; however, small variations in the P2 position have a more dramatic effect on Ki and time-dependent inhibition. A series of pepstatin fragments were also synthesized and tested for inhibition of porcine pepsin.

The synthesis of two new analogues of statine are reported corresponding to analogues with the lysine side chain and the ornithine side chain. These analogues were designed on the basis of substrate specificity and molecular modeling of three-dimensional structures of the penicillopepsin: Iva-Val-Sta-OEt crystal structure. 4,8-Diamino-3-hydroxyoctanoic acid [LySta] and 4,7-diamino-3-hydroxyheptanoic acid [OrnSta] were synthesized respectively from Boc-Lys(Z)-al and Boc-Orn(Bzl,Z)-al by addition of lithio ethyl acetate to the aldehyde group. The [LySta] derivative was converted to the trichloroethoxycarbonyl derivative and separated into the corresponding 3S,4S and 3R,4R diastereomers. The [OrnSta] derivative was used as a mixture of 3-position diastereomers. These new amino acids were used to prepare the following inhibitors: Iva-Val-Val-[LySta]-OEt and Iva-Val-Val-[OrnSta]-OEt as well as the corresponding synthetic intermediates. Inhibition constants (Ki values) were measured for inhibition of porcine pepsin and penicillopepsin. Both compounds were potent inhibitors of penicillopepsin with Ki values 10-100 times smaller (2.1 and 1.1 nM, respectively) than the Ki of Iva-Val-Val-Sta-OEt (47 nM). In contrast both inhibitors are exceptionally weak inhibitors of porcine pepsin with Ki values greater than 1 microM. These results are correlated with the ability of the basic group in the new inhibitors to bind to aspartic acid-77 in penicillopepsin.

The synthesis of 10 analogues of pepstatin modified so that statine is replaced by 4-amino-3-hydroxy-3,6-dimethylheptanoic acid (Me3Sta) or 4-amino-3-hydroxy-3-methyl-5-phenylpentanoic acid (Me3AHPPA) residues is reported. Both the 3S,4S and 3R,4S diastereomers of each analogue were tested as inhibitors of the aspartic proteases, porcine pepsin, cathepsin D, and penicillopepsin. In all cases the 3R,4S diastereomer (rather than the 3S,4S diastereomer) of the Me3Sta and Me3AHPPA derivatives was found to be the more potent inhibitor of the aspartic protease (Ki = 1.5-10 nM for the best inhibitors), in contrast to the results obtained with statine (Sta) or AHPPA derivatives, where the 3S,4S diastereomer is the more potent inhibitor for each diastereomeric pair of analogues. The Me3Sta- and Me3AHPPA-containing analogues are only about 10-fold less potent than the corresponding statine and AHPPA analogues and 100-1000-fold more potent than the corresponding inhibitors lacking the C-3 hydroxyl group. Difference NMR spectroscopy indicates that the (3R,4S)-Me3Sta derivative induces conformational changes in porcine pepsin comparable to those induced by the binding of pepstatin and that the (3S,4S)-Me3Sta derivatives do not induce the difference NMR spectrum. These results require that the C-3 methylated analogues of statine-containing peptides must inhibit enzymes by a different mechanism than the corresponding statine peptides. It is proposed that pepstatin and (3S)-statine-containing peptides inhibit aspartic proteases by a collected-substrate inhibition mechanism. The enzyme-inhibitor complex is stabilized, relative to pepstatin analogues lacking the C-3 hydroxyl groups, by the favorable entropy derived when enzyme-bound water is returned to bulk solvent.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthetic details for the preparation of a series of hydroxyethylene and ketomethylene dipeptide isosteres with control of stereochemistry at C(2) are described. Incorporation of the isosteres into peptide sequences derived from pepstatin afforded potent inhibitors of the aspartic protease porcine pepsin. When Leu-OH-Ala or Leu-OH-Phe was substituted for statine [3S,4S)-4-amino-3-hydroxy-6-methylheptanoic acid), inhibitors equipotent to the parent compound were obtained, whereas Leu-OH-Gly was a much less effective replacement for statine. A similar trend was evident in the corresponding ketones. The finding that structural features for good substrates do not closely parallel those for good inhibitors is discussed.

Synthetic details for the preparation of a series of hydroxyethylene and ketomethylene dipeptide isosteres with control of stereochemistry at C(2) are described. Incorporation of the isosteres into peptide sequences derived from pepstatin afforded potent inhibitors of the aspartic protease porcine pepsin. When Leu-OH-Ala or Leu-OH-Phe was substituted for statine [3S,4S)-4-amino-3-hydroxy-6-methylheptanoic acid), inhibitors equipotent to the parent compound were obtained, whereas Leu-OH-Gly was a much less effective replacement for statine. A similar trend was evident in the corresponding ketones. The finding that structural features for good substrates do not closely parallel those for good inhibitors is discussed.

The synthesis of two new analogues of statine are reported corresponding to analogues with the lysine side chain and the ornithine side chain. These analogues were designed on the basis of substrate specificity and molecular modeling of three-dimensional structures of the penicillopepsin: Iva-Val-Sta-OEt crystal structure. 4,8-Diamino-3-hydroxyoctanoic acid [LySta] and 4,7-diamino-3-hydroxyheptanoic acid [OrnSta] were synthesized respectively from Boc-Lys(Z)-al and Boc-Orn(Bzl,Z)-al by addition of lithio ethyl acetate to the aldehyde group. The [LySta] derivative was converted to the trichloroethoxycarbonyl derivative and separated into the corresponding 3S,4S and 3R,4R diastereomers. The [OrnSta] derivative was used as a mixture of 3-position diastereomers. These new amino acids were used to prepare the following inhibitors: Iva-Val-Val-[LySta]-OEt and Iva-Val-Val-[OrnSta]-OEt as well as the corresponding synthetic intermediates. Inhibition constants (Ki values) were measured for inhibition of porcine pepsin and penicillopepsin. Both compounds were potent inhibitors of penicillopepsin with Ki values 10-100 times smaller (2.1 and 1.1 nM, respectively) than the Ki of Iva-Val-Val-Sta-OEt (47 nM). In contrast both inhibitors are exceptionally weak inhibitors of porcine pepsin with Ki values greater than 1 microM. These results are correlated with the ability of the basic group in the new inhibitors to bind to aspartic acid-77 in penicillopepsin.

A series of pepstatin analogues having structural variations in the P2', P1', and P2 positions have been synthesized and tested for inhibition of porcine pepsin. The standard peptide for this study was Iva-Sta-Val-Ala-Iaa. Structural variations in the P2' and P1' positions have relatively little effect on Ki; however, small variations in the P2 position have a more dramatic effect on Ki and time-dependent inhibition. A series of pepstatin fragments were also synthesized and tested for inhibition of porcine pepsin.